**2. Material and methods**

lated through the massive stress connected with a first diagnosis of cancer. We also assumed that the immunotherapy of advanced cancer patients with a psychogenic medical history should be preceded with an effective correction of psychoemotional disorders in order to avoid a psychogenic immunosuppressive influence. The objective of this work is the development and implementation of a psychoimmunological approach to the treatment of advanced cancer

**1.1. Theoretical justification for a psychoimmunological approach to the treatment of**

Today there is no doubt that immune dysfunctions are the backbone of tumour pathogenesis. This immune dysfunction, along with destruction of the cellular genetic apparatus in the malignant cells, occurs in the form of the cell component of the immune system dysfunction along with the malfunction of cell control and cell differentiation mechanisms, immune tolerance, and inability to provide an effective immune response to a developing tumour [3, 4]. In this connection it was logical to use the wide range of immunotherapy methods in modern oncology. Despite being a new step in malignant tumour treatment it has not solved

At the same time, laboratory and clinical trials have shown linked and multi-functional interconnections between the two most important integrative systems of the human organism, which are the immune and the nervous systems [7-9], which formed the basis of the develop‐ ment of modern scientific trends of the psychoneuroimmunology and psychoimmunology of cancer [10]. The conclusions of the scientific research on the influence of chronic psychoemo‐ tional stress (CPES) to an organism of healthy and unhealthy individuals, including those with cancer, are most interesting from the practical point of view. Nowadays the somatic outcomes of CPES are well known: the CPES is able to damage the cell's DNA and inhibit DNA repair through the activation of endogenous mutagens, which are the reactive species of oxygen, nitrogen, etc. This leads to genome instability [11, 12]. CPES is always followed by immuno‐ suppression, a decrease in the quantity and cytotoxic activity of CD8+ and NK-cells, and dysfunction of their supervising functions, processes of apoptosis, activation of proinflamma‐ tory cytokines and sustentation of the non-cropped areas of chronic inflammation [13-15]. All of these lead to a concentration of malignant cells in the body with an increase in their invasive potentiality [16]. CPES is linked with a high risk of development, progress and recurrence of malignant tumours, and the high mortality rate of cancer patients [17, 18]. The CPES also leads to hippocampal neuronal degeneration as well as amygdala atrophy [19], prolonged hyper‐ activity of the hypothalamo-pituitary-adrenocortical axis [20], and accelerated aging of the human body [21]. Therefore, the extensive and deep somatic damaging effect of CPES suggests a significant role of psychogenic factors in the development, recurrence and progression of cancer disease in some cancer patients with a psychogenic medical history. On the basis of the above, it is logical to assume that it is difficult to eliminate the factors compromising the immune system due to psychogenic influence and the suppression of anti-tumour immunity without effective elimination of persistent tonic descending influences of the central nervous system and the higher nervous activity to the body of cancer patients with a psychogenic medical history. The confirmation of the dependency of anti-tumour immune activity to higher nervous activity is presented by therecently discovered phenomenon of the spontaneous

patients with a psychogenic medical history.

34 Updates on Cancer Treatment

the problem of its effective treatment [5, 6].

**advanced cancer patients with a psychogenic medical history**

This study had local ethical committee approval (the Ethical committee of the Institute of Clinical Immunology, Siberian Branch, Russian Academy of Medical Sciences, Novosibirsk, Russian Federation, protocol № 29, August 18, 2004). All patients gave written informed consent.

#### **2.1. Characteristics of advanced cancer patients**

The special psychoimmunological cancer treatment was offered to 17 patients with advanced cancer (13 women and four men). These patients, over seven years (2005-2012),were selected by us from the cancer patients using the following criteria: 1) the progression of the disease, despite the ongoing standard combination therapy of cancer provided, 2) the presence of distant metastases (stage IV of cancer), 3) the obvious massive psychotrauma that occurred in the lives of patients before being diagnosed with cancer (psychogenic medical history), and 4) informed consent of cancer patients for carrying out cancer treatment. A special clinical case was associated with patient № 9 (Table 1), who strictly refused to take a standard combination therapy for cancer, but expressed the strong desire to receive psychological and psychother‐ apeutic aid. Individual characteristics of cancer patients are presented in Table 1.



**Table 1.** Characteristics of advanced cancer patients (n=17)

#### **2.2. Psychogenic medical history investigation**

**Patient**

36 Updates on Cancer Treatment

**№ Age Sex Cancer types Primary tumour**

7 38 F Melanoma Skin of temporal

8 53 M Melanoma Back skin

9 55 F Melanoma Neck skin

10 76 F Kidney cancer Right kidney

Stomach

Stomach& pancreas cancer

15 54 F Breast cancer Right breast Ribs, shoulder

cancer Right ovarian

SUR – surgery; CHT – chemotherapy; RT – radiotherapy; HYP – hyperthermia

Ovarian

**Table 1.** Characteristics of advanced cancer patients (n=17)

14 50 F Breast cancer Left breast

12 53 F

13 28 M

16 45 F

6 51 M Melanoma

**localization**

5 46 F Melanoma Back skin Porta hepatis IV Hypertensive

11 58 F Kidney cancer Right kidney Left kidney IV Hypertensive

Stomach& pancreas

Anterior abdominal wall

region

4 43 M Melanoma Uveal Liver IV - SUR, CHT

**Metastasis**

Subcutaneous hands & feet

Retroperito-

Subcutaneous

Supraclavicular & axillary lymph nodes

Both lungs, mediastinal & neck lymph nodes, ribs

cancer Stomach Porta hepatis IV - SUR, CHT

Paraaortic lymph nodes

Skull, ribs, sternum, clavicles, spine,

pelvis

joint

17 48 F Lung cancer Right lung Left lung IV - SUR, CHT

Mediastinal lymph nodes IV

**localization Stage Comorbidities Treatment**

Chronic

neum IV - SUR, CHT

hands & feet IV Stomach ulcer SUR, CHT

Hypertensive heart disease, coronary artery disease

heart disease SUR, CHT

IV - SUR, CHT

IV - SUR, CHT, RT

heart disease SUR, CHT

IV - SUR, CHT

IV Hypertensive

IV -

IV

heart disease SUR, CHT

cholecystitis SUR, CHT

Refuse standard treatment

SUR, CHT

Psychogenic medical histories were taken for each patient in the clinical trial (anamnesis morbi) during the first visit, and these histories included the presence of massive psychotraumatic events (death of close person, divorce, etc.) with the development of helplessness and despair.

#### **2.3. The mental status examination of advanced cancer patients**

The mental status examination of cancer patients was conducted by the psychiatrist with supplemental usage of the following psychometric test systems (e.g., Symptom Checklist 90 (SCL-90) and rates: Somatization, Obsessive-Compulsive, Interpersonal Sensitivity, Depres‐ sion, Anxiety, Hostility, Phobic Anxiety, Paranoid Ideation, Psychoticism, Global Severity Index. The psychiatrist detected the presence or absence of mental disorders in cancer patients in accordance with International Classification of Diseases (ICD-10). Normal values of SCL-90 parameters for healthy people are presented in Table 2 [23]. The mental status examination was administered in the following stages of research: 'before' – before psycho-correction; 'after' – after hypnotherapy session; and 'one month later' –hypnotherapy sessions one month later.

#### **2.4. The pharmacotherapy of psychoemotional disorders**

The pharmacotherapy of mental disorders in advanced cancer patients was conducted immediately after diagnosis. Antidepressants (tianeptine, venlafaxin), anxiolytics (afobazo‐ lum, microdoses of diazepam) and their combination were used. The psychotropic drugs were prescribed for a period of three to six months to enhance and prolong the therapeutic effect of hypnosuggestive psychotherapy (HSP).

#### **2.5. Method of hypnosuggestive psychotherapy**

The method of hypnosuggestive psychotherapy is based on strict successive, interconnected, figurative, pathogenically substantiated suggestive influences in hypnotic states. This method has previously been described in detail [24]. It includes: 1) Establishment of hypno-rapport between patient and physician; 2) Hypnotic de-actualization of psychotraumatic emotions and experience including the fact of cancer diagnosis; 3) Hypnotic lockout of dreams connected with known stress situations which are regularly reproduced in sleep with the corresponding psycho-vegetative reactions – this is needed for the subject's exhaustion of psychogenic disorders and to prevent their lingering course; 4) Hypnotic reproduction of a personal 'health standard' or 'health syndrome' – a key session of the whole course of HSP. This 'syndrome' is based on using the known phenomenon of hypnotic hypermnesia (increased memory under hypnosis), generally used for the restoration of psychogenic abnormalities of memory. However, it is possible to restore the memory of the heartbeat, respiratory rate, glycaemic rate, enzyme reaction activity, stereotype of digestive system functions, etc. from a specific time in the past. The patient recollects the concrete day (date, month, year) from the past – the 'model standard' of his health when there was no tumour and he felt well, mentally and physically. In a hypnotic state suggestions were conducted using the images required to retrieve the 'records' that are well known to the body as 'health standard' from the memory of cancer patients. It should be noted that in this key session HSP hypnotic suggestions related to the activation of a great desire and the need for further self-realization in their lives were con‐ ducted. In fact, these suggestions were aimed at restoring of life purpose dominant loss [25]; 5-6). The last HSP sessions focused on patients' education in self-hypnosis (autohypnosis) under suggestive influence. The patients were then given detailed instructions to use selfhypnosis for the prolongation of the medicinal effect, as well as keeping the psychic and vital tone of cancer patients. The duration of the individual course of the HSP was 14-16 days.

#### **2.6. Anti-cancer diagnostic skin test for evaluation of specific anti-tumour activity of the immune system (non-stimulated native activity) in advanced cancer patients**

The anti-tumour activity of the immune system was assessed by a skin test of the delayed type hypersensitivity (DTH) reaction on tumour-associated antigens (TAA), which were used as a lysed human melanoma cell line BRO [26] of 25,000 cells in a test (see Figure 1). Human melanoma cell line BRO was obtained at the Institute of Cytology of the Russian Academy of the Sciences (St. Petersburg, Russia). We investigated the DTH skin reaction after the intra‐ dermal administration on the forearm at 9, 12 and 24 hours, to identify the peak responses. The peak response in most cases (the diameter of redness in mm) was observed after 12 hours. Selection of the human melanoma cell line BRO was determined by the need to use in one test the maximum range of TAA, in order to assess the anti-tumour activity of the immune systems of patients with different cancers. As shown above, there exist all kinds of TAA characteristics of solid tumours on the melanoma cells [27].

**Figure 1.** Anti-cancer diagnostic skin test: evaluation of specific anti-tumour activity of the immune system in advanced cancer patients (Patient № 11 after psycho-correction). (a) Intradermal introduction of tumour-associated antigens: lysed cells of human melanoma line BRO 25103 per doze (50 µL); (b) Evaluation of DTH skin reaction on TAA (12 hours later): 0- 5 mm – low activity, 5-10 mm – average activity, >10 mm – high activity. **Figure 1.** Anti-cancer diagnostic skin test: evaluation of specific anti-tumour activity of the immune system in ad‐ vanced cancer patients (Patient № 11 after psycho-correction). (a) Intradermal introduction of tumour-associated anti‐ gens: lysed cells of human melanoma line BRO 25×103 per doze (50 µL); (b) Evaluation of DTH skin reaction on TAA (12 hours later): 0-5 mm – low activity, 5-10 mm – average activity, >10 mm – high activity.

The selection of the minimal quantity of TAA for the anti-cancer diagnostic DTH skin test (according to our preliminary studies) was used in order to obtain a physiological specific antitumour immunological response (non-stimulated native DTH skin reaction on TAA) as well as to exclude the possibility of the vaccine's effect on the diagnostic test itself. In comparison with our colleagues, who used the DTH skin reaction on the TAA of human melanoma cell lines in a study of the clinical efficacy of the anti-tumour polyvalent vaccine 'CancerVax' (developed from three allogeneic human melanoma cell lines) [28], we used a diagnostic dose that was nearly 100 times smaller (2.5 × 104 cells vs. 2.4 × 106 cells). In addition, our patients did not receive any immunotropic therapy during the study. The selected dose does not cause allergic and other pathological reactions. It is known that the delayed-type hypersensitivity reaction is a specific immune response and begins to manifest in eight to 12 hours after ingestion of The selection of the minimal quantity of TAA for the anti-cancer diagnostic DTH skin test (according to our preliminary studies) was used in order to obtain a physiological specific antitumour immunological response (non-stimulated native DTH skin reaction on TAA) as well as to exclude the possibility of the vaccine's effect on the diagnostic test itself. In comparison with our colleagues, who used the DTH skin reaction on the TAA of human melanoma cell lines in a study of the clinical efficacy of the anti-tumour polyvalent vaccine 'CancerVax' (USA) (developed from three allogeneic human melanoma cell lines) [28], we used a diagnostic dose

> In our case the peak responses, due to the absence of prior immunization of cancer patients and the largest contribution of cellular reactions in the DTH skin test, were achieved early (within 12 hours) [30]. An evaluation of a specific anti-tumour activity of the immune system in advanced cancer patients was conducted through the research phases: 'before' – before psycho-correction, 'after' – after completing a course of hypnotherapy and 'one month later' –

> **2.7. Preparation of tumour-associated antigens for diagnostic test - DTH skin reaction** The lysed cells of the human melanoma cell line BRO were used as the TAA for diagnostic test. The human melanoma cell line BRO were maintained in RPMI 1640 supplemented with 10% heat-inactivated foetal calf serum, L-glutamine (2 mmol/ml), 25 mmol HEPES buffer, and 25 μg/ml gentamicin at 37°C in 5% CO2 humidified air. Cells were detached from the dish by treating with trypsin-EDTA followed by washing three times with Dulbecco's phosphatebuffered saline, precipitated by centrifuging, counted, and diluted with 0.9% saline solution with 0.1% EDTA. Cells were lysed by repeated (eight times) freezing and stored at -80ºC until

> > lysed cells line BRO in 50 microliters were used.

antigen, and in most cases the reaction reaches a peak after 48-72 hours [29].

after one month following the completion of the course of hypnotherapy.

use. As a diagnostic test 2.5 × 104

that was nearly 100 times smaller (2.5 × 104 cells vs. 2.4 × 106 cells). In addition, our patients did not receive any immunotropic therapy during the study. The selected dose does not cause allergic and other pathological reactions. It is known that the delayed-type hypersensitivity reaction is a specific immune response and begins to manifest in eight to 12 hours after ingestion of antigen, and in most cases the reaction reaches a peak after 48-72 hours [29].

patients. It should be noted that in this key session HSP hypnotic suggestions related to the activation of a great desire and the need for further self-realization in their lives were con‐ ducted. In fact, these suggestions were aimed at restoring of life purpose dominant loss [25]; 5-6). The last HSP sessions focused on patients' education in self-hypnosis (autohypnosis) under suggestive influence. The patients were then given detailed instructions to use selfhypnosis for the prolongation of the medicinal effect, as well as keeping the psychic and vital tone of cancer patients. The duration of the individual course of the HSP was 14-16 days.

**2.6. Anti-cancer diagnostic skin test for evaluation of specific anti-tumour activity of the**

The anti-tumour activity of the immune system was assessed by a skin test of the delayed type hypersensitivity (DTH) reaction on tumour-associated antigens (TAA), which were used as a lysed human melanoma cell line BRO [26] of 25,000 cells in a test (see Figure 1). Human melanoma cell line BRO was obtained at the Institute of Cytology of the Russian Academy of the Sciences (St. Petersburg, Russia). We investigated the DTH skin reaction after the intra‐ dermal administration on the forearm at 9, 12 and 24 hours, to identify the peak responses. The peak response in most cases (the diameter of redness in mm) was observed after 12 hours. Selection of the human melanoma cell line BRO was determined by the need to use in one test the maximum range of TAA, in order to assess the anti-tumour activity of the immune systems of patients with different cancers. As shown above, there exist all kinds of TAA characteristics

> **Figure 1.** Anti-cancer diagnostic skin test: evaluation of specific anti-tumour activity of the immune system in advanced cancer patients (Patient № 11 after psycho-correction). (a) Intradermal introduction of tumour-associated antigens: lysed cells of human melanoma line BRO 25103 per doze (50 µL); (b) Evaluation of DTH skin reaction on TAA (12 hours later): 0-

**Figure 1.** Anti-cancer diagnostic skin test: evaluation of specific anti-tumour activity of the immune system in ad‐ vanced cancer patients (Patient № 11 after psycho-correction). (a) Intradermal introduction of tumour-associated anti‐

**a b**

The selection of the minimal quantity of TAA for the anti-cancer diagnostic DTH skin test (according to our preliminary studies) was used in order to obtain a physiological specific antitumour immunological response (non-stimulated native DTH skin reaction on TAA) as well as to exclude the possibility of the vaccine's effect on the diagnostic test itself. In comparison with our colleagues, who used the DTH skin reaction on the TAA of human melanoma cell lines in a study of the clinical efficacy of the anti-tumour polyvalent vaccine 'CancerVax' (developed from three allogeneic human melanoma cell lines) [28], we used a diagnostic dose that was

The selection of the minimal quantity of TAA for the anti-cancer diagnostic DTH skin test (according to our preliminary studies) was used in order to obtain a physiological specific antitumour immunological response (non-stimulated native DTH skin reaction on TAA) as well as to exclude the possibility of the vaccine's effect on the diagnostic test itself. In comparison with our colleagues, who used the DTH skin reaction on the TAA of human melanoma cell lines in a study of the clinical efficacy of the anti-tumour polyvalent vaccine 'CancerVax' (USA) (developed from three allogeneic human melanoma cell lines) [28], we used a diagnostic dose

cells vs. 2.4 × 106

antigen, and in most cases the reaction reaches a peak after 48-72 hours [29].

after one month following the completion of the course of hypnotherapy.

receive any immunotropic therapy during the study. The selected dose does not cause allergic and other pathological reactions. It is known that the delayed-type hypersensitivity reaction is a specific immune response and begins to manifest in eight to 12 hours after ingestion of

In our case the peak responses, due to the absence of prior immunization of cancer patients and the largest contribution of cellular reactions in the DTH skin test, were achieved early (within 12 hours) [30]. An evaluation of a specific anti-tumour activity of the immune system in advanced cancer patients was conducted through the research phases: 'before' – before psycho-correction, 'after' – after completing a course of hypnotherapy and 'one month later' –

**2.7. Preparation of tumour-associated antigens for diagnostic test - DTH skin reaction** The lysed cells of the human melanoma cell line BRO were used as the TAA for diagnostic test. The human melanoma cell line BRO were maintained in RPMI 1640 supplemented with 10% heat-inactivated foetal calf serum, L-glutamine (2 mmol/ml), 25 mmol HEPES buffer, and 25 μg/ml gentamicin at 37°C in 5% CO2 humidified air. Cells were detached from the dish by treating with trypsin-EDTA followed by washing three times with Dulbecco's phosphatebuffered saline, precipitated by centrifuging, counted, and diluted with 0.9% saline solution with 0.1% EDTA. Cells were lysed by repeated (eight times) freezing and stored at -80ºC until

lysed cells line BRO in 50 microliters were used.

cells). In addition, our patients did not

per doze (50 µL); (b) Evaluation of DTH skin reaction on TAA

5 mm – low activity, 5-10 mm – average activity, >10 mm – high activity.

(12 hours later): 0-5 mm – low activity, 5-10 mm – average activity, >10 mm – high activity.

**immune system (non-stimulated native activity) in advanced cancer patients**

of solid tumours on the melanoma cells [27].

38 Updates on Cancer Treatment

nearly 100 times smaller (2.5 × 104

gens: lysed cells of human melanoma line BRO 25×103

use. As a diagnostic test 2.5 × 104

In our case the peak responses, due to the absence of prior immunization of cancer patients and the largest contribution of cellular reactions in the DTH skin test, were achieved early (within 12 hours) [30]. An evaluation of a specific anti-tumour activity of the immune system in advanced cancer patients was conducted through the research phases: 'before' – before psycho-correction, 'after' – after completing a course of hypnotherapy and 'one month later' – after one month following the completion of the course of hypnotherapy.

#### **2.7. Preparation of tumour-associated antigens for diagnostic test - DTH skin reaction**

The lysed cells of the human melanoma cell line BRO were used as the TAA for diagnostic test. The human melanoma cell line BRO were maintained in RPMI 1640 supplemented with 10% heat-inactivated foetal calf serum, L-glutamine (2 mmol/ml), 25 mmol HEPES buffer, and 25 µg/ml gentamicin at 37°C in 5% CO2 humidified air. Cells were detached from the dish by treating with trypsin-EDTA followed by washing three times with Dulbecco's phosphatebuffered saline, precipitated by centrifuging, counted, and diluted with 0.9% saline solution with 0.1% EDTA. Cells were lysed by repeated (eight times) freezing and stored at -80ºC until use. As a diagnostic test 2.5 × 104 lysed cells line BRO in 50 microliters were used.

#### **2.8. Preparation of tumour-associated antigens for epicutaneous activation of specific antitumour immunity**

The lysed cells of the human melanoma cell line BRO were used as the TAA for epicutaneous activation of specific anti-tumour immunity. The human melanoma cell line BRO was maintained in RPMI 1640 supplemented with 10% heat-inactivated foetal calf serum, Lglutamine (2 mmol/ml), 25 mmol HEPES buffer, and 25 µg/ml gentamicin at 37°C in 5% CO2 humidified air. Cells were detached from the dish by treating with trypsin-EDTA followed by washing three times with Dulbecco's phosphate-buffered saline, precipitated by centrifuging, counted and diluted with 0.9% saline solution with 0.1% EDTA. Cells were lysed by repeated freezing (eight times) and stored at -80ºC until use. The one epicutaneous activation of specific anti-tumour immunity was performed with the use of a 2.5 × 106 lysed cells line BRO in 0.5 ml.

#### **2.9. Preparation of tumour-associated antigens for extracorporeal activation of specific antitumour immunity**

Lysed placental domestic pig cells were used as the TAA for extracorporeal activation of specific anti-tumour immunity. They were obtained by careful mechanical homogenization of placenta without trypsin. Cells were diluted in saline solution supplemented with 0.1% EDTA and 25 µg/ml gentamicin up to a concentration of 50 × 106 placental cells in 1.0 ml. Cells were lysed by repeated freezing (eight times) and stored at -80ºC until use.

#### **2.10. Extracorporeal activation of specific anti-tumour immunity**

A peripheral blood mononuclear cell(PBMC), separated from 25 ml of heparinized cancer patient blood was diluted in RPMI 1640 and supplemented with 20% autological plasma, Lglutamine (2 mmol/ml), 25 mmol HEPES buffer, and 25 µg/ml gentamicin. PBMC in concen‐ tration 4 × 106 cells/ml was placed in a cell culture dish in the proportion 1-2 × 106 cells/sm2 . The lysed domestic pig placental cells were used as the antigen at a proportion of 1/6 (lysed cells of pig placenta/PBMC). This proportion was found to be optimal in previous research. Further PBMC with added antigen was placed into a CO2 incubator and incubated at 37°C in 5% CO2 humidified air. The incubation time was six to eight hours for antigen processing by monocytes of PBMC. The PBMC was collected after completion of incubation by rubber policeman and was triplewashed in the phosphate buffer solution with the addition of 5% autological plasma of the cancer patient. The washed incubated PBMC was diluted in 2 ml of autological plasma from the cancer patient and shared between two 1 ml syringes. Incubated PBMC was administered subcutaneously in the subscapular fossa area and in the area of the lower abdomen laterally from the umbilicus (palm width sinistral or dextral). A total of three procedures were provided, the second after two weeks and the third after one month from the first procedure.

#### **2.11. Epicutaneous (scarification) activation of specific anti-tumour immunity**

The superficial line scarifications were applied after skin disinfection on the area of 4 cm2 with a gap in between lines of 2-3 mm wide by blood lancet (scarificator) (see Figure 2). The damaged area was covered by sterile patch underneath which a solution with ТАА (2.5 × 10<sup>6</sup> lysed cells line BRO in 0.5 ml) was administered by syringe. The subclavicular and subscapular areas were used for the epicutaneous application of ТАА. The exposition of patch was left for three days. Four sessions of epicutaneous activation of specific anti-tumour immunity were administered with a 14-day break and three sessions were provided with a 30-day break.

**Figure 2.** Epicutaneous (scarification) activation of specific anti-tumour immunity. (A) The superficial line scarifica‐ tions plotted after skin disinfection by blood lancet (scarificator); (B) Patch (with viscose bandage) is superimposed on the network of surface scarification and impregnation of bandage patch by solution of tumour-associated antigens.

#### **2.12. Statistical analysis**

**2.10. Extracorporeal activation of specific anti-tumour immunity**

tration 4 × 106

40 Updates on Cancer Treatment

first procedure.

A peripheral blood mononuclear cell(PBMC), separated from 25 ml of heparinized cancer patient blood was diluted in RPMI 1640 and supplemented with 20% autological plasma, Lglutamine (2 mmol/ml), 25 mmol HEPES buffer, and 25 µg/ml gentamicin. PBMC in concen‐

The lysed domestic pig placental cells were used as the antigen at a proportion of 1/6 (lysed cells of pig placenta/PBMC). This proportion was found to be optimal in previous research. Further PBMC with added antigen was placed into a CO2 incubator and incubated at 37°C in 5% CO2 humidified air. The incubation time was six to eight hours for antigen processing by monocytes of PBMC. The PBMC was collected after completion of incubation by rubber policeman and was triplewashed in the phosphate buffer solution with the addition of 5% autological plasma of the cancer patient. The washed incubated PBMC was diluted in 2 ml of autological plasma from the cancer patient and shared between two 1 ml syringes. Incubated PBMC was administered subcutaneously in the subscapular fossa area and in the area of the lower abdomen laterally from the umbilicus (palm width sinistral or dextral). A total of three procedures were provided, the second after two weeks and the third after one month from the

**2.11. Epicutaneous (scarification) activation of specific anti-tumour immunity**

area was covered by sterile patch underneath which a solution with ТАА (2.5 × 10<sup>6</sup>

The superficial line scarifications were applied after skin disinfection on the area of 4 cm2 with a gap in between lines of 2-3 mm wide by blood lancet (scarificator) (see Figure 2). The damaged

line BRO in 0.5 ml) was administered by syringe. The subclavicular and subscapular areas were used for the epicutaneous application of ТАА. The exposition of patch was left for three days. Four sessions of epicutaneous activation of specific anti-tumour immunity were administered with a 14-day break and three sessions were provided with a 30-day break.

**Figure 2.** Epicutaneous (scarification) activation of specific anti-tumour immunity. (A) The superficial line scarifica‐ tions plotted after skin disinfection by blood lancet (scarificator); (B) Patch (with viscose bandage) is superimposed on the network of surface scarification and impregnation of bandage patch by solution of tumour-associated antigens.

cells/ml was placed in a cell culture dish in the proportion 1-2 × 106 cells/sm2

The statistical data processing was performed using 'BioStat 2009 Professional 5.8.4', which is publicly available. The level of statistical significance (so-called alpha level for a p-value) was accepted as 0.05. All parameters of investigation were normally distributed (by Kolmogorov-Smirnov test), so in general the parametric tests were used. In order to compare two inde‐ pendent statistical samples, the non-parametric Mann-Whitney test was used. The relationship study between psychometric (SCL-90) and immunological (DTH skin reaction on TAA) parameters was carried out using Pearson's correlation test.
