Elke Winter

Presence of mycobacterial dna in sarcoidosis

In 11 of 35 clinically proven cases of sarcoidosis, we detected DNA sequences coding for the mycobacterial 65-kDa antigen. In four cases, the sequences were homologous to Mycobacterium avium; seven sequences were related to other nontuberculous Mycobacteria. The insertion sequence 1110, characteristic for Mycobacterium avium, was present in three cases. The insertion sequence 6110 of the Mycobacterium tuberculosis complex (M tuberculosis, africanum, bovis, BCG) was not detectable in any of the 11 cases, ruling out the presence of members of the Mycobacterium tuberculosis complex. Therefore, it seems reasonable to speculate about a mycobacterial cause in some cases of sarcoidosis.

MiR-200a regulates epithelial to mesenchymal transition-related gene expression and determines prognosis in colorectal cancer patients

Background:MicroRNAs (miRNAs) regulate the biological properties of colorectal cancer (CRC) cells and might serve as potential prognostic factors and therapeutic targets. In this study, we therefore globally profiled miRNAs associated with E-cadherin expression in CRC cells in an attempt to identify miRNAs that are associated with aggressive clinical course in CRC patients.Methods:Two CRC cell lines (Caco-2 and HRT-18) with different E-cadherin expression pattern were profiled for differences in abundance for more than 1000 human miRNAs using microarray technology. One of the most differentially expressed miRNAs, miR-200a was evaluated for its prognostic role in a cohort of 111 patients and independently validated in 217 patients of the Cancer Genome Atlas data set. To further characterise the biological role of miR-200a expression in CRC, in vitro miR-200a inhibition and overexpression were performed and the effects on cellular growth, apoptosis and epithelial–mesenchymal transition (EMT)-related gene expression were explored.Results:In situ hybridisation specifically localised miR-200a in CRC cells. In both cohorts, a low miR-200a expression was associated with poor survival (P<0.05). Multivariate Cox regression analysis identified low levels of miR-200a expression as an independent prognostic factor with respect to cancer-specific survival (HR=2.04, CI=1.28–3.25, P<0.002). Gain and loss of function assays for miR-200a in vitro led to a significantly differential and converse expression of EMT-related genes (P<0.001.) A low expression of miR-200a was also observed in cancer stem cell-enriched spheroid growth conditions (P<0.05).Conclusions:In conclusion, our data suggest that low miR-200a expression is associated with poor prognosis in CRC patients. MiR-200a has a regulatory effect on EMT and is associated with cancer stem cell properties in CRC.

MiR-181a is associated with poor clinical outcome in patients with colorectal cancer treated with EGFR inhibitor

Aims miR-181a expression is frequently altered in different types of cancer. Members of the Wnt/β-catenin signalling pathway, which is commonly altered in colorectal cancer (CRC), have been reported as molecular interaction partners of miR-181. However, the role of miR-181a expression in CRC and its ability to predict survival and response to agents targeting the epidermal growth factor receptor (EGFR) have not been explored yet. Methods In this study, we analysed 80 patients with wild type KRAS CRC undergoing treatment with the EGFR-targeting monoclonal antibodies cetuximab and panitumumab for metastatic CRC. The KRAS mutational status was determined by pyrosequencing and miR-181a expression was measured by quantitative RT-PCR in CRC tumour tissue and corresponding non-neoplastic colon tissue. The microRNA expression levels were correlated with clinicopathological characteristics. Cancer-specific survival was calculated by univariate and multivariate analyses, and progression-free survival (PFS) during treatment with EGFR-targeting agents was also evaluated. Results A low miR-181a expression level was associated with poor differentiation of CRC (p=0.04). A Kaplan-Meier curve showed a decreased survival time for patients with low miR-181a expression (p=0.019). Low miR-181a expression was furthermore associated with poor PFS (p=0.015). Conclusions In conclusion, our data suggest that the miR-181a expression level is associated with poor survival in patients with CRC. Furthermore, miR-181a expression might predict PFS in EGFR-targeted therapy.

Two major pathways of penile carcinogenesis: HPV-induced penile cancers overexpress p16ink4a, HPV-negative cancers associated with dermatoses express p53, but lack p16ink4a overexpression

BACKGROUNDnPenile squamous cell carcinomas (SCC) arise either through transforming infections with human papillomavirus (HPV) or independent of HPV, often in the background of lichen sclerosus (LS) and lichen planus (LP). Despite impact on therapy and prognosis, etiologic stratifications are missing in most histological diagnoses and publications about penile cancers/precursors.nnnOBJECTIVEnClassification of penile lesions into HPV-induced or HPV-negative via immunohistochemical demonstration of p16(ink4a) overexpression, a surrogate marker for transforming HPV-high-risk infections, and p53 expression in the absence of p16(ink4a) overexpression.nnnMETHODSnArchival formalin-fixed material of 123 invasive penile cancers and 43 pre-invasive lesions was evaluated for the presence of LS, LP, 28 HPV genotypes, and expression of p53 and p16(ink4a).nnnRESULTSnSeventy-two of 123 SCCs and 33 of 43 pre-invasive lesions showed p16(ink4a) overexpression independent of HPV-HR genotypes involved; 66 of 72 SCCs and 29 of 43 precursor lesions revealed a single HPV-high-risk-genotype (HPV-HR16 in 76% followed by HPV33, HPV31, HPV45, HPV18, HPV56); 5 of 72 SCCs and 4 of 43 precursor lesions revealed multiple HPV-HR-genotypes. One SCC revealed HPV-LR and HR-DNA. Fifty-one of 123 SCCs and 10 precursor lesions were p16(ink4a) negative, but showed nuclear p53 expression in tumor cells and basal keratinocytes. Forty-nine of 51 SCCs and 10 of 10 precursor lesions lacked HPV DNA. Two of 51 SCCs contained HPV18 and HPV45 DNA, respectively, but p16(ink4a) negativity classified them as non-HPV-induced. Twenty-seven of 51 SCCs showed peritumoral LS, 13 of 51 SCCs showed peritumoral LP, and 11 SCCs revealed no peritumoral tissue. Histologically, HPV-negative precursors showed hyperkeratotic, verrucous, atrophic, and basaloid differentiation.nnnLIMITATIONSnThis was a retrospective study.nnnCONCLUSIONSnp16(ink4a) overexpression identifies HPV-HR-induced penile carcinogenesis independent of HPV-HR genotype. p53 expression along with p16(ink4a) negativity identifies HPV-negative cancers. Correct etiologic classification of penile lesions during diagnostic work-up allows optimal therapy decisions.

Genome-wide microRNA analysis identifies miR-188-3p as novel prognostic marker and molecular factor involved in colorectal carcinogenesis.

Purpose: Characterization of colorectal cancer transcriptome by high-throughput techniques has enabled the discovery of several differentially expressed genes involving previously unreported miRNA abnormalities. Here, we followed a systematic approach on a global scale to identify miRNAs as clinical outcome predictors and further validated them in the clinical and experimental setting. Experimental Design: Genome-wide miRNA sequencing data of 228 colorectal cancer patients from The Cancer Genome Atlas dataset were analyzed as a screening cohort to identify miRNAs significantly associated with survival according to stringent prespecified criteria. A panel of six miRNAs was further validated for their prognostic utility in a large independent validation cohort (n = 332). In situ hybridization and functional experiments in a panel of colorectal cancer cell lines and xenografts further clarified the role of clinical relevant miRNAs. Results: Six miRNAs (miR-92b-3p, miR-188-3p, miR-221-5p, miR-331-3p, miR-425-3p, and miR-497-5p) were identified as strong predictors of survival in the screening cohort. High miR-188-3p expression proves to be an independent prognostic factor [screening cohort: HR = 4.137; 95% confidence interval (CI), 1.568–10.917; P = 0.004; validation cohort: HR = 1.538; 95% CI, 1.107–2.137; P = 0.010, respectively]. Forced miR-188-3p expression increased migratory behavior of colorectal cancer cells in vitro and metastases formation in vivo (P < 0.05). The promigratory role of miR-188-3p is mediated by direct interaction with MLLT4, a novel identified player involved in colorectal cancer cell migration. Conclusions: miR-188-3p is a novel independent prognostic factor in colorectal cancer patients, which can be partly explained by its effect on MLLT4 expression and migration of cancer cells. Clin Cancer Res; 23(5); 1323–33. ©2016 AACR.

MiR‐96‐5p influences cellular growth and is associated with poor survival in colorectal cancer patients

Expression of miR‐96‐5p is frequently altered in various types of cancer and the KRAS oncogene has been identified as one of its potential targets. However, the biological role of miR‐96‐5p expression in colorectal cancer (CRC) and its ability to predict the clinical course of patients have not been investigated yet. In this study, we explored miR‐96‐5p expression in 80 CRC patients and evaluated the impact on clinical outcome by Kaplan‐Meier curves and multivariate Cox proportional models. In vitro miR‐96‐5p inhibition and overexpression were performed in CRC cells and the effects on cellular growth, anchorage‐independent growth, apoptosis, and epithelial‐mesenchymal transition (EMT)‐related gene expression were explored. Low miR‐96‐5p expression levels in tumor tissue were associated with distant metastasis (Pu2009=u20090.025) and multivariate Cox regression analysis identified low levels of miR‐96‐5p as an independent prognostic factor with respect to cancer‐specific survival (hazard ratiou2009=u20091.78, 95%CIu2009=u20091.03‐3.03, Pu2009<u20090.038). In vitro overexpression of miR‐96‐5p led to a reduced cellular growth rate (Pu2009<u20090.05), reduced colonies in soft agar (Pu2009<u20090.05), corroborated by a decreased cyclin D1 and increased p27‐CDKN1A expression (Pu2009<u20090.05). Forced expression of miR‐96‐5p in CRC cells entailed no effects on apoptosis or EMT‐related genes but decreased the expression levels of the KRAS oncogene (Pu2009<u20090.05). Despite regulating KRAS expression, there was no significant association in miR‐96‐5p expression levels and response rates to EGFR‐targeting agents. In conclusion, our data suggest that miR‐96‐5p influences cellular growth of CRC cells and low expression of miR‐96‐5p seems to be associated with poor clinical outcome in CRC patients.

Familial hemophagocytic lymphohistiocytosis associated with disseminated T-cell lymphoma: A report of two siblings

SummaryTwo siblings with evidence of disseminated T-cell lymphoma at the time of diagnosis of familial hemophagocytic lymphohistiocytosis (FHL) are reported, an association which has not been described previously. The first child with typical clinical and laboratory features of FHL died shortly after admission, before diagnosis could be established. Retrospective analysis of autoptic tissue revealed marked hemophagocytosis as well as morphological and immunohistochemical features suggestive of disseminated T-cell lymphoma. In the second child, FHL was diagnosed in time. Subsequent histologic investigation of bone marrow biopsies displayed a focal infiltration by T-cell lymphoma. DNA hybridization studies provided evidence of a monoclonal T-cell receptor beta chain gene rearrangement. Following conventional chemotherapeutic induction for FHL, the patient received an allogeneic bone marrow transplant (BMT) from a related healthy donor. Currently, 17 months after BMT, the boy is in unmaintained remission from FHL and T-cell lymphoma. The current pathogenetic concepts for FHL and a possible relationship between T-cell lymphoma and FHL are discussed.

Mutation Profiling of Usual Ductal Hyperplasia of the Breast Reveals Activating Mutations Predominantly at Different Levels of the PI3K/AKT/mTOR Pathway

Usual ductal hyperplasia (UDH) of the breast is generally regarded as a nonneoplastic proliferation, albeit loss of heterozygosity has long been reported in a part of these lesions. To gain deeper insights into the molecular drivers of these lesions, an extended mutation profiling was performed. The coding regions of 409 cancer-related genes were investigated by next-generation sequencing in 16 cases of UDH, nine unassociated with neoplasia (classic) and seven arising within papillomas. Phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (mTOR) activation was investigated by phosphorylated AKT, mTOR, and S6 immunohistochemistry. Of 16 lesions, 10 (63%) were mutated; 56% of classic lesions were unassociated with neoplasia, and 71% of lesions arose in papillomas. Fourteen missense mutations were detected: PIK3CA [6 (43%) of 14], AKT1 [2 (14%) of 14], as well as GNAS, MTOR, PIK3R1, LPHN3, LRP1B, and IGF2R [each 1 (7%) of 14]. Phosphorylated mTOR was seen in 83% and phosphorylated S6 in 86% of evaluable lesions (phospho-AKT staining was technically uninterpretable). In conclusion, UDH displays mutations of the phosphatidylinositol 3-kinase/AKT/mTOR axis at different levels, with PIK3R1, MTOR, and GNAS mutations not previously described. Specifically, oncogenic G-protein activation represents a yet unrecognized route to proliferation in UDH. On the basis of evidence of activating mutations, loss of heterozygosity, and a mass forming proliferation, we propose that UDH is most appropriately viewed as an early neoplastic intraductal proliferation.

HPV-negative penile squamous cell carcinoma: disruptive mutations in the TP53 gene are common

The majority of penile squamous cell carcinomas is caused by transforming human papilloma virus (HPV) infection. The etiology of HPV-negative cancers is unclear, but TP53 mutations have been implicated. Archival tissues of 108 invasive squamous cell carcinoma from a single pathology institution in a low-incidence area were analyzed for HPV-DNA and p16ink4a overexpression and for TP53 mutations by ion torrent next-generation sequencing. Library preparation failed in 32/108 squamous cell carcinomas. Institutional review board approval was obtained. Thirty of 76 squamous cell carcinomas (43%; average 63 years) were HPV-negative with 8/33 squamous cell carcinomas being TP53 wild-type (24%; average 63 years). Twenty-five of 33 squamous cell carcinomas (76%; average 65 years) showed 32 different somatic TP53 mutations (23 missense mutations in exons 5–8, 6 nonsense, 1 frameshift and 2 splice-site mutations). Several hotspot mutations were detected multiple times (R175H, R248, R282, and R273). Eighteen of 19 squamous cell carcinomas with TP53 expression in immunohistochemistry had TP53 mutations. Fifty percent of TP53-negative squamous cell carcinomas showed mostly truncating loss-of-function TP53 mutations. Patients without mutations had longer survival (5 years: 86% vs 61%; 10 years: 60% vs 22%), but valid clinically relevant conclusions cannot be drawn due to different tumor stages and heterogeneous treatment of the cases presented in this study. Somatic TP53 mutations are a common feature in HPV-negative penile squamous cell carcinomas and offer an explanation for HPV-independent penile carcinogenesis. About half of HPV-negative penile cancers are driven by oncogenic activation of TP53, while a quarter is induced by loss of TP53 tumor suppressor function. Detection of TP53 mutations should be carried out by sequencing, as immunohistochemical TP53 staining could not identify all squamous cell carcinomas with TP53 mutations.

Quantitative short-tandem repeat analysis of recipient-derived cells as an additional tool for diagnosing cardiac allograft rejection.

Background. Diagnosis of cardiac allograft rejection is currently based on histologic exploration of endomyocardial biopsies. Moderate interobserver reproducibility in the estimation of number and distribution of inflammatory cells leads to disagreements in the assignment of rejection grades. Short-tandem repeat (STR) analysis is routinely used after hematopoietic stem-cell transplantation to determine the proportions of donor cells. We compared the amount of recipient-derived cells with the histopathologic grade of rejection in cardiac allografts to determine whether this method might be useful for the assessment of rejection episodes. Methods. One hundred forty-three endomyocardial biopsies from 18 patients were investigated for the percentage of recipient-derived cell content by polymerase chain reaction-based STR analysis and correlated with rejection grades determined according to the International Society for Heart and Lung Transplantation grading system. Y-chromosome chromogene in situ hybridization was performed in gender-mismatched (female-to-male) heart transplants to explore the influence of cardiomyocyte cell chimerism. Results. The mean percentages of recipient-derived cells associated with various degrees of rejection were 13% in grade 0, 24% in grade 1A, 29% in grade 1B, 35% in grade 2, and 50% in grade ≥3A. Samples lacking signs of rejection (grade 0) had a significantly lower (P<0.001) amount of recipient-derived cells than higher degrees of rejections. Chromogene in situ hybridization analysis revealed that the recipient-derived cells were mainly inflammatory. Conclusions. The results of STR-analysis indicate that rejection is correlated with a higher proportion of recipient-derived cells. This assessment is observer independent and may thus represent an additional diagnostic tool for the assessment of rejection and management of immunosuppressive treatment.