Konstanty Korski

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.

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

ObjectiveDiabetes 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 β-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.MethodsThe expression of ER, PR, IGF-1R, β-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.ResultsComparing 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, β-catenin and PAX-2 expression among women receiving metformin and other hypoglycemic treatment.ConclusionAlthough 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.

A case of primary testicular germ cell tumor with rhabdomyosarcoma metastases as an example of applying the FISH method to diagnostic pathology.

We present the interesting case of a 38‐year‐old man with a primary malignant tumor of the right testis that metachronously metastasized to the urinary bladder and the stomach. Histologically, the testicular tumor was a mixed germ cell tumor composed of teratoma and embryonal carcinoma, but it also contained a sarcoma component of somatic type malignancy. Metastases showed rhabdomyoblastic differentiation histologically identical to the sarcoma component of the testicular tumor that was diagnosed as rhabdomyosarcoma. By applying fluorescence in situ hybridization (FISH) to the cytogenetic examination of cells taken from the periventricular lymph node metastases, we demonstrated a structural chromosomal aberration characteristic of testicular neoplasms, i.e. the presence of isochromosome 12p (i(12p)). Additionally, the diagnosis was supported by immunohistochemistry.

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

Metastatic apocrine adenocarcinoma of the axillary area

Summary Background Apocrine adenocarcinoma of the skin is a rare entity. It is characterized by slowly enlarging, painless, indurate nodules or plaques and often misdiagnosed as benign skin tumours. Although these tumours show characteristic tubular structures mixed with cellular cords and have some pattern of cytokeratins, primary apocrine carcinoma is indistinguishable from metastatic mammary ductal carcinomas. Like other apocrine carcinomas it is radioresistant and therefore surgical resection is the method of choice in treatment of patients. Distant metastases have been reported in a limited number of published cases. Aim We present the case of a 66-year-old woman with apocrine adenocarcinoma of the left axillary area with local lymph node and distant metastases. Case Report A 66-year-old woman was admitted to hospital because of a tumour located in the skin of the axillary area. After incision biopsy lobular breast carcinoma was initially suspected. Correlation of clinical, histological and immunohistochemical data allowed the rare apocrine adenocarcinoma to be diagnosed. The tumour was excised with axillary lymph nodes. Next chemotherapy was applied as palliative treatment. Conclusions Our observations lead us to following conclusions: (I) apocrine adenocarcinoma is a rare and difficult to diagnose tumour requiring special examination; (II) metastases to lung, liver and bones cause worse prognoses; (III) a wide surgical excision is the treatment of choice.

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, single‑stranded nucleic acids, which have been investigated as potential molecular markers for various types of cancer. The gold‑standard and most sensitive method for comparing miRNA levels in cancer tissues is reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). 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 RT‑qPCR results. A total of six known miRNAs (let‑7a, miR‑17, miR‑27b, miR‑125a, miR‑125b and miR‑206), RNU6B RNA and five mRNAs [erb‑b2 receptor tyrosine kinase 2 (ERBB2), hydroxymethylbilane synthase and polymerase (RNA) II (DNA directed) polypeptide A] were analysed pair‑wise, in order to determine which biomarker pairs best correlated with the histological groups of 27 breast cancer samples. The lowest P‑values 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, miR‑17/miR‑27b and miR‑125a/RNU6B best discriminated three groups of samples with different tumour grades (G classification). miR‑125b/miR‑206 best discriminated two groups of samples with different tumour sizes (pT), let‑7a/RNU6B best discriminated two groups of samples with different lymph node status (pN), and let‑7a/miR‑125b 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, miR‑125a/ERBB2, discriminated HER2‑negative from HER2‑positive 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: miR‑125a/miR‑125b and miR‑125a/miR‑206. In conclusion, the pair‑wise method of RT‑qPCR 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.

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.

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.

Abstract 4272: Applying TCGA data for breast cancer diagnostics and pathway analysis

The main objective of our project is to obtain a comprehensive insight into oncogenic signaling in order to develop novel diagnostic tools for molecular subtypes of breast cancer (BRCA). We have provided about 100 BRCA samples along with full pathology and clinical annotations to the TCGA program. These cancer samples have been extensively characterized using all available genomics platforms and the obtained molecular data contributed to the molecular characterization of BRCA. This integrated effort identified four major molecular subtypes of breast cancer that can be identified across five profiling platform including genomics, transcriptomics and proteomics (Nature, 490 (7418):61-70). Building upon these datasets we aim to translate these genomics profiles into diagnostic tools that can be used in every day medical practice. In the first step, we have generated tissue microarrays (TMAs) from FFPE BRCA samples obtained from the patients enrolled into the TCGA project. Next, the TMA slides were used for immunohistochemistry (IHC) using about 100 antibodies specific for major cancer markers, including oncogenic kinases frequently activated or overexpressed in BRCA. The obtained IHC results have been scored and verified independently for each marker by two board-certified pathologists. To identify markers that correlate with or are specific to the molecular subtypes of BRCA, the TMA/IHC read-outs from each tumor sample were correlated with all TCGA genomic data. In addition, we integrate the pathology and genomics results with clinical data obtained from the TCGA-enrolled patients, including up to 4 years follow-up. Finally, we apply system biology tools to integrate the genomic data from TCGA with the proteomic analysis to understand causalities between changes in DNA, transcriptome and signal transduction pathways. Our long-term goal is to identify novel diagnostic biomarkers that will precisely identify each molecular subtype of BRCA and which may be predictive of patient response to therapy thus paving the way for novel personalized therapies for cancer. Citation Format: Jannik Andersen, Parantu Shah, Konstanty Korski, Matthew Ibbs, Violetta Filas, Michal Kosiedowski, Juliusz Pukacki, Cezary Mazurek, Yuanqing Wu, Edward Chang, Carlo Toniatti, Giulio Draetta, Maciej Wiznerowicz. Applying TCGA data for breast cancer diagnostics and pathway analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4272. doi:10.1158/1538-7445.AM2014-4272

Abstract 254: Expression of the putative stem cell marker CD44 in prostate biopsies correlates with Gleason score in radical prostatectomies in patients with prostate cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC AIM: Prostate cancer is one of the leading causes of death among male oncology patients. There is growing evidence to support the hypothesis that cancer cells in the prostate are of stem cell origin. In this study we used CD44, as one of the most promising putative stem cell markers, to evaluate the prevalence of prostate cancer cells with stem cell like features in prostate biopsies and in radical prostatectomy specimens. We tested both types of specimen for the existence of a correlation between the expression of CD44 and commonly used prostate cancer prognostic factors such as Gleason grade, pathological stage (pT) according to TNM, patient age and preoperative plasma PSA levels. M&M: Formalin fixed paraffin embedded tissue samples from fifty two prostate biopsies and matched radical prostatectomy specimens were retrieved from the archives of the Pathology Department at the City Hospital, Poznan, Poland or from a collection of cases sent for consultation in the private laboratory of one of the authors (JB). Immunohistochemistry was performed on sections from all paraffin blocks using an anti-CD44 primary antibody (Abcam) according to the manufacturers protocol. Using an OLYMPUS BX41 microscope, we counted at least 100 cancer cells from core biopsies and 500 cancer cells from radical prostatectomy specimens. Only cells with clearly visible membranous staining at 10x magnification were called positive for the expression of CD44. The percentage of cancer cells expressing CD44 was used for further analysis. The statistical analysis was based on Spearman and Person correlations performed in Microsoft Office Excel 2007. RESULTS: CD44 positive cancer cells were identified in forty three prostate biopsies (82%) and forty seven radical prostatectomy specimens (90%). There was a positive correlation between the expression of CD44 in cancer cells from prostate biopsies and in radical prostatectomy specimens. We also observed a negative correlation between CD44 expression in cancer cells and Gleason score, both in prostate biopsies and in radical prostatectomy material. When other clinical data were analysed, such as patient age, preoperative PSA serum levels or tumour stage (pT), only the first parameter was found to correlate positively with CD44 expression in prostate biopsies. CONCLUSION: To the best of our knowledge this study showed for the first time, that the level of CD44 expression in prostate biopsies correlates with that observed in matched radical prostatectomy specimens. Additionally, we confirmed the results of other authors which show that the percentage of CD44 expressing cancer cells in prostate biopsies negatively correlates with Gleason grade of tumours, both in prostate biopsies and in radical prostatectomy material. Citation Format: Konstanty Korski, Anna Malicka, Jan Breborowicz. Expression of the putative stem cell marker CD44 in prostate biopsies correlates with Gleason score in radical prostatectomies in patients with prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 254. doi:10.1158/1538-7445.AM2013-254