AntiNeoplasms - Interesting reading

One interesting read in the field of Oncology is " Comparative Oncology", Specifically, Chapter 19, Principles of Anticancer Therapy" found at http://www.ncbi.nlm.nih.gov/books/NBK9546/ . Under a claim of "Fair Use" , since I do not hold the Copyright to this fine work, I quote from this work from Baba AI, Câtoi C. (The following extended quote is from that work...)

"Anticancer therapy, especially in veterinary medicine, was based and still relies almost exclusively on surgical therapy, although associated therapy has developed over the past decades: surgery and/or chemotherapy and/or radiotherapy, with the development of cryotherapy, immunotherapy and in general, the adoption of techniques and methodologies used in human oncology.

The therapeutic strategy should take into consideration three indispensable elements:

• – the histological nature of the lesion;
• – the assessment of the extension of the tumor process;
• – the evaluation of the general disease state.

The histological nature of the lesion should be known at least for the following reasons: not all tumors show the same sensitivity to chemotherapy or radiotherapy; antimitotic substances that possess an activity against some cell families and hematolymphopoietic organ tumors are not active against sarcomas; anticancer therapy should be capable of anticipating the probable evolution of the tumor process against which action should be taken [6].

Anticancer therapy should be evaluated for each individual patient, with all peculiarities involved. In this sense, therapy in neoplastic disease can be aimed at:

• – curative treatment, which determines recovery;
• – adjuvant treatment, which helps other complex therapies or prevents unfavorable evolution (recurrences, metastases) and participates at the same time in hopefully curative polytherapy;
• – palliative treatment, which cannot be aimed at recovery but only an improvement in symptomatology and/or the prolongation of life.

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19.1 SURGICAL TREATMENT

Cancer surgery has rules and principles specific for neoplastic disease. This involves not only the removal of the tumor as a local pathological formation, but also the involvement of the whole organism. In numerous situations, the simple excision with extensive resection beyond the tumor limits does not solve the problem of recurrence and metastasis. Surgery remains a basic method for oncology, although there are numerous limits and contraindications such as adenopathies, metastases, etc. This method is the key to success in many cases of animal tumors, because it is effective, easy to perform and economical. Contraindications can be synthesized for the following situations: multicentric cancer (leukosis); cancer with special locations: mesenteric lymph node tumors, facial, spinal, pelvic osteosarcomas; cancer characterized by important local extensions; tonsillar epidermoid carcinoma, bilateral thyroid adenocarcinoma, disseminated pancreatic insulinomas [22].

The therapeutic approach should include a well conceived plan: in the first place, this should be individualized, particularized for each patient, then it should involve the simultaneous treatment of metastases, paraneoplastic syndromes and side effects. The evaluation of the patient’s general state is essential, continuous maintenance over the whole duration of treatment being required or, if possible, even preoperative preparation.

It has been found that a successful operative act does not exclusively depend on the extension of resection, which has led to the concept of adequate excision. This concept includes, in addition to surgery proper, local and systemic factors. Local factors are represented by the characteristics of the neoplasm: size, location, invasion depth and integrity of surrounding tissue. At the same time, some factors will be considered that cannot be controlled by the surgical act, but which contribute to or may even have a determining role in the evolution and outcome of the disease. Among these factors, the following can be mentioned: the general state, immune status, metastatic potential and individual particularities (age, sex, breed, etc.). The understanding of the limits of the operative act allows the surgeon to individualize the extension of resection and avoid useless mutilating or massive surgery[12].

The attitude of the veterinary oncologist towards the animal affected by cancer should concern the subject’s condition, with the severity of the neoplasm stage and the sensitivity and attitude of the owner. In the case of a patient with bone cancer disease, the owner should be explained the situation and posttherapeutic prognosis, as well as the costs involved by a complex treatment, which can lead to the owner’s consent to euthanasia.

The preoperative evaluation of patients with cancer should include history, a thorough clinical examination, hematological, serological, urine and radiological examination. Based on all these data, the veterinary oncologist will present the situation to the owner, and their common decision will determine the therapeutic approach. The aim of each veterinary oncologist should be to satisfy the needs of each client and of the affected animal. A reasonable perspective should always exist, in the sense of a therapeutic approach that should relieve pain, improve symptoms and prolong life.

The knowledge of the tumor type influences the therapeutic approach. Mastocytomas are biologically active tumors that produce vasoactive amines, such as histamine and heparin. This can determine postoperative hypotension, hemorrhagic diathesis, and gastrointestinal ulcers, all with acute evolution. Another example is represented by the local excision of soft tissue sarcomas (fibrosarcomas, hemangiosarcomas, neurofibrosarcomas), which are associated with brutal acute recurrences. For the same neoplasms, treatment by the amputation of the affected limb may be curative [12].

The mechanics of surgical intervention influences the cancer recovery rate much less than other factors, such as the tumor type, location, growth type, biological behavior, as well as the patient’s general condition. In this context, it is essential that the veterinary oncologist should understand the nature of cancer, its development and dissemination, as well as the different therapeutic possibilities available for each type of neoplasm.

The therapeutic approach in surgical mechanics is based on several principles: all lesions suspected to be neoplastic will be histologically examined.

Surgery remains the most used therapeutic method in veterinary oncology, with peculiarities from one case to another. The operative decision depends in the first place on the owner, the animal’s chance of survival, and the surgeon. The cancer diagnosis suggests to the owner that the animal is doomed to die, sometimes in a short time, at other times by treatment, with the possible prolongation of life, but always with an inexorable end, death. In this situation, the owner is confronted with the dilemma of choosing between the initiation of treatment and euthanasia. The surgeon’s role is essential in explaining the patient’s situation, with all the possibilities and chances that exist in the particular case of each subject.

Another requirement of the surgical decision is to ensure a satisfactory quality of life before and after the intervention. The preparation of the owner is part of the surgeon’s obligation and ability to explain the advantages and disadvantages of surgery. There are some facts of which the owner should be informed, such as the situation of the amputation of a limb in the case of an osteosarcoma or the impossibility of surgery in the case of a multifocal neoplasm with dissemination in the organism. Sometimes, surgery is an alternative to euthanasia [2].

According to BARON and VALIN (1990), the main rules that should be respected in anticancer surgery are the following:

1. Evaluation of tumor extension and establishment of adequate reaction. Determination of tumor margins, envisaged behavior, and barrier formed by surrounding tissue. The routes of previous biopsies will be eliminated.
2. Protection of healthy tissue from possible tumor contamination. Contaminated fields, gloves and instruments will be changed.
3. The beneficial washing of the parietal wound is risky in cavitary surgery.
4. Minimal manipulation of the tumor during the placement of support sutures in healthy tissue.
5. Ligaturing of lymphatic and blood vessels as soon as possible and always before their sectioning.
6. Scraping of exposed tumor areas.
7. If drainage lymph nodes are invaded, block resection is performed, starting with the most peripheral one up to the primitive tumor.
8. Elimination of the whole tumor tissue even if cicatrization “per secundam” or by pedicled flaps occurs.
9. Avoidance of hematomas and edemas (sutures, drainage) that diminish local immunity, favoring recurrences.
10. Preservation of the margins of excised tissue for histopathological examination.

Types of excision in oncology (according to BARON and VALIN, 1990)

Excision type	Dissection	Source of failure	Lesional type	Examples
Intracapsular excision	Elimination of tumor mass Intracapsular section plane	Remaining microfragments	Benign lesions	Abscesses, bone cyst
Marginal excision	Block elimination of tumor and pseudocapsule Direct section plane of reactive area	Satellite or skip metastases	Benign tumors	Enucleation of lipoma
Extensive excision	Block resection of tumor, pseudocapsule from the reactive area, and a margin of healthy tissue Direct intracompartmental section plane of normal tissue	Skip metastases	Well localized or low malignant tumors	Amputation of auricular conchal area (squamous cell carcinoma) Partial mandibulectomy (oral fibrosarcoma)
Radical excision	Block resection of tumor and anterior compartment Extracompartmental section plane, in healthy tissue	Distant metastasis	Poorly localized or highly malignant tumors	Finger amputation (ungual carcinoma) Limb amputation (bone sarcoma)

Explanation:

Pseudocapsule: macroscopically visible membrane around the tumor that includes normal compressed cell layers and tumor cell layers compressed by tumor growth.

Reactive area: inflammatory tissue layer surrounding the pseudocapsule, mesenchymatous cell proliferation, inflammatory vessels and infiltrates.

Satellite metastases: tumor nodules separated from the primary tumor, but located in the reactive area (extension by extravascular contiguity).

Skip metastases: tumor nodules separated from the primitive tumor, located in the normal tissue, outside the reactive area, but in the same tissue compartment (vascular extension).

Tissue compartment: anatomical compartment defined by barriers that usually resist to tumor invasion. The resistance of these barriers varies depending on the tissue type, aggressiveness and tumor type.

The postoperative approach is focused on cicatrization, involving complementary treatments. Chemotherapy will be avoided during seven days before and after surgery, and radiotherapy during two or three weeks before surgery. Postoperative pain is most frequently neglected in the case of animals. Moderate pain will be successfully treated with aspirin and phenylbutazone. Great pain will be treated parenterally with narcotics: oxymorphine, butorphanol, meperidine or combinations of these [2].

Biopsy will be performed whenever it is possible. Incisional biopsy is only recommended when the excision of the whole tumor mass is not possible. Incisional biopsy causes changes in pressure, associated with intratumoral edema, the multiplication of malignant cells and the migration of these cells in veins and regional lymphatic vessels.

The manipulation of the tumor mass during the operation will be minimized, in order not to facilitate the diffusion of malignant cells in adjacent tissues. The washing of the wound immediately after the tumor excision reduces the number of malignant cells released from structures during the surgical act. The exfoliation of malignant cells after tumor excision can become an important cause of local recurrences of an otherwise completely excised neoplasm [12].

The elective removal of lymph nodes seems to have no direct effect on survival, although in some neoplasms, such as melanoma and mammary carcinoma, lymph node removal is recommended and beneficial. It should be mentioned that in the case of lymph nodes with metastatic signs, their removal is not mandatory. In addition, the histopathological examination of lymph nodes determines the adaptation of an adequate therapeutic approach.

Cancer surgery includes adjustments for each situation, taking into consideration all elements regarding the neoplasm as such and the patient in all his/her complexity. Thus, the following situations are distinguished: curative resection, palliative surgery, preventive surgery, diagnostic surgery, cytoreductive surgery.

Curative resection can only be used in the case of the absence of metastases, and the tumor is circumscribed, with the possibility of total excision. This situation is rarer in veterinary medicine, especially due to owners that bring animals late for consultation. In addition, advanced age makes the veterinary doctor delay surgery.

Palliative surgery aims to treat and prevent symptoms, to prolong the patient’s comfortable life. Thus, palliative treatment is considered the leg amputation of a dog with osteosarcoma. In this way, metastases and some complications can be avoided. In other situations, when recovery is unlikely, palliative surgery can bring improvements for a time period and/or the prolongation of life. The procedure of tumor fighting can be successfully applied to tumors with slow development and low metastatic rate. An example in this sense is oral fibrosarcoma in dogs. Sometimes, fibrosarcoma recurs after removal, but several interventions are possible without the appearance of metastases. Life will be prolonged by slowing down the normal tumor progression.

Preventive surgery can be applied by the excision of the benign, premalignant or in situ tumor, which prevents the possible onset of invasive malignancy. Preventive surgery can be performed by the removal of tissues or organs that can contribute to the possible appearance of neoplasms or present a higher predictable malignization rate than normal. Thus, early ovariohysterectomy can be performed in bitches, which prevents the occurrence of mammary carcinoma. The removal of testes in cryptorchid dogs reduces the appearance of neoplasms in these organs.

Diagnostic surgery is frequently performed under the form of excisional biopsy, when in addition to tumor removal, material is harvested for histological examination. Incisional biopsy provides material for microscopic diagnosis, without the complete removal of the tumor. Natural, especially peritoneal cavities, can be used by surgical route for the purpose of visualization, but also in order to sample material for microscopic examination. The tumor can also be totally removed. Endoscopic techniques, such as gastroscopy, colonoscopy and laparoscopy can avoid the performance of laparotomy.

Cytoreductive surgery results in a reduction of tumor cell pressure, so that other simultaneous therapeutic methods can be more effective. Thus, surgery improves the efficacy of adjuvant therapy by the mechanical reduction in the number of cells that will be treated. Observations show that, following the reduction of the large tumor mass, the cell duplication time decreases, and cells proliferate much more rapidly. Rapid cell divisions, with an increased metabolic rate, are much more sensitive to cytotoxic medication. The decrease in volume of the tumor also allows the reduction in the amount of “blocking antibodies”, allowing a significantly more effective functioning of endogenous immunological mechanisms.

Oral cavity tumors in dogs have a high incidence, and in most cases they are treated by surgery. As shown by the synthesis performed by MOISSONNIER and DELISLE (1990), the result of exeresis depends almost exclusively on the histological nature of the tumor. This requires the establishment of the histological type before surgery.

Benign tumors:

• – viral papillomas, treated by exeresis with an electric or mechanical scalpel, good prognosis;
• – epulis, after bone resection, prognosis is good;
• – ameloblastoma, after bone resection, prognosis is good.

Malignant tumors:

• – melanoma, after “convenience” local surgery, prognosis is gloomy;
• – non-tonsillar squamous cell carcinoma, after extensive resection, prognosis is severe;
• – tonsillar squamous cell carcinoma, after extremely early extensive resection, prognosis is gloomy;
• – fibrosarcoma, after extremely early extensive resection, local prognosis is severe;
• – osteosarcoma, after bone resection, prognosis is severe.

In conclusion, veterinary cancer surgery should respect several basic principles:

• – biopsy-exeresis is preferable to biopsy, especially when a melanoma is involved, which can easily disseminate;
• – the ablation of cutaneous tumors will not be performed under local anesthesia, since this attenuates the tumor limits;
• – preoperative and intraoperative manipulation will be minimized, in order to reduce the risk of emboli;
• – as early as possible ligaturing of venous vascular pedicles, in order to reduce the risk of metastasis.
• – maximum detachment of tissue around the tumor.

The therapy of cancer disease recognizes that surgical intervention is the most effective and practical method, which requires an adequate control of the technique specific for each case. It should be mentioned that surgery is local therapy in a disease that is essentially systemic. This requires a conceptual understanding of the biology of malignant disease by the surgeon, and the need to consider the extensive implications that the development of the neoplasm may cause.

Cryosurgery. Cryosurgery is defined as a method that uses the local application of cold generated in order to destroy abnormal tissues, with the possibility of the maintenance of normal adjacent tissue. Normal tissue can be maintained because the extension of the ice ball can be controlled. Cryosurgery cannot replace conventional techniques, but in certain situations, freezing can represent the most effective method to destroy neoplastic tissues [29].

Cryobiology requires several rules in the use of cold: the crystallization of interstitial water starts at 0°C, and this can be performed by slow freezing and rapid freezing.

Slow freezing allows the formation and development of crystals in intercellular spaces as temperature decreases, and the unfrozen solution on the surface of these crystals becomes much more concentrated with the transformation of water in ice. The pressure of ice vapors and the development of a high osmotic gradient force the water out of cells and make them dilute the remaining solution. The development of ice crystals and cellular dehydration continues until all the salt and water have frozen at a temperature of −22°C. Before freezing is complete, the salt concentrations from the unfrozen electrolyte portion denature lipoproteins, causing the disappearance of cell compartmentalization, as well as a free movement of salts and water over the membranes of damaged cells. The additional effect of distortion and mechanical destruction as ice crystals increase contributes to complete cell death.

Rapid freezing does not allow the time needed for the increase of large crystals or the loss of compartmentalization, small ice crystals being thus formed all through the system. The intercellular fluid and cytoplasmic components freeze simultaneously, producing small ice crystals, which pass through the cell membranes, causing severe physical lesions in the rapidly frozen tissue.

Defreezing also has a special importance, i.e. slow defreezing causes a maximum of cell death. Slow thawing allows the recrystallization of small ice crystals into large crystals, stimulating the tissue fragmenting effect of ice. After the lysis of cell membranes, electrolytes and water are moved from the intercellular fluid into and out of the cells.

Rapid freezing does not allow the time needed for recrystallization or for the loss of cell compartmentalization, which allows less damaged cells to survive the freezing process. The rapid defreezing of tissues that have been progressively frozen will reduce the time needed for concentrated saline solutions to act on cell membranes, favoring in this way cell survival.

Regarding cryobiology, ROBINSON (1987) synthesizes the effects of cold on cells as follows:

• – thermal shock occurs as a result of the acute action of temperature; although the biological effect is not sufficiently elucidated, it contributes to cell death;
• – ice crystals physically destroy the cell;
• – cell dehydration occurs by the blockage of water during freezing;
• – toxic electrolyte concentrations among and around the cells appear at temperatures between −2°C and −10°C. Hypertonicity due to increased salt concentrations leads to the precipitation of proteins and deregulation of the solubility balance of lipoproteins;
• – true proteolysis occurs; lipoproteins are maintained together by a poor association of some chemical bonds, which are easily broken with the rapid change of temperature;
• – pH changes are caused by the loss of buffer salts in the course of crystallization, which allow the appearance of toxic gas concentrations and dissolved urea;
• – anoxia occurs as a result of vascular stasis.

Although frozen, after thawing the main blood vessels will continue to supply with blood the tissues incorporated in the ice block, while arterioles, venules and capillaries are seriously affected. During thawing, arterioles and venules contract immediately and resume a reduced blood flow with the almost instantaneous and continuous production of thrombi. Momentary vasoconstriction is followed by vasodilation. Stasis starts from the venous part, the capillary flow is diminished and consequently, the arteriolar flow is slowed down. Damaged blood platelets, coagulation factors and cell elements adhere to vascular walls during thawing. Since the blood flow is slowed down by a partial or complete vascular occlusion, the adhesion of thrombi is much more marked, causing complete stasis. This process induces a complex infarction of the cryolesion within 24 hours.

Cryosensitivity is extremely high for cells with a high mitotic activity, with a high oxygen and fluid content. Tumors have a different cryosensitivity; thus, melanomas and adenomas are cryosensitive, while osteosarcmas are cryoresistant; a highly vascularized tissue manifests cryoresistance.

Main clinical changes after surgery: edema appears within 1–6 days; necrosis occurs within 3–8 days, the color of the area is black; the demarcation is visible at 4–21 days, and healing is completed by day 28; the color of skin is restored after a few months, but the color of hair remains white. The histological changes of tissues submitted to cryosurgery consist of the following: during the first 10 minutes, nuclear pyknosis occurs; within 1 hour edema and dilation of capillaries appear; at 1–3 days, cell dissociation is initiated, with pyknosis and karyolysis; after 1 week, coagulation necrosis appears; at 2 weeks, granulation tissue is formed, and at 4 weeks, the epidermis is restored.

Cryosurgery causes the rapid injury of sensitive nerves, which makes pain disappear; the treatment of dogs requires only manual restraint or sedation; cats are treated under general anesthesia. The most used substance is liquid nitrogen, which determines a temperature of −195.6°C and is applied by direct spraying on the affected area, or indirectly, by the contact of tissues with metal applicators through which a cryogen passes. The neighboring tissues will be protected, but along with the treatment of tumors, a safety area of healthy tissue is cryogenized, 3–5 mm for benign tumors and 10 mm for malignant tumors. In the case of malignant tumors, 3 consecutive freezing-defreezing cycles are performed.

In 1996, SIEBERT and BEHRENS showed the advantages and disadvantages of the use of cryosurgery; advantages include: absence of pain, no risk of dissemination of tumor cells, low risk of infection, minimal hemorrhage, possibility of repeating the intervention, physiological functions are maintained, no scars are formed, the procedure is easy and rapid to perform, low costs; disadvantages are few and minimal: no direct estimation of the effect is possible, the lesion is unpleasant to see and smell, postoperative edema occurs, and the area remains depigmented [39].

An optimal freezing requires the following conditions [29]:

1. Rapid freezing to produce small intercellular crystals.
2. Slow thawing to allow recrystallization and prolong the effects of increased electrolyte concentrations.
3. Repetition of the freeze-thaw cycle, at least two times, in order to reduce the possibility of survival of cells that are not severely damaged by the first freeze-thaw cycle.
4. The successful fighting of highly malignant tumors requires freezing for several minutes at temperatures of −50°C to −60°C before complete thawing and then refreezing. Previous literature data recommended temperatures of −20°C to −30°C [22].

The ice ball starts to form when the cryoprobe reaches the temperature of 0°C, continuing to radiate around under a spherical form, as the tissue reaches the temperature of 0°C. Multiple, successive freezings in the same location will interfere with the blood flow, inducing a decrease in the heat flow from the ice ball. By this phenomenon, the ice ball becomes larger with each successive freezing.

Cells from the rapid freezing area that is closest to the cryoprobe are exposed to the most serious degradation. Here occurs the most rapid heat loss. The thawing duration is prolonged, and the tissue temperature is the lowest. Cells from other areas, especially from the slow freezing region, freeze more slowly, thaw more rapidly and have a better chance of survival. In these areas, multiple freezings offer the maximum effects of optimization of cell destruction.

Cells from peripheral areas will not be frozen, but just refrigerated at the maximum level. During refrigeration, temperature decreases slowly enough to maintain the balance between the cytoplasm and the intercellular fluid and ice. Cell structures are not lost and the effects of severely damaged electrolyte concentrations and pH changes do not manifest. These cells are not severely damaged in the case of rapid thawing, and many of them will survive the freezing process.

The most effective techniques for the rapid freezing of neoplasms will include all areas, even the deepest ones. In veterinary medicine, where large neoplasms are frequently found, this process is difficult to carry out. It has been estimated that the temperature level of 10°C/mm is usual at the periphery of the ice ball; this is why, in order to include all suspect tumor cells from the temperature range between −10°C and −60°C, the ice ball will be extended to at least 10 mm beyond the external limit of the neoplasm.

A remark should be made: the owner must be warned that necrosed tissues have an unpleasant appearance and smell, which does not involve a complication. In order to reduce these phenomena, the lesion can be washed and cleaned with oxygenated water [22].Cicatrization occurs by granulation tissue. The mentioned authors indicate the use of cryosurgery in the case of eyelid and skin tumors, for multiple benign tumors (sebaceous adenomas, papillomas, etc.) and malignant tumors: spindle cell carcinomas; malignant mastocytomas; fibrosarcomas. Among oral tumors, the following are mentioned: melanomas; epidermoid carcinomas; fibrosarcomas. The same authors remark the advantages of cryosurgery in relation to its shortcomings:

• – general anesthesia is useless, lesions are small and duration is short;
• – it treats lesions that are difficult to treat by classical surgery: anal margin, bone invading oral tumors, etc.;
• – it is accompanied by immunostimulation;
• – there is a hemorrhage risk at the end of thawing;
• – the cryonecrosed tissue progressively eliminates the unpleasant appearance and smell;
• – the scar and surrounding skin remain depigmented;
• – special equipment is needed.

SIEBERT [39] recommends the use of cryotherapy especially for tumors located in the skin and its adnexae. In veterinary medicine, cryosurgery is used in malignant and benign skin tumors. In general, 2 freezing-defreezing cycles are applied for benign tumors and 3 cycles in the case of malignant tumors. Infiltration anesthesia is recommended in the intervention area.

Mast cell tumors with a diameter larger than 1 cm are not treated by cryosurgery because they induce cell degranulation resulting in anaphylactic shock.

In human medicine, cryosurgery is successfully used for malignant skin tumors; in the case of inoperable malignant forms, it can be used to prolong the patient’s life. The operation can be repeated several times; for example in the case of recurrent or inoperable squamous cell carcinoma, cryosurgery allows to control the neoplasm for years, most frequently until the natural death of the animal [39].

Cryosurgery is recommended in the case of eyelid tumors, such as Meibomian gland adenoma, papillomas, without any recurrences, as well as in squamous cell carcinoma of the eyelid in the cat [39].

Cryosurgery is recommended in the benign forms of the oral cavity (multiple fibromatous epulides); in the benign tumors of the external auditory duct; in perianal adenomas, but not in invasive malignant forms; it is recommended in the transmissible venereal tumor of the dog [39].

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19.2 RADIOTHERAPY

Radiotherapy is based on the fact that ionizing radiation destroys tumor cells. X rays and gamma rays are able to penetrate the tissue depth, destroying tumor cells even from deep layers. Radiotherapy induces direct lesions in the DNA or biological molecules, which eventually affect DNA. These changes deregulate cell division, and daughter cells finally die.

In veterinary medicine, radiotherapy has a rather limited use due in the first place to the reduced number of specialists, veterinary oncologists, but also to the high costs of necessary equipment. It is indicated in the case of voluminous or extensive tissues, in the case of anatomical locations that are inaccessible to surgical excision, and especially in the case of metastases. Radiotherapy is envisaged as a treatment that reduces the volume of the primary tumor and/or extensive metastases, or as preventive therapy in the case of some filter organs threatened by neoplasms [11].

Radiotherapy uses corpuscular or electromagnetic radiation emissions that penetrate the tissues, causing the ionization of the environment, determining more or less important molecular lesions and variable biological manifestations. The effect on cells is that of the loss of the multiplication capacity, death, or lesion repair. Corpuscular radiation or particle radiation with directly ionizing particles can also be used, such as: electrons and electromagnetic radiation, photons, which are indirectly ionizing; X rays andgamma rays, which act on the constitutive atoms of cells. X and gamma rays are radiations with reduced linear energy transfer, while neutron and alpha particle radiations are radiations with high linear energy transfer.

Radiotherapy can be used in three modalities:

• – curative radiotherapy;
• – adjuvant radiotherapy;
• – palliative radiotherapy.

Curative radiotherapy is indicated to be used in: Sticker’s sarcoma, mastocytomas with particular locations, such as ears, nose, extremities, eyelids, etc.; acanthomatous epulis; ameloblastoma; nasal cavity adenocarcinoma and anal gland adenocarcinoma. Success depends on early diagnosis, the volume and histological nature of the tumor.

Adjuvant radiotherapy is associated with surgical therapy and/or chemotherapy.

Radiotherapy associated with surgery can be used preoperatively, in order to reduce the tumor volume before excision and to limit the diffusion of metastases. Postoperative radiotherapy is indicated in the case of recurrent tumors or early diagnosed metastases. This procedure is recommended in recurrent connective tumors: hemangiopericytoma; malignant fibrohistiocytoma; neurofibrosarcoma; fibrosarcoma; and schwannoma. In soft tissue sarcoma in dogs, resection followed by radiotherapy led to an excellent survival period [21]. This therapeutic protocol did not require amputation. Acute toxicity and postradiotherapeutic phenomena were minimal, in the case of the protocol used. The 5 year survival rate was 76%, without being influenced by the tumor type or location. In dogs that developed metastases, the mean survival time was 250 days. The mean interval in all unaffected dogs was 1082 days. All treatments used cobalt 60; treatment was applied on alternative days (three fractions of 3 Gy/WK), the number of administered fractions being up to 21. The metastatic epithelial or glandular tumors in which postoperative radiotherapy can be used are: mammary, thyroid, tonsillar adenocarcinomas and squamous cell carcinoma located in the finger, lip and nostril.Intraoperative radiotherapy is used in the case of exploring laparotomy in order to treat urinary bladder, prostatic or pancreatic carcinomas.

Radiotherapy associated with chemotherapy is used in neoplasms with a high dissemination capacity. This therapy increases locoregional activity (mediastinal lymphoma, bone tumors, lung carcinomas) and therapeutic activity at distance, being also used against the diffusion of metastases (mammary tumors, melanoma, tonsillar carcinoma, etc.).

Palliative radiotherapy, less used in veterinary medicine, can control advanced or extremely late diagnosed tumor processes [8].

The main radiotherapeutic methods can be classified in: teleradiotherapy, brachyradiotherapy, and metabolic radiotherapy.

Teleradiotherapy is also termed external transcutaneous or distant radiotherapy , in which case the radiation source is situated at a certain distance from the tissue to be treated. In veterinary medicine, teleradiotherapy preferentially uses X rays, cobalt 60, strontium 90, and electrons.

X rays are absorbed by several centimeters of soft tissue, being recommended in the treatment of cutaneous tumors, in general with 220 Kpv (200–500 Kpv).

Cobalt 60 is recommended in less deep tumors, and the source is decreasing to 10% in order to be frequently applied.

Strontium 90 has a limited penetration, of 2–3 mm, and is used in the treatment of superficial ocular lesions, such as epidermoid carcinoma, without the risk of crystalline lens radiation.

Electrons allow by circular and linear accelerations the use of high energy electron bundles. The therapeutic advantage of good penetration and focalization is countered by the economical factor, which limits their use in veterinary oncology.

Brachytherapy is also termed short direct radiotherapy or curitherapy. The radiation source can be placed in contact with the tumor or it can be implanted in the tumor.

In veterinary medicine, direct radiotherapy uses radioimplants (needles), most frequently cobalt 60, cesium 137, strontium 90, more rarely iridium 192. The application of radioimplants is recommended in particular in:

• – large animals, in which teleradiotherapy is difficult to perform;
• – small animals, in the case of small and surgically inaccessible tumors;
• – tumors justifying radiotherapy, situated in the proximity of highly radiosensitive organs (periocular carcinomas and sarcomas in large animals), in which the use of teleradiotherapy is dangerous.

The advantages of direct therapy would be continuous radiation, and the accumulation of a higher dose by neoplastic tissues compared to healthy adjacent tissues. A major inconveniency is the exposure of the surrounding environment by the subject that carries radioactive material.

Metabolic radiotherapy represents the administration of isotopes or isotope carrying molecules that selectively concentrate in a certain tissue into which they enter as metabolites (iodine 131, in thyroid cancer; phosphorus 32, in myeloproliferative disorders)[8].

The radiation of the whole body is especially used in the treatment of malignant lymphomas in dogs and cats. The administration of 10–12 Gy as a single treatment or 7–9 Gy repeated after 2 days is considered. The basic principles of this therapy consist in the destruction of neoplastic cells, sterilization of the infiltrated bone marrow and bone marrow transplantation. Without bone marrow transplantation, survival is reduced (2–42 weeks) [36].

The radiosensitivity of non-tumor tissues is different, with an increased sensitivity of cells in division. The tolerance of normal tissues varies considerably, being influenced by the tissue type, the intensity and duration of treatment, the fractioning of radiation and other local and general situations. The radiosensitivity of a normal tissue is proportional to the mitotic index. Cells in mitosis are most sensitive to ionizing radiation, while the S cell phase ensures a high resistance. As cells pass through 62 and M, they become more sensitive to radiation. The response of a tissue exposed to radiation includes two phases, an immediate or acute phase and a later phase. Thus, the oral mucosa exposed to radiation responds by an exaggerated mucus secretion, while a marked wet desquamation occurs at skin level. Intestinal radiation in sufficiently high doses can cause death.

Nerve and muscle cells are relatively radioresistant, since they do not divide, while bone marrow, intestinal and skin cells are radiosensitive, as they normally have a higher mitotic index. Endothelial cells and fibroblasts have an intermediate sensitivity to radiation. Subsequent effects can be extremely important, because the proliferation of arteriolar endothelium reduces the vascular lumen, and the proliferation of perivascular fibroblasts determines tissue hypoxia.

The radiosensitivity of tumor tissues differs depending on the cellular component and to a large extent, on vascularization, with the mention that tumor cells are not significantly more sensitive to radiation than normal tissues. The radiosensitivity of neoplastic tissues depends on three factors: the mitotic cycle phase in which irradiated cells are found; the pericellular environment; and the histological type [22].

The mitotic cycle phase increases or decreases radiosensitivity, although radiations act on cells regardless of the mitotic cycle phase in which they are. The most intense action is exerted in phases G2 and M characterized by the presence of 4 nchromosomes. The most resistant cells are those that are found in the S phase. The second radiation is more effective than the application of a single radiation, cells in phase S that have survived and pass to 62 and M phases during the time interval between the two radiations being much more sensitive.

The pericellular environment influences radiosensitivity, in the sense that voluminous tumors and necroses from their center determine a decrease in radiosensitivity. Due to poor oxygenation, in highly cellularized tumors, solid tumors, anoxic cells have a resistance to radiotherapy 2–3-fold higher compared to well vascularized cells. Oxygen and in general all oxidants are radiosensitizers, hyperthermia acting in the same direction (41–43°C). In contrast, the presence in the environment of SH and NH+group molecules (the case of cysteamine) captures OH− free radicals formed on the occasion of substance ionization, consequently having a radioprotective effect.

The histological type should be established by biopsy and histological examination, in order to assess the metastatic potential and the radiosensitivity of the tumor. An increased radiosensitivity have lymphomas, testicular seminomas, carcinomas, while malignant connective tumors (sarcomas) are much less sensitive.

Consequently, combined therapy (radiation, chemotherapy and hyperthermia) is recommended in order to reduce the heterogeneity of tumor response. This heterogeneity is a significant factor, even for tumors of the same histological type, with the same location and at the same stage.

The methodology of radiotherapy requires the clarification of some terms and the definition of the units that will be used in the therapeutic approach [22]:

• – rad is a unit equal to 100 ergi, absorbed in one gram of tissue (universal unit of ionizing radiation absorption);
• – Gray (Gy) is 1 joule ionizing energy absorbed in one kilogram of matter (1 Gy = 100 rad);
• – Curie (Ci) represents the activity of bodies that provide 3.7 × 1010 disintegrations per second;
• – electron-volt (ev) is the energy released by an electron submitted to a potential difference of 1 volt: 1 ev = 1.6 · 10−9Joule = 1.6 · 10−12 erg. Multiples of ev are: kilo-electron-volt (Kev); million electron volt (Mev).
• – Roentgen (R) is a unit of exposure (radiation output) that corresponds to the measure of the ionizing radiation capacity of air ionization. Practically, only rad and Gray are absorbed dose units, in the therapeutic effect.

The use of radiation in the treatment of cancer is based on: physical radiation methods; tumor characteristics; different approaches in cancer therapy [36].

Radiotherapeutic protocols should be preceded by complete clinical examination, accompanied by pulmonary radiography and tumor biopsy. The results of clinical investigations will allow to choose radiation, the lethal dose, exposure and therapeutic fractioning.

Radiotherapy can be administered using extremely varied equipment. Radiation emitted from a distance of 35–100 m is called teletherapy or external beam radiotherapy and is emitted by orthovoltage, cesium 137 and cobalt 60 X rays, with linear accelerator or heavy particle accelerator (cyclotron). Each unit is characterized by the type and energy of radiation produced, the difference consisting in the penetrability and absorption of the different tissue radiations. In the case of orthovoltage X rays, the maximum radiation absorption occurs at the surface of the region, with a differentiated absorption in tissues. Thus, for bones, a 4-fold higher dose will be administered compared to soft tissues. This treatment is recommended for dermal and superficial soft tissue tumors. Cesium 137 emits gamma rays whose energy is approximately half the energy of gamma rays produced by cobalt 60. Apparatuses that use cobalt 60 are preferred because they have a higher dose rate and a better distribution dose than those using cesium 137. Both cobalt 60 and cesium 137 are skin protective, which is not found in X ray treatment; in addition, the two sources of radiation are recommended to be used in bone, soft tissue and deep tumors [36].

The selection of the radiation type will be conditioned by the nature of exposure: of surface or depth. Then, the dispersion of radiation will be taken into consideration. Thus, for photons, the dispersion of radiation is considered to be reversely proportional to the square of distance, which is extremely important for the treatment of large tumors. The distance between the radiation source and skin, as well as the distance between the source and the tumor are elements that will be defined before the initiation of treatment.

A thorough clinical examination will establish the volume of the tumor to be radiated, of the infiltrated adjacent tissue and adjacent lymph nodes, as well as of the healthy tissue around the tumor. For a maximal efficacy, the technique of multiple radiation beams can be used, with the protection of healthy tissues.

The final therapeutic scheme consists of the administration of a maximal dose in the tumor center, a “sterilizing” dose in the surrounding tissues and a minimal dose in healthy tissues [22].

The selection of the total dose or eradication dose depends on the tumor radiosensitivity and volume. The histological type significantly influences radiosensitivity. Tumors with an extremely high radiosensitivity are known: lymphomas, Sticker’s sarcoma, canine circumanaloma; other tumors have a moderate radiosensitivity: malpighian and glandular carcinomas and radioresistant tumors, such as support tissue sarcomas.

Extremely large tumors require a complementary dose of 500–1500 rad. Dose fractioning leads to an increased tolerance to radiotherapy, and efficacy also improves. Thus, between each radiation:

• – cells follow their synchronized cycle;
• – cells in the G0 phase are recruited;
• – hyperemia favors tumor oxygenation;
• – the destruction of cells situated in the oxygenated segment allows cells in anoxia to “migrate” in the capillaries;
• – the proliferation of normal unaffected cells ensures tissue repair.

Total doses range for animals between 1500 and 5500 rad, administered in 2 or 3 fractions per week, of 1000–5000 rad each [22].The same authors mention some practical data regarding the radiosensitivity of tumors and healthy tissues, and total doses also involve lesions secondary to radiotherapy.

Radiosensitivity of tumors:

• – extremely sensitive: cancers of embryonic origin; lymphomas; Sticker’s sarcomas; circumanalomas; seminomas-goniomas;
• – sensitive: cutaneous-mucous carcinomas; glandular carcinomas;
• – poorly sensitive: various sarcomas; osteosarcomas.

Taking into account the radiosensitivity of healthy tissues, the total dose cannot exceed for:

• – cartilage of conjugation and bone marrow: 500 rad;
• – digestive tract, testes: 1500 rad;
• – liver, lung, kidney: 3000 rad;
• – eye, spinal cord: 4000 rad;
• – heart, bone: 5000 rad;
• – skin, bladder: 6000 rad;
• – muscles; 8000 rad.

Total doses in the treatment of some tumors in humans and animals:

in humans:
seminoma:	2500–3000 rad
lymphoma:	4000–5000 rad
epithelioma:	5500–6000 rad
glandular carcinoma:	6000–8000 rad
osteosarcoma:	≥ 8500 rad
connective sarcoma:	≥ 8500 rad
in animals:
circumanaloma:	1000–2000 rad
myeloid leukosis:	1500 rad
Sticker’s sarcoma:	1500 rad
epidermoid carcinoma:	4000 rad
glandular carcinoma:	4000 rad
mastocytoma:	5300 rad
fibrosarcoma:	5500 rad
melanoma:	5000 rad

In humans, total doses are of about 1000 rad per week, administered in 3–4 sessions.

Manifestations secondary to radiolesions of different tissues or organs:

• – skin: local erythema, pruritus, hair loss, pigmentations, dry or wet dermatitis;
• – intestinal mucosa: aplasia, vomiting, incoercible diarrhea, stenosis, perforation;
• – bone marrow: medullary aplasia, pancytopenia;
• – nerves: neuritis;
• – eye: cataract;
• – subcutaneous connective tissue: sclerosis;
• – muscles: sclerosis, atrophy;
• – neck region: laryngeal edema, cartilage necrosis;
• – head region: nasal cartilage necrosis, osteonecrosis.

Radiotherapy in veterinary oncology is a more recent method, which requires investigations of different tumor types, for each species. In principle, radiotherapy should be considered as a viable treatment for any animal with non-resectable tumors or in the case of recurrences.

Radiotherapy will only be used after the following parameters have been known and evaluated:

• – the histological nature of the tumor;
• – locoregional extension;
• – general extension;
• – the volume of the target tissue [8].

The remarks regarding the adoption of an individual approach, specific for each affected subject, depending on the histological type and subtype of the neoplasm, maintain their validity. However, some more general aspects should be defined, especially in dogs and felines [36].

Canine tumors. These are presented in what follows.

Acanthomatous epulis and adamantinoma are periodontal and odontogenic tumors, respectively. Both are benign and respond favorably to radiotherapy. Radiation doses higher than 36 Gy are salutary in more than 85% of cases.

Central nervous system tumors include meningiomas, astrocytomas, gliomas, oligodedrogliomas, choroid plexus tumors and pituitary tumors. The cited author mentions that cobalt 60 radiation with a total dose of 30–40 Gy led to a mean survival of 5 months. A month after treatment, the majority of dogs showed a marked improvement in clinical signs, with a reduction in tumor size, controlled by tomography.

Chemodectoma, the carotid and aortic body tumor, is extremely sensitive to radiotherapy in humans. In dogs, the few cases treated by radiation have responded favorably.

Fibrosarcoma is a highly malignant tumor and, in most cases, surgical resection results in recurrences and metastases. Cases show that radiation does not give good results either. The explanation consists in the presence of a high number of hypoxic tumor cells, resistant to radiotherapy. Encouraging data in the fight of canine sarcoma are obtained from the combined use of hyperthermia and radiation, with good results in more than 70% of cases.

Hemangiopericytoma has been treated with good results by surgical excision, and radiation has been successful in the case of metastases and incomplete resections. The use of orthovoltage X rays, by both teletherapy and interstitial brachytherapy, has led to encouraging results. In the case of the combination of hyperthermia with orthovoltage radiation in low radiation doses, the objective response of the tumor has been found in 91% of cases, and the tumor has been successfully fought in 54% of cases.

Lymphoma requires combined treatment, radiotherapy associated with chemotherapy, although lymphoid tumors are extremely sensitive to radiation. The areas with lymphomatous infiltrations that induce dysfunctions and compressions can be successfully treated by applying radiotherapy with 5–20 Gy doses. Marked regressions are obtained even within 12 hours.

Melanoma is a relatively radioresistant tumor, but high radiation doses cause a complete tumor regression, in 88% of the treated dogs. Subjects with melanomas die due to recurrences and generalized metastases.

Mastocytoma is a tumor that responds positively to radiotherapy. The recovery rate at one year is estimated between 51 and 100%, and the survival duration in treated dogs is much longer compared to surgical excision. The results reported by the literature clearly prove that radiotherapy is necessary in incompletely excised or inoperable mast cell tumors.

It is estimated that intranasal tumors in dogs can be successfully treated by radiotherapy. The use of orthovoltage X rays is recommended, which ensures a mean survival time of 23–25 months, and more than 50% of treated subjects survive after 1 year. In conclusion, radiotherapy with or without surgical resection is recommended for dogs with nasal tumors.

Perianal gland adenoma and adenocardnoma. The benign form, adenoma, can be successfully treated with low radiation doses. Because these tumors are hormone dependent, being stimulated by androgenic hormones, castration is recommended. The adenocarcinoma of this gland is more or less sensitive to radiation. In dogs with adenocarcinomas, but without metastases, surgical resection followed by radiotherapy results in a control of tumors in approximately 60% of cases.

Prostate carcinoma. The literature shows that radiotherapy has not been used in this neoplasm. Intraoperative radiation [36] was used in 9 patients with prostate carcinoma, the survival duration in 6 dogs being 6 months, without the detection of metastases.

Dermal squamous cell carcinoma appears under the form of a wound in the unpigmented skin. Superficial X rays or electron radiations represent adequate therapy, these tumors being considered as radiosensitive tumors. Squamous cell carcinoma with nasal location did not respond favorably to orthovoltage teletherapy. In contrast, interstitial brachytherapy with iridium 192 was efficient, tumor growth being successfully fought in the case of 5 dogs, with a tumor-free period between 3 and 23 months.

Squamous cell carcinoma with oral location is relatively radiosensitive in dogs. With doses higher than 40 Gy, the control of tumors was found in 46% of dogs, after one year. Tonsillar squamous cell carcinoma benefits from a palliative result, following fractioned teletherapy. In general, squamous cell carcinomas with minimal bone involvement and rostral location in the oral cavity seem to be significantly more radiosensitive than carcinomas located much more caudally.

Thyroid adenoma and adenocarcinoma in dogs are accompanied or not by hyperthyroidism. Thyroid adenomas are easy to remove by surgery when the tumor is accompanied by hyperthyroidism, radioactive iodine treatment being successful. In dogs, most cases of thyroid tumors are malignant, usually being non-functional or hyperfunctional, and radioactive iodine treatment is not indicated. The indication is to use external radiation for incompletely excised thyroid tumors.

Transmissible venereal tumor is particularly sensitive to radiation, doses of 10–30 Gy being sufficient to obtain a complete control of the tumor, in up to 100% of the treated cases.

Tumors in felines. The most common tumors are described in what follows.

Feline lymphoma is a neoplasm with multiple locations, which recommends either local radiotherapy or the radiation of the whole body. In the case of large tumor masses, which cause compressions or obstructions, prompt treatment can save the cat’s life, especially in the case of neoplasms that induce obstructions or compressions in the trachea, esophagus or intestine. In the case of thymic, orbital, nervous system and renal lymphomas, clinical distress signs disappear very soon. Usually, remission is obtained even after the administration of low doses (5–20 Gy).

Nasal lymphoma in cats is more sensitive to radiation than in dogs. Following the administration of low doses, lymphoma regresses and prior partial or total excision can be sometimes performed.

Solitary mastocytoma with cutaneous location responds to radiation by superficial teletherapy. Eosinophilic granulomas of the lip and skin respond favorably to radiation.

Squamous cell carcinoma with dermal location, more frequently in the nose, nostrils, eyelids, ears and preauricular areas is more difficult to access for surgery. This neoplasm is highly radiosensitive, which is why radiotherapy is considered most efficient; in addition, it does not cause mutilations. The superficial neoplasm, with a depth of less than 3 mm, can be treated with strontium 90. It is recommended to initiate superficial teletherapy using orthovoltage X rays or electrons produced by a linear accelerator.

Squamous cell carcinoma with oral location is less sensitive to radiotherapy than dermal squamous cell carcinoma. Even with high radiation doses, the control rate of tumors at 1 year is only 10–20% in cats with squamous cell carcinoma of the gums, tongue or tonsils. The attempts to improve the therapeutic approach by the use of brachytherapy associated with interstitial hyperthermia have led to little encouraging results.

Thyroid adenoma and adenocarcinoma are associated in old cats with endocrine disorders and hyperplasia with thyroid hyperfunction. Excellent results are obtained after iodine 131 therapy, sometimes one injection being sufficient for cats to become euthyroid (over 85%). Cats that remain hyperthyroid recover after the second iodine 131 injection.

Tumors in horses. Three types of tumors are described.

Bone tumors: osteomas, fibromas, osteosarcomas and ossifying chondrosarcomas have a low incidence, as well as adamantinomas and odontomas. Surgery is usually performed, after which radiotherapy is recommended, especially when the tumor has not been completely removed.

Equine sarcoid benefits from interstitial brachytherapy, with good results. The literature mentions a particular sensitivity to radiotherapy, in the case of radon 222, cobalt 60, gold 198, and iridium 192 in doses higher than 40 Gy, with a control rate of 83–96%. Primary surgical and cryosurgical treatment, as well as interstitial brachytherapy, are definitely indicated, especially that the sarcoid manifests frequent recurrences, aggressive behavior and locations that exclude surgical excision.

Squamous cell carcinoma is more common in non-pigmented areas, periorbitally and in the genital organs. Squamous cell carcinoma is a radiosensitive neoplasm that reacts to the different radiotherapeutic procedures. Superficial tumors, less than 3 mm deep, can be efficiently treated with strontium 90, with doses between 100 and 200 Gy. Because of the limited penetrability of beta particles emitted by strontium catheters, surgical resection may be needed to reduce the tumor thickness. Good results are mentioned, with a control rate of 89% of squamous cell carcinomas with periocular location, and in the case of deeper tumors, interstitial brachytherapy has led to a success rate of more than 80%, with a dose of more than 50 Gy. Squamous cell carcinomas located in the vulva, prepuce and penis are adequately treated by interstitial brachytherapy, although surgery may be necessary for large tumors.

Tumors in cattle. Squamous cell carcinoma is a radiosensitive tumor, located in the eyes, eyelids, and the non-pigmented skin of the mouth. Interstitial brachytherapy with gold 198 determines a favorable response in 90% of cases. The use of iridium 182 has resulted in a control of carcinoma of approximately 60%, at 1 year. In some cases, surgery is considered, with the enucleation of the ocular globe, and implantation of iridium 192. In the case of superficial ocular or palpebral tumors, 100–250 Gy by strontium 90 administration gives favorable results.

Bone and connective tissue tumors can be treated by surgery, which should be followed by radiotherapy.

Tumors in other species. Radiation is recommended to be used in all domestic or wild species, by analogy with the indications presented and considered for the situation of other species.

Tissues exposed to normal or neoplastic radiation will develop lesions, depending on their sensitivity and radiation doses. Special attention should be given to both acute lesions and lesions secondary to radiation exposure. The effects of acute radiation usually appear in the course of the last two weeks of treatment. The most severely affected tissues are those that divide more rapidly, such as the skin and mucosae. However, after the cessation of radiation, cell repair in these tissues is rapid, recovery occurs within 2 weeks post-treatment. Significant delays or serious complications may occur in the case of autotraumas, or when the animal is not given maintenance care, in the immediate post-radiotherapeutic period.

Chronic effects develop weeks, months or years after radiotherapy. These lesions especially occur in endothelial and/or connective tissues. Lesions consist of fibrosis, atonic wounds, formation of fistulas, and increased sensitivity to infections. A shortcoming of radiotherapy is represented by the difficulty of the individual assessment of sensitivity to radiation.

A late reported effect is carcinogenesis caused by radiation. Tumors induced by radiation occur in animals 30–78 months after treatment. These vary depending on the radiated territory and are the response of tissues to slow renewal. The radiated lung responds by fibrosis with severe respiratory failure; myelitis with tetraplegia occurs; osteonecrosis with spontaneous fractures [8].Incidence with an inconsistent frequency of tumors after radiotherapy should not discourage the use of this means of fighting tumors, since benefits exceed by far the potential of the development of a second neoplasm, after several years [36].

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19.3 CHEMOTHERAPY

Chemotherapy uses chemical substances that act electively on cells in mitosis, and antimitotic agents finally aim to destroy cancer cells. These substances have the great advantage that they do not act strictly locally on the primary neoplasm, and antimitotic agents perform a therapy of the potential or disseminated systemic disease. Chemotherapy is the most effective therapeutic approach, it relieves painful symptoms, prolongs life and/or even heals the disease. Thus, chemotherapy can cure a clearly diagnosed metastatic disease but, at the same time, a strategy can also be established for the control of occult metastases.

Chemotherapy has been used since the antiquity, or maybe even earlier, to fight tumors. In human medicine, chemotherapy has evolved, and the results of anticancer chemotherapy have been remarkably successful [31]. In this sense, the following can be mentioned:

• – the perspective of a normal life for some patients with different types of metastasized tumors;
• – increased recovery rates, in the case of the use of an adjuvant in surgical therapy or radiotherapy;
• – total remission in more than 25% of the treated patients;
• – an increased rate of response, with a significant prolongation of life duration;
• – objective regression in 30–50% of patients treated for the first time with a chemical product.

Many tumors only respond to chemotherapy in a low proportion, in 10–15% of patients.

At the same time, the complex phenomena that occur in cells need to be understood, especially in neoplastic cells, under the action of chemical substances used in the treatment of tumors. In this sense, the principles of chemotherapy will be presented, according to CARTER (1987 b).

The continuous biological changes in the neoplastic cell population represent an essential characteristic of each neoplasm, with infinite possibilities of transformation. The heterogeneity of neoplastic cells is a property that is amplified with the evolution of cancer disease, oncogenes being at the basis of this phenomenon.

Chemotherapy is a modality that is predominantly used in the treatment of cancer disease, with metastases and dissemination. Depending on the primary location and the extension of the disease, chemotherapy can be curative or palliative. In the majority of cases, chemotherapy leads to the prolongation of survival, and in other cases it results in the eradication of the disease. Chemotherapy can be in most cases a major adjuvant to surgical therapy and/or radiotherapy.

Cancer chemotherapy in the case of metastasized forms requires adaptations depending on:

• – the target, which is a macroscopically or microscopically detectable formation;
• – the location of the primary tumor from which the metastasis has developed;
• – the clinical and morphological stage of the disease, at the time of treatment.

The aim of chemotherapy can be curative or palliative depending on the therapeutic scheme; chemotherapy can use a single therapeutic agent, a combination of substances or it can be an adjuvant to surgical and/or radiation therapy.

The application of cytotoxic chemotherapy does not result in a tumor regression in all patients, also having undesired side effects. The heterogeneity of the neoplasm as such, the possibility of locations in different tissues, organs and body regions are as many reasons for a response to chemotherapy that is not the one expected. Anticancer drugs have each their own type of sensitive and resistant primary tumors, and the use of combined drugs leads to an extreme complexity of possible therapeutic schemes.

The biological heterogeneity of a primary tumor consists in its invasive capacity, metastatic potential, growth rate, immunogenicity, antigenicity, and intrinsic response to specific drugs. The heterogeneity of the response to a therapeutic scheme explains why some patients have the same tumor type and why, during the same evolution stage, some have favorable responses, and others not. In general, therapy is selected based on data regarding the response obtained in a population of treated patients. This database is far from being complete and is full of examples of different opinions offered by contradictory results obtained in the clinic.

The most successful chemotherapy in clinically evidenced metastatic tumor disease is the combined one. The selection of drugs to be included in a combination specific for the disease follows some guidelines:

• – drugs should be active when they are used alone, in the disease concerned;
• – drugs should have different postulated or known mechanisms of action;
• – drugs should not have a toxicity grade higher than a certain admissible grade.

An as weak as possible myelosuppressive action is added to these conditions for the selection of a drug.

An objective regression of the primary tumor and metastases can be a complete or partial regression, evaluated based on generally accepted criteria that are subject to extremely varied interpretations. In medical oncology, a partial regression of 20% is generally accepted as a minimal activity level.

The complete evaluation of the impact of chemotherapy on survival is difficult to perform. However, in some situations such as leukemias, lymphomas and testicular neoplasms, the positive impact of chemotherapy is easy to observe. It is difficult to establish the survival benefit from partial responses of 20–50%, as it is usually reported in the majority of solid tumors. Survival is influenced by a series of factors that are not exclusively related to the chemotherapy used. These variables include the performance status, the extension of metastatic disease before therapy, the nutrition status related to weight loss and the presence of more or less clinically evidenced diseases. Consequently, patients who respond to chemotherapy have the perspective of a positive prognosis for survival.

The selection and excessive development of neoplastic cells that are specifically resistant to drugs is according to SKIPPER (1982) the major cause of chemotherapeutic failure, and the causes of the excessive growth of drug-resistant neoplastic cells are multiple [4, 5].

Part of the cellular factors that can explain why some tumor cells are sensitive, and others are resistant, includes:

• – the transmembrane transport of the drug in the cell;
• – the limits of the transphosphorylation of purine and pyrimidine analogues;
• – the limits of catabolism, such as desamination, decarboxylation, phosphorolysis, hydrolysis, reduction, oxidation or esterification of the drug in inactive forms;
• – the changed affinity of target enzymes for inhibitory drugs;
• – different modalities of using precursors for DNA synthesis by tumor cells;
• – proportions of lesions caused by drugs and their repair;
• – drug induction of enzymatic activity in tumors or in normal tissues;
• – distribution of drug receptors on the cell, cytoplasm or nucleus surface;
• – the immune inactivation of antigenic drugs;
• – the fractioning of the cell population in sensitive mitotic cycle, by drugs.

Resistance to chemotherapy results from the adaptation at cellular level, by which enzymes are rapidly induced in cells to change the drug transport or distribution, or to repair or protect the cancer cell from specific drug effects. The conception on resistance to chemotherapy has evolved due to researches over the past decades. Drug resistance can be derived from the heterogeneity of tumor cells, in which resistant cell colonies or subpopulations are selected for continuous growth by non-response to treatment. This heterogeneity can be due to both genetic and epigenetic phenomena.

Drugs have been classified according to their preferential destruction effect in the cell cycle, based on kinetic cell culture studies. The most resistant cell has been considered the non-proliferative cell, in the G0 phase, in order to separate it from the cells that pass through critical proliferating phases of the cell cycle (M, G1, S, G2, M). Solid tumors have been considered to be more resistant, compared to hematogenic tumors, since they have a higher percentage of cells in the G0 phase, and the growth rate is low; thus, their duplication time is longer. Researches have been focused on the discovery of drugs that are able to destroy G0cells. However, these researches have been limited by the fact that usual in vivo selection systems have high growth rates during short duplication periods compared to solid tumors.

The biochemical resistance of cells is invoked as a factor that opposes the destruction of neoplastic cells during chemotherapy. A high number of cytotoxic drugs have antimetabolic effects, tested in cell cultures. These drugs have not had the expected effect in practice.

Another concept that attempts to explain the resistance of neoplastic cells to chemotherapy is the need for the drug to reach an adequate concentration for an adequate time period. Like all drugs, anticancer drugs pass through some phases or all the classical phases of: absorption, activation, inactivation, degradation and excretion. Pharmacokinetic studies attempt to establish each of the successive stages by which a drug can have a maximum efficacy on neoplastic cells.

Cancer resistance to chemotherapy can be understood if the concepts of pharmacology, biochemistry and proliferation are accepted and integrated. In this way, the success of chemotherapy in the fight of cancer can be explained, which opens the perspective for the formulation of combined therapeutic schemes, adjuvant and/or radical treatment, with the blocking of toxicity. Increased drug concentrations have not led to a proportional destruction of tumor cells, but to an increase in the toxicity grade in particular, with undesired side effects. The strategy of adjuvant chemotherapy has aimed to establish a standard dose with effect on small tumor masses, micrometastases, which are supposed to be kinetically much more sensitive.

The appearance and development of some chemotherapy-resistant tumor cells has used the analogy with microbial antibiotic resistance. It has been demonstrated that cells acquire their drug resistance with the repeated use of the same substances and they are more sensitive at the first contact. The appearance, development and perpetuation of a genetic mutation in a cell population make permanent the cell resistance to the drugs used.

In a first phase, tumor growth occurs without the development of resistant cells, followed by the appearance of a resistant, phenotypically stable cell clone, leading to the critical growth phase. This is also noted in the treatment efficacy, and the transition from curability to incurability with drugs is sudden discontinuous. The tumor that has reached a certain size, with a high mutation rate, will most probably contain resistant cells as well.

The GOLDIE-COLDMAN model [31] of associating tumor drug sensitivity with the incidence of spontaneous mutations offers some suppositions:

1. The resistance mutation rate in a variety of antineoplastic agents, in a variety of mammalian tumor cells, is estimated between approximately 104 and 107, this rate being similar in humans.
2. All tumor cells have the capacity of peduncle cells, which means that they are capable of infinite self-renewal, without the loss of cells due to the formation of new generations, with a limited proliferation capacity. Any detailed mathematical model is supposed to offer “the same general conclusions as those obtained from the minimal model”.
3. Resistant phenotypes derived from the tumor population are totally resistant to maximal tolerated drug doses. The complexity of partial resistance is also recognized, which does not seem to change the conclusions drawn from the model.
4. Resistant phenotypes will be kinetically identical to those of the related sensitive line.

The conclusion of this model could be synthesized as follows: cell differentiation and destruction contributes not only to a slow tumor growth, but also to a marked proportional growth of peduncle cells resistant to several drugs for a given tumor volume. This is why the size, growth rate and time of the tumor in relation to its response to the drug could reflect the natural selection of drug-resistant permanently mutant peduncle tumor cells.

In clinical observations, long remission periods have been noted, followed by progressive recurrences. These prolonged remissions are caused by latent tumor cells that, for unknown reasons, cause a recurrent tumor form by proliferation. The limitation of tumor growth for a time period can be achieved by active or passive mechanisms. Active mediation occurs by cytostatic or cytolytic humoral mechanisms, or by cell mediation.

Three main mechanisms have been identified for the latent state in animals:

• – lack of vascularization and isolation of tumor cells;
• – constitutive dependence of tumor cells on growth factors;
• – immunological limitation.

In order to understand clinical results, but also for the development of further studies on chemotherapeutic functions, SKIPPER (1982) has synthesized several concepts that have been commented and completed by THEILEN et al. (1987):

1. Reasons for which surgery and/or radiation therapy frequently cure local cancer, but fail to cure widely distributed neoplasias.
   The concept according to which cancer can be clinically localized, but microscopically disseminated, is the cornerstone of the modern therapeutic approach of cancer. This determines the application of treatment with a systemic adjuvant drug. It has been demonstrated that a single tumor cell left outside the radiotherapeutic and/or surgical field can result in a metastatic recurrence.
2. First order rate of cell destruction by certain chemical and drug products, and the logarithmic order of the destruction of neoplastic cells by anticancer drugs.
   This concept establishes that an effective dose of an anticancer drug will destroy a definite percentage of tumor cells, regardless of the number of tumor cells present in the host body. This is true in the following circumstances: a) when all tumor cells are similarly exposed to the active drug portion; b) when the growth rate of all tumor cells is the same; c) when the ratio between sensitive tumor cells and resistant cells is the same in all tumors. It is obvious that the heterogeneity of tumor cells significantly reduces the impact of this concept in the planning of treatment. This concept is especially true in experimental models, in leukemias. There is a wide range of natural heterogeneity, which involves kinetic, blood flow, genetic and biochemical heterogeneity, which probably defines the sensitivity or lack of sensitivity to a certain drug.
   This concept provides the major reason for the use of anticancer drugs in the highest possible dose. The higher the dose, the higher the functional cell destruction and the more rapidly the zero viable tumor cell level is obtained.
3. Haber’s toxicological principle (CxT=K) states that for extremely high drug concentrations (C) and exposure time (T), the same toxicity or cytotoxicity grade results from the same CxT (μg/ml/minute).
   This concept is most adequately tested when all cell exposed to a given drug concentration are susceptible of being destroyed. The action limits are easy to identify in drugs with antimetabolic action (e.g. arabynosil cytosine), which can destroy cells that actively synthesize DNA. While DNA alkylating and binding agents are able to destroy cells in all cell cycle phases, kinetic sensitivity is not equal in all phases, cells at rest (G0) being the less sensitive. Another major limitation of this concept in cancer chemotherapy is the fact that the critical factor is CxT of the active half of the drug at the level of tumor cells and not necessarily plasma CxT. For anticancer drugs, tissue pharmacology is much more relevant than plasma studies, and studies on blood cannot be easily extrapolated to what occurs at tissue level.
4. Logarithmic order of neoplastic cell destruction with anticancer drugs before the action of limitations due to cell population heterogeneity.
   This concept establishes that a given dose of a certain drug will destroy the same percentage, the same number of cancer cells from tumors of extremely different sizes, as long as they are similarly exposed, and both the growth fraction and the ratio between sensitive cells and permanently resistant cancer cells is the same.
5. Invariably reverse ratio between the loading of tumor cells at initiation and curability of chemotherapy.
   The concept is supported by numerous experimental data, and it is intuitively logical. Theoretical support is obtained from both the hypothesis of cell destruction based on kinetics and the somatic mutational theory, currently receiving high relevance. The kinetic theory establishes that the higher the loading of tumor cells, the lower the growth fraction, and consequently the lower the percentage of cells in the most sensitive phases of the cell cycle. The theory of the somatic mutation, Goldie-Coldman, establishes that the higher the number of tumor cells, the higher the probability of the presence of drug-resistant sub lines.
6. Influence of growth fraction and cell death, and tumor growth change rate.
   This concept is part of older explanations based on kinetics, which represented the foundation of the cell destruction hypothesis. The growth fraction is that fraction of cells that replicate DNA in order to prepare division at a certain time. The tumor growth rate is considered to be the result of the interaction of the growth fraction and the cell destruction rate. In this conceptual approach, small tumors found at microscopic level have a high growth fraction and develop exponentially. As tumors become larger, the growth rate becomes slower due to the reduction in the growth fraction or the increase in cell destruction or both aspects. Tumors have a rapid initial growth and a flattening of the growth curve occurs. Anticancer drugs have a high efficacy on actively replicated cells, consequently in the case of early and adjuvant treatment of micrometastases, after the local fight of the primary disorder.
7. Mechanism of action of different anticancer agents, at molecular level.
   The exact knowledge of the molecular mechanism of cell destruction will allow a much more rational and effective use of these cytotoxic preparations. Many drugs act on the synthesis or functioning of the DNA, in a certain way. The mechanisms of action have been predominantly found by in vitro laboratory studies, which can be or not totally extrapolated to both humans and animals. The usual classification of anticancer drugs into alkylating, antimetabolic agents, mitotic inhibitors, synthetic, random antibiotics, etc. is inconsistent and only minimally useful. For many preparations, several mechanisms are postulated, without the possibility of establishing the certain dominance of a single mechanism for tumor cell destruction.
8. Inactive cells are temporarily refractory to a number of agents, and there is a direct relation between the growth fraction and (neoplastic and normal) cell destruction by the exposure to one or more drugs.
   This concept is a synthesis and almost a redefinition of the majority of the mentioned concepts and is based on numerous experimental data in vitro: inactive bacteria refractory to penicillin or other antibiotics; inactive mammalian cells, which are extremely refractory to antimetabolites and refractory to other anticancer drugs, when these inactive cells do not initiate a DNA replication before DNA reporting; inactive cells, which become drug-sensitive when they cause DNA replication.
9. Correlation of data regarding the toxicity of anticancer drugs in different species, including humans (mg/m 2 ratio).
   The older use of mg/kg as a basis of specific dosage in patients does not allow an easy correlation in toxicological studies performed on rodents and large animals, on the one hand, and on humans, on the other hand, because of factors such as size, cardiac output, and delivery of half the output to organs such as the kidneys and liver. On the other hand, 10 mg/m2 in mice and humans are roughly comparable doses. In addition to this superiority in the interaction of experimental and clinical data, the mg/m2 dose allows the administration of a safer dose where there are extreme values in size, like in the case of animals and humans, children, for example, extreme variations in breed sizes, e.g. dogs and adult obese animals.
10. Pharmacokinetic behavior of anticancer agents in consanguine animals and humans, in relation to the anticipated use of different doses of a single drug or a drug combination.
    This concept involves the capacity to determine minimal cytotoxic levels in the serum and any possible therapeutic benefit. Based on this information, models can be developed for the achievement of optimal CxT, with the knowledge of efficient concentrations. A much more rational dosage of drug combinations is being attempted in order to ensure for each one the minimal efficient concentration for a reasonable time period. This hopefully remains to be done in a not too far future. It is possible to obtain efficient concentrations for the destruction of cells in cell cultures, but the extremely varied situations existing in vivo make difficult the use of these results in medical practice. The gaps in the knowledge of tissue pharmacology in anticancer preparations determine discrepancies between the results of experimental investigations and their application in anticancer therapy. This concept should be considered as a future aim rather than as a present reality.
11. Consanguine animals as “screening models” are useful, when they are adequately used, in the selection of drugs and drug combinations, which can be useful in the treatment of cancer in humans (administered separately or as adjuvants).
    Animal models can be useful, with predictable results in terms of efficacy for new chemotypes. Each efficient anticancer drug is active at least in one of the transplantable tumor models used in rodents. The value of these models has been proved by the use of combined therapeutic schemes and the selection of complex modalities, at least in a predictable way.
12. Acute lymphocytic leukemia in children represents a useful model for the development and documentation of important principles in the treatment of some cancer varieties in humans and animals.
    Acute lymphocytic leukemia has remained an example of the greatest triumph in cancer chemotherapy. Some practical therapeutic concepts considered as certain have been initially elucidated by pioneering clinical investigations in the chemotherapy of acute lymphocytic leukemia. In this sense, the following are mentioned: the importance of obtaining complete remission, the importance of maintaining therapy, the superiority of combined active agents compared to the use of a single agent, the use of different preparations and schemes for induction and maintenance, the importance of using the whole pharmacological armamentarium (CNS therapy), and the importance of support therapy (therapy of an infectious disease and blood transfusion). What has been obtained in acute lymphocytic leukemia will probably be valid for any disseminated tumor for which effective chemotherapy, equivalent to that developed for acute lymphocytic leukemia, has not yet been discovered.
13. Bacteria can pass to a resistance state under certain conditions or after the contact with some drugs; neoplastic cells spontaneously pass to a phase of specific resistance to a large spectrum of anticancer drugs.
    This concept leads to the Goldie-Coldman hypothesis discussed above.
14. In essence, all anticancer drug categories select and allow an overgrowth of specific drug-resistant cells.
    The selection rate of a subline of drug-resistant cancer cells is directly correlated with the eradication rate of drug-sensitive cells and is influenced by the percentage of tumor cells, as well as by the intensity and duration of treatment. Clinical resistance appears when this selection has reached the point in which the critical percentage of surviving cells (1–50%) resists to the agent(s) used. The higher the tumor mass, the higher the chance of resistance to one or more agents prior to therapy. The mutation rate in the stage of resistance to two different drug classes is not proportional to the product of the mutation rate of the resistance to each drug, but is higher.
    The clear implication of this concept is that combined chemotherapy will be superior to the use of a single agent in the tumor, regardless of the tumor size. Consequently, the dosage and application scheme of combined drugs will be extremely important, although there are no complete investigation means available to obtain an optimal application scheme.
15. The number of anticancer drugs, which manifest low or no cross resistance, is higher than usually expected.
    Neoplastic cells resistant to drugs that have been selected by antimetabolites or DNA alkylating or binding agents do not present cross resistance beyond these classifications, but will cause cross resistance within the same classification. The main exception is in the case of the extensive classification of antimetabolites. For example, cells selected by methotrexate do not manifest cross resistance to purine and pyrimidine antagonists. Cells selected by different purine analogues do not present cross resistance to all purine analogues. This is increasingly recognized in other classes as well, allowing for new combinations which are supposed to include more than one drug of the same class.
16. The combined treatment modality involves chemotherapy associated with surgery and/or radiation.
    This concept establishes that the major percentage of tumor cells is frequently contained in the local tumor mass and its regional distribution. When the locoregional disease is fought by surgery and/or radiation, chemotherapy will only be used to attack the remaining micrometastatic disease.
    The mentioned concepts show that micrometastases may have a higher growth fraction and contain fewer or possibly no neoplastic cells with specific drug resistance, if the residual percentage after local treatment is low.

Application of cancer chemotherapy. Chemotherapy is expanding as new efficient anticancer drugs are discovered, in correlation with the histological determination of the malignancy grade. In this sense, the following parameters are considered: tumor mass determination; quantitative determination of tumor cells in blood or bone marrow, in the case of leukemia; dispersion of distribution; reduction of serum calcium content, in the case of lymphoma; reduction of Bence-Jones protein content in the urine, in multiple myeloma. The objective of chemotherapy in cancer is the selection of drugs and dose levels, which will eradicate disseminated cancer cells, without causing severe host toxicity.

Anticancer drugs particularly affect cells in active division and, in general, normal and neoplastic cells cannot be differentiated. The maximal therapeutic effect is obtained in the case of moderate drug toxicity, a careful monitoring of the patient being required in the long term. This monitoring includes behavior, appetite, body temperature, mucous membrane integrity, and serum and blood biochemical examination. The prevention of infections will be attempted and if these occur, prompt antibiotic therapy will be instituted [32, 33].

The toxic effects of anticancer drugs are exerted on normal cells in the following order:

1. peduncle cells of the bone marrow and lymphocytes;
2. mucous cells of the gastrointestinal tract;
3. liver and kidney cells;
4. cells of the basal epithelial layer;
5. nervous system cells.

The usual toxic hematological signs include: thrombocytopenia, neutropenia, reticulocytopenia; anemia and lymphopenia. The effects of toxicity on the gastrointestinal tract are anorexia, nausea, vomiting, diarrhea, stomatitis and intestinal mucosal ulcers; hair loss, especially in dogs with curly hair.

Anticancer agents act by disturbing cell multiplication or normal functioning, DNA synthesis or chromosomal migration, and by blocking or changing RNA and protein metabolism. In this sense, depending on their action, chemotherapeutic substances can be classified as follows:

• – alkylating agents, capable of denaturing certain macromolecules such as DNA macromolecules; the main representative is cyclophosphamide;
• – intercalating agents, which interact with DNA and are intercalated between two bases, inducing a structural change and a functioning of this molecule; these are represented by adriamycin;
• – cleaving agents, capable of breaking DNA molecules, represented by bleomycin;
• – antimetabolites that can be structural analogues of purines or pyrimidines; they block the synthesis of the corresponding bases (5 FU), or folate analogues, such as methotrexate, which blocks the synthesis of the same bases;
• – mitostatic agents that inhibit tubulin synthesis, these being cell spindle poisons, of which the main representative isvincristine;
• – platinum derivatives, cisplatin, a separate class, which plays a role by DNA binding;
• – corticoids that possess a cytolytic action that can be profitable [7].

Following the administration of chemical substances, a destruction and reduction of neoplastic cells occurs in the tumor tissue on the one hand, and, on the other hand, a reaction of multiplication and increase in the number of viable cells. This requires the establishment of a polychemotherapeutic protocol that should increase in a first stage the percentage of cells in the active cell cycle phase, and subsequently, the use of an active chemotherapeutic agent for the destruction of these cells.

This therapeutic scheme is widely used in human oncology, where polychemotherapy is a rule, and the administration at certain hours is well established. Chronotherapy in oncology aims precisely to coordinate the administration of a drug with the optimal phases of tumor cells for the lytic action of that drug.

The effect of chemotherapy is not strictly focused on cells or neoplastic tissue, the substances used having a toxic action on the majority of body cells, but especially on regions in which a normal physiological replacement of non-tumor cells occurs at a rapid rate (epithelia, hematopoietic stem cells). This is non-specific toxicity, while some chemotherapeutic agents act on metabolism, increasing concentrations in certain tissues or organs, inducing specific toxicity [7].

The hematological toxicity of antimitotic drugs particularly and drastically manifests by leukopenia and thrombopenia. The toxic aggression on the bone marrow results in the destruction of cells in multiplication, in maturation and on reserve cells. Substances from the alkyl group act on the stem cell population.

Most of the drugs used manifest their toxic action around the fourth day of treatment by a diminution in the number of leukocytes and blood platelets. This effect is progressively exerted until the 10th day, then it becomes stable. The drastic reduction in the number of granulocytes significantly increases the risk of intercurrent infections. The majority of antimitotics also show toxicity for erythrocytes and blood platelets, but always to a smaller extent than for granulocytes.

Aggressive toxicity, in dogs, manifests by vomiting, following the use of cyclophosphamide, adriamycin and cisplatin. Diarrhea is another manifestation of antimitotic toxicity, due to lesions caused in the gastric and intestinal mucosa.

Alopecia appears exceptionally in carnivores, especially after the prolonged administration of cyclophosphamide and vincristine. The Caniche, Afghan, and Scottish Terrier breeds are more sensitive.

The main antimitotics used in veterinary oncology are according to DELISLE (1990) the following: Cyclophosphamide (Endoxan ND), which is an alkylating agent; Vincristine (Oncovin ND), a cell spindle inhibitor; Adriamycin (Adriablastin ND), an antibiotic; 5-Fluorouracil (Fluorouracil ND), an antimetabolic; Cisplatin (Cisplatyl ND), which acts by its fixation on DNA.

Groups of chemotherapeutic substances and main representatives (according to RUSLANDER, 2000)

Substance group	Representatives	Mode of action
Alkylating agents	Cyclophosphamide Ifosfamide Meclorethamine Chlorambucil Melphalan Lomustine Thiotepa Dacarbacine (DTIC)	Crosslinking of DNA chains
Antitumor antibiotics	Doxorubicin Actinomycin D Mitoxantrone Bleomycin	Inhibition of topoisomerase II Intercalation
Antimetabolites Vinca alkaloids	Cytosine – arabinoside 5-Fluorouracil Methotrexate Vincristine Vinblastine	Inhibition of enzymes Introduction of false substances in DNA “poisons of the division spindle” –inhibition of the division spindle
Enzymes	L-Asparaginase	Enzymatic cleavage of asparagin
Unclassified chemotherapeutics	Platinum bonds (Cisplatin, Carboplatin) Hydroxyurea Taxol	Crosslinking of DNA chains (similarly to alkylating agents) Destruction of enzymes

Alkylating agents are dose-dependent non-specific drugs that act in the cell cycle, replacing a hydrogen atom from another molecule by an alkyl radical (R-CH2-CH2+). The majority of demonstrations suggest that alkylation is responsible for the main toxic effects of these compounds and occurs by the alkylation of nucleic acids. This alkylation causes ruptures in DNA nuclei and in the cross links of the two double DNA spirals, interfering with RNA replication and transcription. Similar effects are produced by certain ionizing radiations, so that alkylation is considered to be radiomimetic.

The differences in the activity of alkylating agents are attributed to differences in absorption, metabolic rate and tissue affinity rather than to differences in the mode of action. With rare exceptions, the resistance of tumor cells to a certain alkylating agent indicates resistance to other alkylating agents. Alkylating agents cause different pharmacological effects, including the interference with mitosis, mutagenesis, immunosuppression, and, paradoxically, carcinogenesis.

There are five chemical classes of clinically useful alkylating agents:

1. nitrogen mustard derivatives (mechlorethamine, cyclophosphamide, chlorambucil, melphalan);
2. ethylene derivatives;
3. alkyl sulfonates (busulfan);
4. triazine derivatives (dacarbazine);
5. nitrosoureases (carmustine, lomustine, semustine).

Chemical groups of alkylating agents (according to RUSLANDER, 2000)

Group of substances	Representative
Nitrogen derivatives	Cyclophosphamide Ifosfamide Meclorethamine Chlorambucil Melphalan
Alkyl sulfonates	Busulfan
Ethylene derivatives	Thiotepa
Triacetic derivatives	Dacarbazine
Nitrosourea derivatives	Lomustine Carmustine

Cyclophosphamide is the most widely used alkylating agent and is unique among antitumor agents by the fact that it requires the activation of an alkylating metabolite by the liver. It is a nitrogen derivative that through the microsomal and enzymatic oxidases of the hepatic cell turns into an active metabolite, i.e. aldophosphamide; another metabolite is acrolein, which causes hemorrhagic cystitis. Cyclophosphamide resistance is acquired by the reduction of the cell entry, changes in the sulfhydryl content (inactivation of thiol), enzymatic intracellular detoxification, and enzymatic repair of lesions. The cross-resistance between various alkylating substances does not reach 100%. Cyclophosphamide is effective both orally and intravenously, being useful in the treatment of leukemias and lymphomas in dogs and cats, as well as of solid tumors. The toxicity of cyclophosphamide in dogs manifests by vomiting, hemorrhagic diarrhea, salivation, oscillations of the body temperature, hematuria, anorexia and weight loss. Hematological changes include: leukopenia, thrombocytopenia and reticulocytopenia, and finally, anemia. Biochemical serum changes consist of: increase in transaminase values, prothrombin time, alkaline phosphatase concentrations, urea nitrogen and creatinine.

Other toxic manifestations of cyclophosphamide, unpleasant especially for humans, are: alopecia, sterility, fatal lesions and others. Sterile necrotizing hemorrhagic cystitis associated with chronic cyclophosphamide administration can cause mucosal ulceration, hemorrhages and edemas in all tissues, as well as the necrosis of smooth muscles and small arteries. Urinary bladder lesions associated with the use of cyclophosphamide include acute hemorrhagic cystitis, chronic cystitis, anomalies of exfoliative cytology from urine, interstitial fibrosis and transitional cell carcinoma. The treatment of cyclophosphamide toxicity involves the early detection of symptoms and the suppression of treatment. Cyclophosphamide-induced cystitis can be prevented by forced diuresis. Severe hemorrhage in cystitis can be clinically treated by the intravesical instillation of a diluted formalin solution (1%). Complications are minimized by corticoids in combination with cyclophosphamide or by a saline diuretic drug and cyclophosphamide administration in the morning every other day, allowing the animal to evacuate urine that contains metabolic residues, before the administration of an additional drug. Chronic cyclophosphamide use results in secondary neoplasms, such as transitional cell carcinoma of the urinary bladder.

Cyclophosphamide – CPM (Endoxan ND) is successfully used in human oncology, and is of interest in veterinary medicine, since it is easy to administer orally and is less expensive. The drug does not traverse the hematoencephalic barrier, and the elimination of active metabolites occurs by renal route and manifests toxicity on the urinary bladder.

The product can be used in domestic carnivores in two ways:

• – it is administered semicontinuously in small doses of 50 mg/m2 per day;
• – it is administered discontinuously in high doses, 250 mg/m2 over all three weeks, this administration mode being more effective.

Non-specific toxicity constantly manifests by myelosuppression, with a decrease in neutrophils and lymphocytes, with a maximum peak between days 5 and 7 of administration, with a gradual restoration of values. In cats, depletion occurs at a later stage, between days 9 and 10, and recovery is slower. Anemia is moderate, and skin toxicity is maintained for a long time.

Specific toxicity in dogs and cats manifests at the level of the urinary bladder, and high doses can determine acute hemorrhagic cystitis. In this case, treatment is interrupted, and in more serious cases, vesical instillation with 1 % formol solution can be performed. Corticoid-induced polyuro-polydipsia may occur.

The major indication of cyclophosphamide, which is efficient in carnivores, is in malignant lymphomas or in polychemotherapeutic protocols. Cyclophosphamide is frequently used in hematopoietic tumors and is part of several combined protocols for the treatment of carcinomas and sarcomas. Dosage: 150–300 mg/m2 body surface area, once every 3 weeks, i.v. or per os; or: 50 mg/m2 body surface area per os, daily, 4 days a week; or 50 mg/m2 body surface area per os every other day. The exact dose depends on the other drugs of the therapeutic protocol.

The use of cyclophosphamide in squamous cell carcinoma in sheep has caused partial or total tumor regression, the regression rate varying proportionally to the dose used. Cyclophosphamide has beneficial effects by its immunosuppressive action on lymphocytes, many of which have a direct cytotoxic action on tumor cells [16,37].

Ifosfamide is similar to cyclophosphamide in terms of action, pharmacological behaviour and resistance. It has an effective action on malignant lymphoma in the dog; it has as a side effect hemorrhagic cystitis, which requires the administration of mesna.

Ifosfamide is used: 350–375 mg/m2 body surface area, i.v., once every 2×3 weeks.

Therapeutic protocol of ifosfamide and mesna:

1. Mesna (dose in mg = mg ifosfamide/5) in 0.9% NaCl (volume = mg mesna × 0.04),
2. Diuresis with 0.9% NaCl solution, 18 ml/kg for 30 minutes,
3. Ifosfamide in a 0.9% NaCl solution, 18 ml/kg/h after 30 minutes,
4. Diuresis with 0.9% NaCl solution, 18 ml/kg for 5 hours,
5. Mesna 2 hours after ifosfamide (dosage and administration as for item 1),
6. Mesna 5 hours after ifosfamide (dosage and administration as for item 1),
7. The cannula is removed and the dog is walked [37].

Chlorambucil is the alkylating agent with the slowest action and the lowest toxicity, and is used in the treatment of canine lymphoma. It is a substitute of cyclophosphamide in any protocol, if animals develop hemorrhagic cystitis or if several cyclophosphamide treatments have been administered (8–15 weeks). Chlorambucil is widely used in the treatment of chronic lymphoid leukemia, and busulfan is administered in the treatment of chronic granulocytic leukemia. Chlorambucil induces mild myelosuppression, but a cumulative bone marrow myelosuppression may also appear. Gastrointestinal toxicity is rare, and hemorrhagic cystitis does not occur. Dosage in chronic lymphocytic leukemia: 0.2 mg/kg per os for 3–10 days, after which 0.1 mg/kg per os, daily. In malignant lymphoma, it is used as a component of the therapeutic protocol, associated with other chemotherapeutics. Dosage: 1.5 mg/kg per os, once every 4–8 weeks, or 2–8 mg/m2 body surface area once every other day. The dosage depends on the number of neutrophils [37].

Melphalan is most extensively administered in the treatment of multiple myeloma and monoclonal gammopathies, as well as in ovarian carcinoma. It is a nitrogen derivative, whose mode of action is comparable to that of cyclophosphamide. It is used in veterinary medicine for the treatment of multiple myeloma. Its toxicity manifests through myelosuppression. Dose used in multiple myeloma: 0.1 mg/kg per os, daily, for 10 days, after which the dose is reduced to 0.05 mg/kg per os, daily, for maintenance. In cats – alternatively 1.5 mg/m2 body surface area, daily, for 10 days.

Lomustine is an alkylating nitrosourea substance which, similarly to the other alkylating compounds, results in crosslinking of DNA. It is metabolized through several stages in the liver, being transformed into the active form. Its use in veterinary medicine is limited to some central nervous system tumors and to lymphomas. Side effects: myelosuppression, which is more marked on days 7–10; in dogs, thrombocytopenia occurs; in humans, pulmonary fibrosis, hepatic and renal toxicity have been found.

Dosage is 90 mg/m2 body surface area, per os, once a month [37].

Dacarbazine induces the direct destruction of DNA, after its activation; it interferes with purine synthesis, RNA and protein synthesis. The substance is metabolized in the liver and is renally excreted. The drug appears to be more active in cells in late G2phase, but has activity throughout the cell cycle and is not cell cycle-phase specific. It is not for use in cats.

In veterinary medicine, it is used in combination with doxorubicin in the treatment of refractory lymphomas and sarcomas in dogs. In humans, it is also used in malignant melanomas. Side effects consist of gastrointestinal toxicity and myelosuppression. The dose is 200 mg/m2 body surface area, i.v., for 5 days, once every 3 weeks.

Mode of administration of dacarbazine:

1. The drug is diluted with a 0.9% NaCl solution (20 ml/kg BW)
2. Premedication with metoclopramide 0.2–0.4 mg/kg s.c.
3. The catheter is introduced
4. An infusion with dacarbazine is initiated after 8 hours
5. Antiemetic treatment is performed
6. The catheter is washed with 2–4 mg dexamethasone in order to minimize vascular spasm and the appearance of phlebitis.

Therapeutic protocol for recurrent sarcomas, melanomas and lymphomas, using combined chemotherapy, doxorubicin-dacarbazine. Both drugs will be administered on the first day, and the administered dose will be repeated once every 3 weeks. On day 8, leukocyte count is required. If the number is less than 3000, the next dose is reduced by 20%.

First day: – doxorubicin 30 mg/m2 body surface area, i.v.

– dacarbazine (DTIC), 800–1000 mg/m2, i.v., after 8 hours [37].

Antimetabolites directly interact with special enzymes, leading to the inhibition of that enzyme or the subsequent synthesis of an aberrant molecule that cannot function normally. These antimetabolites are structurally analogues of normal metabolites needed for purine and pyrimidine biosynthesis. When introduced in the cell in the place of the physiological substance, they result in an inhibition of the cellular function. Cell lesions occur through the competitive inhibition or feedback of enzymes that are still indispensable for DNA. Antimetabolites are specific for cell cycle phases and act during DNA synthesis (cell S phase).

Main antimetabolites:

1. Pyrimidine analogues: 5-fluorouracil; cytosine-arabinoside
2. Purine analogues: 6-mercaptopurine; 6-thioguanine
3. Folic acid antagonists: methotrexate [37].

6-mercaptopurine, 6-thioguanine, and azathioprine are purine analogues, which inhibit RNA and DNA synthesis. 6-mercaptopurineand 6-thio-guanine are used in the treatment of acute leukemia and occasionally of chronic granulocytic leukemia in humans. Their main toxicity is due to myelosuppression, as well as to hepatocellular lesions and cholestases. Purine analogues are used in animals, in the treatment of canine lymphoma and feline myeloproliferative diseases, while azathioprine is most frequently used in the treatment of inflammatory neoplastic disorders.

Cytosine arabinoside is a synthetic compound, which blocks the conversion of cytidine into deoxycytidine, resulting in a blockage of DNA synthesis. Cytosine arabinoside is used in the first place in human patients in the treatment of myelogenic leukemia and lymphomas. Administration in dogs has revealed that its toxic action is aimed at the rapid proliferation of bone marrow and gastrointestinal epithelial tissue. In veterinary oncology, the drug is used in the treatment of lymphoma and canine and feline myeloproliferative disease. In veterinary practice, it is used in the treatment of central nervous system lymphoma, in dogs. The dose administered is of 20 mg/m2 body surface, injected intracranially after the extraction of an equal cerebrospinal fluid volume. The total dose is diluted in 2–4 ml lactated Ringer’s solution, injected 2 times per week, for 6 weeks. The response obtained in affected animals is the reduction of nervous signs.

The administration of the drug by intravenous perfusion is much more efficient. A dose of 5–10 mg/m2 body surface, administered subcutaneously or intravenously, 2 times daily, gives good results in acute myelogenic leukemia and hemopoietic dysplasia, in dogs and cats.

Therapeutic protocol recommended by RUSLANDER (2000):

1. 100 mg/m2 body surface area, s.c., 3–4/day, once every 5–9 days
2. 10 mg/m2 body surface area, s.c., 2/day, until remission
3. 300 mg/m2 body surface area, as DTI, i.v., for 2 days, in lymphoma cases.

Another antimetabolite, 5-azacytidine, is a cytidine analogue and is used in humans in the treatment of infant and adult leukemia, with a potential that remains to be used in veterinary oncology.

Methotrexate is a folic acid antagonist, which competitively inhibits the dihydrofolate reductase enzyme. The inhibition of this enzyme reduces the biologically active folate reserve, which is an essential cofactor for DNA, purine and protein synthesis. Methotrexate is used in human oncology practice, in the treatment of acute lymphoid leukemia, in non-Hodgkin lymphoma, in epidermoid head and neck carcinoma, in breast carcinoma, in choriocarcinoma and in osteosarcoma. Proliferated bone marrow cells and gastrointestinal epithelial cells are the main targets of toxicity. Methotrexate is eliminated by renal excretion. The primary side effects of methotrexate in dogs and cats are gastrointestinal in nature (diarrhea, nausea, vomiting, and anorexia). Leukopenia, thrombocytopenia, temporary hair loss, pyrexia, skin rashes or discoloration, and oral lesions may occur less commonly.

In combined protocols for the treatment of lymphomas and osteosarcomas, the recommended dose is 2.5 mg/m2 per os daily or 0.6–0.8 mg/m2 i.v., once every 3 weeks [37].

Methotrexate is used in veterinary oncology in the treatment of lymphoma and osteosarcoma, in doses of 3–6 g/m2 body surface. Treatment does not seem to influence the duration of appearance of lung metastases.

Plant alkaloids. The most important ones are presented in what follows.

Vincristine and vinblastine sulfate are alkaloids extracted from the periwinkle (Vinca rosea) plant. Vinca alkaloids exert their antitumor effect by binding to cytoplasmic microtubular proteins, inhibiting the formation of the mitotic spindle and stopping cell division in the metaphase. The alkaloids from the vinca plant are large complex molecules; despite similarities between these alkaloids, the resistance of a certain tumor to a certain drug does not exclude the sensitivity to another alkaloid. These alkaloids are successfully used in the treatment of malignant lymphohemopoietic disease and other canine and feline neoplasms.

The drug is used in 0.75 mg/m2 doses per week by strictly intravenous route. This rhythm can be maintained for four weeks, when toxicity manifests by weight loss, medullary hypoplasia, intestinal mucosal necrosis. A particularly high sensitivity have neutrophil granulocytes. Clinical intoxication signs are: anorexia, diarrhea, vomiting and alopecia. Polyneuritis manifests in cats. If 0.37–0.50 mg/m2 are administered two times per week for 12 weeks, the following may be noted: ataxia associated with unstable walking, body oscillations, limbs spread apart, abolishment of Achilles reflex, and a posterior flexed position.

Vincristine is effective and controls malignant lymphomas and Sticker’s sarcoma, being a drug that leads to encouraging results in acute lymphoblastic leukemia of the dog. The product can be used in all polychemotherapeutic schemes.

Vincristine toxicity experimented on Beagle dogs has shown that overdosage induces intestinal tract mucosal necrosis, lymphoid hypoplasia and blockage of spermatogenesis. These lesions are supplemented by clinical signs of anemia, leukopenia and increased serum aspartate aminotransferase, alkaline phosphatase and lactic dehydrogenase content. Alopecia, stomatitis, constipation and peripheral neurological signs are reported. Clinically, vincristine neurotoxicity manifests by a reduced sensory and motor function, usually involving the extremities. Signs disappear after the cessation of treatment.

Perivenous administration causes drastic local reactions: painful phlebitis, edema, desquamation and necrosis. Sometimes, lesions become deep, such as deep ulcers, necrosis of tendons and exposed bone.

Taxol is a complex diterpene ester and is derived from the Taxus brevifolia tree. Its action is based on the binding of microtubules. Drug resistance is formed as part of multidrug resistance. It is used alone in the treatment of mammary carcinomas of the dog. Side effects: myelosuppression, gastrointestinal toxicity, alopecia, and allergic reactions.

The dose in dogs is 165 mg/m2 body surface area for 2.5 months. Premedication with HI blocking agents (diphenhydramine), dexamethasone, cimetidine is required [37].

Acemannan (Carrisyn) is a galactomannan extracted from aloe (a decorative plant of the Aloe genus); it stimulates the release of IL-1, IL-6, TNF, IFN from macrophages and increases NK cell activity. Administered in cats with FeLV, it has been found to delay the symptoms of the disease.

In dogs and cats with different neoplasias, acemannan associated with other therapeutic modalities has a positive effect. In a study performed on 8 dogs and 5 cats with fibrosarcomas, HARRIS et al. (cited by 38) obtained a prolongation of life by 372 days using acemannan in combination with surgical excision and radiation therapy, as compared to a survival of 161 days in the group without acemannan. The therapeutic protocol consisted of the administration of 1 mg/kg i.p. once a week for 6 weeks, then once a week for 1 year; in addition, patients received 2 mg acemannan intraneoplastically once a week, for 6 weeks. Tumors became necrosed, lymphocytic infiltration or edema appeared. No animal presented any side effects, and on necropsy, the peritoneum presented no changes.

Antitumor antibiotics. They act by the formation of stable complexes with DNA, inhibiting in this way DNA and RNA synthesis or both. Antitumor antibiotics used in clinical practice include bleomycin, actinomycin-D and anthracycline antibiotics, doxorubicin and daunomycin. There are other antitumor antibiotics, but these are not used, due to their high toxicity.

Bleomycin is extremely active in the G2 and M phases of the cell cycle. It is extracted from the fungus Streptomyces verticillm, it combines with DNA and directly induces an oxidative cleavage by the production of free radicals. Bleomycin is activated at hepatic level by the microsomal enzymatic system and is excreted in the first 24 hours by the kidneys. In humans, it is successfully used in lymphoma, testicular carcinoma and squamous cell carcinoma. In dogs and cats, bleomycin can be used in squamous cell carcinoma, all the more so as it has no toxic effects on the bone marrow. In dogs, toxic effects manifest by interstitial pneumonia, followed by pulmonary fibrosis and pleural scars.

The dose is 0.2 to 0.6 mg/m2 body surface area, s.c. or i.v., daily, for 5 days, then twice/week for 5 weeks.

Actinomycin-D is used in human oncology, being capable of healing gestation choriocarcinoma and increasing the recovery rate of embryonic nephroblastoma; it is useful in the treatment of embryonic rhabdomyosarcoma and testicular tumors. It is the product of the Streptomyces fungus and it acts through the binding of DNA, which results in the reduction of DNA, RNA and protein synthesis; it is mainly excreted by the kidneys. It is used in the treatment of lymphosarcomas, soft tissue sarcomas (canine nephroblastoma).

The dose is 0.9 mg/m2 body surface area, i.v., once every 3 weeks [37]. It is important that a patent i.v. catheter be used. To administer the drug, flush the catheter with 5–10 ml of sterile saline, inject the drug, then follow with another flush of 5–10 ml sterile saline. Prior to administration, draw up the calculated dose, dilute that into 100 ml of sodium chloride and administer this diluted dose as a slow i.v. drip over one hour.

The toxicity of actinomycin-D has been tested in dogs, the following being noted: diarrhea, anorexia, weight loss, as well as reticulocytopenia, lymphopenia, eosinopenia and hypochloremia. Intestinal and hematopoietic tissue lesions are constantly present.

Anthracycline antibiotics: doxorubicin (adriamycin) daunomycin are products of a mutant fungal strain, Streptomyces peucetius. Drugs rapidly penetrate the cells, become fixed in the nuclear structures and are intercalated with DNA, inhibiting in this way DNA-dependent RNA synthesis, as well as DNA duplication.

Clinically, adriamycin has a significant therapeutic activity in a considerable number of tumors, even in some cases refractory to other drugs. In human medicine, it is used in breast tumors, in bronchogenic carcinoma, in thyroid carcinoma and hepatocellular carcinoma, neuroblastomas, osteosarcomas, soft connective tissue sarcomas, solid tumors and acute leukemias.

Adriamycin – ADM (Adriablastin ND) is an intercalating agent, which does not exceed the hematoencephalic barrier, being inactivated in the liver and eliminated by biliary route.

Non-specific toxicity can manifest by histamine shock at the time of injection, phlebitis in the case of perivenous administration, and specific toxicity may be cardiac, by cumulation.

Shock is caused by basophil degranulation, during or immediately after injection, manifesting by salivation, tremor, nausea, skin hyperemia with the reddening of glabrous areas. Histamine shock can be fought by antihistamine drugs (Phenergan ND); warning: no corticoids will be used, since they have no effect.

In humans and dogs, adriamycin results in cardiomyopathy, because toxicity is cumulative, it develops after discontinuous administration and can manifest a long time after the cessation of treatment. In principle, a cumulated dose of 180–200 mg/m2should not be exceeded.

The administration scheme, in dogs and cats, is of 30 mg/m2 for three weeks, with the possibility of a total number of 6–7 administrations, or 5 months of treatment if adriamycin is used as monochemotherapy.

In veterinary cancerology, the use of adriamycin is extending, alone or in sequential association with cisplatin, in osteosarcomas and soft tissue osteosarcomas, as well as in canine malignant lymphomas. In animals, adriamycin has been used as an adjuvant agent in soft tissue sarcomas in dogs and cats, and in lymphomas and osteosarcomas.

The toxicity of adriamycin and daunomycin is extremely high. In the first place, they block the bone marrow, then alopecia, vomiting, stomatitis, cardiotoxicity and severe lesions occur when they are administered perivascularly. Cardiac arrhythmias include atrioventricular dissociation, ventricular tachycardia, atrial fibrillation and other manifestations due to cardiomyopathies. Other toxic manifestations are cutaneous reactions, such as urticaria, pruritus, sometimes collapse, urinary epithelial lesions, stomatitis, alopecia and pancytopenia. Urticaria may appear at the injection site. These phenomena can be prevented by corticosteroid and antihistamine premedication.

Partial or total remission was obtained in 41% of 157 tumors in dogs, which were assessed 3 weeks after the second doxorubicin dose (30 mg/m2). The following tumors had a partial or total regression: lymphoma (42/63); fibrosarcoma (1/14); solid follicular thyroid carcinoma (3/13); mammary carcinoma (2/8); hemangiosarcoma (2/8); osteosarcoma (1/16); perianal carcinoma (3/5); synovial cell sarcoma (2/3); undifferentiated sarcoma (2/3); nasal adenocarcinoma (1/2); liposarcoma (1/2); scirrhous mesothelioma (1/1) and neurofibrosarcoma (1/2). It was conclusive that 2 doxorubicin doses caused the regression in the size of different malignant tumors in dogs [25]. Intoxication signs, in dogs treated with intravenous doxorubicin, 1–2 doses of 30 mg/m2, at 2 days interval, were: diarrhea, vomiting, anorexia and pruritus [26].

Daunomycin is efficient in acute leukemias.

Cisplatin-CISDDP (Cisplatyl ND, Platinol) has a mode of action similar to that of alkylating agents. It is excreted at the level of the proximal tubule epithelium and is eliminated by renal excretion.

Toxicity is poor, compared to other antimitotic drugs, but in high doses in continuous perfusion it causes constant myelosuppression. Acute gastrointestinal toxicity manifests in humans and dogs. At the same time, it acts by stimulating the vomiting centers, as well as by a direct gastrointestinal action. The nausea and vomiting manifestations occur within 5–6 hours after injection, and anorexia may persist for 48 hours. These manifestations are easy to prevent and/or treat, by using metoclopramide (Primperan ND).

The major toxicity is specific acute renal, inducing tubular epithelial necrosis. The clinical manifestation is a transient increase in urea and creatinine, with irreversible renal failure. This can be improved by diuresis induced by mannitol associated with hyperhydration, in dogs that have received a nephrotoxic dose.

The indication of cisplatin treatment in dogs is in bone or soft tissue sarcomas, with high malignancy manifestations and metastasis. The drug can be associated with surgical or radiation therapy.

Cisplatin should not be administered in cats, since it induces fatal acute pulmonary edema even in low doses.

The recommended cisplatin dose in dogs is 70 mg/m2, with an administration protocol:

perfusion:	– NaCl 9 p/1000	20 ml/h for 4 h
	– mannitol	10 ml/kg in ½ h
	– cisplatin	70 mg/m2 in 250 ml NaCl in ½h
	– Na Cl 9 p/1000	20 ml/kg/h for 3 h

In order to avoid vomiting, intravenous metoclopramide (primperan) is administered in a dose of 0.1 mg/kg before cisplatin administration, and it may be repeated if necessary [7].

In the treatment of cutaneous tumors in horses, intratumoral cisplatin can be used, 1 mg cisplatin/cm3 tissue, 4 intratumoral treatments, at 2 weeks interval [34].

RUSLANDER (2000) proposes the following therapeutic protocols:

1. Diuresis with 0.9% NaCl solution 18 ml/kg/h for 4 hours.
2. Ondansetron (Zofran) 2–4 mg/dog, per os, 3 hours after the initiation of diuresis (optional)
3. Cisplatin as DTI after 20 min, in 18 ml/kg/h 0.9% NaCl.
4. Butorphanol (0.4 mg/kg i.m.), metoclopramide (0.2–0.4 mg/kg i.m.), after cisplatin
5. Diuresis with 0.9% NaCl solution 18 ml/kg/h for 2 hours
6. Antiemetic treatment (ondansetron, metoclopramide)

1. Diuresis with 0.9% NaCl solution 25 ml/kg/h for 3 hours
2. Ondansetron (Zofran) 2–4 mg/dog, per os, at the initiation of diuresis (optional)
3. Cisplatin as DTI after 20 min, in 25 ml/kg/h 0.9% NaCl solution
4. Antiemetic drugs
5. Diuresis with 0.9% NaCl solution 25 ml/kg/h for 1 hour
6. Antiemetic treatment (ondansetron, metoclopramide).

Carboplatin is a cisplatin analogue which shows no toxicity; its action is similar to that of cisplatin, but less marked. In the dog and cat it acts on the following neoplasms: osteosarcomas, epithelial carcinomas and sarcomas, and other tumor types, especially carcinomas. The drug induces a decrease in the number of neutrophils, which is most marked on days 7–10 and on day 21 post-administration. In cats, a neutrophil count is recommen-ded before each administration. Other side effects: thrombocytopenia, gastrointestinal disorders (vomiting, diarrhea). It is less nephrotoxic than cisplatin; in cats, it does not cause pulmonary edema.

It is recommended:

• – in dogs: 250–300 mg/m2 body surface area, i.v., once every 3 weeks. In large dogs (more than 0.7 m2 body surface area), the dose reaches 350 mg/m2 body surface area.
• – in cats: 210 mg/m2 body surface area, i.v., once every 3–4 weeks.

Carboplatin should be administered in 5% dextrose solution, in order to avoid a transformation into cisplatin, as it can cause problems in cats and animals with renal diseases. In animals with kidney diseases, the dose should be reduced proportionally to the kidney function, which is established based on creatinine clearance [37].

5-Fluorouracil FU is a pyrimidine antimetabolite that blocks DNA synthesis, and secondarily, RNA synthesis. It is metabolized in the liver and is partially excreted by the kidneys.

The non-specific toxicity of the product is weak. The product is poorly myelodepressive and does not induce gastrointestinal disorders or alopecia. In contrast, it has specific drastic toxicity, being sporadic in dogs and constant in cats, which forbids the use of this drug in cats. In dogs, toxicity depends on the dose, which is recommended not to exceed 150 mg/m2 per week, although some authors admit 200 mg/m2 per week as a maximal dose. The toxic action manifests by hallucinations, dromomania, hyperexcitability, tremor, seizure episodes and death. Neurological toxicity also manifests after intravesical instillations (500 mg/m2in 10 minutes) for the treatment of transitional carcinoma.

Fluorouracil is indicated in adjuvant monochemotherapy after tumor exeresis (mammary gland, thyroid, lung).

Hormonal agents. Adrenal corticosteroids are used in the treatment of hematogenic tumors, resulting in tumor size reduction by the direct destruction of neoplastic cells. Then, glucocorticoids are used for the treatment of secondary complications of malignant diseases, such as hemolytic anemia, thrombocytopenia and hypercalcemia.

Corticoids, with their main representative, Prednisone (PDN, Cortancyl ND), induce cytolysis, selectively thymocytes and some tumor or non-tumor lymphocytic subpopulations.

In the liver, prednisone is transformed into prednisolone, which can traverse the hematoencephalic barrier. The toxicity of this product manifests by polyuro-polydipsic syndrome, digestive ulcers, acute pancreatitis, diffuse alopecia, diabetes mellitus, all clinical and biological signs of iatrogenic hypercorticism, and may even result in death.

Indications for carnivores are in malignant lymphomas, acute lymphoblastic leukemia and mastocytomas, with real anticancer properties in these tumors. Prednisone is less used alone, being administered in polychemotherapy, in doses of 1mg/kg per day per os.

Corticoids can be successfully used in the fight of paraneoplastic syndromes such as: the control of hypercalcemia, autoimmune hemolytic anemia, edema that is associated with intracranial tumors, and respiratory failure induced by pulmonary lymphatic tumor infiltrations (metastasized mammary tumors).

Prednisone is relatively currently used in the treatment of malignant disorders. This product retains salts much more effectively than cortisol, also having an extremely high glucocorticoid activity, not to mention its extremely low cost.

Corticosteroids in high doses are antimitotic in mammalian and cytolytic cell cultures and are antimitotic for lymphoid tissues. In vitro, they can inhibit RNA and protein synthesis in lymphoid cells and they can induce an accumulation of free fat acids in sensitive cells, which results in nuclear membrane lysis.

Glucocorticoids are particularly useful in the treatment of malignant lymphomas. In dogs and cats, they cause an impressive regression and reduction of lymph node hypertrophies. Glucocorticoids are used in association with other cytotoxic drugs that are myelosuppressive. Glucocorticoids are also highly efficient in the treatment of leukemias and SNC lymphomas, since they penetrate the cerebrospinal fluid.

The fact that the glucocorticoid action is exerted on lymphoid cells, whether the target cell is at a certain proliferative stage or not, is particularly important. It should be mentioned that lymphoid cells shortly become resistant to glucocorticoids, and resistance to one of them involves resistance to all glucocorticoids.

Sex hormones have been used in the treatment of breast, prostate and endometrial tumors in humans. They have a palliative effect on endocrine dependent carcinomas. In veterinary medicine, estrogenic steroids are used in the treatment of prostatic hyperplasia and in perianal gland neoplasms, as well as in mammary tumors. Toxic effects manifest by thrombocytopenia, gynecomastia and fluid retention in tissues and cavities.

Diethylstilbestrol is a drug that is frequently used, with good results. Side effects are weak, and they can be prevented or treated.

The administration of androgenic compounds in dogs leads to an increase in the hematocrit, reticulocytosis and erythroid hyperplasia in the marrow; the stimulation of myelopoiesis and thrombopoiesis can appear at a lower level.

Other anticancer drugs. L-asparaginase is included along with asparaginase and amidohydrolase in the category of E. coli products. Unlike normal cells, neoplastic cells are L-asparaginase synthetase deficient, i.e. they cannot synthesize the amino acid L-asparagine and consequently rely on external sources for protein, DNA, RNA synthesis. L-asparaginase catalyzes asparagine cleavage and thus decreases the L-asparagine source for tumor cells. Resistance mechanisms appear as a result of mutations in the population of tumor cells that may produce asparagine synthesis; antibodies may also develop, which result in the drug destruction.

L-asparaginase is used for the treatment of lymphatic neoplasms, especially malignant lymphoma and lymphoblastic leukemia in dogs and cats, but other tumors such as melanoma and mastocytoma also have a favorable response. Side effects have been found: hypersensitivity and anaphylactic shock, which can be improved by the administration of dexamethasone sodium phosphate (hexadreson 1.0 mg/kg i.v.) and diphenhydramine (benadryl 0.5 mg/kg i.v.). When the hypersensitivity reaction appears, the administration of this drug is suppressed because the reaction will be much stronger. Hypersensitivity and anaphylactic reactions rarely appear in animals, but when they occur, they cause many problems. The following undesired effects are cited: urticaria, agitation, abdominal spasms, vomiting and diarrhea, dyspnea, hypotension, pruritus, and loss of consciousness. These effects disappear when the drug is suppressed. Other effects: more rarely, pancreatitis (in overweight dogs), decreased protein synthesis, coagulation disorders. Myelosuppression does not occur, this is why it is indicated for the treatment of patients with lymphomas, who present neutropenia.

Dose: it is not administered intravenously because of the risk of anaphylactic shock. It is administered i.m., s.c., i.p., the dose being 10,000 IU/m2 body surface area or 400 IU/kg. A deep i.m. administration reduces the risk of an anaphylactic reaction. No more than 5000 IU/injection site are administered. It can be administered weekly, drug resistance develops very quickly. The dose for PEG-L-asparaginase is 30 IU/kg i.v. [37].

Imidazole carboxamide is a drug whose actions are similar to those of alkylating agents, being active in the treatment of human melanoma. Toxic symptoms in dogs manifest by debility, anorexia, thrombocytopenia, leukopenia and anemia. Lesions are hemorrhagiparous, pulmonary edema, bone marrow hypoplasia and lymphoid depletion.

Hydroxyurea is a product that inhibits ribonucleotide-reductase, being effective in polycythemia vera.

Cis-diamminedichloroplatinum (cisplatin) inhibits DNA synthesis by cross-linking of complementary DNA strands. This product acts as a bifunctional alkylating agent. In human oncology, it is used in the treatment of ovarian, testicular, urinary bladder and cervical carcinomas. Good results have been obtained in the therapy of head and neck neoplasms, as well as in osteosarcomas.

Chemotherapy in veterinary medicine is still little used, since local treatment is preferred, especially the surgical excision of the tumor. Chemotherapy can be used for curative purposes, to eliminate the tumor, or as palliative treatment, to improve symptoms and prolong survival.

a) Chemotherapy used alone or primary chemotherapy is recommended to be used: in malignant lymphomas, acute lymphoblastic leukemia, Sticker’s sarcoma and canine mastocytomas; in felines, in malignant mediastinal and renal lymphomas. The substances that can be used in the mentioned neoplasms are: vincristine, cyclophosphamide, corticoids, adriablastin. These substances are included in sequential polychemotherapeutic regimens that allow the maximal use of the cytotoxic properties of different agents with minimal toxic effects, with an adequate administration rate, allowing the repair of normal tissues (bone marrow, epithelia).

The treatment of malignant lymphoma in dogs extends, according to a protocol proposed by COTTER [7], over three weeks:

• – day 1, intravenous vincristine 0.75 mg/m2, for three weeks;
• – days 1–7, oral prednisone 1mg/kg, for three weeks;
• – day 1, oral cyclophosphamide 300 mg/m2, in the first week.

The maintenance protocol starts with week 4:

• – day 1, intravenous vincristine 0.75 mg/m2, every three weeks;
• – day 1, oral cyclophosphamide 300 mg/m2, every three weeks;
• – day 1, oral prednisone 1 mg/kg, every other day.

If the remission period exceeds one year, the two mitotics are administered during all 4 weeks, for another 6 months. If signs disappear, prednisone is gradually stopped. Toxicity remains moderate.

Acute lymphoblastic leukemia of the dog is a disease with uncertain treatment, due especially to rapid evolution. An associated treatment is proposed, vincristine 0.75 mg/m2, once per week, and prednisone 2 mg/kg per day. If remission occurs (diminution in the number of lymphocytes by more than 50%), vincristine is administered every three weeks and prednisone will be administered in decreased doses (1 mg/kg per day).

Sticker’s sarcoma can be fought by microchemotherapy, using vincristine 0.750 mg/m2 once per week, continuing two weeks after clinical recovery. If treatment exceeds 5 weeks, the toxic effects on the bone marrow will be major.

Mastocytomas are frequent in dogs, and glucocorticoids are the most active substances in their treatment. In the case of multicentric mastocytomas with a low malignancy grade, prednisone 40 mg/m2 can be attempted for 15 days, then every other day for 4–6 months. Therapeutic regimens that associate cyclophosphamide, vincristine and prednisone do not give favorable results.

Feline malignant lymphoma can be successfully treated by using antimitotic substances. Mediastinal and renal locations respond very well to treatment, and complete recovery can be obtained. The association of vincristine and prednisone gives extremely good results. Prednisone can be administered in doses of 2 mg/kg per day in the induction phase. Vincristine is administered intravenously 0.70 mg/m2 per week for four weeks, then every three weeks.

Cyclophosphamide is administered in doses of 250 mg/m2, and it can be used over all three weeks in association with vincristine, but it causes intense granulopenia in many cats, and real efficacy is uncertain. If remission can be obtained without vincristine, prognosis is better.

b) Adjuvant chemotherapy is used after a local or locoregional surgical exeresis of the primary tumor. After surgery, even in favorable local evolutions, the risk of metastases remains a major problem. Tumors with a high postoperative metastatic risk are mainly bone, thyroid neoplasms, splenic hemangiosarcomas, mammary tumors and most malignant tumors. Adjuvant chemotherapy prevents the risk of metastasis of tumors that possess a high generalization degree. Adjuvant radiotherapy may be used in order to prevent the risk of postoperative local recurrences.

The rules for the use of adjuvant chemotherapy are based on some principles [7]:

1. Chemotherapy is more effective when the number of destroyed normal cells is smaller.
2. Chemotherapy has higher chances of being eradicating when its intensity is higher.
3. The number of chemotherapy-resistant cell clones is higher when the tumor is more developed.

Adjuvant chemotherapy should begin as soon as possible after surgery, it should be performed with efficient doses, polychemotherapy being preferred to monochemotherapy.

Adjuvant chemotherapy in veterinary oncology is indicated in major neoplasms:

• – thyroid tumors: fluorouracil (5 FU) or adriamycin;
• – mammary tumors: fluorouracil or adriamycin;
• – (splenic) hemangiosarcoma: cisplatin and adriamycin, alternatively;
• – soft tissue sarcomas with a high metastasis degree: cisplatin and adriamycin, alternatively;
• – second grade mastocytomas: prednisone;
• – third grade mastocytomas: vincristine and prednisone, in association;
• – bone sarcomas: adriamycin – cisplatin, pre- and postoperation.

Adjuvant chemotherapy is a new therapy in veterinary oncology, with perspectives of development and diversification, whose effects will not fail to appear, concomitantly with the development of the training and specialization of veterinary doctors in the field of oncology.

c) Palliative chemotherapy aims to improve symptomatology and prolong the patient’s survival. In the presence of a lesion, even at an extremely advanced stage, treatment can be proposed, under the conditions of low acceptable toxicity. In this case, the administration doses and rate should allow the maintenance of a satisfactory general state, under the conditions of a balance between toxicity and the therapeutic benefit.

In veterinary cancerology, it is difficult to objectively evaluate the response of a palliative treatment, since the criteria and observations that allow to assess an improvement in the patient’s condition are subjective, and sometimes it is difficult to persuade the owner to initiate a treatment that does not lead to recovery.

The indications of palliative chemotherapy are almost identical to those of adjuvant chemotherapy, when it has not been applied and/or when it has been instituted too late (metastatic tumors). Acute low toxicity drugs (5 FU) will be used, as well as all therapies that are extremely badly tolerated by the patient for a poor benefit [7]

In oncological therapy, doses are calculated per body surface, not per body weight, since considerable differences might exist in the optimal drug amount administered, especially in small sized animals. Thus, in dosages given based on body weight, small sized subjects of a species or breed will receive lower doses, compared to obese subjects that will be overdosed.

The formula that can establish body surface is:

m2=K×body mass (in grams)2/3104

where: the K factor is constant depending on species, thus: for dogs = 10.0; cats = 10.1; cattle = 9.4; horses = 10.5; monkeys = 11.8; mice = 7.9; and rats = 9.5. Body weight W is expressed in grams.

Body surface area for cats

kg	m2	kg	m2
0,25	0,040	4,0	0,255
0,50	0,046	4,5	0,275
0,75	0,083	5,0	0,295
1,00	0,101	5,5	0,315
1,25	0,117	6,0	0,333
1,50	0,132	6,5	0,352
1,75	0,147	7,0	0,370
2,00	0,160	7,5	0,387
2,25	0,173	8,0	0,404
2,50	0,186	8,5	0,421
2,75	0,198	9,0	0,437
3,00	0,210	9,5	0,453
3,25	0,222	10	0,469
3,50	0,223	11	0,500
3,75	0,244	12	0,530

Body surface of dog

kg	m2		kg	m2	kg	m2
0,25	0,0397		13	0,553	43	1,23
0,5	0,0630		14	0,581	44	1,25
0,75	0,0825		15	0,608	45	1,27
1	0,100		16	0,635	46	1,28
1,25	0,116		17	0,661	47	1,30
1,5	0,131		18	0,687	48	1,32
1,75	0,145		19	0,712	49	1,34
2	0,159		20	0,737	50	1,36
2,25	0,172		21	0,761	51	1,38
2,5	0,184		22	0,785	52	1,39
2,75	0,196		23	0,809	53	1,41
3	0,208		24	0,832	54	1,43
3,25	0,219		25	0,855	55	1,45
3,5	0,231		26	0,878	56	1,46
3,75	0,241		27	0,900	57	1,48
4	0,251		28	0,922	58	1,50
4,5	0,273		29	0,944	59	1,51
5	0,292		30	0,965	60	1,53
5,5	0,312		31	0,987	61	1,55
6	0,330		32	1,01	62	1,57
6,5	0,348		33	1,02	63	1,58
7	0,366		34	1,05	64	1,60
7,5	0,383	35		1,07	65	1,62
8	0,4	36		1,09	66	1,63
8,5	0,416	37		1,11	67	1,65
9	0,433	38		1,13	68	1,67
9,5	0,449	39		1,15	69	1,68
10	0,464	40		1,17	70	1,70
11	0,495	41		1,19
12	0,524	42		1,21

d) Adjuvant or prophylactic chemotherapy and drug combinations. Local surgical treatment by the excision of the primary tumor does not lead to the expected results, the eradication or improvement of neoplastic disease, due to the presence of micrometastases. The increased incidence of local recurrences and metastases in dogs, following surgery in some neoplasms (osteosarcomas, mammary carcinomas, melanomas) suggests that micrometastases have been present at the time of surgery. These practical data have led to the concept that the treatment of micrometastases by chemical substances can prolong the time interval in which the animal manifests no disease signs, can prolong survival or even increase the recovery rate. In humans, prophylactic regimens have been established with good results, in a number of neoplasms, including breast carcinoma and osteosarcomas.

Adjuvant chemotherapy is a logical, systematic and objective concept in the treatment of malignant diseases in humans [32, 33]. Adjuvant therapy programs have proved their efficacy by the fact that: 1) the metastatic disease incidence is directly correlated with the tumor mass; 2) the surgical recovery rate decreases with the increase in the size of the tumor mass at the time of surgery; 3) obvious primary tumors are generally not curable by drug therapy; 4) chemotherapy adjuvant to surgery increases the long-term recovery rates of many tumors.

This concept of establishing therapeutic programs of surgery supplemented by adjuvant chemotherapy should be introduced in veterinary oncology, and beneficial results will not fail to appear.

The use of combined drugs in the treatment of cancer has significantly prolonged the survival time in many human neoplasms. The use of complex regimens containing several drugs aims to induce different biochemical lesions in cancer cells, in order to block the systems that facilitate the growth and multiplication of neoplastic cells. In general, combined drug regimens only include drugs known to be specifically active in relation to a tumor, and these act by different mechanisms, in order to prevent tumor resistance to drugs. In addition, the drugs included in combinations have different toxicity limiting doses, allowing the administration of the whole or almost whole dose of each drug.

In veterinary medicine, by the development of combined therapeutic programs, the response of a neoplasm to a single chemotherapeutic agent should be determined in the first place. The drugs that have demonstrated activity as individual agents are subsequently combined with other active drugs in order to optimize the response to treatment. The combined chemotherapy of hemolymphopoietic neoplasms in dogs can serve as an example, being superior to treatment with a single chemical agent. Combined chemotherapy is used as a routine procedure in a number of neoplasms, such as lymphomas, leukemias and solid tumors.

The synchronization of drug doses is important, and the effect depends on a repair of normal cells before the patient reaches the critical intoxication phase because of a drug overdose. The induction of remission is the first phase of chemotherapy and is much more intense than maintenance therapy. The duration of remission induction varies between 6 and 16 weeks, depending on the protocol and type of the neoplasm treated. Maintenance therapy is the second phase of chemotherapy, which can last between 6 and 9 months and includes low drug doses and/or low administration frequencies. If the patient does not manifest a malignant disease after one year, chemotherapy is usually interrupted.

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19.4 HYPERTHERMIA THERAPY

Hyperthermia therapy is used due to its cytotoxic effects and because it can be an adjuvant to chemotherapy and radiotherapy. However, it should be mentioned that the use of hyperthermia may also have undesired effects (inefficiency, toxicity, increased tolerance to heat and even the appearance of resistant cells).

The biological efficacy of hyperthermia is closely dependent on both temperature and time; in addition, variable results also occur due to the tumor type and structure, as no uniform temperature can be maintained in the tumor mass.

Temperatures higher than 41°C induce lesions in tumor tissues, directly by cytotoxicity, and indirectly, by microcirculatory lesions.

The thermotolerance of tissues or tumors may occur when constant heating is performed at temperatures lower than 43°C; in the case of heating at temperatures higher than 43°C, followed by cooling to body temperature and in the case of heating at sublethal temperatures (40–41°C), the thermo-tolerance degree is positively correlated with the intensity and extension of cell destruction. So, a cell population that survives exposure to a high temperature will be much more thermotolerant than one that has survived a low temperature. Thermotolerance is a transient reversible phenomenon; with the recovery of a normal thermal state of cells or tissues, thermotolerance will decrease. The kinetics of the induction and decrease of thermoresistance depends on the tissue and cells, as well as on the temperature/time ratio. Thermotolerance reduces the efficacy of cell destruction in the case of thermotherapy associated with radiotherapy [9]. Hyperthermia can be applied in multiple fractions, the optimal interval being over 72 hours, in order to avoid the treatment of thermotolerant tissues. The thermotolerance induction degree depends on temperature, so that surviving cells situated in the (probably poorly vascularized) thermal tissue area of a tumor will be the most thermally tolerant, and duration until its reduction will be relatively long. In contrast, cells situated at the tumor limit, being well vascularized in the normal adjacent tissue as well, will be poorly thermotolerant, because of the lower temperature maintained during the thermal process. In this case, the heat sensitivity of the normal tissue will be increased, resulting in the loss of therapeutic efficacy. The attempts to differentially increase thermotolerance in normal tissue in relation to tumor tissue, by a change in the fractioning of the thermal action, have had a relatively low success rate.

Neoplasm microvascularization is structurally and physiologically ab-normal. Tumors have a fixed arteriolar flow and derive their vascularization from the venous portion of the vascular network. Consequently, they are typically hypotensive, with areas in which the blood flow is intermittent and slow. In addition, tumor vascularization has an abnormal structure, large sinusoidal capillaries, blood pools bordered by discontinuous epithelium and hemorrhages. This abnormal morphofunctional state determines hypoxia and acidosis in the tumor tissue. Acidosis is enhanced by the accumulations of residues derived from anaerobic metabolism. These conditions make the tumor extremely sensitive to hyperthermia induced lesions:

• – high temperatures are preferentially cytotoxic for cells from an acid environment;
• – high temperatures (42–46°C) will preferentially destroy tumor vessels, which determines a pH decrease and increased anoxia and ischemic necrosis;
• – normal adjacent tissues are warmed, while the tumor tends to become warmer, due to its relative hypotension.

In spite of all these strong biological reasons, heat alone is a relatively inefficient treatment. This deficiency is rather due to technical matters, but also to the non-uniform tumor structure, which makes difficult to ensure an optimal uniform temperature in the tumor mass.

The parenteral administration of glucose in order to decrease the pH has led to the potentiation of the antitumor effects of hyperthermia in the case of the association with cyclophosphamide.

Topical lidocaine anesthesia stimulates the efficacy of hyperthermia, due to the increased fluidity of the membrane. The use of this procedure has given good results, with a complete response in 90% of the cattle with ocular squamous cell carcinoma.

The induction of temporary ischemia in order to diminish the pH and increase thermal sensitivity has led to favorable results in murine squamous cell carcinoma. Cooling before heating has also been associated with increased in vitro cytotoxicity and a reduced vascular flow in normal arterioles.

In conclusion, hyperthermia as a therapeutic method has its limits, but it is certain that the heating of the whole tumor can be an adjuvant treatment to surgery, radiotherapy and chemotherapy [9].

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19.5 COMBINED TREATMENT

19.5.1 Combined hyperthermia-radiotherapy treatment

The separate studies of the effects of hyperthermia and radiotherapy on tumor cells have led to the conclusion of the combination of the two beneficial results, based on the following reasons:

1. High temperatures have cytotoxic effects on acidophilic cells in hypoxic condition. These findings correlated with the fact that hypoxic cells manifest a 2.5–3-fold increased resistance to destruction by radiation have determined the association of the two therapies.
2. Hyperthermia increases the radiosensitivity of all cells and diminishes the repair of sublethal lesions induced by radiation.
3. Hyperthermia is preferentially cytotoxic for cells in the S phase of the cell cycle, in contrast, these cells being almost radioresistant.

The use of an adequate combination of hyperthermia with radiotherapy shows that hyperthermia directly destroys or sensitizes poorly vascularized cells to radiation, while radiation destroys the cells from the well vascularized tumor surface. When hyperthermia is applied before radiation, there is also the risk of inducing transient hypoxia, with the development of resistance to radiation in cells that survive hyperthermal treatment [9].

The highest thermal stimulation ratio is obtained when heat is administered at the same time with radiation. However, in this case, the thermal stimulation ratio for the tumor and normal tissue is identical and no therapeutic benefit is obtained. The optimal effect is obtained when an interstitial heating method is used or when the hyperthermia generator is in contact with the tumor tissue alone.

In the case when a significant proportion of normal adjacent tissue is heated, radiation followed by heating after 3–4 hours produces the most consistent and important therapeutic gain factors. Data on human melanoma support the idea of heating after radiotherapy, in order to obtain the greatest therapeutic gain.

The thermal stimulation ratio depends on both the normal tissue and the tumor, temperature and heating duration. This dependence is probably related to the combined effects of direct destruction by heat and vasodilation by hyperthermia. It has been noted that the coldest tumor portion will manifest the lowest heat radiosensitivity, as well as a relatively reduced direct destruction by heat [9].

Clinical studies show that hyperthermia added to radiation will improve, under strictly defined conditions, local tumor control, compared to radiation alone. Combined treatment should be individualized for each subject.

It is ideal, from the point of view of the clinical objective, to obtain the permanent eradication of the tumor, so that it does not recur during the patient’s life, but the usual aim is the long-term response and survival without the disease. It is important to investigate the prognostic factors that influence the complete initial response, as well as those factors that influence the response duration. The combined use of hyperthermia and radiation gives favorable results in the treatment of canine tumors [35]. In 51 dogs with naturally occurring tumors, of which 35 located in the oral cavity, the authors used 4 radiation fractions, weekly, in a total dose of 3600–4000 Gy and 1 or 2 treatments with microwave hyperthermia, heating the tumor at 44°C, for 30 minutes each time. Results showed that 25 cases, 51 % of tumors, respectively, had a complete response to this therapeutic protocol, 18 cases, 35% respectively, significantly regressed, with a total response rate of 86%. Sixteen tumors developed recurrences, within 11–50 weeks after treatment. It may be concluded that hyperthermia combined with radiotherapy is useful in the treatment of certain malignant tumors in dogs.

The tumor volume significantly influences the complete response rate. Volume has a more reduced influence on tumors treated by a combined modality than on tumors treated by radiotherapy alone. The risk of local recurrence is approximately 1.5–2-fold higher in tumors treated by radiotherapy alone, compared to those treated by combined therapy. There are also exceptions: in malignant melanoma, animals treated by radiotherapy alone manifest a 3-fold better chance of maintaining response, compared to combined treatment. In contrast, combined therapy increases the chance of maintaining response in many other tumors, such as: fibrosarcomas, mastocytomas, squamous cell carcinomas and mammary carcinomas. In general, the minimal thermal dose positively correlates with both the complete response rate and the response duration.

Lesions of normal tissues submitted to hyperthermia consist of direct burns, which are rarer, and the most common lesion is skin infarction. The careful control of surface temperature can reduce thermal burns, but it does not influence the development of infarctions. The formation of infarctions seems to be related to rapid tumor necrosis, which sometimes appears deep under the cutaneous layer. In dogs and cats, a peculiarity of cutaneous vascularization occurs, which consists in the limited presence of collateral vascularization.

Skin burns and infarctions have a delayed repair, since they have been radiated. The surgical removal of tissue residues is not efficient, which is why conservative treatment is recommended. The routine cleaning of the surface is performed and treatment with antibiotics and/or drugs that favor cicatrization by granulation tissue, in 2–3 months, is administered. Thermal lesions are healed without complications in 85% of the treated cases.

Lesions induced by radiotherapy used alone or in association with hyperthermia have not been significantly increased. These lesions consist of wet cutaneous desquamations and radiation-induced burns, and subsequent complications are represented by skin fibrosis, bone necrosis and peripheral neuropathies. No increase in the incidence of all these effects could be demonstrated, as a result of hyperthermia, when direct thermal lesions have been eliminated.

The results obtained by DEWHIRST (1987) show that the therapeutic benefit depends to a great extent on the application of hyperthermia on the whole tumor volume. Then, an excessive maximal thermal dose will result in an increased incidence of thermal lesions, reducing in this way the chance of a benefit of hyperthermia.

19.5.2 Combined hyperthermia-chemotherapy treatment

The reason for a combined hyperthermia-chemotherapy treatment is the fact that systemic chemotherapy with local or regional hyperthermia can increase the efficacy of therapy, without increasing systemic toxicity. The chemical preparations used act synergically in the case of the use of hyperthermia provided that temperature is precisely regulated. Thus, bleomycin and adriamycin do not manifest a synergic action at temperatures below 42°C; in contrast, at temperatures higher than 43°C, their synergic action is highly effective. Cysteamine and amphotericin B do not manifest cytotoxicity at 37°C, but only at temperatures higher than 42°C. There are also situations in which high temperatures do not favor cytotoxicity, like in the case of 5-fluorodeoxyuridine, methotrexate and Vinca alkaloids.

Tumor fight can be stimulated by the combination of heat with cytoxan administration, if hyperthermia has been previously used. Hyperglycemia leads to a reduction in intratumoral pH by anaerobic glycolysis, coupled with vascular stasis.

The highest efficacy of heat combined with chemotherapy will be obtained when:

• – the therapeutic agents used are those that show synergy with hyperthermia;
• – hyperthermia and chemotherapy are administered simultaneously or shortly after one another. In the case of several drugs, this can mean synchronized drug administration, so that the highest tissue level is reached at the time of hyperthermia;
• – hyperthermia is performed by a route that does not interfere with the obtaining of reasonable intratumoral concentrations of the preparation. This will be possible by the use of and exposure to high hyperthermia, in order to diminish the intratumoral blood flow, which causes a reduction in the concentration of the administered drug that penetrates the tumor;
• – hyperthermia is applied at a temperature that will allow a maximal efficacy of the studied drug, without compromising the normal tissue. Thus, whole body hyperthermia is limited to temperatures between 41 and 42°C, since temperatures above this level are highly toxic.

Hyperthermia, even at a relatively low temperature of 41°C, improves melphalan efficacy; in addition, an increased blood flow in the tumor is obtained, which stimulates the drug level. Chemotherapy combined with local low temperature hyperthermia (40–41°C) has been used in extremity tissues in humans. Therapy has used thermal perfusates, with spectacular results [9]. The same author recommends the use of the limb perfusion technique in veterinary medicine, since the cost of equipment is reasonable and the technique is without complications. The main advantage of this procedure is the possibility of saving the limb. It is not known whether this procedure influences distant metastatic rate.

The work technique consists of the local application of heated paraffin, warm water compresses, heat radiating devices, etc. General anesthesia is necessary in the majority of the methods used, less in the case of heat radiating devices. Studies have been so far performed on human patients at advanced disease stages. Adverse reactions consist of cerebral and pulmonary edemas.

In dogs, whole body hyperthermia has been used in the fight against disseminated mastocytoma. The complications found include surface burns at pressure points and perforating gastroduodenitis. Whole body hyperthermia has been used in dogs with malignant lymphoma, the following complications being found: pulmonary edema, intrinsic coagulation factor disorders and thrombocytopenia.

Whole body hyperthermia in animals, even in company animals, is little used. Consequently, some criteria will be exposed that are applied in humans in order to avoid excessive cardiovascular and pulmonary stress, which should be considered in the case of experiments or even practice in animals [9]:

• – detection of the absence of nerve metastases;
• – absence of congestive heart failure, which cannot be controlled by adequate medication;
• – minimal ascites and normal serum bilirubin concentrations;
• – serum creatinine under 2.0 mg/dl;
• – lack of thrombocytopenia.

Body hyperthermia used alone has a reduced therapeutic value, which is why it is recommended to be used in combination with chemotherapy and/or radiotherapy. In companion animals, the associated technique is recommended in the treatment of the occult metastases of osteosarcomas and melanomas. A previous thorough clinical examination is compulsory.

The methods for the production of hyperthermia are varied, each having its own advantages and disadvantages, depending on the tumor type and location. Many of these methods, with their technical means, are relatively expensive to be currently used.

Local heating for accessible peripheral tumors can be carried out by microwave equipment, ultrasonographs, magnetic induction devices and radiofrequency applicators. Interstitial methods of local heating include microwave antennas and loaded metal needles – radiofrequences.

External local devices have the advantage of being non-invasive and of requiring a minimal time to be set up. The major disadvantage consists of fixed sizes and configurations, which frequently cause difficulties in the coupling to the tissue, especially when the surface is not smooth, and in the covering of the tumor. Thus, head and neck tumors are difficult to treat by these means. Moreover, the presence of bones in these regions complicates accessibility, leaving tumor portions difficult to approach. The depth of penetration is also limited, so that at a depth of over 4 cm the tumor cannot be effectively treated.

Interstitial heating has the advantage of virtually covering any geometry, and penetration depth is only limited by the skills of the surgeon who performs the technique.

Regional heating has been experimented by different devices, for body regions (pelvis, abdomen, thorax), but the application of these methods has not been proved to be technically possible.

The use of hyperthermia in veterinary practice depends on the development of valid, practicable, cost-effective techniques and equipment. In the field of hyperthermia, this involves safety for users and electronic reliability of equipment, in the sense of eliminating electrocution and burning risks.

The problem of efficacy depends on the aim pursued in the use of equipment. For example, the thermal pistol has been proved to be effective in the treatment of bovine squamous cell carcinomas with ocular location. In general, it is estimated that thermal treatment used alone is relatively ineffective.

Hyperthermia can be useful as a surgical pretreatment, by tumor volume reduction, and as a local adjuvant to systemic chemotherapy. Whole body hyperthermia may represent a somewhat promising technique in systemic tumor disease.

In conclusion, it can be estimated that hyperthermia is a promising therapy, whose potential has not been completely discovered, being a new, highly interesting modality in the treatment of cancer. The solution of some technical problems will allow to extend the use of this therapy in cancer disease.

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19.6 PHOTOTHERAPY

Dynamic phototherapy refers to the use of hematoporphyrin or photofrin II preparations, at specific light wavelengths, in the detection and treatment of malignant solid tumors. Both the detection and treatment of tumors by the dynamic phototherapy technique depend on the character of the neoplasm and on the location of the preparation in the tumor. So, at a certain time after the intravenous injection of the preparation, this accumulates and/or is retained in a higher concentration in malignant tissues compared to normal tissues [14].

Hematoporphyrin used in oncology is prepared according to the DOUGHERTY et al. and GOMER method of [14], which consists of the following: hematoporphyrin HCL is solved in glacial acetic acid and sulfuric acid (in a 1:19 proportion, per volume) and is maintained for 12–16 hours at the temperature of 20°C. The product is filtered with a Whatman filter paper and is adjusted to a pH of 6.0, using a 35% sodium acetate solution. After repeated stirrings and vacuum drying, an injectable solution is prepared (pH = 7.2–7.4) in isotonic physiological serum at a final concentration of 5 mg/ml and sterilized by millipore filtration.

Hematoporphyrin, prepared according to the described procedure, is a mixture of several porphyrins whose locations in the tumor vary depending on the neoplasm structure. The active component of hematoporphyrin has been isolated and its structure has been established, dihematoporphyrin ether, experimentally tested under the designation of photofrin II.

The tumor can be located using hematoporphyrin, by lighting with 405 nm light, which has a fluorescence of approximately 630 nm (red-orange).

The hematoporphyrin-light interaction has therapeutic effects by lighting with specific visible light wavelengths (especially 632 nm, red), a photochemical reaction taking place, from which pure oxygen, a strong oxidant agent, results. Biological lesions secondary to the action of active oxygen consist of the direct cytotoxicity of malignant cells and the destruction of tumor microcirculation.

It can be estimated that photodynamic therapy depends on the hematoporphyrin concentration, molecular oxygen availability and visible light. The preferential distribution of hematoporphyrin derivatives in tumors offers a selectivity level in the establishment of a positive therapeutic ratio, a more marked effect on the tumor compared to normal tissue. The improvements in the technology of light emission systems allow activating light to be directed towards the lesion in the first place, avoiding normal tissues. In most tissues, approximately 1% of the activating red light will penetrate 1–2 cm deep, ensuring a sufficient level to produce the reaction. While this level also limits the treatment volume or at least complicates the treatment procedure, the necessary protection for normal tissue structures is a major advantage of this therapeutic procedure.

The location of the lesion can be detected using a light source that produces 405 nm light. For superficial lesions, any “black light” can be used in a dark room. Much more sophisticated systems can be built, in association with conventional cytoscopes and endoscopes, using ultraviolet lasers and quartz optical fibers.

Superficial lesions are treated using high intensity xenon arc lamps (2.5–5.0 kW), with filtration in order to eliminate inconvenient wavelengths. Usual lamps produce a high intensity beam (> 100 mW/cm3) within spectral limits of 620–650 nm; in this situation, the disadvantage consists of the maximal optical power density that is deposited at the surface of the target region (frequently at the normal skin surface) and effective penetration is limited to a 1–2 cm surface.

For therapeutic purposes, a 5–19 W argon laser is currently used for the activation of a second coloring laser that produces a beam of the order of 635 ± 5 nm, and continuous power of approximately 2 W, using glass optical fibers.

The results obtained by THOMA et al. [14] by using phototherapy are more than encouraging. In malignant solid tumors, the authors intravenously inject a dose of 5.0 mg/kg hematoporphyrin, considering the date of injection as day zero (starting with which the whole period is measured). The patient will be protected from intense solar radiation for 2–4 weeks. On day 3, the lesion will be submitted to surgery under anesthesia, in the case of deep locations, or hair is removed from the tumor surface. In superficial lesions, the fiber is positioned so as to light the target area, and for deep lesions, the fiber or fibers are placed in 18–20 needles, in order to offer an adequate optical geometry. When hemorrhage appears, since blood significantly reduces light penetration, the fiber must be repositioned. After treatment, the animal will be monitored for 24 hours, then it will be followed up weekly. The necrotic mass and the superficial crust are monitored, and if necessary, antibiotic therapy will be initiated. On day 10, the second light treatment may be performed, which significantly improves the disease evolution; sometimes, depending on the case, the whole process can be resumed after 3 weeks.

Of a number of 75 animals, the cited authors have obtained an objective response in 90% of cases. Deeper lesions have a late response, regression being due to injured local microcirculation, in regions closely adjacent to the lesion. Except for transient photosensitivity, for at most 4 weeks, no other side effects of photodynamic therapy have been noted.

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19.7 IMMUNOTHERAPY

Regarding cancer disease therapy, JASMIN’S statement (1987) is highly suggestive, by which the three currently used weapons,surgery, radiotherapy and chemotherapy, are symbolically compared to iron, fire and poison. The opposite of these therapies, which attempts to compensate and replace these “brutal” treatments by a less aggressive treatment, remains the mobilization of the inner defense or repair systems of affected cells. Biotherapies, which use natural biological substances and have as a unique target anticancer cells remain a desideratum and a hope for the solution of the problem of cancer disease. The techniques of creating hybridomas able to produce monoclonal antibodies that might recognize a single antigen also give hope for both the early diagnosis and therapy of cancer metastases. Antigens recognized in tumors by cytolytic T lymphocytes may serve as targets for immune responses that destroy the tumor without affecting normal tissues. Studies performed on experimental cancer induced by chemical carcinogens or ultraviolet rays have proved that the majority of these tumors possess antigens capable of inducing an immune rejection response [3]. The authors remark in spontaneous tumors the presence of non-immunogenic cells, which bear too weak an antigen to induce an immune response, but which can serve as a target for a previously induced immune response. This response involves T lymphocytes. The authors reach the conclusion that all mouse tumors possess tumor rejection antigens. According to TOWNSEND et al. [3], viral proteins can be recognized by T lymphocytes, even if they do not appear on the cell surface.

Immunotherapy is another modality of stimulating the host defense mechanisms, an anticancer strategy. Experiments and practical results have proved that immunotherapy alone has an inconsistent efficacy, while preceded by surgery for tumor volume reduction, by radiotherapy or chemotherapy, it has proved to be a valuable adjuvant in the treatment of neoplastic disease.

Immunotherapy has demonstrated a particular efficacy in the case of tumor remnants, of residual neoplastic cells, inducing their destruction.

In time, the understanding of the complex phenomena occurring as a result of the reaction of the neoplastic organism and of tumor cell behavior has led to the progress, diversification and increased efficacy of immunotherapeutic means. This has allowed the development and use of therapies based on cytokines, monoclonal antibodies, chemical immunostimulators, interferon, tumor necrosis factor, anti-tumor growth factor antibodies, antiangiogenesis factors, and many others.

Veterinary oncology in spontaneous or experimental cases in the field of immunotherapy offers an extensive research area, which will eventually prove to be beneficial in human cancer therapy. Immunity ontogenesis involves numberless cell and protein interactions, such as macrophages, natural killer (NK) cells, lymphoid cells (T and B lymphocyte), granulocytes, antibodies and complement. The reactions occurring in the tumor host prove that neoplastic cells are non-self cells, and neoantigens are present on their surface. These antigens seem to be tumor specific transformation antigens or tumor associated transformation antigens, which are not necessarily specific.

The non-specific natural defense of the organism is carried out by natural-killer cells, macrophages and their biological products. In the specific response to tumor development, killer cells are stimulated to respond to specific soluble transformation antigens and tumor associated transformation antigens, in the attempt to destroy newly formed tumor cells. The complement also plays an important role in tumor immunity, inducing complement mediated cell cytotoxicity (killer T cells) or antibody dependent cell cytotoxicity.

Tumors caused by viruses may be prevented by vaccination (Marek disease, viral papillomatoses, etc.). In many tumor disease situations, the interdependence between cell mediated immunity and humoral immunity has been demonstrated.

The presence of tumor immune control exerted by the host is proved in numerous cases, such as patients receiving immunosuppressive drugs, subjects with immune deficiencies, advanced age patients, etc., which are exposed to a higher risk for tumor disease. Tumor progress is also correlated with factors elaborated by the tumor, poorly antigenic mutant tumor cells allowing the development of metastases. The protein structures of tumor antigens can determine a reduced immune response; angiogenesis and growth factors stimulate the onset and dissemination of the tumor.

Efficient immunotherapy will be theoretically carried out if immune interferences are eliminated, allowing the restoration of cancer immune control. This process, known as tumor “cytoreduction”, stimulates natural immune defense, but is of short duration in the case of neoplastic recurrences [32, 33].

Antitumor immunotherapy modalities. Immunotherapy methods can be grouped into: preventive, passive, adoptive and active,and depending on the type of reactions induced, they can be specific and non-specific [23, 32, 33].

Specific and non-specific active methods are the most frequently used, but all are used in practice.

19.7.1 Preventive immunotherapy

Preventive immunotherapy aims to limit the use of immunosuppres-sive drugs in the following situations: the immunodepression state existing between the limitation of antilymphocyte serum, developing immunode-pression and the high incidence of certain cancer diseases (immunocancerology). In dogs, malignant lymphomas are not altogether exceptional after prolonged corticotherapy used in pruriginous dermatosis.

19.7.2 Non-specific active immunotherapy

Non-specific active immunotherapy has been investigated in laboratory animals and used in both human and veterinary medicine. Non-specific immunostimulants used in cancer therapy are biological substances and chemical immunopotentiators. The more frequently used biological substances are: Bacillus Calmette Guérin; C. parvum and C. granulosum; B. pertussus; viral vaccines; purified protein derivatives; Freund adjuvant (mycobacterial fractions); mixed bacterial toxins; lipopolysaccharides and polysaccharides.

Chemical immunopotentiators: levamisole, dinitrochlorobenzene and synthetic RNA homopolymers. Synthetic immunopotentiators: azimexon, tuftsin and thymosin.

Biological substances were and are used in non-specific active immunotherapy based on the observation that some bacteria, bacterial substances (toxins and bacterial membranes) stimulate host resistance and immune responses to numerous infections and tumor associated transformation antigens.

The Calmette Guérin bacillus (CGB) is an attenuated and avirulent Mycobacterium bovis strain, and the active substance is muramyl dipeptide, which is a macrophage activator. In humans, CGB alone or in association with IL-2 is used in the early treatment of urinary bladder tumors, with intravesical administration. In principle, preparations containing high doses of living organisms, 107–108 organisms, have a more marked effect than those containing less than 106 organisms [38]. CGB administration to human and animal subjects cancer has frequently caused tumor regression. Practical results with show a decrease of pulmonary metastases in dogs with osteosarcomas or mammary carcinomas following intravenous CGB administration. Among possible side effects, transient CGB infection, indisposition, fever and anorexia are mentioned. Intratumoral CGB administration in patients with melanoma, with pulmonary metastases, has led to encouraging results in humans. CGB stimulates immunologic cell and humoral response to non-related antigens, in addition to their antigenic determinants. This includes T cell proliferation, the amplification of T cell dependent humoral responses and an enhanced cell response of lymphocytes and macrophages, i.e. delayed hypersensitivity, skin allograft rejection, increased phagocytic macrophage capacity, and decomposition tolerance. It activates the macrophage activity of natural killer (NK) cells.

Intralesional CGB administration in ocular squamous cell carcinoma in cattle has a moderate percentage of positive results. In contrast, CGB is successfully used in equine sarcoids, especially associated with cryosurgery [17, 28]. In the eyelid squamous cell carcinoma of a pony, cryotherapy and CGB immunotherapy as multiple injections were successfully used over 17 weeks. The tumor regressed, and lymphatic nodules recurred after 7 months. Over the following 18 months, there were no recurrences [20].

Mycobacterial membrane residues have reduced side effects compared to CGB, having at the same time a polyclonal lymphocyte mitogenic potential. These mycobacterial residues have been successfully used in the intralesional treatment of numerous experimental and spontaneous neoplasms. Good results have been obtained in human melanoma, bovine ocular squamous cell carcinoma and equine ocular sarcoids.

Muramyl dipeptide (MDP) is the structural component of the Mycobacterium cellular wall. In vitro, monocyte – macrophage activity increases, but in vivo, the activity of MDP is limited because it is metabolized in 60 minutes; it also has marked nephrotoxicity and may induce vasculitis.

Muramyl tripeptide phosphatidyl ethanolamine (MTP-PE) is the lipophilic derivative of MDP, which for a better administration is encapsulated in liposomes. This liposome- encapsulated MTP-PE (L-MTP-PE) causes a non-specific stimulation of macrophages and monocytes, thus having an antineoplastic activity. In humans, after L-MTP-PE administration, a release of cytokines, especially IL-1, TNF, IL-6, was observed. Antitumoral effects were found in metastasizing osteosarcoma, melanoma, renal cell carcinoma. In dogs with osteosarcoma, the survival duration after amputation and i.v. L-MTP-PE administration was significantly longer than in the control group (222 days, 77 days, respectively). When after amputation and L-MTP-PE administration, cisplatin was also used, the survival duration was 14.5 months compared to the control group, in which cisplatin was not used and the survival duration was 10 months. Dogs with hemangiosarcoma of the spleen had, after surgery and administration of cytostatic drugs, a survival duration of 277 days. Good results were obtained in dogs with oral melanoma; in cats with mammary tumors, no improvement was found.

Antitumor immunotherapy (according to MAGNOL and ACHACHE, 1983)

	Passive immunotherapy	Adoptive immunotherapy	Active immunotherapy
			Specific	Non-specific
				Biological adjuvants	Chemical adjuvants
Modalities	Serum or Ig from subjects in remission or tumor-rejecting patients	Lymphocyte transfusion from normal subjects or subjects in remission	Allogenic tumor cell graft	Live CGB	Polynucleotides
	Heterologous antitumor serum	Allogenic bone marrow graft from normal subjects	Inactive autologous or allogenic tumor cell graft	Reticulostimulines Mixed bacterial vaccine	Levamisole
	Heterologous antilymphocyte serum (in the case of a malignant hemopathy)		Changed inactive autologous or allogenic tumor cell graft	Corynebacterium parvum
Risks	Facilitation Pathology related to the use of heterologous sera	Host versus graft reaction	Graft acceptance Facilitating phenomena	Facilitation Local and/or general reactions

L-MTP-PE is a stable preparation, commercially available for clinical studies as CGP-19835A Lipid, Novartis Basal, CH. The general dose is 2 mg/m2, twice a week, for 8 weeks, in slow i.v. administration. L-MTP-PE is well tolerated by dogs; the side effect consists of increased body temperature by 1–2°C, 2–4 hours after administration [38].

Mixed bacterial toxins obtained from Streptococcus and Serratia spp. have been used in some human neoplasms, but in dogs and cats they have led to unconvincing results.

Corynebacterium parvum, an anaerobic gram-positive bacillus, has been prepared as a suspension inactivated with phenol. The preparation can immunologically potentiate the activity of macrophages and some lymphocytes by the intense stimulation of the reticuloendothelial system. The activity of macrophages exerts a cytostatic and cytocidal action. Activated macrophages induce an increased production of T cell dependent and independent antigen antibodies. Corynebacterium parvum can stimulate the specific tumor immunity, mediated by T cells by the cross reaction of antigenic determinants. Corynebacterium parvum is not cytotoxic in vitro for tumor cells, although the treatment result consists of an increased production of killer and suppressor T cells, as it results from graft-host reactions. CGB and Corynebacterium parvum seem to act similarly in antitumor activity, i.e. by macrophage activation and increased killer cell activity. Each microbiological preparation demonstrates a non-specific antitumor activity in vitro, and the in vivo antitumor effects of both partly result from the stimulation of the immune response towards the organisms themselves. The effects of Corynebacterium parvum preparations have been studied in experimental tumors in animals and in spontaneous human and animal tumors. Local inoculations with Corynebacterium parvum in hemangiopericytoma and mastocytoma in dogs and mammary carcinoma in cats have not led to encouraging results [23]. C. parvum directly injected in the lesions of humans with melanoma has resulted in a longer survival duration than in the case of CGB [38].

Freund’s adjuvant: the immunogenicity of soluble proteins is stimulated in the case of their persistence in tissues. An efficient mechanism in order to obtain this effect consists of water and oil emulsions, developed by Freund, in particular those containing live or inactivated mycobacterial suspensions. This adjuvant is used in clinical human and veterinary examinations, especially as a non-specific active immunogen associated with “cytoreductive” procedures.

Lipopolysaccharides are responsible for the characteristic biological properties of endotoxins derived from gram-negative bacteria and they stimulate each type of cells known to be involved in immune response. Polysaccharides cause an acute hemorrhagic necrotic reaction in some induced tumors, but they are not toxic for the cells of the same tumor developed in vitro, consequently a direct action has been excluded. Lipopolysaccharides are supposed to be able to induce the secretion of a factor or mediator from macrophages, which is responsible for the tumor necrosis factor.

Polysaccharides are immunostimulators due to their function of activators of an alternative complement pathway, being in this way effectors of non-specific immune stimulation. This includes the initiation of inflammation, which attracts polymorphonuclear leukocytes and macrophages. By immune adherence to macrophages, platelets, antigens and the antigen-antibody complex (stimulating in this way immune complex phagocytosis) and an increased susceptibility to the attack of cytotoxic T cells, as well as viral neutralization, polysaccharides participate in antitumor defense. Like lipopolysaccharides, polysaccharides have been little studied and used in veterinary oncology.

The development of gene transfer techniques represents a new direction of active immunotherapy. The intratumoral expression of transferred genes, which locally stimulates the immune system, allows the tumor diminution by general route; gene therapy also allows the modification of the protein composition of tumor cell membranes. The combined transfer with several therapeutic genes in the same cell allows to conceive combinations susceptible to induce a better local stimulation of the immune system [10]. The reviewing of the literature, as well as their own experiments, make the mentioned authors reach certain conclusions regarding antitumor vaccination and gene therapy. A major problem of direct in vivo gene therapy remains the target of transferred cells, but in the case of solid tumors, intratumoral injection allows a physical targeting of the vector. Transfer methods should meet criteria of efficacy, gene expression level, and treatment. The development of the best vectors remains a priority and a desideratum of the future investigations.

The treatment of cancer by gene therapy should be adapted to each clinical situation. Different protocols can be imagined with in vivo or ex vivo gene transfer associated or not in conventional or transformation treatments.

Vaccination should induce an immune response, an active antigen-specific immunity. Vaccination involves two notions:

1. Curative vaccination, with the development, following the contact with the antigen (antigens), of immunity that allows to fight a disease in evolution, active immunity.
2. Preventive vaccination, with the development of protective immunity, such as viral vaccinations, by which persons are immunized against viral antigens before coming into contact with them. This type of vaccine requires further investigations in order to be improved, in cancer.

The response of T lymphocytes, a cell mediated response, is essential and has become the main target of antitumor immunization strategies. The response of T lymphocytes is complex and may be synthesized in the following way:

1. The initiation of the response, which triggers the action of TCD4+ lymphocytes and cells that present the antigen (antigen presenting cells – APCs) such as dendritic cells or macrophages. There is a system with two signals, one specific for the antigen, and the other, the costimulation signal, which does not depend on the antigen. The first signal involves the molecules of MHC (major histocompatibility complex) class II which, associated to an antigen peptide integrated by endocytosis and degraded, binds the T lymphocyte receptor (TcR, T cell receptor). The second signal triggers among others the B7 molecule, which binds the CD28 receptor, also situated on T lymphocytes. This double interaction induces the secretion of numerous cytokines by the two cell partners, of which interleukin 2 (IL2), secreted by the TCD4+lymphocytes. L2 will allow the proliferation of the T clone, which recognizes the antigen and, consequently, the amplification of the response. It is important to emphasize that the occupation of TcR in the absence of the costimulation signal causes T cell anergy, a mechanism potentially responsible for the induction of tolerance.
2. The specific cytotoxic effector response of the antigen is restricted by MHC, since this is changed by TCD8+ lymphocytes that also require two signals: the first is the recognition by TcR of the antigen associated to a membrane of MHC class I; the second signal is more complex, being ensured by different cytokines, such as IL2, which allows CD8+ differentiation and proliferation in cytotoxic lymphocytes (CTL). These cytotoxin-producing T lymphocytes will act on different cell categories and ensure the response amplification.

The absence of a spontaneous effective immune response against cancer cells that possess tumor antigens may have different origins such as: the absence of antigenic peptides presented by the molecules of MHC class II, an antigen deficiency of APCs (antigen-presenting cells), a quantitative insufficiency of cytokines secreted by TCD4+ lymphocytes. A deficient effector function may be due to a defect in the molecular expression of class I at the tumor cell surface, to incorrectly processed or deficient tumor antigens, which results in a deficiency or the absence of the presented antigenic peptides. All these phenomena may lead to an anergy of tumor antigen-specific T lymphocytes or a tolerance by non-recognition [10].

Chemical and synthetic immunopotentiators. These chemical substances are used as adjuvants, which stimulate macrophage activity, potentiating the action of other therapies used in cancer disease.

Levamisole is an imidazole compound, with immunorestoring capacity, which stimulates macrophage and T lymphocyte functions. Levamisole increases the production of antibodies in immunodeficient animals, as well as lymphocyte-mediated antitumor toxicity and the chemostatic response of neutrophils and monocytes.

Levamisole can be used as an immune modulator associated with chemotherapy after tumor volume reduction; associated with corticoids, it is effective in the treatment of eosinophilic granulomas in cats [32, 33]. In dogs and cats with mammary tumors and in dogs with lymphosarcomas, following levamisole treatment in a 5 mg/kg dose administered orally once every other day, no positive effects have been found. In humans with colon carcinoma, levamisole is used in combination with 5-fluorouracil and it positively influences survival [38].

Dinitrochlorobenzene stimulates local cell-mediated DTH reactions (allergic contact dermatitis), which determines the local destruction of skin and subcutaneous tumor cells. In humans, this chemical product is successfully used in the treatment of subcutaneous and skin neoplasms. In dogs, it gives good results in the treatment of squamous cell carcinoma. The treatment scheme proposed by KLEIN [17] involves a sensitization by the local application of 1:100–1:1000 dilutions, in lanolin cream with water, at 10–14 days intervals, after which dinitrochlorobenzene is applied on the tumor surface, inducing DTH reactions. In humans, sensitization is performed by lower dinitrochlorobenzene concentrations (dilutions in doses of 1 to 105–1 to 106); the direct contact with dinitrochlorobenzene should also be avoided in the treatment of canine tumors.

Azimexon is a synthetic immunoadjuvant, which increases DTH reactions, by the reaction of lymphocytes to the antigen, of T lymphocytes and monocytes under the control of suppressor T lymphocytes. This immunoadjuvant potentiates the responses of T cell dependent and independent antigens, but also activates macrophages in their cytostatic action for tumor cells. Azimexon also reduces the immunosuppressive effects of cyclophosphamide or effects caused by radiation.

Tiftsin is a tetrapeptide that potentiates the responses of antibodies to T cell dependent antigens. It also acts on macrophages and stimulates antibody dependent cell cytotoxicity.

Pyroxicam is a prostaglandin (PG–E2) antagonist, which gives it antineoplastic properties in clinically induced tumors, transplanted tumors in rats, metastasizing tumors in humans.

In the dog, partial remission was found in the case of pyroxicam monotherapy in mammary adenocarcinoma (1 of 3 dogs), pavement epithelium carcinoma (3 of 5 dogs) and Sticker sarcoma (1/1). In a study performed on 34 dogs with epithelial carcinoma of the urinary bladder, KNAPP et al., cited by NEIGER [38], found the following: 2 animals had complete remission, 4 animals had partial remission, in 18 animals the disease was stabilized, and in 10 animals the disease was aggravated.

The relatively safe dose is 0.3 mg/kg per os once a day. The most frequent side effects are gastrointestinal symptoms (melena, vomiting), nephropathies (interstitial nephritis).

Cimetidine (tagamet) is an antagonist of H2 receptors and is used in peptic ulcer. It may influence immune functions in humans and animals, both in vitro and in vivo, it reinforces cell mediated immunity. The action mechanism seems to be an inhibition of suppressor T lymphocytes, resulting in an increased activity of cytotoxic T lymphocytes in animals with experimentally induced tumors and in humans with malignant melanoma. In horses with cutaneous melanoma, regression was described following cimetidine administration [38].

Synthetic adjuvants do not include non-specific tumor suppressor cells.

In the treatment of cancer disease, the pharmaceutical industry is continuously bringing new products on the market, which stimulate the local or general reactivity of tumor organisms.

19.7.3 Specific active immunotherapy

Specific active immunotherapy is the most used in practice.

Specific active immunotherapy aims to focus immune host reactions, which are supposed to be potentially intact, on the single membrane neoantigens of neoplastic cells. For this purpose, autologous or allogenic tumor cells that are most frequently inactivated or changed can be grafted, as well as antigenic extracts of these cells, tumor cells that have been changed in order to become much more immunogenic.

Vaccines used in veterinary medicine are prepared by the phenolization or formolization of autogenic tumor cells, with good results in the treatment of squamous cell carcinoma located at the horn base or in the eyes of cattle.

Extremely good results have been obtained in the treatment of bovine ocular squamous cell carcinoma by using different specific active immunotherapy formulas. Thus, small pieces of fresh carcinomatous tissue in phenol-physiological serum induce tumor regression, by a single injection. Results are better in the case of incipient forms of ocular squamous cell carcinoma, following the administration of approximately 500 mg tumor extract in physiological serum-phenol. The same methodology is used with good results in horn carcinoma. The formolized tumor tissue from cutaneous papillomas is used in the fight against generalized cutaneous papillomatosis.

Neuraminidase is used in order to increase immunogenicity. Neuraminidase is an enzyme capable of changing sialic acid residues from cell membranes, releasing immunostimulating antigens. Cases are reported in which, following the surgical excision of canine mammary carcinoma, neuraminidase administration causes the reduction of small tumor tissues and decreases the chances of recurrence. In canine mammary tumors, a mixture of tumor cells and Vibrio cholerae neuraminidase (VCN) has been used in intradermal injections. The protocol consists of a variable number of autologous tumor cells (105, 106, 107, and 108) treated with mitomycin (M-TC) and mixed with various VCN amounts (10, 50 and 100 ml). Vaccination has caused the regression of the spontaneous mammary tumor in 6 of 23 cases [30]. The authors remark the fact that canine mammary tumors are immunogenic. Intravenously administered non-specific CGB-like immunostimulators prolong survival; in contrast, intratumoral inoculation has no therapeutic effect. Vaccination with autologous tumor cells and active enzymatic VCN induces the regression of benign and malignant tumors, preventing the development of metastases.

In conclusion, the authors emphasize the fact that “chess board” vaccination using M-TC and VCN is an efficient therapy in canine mammary tumors, and the therapeutic effect seems to be tumor antigen-specific.

The success of active immunotherapy is ensured by the potentiation of weak tumor-associated transformation antigens, like in the case of bovine ocular squamous cell carcinoma. Cytoreduction results in the release of interfering antigen-antibody complexes or in a reduced number of suppressor T cells, in the case of horn cancer or equine sarcoid. Bovine ocular squamous cell carcinoma at an early stage can be successfully treated by radiotherapy, hyperthermia or cryosurgery; other therapeutic formulas, can induce, alone or in association, the release of antigens, determining autovaccination.

19.7.4 Specific passive immunotherapy

Specific passive immunotherapy may be considered a serum therapy, a passive transfer of serum or immunoglobulins from a cancerous organism, from patients in remission or tumor rejecting patients. Heterologous sera can also be used, but there are some risks, such as the facilitation phenomenon, pathology related to the use of heterologous sera, etc.

Specific antibodies can be coupled with cytotoxic substances, so that the target cell, i.e. the cancer cell, will be recognized through the membrane neoantigens and submitted to a double lytic action. The injection of specific antibodies will allow the activation of K cells, releasing the phenomenon of antibody-dependent cell-mediated cytotoxicity [22].

Although it has not led to spectacular results in veterinary medicine, specific passive immunotherapy is successfully used in some tumor forms. Thus, in bovine generalized cutaneous papillomatosis, autohemotherapy is used. In cats with leukemia, a large amount of fresh whole heparinized blood can be administered, in transfusion from normal cats.

Specific monoclonal antibodies produced against tumor specific or tumor associated transformation antigens open perspectives in diagnosis, prognosis, therapy and direct serum therapy. Antitumor antibodies can be used to correlate the presence of some cell surface antigens with prognosis, offering information on the evolution of leukemia. Specific monoclonal antibodies can be used to differentiate between malignant and benign neoplasms; neoplastic cells may be identified in histological and metastatic sections of unknown origin, as well as in the analysis of serum for tumor associated antigens. Specific monoclonal antibodies can potentially identify the location of tumor metastases, by the injection of antibodies labeled with radioisotopes.

Specific monoclonal antibodies pose some difficulties in therapy due to the lack of an absolute tumor specificity and cross reactivity of normal cells. Therapy requires the association of the effects of specific monoclonal antibodies with complement-dependent cytotoxicity, antibody-dependent cell cytotoxicity and the direct inhibitory effects of antibodies associated with neoplastic cells.

Specific monoclonal antibodies have been used with some positive responses in the treatment of lymphomas, leukemias, carcinomas and melanomas.

The associated use of specific monoclonal antibodies is particularly promising, based on the results obtained. By combining or adding specific monoclonal antibodies to cytotoxic preparations, the therapeutic chemical products can be directly transmitted to tumor cells. In this way, toxic effects will be reduced, allowing the preparations used to be preferentially directed to the tumor. Specific monoclonal antibodies associated with antitumor antibodies can also be labeled with radioisotopes, which allows to focus the lesional action of radiation on the tumor mass.

Immunotoxins are used in in vivo cancer treatment or in the in vitro treatment of bone marrow in patients with leukemias, in order to destroy tumor cells before the reinjection of the bone marrow into the tumor host, to which high chemotherapeutic doses have been previously administered. Toxins of plant origin, ricin, can be conjugated with an antibody that is directed against the tumor antigen.

19.7.5 Chemoimmunotherapy

Cytoreductive preparations have been clinically used in order to reduce tumor volume, and specific or non-specific active immunotherapy has been used to stimulate immune defense mechanisms. The method has been successfully applied in human leukemias and solid tumors. Canine lymphomas are more successfully treated by chemoimmunotherapy compared to chemotherapy. The survival rate has been significantly longer in patients treated by chemoimmunotherapy, compared to the survival rate in non-treated dogs or dogs treated by chemotherapy using one or more preparations. In this sense, the technique employed consists of the KC1 extraction of autochthonous canine lymph node cells changed with diketen plus complete Freund’s adjuvant, unchanged tumor cell extract plus complete Freund’s adjuvant, or complete Freund’s adjuvant alone. In the experiments that have followed, the active ingredient from the autologous vaccine has probably been complete Freund’s adjuvant, and not the unchanged cells of the lymphoma. The route of administration of specific and non-specific tumor immunogens has proved to be important in the treatment of canine lymphomas.

Not all cytoreductive preparations act exclusively as immuno-depressants. The antineoplastic activity of adriamycin increases in the case of an increased immunogenicity of the treated tumors. Adriamycin, cyclophosphamide, cytosine-arabinose and melphalanpreferentially act by the destruction of suppressor T cells (suppressor T lymphocytes that interfere with killer T cells), which allow lymphoid cells involved in destruction to be more effective. Adriamycin does not seem to regulate the function of natural killer cells or macrophages, unlike azathioprine and cyclophosphamide.

Chemoimmunotherapy is used in combination with surgery and radiotherapy in the treatment of different solid tumors. Before using immunotherapy, other methods and techniques for tumor volume reduction should be used, which helps to restore tumor immunity control and allows a higher efficiency in the approach of immunology in cancer therapy [32, 33].

Interferon is a glycoprotein that has two types: one is virally induced and will inhibit virus replication, the other is triggered by T cells during antigen exposure (immune interferon).

Immune interferon can be induced by antilymphocyte serum, bacteria, RNA, poly I:C, mitogens and immunologic mechanisms. Immune interferon influences infracellular processes and slow cell proliferation, affects cell division and modulates immune responses. Interferon administered during the late phase of immune response increases the number of cells that form antibodies. In cancer disease, interferon acts as a regulating factor of cell functions.

Based on physico-chemical, antigenic and biological differences, three classes of interferons have been distinguished: α, β, and γ. By DNA recombination techniques, the complete nucleotide sequences of interferon α and β have been defined. The analysis of the restrictive endonuclease of cloned DNA and interference analysis have allowed to determine considerable differences, which suggest the presence of several types of interferon α. The interferons produced by the DNA recombination technique through the insertion of genes for an interferon of each class, in E. coli, have been purified for homogenization. This has allowed the production of large amounts of pure interferons from prokaryotic cells, for clinical and biological studies.

The successful production of interferon has made possible the determination of maximal tolerated doses and the optimal biology of this product. In a subsequent stage, the sensitive tumor type has been determined, and the therapeutic role of interferon has been finally defined.

Interferons regulate gene expression, change surface cell proteins and induce the synthesis of new enzymes. This results in changes in other cytokine receptors, regulation proteins on the surface of immunocompetent cells, and enzymes that influence cell growth. The antineoplastic effect of interferons relies on an increased activity of cells that can destroy neoplastic cells: cytotoxic T cells, NK cells, and monocytes. In humans, recombinant interferon α was introduced for the therapy of leukemia, non-Hodgkin malignant lymphoma, and renal cell carcinoma [38]. The side effects after interferon treatment in humans are the following: anorexia and fatigue; weight loss; fever that usually appears in the first days, then disappears; leukocytopenia, etc. The therapeutic schemes in which interferon is associated with other therapeutic means or the administration before or after other therapeutic forms give good results.

Interferon, like other therapeutic means in cancer disease, should be administered according to a scheme adapted for each patient in terms of dose, tolerance and combinations in complex therapeutic regimens.

In canine mammary carcinoma, good results have been obtained after protein A perfusion and cytosine-arabinose administration, the response to this combined treatment occurring after 12 hours. This method is used in order to eliminate immune complexes or other factors from the plasma [32, 33]. Interferon-α was clinically tested in positive cats with FeLV, the dose being 30 U/kg orally, once a day, for 7 days, with a 7 day break. This dosage significantly improved the quality of life, and increased appetite, as well as life duration, even if cats remained positive. The use of IFN-α 2b in a dose of 3 million IU/m2 body surface area, s.c., once a week, resulted in complete, long duration remission in dogs with cutaneous lymphoma and systemic histiocytosis [38].

Cytokine treatment has been introduced in cancer therapy after the obtaining of cytokines by genetic engineering. Mammalian cells produce a high number of cytokines (lymphokines and monokines), in response to different stimuli. Macrophages and lymphocytes are important cells in the production of active biological factors. Some lymphokines and monokines seem to be limited by the major histocompatibility complex, which may be characterized as specific and non-specific. The majority of cytokines are not restricted by the major histocompatibility complex and are not antigen-specific. Based on biological activity, the following substances have been identified: IL-1 lymphocyte activity factor; thymocyte differentiation factor; antibody inhibitory material; fibrinogen degradation products; leukocyte inhibition factor; lymph node permeability factor; osteoclast activation factor; pyrogens; mitogenic or blastogenic factor; IL-2 T cell growth factor; tumor necrosis factor. These substances can be isolated from normal cell products, from cell lines derived from normal tissues or from tumor-derived cell lines. There is a perspective for cloning these biological substances and making them available in sufficient amounts to be experimentally and clinically evaluated. The use of these products in therapy will not be limited to cancer, since many of these agents have qualities that recommend them in autoimmune, inflammatory and even infectious disorders [32, 33].

Interleukins 1-α and 1-β are used in human oncology as myeloprotective substances. IL-1 is produced by macrophages, stimulates in vitro and in vivo bone marrow stem cells, has a synergic effect in combination with cytostatics, and protects the bone marrow; it activates T lymphocytes after meeting the antigen, causes the decrease or even disappearance of fever.

IL-2 is synthesized by helper T cells after they are activated. It is the most important enzyme that regulates the immune system response and induces in particular the proliferation and differentiation of lymphocytes and other cells. The action of IL-2 in oncology is based on an increase in cytotoxic T lymphocytes, as well as a decrease in the activity of NK cells. Through IL-2, these lead to the production of lytic substances. IL-2 regulates the synthesis of other cytokines that may directly or indirectly influence the destruction of tumor cells. It is frequently used in combined treatments. IL-2 is used together with lymphocytes extracted from patients with cancer, which are incubated in vitro with IL-2 in order to obtain LAK cells, which will be administered to the patient. Lately, LAK cells and IL-2 have been combined with monoclonal antibodies. LAK cells express Fc receptors on the cell membrane. The antibodies that are attached to tumor cells can be used as connecting elements of the Fc receptors of the LAK cells.

Human recombinant IL-2 (hr IL-2) stimulates in the dog the proliferation of peripheral lymphocytes and induces in lymphocytes in vitro the same LAK phenomenon as in humans. Good results have been obtained in oral melanoma and in mast cell tumor in the dog. The side effects in dogs consist of digestive manifestations [38].

Interleukin-3 is one of the four factors of hematopoietic growth and supports the proliferation of pluripotent stem cells. In humans, recombinant IL-3 is used for clinical studies in neutropenia, thrombocytopenia, after cytostatic treatment. Unfortunately, IL-3 is also an important factor of some neoplasias, it stimulates in vitro and in vivo mast cell proliferation [38].

Interleukin-4 is produced by a subgroup of helper T cells and has both stimulating and suppressive effects. It has an antitumor action against tumor B cells. Other neoplasms in which IL-4 has shown positive effects in monotherapy or therapy combined with TNF or IFN are melanoma, mammary cancer, renal cell carcinoma [38].

Interleukin-6 is synthesized by macrophages, monocytes, activated T lymphocytes, and fibroblasts. During an inflammation, it stimulates the development of B cells into immunoglobulin-producing plasma cells. Hypergammaglo-bulinemia that may be found in neoplasias can be due to an IL-6 overproduction of tumor cells. IL-6 acts as an autocrine or paracrine growth factor in different neoplasms, such as multiple myeloma. High IL-6 concentrations have also been found in cats with feline infectious peritonitis. IL-6 possesses antitumor properties, the tumor type dictates whether IL-6 activity is suppressed by therapy or an (exogenous) IL-6 dose is introduced [38].

The tumor necrosis factor was first reported by CARSWELL et al. (1970). The serum of mice, rabbits or rats treated with endotoxin and sensitized to CGB proved to contain a factor that, injected in mice with transplanted tumors, induced extensive tumor hemorrhage, with no side effects. This was termed tumor necrosis factor (TNF).

The tumor necrosis factor, purified from the rabbit and mouse serum, contains a glycoprotein with a molecular weight of 39–55 K and an isoelectric pH point of 5.1–5.2. The tumor necrosis factor has characteristics similar to those of lymphotoxin, but lymphotoxin is produced by B and T cell lines, while TNF is produced by cells of macrophagic origin. Recombinant TNF can be obtained by DNA expression or by complementarity in E. coli, and determines necroses in methylcholanthrene-induced sarcoma in syngeneic mice. Studies performed by WANG et al. have shown that TNF can be purified and manifests cytotoxicity against in vitrotumor cells, causing necrosis in some tumors transplanted in laboratory mice, while it is not toxic for normal human and mouse cells [32, 33].

The antiproliferative effects of TNF can be enhanced by the administration of other cytokines; this factor seems to act by the formation of pores in the cytoplasmic membrane of the tumor cell, which results in lysis. Due to its extremely high toxicity, its use is limited and it is associated with other cytokines, cytostatics, by hyperthermia or other mode of administration, such as in the portal vein, in the case of hepatic metastasis. TNF has been used in oral melanoma and in mastocytomas, in the dog [38].

The biologically active tumor growth factor has been detected in different tumors and is produced by cells transformed by different retroviruses, but not by cells transformed by DNA viruses. This factor stimulates cell division, binds to the epidermal growth factor and activates a factor associated with tyrosine kinase. The tumor growth factor meets the criteria for tumor-specific cell surface antigens and marks tumor cells that express TGF as a target for the cell mediated immune response, such as the T cell dependent cytotoxic response and the lysis mediated by complement dependent antibodies. TGF antibodies are tested for their capacity to inhibit tumor growth in both cell cultures and experimental animals with tumors [32,33].

The classification of the different substances used in immunotherapy, which are biological response modifiers, may be synthesized according to MAC EWEN (1985) as follows:

1. Non-specific active immunostimulation or immunomodulation by bacterial extracts and chemical substances:
   • – Calmette-Guérin bacilli (CGB);
   • – Corynebacterium parvum (CP) (Proprionibacterium acnes);
   • – Levamisole, Thiobendazole;
   • – Muramyl tripeptide-phosphatidylethanolamine (MTPPE);
   • – Staphylococcus aureus Cowan I (SAC);
   • – Mixed bacterial vaccine (MBV).
2. Interferons.
3. Lymphokines and cytokines:
   • – interleukins (1,2, and 3);
   • – tumor necrosis factor;
   • – colony stimulating factor.
4. Monoclonal antibodies.
5. Antigens:
   • – tumor cell vaccines.
6. Various agents:
   • – bone marrow graft;
   • – blood component therapy;
   • – plasmapheresis.

Clinical results obtained by immunotherapy in dogs

Changes in the biological response	Administration route	Type of tumor disease	Used in association with	Results
Calmette-Guérin bacillus(CGB)	Intravenous	Osteosarcoma	Amputation	Survival
	Intravenous	Mammary tumors	Surgery	Survival
CGB and Corynebacterium parvum (CP)	Intratumoral	Mammary tumors	Surgery	Inefficient
Corynebacterium parvum(CP)	Intratumoral	Mammary tumors	Surgery	Inefficient
	Intravenous	Oral cavity melanoma	Surgery and/or cryosurgery	Little efficient
Levamisole	Oral	Mammary tumors	Surgery	Inefficient
	Oral	Lymphoma	Chemotherapy	Inefficient
	Oral	Nasal cavity cancer	Surgery	Survival
Muranyl tripeptide phosphatidyl- ethanolamine (MTPPE)	Intravenous	Osteosarcoma	Amputation and chemotherapy (cisplatin)	Survival
Staphylococcus aureusCowan I (SAC)	Plasmapheresis	Mammary tumor	-	Tumor necrosis
Mixed bacterial vaccine(MBV) (Serratia marcescins andStreptococcus pyogenes)	Intramuscular	Oral cavity melanoma	Surgery or cryosurgery	Inefficient
	Intramuscular	Lymphoma	Chemotherapy	Remission, survival, unchanged
Monoclonal antibodies mouse-derived canine antilymphoma	Intravenous	Lymphoma	Chemotherapy until clinical remission is obtained, then maintenance of remission with monoclonal antibodies	Remission, survival
Calmette-Guérin bacillus(CGB)	Intravenous	Osteosarcoma	Amputation	Survival
	Intravenous	Mammary tumors	Surgery	Survival
CGB and Corynebacterium parvum (CP)	Intratumoral	Mammary tumors	Surgery	Inefficient
Corynebacterium parvum(CP)	Intratumoral	Mammary tumors	Surgery	Inefficient
	Intravenous	Oral cavity melanoma	Surgery and/or cryosurgery	Little efficient
Levamisole	Oral	Mammary tumors	Surgery	Inefficient
	Oral	Lymphoma	Chemotherapy	Inefficient
	Oral	Nasal cavity cancer	Surgery	Survival
Muranyl tripeptide phosphatidyl- ethanolamine (MTPPE)	Intravenous	Osteosarcoma	Amputation and chemotherapy (cisplatin)	Survival
Staphylococcus aureusCowan I (SAC)	Plasmapheresis	Mammary tumor	-	Tumor necrosis
Mixed bacterial vaccine(MBV) (Serratia marcescins andStreptococcus pyogenes)	Intramuscular	Oral cavity melanoma	Surgery or cryosurgery	Inefficient
	Intramuscular	Lymphoma	Chemotherapy	Remission, survival, unchanged
Monoclonal antibodies mouse-derived canine antilymphoma	Intravenous	Lymphoma	Chemotherapy until clinical remission is obtained, then maintenance of remission with monoclonal antibodies	Remission, survival
Tumor cell vaccines	5 vaccines in 7 weeks (administration route not mentioned)	Lymphoma	Chemotherapy, in animals in remission, injection of irradiated tumor cells	Remission, survival, unchanged
	Intralymphatic	Lymphoma	Chemotherapy in order to obtain remission + whole body radiation	Remission, survival, unchanged
Bone marrow graft	Intravenous	Lymphoma	Chemotherapy in order to obtain remission + whole body radiation	25% of dogs with long survival
Blood component therapy	Intravenous, total blood or blood plasma transfusion	Lymphoma and/or leukemia	-	Complete remission: 4 cases; regression > 50%: 8 cases. No regression: 4 cases, but transient efficacy

Clinical results obtained by immunotherapy in cats

Changes in the biological response	Administration route	Type of tumor disease	Used in association with	Results
Calmette-Guérin bacillus (CGB)	Intralymphatic	Mammary tumor	Surgery	Inefficient
Corynebacterium parvum (CP)	Intravenous	Healthy carriers of FeLV	-	Absence of antiviral effects
Levamisole	Oral	Mammary tumor	Surgery	Inefficient
	Oral	Healthy carriers	-	Survival
Staphylococcus aureus Cowan I (SAC)	-	Healthy carriers of FeLV + lymphoma + leukemia	-	Short duration efficacy
Mixed bacterial vaccine (MBV)(Serratia marcescins andStreptococcus pyogenes)	Intramuscular	Mammary tumor	Surgery	Little efficient
P. Interferon (of bovine origin) and interferon (of human origin)	Oral	Carriers of FeLV + anemia and leukopenia	-	Increase in the level of red and white cells up to normal values
Tumor necrosis factor	Intramuscular	Oral cavity epithelioma	Local radiotherapy and hyperthermia	Evaluation of clinical results underway
Colony stimulating factor	Intravenous and subcutaneous	Carriers of FeLV + aregenerative anemia (hemocrit <12%) and panleukopenia (white elements < 2000)	-	Little efficient
Anti-FOCMA(Feline Oncornavirus Associated Cell Membrane Antigen)antibodies	Intravenous	Lymphoma	Chemotherapy until clinical remission, then maintenance by immunotherapy	Inefficient
Tumor cell vaccines	Intralymphatic	Mammary tumors	Surgery, injection of irradiated tumor cells + CGB	Inefficient
Bone marrow graft	Intravenous	Healthy carriers of FeLV	Cyclosporin Prednisone	Inefficient
Blood component therapy	Intravenous transfusions of: – feline blood – feline blood plasma – feline serum of a feline plasma cryoprecipitate – feline fibronectin	Carriers of FeLV + lymphoma and/or leukemia	-	Complete remission: 19 cases; regression > 50%: 16 cases; no regression: 7 cases; short duration tumor regressions, malignant lymphocyte lysis associated with disseminated intravascular coagulation

The actions of immunotherapy according to HAYES (1990), based on results obtained in anticancer therapy practice in dogs and cats in particular, are presented in the tables previous.

Immunotherapy is still an aleatory therapeutic method, and positive results continue to be considered with circumspection, but the improvement of techniques and the deep knowledge of the relationships between the host and tumor cells will lead to progress, and results will not fail to appear. Immunotherapy has all the chances to turn from a hope to a certainty in the fight against malignant disease.

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19.8 METABOLIC DISORDERS AND NUTRITIONAL THERAPY IN CANCER PATIENTS

In both humans and animals, cancer in the preclinical or quiet phase does not manifest any disease signs, then it manifests as a local lesion, subsequently inducing changes with general disturbances in the whole organism, which are called cancer disease. In cancer disease, the patient undergoes deep metabolic changes, with severe consequences. In veterinary medicine, there are two major requirements: a balanced diet, adequate for the patient’s state, and the avoidance/relief of pain, without omitting the information and preparation of the owner.

Cancer cachexia. In cancer disease, due to metabolic disorders (hydrocarbons, lipids and proteins), animals rapidly reach an advanced weight loss stage. Patients with weight losses have been found to present a shorter survival duration, compared to patients without weight losses. Weight loss depends on the patient’s general state (age, associated diseases) and on the type of neoplasm (affected organ or tissue, evolution in time). Very frequently, patients have anorexia.

By synthesizing the main causes of anorexia in dogs and cats, as well as the therapeutic possibilities, LINK (40) mentions the following:

• – the mechanical obstruction of the digestive tract induced by the tumor, which can be treated by surgery and/or chemotherapy or by bypass with a feeding tube;
• – changed tasting sense, food aversion, especially in cats, which can be remedied by food as varied as possible;
• – nausea, when intestines are affected due to cytostatic drugs, at other times nausea is of central nature, being induced by excess drugs. In this case, drug therapy with one of the following products can be administered:
• – metoclopramide, 0.5 mg/kg, 3 times a day, per os, s.c.;
• – butorphanol, 0.3 mg/kg s.c.;
• – prochlorperazine, 0.3 mg/kg, 3 times a day, s.c.;
• – diphenhydramine, 2–4 mg/kg, 3 times a day, per os;
• – ondansetron, 2–4 mg/animal, 2 times a day.
• – central anorexia (of the central nervous system) – can be improved by:
• – diazepam, 0.2 mg/kg, i.v., in the cat;
• – oxazepam,2.5 mg/cat/day, per os;
• – flurazepam, 0.3 mg/kg, once every 5 days, per os;
• – iatrogenic anorexia can be improved by special attention given to the patient, patting, a "familial" environment, ambulatory treatment;
• – fever will be treated depending on the cause (infectious, paraneoplastic);
• – pain caused by a tumor will be treated by analgesics, with peculiarities for each patient.

Carbohydrate metabolic disorders are extremely severe, glucose being the substrate used by tumor cells; these partially metabolize glucose anaerobically (glycolysis). It is known that aerobic glucose degradation occurs with an energy of 36 mol ATP/mol glucose, while anaerobic glycolysis only produces 2 mol ATP/mol glucose, and the lactate metabolite is formed; in the liver, lactate is transformed into glucose. In order to meet the glucose requirements of tumor cells, fats and proteins are degraded to gain energy.

Increased lactate and insulin levels in the blood appear in humans as well as dogs with cancer, and show an increased anaerobic transformation of glucose into lactate. Hyperinsulinemia (and relative insulin resistance) is due to a hyper-secretion of beta cells or to a receptor deficiency. The above mentioned metabolic changes remain unchanged even after the total remission of the tumor, and clinically manifest similarly to insulin dependent diabetes mellitus [40].

Protein metabolism in patients with tumors is disturbed by the onset of a disequilibrium between protein synthesis and degradation. Protein degradation results in albumin loss and implicitly, muscular atrophy. Certain amino acids are used by tumor cells for synthesis and gluconeogenesis, which causes the reduction of some amino acids, such as glutamine, cystine, arginine, threonine, valine and threamine. A diet that has moderate amounts of highly bioavailable proteins may be of value to the cancer patient, and certain amino acids such as glutamine, cystine, and arginine may also be beneficial for some cancer patients [40, 41].

Lipid metabolism is disturbed in cancer patients, as there is a high rate of fat oxidation, especially when acute weight losses occur. The deregulation of carbohydrate metabolism results in an increased mobilization of body fats, which are needed for glucose production. In the healthy organism, fat acid oxidation is inhibited by glucose, which does not occur in patients with tumors. In both humans and dogs with cancer, characteristic changes in the composition of plasma lipids occur. Extremely low HDL (high density lipoprotein cholesterol) concentrations have been found, which are restored to normal after tumor remission; increased triglyceride and VLDL (very low density lipoprotein cholesterol) serum levels have also been found, which in humans are restored to normal after tumor remission, but not in dogs [40].

Studies of polyunsaturated fatty acids of the n-3 series, especially eicosapentaenoic and docosahexaenoic acid, indicate that these fatty acids may prevent the development of carcinogen-induced tumors, the growth of solid tumors, as well as the occurrence of cachexia and metastatic disease in experimental tumor models. The bottom line is that n-3 fatty acids in moderate amounts appear to benefit the cancer patient. A diet relatively high in n-3 fatty acids and relatively low in simple carbohydrates has been shown not only to improve alterations in metabolism associated with cancer, but also has been shown to improve response to chemotherapy and to decrease the adverse effects associated with radiation therapy [41].

Fiber, soluble and insoluble, are both important to prevent cancer and to enhance bowel function. A diet with adequate amounts of soluble and insoluble fiber may be indicated for many dogs and cats with cancer [41].

In addition, OGILVIE [41], proposes the following guidelines may be considered early for each patient:

1. Provide clients with appropriate information, dietary plans, and appetite stimulants such as cyproheptadine and megesterol acetate from the very beginning. The goal is to prevent anorexia and weight loss from ever happening.
2. Consider foods that are highly bioavailable, easily digested, and highly palatable with a good smell and taste.
3. Consider foods that are relatively low in simple carbohydrates, have moderate amounts of good-quality sources of proteins, include soluble and insoluble fiber, and have moderate amounts of fats. Fats of the n-3 fatty acid series may be effective in reducing or eliminating some of the metabolic alterations associated with cancer cachexia. Antioxidants, such as vitamin E, are essential whenever n-3 fatty acids are used.
4. Enhanced quantities of arginine, cystine, and glutamine may be of value in maintaining a more normal immune, hematologic, and gastrointestinal tract.
5. Fiber, both soluble and insoluble, is essential to maintain normal bowel health. A diet with adequate amounts of fiber is essential to prevent or to treat various problems of the gastrointestinal tract.

The nutrition of cancer patients requires the calculation of the necessary energy and a special diet.

The formula for the calculation of the necessary energy (Kcal/day non-protein calories, according to LINK [40]:

• – animals with a body weight less than 2 kg, required energy (Kcal) 70 x kg body weight 0.75
• – animals with a body weight more than 2 kg, required energy (Kcal), 30 x kg body weight +70.

The specific factor in the dog is 1.5–2, during the first disease stages this factor ranges between 0.8 and 0.2, and in more advanced stages it is 1.1–2. In the cat, this factor is 1.25–1.5; in cats, overnutrition causes hepatic lipidosis.

A diet containing easily digestable proteins is recommended; carbohydrates will be replaced by lipids; simple carbohydrates will be avoided, as they increase abnomal carbohydrate metabolism, which results in non-favorable energy relationships. A large part of the required energy (40–60% of non-protein calories) will be provided as fats, because these are extremely difficult or impossible to metabolize by tumors. It is very important that the diet contains essential lipid acids such as linoleic and linolic acids, omega-3 lipid acids with an antitumor and anticachectic effect, which act against lactic acidosis in canine lymphoma. The required protein intake in dogs with tumors is 3–4(-6) g/100 Kcal/day, and in cats 5–6(-9) g/100 Kcal/day. In patients with renal or hepatic failure, the albumin amount is reduced (in dogs less than 3 g/100 Kcal, in cats less than 4 g/Kcal) [40].

The ration composition in patients with tumors should take into consideration certain species peculiarities, as well as the preferences of each subject. Thus, for cats, fat fish and meat, eggs, cheese and fowl meat are recommended as a source of proteins. Food rations will be supplementd with arginine, taurine, selenine, vitamins A and B, and for essential lipid acids, food oil will be added. Food will be administered at body temperature. Dogs are less food selective, but prefer heated food; individual preferences should be considered, and essential fat acid, arginine and taurine supplements are extremely important.

According to LINK [40], diets for dogs and cats with tumors must have the following structure:

Dog	Dog	Cat
70% fowl or pork meat	46% fowl or pork meat	48% fowl meat
12% wheat germ oil	25% red perch	25% mackerel
10% oat flakes	10% oat flakes	2% liver
5% wheat bran	10% wheat bran	13% rice
3% vitamin mineralized food	5.5% dehydrated carrots	5% wheat germ oil
-	1% beer yeast	5% wheat bran
-	2.5% vitamin mineralized food	2% vitamin mineralized food

The stimulation of appetite is first attempted by varied food, heated to body temperature, and if anorexia persists, drugs will be administered. In the cat, benzodiazepines, diazepam, 0.2 mg/kg i.v., maximum dose 5 mg/patient, and oxazepam, 2.5 mg/cat per os, have good effects. After intravenous administration, the effect lasts for 20 minutes, while the patient is eating. In the case of the administration of serotonin antagonists, cyproheptadine (peritol), 2–4 mg/cat daily; 4 mg/dog daily, appetite is regained only after a few days. After cyproheptadine administration, 20% of the treated cats show increased excitability and become aggressive. The increase in appetite is also obtained following corticosteroid administration: prednison 0.25–0.5 mg/kg daily;megestrolacetate, megecat, vetoquinol, 1 mg/kg daily or 2.5 mg/cat daily for 4 days, then once every 2, 3 days, induce appetite and result in weight gain in animals; oral administration of metoclopramide increases appetite. If these methods are not sufficient, if a weight loss of 10% or more occurs, artificial enteral or parenteral nutrition will be initiated [40].

Artificial nutrition is performed in anorexic patients with survival chances (tumor regression, healing). Artificial enteral nutrition is preferred to parenteral nutrition, because the food administered to the intestine stimulates motility and blood circulation, the secretion of enzymes, bile and hormones. Artificial parenteral nutrition is used in patients with functional or mechanical disorders (tumor obstructions).

Enteral tube nutrition can be administered to cancer patients with a nasoesophageal, esophageal, gastric, jejunal tube. These tubes are used depending on the established diagnosis, on the patient, and the food to be administered. The technique for the insertion of the tube is known by every clinician specialized in the pathology of companion animals [40].

Total parenteral nutrition is extremely expensive and creates more serious complications than enteral nutrition; it is used in the following cases: before and after surgery, in severe vomiting, and ileus. In these cases, the composition of the administered solutions will be rich in proteins, fats and poor in carbohydrates [40].

Tumor-associated pain can be somatic, visceral, and deafferent. Somatic pain is located, it occurs following the activity of nociceptors in the skin and tissues; it appears in relation to bone metastases, postoperation or as muscle pain. Visceral pain is the result of infiltration, compression or twisting of internal organs, induced by tumor or metastatic growth. Deafferent pain appears as a result of central or peripheral nervous system lesions, caused by tumor compression or infiltration, radiation, surgery or chemotherapy.

Behavioral changes may indicate pain: restlessness, insomnia, inappetence, anorexia, immobility or stiff movements, howling. The intensity of pain can be mild, moderate, violent. There is no objective method for assessing pain in animals.

The prevention or fighting of pain in veterinary medicine is done using non-steroidal antiphlogistics, agonist opioids, agonist-antagonist opioids.

Non-steroidal antiphlogistics prevent the production of prostaglandins and are most effective when they are used before surgery, before the appearance of inflammation. They are not effective after surgery or in violent pain, this is why they are combined with opiates. Possible side effects: gastrointestinal ulcers, renal ischemia, prevention ofthrombocyte aggregation. Antiprostaglandins should not be administered to patients with renal failure, dehydration, hypotension, thrombocytopenia, gastrointestinal ulcers. Non-steroidal antiphlogistics used in veterinary medicine are pyroxicam, acetylsalicylic acid, meloxicam, vedaprofen, karprofen.

Opioid analgesics are most effective in the treatment of violent pain. The efficacy of these drugs varies in the different animals depending on the intensity and the cause of pain, but also depending on the presence of other diseases. In dogs, agonist opioids induce a dose-dependent CNS depression, and in cats, they cause a CNS excitement. Other side effects: respiratory depression, bradycardia and hypotension; in dogs, vomiting is frequent, in cats it is more rare; prolonged use induces constipation.

Agonist-antagonist opioids (Nalokon) induce minimal systemic side effects, and the dose-action relationship is linear, i.e. at increased doses the efficacy of the drug increases, and overdosage is not possible; they have a low analgesic potential and they cannot be used in violent pain.

The doses proposed by OGILVIE and VAIL (1996), as well as by STÖCKLIN (1998) (cited by 40) are the following:

Non-steroidal antiphlogistics:

• acetylsalicylic acid: – dog, 10–15 mg/kg 3 times a day, per os
  – cat, 10 mg/kg once every 48 hours, per os
• meloxicam: – dog, cat, 0.2 mg/kg on the first day, after which O.lmg/kg daily, per os or s.c.
• vedaprofen: – dog, cat, 0.5 mg/kg, daily, per os
• karprofen: – dog, 4.4 mg/kg once a day.

Agonist opioids:

• morphine: dog, 0.05–"0.4 mg/kg once every 1–4 hours, i.v.
  0.2–1.0 mg/kg once every 2–6 hours, i.m., s.c.
  cat, 0,1 mg/kg, i.m. with a sedative
• oxymorphon: dog, 0.02–0.1 mg/kg, once every 2–4 hours, i.v.
  0.05–0.2 mg/kg once every 2–6 hours, s.c.
  cat, 0.02–0.05 mg once every 2–6 hours, i.m., s.c.

Agonist-antagonist opioids have the advantage that they can be applied epidurally and in this way, they have fewer side effects:

• butorphanol: dog, 0.2–1.0 mg/kg
  cat, 0.1–0.4 mg/kg once every 1–4 hours, s.c., i.m., i.v.
• buprenorphin: dog, 0.005–0.02 mg/kg, i.m., i.v.
  cat, 0.005–0.01 mg/kg once every 4–12 hours, i.m., i.v.
• morphine, 0.1 mg/kg, duration of analgesia up to 24 hours, administered epidurally.

The use of epidural analgesia is recommended perioperatively and postoperatively after amputation in osteosarcomas.

The analgesics used in veterinary medicine are much more varied, it is important to know their action and side effects."

( Note, there is an extensive bibliography / footnote section included in the source material )

This should help to introduce the neophyte in the area of oncology into some of the types of cancer and some procedures that are used. `Anon99