[Immunoscore and Its Predictive Value for Colorectal Cancer]. / Immunoscore a jeho prediktivní hodnota u kolorektálního karcinomu.

Recent studies suggest that immune  classification (immune-score) in cancer patients has a prognostic value in some cases that seems to be superior to the AJCC/ UICC TNM  classification. The clinical outcome can vary significantly among patients with a particular diagnosis within the same TNM stage. Immunoscore methodology quantifies and detects different types of immune cells in tumor tissue, and also determines the density of their infiltration and localization at the tumor site. Currently within an international collaboration of 23 centers in 17 countries (including our department), immunoscore is being evaluated in more than 7,000 colorectal cancer patients in terms of the tumor microenvironment, focusing on the presence of immune cells both in the tumor tissue and the tumor invasive margin. Immunoscore results are assessed in correlation with: 1. patients response to the treatment, 2. rate of progression, disease prognosis and other immune parameters. It appears that the TNM classification and tumor invasiveness is statistically dependent on the immune response of the patient (there is an inverse correlation between the density of the infiltration of CD8⁺, CD3⁺ lymphocytes and the tumor stage). High densities of T-lymphocytes (CD8⁺, CD3⁺) both in the core and the invasive margin of the primary tumor are associated with longer term asymptomatic survival, overall survival, lower risk of relapse and reduced likelihood of metastases. The project of the international collaboration aims to introduce immunoscore in routine diagnostics.

Cellular and humoral immunodeficiency in breast cancer patients resistant to hormone therapy.

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.

Preoperative transforming growth factor-beta 1 (TGF-beta 1) plasma levels in operable breast cancer patients.

OBJECTIVES: The aim of this project was to search for new risk prognostic markers in the early stage of breast cancer. We tested preoperative plasma transforming growth factor - beta 1 (TGF- beta 1) levels in patients with operable breast cancer. Correlation with traditional prognostic markers and with positivity/negativity sentinel lymph node was evaluated. MATERIALS AND METHODS: Between 2003 and 2005, 36 patients with operable breast cancer (T1-2, N0-1, M0) with positive or negative sentinel lymph nodes were evaluated for their plasma TGF-beta 1. Twenty-seven healthy individuals (9 premenopausal and 18 postmenopausal) served as controls. Patients were evaluated for the traditional prognostic markers including tumor characteristics, positivity and negativity of sentinel lymph node, TNM, tumor grade, expression of tumor markers CA 15-3 and CEA, hormonal status (pre- or postmenopausal patients, estrogen and progesteron receptor expression), ERB and p53 expression. Predictive value of TGF-beta 1 level and correlation with either of the assessed parameters was tested by one way ANOVA analysis. RESULTS: Measurements of preoperative plasma TGF-beta 1 levels in patients with operable breast cancer were significantly higher compared with healthy individuals (median 15293 and 3983 pg/ml p < 0.0001). TGF-beta 1 level in plasma of patients with a positive sentinel lymph node was significantly higher than in patients with negative sentinel lymph nodes (high vs low, median 18,9 and 14,5 ng/ml, respectively, p = 0.05). CONCLUSION: The determination of TGF-beta 1 status might help to identify a high-risk population early in tumor progression, for which a more appropriate therapy should be established. In the node-negative population, the up-regulation of TGF-beta 1 might constitute an early event that promotes further progression of breast tumors.

[Transforming growth factor-beta 1 as a marker in patients with operable breast cancer]. / Transformacní rustový faktor--beta 1 jako marker u pacientek s operabilním karcinomem prsu.

UNLABELLED: OBJECTIVE AND DESIGN OF STUDY: Determination of TGF-beta 1 levels in plasma of patients with operable breast cancer. The correlation of TGF levels with the stage of disease and other prognostic markers. Prospective study. METHODS: There were 36 patients fulfilling the entrance criteria included in the study. The blood samples were taken to set the plasma levels of TGF-beta before the operation, with no adjuvant therapy. 27 age matched healthy volunteers served as controls. The study was approved by ethical board and patients signed informed consent regarding blood sampling and results presentation. Differences between groups were determined using the Mann-Whitney U-test. RESULTS: We proved that TGF-beta 1 levels are elevated in patients with operable breast cancer. Moreover, TGF-beta in plasma of patients with positive sentinel lymph node was significantly higher as compared to patients with negative sentinel node. Most important is the fact that patients involved in our study were in very early stages of disease. CONCLUSION: TGF-beta 1 is marker correlating with breast cancer disease risk factors, especially with positive sentinel lymph node. We conclude that TGF is one of the early markers which may help define the risk of disease progression already before the operation.

The effect of ramipril therapy on cytokines and parameters of incipient diabetic nephropathy in patients with type 1 diabetes mellitus.

We compared the levels of transforming growth factor beta1 (TGF-beta1), 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-beta1 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-beta1 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.

Post-treatment plasma transforming growth factor beta 1 (TGF-beta1) level predicts for late morbidity in patients with advanced head and neck cancer.

Between 2001 and 2002, 29 patients with advanced inoperable squamous head and neck cancer treated with radiotherapy with or without simultaneous chemotherapy were evaluated for their plasma TGF-beta1 levels prior to the treatment, in the middle of the radiotherapy course and at the end of the treatment. Patients were assessed for treatment response and late morbidity. Predictive value of TGF-beta1 level on either of the assessed parameters was tested. From 29 eligible patients (pts), 18 achieved complete response, 8 partial response and three pts progressed primarily. After a median follow-up of 16 months we recorded 16 cases of grade >1 late morbidity. We found that post-treatment elevated plasma TGF-beta1 level predicts late morbidity grade >1 (p=0.05) rather than pre-treatment level (p=0.062). Neither pre-treatment nor post-treatment plasma TGF-beta1 level has a predictive value to the treatment response (CR vs. no CR, p=0.125 and 0.252, respectively). The post-treatment plasma TGF-beta 1 level can predict late morbidity grade >1 in advanced head and neck cancer treated with radio(chemo)therapy. This could make a basis for dose escalation in selected patients.

Maturation of dendritic cells from ovarian cancer patients.

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 alpha 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.

Oral therapy with proteolytic enzymes decreases excessive TGF-beta levels in human blood.

UNLABELLED: 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; P<0.0001). 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 (P < 0.005), in OMF (P < 0.05) and in HZ (P < 0.05). CONCLUSION: These results support the concept that OET is beneficial in diseases characterized in part by TGF-beta1 overproduction.

The role of macrophages in antitumor defense of patients with ovarian cancer.

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.

Polyenzyme preparation Wobe-Mugos inhibits growth of solid tumors and development of experimental metastases in mice.

Long-term rectal administration of enzyme mixture containing papain, trypsin and chymotrypsin in the same ratio as the preparation Wobe-Mugos E (Mucos Pharma, Germany) was evaluated for their antitumor effects in C57Bl6 inbred mice inoculated with Bl6 melanoma cells. 30% of animals in the test group (3 pcs) have been cured of cancer. In the rest of animals (70%) the survival time was prolonged by 58.3% compared to the control group (from average survival time of 24 days in control group to 38 days in the test group). Based on histological and immunohistochemical evaluation a faster process of metastasizing was found in control group than in the group treated with the polyenzyme preparation. In the case of melanoma Bl6 an antimetastatic effect of the preparation was thus proved.

Alveolar macrophages of patients with adult respiratory distress syndrome express high levels of heat shock protein 72 mRNA.

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.

Stimulation of reactive oxygen species production and cytotoxicity in human neutrophils in vitro and after oral administration of a polyenzyme preparation.

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:p < 0.01). Exposure of PMN to Wobenzym caused a time-dependent significant (p < 0.02) increase in release of ROS. Similarly, oral administration of Wobenzym to healthy volunteers (n = 28) resulted in significant increases (p < 0.01) 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.