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Aim: To evaluate the effect of pleuran (β-glucan from Pleurotus ostreatus) administration on the immune profile of patients with endocrine-dependent breast cancer (clinical stages I–II) in clinical and imaging remission. Methodology: Antitumor cellular immunity (CD19+, CD3+, CD4+ and CD8+ T lymphocytes and natural killer cells) of 195 patients (49 in the pleuran group and 146 in the control group) was measured by flow cytometry. Results: We observed a significant increase in the absolute number of CD3+, CD19+, CD4+ and CD8+ T lymphocytes in the pleuran group compared with the control group. Conclusion: Our results suggest potential benefit of continuous pleuran administration on immune rehabilitation of cellular antitumor immunity and better prognosis in breast cancer patients in remission.

Lay abstract We aimed to evaluate the effect of pleuran (β-glucan from oyster mushroom) on the selected immune parameters of patients with breast cancer in remission. We studied antitumor cellular immune parameters of 195 patients (49 in the pleuran group and 146 in the control group) by means of flow cytometry. After 12 months, we measured a significant increase of cytotoxic T lymphocytes in the pleuran group compared with a significant decrease in the control group. Our results suggest potential benefit of long-term administration of pleuran on antitumor cellular immunity and better prognosis in breast cancer patients in remission.

Background The present study was performed to evaluate the predictive capacity of the TNM-Immune (TNM-I) classification staging system vs. the 8th edition of the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC) tumor-node-metastasis (TNM8) staging system for survival of patients with colon cancer. Methods Data of eligible patients from the Society-for-Immunotherapy-of-Cancer (SITC) international-consortium from 13 countries were evaluated for the consensus Immunoscore in 3539 patients with AJCC/UICC-TNM stages I-III colon cancer. The primary-endpoint time-to-recurrence (TTR) and secondary-endpoint overall-survival (OS) were evaluated for 2650 patients with complete TNM staging and Immunoscore information. The consensus Immunoscore was predefined and previously published (Pages et al. Lancet 2018), and TNM-I categories (IA to IIID) were performed based on TNM8 plus consensus Immunoscore categories. Results A total of 1737 (64.8%) patients presented stage migration, including 1495 (55.8%) migrating to a lower stage and 242 (9.0%) to a higher stage. In TNM8 stage IIIB, patients migrated to all possible TNM-I stages from IA to IIID. A total of 563 Stage IIIB (67.0%) patients presented stage migration, including 181 (32.1%) migrating to a lower stage and 196 (34.8%) to a higher stage. TNM-I Hazard ratio for TTR between TNM-I stage IA and IIID was HR= 16.9 (95%CI 7.75-36.83), in contrast TNM8 TTR between IA and IIIC was only HR= 12.77 (95%CI 7.99-20.4), all P< 0.0001. TNM-I Hazard ratio for OS between TNM-I stage IA and IIID was HR= 5.95 (95%CI 3.17-11.19), in contrast, TNM8 OS between IA and IIIC was only HR= 3.16 (95%CI 2.38-4.18), all P< 0.0001. The TNM8 staging system exhibited prognostic discrepancies in discriminating stage IIB and IIC and between IIC and IIIA on survival curves from TTR and OS, which were improved in the TNM-I staging system. In cox multivariate analysis, the relative contribution of TNM-I was 90.3% in comparison to gender, sidedness, MSI, grade of differentiation, mucinous colloid, VELIPI. The TNM-I had a better predictive capability of survival, as evidenced by higher likelihood ratio scores, and smaller AIC values for TNM-I compared with those for TNM8 edition. Conclusion Therefore, the present study showed the importance of immuno-pathology and demonstrated that the TNM-Immune (TNM-I) classification staging system is superior (log-likelihood ratio test P< 0.0001) to the 8th edition of the AJCC/UICC-TNM staging system in predicting the TTR and OS of patients with colon cancer.

PURPOSE The purpose of this study was to evaluate the prognostic value of Immunoscore in patients with stage III colon cancer (CC) and to analyze its association with the effect of chemotherapy on time to recurrence (TTR). METHODS An international study led by the Society for Immunotherapy of Cancer evaluated the predefined consensus Immunoscore in 763 patients with American Joint Committee on Cancer/Union for International Cancer Control TNM stage III CC from cohort 1 (Canada/United States) and cohort 2 (Europe/Asia). CD3+ and cytotoxic CD8+ T lymphocyte densities were quantified in the tumor and invasive margin by digital pathology. The primary end point was TTR. Secondary end points were overall survival (OS), disease-free survival (DFS), prognosis in microsatellite stable (MSS) status, and predictive value of efficacy of chemotherapy. RESULTS Patients with a high Immunoscore presented with the lowest risk of recurrence, in both cohorts. Recurrence-free rates at 3 years were 56.9% (95% CI, 50.3% to 64.4%), 65.9% (95% CI, 60.8% to 71.4%), and 76.4% (95% CI, 69.3% to 84.3%) in patients with low, intermediate, and high immunoscores, respectively (hazard ratio [HR; high v low], 0.48; 95% CI, 0.32 to 0.71; P = .0003). Patients with high Immunoscore showed significant association with prolonged TTR, OS, and DFS (all P < .001). In Cox multivariable analysis stratified by participating center, Immunoscore association with TTR was independent (HR [high v low], 0.41; 95% CI, 0.25 to 0.67; P = .0003) of patient’s sex, T stage, N stage, sidedness, and microsatellite instability status. Significant association of a high Immunoscore with prolonged TTR was also found among MSS patients (HR [high v low], 0.36; 95% CI, 0.21 to 0.62; P = .0003). Immunoscore had the strongest contribution χ2 proportion for influencing survival (TTR and OS). Chemotherapy was significantly associated with survival in the high-Immunoscore group for both low-risk (HR [chemotherapy v no chemotherapy], 0.42; 95% CI, 0.25 to 0.71; P = .0011) and high-risk (HR [chemotherapy v no chemotherapy], 0.5; 95% CI, 0.33 to 0.77; P = .0015) patients, in contrast to the low-Immunoscore group ( P > .12). CONCLUSION This study shows that a high Immunoscore significantly associated with prolonged survival in stage III CC. Our findings suggest that patients with a high Immunoscore will benefit the most from chemotherapy in terms of recurrence risk.

Background Immunoscore® is an in vitro diagnostic test predicting the risk of relapse in early-stage Colon Cancer (CC), by measuring the host immune response at the tumor site. This risk-assessment tool provides independent and superior prognostic value than the usual risk parameters and is intended to be used as an adjunct to the TNM classification for clinical decision. In the present study, we investigated Immunoscore® clinical performance in the Asian population from the international SITC-led validation study (Pagès et al. The Lancet 2018). Methods Out of the 2681 eligible stage I-III patients of the international Immunoscore® study, 423 were collected from 4 expert centers in Asia including Japan (n=330), China (n=35), and India (n=58). Patients were classified by Immunoscore® based on pre-defined cutoffs, either in 5 (IS 0-4) or 2 categories: IS Low (IS 0-1) and IS High (IS 2-4). Time to recurrence (TTR) was compared between Immunoscore® categories. Results Immunoscore® Low and High were observed in 37% (n=158) and 63% (n=265) of the Asian cohort, respectively. Immunoscore® was positively and significantly correlated with TTR. After 5 years, 86.9% (95% CI 82.7-91.4), and 77% (95% CI 70,5-84,1) of Immunoscore -High and -Low patients respectively were event free (HR =0.52; 95% CI 0.32-0.86; p=0.0085). When adjusting the model with Immunoscore®, age, gender, T-stage, N-stage, sidedness and MSI, and when stratified by center, Immunoscore® remained a significant parameter (HR=0.45; 95% CI 0.22-0.91; p=0.027). When stratified into 5 Immunoscore® categories, TTR rates at 5 years were 100%, 96%, 84%, 80%, and 73.5% for IS4, IS3, IS2, IS1, IS0, respectively. These results were similar to those found in European and North American patients. Conclusions Immunoscore® is a strong prognostic indicator of the risk of recurrence in stage I-III CC patients who receive standard of care treatment in real-life clinical practice in Asia. This first standardized immune-based assay risk assessment tool can be used reliably to guide clinical decision according to each patient information.

Background Immunoscore® is an in vitro diagnostic test that predicts the risk of relapse in patients with Colon Cancer (CC) by measuring the host immune response at the tumor site. It is an immune risk-assessment tool providing independent and superior prognostic value than traditional histopathological risk parameters, and is intended to be used as an adjunct to the TNM classification. Currently, Immunoscore plays a critical role to guide post-surgery decisions in stage II & III CC patients. In stage I, survival rates are high and adjuvant chemotherapy is not typically recommended. However, approximately 10% of stage I CC tumors will recur even after surgical resection. Methods A subgroup analysis was performed on the stage I patients (n = 451) from the Immunoscore international validation study (Pages et al. The Lancet 2018). Patients were classified by Immunoscore based on pre-defined cutoffs, either in 5 (IS 0-4) or in 3 categories: IS Low (IS0-1), Intermediate (IS 2), High (IS 3-4). Time to recurrence (TTR) was compared between Immunoscore categories. Results Immunoscore Low, Intermediate and High were observed in 14%, 47% and 39% of the cohort, respectively. Immunoscore was positively and significantly correlated with TTR. When adjusting the model with Immunoscore, age, gender, T-stage sidedness and MSI, Immunoscore remained the sole significant parameter (stratified by participating center HRlow vs high=7.82; 95% CI 1.49 − 41.01; p = 0.015). In multivariate analysis, the variable with the most important relative contribution to the risk (Chi2) was Immunoscore. The robust correlation between Immunoscore classification and TTR was further corroborated by a separate analysis of the same cohort distributed into five IS categories. In MSS stage I patients, TTR rates at 5 years were 100%, 98.1%, 93.5%, 85.4%, 87.5% for IS4, IS3, IS2, IS1, IS0, respectively. Conclusions Immunoscore® is a robust prognostic indicator of the risk of recurrence in stage I CC. This risk assessment tool reliably identifies a sub-group of patients with an increased risk of relapse for whom a more intensive surveillance program after curative resection may be recommended. Legal entity responsible for the study SITC (Society for ImmunoTherapy of Cancer) / INSERM. Funding Grants from National Institute of Health and Medical Research (INSERM), the LabEx Immuno-oncology, the Transcan ERAnet European project, Association pour la Recherche contre le Cancer, CARPEM, AP-HP, INCA translationnel, Japan-AMED (P-DIRECT) and MEXT (Grants-in-aid for Scientific Research-S), and Ministry of Health of the Czech Republic (AZV CR 15-28188A) Progres Q25-LF1. Disclosure J. Galon: Advisory / Consultancy, Shareholder / Stockholder / Stock options, Co-founder : HalioDx; Research grant / Funding (institution): Perkin-Elmer; Advisory / Consultancy, Research grant / Funding (institution): IObiotech; Advisory / Consultancy, Research grant / Funding (institution): MedImmune; Research grant / Funding (institution): Janssen; Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Imcheck Therapeutics; Advisory / Consultancy: BMS; Advisory / Consultancy: Northwest Biotherapeutics; Advisory / Consultancy: Amgen; Advisory / Consultancy: Gilead; Advisory / Consultancy: CatalYm GmbH; Advisory / Consultancy: Sanofi; Licensing / Royalties, Patent holder: INSERM. F. Hermitte: Shareholder / Stockholder / Stock options, Full / Part-time employment, Co-founder : HalioDx. B. Mlecnik: Licensing / Royalties, Patent holder: INSERM. F. Pages: Licensing / Royalties, Patent holder: INSERM. All other authors have declared no conflicts of interest.

Contains fulltext : 193579.pdf (Publisher’s version ) (Closed access) BACKGROUND: The estimation of risk of recurrence for patients with colon carcinoma must be improved. A robust immune score quantification is needed to introduce immune parameters into cancer classification. The aim of the study was to assess the prognostic value of total tumour-infiltrating T-cell counts and cytotoxic tumour-infiltrating T-cells counts with the consensus Immunoscore assay in patients with stage I-III colon cancer. METHODS: An international consortium of 14 centres in 13 countries, led by the Society for Immunotherapy of Cancer, assessed the Immunoscore assay in patients with TNM stage I-III colon cancer. Patients were randomly assigned to a training set, an internal validation set, or an external validation set. Paraffin sections of the colon tumour and invasive margin from each patient were processed by immunohistochemistry, and the densities of CD3+ and cytotoxic CD8+ T cells in the tumour and in the invasive margin were quantified by digital pathology. An Immunoscore for each patient was derived from the mean of four density percentiles. The primary endpoint was to evaluate the prognostic value of the Immunoscore for time to recurrence, defined as time from surgery to disease recurrence. Stratified multivariable Cox models were used to assess the associations between Immunoscore and outcomes, adjusting for potential confounders. Harrell's C-statistics was used to assess model performance. FINDINGS: Tissue samples from 3539 patients were processed, and samples from 2681 patients were included in the analyses after quality controls (700 patients in the training set, 636 patients in the internal validation set, and 1345 patients in the external validation set). The Immunoscore assay showed a high level of reproducibility between observers and centres (r=0.97 for colon tumour; r=0.97 for invasive margin; p

In view of the fact that insufficiency in immune response often correlates with poor prognosis, research in recent years has focused on the task of describing the precise status and function of the immune system and its possible effect on cancer patients. Although more than two thirds of treated patients respond to endocrine therapy, most patients with metastatic breast cancer develop a resistance to it. Estrogen modulates angiogenesis, partially through its effects on vascular endothelial growth factor (VEGF). It also appears that transforming growth factor-beta (TGF beta) could be another factor contributing to this resistance. TGF beta is a highly immunosuppressive factor that inhibits natural and specific immunity against tumors and stimulates the production of VEGF. The purpose of the study was to monitor immune responses in patients with hormone receptor-positive breast cancer who were resistant to hormone therapy. The examination of cellular components (CD4, CD8, HLA-DR, NK cells) and humoral immunity (IgG, IgG subclasses, IgA, IgM,). TGF beta and VEGF production were monitored with special attention, along with an analysis of the changes that occurred during the hormonal treatment. 68 patients included in the research project were implemented with routine cancer treatment with endocrine therapy. Basic parameters (the histological type and grade, the degree of expression of estrogen receptors (ER) and progesterone receptors (PR), human epidermal growth factor receptor 2 (HER2), and the proliferative marker) were established. Patients were evaluated by a cancer clinical immunologist to exclude immune disorders, allergic or autoimmune origin. TGF beta and VEGF were measured by ELISA and antitumor cellular immunity (CD4, CD8) was measured by flow cytometry. Patients who failed in the first line of hormone therapy treatment were considered as resistant to hormone therapy.Depression in cellular immunity was found especially in patients with resistance to endocrine therapy. In addition, immunoglobulin plasma levels were decreased (mainly IgG4 subtype). Most patients showed clinical symptoms of immunodeficiency (frequent infections of respiratory or urinary tract, herpetic infections). Significant increases in TGF beta and VEGF plasma were also detected.The correlation of these factors with resistance to hormonal therapy and the state of anticancer immunity could be helpful in the task of predicting resistance to hormonal therapy and could contribute to the selection of targeted immune therapy in cancer patients in the future.

O1 Combinatorial CD8+ and PD-L1+ cell densities correlate with response and improved survival in non-small cell lung cancer (NSCLC) patients treated with durvalumab #### Sonja Althammer1, Keith Steele2, Marlon Rebelatto2, Tze Heng Tan1, Tobias Wiestler1, Guenter Schmidt1, Brandon Higgs2, Xia

487 Background: Immunoscore Colon is an IVD test predicting the risk of relapse in early-stage colon cancer (CC) patients, by measuring the host immune response at the tumor site. It is a risk-assessment tool providing independent and superior prognostic value than the usual tumor risk parameters and is intended to be used as an adjunct to the TNM classification. Risk assessment is particularly important to decide when to propose an adjuvant (adj.) treatment for stage (St) II CC patients. High-risk stage II patients defined as those with poor prognostic features including T4, lymph nodes < 12, poor differentiation, VELIPI, bowel obstruction/perforation can be considered for adj. chemotherapy (CT). However, additional risk factors are needed to guide treatment decisions. Methods: A subgroup analysis was performed on the St II untreated patients (n = 1130) from the Immunoscore international validation study (Pagès The Lancet 2018). The high-risk patients (with at least 1 clinico-pathological high-risk feature) were classified in 2 categories using pre-defined cutoffs: Low Immunoscore versus High Immunoscore and their five-year time to recurrence (5Y TTR) was compared to the TTR of the low-risk patients (without any clinico-pathological high-risk feature). Results: Among the patients with high-risk features (n = 630), 438 (69.5%) had a High Immunoscore with a corresponding 5Y TTR of 87.4 (95% CI 83.9-91.0), statistically similar (logrank pv not stratified p > 0.42, wald pv stratified by center p > 0.20) to the TTR 89.1 (95% CI 86.1-92.1) observed for the 500 low-risk patients (with no clinico-pathological feature). Furthermore, 5Y TTR for these patients were statistically similar to those of St II patients with high-risk features and a High Immunoscore (n = 438), who received adj. CT (n = 162) (5Y TTR of 83.4 (95% CI 77.6-89.9). Conclusions: These data show that despite the presence of high-risk features that usually trigger adj. treatment, when not treated with CT, a significant part of these patients (69.5%) have a recurrence risk similar to the low risk patients. Therefore, the Immunoscore test could be a good tool for adj. treatment decision in St II patients.

4 Background: A growing body of evidence over the past few years suggests that the presence of immune elements within the tumor or in the tumor stroma has prognostic and predictive value in breast cancer. Immunotherapy is successfully used in many types of cancer, and modulation of immune system became standard part of the cancer patients treatment Responses in breast cancer have been more recently reported. However it has yet to be determined whether predictable biomarkers of response can be identified. Therefore in recent years, research focused on a precise description of the status and function of the immune system.The purpose of the study was to monitor immune responses in patients with breast cancer, particularly the examination of cellular (CD4, CD8, B cells) as well as humoral immunity (IgG, IgG1, IgG2, IgG3, IgG4. It appears that a factor contributing to the immunosupression may be a transforming factor-beta (TGF-beta).It is highly immunosuppressive factor that inhibits the natural and specific immunity against tumors. Methods: 50 patients included in the research project were implemented routine cancer treatment. Basic parameters (histological type and grade, the degree of expression of ER and PR, HER2, and the proliferative marker) were established. Patients were evaluated by a cancer clinical immunologist to exclude immune disorders, allergic or autoimmune origin. Anti-tumor cellular immunity (CD4, CD8, CD19) was measured by flow citometry, humoral immunity (IgG, IgG1, IgG2, IgG3, IgG4) was measured and TGF beta and VEGF production was monitored by ELISA. Results: In breast cancer patients mainly depression in cellular immunity was found. Immunglobuline plasma level was decreased as well (mainly IgG4 subtype). TGF beta as well as VEGF plasma level were increased. Most patients have shown clinical symptoms of immunodeficiency (frequent infections of respiratory or urinary tract, herpetic infections).Those patient could benefit from immunomodulation. Conclusions: The state of anticancer immunity could contribute to the selection of targeted immune therapy in breast cancer patients and to help to find optimal combination of immunotherapy. This project was supported by governmental grant AZV CR 15-28188A.

We compared the levels of transforming growth factor β1 (TGF-β1), vascular endothelial growth factor (VEGF) and other biochemical parameters in patients with type 1 diabetes mellitus with and without incipient diabetic nephropathy (iDN) and compared them with healthy control subjects. We also measured the effect of 3 and 6 months of ramipril treatment in diabetes patients with iDN. Compared with healthy controls, TGF-β1 levels were increased in both groups of diabetes patients, whereas VEGF was only elevated in patients with iDN. Ramipril did not have a significant effect on TGF-β1 or VEGF levels. We observed a significant decrease in microalbuminuria and cystatin C following ramipril treatment. Increased VEGF levels in patients with iDN suggest a role for this cytokine in the pathogenesis of diabetic nephropathy. Cystatin C would make a suitable marker for the screening and assessment of iDN, and for the evaluation of the therapeutic efficacy of drugs.

To assess the plasma TGF-beta1 level during radio(chemo)therapy and to test the predictive power of TGF-beta1 for treatment response in patients with advanced head and neck cancer. Twenty nine patients with advanced head and neck cancer were treated with curative radio(chemo)therapy. Plasma TGF-beta1 level was established at the beginning, in the middle and at the end of the therapy. The dynamics of the TGF-beta1 level was assessed separately for patients with and without chemotherapy. Treatment response was correlated to the TGF-beta1 level. Eighteen patients achieved complete remission, eight partial remission and three patients progressed. Patients treated with radiotherapy had significantly higher initial plasma TGF-beta1 level compared to radiochemotherapy patients (p=0.044). During the treatment, there was a significant decrease in patients treated with radiochemotherapy (p=0.008) but not in radiotherapy patients (p=0.34). Tumor burden did not correlate with plasma TGF-beta1 level (p=0.07). TGF-beta1 has no predictive value for treatment response (CR vs. PR and PD, p=0.12). The combination of radiotherapy and chemotherapy significantly decreases plasma TGF-beta1 level in patients with advanced head and neck cancer. Treatment response cannot be predicted using TGF-beta1.

e23038 Background: Colorectal cancer (CRC) is the second leading cause of cancer death worldwide. Immunoscore has been shown to be a very powerful prognositc indicator in patients with clinically localized colorectal cancer, with no detectable tumour spread to lymph nodes or distant organs. These patients are usually treated with only surgical removal of the tumour; however, approximately 25% of these patients will have recurrence of their disease, indicating that occult metastases were already present at the time of curative surgery. VEGF is the factor responsible for neoangiogenesis and it is being considered as a possible prognostic marker of disease progression. Transforming growth factor-beta (TGF-beta) is also neoangiogenic and a highly immunosuppressive factor. The purpose of this study was to monitor the immune response in patients with stage II and III CRC, with a focus on cellular as well as humoral immunity. TGF-beta and VEGF levels were followed. Methods: 25 patients with stage II and III colon cancer included in the research project received routine cancer teratment. Basic parameters - histological type and grade - were established at baseline. Patients were evaluated by a clinical immunooncolgist to exclude any immune disorders of allergic or autoimmune origin. TGF-beta and VEGF were measured using ELISA, and anti-tumour cellular immunity (CD4, CD8, T-reg, B cells ) were measured via flow cytometry. Results: In patients with stage II and III colorectal cancer, predominantly a depression in cellular immunity was seen. Plasma levels of immunglobulins were also reduced, particularly the IgG4 subtype. Most patients showed some clinical symptoms of immunodeficiency, such as frequent respiratory tract infections and/or herpetic infections. TGF-beta and VEGF plasma levels were increased. Conclusions: The correlation of these neoangiogenic and immunosuppressive factors, as well as the state of anticancer immunity with the clinical outcome of the patients, could help in the future as a prognostic marker and contribute to the selection of targeted immune therapy in patients with colorectal cancer. Dedication: Ggrant of Czech Ministery of Health, 15-28188A, League against cancer and PRVOUK-P-27/LF1/1

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression-based stratification. These parameters rely on tumour-cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named ‘Immunoscore’ has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune). © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Purpose: Dendritic cells (DC) are the most potent antigen-presenting cells of the immune system. We have shown that DC, defined as LN–DR+ leukocytes from the ascites of patients with ovarian or peritoneal carcinoma, have the cell surface characteristics of immature cells. Moreover, p70 interleukin-12 has not been detected in the ascites of ovarian cancer patients in vivo. In the current study, we examined the effects of in vitro treatment of DC from peripheral blood and ascites samples of patients with ovarian cancer with either cytokines or proteolytic enzymes (polyenzyme preparation). Methods: Mononuclear leukocytes from the ascites of 15 patients or peripheral blood from 9 patients with epithelial ovarian cancer were cultured with tissue culture medium containing either papain, trypsin and chymotrypsin for 5–7 days or recombinant human granulocyte-macrophage colony-stimulating factor, tumor necrosis factor α and interleukin-4 (complete medium) or tissue culture medium alone. Results: Phenotypic analysis of DC obtained on days 5–7 of the culture showed higher proportions of CD83+, CD40+ and CD80+ cells when cultured with cytokines or enzymes as compared with DC cultured with medium alone. Stimulation of allogeneic T cells was detected by the mixed leukocyte reaction (MLR) and higher concentrations of IL-12 were detected after growing these cells in the presence of cytokines or enzymes as compared to tissue culture medium alone. Conclusion: Our studies demonstrate for the first time that DC obtained from the peritoneal cavity and peripheral blood of ovarian cancer patients after culturing in the presence of a polyenzyme preparation, will undergo maturation. Further studies are warranted to determine whether polyenzyme preparations facilitate DC maturation in vivo.

Background Colorectal cancer is the second leading cause of cancer death in the world. The cause of high mortality is that almost half the cancers are detected at an advanced stage of disease. In addition to surgical and chemotherapy treatment in recent years, applied monoclonal antibody therapy may target against growth factors, and particularly against the receptors for growth factors. In clinical practice, an antibody against vascular endotelial growth factor (VEGF) - bevacizumab-is already used. VEGF is the factor responsible for neoangiogenesis and it was considered as a possible prognostic marker of disease progression. The correlation of higher levels of VEGF with the response to applied therapy, disease progress levels and a poor prognosis in patients with solid tumors is recently discussed. Transforming growth factor - beta (Transforming growth factor - beta, TGF-beta) is also neoangiogenetic and highly immunosuppressive factor. TGF-beta suppresses natural immunity against tumors. The TGF is considered as another possible prognostic marker of disease progression. The purpose of the study was to monitor immune responses in patients with colorectal cancer, particularly the examination of cellular as well as humoral immunity. TGF beta, and VEGF production was monitored. Methods

Adult respiratory distress syndrome (ARDS), a multifactorial disease with poor prognosis, is characterized by an accumulation of inflammatory cells within the airspaces of the lungs. There is evidence that alveolar macrophages (AM) are involved in the pathogenesis of this pulmonary disease. It has been demonstrated that AM synthesize heat shock proteins (HSPs) after exposure to certain stress factors. Increasing evidence suggests that HSPs could confer protection against oxidative injury, noxious molecules, and bacterial toxins. In stressed cells HSP 72 appears to be essential for survival during and after exposure to cellular injury. The aim of this study was to evaluate the magnitude of HSP 72 expression by human AM of patients with ARDS and correlate that with respiratory burst activity. Bronchoalveolar lavage was performed in six ARDS patients, 10 patients with high risk for developing ARDS, and two patients who underwent bronchoscopy for other reasons. Spontaneous ex vivo expression of HSP 72 in AM could be demonstrated by immunocytochemistry. Total RNA as well as poly(A)-rich mRNA were extracted from recovered AM and analyzed by Northern blot and slot blot using a human HSP 72-specific probe. Signals of slot blot were analyzed by densitometry and expressed as relative levels of HSP 72 mRNA of stressed (42 degrees C) HT 1080 control cells. Significantly (p < .001) higher levels of HSP 72 mRNA were measured in patients with ARDS (96.2 +/- 9.5 relative levels) in comparison to those not developing this syndrome (46.0 +/- 4.2). With regard to respiratory burst activity of AM in patients with ARDS, there was a negative correlation between HSP 72 expression and reactive oxygen species production. The AM of patients with ARDS with high relative levels of HSP 72 expression showed low respiratory burst activity. A predictive value for disease severity of high level of HSP 72 mRNA in AM in patients at risk for ARDS has to be evaluated by future studies. This demonstration of HSP 72 expression ex vivo suggests a protective role of HSP response against endo/exogenously generated stress factors in AM.

3500Background: Mounting evidence indicates an enhanced lymphocytic reaction as an informative prognostic indicator in colon cancer (CC). The IM methodology has been defined to quantify the in situ immune infiltrate. Methods: A Society for Immunotherapy of Cancer-led international consortium of 23 pathology expert centers from 17 countries was initiated to evaluate the standardized IM assay in routine clinical settings. Patient (pt) eligibility criteria were CC Stages I/II/III and no neo-adjuvant treatment. 1336 pts split into a training set (TS) and internal validation set (IVS) were quantified for IM using standardized procedure, immunohistochemistry using CD3 and CD8 antibodies, and quantification using digital pathology on whole slide section of primary CCs. Statistical analysis plan was pre-defined and all statistical analyses performed by blinded external statisticians. Primary study endpoint was time-to-recurrence (TTR); analyses were by Cox models stratified by enrolling center. Results: Median re...

Polymorphonuclear neutrophils (PMN) can be primed for enhanced release of reactive oxygen species (ROS) by exposure to cytokines and biological response modifiers. ROS are considered to possess tumoricidal activity. The polyenzyme preparation Wobenzym (WE) contains pancreatin, papain, bromelain trypsin and chymotrypsin and is used in adjuvant tumor therapy. We investigated killing of WE-exposed PMN against tumor cells and analyzed WE influence on ROS production in a chemiluminescence assay in PMN in vitro and in vivo. Depending on dose WE stimulates the cytotoxic capacity of PMN in vitro against tumor cells (50 micrograms/ml:p0.01). Exposure of PMN to Wobenzym caused a time-dependent significant (p0.02) increase in release of ROS. Similarly, oral administration of Wobenzym to healthy volunteers (n = 28) resulted in significant increases (p0.01) in ROS production, depending on dose (peak with 20 tablets) and time (peak 4 hours after Wobenzym administration). In contrast, ROS production was not elevated in the PMN of healthy volunteers receiving placebo (n = 8) or no treatment (n = 16). These findings point to an immunomodulatory capacity of WE in adjuvant tumor therapy.

Immunoscore has been shown to be a very powerful prognositc indicator in patients with clinically localized colorectal cancer, with no detectable tumour spread to lymph nodes or distant organs. These patients are usually treated with surgical removal of the tumour only. However, approximately 25% of these patients will have recurrence of their disease. No tumour-associated marker to predict the recurrence of this subgroup of patients and define who could benefit from adjuvant therapy is used in clinical routine[1] In addition to immunoscore, other parameters of the immune response are investigated – mostly cellular immunity and the production of immunosuppressive and neoangiogenic markers. Vascular endothelial growth factor (VEGF) is the factor responsible for neoangiogenesis and it is being considered as a possible prognostic marker of disease progression. Transforming growth factor-beta (TGF-beta) is also neoangiogenic and a highly immunosuppressive factor as it suppresses the body's natural immunity against tumours and is also being considered as another possible prognostic marker of disease progression.

Celebrex and radiotherapy in advanced head and neck cancer. This phase I dose-escalation study seeks to determine the phase II recommended dose of cyclooxygenase type 2 (COX-2) inhibitor in patients with locally advanced squamous cell head and neck (HN) cancer, treated with accelerated radiotherapy. Anti-vasculogenic effect of this treatment on serum vascular endothelial growth factor (VEGF) is examined. Patients were irradiated with curative intent (72Gy in 6weeks). Celecoxib was administered throughout the radiotherapy course. Serum VEGF level were tested during radiotherapy and in follow-up. Tumor specimens were stained to quantify the COX-2 expression. Thirty-two patients completed the treatment. The dose of celecoxib was escalated (200, 400 and 800mg bid, then de-escalated to 600mg bid). The acute toxicity related to the treatment in the first and second cohort did not reach grade III; in the third cohort three patients had grade III radiation toxicity and one had celecoxib-related toxicity. In the last fourth cohort the toxicity was acceptable. Significant VEGF level drop (p=0.011) was found between radiation day 1 and post-treatment visit. Significant decrease (p=0.022) of the VEGF level was shown in patients with high COX-2 expression in the tumor. Phase II recommended dose of celecoxib combined with accelerated radiotherapy in advanced HN cancer was 600mg bid. A significant decrease of the post-treatment serum VEGF level compared to the initial level was noticed only in patients with high COX-2 expression in tumors.

Therapy with oral proteolytic enzymes (OET) with combination drug products containing papain, bromelain, trypsin, and chymotrypsin has been shown to be beneficial in clinical settings such as radiotherapy-induced fibrosis, bleomycin pneumotoxicity and immunosuppression in cancer, all of which are nowadays known to be accompanied by excessive transforming growth factor-beta (TGF-beta) production. It has been demonstrated that proteolytic enzymes reduce TGF-beta levels in serum by converting the protease inhibitor alpha2 macroglobulin (alpha2M) from the "slow" form into the "fast" form, whereby the "fast" form binds and inactivates TGF-beta irreversibly. In this study we have investigated the effect of OET on the concentration of TGF-beta1 in serum of patients with rheumatoid arthritis (RA) (n = 38), osteomyelofibrosis (OMF) (n = 7) and herpes zoster (HZ) (n = 7). Seventy-eight healthy volunteers served as controls. TGF-beta1 levels in serum were assessed by enzyme-linked immunosorbent assay (ELISA). We have demonstrated that in healthy volunteers and in patients there exists a correlation between active and latent TGF-beta1 in serum (r=0.8021; P0.0001). Treatment with OET had no significant effect on TGF-beta1 concentration in healthy volunteers or patients with a normal level of TGF-beta1. In patients with elevated TGF-beta1 concentration (50 ng/ml serum), OET reduced TGF-beta1 in RA (P0.005), in OMF (P0.05) and in HZ (P0.05).These results support the concept that OET is beneficial in diseases characterized in part by TGF-beta1 overproduction.

Macrophages have diverse effects on tumor biology, including neovascularization, growth rate, and stroma formation. Tumor-associated macrophages (TAMs) represent a major subpopulation of the mononuclear leukocytes present in malignant ascites of ovarian cancer patients. TAMs appear to participate in the immunologic antitumor defense mechanism through cytotoxic activities, such as direct cellular cytotoxicity and the release of cytokines, and may represent key targets for a variety of therapeutic interventions.

Defects in the immune response correlated with poor prognosis and therefore in recent years, research focused on a precise description of the status and function of the immune system and its possible effect in cancer patients. Although more than 2/3 of treated patients respond to the endocrine therapy, most patients with metastatic breast cancer will develop resistance.. It appears that other factor contributing to the resistance may be a transforming factor-beta (TGF-beta). It is highly immunosuppressive factor that inhibits the natural and specific immunity against tumors and stimulates production of vascular endothelial growth factor /VEGF. The purpose of the study was to monitor immune responses in patients with hormone receptor positive breast cancer resistant to hormone therapy, particularly the examination of cellular (CD4, CD8, HLA-DR) as well as humoral immunity. TGF beta, and VEGF production was monitored and we analyzed the changes during hormonal treatment. Methods: 85 patients included in the research project were implemented routine cancer treatment with endocrine therapy. Basic parameters (histological type and grade, the degree of expression of ER and PR, HER2, and the proliferative marker) were established. Patients were evaluated by a cancer clinical immunologist to exclude immune disorders, allergic or autoimmune origin. TGF beta, VEGF were measured by ELISA and anti-tumor cellular immunity (CD4, CD8, antigen presenting cells ) were measured by flow cytometry. Results In patients with resistance to endocrine therapy mainly depression in cellular immunity was found. Immunglobulin plasma level was decreased as well (mainly IgG4 subtype). Most patients have shown clinical symptoms of immunodeficiency (frequent infections of respiratory or urinary tract, herpetic infections) TGF beta as well as VEGF plasma level were increased. Significant increase of autoantibodies production was found, particularly in those, involved into blood coagulation process/anti phospholipide and anti cardiolipin autoantibodies. Conclusion Correlation of these factors with resistance to hormonal therapy, and the state of anticancer immunity could help in the future with the prediction of resistance to hormonal therapy and contribute to the selection of targeted immune therapy in cancer patients. Dedication: This project was supported by grant IGA NT11168-3/2010. Citation Format: Eva Zavadova, Bohuslav Konopasek, Michal Vocka, Michaela Miskovicova, Jan Spacek, Terezie Fucikova, Lubos Petruzelka. Abnormalities in cellular and humoral immunity in breast cancer patients resistant to endocrine therapy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B9.

Although more than 2/3 of treated patients respond to the endocrine therapy, most patients with metastatic breast cancer will develop resistance. Estrogen modulate angiogenesis, in part, through the effects on VEGF. It appears that other factor contributing to the resistance may be a transforming factor-beta (TGF-beta). It is highly immunosuppressive factor that inhibits the natural and specific immunity against tumors and stimulates production of vascular endotelial growth factor /VEGF. Defects in the immune response correlated with poor prognosis and therefore in recent years, research focused on a precise description of the status and function of the immune system and its possible effect in cancer patients. The purpose of the study was to monitor immune responses in patients with hormone receptor positive breast cancer resistant to hormone therapy, particularly the examination of cellular (CD4, CD8, HLA-DR) as well as humoral immunity. TGF beta, and VEGF production was monitored and we analyzed the changes during hormonal treatment. Methods: 60 patients included in the research project were implemented routine cancer teratment with hormonal therapy. Basic parameters (histological type and grade, the degree of expression of ER and PR, HER2, and the proliferative marker) were established. Patients were evaluated by a cancer clinical immunologist to exclude immune disorders, allergic or autoimmune origin. TGF beta, VEGF were mesured by ELISA and anti-tumor cellular immunity (CD4, CD8, antigen presenting cells) will be measured by flow cytometry. Results In patients with resistance to hormone therapy mainly depression in cellular immunity was found.Immunglobulin plasma level was dicreased as well (mainly IgG4 subtype). Most patients have shown clinical symptoms of immunodeficiency (frequent infections of respiratory or urinary tract, herpetic infections) TGF beta as well as VEGF plasma level were increased. Significant increase of autoantibodies production was found, particulary in those, involved into blood coagulacion process/anti phospholipide and anti cardiolipin autoantibodies. Conclusion Correlation of these factors with resistance to hormonal therapy, and the state of anticancer immunity could help in the future with the the prediction of resistance to hotmonal therapy and contribute to the selection of targeted immune therapy in cancer patients. Dedication: This project was supported by grant IGA NT11168-3/2010 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5382. doi:1538-7445.AM2012-5382

Even with high therapeutic efficacy of first-line hormonal treatment, most patients with metastatic breast cancer will develop resistance. It appears that a factor contributing to the resistance may be a transforming growth factor-beta (TGF-beta). It is highly immunosuppressive factor that inhibits the natural and specific immunity against tumors and stimulates production of vascular endotelial growth factor (VEGF). Defects in the immune response correlated with poor prognosis and therefore in recent years, research focused on a precise description of the status and function of the immune system and its possible effect in cancer patients. The purpose of the study was to monitor immune responses in patients with hormone receptor-positive breast cancer resistant to hormone therapy, particularly the examination of cellular (CD4, CD8, HLA-DR) as well as humoral immunity. TGF-beta and VEGF production was monitored and we analyzed the changes during hormonal treatment. Methods: Patients included in the research project were implemented routine cancer treatment with hormonal therapy. Basic parameters (histological type and grade, the degree of expression of ER and PR, HER2, and the proliferative marker) were established. Patients were evaluated by a cancer clinical immunologist to exclude immune disorders, allergic or autoimmune origin. TGF-beta and VEGF were mesured by ELISA and antitumor cellular immunity (CD4, CD8, antigen presenting cells) will be measured by flow cytometry. Results: In patients with resistance to hormone therapy mainly depression in cellular immunity was found. Immunglobulin plasma level was decreased as well (mainly IgG4 subtype). Most patients have shown clinical symptoms of immunodeficiency (frequent infections of respiratory or urinary tract, herpetic infections) TGF-beta as well as VEGF plasma level were increased. Significant increase of autoantibodies production was found, particulary in those, involved into blood coagulacion process/antiphospholipide and anticardiolipin autoantibodies. Conclusion: Correlation of these factors with resistance to hormonal therapy, and the state of anticancer immunity could help in the future with the the prediction of resistance to hormonal therapy and contribute to the selection of targeted immune therapy in cancer patients. Acknowledgement: This project was supported by grant IGA NT11168-3/2010. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C68.

The role of tumor-associated macrophages (TAM) as potential effector cells for eradicating malignant cells is not yet entirely clarified. In the present study TAM were isolated from malignant effusions of ovarian, breast and lung cancer patients by gradient separation and phenotypically and functionally characterised by the following parameters: surface epitopes (moAb 27E10, 25F9), respiratory burst activity, cytotoxicity and cytokine production (TNF-α, TGF-β,) measured in culture supernatants by bioassay, ELISA/RIA. Additionally mRNA of these cytokines was detected in TAM by in situ hybridisation. Incubation of TAM with rh-GM-CSF and rh-IFN-γ resulted in an augmentation of cytotoxicity. Furthermore, cell-bound TNF-α was detectable by immunohistochemistry and correlated with mRNA (in situ hybridisation). In contrast, GM-CSF, as well as IFN-γ, reduced the production of TGF-β by TAM, verified by ELISA assay as well as by in situ hybridisation. Our studies show that TAM obtained from malignant effusions of cancer patients can be stimulated by GM-CSF and IFN-γ for cytotoxicity and cytokine production. Though, TGF-β release was reduced. Whether this observation is of therapeutical relevance has to be determined by further studies.

The role of tumor - associated macrophages (TAM) as potential effector cells for eradicating malignant cells is not yet entirely clarified. In the present study TAM were isolated from malignant effusions by gradient separation and phenotypically and functionally characterised by the following parameters: surface epitopes (moAb 27EIO, 2SF9), respiratory burst activity, cytotoxicity and cytokine production (TNF-α., TGF-β, IL-6) measured in culture supernatants by bioassay, ELISA/RIA. Additionally mRNAs of these cytokines was detected in TAM by in situ hybridisation and RT-PCR. Incubation of TAM with rh-GM-CSF and rh-IFN-y resulted in an augmentation of cytotoxicity. Furthermore, cell-bound as well as released TNF- a increased significantly following incubation with GM-CSF. In contrast, OM-CSP, as well as IFN-γ reduced the production of TGF-β by TAM, as verified by ELISA assay as well as by in situ hybridisation and RT-PCR. Our studies show that TAM obtained from malignant effusions of cancer patients can be stimulated by GM-CSF and IFN-γ for cytotoxicity and cytokine production. TGF-β release was reduced. Whether this observation is of therapeutical relevance has to be determined by further studies.

The enzyme inducible Nitric oxide synthase (iNOS) is known to catalyse the release of Nitric oxide from L-arginin. Expression of iNOS is readily induced in macrophases of various mammals, but the presence of this enzyme in human monocytes and macrophages has not been entirely clarified. In this study we tested wbether iNOS mRNA and protein can be detected in human tumor-assiociated macrophages (TAMs). TAMs were isolated from malignant effusions of cancer patients (n = 4) by gradient separation. The reverse transcription polymerase chain reaction (oligonucleotide primers derived from human hepatocyte iNOS mRNA) and immuncytochemistry (anti-macNOS) were used. Stimulalion of TAMs was achieved by incubating the cells with rh-GM-CSF and rh. IFN-g and a combination of these cytokines. iNOS specific mRNA as well as iNOS protein could be detected in TAMs. The mRNA signal was induced in 10/14 samples and increased in 4/14 following stimulation with the cytokines GM-CSF and IFN-g. This stimulalion correlated with enhancement of TNF-a production and ceIl mediated cytotoxicity of TAMs against tumor targets. Our finding that iNOS is detectable and can be stimulated in human TAMs supports the notion that iNOS/NO are important molecules in antitumoral effector mechanisms.

Expression of both P-glycoprotein (P-gp) and multidrug-resistance-related protein (MRP) confers resistance to antineoplastic drugs (multidrug-resistance; MDR). Analogous to the MDR-phenomenon, tumor cells can also develop resistance to toxic impacts of TNFα (cytokine resistance). We investigated the relationship between these resistance types using either P-gp or MRP overexpressing cell lines (CCRF-CEM, HL-60, K562, KB, HeLa, LoVo, CHO, GLC4), selected by various drugs. Our data (MTT-assay) show that the drug-selection process can have an impact on rTNFα-responsiveness leading to both resistance and sensitivity even within the same cell model. A change in TNFα responsiveness was 1) not dependent on P-gp or MRP-expression, 2) not defined by the cell type but 3) more likely a consequence of the selection process. By means of the CCRF-CEM leukaemia model, including both rTNFα-resistant and-sensitive MDR-sublines, we studied whether the modulation of parameters causing rTNFα-resistance (p55, endogenous TNFα, MnSOD, HSP-70, HSP-27) or apoptosis-resistance (bc1-2, bcl-xl, c-myc) parallels rTNFα-responsivencss (RT-PCR, Western blot). Additionally, sensitivity to other apoptosis-inducers (H2O2, UV-light) was investigated. Results give evidence for a role of endogenous TNFα and MnSOD in rTNFα-responsiveness. rTNFα-resistance in CCRF-CEM cells implies no general “apoptosis resistance phenotype”. We conclude that MDR phenotype and rTNFα-responsiveness are not directly linked. The rTNFα-hypersensitivity of some MDR cell lines implicate new therapeutic strategies against drug-resistant tumors.

Conventional chemotherapy with drug combinations is still the preferred treatment for multiple myeloma. Immuno-chemotherapy with Wobe-Mugos (a polyenzyme preparation) and MOCCA/VMPC of MM patients results in a prolongation ofclinical remission and a significant prolongation of survival time in comparison to MM patients who received chemotherapy only (Sakalova and Stauder et al. in prep.). In the present study we measured the serum levels of β2 microglobulin (β2M) and of soluble tumor necrosis factor receptors (sTNF-R: p55 and p75) in serum of 169 patients to determine their value as a monitor of diseases status in untreated, chemotherapy treated and immunochemotherapy treated MM patients. Serum levels of p55 and p75 as well as β2M were elevated in parallel with the clinical stage in untreated patients. sTNF-R and β2 M correlate (β2m/p55: 0.7162 P Immuno-chemotherapy significantly reduced serum levels of p55, p75 and β2M in stages I and II in comparison to chemotherapy alone (p55: 2970 ± 1095 P In stage III the serum concentrations of p55 and p75 were reduced by chemotherapy (p55: P Our results suggest that p55 and p75 concentrations in serum of MM patients correlate well with β2M and may be potential markers for both disease progression and response to therapy.

Polymorphonuclear neutrophils (PMN) can be primed for enhanced release of reactive oxygen species (ROS) by exposure to cytokines and biological response modifiers. ROS are considered to possess tumorici-dal activity. The polyenzyme preparation Wobenzym R (WE) contains pancreatin, papain, bromelain trypsin and chymotrypsin and is used in adjuvant tumor therapy. We investigated killing of WE exposed PMN against tumor cells and analyzed WE influence on ROS production in a chemiluminescence assay in PMN in vitro and in vivo . Depending on dose WE stimulates the cytotoxic capacity of PMN in vitro against tumor cells (50 μg/ml: P P P