Tumor-agnostic transcriptome-based classifier identifies spatial infiltration patterns of CD8+T cells in the tumor microenvironment and predicts clinical outcome in early-phase and late-phase clinical trials.

BACKGROUND: The immune status of a patient's tumor microenvironment (TME) may guide therapeutic interventions with cancer immunotherapy and help identify potential resistance mechanisms. Currently, patients' immune status is mostly classified based on CD8+tumor-infiltrating lymphocytes. An unmet need exists for comparable and reliable precision immunophenotyping tools that would facilitate clinical treatment-relevant decision-making and the understanding of how to overcome resistance mechanisms. METHODS: We systematically analyzed the CD8 immunophenotype of 2023 patients from 14 phase I-III clinical trials using immunohistochemistry (IHC) and additionally profiled gene expression by RNA-sequencing (RNA-seq). CD8 immunophenotypes were classified by pathologists into CD8-desert, CD8-excluded or CD8-inflamed tumors using CD8 IHC staining in epithelial and stromal areas of the tumor. Using regularized logistic regression, we developed an RNA-seq-based classifier as a surrogate to the IHC-based spatial classification of CD8+tumor-infiltrating lymphocytes in the TME. RESULTS: The CD8 immunophenotype and associated gene expression patterns varied across indications as well as across primary and metastatic lesions. Melanoma and kidney cancers were among the strongest inflamed indications, while CD8-desert phenotypes were most abundant in liver metastases across all tumor types. A good correspondence between the transcriptome and the IHC-based evaluation enabled us to develop a 92-gene classifier that accurately predicted the IHC-based CD8 immunophenotype in primary and metastatic samples (area under the curve inflamed=0.846; excluded=0.712; desert=0.855). The newly developed classifier was prognostic in The Cancer Genome Atlas (TCGA) data and predictive in lung cancer: patients with predicted CD8-inflamed tumors showed prolonged overall survival (OS) versus patients with CD8-desert tumors (HR 0.88; 95% CI 0.80 to 0.97) across TCGA, and longer OS on immune checkpoint inhibitor administration (phase III OAK study) in non-small-cell lung cancer (HR 0.75; 95% CI 0.58 to 0.97). CONCLUSIONS: We provide a new precision immunophenotyping tool based on gene expression that reflects the spatial infiltration patterns of CD8+ lymphocytes in tumors. The classifier enables multiplex analyses and is easy to apply for retrospective, reverse translation approaches as well as for prospective patient enrichment to optimize the response to cancer immunotherapy.

Immune contexture of paediatric cancers.

BACKGROUND: The clinical development of immune checkpoint-targeted immunotherapies has been disappointing so far in paediatric solid tumours. However, as opposed to adults, very little is known about the immune contexture of paediatric malignancies. METHODS: We investigated by gene expression and immunohistochemistry (IHC) the immune microenvironment of five major paediatric cancers: Ewing sarcoma (ES), osteosarcoma (OS), rhabdomyosarcoma (RMS), medulloblastoma (MB) and neuroblastoma (NB; 20 cases each; n = 100 samples total), and correlated them with overall survival. RESULTS: NB and RMS tumours had high immune cell gene expression values and high T-cell counts but were low for antigen processing cell (APC) genes. OS and ES tumours showed low levels of T-cells but the highest levels of APC genes. OS had the highest levels of macrophages (CSF1R, CD163 and CD68), whereas ES had the lowest. MB appeared as immune deserts. Tregs (FOXP3 staining) were higher in both RMS and OS. Most tumours scored negative for PD-L1 in tumour and immune cells, with only 11 of 100 samples positive for PD-L1 staining. PD-L1 and OX40 levels were generally low across all five indications. Interestingly, NB had comparable levels of CD8 by IHC and by gene expression to adult tumours. However, by gene expression, these tumours were low for T-cell cytotoxic molecules GZMB, GZMA and PRF1. Surprisingly, the lower the level of tumour infiltrative CD8 T-cells, the better the prognosis was in NB, RMS and ES. Gene expression analyses showed that MYCN-amplified NB have higher amounts of immune suppressive cells such as macrophages, myeloid-derived suppressor cells and Tregs, whereas the non-MYCN-amplified tumours were more infiltrated and had higher expression levels of Teff. CONCLUSIONS: Our results describe the quality and quantity of immune cells across five major paediatric cancers and provide some key features differentiating these tumours from adult tumour types. These findings explain why anti-PD(L)1 might not have had single agent success in paediatric cancers. These results provides the rationale for the development of biologically stratified and personalised immunotherapy strategies in children with relapsing/refractory cancers.

Anti-CSF-1R emactuzumab in combination with anti-PD-L1 atezolizumab in advanced solid tumor patients naïve or experienced for immune checkpoint blockade.

BACKGROUND: This phase 1b study (NCT02323191) evaluated the safety, antitumor activity, pharmacokinetics, and pharmacodynamics of colony-stimulating factor-1 receptor-blocking monoclonal antibody (mAb) emactuzumab in combination with the programmed cell death-1 ligand (PD-L1)-blocking mAb atezolizumab in patients with advanced solid tumors naïve or experienced for immune checkpoint blockers (ICBs). METHODS: Emactuzumab (500-1350 mg flat) and atezolizumab (1200 mg flat) were administered intravenously every 3 weeks. Dose escalation of emactuzumab was conducted using the 3+3 design up to the maximum tolerated dose (MTD) or optimal biological dose (OBD). Extension cohorts to evaluate pharmacodynamics and clinical activity were conducted in metastatic ICB-naive urothelial bladder cancer (UBC) and ICB-pretreated melanoma (MEL), non-small cell lung cancer (NSCLC) and UBC patients. RESULTS: Overall, 221 patients were treated. No MTD was reached and the OBD was determined at 1000 mg of emactuzumab in combination with 1200 mg of atezolizumab. Grade ≥3 treatment-related adverse events occurred in 25 (11.3%) patients of which fatigue and rash were the most common (14 patients (6.3%) each). The confirmed objective response rate (ORR) was 9.8% for ICB-naïve UBC, 12.5% for ICB-experienced NSCLC, 8.3% for ICB-experienced UBC and 5.6% for ICB-experienced MEL patients, respectively. Tumor biopsy analyses demonstrated increased activated CD8 +tumor infiltrating T lymphocytes (TILs) associated with clinical benefit in ICB-naïve UBC patients and less tumor-associated macrophage (TAM) reduction in ICB-experienced compared with ICB-naïve patients. CONCLUSION: Emactuzumab in combination with atezolizumab demonstrated a manageable safety profile with increased fatigue and skin rash over usual atezolizumab monotherapy. A considerable ORR was particularly seen in ICB-experienced NSCLC patients. Increase ofCD8 +TILs under therapy appeared to be associated with persistence of a TAM subpopulation.

T-Cell Heterogeneity in Baseline Tumor Samples: Implications for Early Clinical Trial Design and Analysis.

Background: In early stage clinical trials, changes to levels of tumor infiltrating lymphocytes (TILs) in the tumor microenvironment (TME) are critical biomarkers of the mechanism of action of novel immunotherapies. However, baseline heterogeneity of tumor samples, both between and within patients, and the resultant impact on the validity of clinical trial data is not well defined. Here we identify and quantify the impact of baseline variables on the heterogeneity of FoxP3+ and proliferating CD8+ T-cells levels (MKi67+CD8A+) in the TME both between and within patients for the purpose of informing clinical trial design and analysis. Methods: We compared levels of FoxP3+ and MKi67+CD8+ cell densities (counts/mm2) from >1000 baseline tumor samples from clinical trials and commercially available sources. Using multivariate hierarchical regression techniques, we investigated whether inter-person heterogeneity of activated or regulatory T-cells could be attributed to baseline characteristics including demographics, indication, lesion type, tissue of excision, biopsy method, prior cancer treatment, and tissue type i.e., "fresh" or "archival" status. We also sought to characterize within-patient heterogeneity by lesion type and tissue type. Results: Prior cancer treatment with hormone therapy or chemotherapy that induces immunogenic cell death may alter the TME. Archival tissue is an unreliable substitute for fresh tissue for determining baseline TIL levels. Baseline and on treatment biopsies should be matched by lesion type to avoid bias.

Computational Tumor Infiltration Phenotypes Enable the Spatial and Genomic Analysis of Immune Infiltration in Colorectal Cancer.

Cancer immunotherapy has led to significant therapeutic progress in the treatment of metastatic and formerly untreatable tumors. However, drug response rates are variable and often only a subgroup of patients will show durable response to a treatment. Biomarkers that help to select those patients that will benefit the most from immunotherapy are thus of crucial importance. Here, we aim to identify such biomarkers by investigating the tumor microenvironment, i.e., the interplay between different cell types like immune cells, stromal cells and malignant cells within the tumor and developed a computational method that determines spatial tumor infiltration phenotypes. Our method is based on spatial point pattern analysis of immunohistochemically stained colorectal cancer tumor tissue and accounts for the intra-tumor heterogeneity of immune infiltration. We show that, compared to base-line models, tumor infiltration phenotypes provide significant additional support for the prediction of established biomarkers in a colorectal cancer patient cohort (n = 80). Integration of tumor infiltration phenotypes with genetic and genomic data from the same patients furthermore revealed significant associations between spatial infiltration patterns and common mutations in colorectal cancer and gene expression signatures. Based on these associations, we computed novel gene signatures that allow one to predict spatial tumor infiltration patterns from gene expression data only and validated this approach in a separate dataset from the Cancer Genome Atlas.

Analysis of Spatial Organization of Suppressive Myeloid Cells and Effector T Cells in Colorectal Cancer-A Potential Tool for Discovering Prognostic Biomarkers in Clinical Research.

The development and progression of solid tumors such as colorectal cancer (CRC) are known to be affected by the immune system and cell types such as T cells, natural killer (NK) cells, and natural killer T (NKT) cells are emerging as interesting targets for immunotherapy and clinical biomarker research. In addition, CD3+ and CD8+ T cell distribution in tumors has shown positive prognostic value in stage I-III CRC. Recent developments in digital computational pathology support not only classical cell density based tumor characterization, but also a more comprehensive analysis of the spatial cell organization in the tumor immune microenvironment (TiME). Leveraging that methodology in the current study, we tried to address the question of how the distribution of myeloid derived suppressor cells in TiME of primary CRC affects the function and location of cytotoxic T cells. We applied multicolored immunohistochemistry to identify monocytic (CD11b+CD14+) and granulocytic (CD11b+CD15+) myeloid cell populations together with proliferating and non-proliferating cytotoxic T cells (CD8+Ki67+/-). Through automated object detection and image registration using HALO software (IndicaLabs), we applied dedicated spatial statistics to measure the extent of overlap between the areas occupied by myeloid and T cells. With this approach, we observed distinct spatial organizational patterns of immune cells in tumors obtained from 74 treatment-naive CRC patients. Detailed analysis of inter-cell distances and myeloid-T cell spatial overlap combined with integrated gene expression data allowed to stratify patients irrespective of their mismatch repair (MMR) status or consensus molecular subgroups (CMS) classification. In addition, generation of cell distance-derived gene signatures and their mapping to the TCGA data set revealed associations between spatial immune cell distribution in TiME and certain subsets of CD8+ and CD4+ T cells. The presented study sheds a new light on myeloid and T cell interactions in TiME in CRC patients. Our results show that CRC tumors present distinct distribution patterns of not only T effector cells but also tumor resident myeloid cells, thus stressing the necessity of more comprehensive characterization of TiME in order to better predict cancer prognosis. This research emphasizes the importance of a multimodal approach by combining computational pathology with its detailed spatial statistics and gene expression profiling. Finally, our study presents a novel approach to cancer patients' characterization that can potentially be used to develop new immunotherapy strategies, not based on classical biomarkers related to CRC biology.

Phase Ib study of anti-CSF-1R antibody emactuzumab in combination with CD40 agonist selicrelumab in advanced solid tumor patients.

BACKGROUND: This phase Ib study evaluated the safety, clinical activity, pharmacokinetics, and pharmacodynamics (PD) of emactuzumab (anti-colony stimulating factor 1 receptor monoclonal antibody (mAb)) in combination with selicrelumab (agonistic cluster of differentiation 40 mAb) in patients with advanced solid tumors. METHODS: Both emactuzumab and selicrelumab were administered intravenously every 3 weeks and doses were concomitantly escalated (emactuzumab: 500 to 1000 mg flat; selicrelumab: 2 to 16 mg flat). Dose escalation was conducted using the product of independent beta probabilities dose-escalation design. PD analyzes were performed on peripheral blood samples and tumor/skin biopsies at baseline and on treatment. Clinical activity was evaluated using investigator-based and Response Evaluation Criteria In Solid Tumors V.1.1-based tumor assessments. RESULTS: Three dose-limiting toxicities (all infusion-related reactions (IRRs)) were observed at 8, 12 and 16 mg of selicrelumab together with 1000 mg of emactuzumab. The maximum tolerated dose was not reached at the predefined top doses of emactuzumab (1000 mg) and selicrelumab (16 mg). The most common adverse events were IRRs (75.7%), fatigue (54.1%), facial edema (37.8%), and increase in aspartate aminotransferase and creatinine phosphokinase (35.1% both). PD analyzes demonstrated an increase of Ki67+-activated CD8+ T cells accompanied by a decrease of B cells and the reduction of CD14Dim CD16bright monocytes in peripheral blood. The best objective clinical response was stable disease in 40.5% of patients. CONCLUSION: Emactuzumab in combination with selicrelumab demonstrated a manageable safety profile and evidence of PD activity but did not translate into objective clinical responses. TRIALREGISTRATION NUMBER: NCT02760797.

The Society for Immunotherapy of Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation.

OBJECTIVES: The interaction between the immune system and tumor cells is an important feature for the prognosis and treatment of cancer. Multiplex immunohistochemistry (mIHC) and multiplex immunofluorescence (mIF) analyses are emerging technologies that can be used to help quantify immune cell subsets, their functional state, and their spatial arrangement within the tumor microenvironment. METHODS: The Society for Immunotherapy of Cancer (SITC) convened a task force of pathologists and laboratory leaders from academic centers as well as experts from pharmaceutical and diagnostic companies to develop best practice guidelines for the optimization and validation of mIHC/mIF assays across platforms. RESULTS: Representative outputs and the advantages and disadvantages of mIHC/mIF approaches, such as multiplexed chromogenic IHC, multiplexed immunohistochemical consecutive staining on single slide, mIF (including multispectral approaches), tissue-based mass spectrometry, and digital spatial profiling are discussed. CONCLUSIONS: mIHC/mIF technologies are becoming standard tools for biomarker studies and are likely to enter routine clinical practice in the near future. Careful assay optimization and validation will help ensure outputs are robust and comparable across laboratories as well as potentially across mIHC/mIF platforms. Quantitative image analysis of mIHC/mIF output and data management considerations will be addressed in a complementary manuscript from this task force.

Data-Driven Discovery of Immune Contexture Biomarkers.

Background: Features characterizing the immune contexture (IC) in the tumor microenvironment can be prognostic and predictive biomarkers. Identifying novel biomarkers can be challenging due to complex interactions between immune and tumor cells and the abundance of possible features. Methods: We describe an approach for the data-driven identification of IC biomarkers. For this purpose, we provide mathematical definitions of different feature classes, based on cell densities, cell-to-cell distances, and spatial heterogeneity thereof. Candidate biomarkers are ranked according to their potential for the predictive stratification of patients. Results: We evaluated the approach on a dataset of colorectal cancer patients with variable amounts of microsatellite instability. The most promising features that can be explored as biomarkers were based on cell-to-cell distances and spatial heterogeneity. Both the tumor and non-tumor compartments yielded features that were potentially predictive for therapy response and point in direction of further exploration. Conclusion: The data-driven approach simplifies the identification of promising IC biomarker candidates. Researchers can take guidance from the described approach to accelerate their biomarker research.

TRIM28 multi-domain protein regulates cancer stem cell population in breast tumor development.

The expression of Tripartite motif-containing protein 28 (TRIM28)/Krüppel-associated box (KRAB)-associated protein 1 (KAP1), is elevated in at least 14 tumor types, including solid and hematopoietic tumors. High level of TRIM28 is associated with triple-negative subtype of breast cancer (TNBC), which shows higher aggressiveness and lower survival rates. Interestingly, TRIM28 is essential for maintaining the pluripotent phenotype in embryonic stem cells. Following on that finding, we evaluated the role of TRIM28 protein in the regulation of breast cancer stem cells (CSC) populations and tumorigenesis in vitro and in vivo. Downregulation of TRIM28 expression in xenografts led to deceased expression of pluripotency and mesenchymal markers, as well as inhibition of signaling pathways involved in the complex mechanism of CSC maintenance. Moreover, TRIM28 depletion reduced the ability of cancer cells to induce tumor growth when subcutaneously injected in limiting dilutions. Our data demonstrate that the downregulation of TRIM28 gene expression reduced the ability of CSCs to self-renew that resulted in significant reduction of tumor growth. Loss of function of TRIM28 leads to dysregulation of cell cycle, cellular response to stress, cancer cell metabolism, and inhibition of oxidative phosphorylation. All these mechanisms directly regulate maintenance of CSC population. Our original results revealed the role of the TRIM28 in regulating the CSC population in breast cancer. These findings may pave the way to novel and more effective therapies targeting cancer stem cells in breast tumors.

The molecular basis of breast cancer pathological phenotypes.

The histopathological evaluation of morphological features in breast tumours provides prognostic information to guide therapy. Adjunct molecular analyses provide further diagnostic, prognostic and predictive information. However, there is limited knowledge of the molecular basis of morphological phenotypes in invasive breast cancer. This study integrated genomic, transcriptomic and protein data to provide a comprehensive molecular profiling of morphological features in breast cancer. Fifteen pathologists assessed 850 invasive breast cancer cases from The Cancer Genome Atlas (TCGA). Morphological features were significantly associated with genomic alteration, DNA methylation subtype, PAM50 and microRNA subtypes, proliferation scores, gene expression and/or reverse-phase protein assay subtype. Marked nuclear pleomorphism, necrosis, inflammation and a high mitotic count were associated with the basal-like subtype, and had a similar molecular basis. Omics-based signatures were constructed to predict morphological features. The association of morphology transcriptome signatures with overall survival in oestrogen receptor (ER)-positive and ER-negative breast cancer was first assessed by use of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset; signatures that remained prognostic in the METABRIC multivariate analysis were further evaluated in five additional datasets. The transcriptomic signature of poorly differentiated epithelial tubules was prognostic in ER-positive breast cancer. No signature was prognostic in ER-negative breast cancer. This study provided new insights into the molecular basis of breast cancer morphological phenotypes. The integration of morphological with molecular data has the potential to refine breast cancer classification, predict response to therapy, enhance our understanding of breast cancer biology, and improve clinical management. This work is publicly accessible at www.dx.ai/tcga_breast. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Atezolizumab in combination with bevacizumab enhances antigen-specific T-cell migration in metastatic renal cell carcinoma.

Anti-tumour immune activation by checkpoint inhibitors leads to durable responses in a variety of cancers, but combination approaches are required to extend this benefit beyond a subset of patients. In preclinical models tumour-derived VEGF limits immune cell activity while anti-VEGF augments intra-tumoral T-cell infiltration, potentially through vascular normalization and endothelial cell activation. This study investigates how VEGF blockade with bevacizumab could potentiate PD-L1 checkpoint inhibition with atezolizumab in mRCC. Tissue collections are before treatment, after bevacizumab and after the addition of atezolizumab. We discover that intra-tumoral CD8(+) T cells increase following combination treatment. A related increase is found in intra-tumoral MHC-I, Th1 and T-effector markers, and chemokines, most notably CX3CL1 (fractalkine). We also discover that the fractalkine receptor increases on peripheral CD8(+) T cells with treatment. Furthermore, trafficking lymphocyte increases are observed in tumors following bevacizumab and combination treatment. These data suggest that the anti-VEGF and anti-PD-L1 combination improves antigen-specific T-cell migration.

Immunohistochemical prediction of lapatinib efficacy in advanced HER2-positive breast cancer patients.

UNLABELLED: Molecular mechanisms of lapatinib resistance in breast cancer are not well understood. The aim of this study was to correlate expression of selected proteins involved in ErbB family signaling pathways with clinical efficacy of lapatinib. Study group included 270 HER2-positive advanced breast cancer patients treated with lapatinib and capecitabine. Immunohistochemical expression of phosphorylated adenosine monophosphate-activated protein (p-AMPK), mitogen-activated protein kinase (p-MAPK), phospho (p)-p70S6K, cyclin E, phosphatase and tensin homolog were analyzed in primary breast cancer samples. The best discriminative value for progression-free survival (PFS) was established for each biomarker and subjected to multivariate analysis. At least one biomarker was determined in 199 patients. Expression of p-p70S6K was independently associated with longer (HR 0.45; 95% CI: 0.25-0.81; p = 0.009), and cyclin E with shorter PFS (HR 1.83; 95% CI: 1.06-3.14; p = 0.029). Expression of p-MAPK (HR 1.61; 95% CI 1.13-2.29; p = 0.009) and cyclin E (HR 2.99; 95% CI: 1.29-6.94; p = 0.011) was correlated with shorter, and expression of estrogen receptor (HR 0.65; 95% CI 0.43-0.98; p = 0.041) with longer overall survival. Expression of p-AMPK negatively impacted response to treatment (HR 3.31; 95% CI 1.48-7.44; p = 0.004) and disease control (HR 3.07; 95% CI 1.25-7.58; p = 0.015). IN CONCLUSION: the efficacy of lapatinib seems to be associated with the activity of downstream signaling pathways - AMPK/mTOR and Ras/Raf/MAPK. Further research is warranted to assess the clinical utility of these data and to determine a potential role of combining lapatinib with MAPK pathway inhibitors.

Relative levels of let-7a, miR-17, miR-27b, miR-125a, miR-125b and miR-206 as potential molecular markers to evaluate grade, receptor status and molecular type in breast cancer.

MicroRNAs (miRNAs/miRs) are a class of short, single­stranded nucleic acids, which have been investigated as potential molecular markers for various types of cancer. The gold­standard and most sensitive method for comparing miRNA levels in cancer tissues is reverse transcription­quantitative polymerase chain reaction (RT­qPCR). This technique uses stably expressed genes for normalisation. The aim of the present study was to improve this model of analysis in the context of RT­qPCR results. A total of six known miRNAs (let­7a, miR­17, miR­27b, miR­125a, miR­125b and miR­206), RNU6B RNA and five mRNAs [erb­b2 receptor tyrosine kinase 2 (ERBB2), hydroxymethylbilane synthase and polymerase (RNA) II (DNA directed) polypeptide A] were analysed pair­wise, in order to determine which biomarker pairs best correlated with the histological groups of 27 breast cancer samples. The lowest P­values and the highest area under the curve values in the receiver operating characteristic analysis were used to select the optimum ratios for discrimination among groups. Among the 21 pairs, miR­17/miR­27b and miR­125a/RNU6B best discriminated three groups of samples with different tumour grades (G classification). miR­125b/miR­206 best discriminated two groups of samples with different tumour sizes (pT), let­7a/RNU6B best discriminated two groups of samples with different lymph node status (pN), and let­7a/miR­125b best discriminated groups of samples with negative and positive oestrogen and progesterone receptor status. No pair of miRNAs was found to discriminate well between groups with either a negative or positive human epidermal growth factor receptor 2 (HER2) status. However, one miRNA/mRNA pair, miR­125a/ERBB2, discriminated HER2­negative from HER2­positive groups. The breast cancer samples investigated in the present study were grouped by immunohistological methods into three molecular classes: Luminal, HER2 positive and basal (L, H and B, respectively). In order to discern L from H and L from B, two miRNA pairs were selected: miR­125a/miR­125b and miR­125a/miR­206. In conclusion, the pair­wise method of RT­qPCR data analysis may be a reasonable alternative to the standard method of using stably expressed reference genes, such as RNU6B RNA, for normalisation. This method may increase the classification power of miRNA biomarkers in breast cancer diagnostics.

Role of extended histological examination in the assessment of local recurrence of the oral cancer.

INTRODUCTION: Oral cancer is the second most prevalent head and neck malignancy in Poland. The incidence of these types of cancer is constantly growing. A reason for a treatment failure of the oral cancer is local recurrence. AIM OF THE STUDY: To find out why the rate of recurrence of the oral cancer is so high despite radical surgical treatment combined with radiochemotherapy. MATERIALS AND METHODS: The study comprised a group of 160 oral cancer patients. 30 patients who showed local recurrence were given an extended histopathological examination in keeping to the guidelines of the Royal College of Pathologists. RESULTS: In 9 patients, primary tumours were found to be histologically aggressive as indicated by the proliferation of the vessels, nerves and muscles. In next 9 cases, the cancer infiltrated both the vessels and nerves or the vessels and muscles, and in the 8 other cases, just one of those structures. The tumours which were found to penetrate the vessels, nerves and muscles were also characterised with peri- and intraneural infiltration. The aggressiveness of invasion measured by the extent of damage done to the muscles, vessels and nerves correlated with the depth of invasion from the mucous membrane, the occurrence of embolisms in blood vessels, and a high (score 4) risk assessment as proposed by Margaret Brandwein-Gensler. CONCLUSION: The progression of cancer depends strongly on histopathological features. The incidence of penetration of the vessels, nerves and muscles correlates with aggressiveness of the front of tumour and few other histological features.

The anti-cancer actions of O6-methylguanine-DNA-methyltransferase in relation to colon polyps.

BACKGROUND: Genetic variability in DNA repair genes may contribute to differences in DNA repair capacity and susceptibility to colon polyps and cancer. In this study, we examined the role of MGMT polymorphisms in colon polyps formation. METHODS: PCR-SSCP analysis was performed included 254 patients with colon polyps and 330 controls. RESULTS: The homozygous F84F genotype was significantly more prevalent in study group than in controls. The polymorphic allele 84F was more frequent appeared in group of older patients and in group of smoking patients. On the other hand, there were no association between 84F and gender, size of polyps, cancer family history. CONCLUSIONS: We concluded that high frequency of 84F allele in the group of patients may suggest the role of the MGMT variant in colon polyps etiology.

Does Metformin affect ER, PR, IGF-1R, ß-catenin and PAX-2 expression in women with diabetes mellitus and endometrial cancer?

OBJECTIVE: Diabetes mellitus, as a risk factor for endometrial cancer (EC), causes an increase in insulin and IGF-1 concentrations in the blood serum. The increase in insulin and IGF-1 are considered mitogenic factors contributory to cancer development. Studies suggest that metformin has preventive activity, decreasing mortality and the risk of neoplasms. Since estrogen (ER), progesterone (PR) and IGF-1 (IGF-1R) receptor expression and ß-catenin and PAX-2 mutations are significant in the development of endometrial cancer, it was decided to study these factors in patients with endometrial cancer and type 2 diabetes mellitus (DM2), and to establish the effects of metformin on their expression. METHODS: The expression of ER, PR, IGF-1R, ß-catenin and PAX-2 have been immunohistochemically investigated in 86 type I endometrial cancer specimens. Patients were grouped according to the presence of DM2 and the type of hypoglycemic treatment administered. RESULTS: Comparing EC patients with DM2 and normal glycemic status, we found increased IGF-1R expression in women with DM2. A decrease in ER expression was noted in women with EC and DM2 receiving metformin as compared to women treated with insulin (p = 0.004). There was no statistically significant difference in PR, IGF-1R, ß-catenin and PAX-2 expression among women receiving metformin and other hypoglycemic treatment. CONCLUSION: Although epidemiological studies suggest the beneficial role of metformin in many human cancers, there are still few studies confirming its favorable effect on endometrial cancer. Decreased ER expression in patients receiving metformin needs further research to allow evaluation of its clinical significance.

rs12976445 variant in the pri-miR-125a correlates with a lower level of hsa-miR-125a and ERBB2 overexpression in breast cancer patients.

Expression of MIR125A is diminished in breast tumors, however the reason for the hsa-mir-125a decrease in the cancer is not known. HER2 is encoded by ERBB2, a target for hsa-miR-125a which interacts with the 3'UTR of ERBB2 mRNA. The present study reveals that a polymorphism (rs12976445) within the pri-miR-125a sequence correlates with the amount of mature hsa-miR-125a in breast tumor samples. miRNA, RNA and DNA were extracted from breast cancer samples obtained from 26 patients. Following immunohistological evaluation of the samples, the ERBB2, PGR and ESR1 mRNA profiles were also analyzed using real-time PCR. Genomic DNA was sequenced using MIR125A flanking primers. PCR products were analyzed using a BaeGI restriction enzyme specific to the rs12976445 variant. The rs12976445 variant (C/T and C/C) correlated with a lower level of hsa-miR-125a in comparison with the T/T variant. The expression of HER2 mRNA was increased in tumors with the rs12976445 variant (C/T and C/C) compared with T/T. We conclude that rs12976445 may be a potential prognostic marker of HER2 expression in breast cancer. Its predictive value on the efficacy of trastuzumab treatment in patients with HER2-positive breast cancer warrants further study.

Analysis of O6-methylguanine-DNA methyltransferase methylation status in sporadic colon polyps.

BACKGROUND/AIM: The aim of our study was to check how MGMT methylation status together with known factors influenced the risk of colon cancer development. MATERIALS AND METHODS: We examined patients with colon polyps. Information concerning gender, age, lifestyle, diet, anthropometry and medical information, including cancer and family history of cancer, was analyzed. Polymorphism variety of MGMT gene was investigated in another study. Genetic analysis for MGMT methylation assessment was performed for polyp tissue samples from 143 patients. RESULTS: Positive methylation MGMT status was found in 55 patients. There was no correlation between gender and MGMT methylation status (p = 0.43). We did not find correlation between patients younger and older than 60 (p = 0.87). There was no correlation between smoking and MGMT methylation status (p = 0.36). We did not find correlation between BMI and MGMT methylation status (p = 0.86). We did not find correlation between MGMT methylation status and colon cancer in familial history (p = 0.45). CONCLUSION: Our study showed no correlations between methylation status of MGMT polymorphisms and clinical features like age, gender, polyp localization, smoking status, or obesity. It has been shown previously that MGMT methylation status may show nonspecific methylation in colon polyps. Gene methylation status in adenoma tissues has also been associated by other authors with the adenoma's size, histology, and degree of atypia. In our study, we evaluated the gene methylation status in colon polyps and found no association with adenoma characteristics. The present study showed no correlation for MGMT methylation in polyps in different regions of colon.

Comparison of in situ hybridization methods for the assessment of HER-2/neu gene amplification status in breast cancer using a tissue microarray.

BACKGROUND: This project compared HER-2/neu gene status in breast cancers, as demonstrated by FISH (fluorescent in situ hybridization) and CISH (chromogenic in situ hybridization) and using a tissue microarray (TMA). The study also aimed to show whether the TMA technique could be used in clinical diagnostics, rather than remain a scientific tool. MATERIALS AND METHODS: A TMA was constructed using 121 breast cancer specimens, 6 cores from each specimen. Demonstration and assessment of HER-2/neu gene status was by FISH (Vysis Path) and CISH (DAKO Duo CISH). RESULTS: The 121 breast cancer specimens were divided into 3 groups by HER-2 status, as determined by immunohistochemistry. In the HER-2 negative group no amplification was observed in 36 out of 40 cases. 3 cases showed amplification by both methods and one by CISH alone. The equivocal HER-2 group showed no amplification in 30 out of 41 cases and amplification in 9 cases. One case was FISH negative CISH positive and one was discarded. In the HER-2 positive group, amplification was confirmed in 37 of the 40 cases by both methods. 3 cases were unsuitable for assessment. CONCLUSIONS: This study indicated that CISH is a sensitive alternative to FISH in detecting HER2 gene amplification and may replace FISH in HER2 testing. Good agreement was observed between methods (98.5% - 119 out of 121 cases). Furthermore, as only 4 out of 121 cases were unsuitable for assessment (no signal or missing TMA cores) - it may be feasible to use TMA in diagnostics.