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[Cancer Biology & Therapy 2:2, 123/1-123/6, March/April 2003]; ©2003 Landes Bioscience

Research Paper

Antitumoral Effect of a Vaccination Procedure with an Autologous Hemoderivative

Eduardo Lasalvia-Prisco1,2,3,†,* Silvia Cucchi3 Jesús Vázquez2 Eduardo Lasalvia-Galante2 Wilson Golomar2 William Gordon3

1School of Medicine; University of Uruguay

2National Cancer Institute; Uruguay

3PharmaBlood, Inc.; North Miami Beach, Florida USA

†Former Professor Director, Department of Medicine, School of Medicine, University of Uruguay; Former Director National Cancer Institute, Uruguay; Present Director of PharmaBlood Inc.,Department of Research and Development Florida, USA

*Correspondence to: Dr. Eduardo Lasalvia-Prisco; PharmaBlood Inc, Research and Development Department; 2050 NE 163rd; 2nd Fl. #202; North Miami Beach, Florida 33162 USA; Tel.: 305.944.2544; Fax: 305-944-5244; Email: telemedical@pharmablood.com

Received 00/00/03; Accepted 00/00/03

Previously published online as a CB&T “Paper in Press” at: http://landesbioscience.com/journals/cbt/

KEY WORDS

Cancer vaccine, Autologous vaccine, Hemoderivative vaccine, Cancer immunotherapy, Cancer

ABSTRACT

Lately, the promising results obtained with autologous cancer vaccines are stimulating new research in the old field of cancer immunotherapy. This paper describes the development of a procedure previously reported by us that is used to obtain an autologous hemoderivative with antitumoral properties. The procedure has been tested in a phase I–II, randomized, controlled clinical trial of 28 cancer patients with different primary malignancies in metastatic and chemotherapy-resistant stages. The histology of the lesions that responded to this treatment was consistent with the characteristic histology observed in malignant lesions treated with a similar antitumoral hemoderivative: proliferation of stromal connective tissue, T-lymphocyte infiltration, and a reduction in the amount of tumor cells and blood vessels. We concluded that vaccination had elicited an immune response because a delayed-type hypersensitivity test made with the autologous hemoderivative produced a significantly more intense response in the responding treated patients. We propose that an immune mechanism acting on tumor cells and/or the regulatory system for stromal growth explains the histological results observed. The use of blood to obtain the immunogen allows vaccination to be repeated, so this method could avoid tumor escape responses due to mutations in the antigen library of the tumor. The results of our study justify further research to optimize the antitumoral effect of vaccination.

INTRODUCTION

Cancer vaccines have been a goal of medical research for many years. Lately, several studies have explored the autologous vaccination model in order to consider the antigenic individuality of malignant tumors. In this area of research, some experimental and clinical studies have obtained promising results. The results of melanoma treated with a vaccine manufactured with autologous hapten-modified tumor cells and adjuvant BCG1 and the results of renal-cell carcinoma, pancreatic cancer, and other solid tumors treated with a vaccine prepared from heat-shock proteins (HSP) from autologous tumors2-5 are examples. However, the favorable results reported were temporary in every case. Tumor escape mechanisms from immunological control include changes in malignant cell antigens due to a high rate of spontaneous mutations associated with their rapid proliferative rate, as well as environmental conditions including chemotherapy and the host biological response.6 Most autologous vaccines have been obtained from surgical tumor specimens. These vaccines, when successful, generated an immune response that recognized and controlled tumor cells with the antigen endowment present in the cells from which the vaccine was obtained. However, if the antigenic library of the tumor cells changes, immune mechanisms will be unable to recognize and control the malignant disease, thus resulting in clinical progression. It is not possible to update these vaccines for each and every new antigenic profile of tumor cells because it is not feasible to obtain new surgical specimens frequently. In order to obtain a series of autologous vaccines using frequently updated tumor antigens, we explored autologous blood as an immunogen source in cancer patients. In 1995, we reported the antitumoral effect of an autologous blood protein fraction that was inoculated repeatedly in cancer patients.7,8 This paper reports the procedure used to obtain an autologous blood fraction with immunogenic properties from cancer patients. The procedure can be performed repeatedly.

MATERIAL AND METHODS

Hemoderivative Preparation and Vaccination. The autologous hemoderivative was obtained by modifying a procedure that we developed previously.7 Twenty milliliters of blood was drawn from the femoral artery in a tube containing 5000 IU heparin. The blood was allowed to sediment at 37˚C for 1 hour. Then, cellular lysis was produced by exposing the supernatant of plasma and cells to hypotonic shock with 3 volumes of distilled water for 15 min, and followed by freezing at -20˚C. After 24 hours, the preparation was thawed and incubated at 100˚C for 10 min. After final filtration through a cellulose acetate membrane filter (0.22 µm pore diameter), the preparation was divided into 5 vials: 1 test-vial containing 0.5 ml and 4 vaccine-vials containing equal aliquots of the remaining preparation. All vials were stored at -20˚C until use. The 4 vaccine-vials were used on days 1, 14, 21, and 28 of the vaccination cycle. Each vaccination consisted of a mixture of the vaccine-vial content and 300 µg of recombinant human granulocyte-macrophage colony stimulating factor (rH GM-CSF) and was given by subcutaneous injection. No more than 3 ml was injected in each subcutaneous vaccination site at a time, so several subcutaneous injections had to be made on the abdominal surface in order to inject the whole vaccine-vial. To prevent the development of immune-tolerance,9 cyclophosphamide 300 mg/m2 was given 3 days before beginning the vaccination procedure. Ten days after the last vaccination, an intradermal test of delayed-type hypersensitivity (DTH) was made with the

0.1 ml of preparation conserved in the test-vial. Measures were taken to ensure sterility at each stage of the procedure (arterial blood extraction, vaccine preparation, and vaccination). The procedure was repeated every 45 days (counting from the first day of the previous cycle) until the diameter of the skin indurations elicited in response to the DTH test was at least 5 mm, or for 6 months if the diameter of the induration never reached 5 mm. The DTH test was not performed in the non-treated control group.

Patients and Trial. A phase I-II, randomized, controlled clinical trial was designed. The patient population studied was quite heterogeneous, as is common in phase I trials, but the objective of assessing an antitumoral effect in a controlled protocol is typical of phase II trials. As can be seen in Table 1, 28 cancer patients were included (11 breast, 7 colorectal, 4 ovarian, 4 pancreatic, 2 prostate) and distributed equally into two groups. The eligibility criteria were: adults up to 70 years old with a solid tumor and metastatic disease, at least one measurable mass, previous acquired resistance to chemotherapy as defined by disease progression after receiving the chemotherapy recommended in the PDQ database, ECOG performance status 2 or less, and an estimated survival of more than 6 months. Eligible patients also had abnormally high concentrations of the respective serum tumor markers released into blood by malignant cells: CA15-3, CEA, CA125, CA19-9 and PSA respectively in breast, colorectal, ovarian, pancreatic, and prostate cancer. The tumor burden in all cases was not minimal, it was evident in imaging studies and clinical assessment, but it did not affect the eligibility criteria in terms of performance status. Patients were registered centrally, stratified by primary malignant site, and then randomly distributed to one of the two study groups. In all cases, informed consent was obtained from patients or their guardians. An institutional review board approved the clinical trial.

  • The first group underwent the vaccination procedure as described.

  • The control group received cyclophosphamide 300 mg/m2 three days before starting a series of subcutaneous injections of rH GM-CSF given on days 1, 14, 21, and 28. This procedure was repeated 4 times, beginning 45 days after the first day of the previous cycle (Total treatment time: 6 months). Vials were prepared with 300 µg of rH GM-CSF diluted in NaCl 0.9% to obtain the same final volume as the volume contained in the vaccine-vials. Vials were used to make control subcutaneous injections when vaccinations were scheduled. No more than 3 ml was injected per site at a time. The control group received no other antitumoral treatment, but did receive symptomatic treatment.

  • Results were analyzed with the Student-t test.

Assessment of the Antitumoral Effect. The RECIST system10 was used every month to evaluate response in terms of tumor size. Performance status was also evaluated monthly. Follow-up lasted for 8 months from onset of vaccine treatment in all cases. The best RECIST-status maintained in 2 successive assessments (one month) was recorded as the response obtained in each patient. A histological study of the response was made in the treated patients; biopsies of accessible metastatic lesions were studied before and after treatment in 5 responders and in 4 non-responders.

Hematoxylin-eosin stain was complemented with more specific stains for connective tissue components: light green, Van Giesen (collagens), and

Table 1 PATIENTS INCLUDED IN A RANDOMIZED, CONTROLLED
CLINICAL TRIAL*
Group 1a Group 2b
Number 14 14
Primary
Breast 6 5
Colorectal 3 4
Ovary 2 2
Pancreas 2 2
Prostate 1 1
Agec 61.1 (51–-67) 60.3 (54–65)
PSc 1.8 (1–2) 1.5 (1–2)
Evaluable 14 13d

*Patients had metastatic, chemotherapy-resistant cancer and were distributed into two groups: treated and not treated by vaccination with the autologous hemoderivative. aTreated group; bNon-treated group; cMean (range); dA patient with pancreatic cancer died before assessment.

Wilder (reticular fibers). Immunocytochemical studies were made of the lymphocytes infiltrating the tumoral stroma to identify the activated populations. Monoclonal antibodies, CD3+ (Ventana) for T-lymphocytes, L26 for B (CD20+) lymphocytes, PGM-1 for macrophages (DAKO), and CD8 (Novocastra) for cytotoxic suppressor T cells were used at a dilution of 1:50. Microvessels in the tumor tissue were detected by immunohistochemical staining with antifactor-VIII antigen-related polyclonal antibody (DAKO). The avidin-biotin complex staining method was used for all immunohistochemical stains, with diaminobenzidine as a chromogen. Apoptotic tumor cells were assessed in representative microscopic fields by 3'-end labeling of apoptotic DNA using an ApopTag in situ apoptosis detection kit (Oncor).

Immunization Assessment. The DTH intradermal tests were read at 48 hours. In each case, we measured the largest and smallest diameters of the induration that appeared. Because the resulting induration had an almost circular shape, the distribution between positive and negative responses was the same regardless of whether the induration was assessed using the average of these two dimensions or the longest diameter. Therefore, positive DTH response was identified using only the longest diameter of the induration, expressed in millimeters.

Protein Vaccine Assessment. Samples of each final preparation were tested for total protein content using the Bradford assay. Chromatography by gel filtration (Sephadex G100 and G200, superfine, Pharmacia) was used to determine the molecular weight of protein fractions found in the vaccine. The respective serum tumor marker was tested in each preparation, before and after thermal fractionation procedures. Because of reports that HSPs participate in autologous cancer immunotherapy,2-5 several HSP samples were tested by immunoblotting before and after thermal fractionation.

Toxicity Assessment. Toxicity was evaluated using the WHO (T) criteria and high levels of each detected toxicity type were recorded.

Performance Status (PS) Assessment. Performance status was evaluated in each patient before and after treatment and scored according to ECOG scale. The variation in PS was recorded for each patient. The mean and standard deviation were calculated for each group.

RESULTS

As can be seen in Table 1, the two groups were comparable.

Figure 1 shows the results of the RECIST status evaluation. At the end of the 8-month follow-up period, 1 patient from the non-treated group had died of progressive disease before completing follow-up, leaving 27 evaluable patients (14 treated and 13 non-treated). At the end of the follow-up period, we obtained the following findings. In Group 1, 5 patients (3 breast, 1 ovary, 1 pancreas) had progressive disease; 9 patients showed some type of

Figure 1. Assessment of tumor response with the RECIST system. The sum of the largest diameters in measurable selected target lesions was recorded before and after each treatment month. A reduction of this value of more than 30%, maintained more than 1 month, was considered a partial remission. An increase in this value of more than 20% is considered progressive disease. The interval between these states was considered stable disease. Favorable responses (Partial Remission + Stable Disease) were significantly more frequent in the treated group than in the non-treated group (Control): P<0.001.

favorable tumor response: 3 of 9 patients (1 breast, 1 colorectal, 1 ovary) reached levels of partial remission and 6 of 9 patients (2 breast, 2 colorectal, 1 pancreas, 1 prostate) had stable disease. In Group 2, 12 patients had progressive disease (5 breast, 4 colorectal, 2 ovary, 1 pancreas) and 1 patient had stable disease (1 prostate). Progressive disease was significantly more frequent in the non-treated group than in the treated group (p<0.01). The total number of favorable responses (partial remission + stable disease) was also significantly higher in the treated group (p<0.001). The median delay in response from the end of treatment to the first signs of a response was

12.7 weeks (range 5 to19) in the 9 patients who had favorable responses. In treated patients (Group 1), the mean change in performance status in the 9 responders was -0.165 versus +1.750 in the 6 non-responders (p<0.02).

Figure 2 shows the results of the DTH test during the follow-up of Group 1: 7 of 9 patients with favorable responses and 1 of 5 patients with progressive disease had indurations of at least 5 mm.

Figures 3, 4, and 5 show histological preparations of tissue from accessible metastatic lesions after treatment in 5 responders in the treated group (4 skin, 1 peritoneal; 3 partial remissions, 2 stable disease). The histological findings were similar in all these lesions, which is consistent with a previously reported favorable response:7 tumoral stromal fibrosis with abundant collagen deposits, T-lymphocyte (CD8+) infiltration, and reduction in the number of vessels and tumor cells, with apoptotic tumoral cells visible. No fibrosis or reduction in the number of vessels and tumor cells were observed in the pre-treatment biopsies of the same patients. In 4 treated non-responders (3 breast, 1 ovary), accessible metastatic lesions (3 skin, 1 peritoneal; progressive disease) could be studied before and after treatment. In these cases, the histological findings before and after treatment were similar and no fibrosis or reduction in vessels or tumor cells was evident. Clinically relevant systemic or local toxicity was not reported. No patient had to discontinue or modify treatment because of toxicity or side effects. The systemic toxicities recorded were all grade 1, according to WHO (T) criteria, and some of them were attributed to cyclophosphamide. Toxicity was hematological (leukopenia that appeared in 10 of 14 treated and 10 of 13 non-treated patients, respectively), gastrointestinal (9 of 14 treated and 10 of 13 non-treated patients experienced nausea), and hair loss (6 of 14 treated and 4 of 13 non-treated patients had minimal hair loss). Among the known systemic side effects of rH GM-CSF, only mild fever (37.5˚C–38.5˚C) was observed, after the third injection (7 of 14 treated and 5 of 13 non-treated patients). In the injection sites, a grade 1-2 local reaction (pain or pain + inflammation) was seen in all patients of both groups, which was attributed to rH GM-CSF. No evidence of any autoimmune phenomenon was evident.

The characterization of the vaccine preparations was consistent with the characterization of an antitumoral autologous hemoderivative previously described.8 Briefly, the antitumoral effect was associated with a final vaccine whose composition included at least 5 protein fractions: a large homogeneous fraction (>50%), MW~50,000, and 4 non-homogeneous protein fractions. Unmodified structural HSPs and tumor markers were not identified in the final vaccines. Prior to the thermal fractionation process, among the many biomolecules present, increased serum concentrations of each patient's respective tumor marker were identified, as well as several HSPs (Hsp70/72, Hsp90, Gp 96/94, and Hsp 47).

DISCUSSION

This phase I–II clinical trial had limitations due to the heterogeneity of the patient population and small number of cases. However, the results indicate the following innovative findings that merit further research in a trial of more advanced design.

Antitumoral Effect. The results categorically show that an autologous hemoderivative obtained by means of the procedure described had antitumoral activity. It inhibited tumor growth in a significant number of patients, according to assessment of tumor measures using the recommended RECIST system. It is known that assessment of tumor size by direct measurement of accessible tumors or measurement of CT-scan images cannot discern differences between viable tumor tissue and tumor fibrosis. Using tumor size alone to assess antitumoral effect in tumors that respond by producing fibrosis could be of questionable value. Vaccine-induced fibrosis can obscure the apparent antitumoral effect by increasing the size of tumors after treatment. Therefore, if fibrosis could be excluded from tumor measurements, the antitumoral effect might be even more significant. This is one reason why institutional references in oncology recommend the use of RECIST.10 This method is recommended to assess antitumoral effects, including those of therapeutic agents that induce tumor fibrosis. In addition, performance status, which is not recognized as a valid measure of response to antitumoral treatment, was significant in our results because it was linked to a reduction in targeted tumor mass growth. Three findings anticipate the potential clinical implications of this antitumoral effect:

a.
the antitumoral effect becomes evident after a delay of several weeks from the beginning of treatment;
b.
it is not specific to the primary tumor site in the tumors studied (breast, colon, ovary, prostate, and pancreas); and
c.
it was observed in metastatic stages of the tumors studied.

Characterization of the Vaccine. We have shown that the vaccine is a preparation containing several species of proteins. It did not differ in composition from an autologous antitumoral hemoderivative previously described. According to filtration chromatography, it contained a pool of five protein fractions with a homogeneous main fraction of molecular weight ~50.000. The vaccine lost its antitumoral properties with further purification of its poly-protein composition.8 The aim of this paper is to report the antitumoral effect of vaccinations of an autologous hemoderivative, so only a preliminary characterization of the vaccine preparation has been included. As in tumor vaccines using tumor cells as an immunogen, the chemical complexity of the hemoderivative vaccine described here, which contains several, mostly non-homogeneous, protein fractions, is difficult to define. In this study, the antitumoral effect elicited by an autologous hemoderivative, as demonstrated by immunological and histological evidence, is as appealing conceptually as the reported antitumoral effect of vaccines containing tumor cells, bacteria, viruses, biological adjuvants, and other immunogens that are as yet undefined chemically.

Mechanism of Action. Our results suggest two mechanisms that could be involved in the antitumoral effect shown:

Immune Response. The patients in the treated group who had DTH reactions also had a favorable antitumoral response more frequently. Therefore, even though the study design did not allow direct evidence of the involvement of antitumoral immunity to be obtained, we cannot disregard the possibility that such an involvement could link DTH results to the RECIST results. Our vaccine could elicit a direct or cross-immunization response against tumor-associated antigens (TAAs) released by malignant cells into the blood.

Stromal Response. Histopathological analysis was limited by the accessibility to biopsy of the tumor lesions before and after treatment. Therefore, in the treated group, the comparability of the histological results in responders and non-responders must be considered as just a preliminary finding. However, only patients with a favorable response to treatment showed an unusual histological image that was not found before treatment in any patient or in non-responders after treatment. Metastatic lesions in responders showed low T-lymphocyte infiltration. However, the most prominent histological finding was increased stromal fibrosis. This histological picture is not characteristic of a pure DTH immune response, and it suggests that our vaccine acts directly or indirectly on the growth of tumoral stroma components. Stromal fibrosis, changes in the blood vessel-to-tumor cell ratio, and the development of a more extensive barrier against tumor cell migration could reasonably have an antitumoral effect.

Immunological and stromal responses may both contribute to the antitumoral effect of this vaccine. The antitumoral effect was associated with DTH and also with stromal fibrosis. DTH to autologous hemoderivatives in the vaccine is not a direct sign of DTH against the tumor. This could have been tested if DTH testing had been performed with tumor cells or tumor extracts. However, such intradermal tests with tumor cells or tumor extracts were not feasible in the study design because repeated testing with extracts or cells from successive surgical tumor specimens cannot be used to control the effects of a sequence of vaccinations of a hemoderivative. However, it must be assumed that autologous blood can contain cells and molecules released by tumors. Several authors have identified cancer cells in the blood of cancer patients. Molecules synthesized in cancer cells, released into the blood, and recognized as tumor markers have also been found in the blood of cancer patients.11-13 All of the patients included in this study had abnormally high serum levels of tumor markers, which is evidence of disease with active release of malignant cell contents to blood. Under such circumstances, other molecules could be released by cancer cells. In this study, the respective tumor marker was present in the lysed blood supernatant from which the vaccine was obtained. As these molecules were not identified in the final vaccine, the only significance that we attributed to them is that they were markers of the presence of molecules released from tumor cells in the blood samples from which the vaccine was obtained. Additional studies must be made to determine if tumors release the molecules responsible for the antitumoral effect of these vaccines in the absence of elevation of serum tumor markers. It is known that the content of tumor cells released at cell death can be immunogenic for the host when this release is the result of the apoptosis of previously stressed tumor cells. A well known cellular stressor is chemical stress induced by oncological chemotherapy. It is known that stress-induced immunogenesis in tumor cells is associated with HSP synthesis.14 All our patients had received previous chemotherapy. HSPs have been very well studied in cancer immunotherapy15-17 as antigens, adjuvants, or antigen chaperones.18-20 Hsp70/72, Hsp90, Gp 96/94, and Hsp 47 were demonstrated in vaccine preparations before thermal fractionation. This finding may be relevant to discussions of the DTH response and stromal fibrosis elicited because the first three HSPs detected are the most immunogenic of the HSPs.3 Hsp 47 has known interactions with type I procollagen.21-23 As these molecules were not identified in the final vaccine, their only significance was the presence, in the samples from which the vaccine was obtained, of immunization and fibrosis-inducing molecules. Further studies should be made to determine if products of molecular modifications of these agents are necessary for the immune and/or stromal response elicited by the vaccine.

Antigenization. As was shown with tumor markers and HSPs, protein components of tumor cells and mediators of stromal fibrosis are present in the lysed hemoderivative before thermal fractionation but are not identified in the final vaccine after thermal fractionation. These molecules are potential targets for an antitumoral immune response. This finding is consistent with properties of the thermal fractionation process that have been reported in other models. Exposure to 100˚C for ten minutes changes the molecular structure of proteins with two known consequences:

a.
the procedure can be used to concentrate proteins with a molecular structure resistant to these conditions, thus resulting in a predominance of specific functional molecular forms of growth factors, as has been shown for platelet-derived growth factor;24 and
b.
it maintains or increases the immunogenic properties of many proteins, with loss of biological properties, as described in toxicological models.25

Therefore, if thermal fractionation is applied to a lysed hemoderivative containing components of tumor cells, inductors of tumor-elicited immune response, and tumor stromal growth modifiers, this may produce molecular structural modifications, with the following consequences:

a.
parental molecules may not be identifiable in the final vaccine;
b.
the remnant molecular forms could have a different biological function than parental molecular forms; and
c.
antigenic molecular structures could be generated that elicit direct or cross-reactivity against targets in tumor cells or regulators of stromal fibrosis.

Given the molecular complexity of blood from cancer patients, thermal antigenization can produce a vaccine with an antigenic polyvalence similar to that of autologous vaccine

obtained from tumor cells. As a consequence, the immune response elicited by tumor-cell vaccines could be similar to that of vaccine obtained by the procedure described. Therefore, hemoderivative vaccines could complement tumor cell vaccines when tumor tissue is not available.

Antigen Empowerment. In order to explain how small amounts of antigens present in the blood sample can induce a clinical tumor response, it must be considered that the method for vaccine preparation described has some steps used to amplify the immune response elicited. These steps are the subcutaneous inoculation of immunogen, activation of dendritic cells with GM-CSF,26 and prevention of tolerance with cyclophosphamide.27 All these steps are already being used by other favorable cancer vaccine trials to enhance the antitumoral immune response. In addition, our procedure, as reported earlier, uses arterial blood instead of venous blood7,8 as a source of immunogen because most of the molecules delivered by tumor cells to the blood enter a deep vein, then travel

cer (skin metastasis) that showed a favorable response in the antitumoral assessment.

through the right and left heart before reaching the

Histological sections from pre-treatment and post-treatment biopsy specimens were immuno

arterial tree. Drawing blood from the arterial tree

histochemically stained for CD3. Immunohistochemical studies were made on formalin-fixed, paraffin-embedded sections using antibodies against the pan T-cell marker CD3 (Ventana). A avoids the first-pass clearance in peripheral micro- minimal pre-treatment (A) and brisk post-treatment (B) T-lymphocyte (CD3+) infiltrate (x 400) circulation and tissue that occurs in blood drawn is visible. The presence of B-lymphocytes (CD20+) in the infiltrate was minimal before and from peripheral veins. after treatment.

Finally, our vaccination procedure, because of the repeated preparation of the vaccine from autologous blood, updates the antigens released by tumors into the blood. Consequently, it could be used to avoid the tumor escape mechanism due to antigenic mutations or changes in the biological response or stroma regulators.

CONCLUSIONS

We conclude that with a procedure developed from an earlier one, it is possible to obtain an autologous hemoderivative that can be inoculated repeatedly to produce an antitumoral effect in patients with advanced cancer. A phase I- II clinical trial has shown that this antitumoral effect is statistically significant and can be elicited in advanced stages of different primary solid tumors. It takes several weeks of treatment for this antitumoral response to become evident. DTH to the hemoderivative was demonstrated by intradermal tests in most responding treated patients, suggesting a link between response to this vaccination and immune response. We discuss possible mechanisms of action of this antitumoral treatment, such as an immune response to modified proteins released into the blood by tumors, as well as stromal fibrosis associated with this immune response. Using autologous blood as the source material for the preparation of a poly-antigenic vaccine for cancer patients makes it feasible to make new vaccine preparations and continuously update the immunogen with the latest mutations of malignant cell antigens. The results of our study justify further research to optimize vaccine characterization, identify the primary molecular target of the immune and stromal responses elicited, and explore the clinical relevance of this therapeutic alternative.

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