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The molecular causes of myeloproliferative neoplasms (MPNs) have not yet been fully elucidated. Approximately 7% to 8% of the patients carry predisposing genetic germline variants that lead to driver mutations, which enhance JAK-STAT signaling. To identify additional predisposing genetic germline variants, we performed whole-exome sequencing in 5 families, each with parent-child or sibling pairs affected by MPNs and carrying the somatic JAK2 V617F mutation. In 4 families, we detected rare germline variants in known tumor predisposition genes of the DNA repair pathway, including the highly penetrant BRCA1 and BRCA2 genes. The identification of an underlying hereditary tumor predisposition is of major relevance for the individual patients as well as for their families in the context of therapeutic options and preventive care. Two patients with essential thrombocythemia or polycythemia vera experienced progression to acute myeloid leukemia, which may suggest a high risk of leukemic transformation in these familial MPNs. Our study demonstrates the relevance of genetic germline diagnostics in elucidating the causes of MPNs and suggests novel therapeutic options (eg, PARP inhibitors) in MPNs. Furthermore, we uncover a broader tumor spectrum upon the detection of a germline mutation in genes of the DNA repair pathway.

Acute myeloid leukemia (AML) is characterized by an expansion of leukemic cells and a simultaneous reduction of normal hematopoietic precursors in the bone marrow (BM) resulting in hematopoietic insufficiency, but the underlying mechanisms are poorly understood in humans. Assuming that leukemic cells functionally inhibit healthy CD34+ hematopoietic stem and progenitor cells (HSPC) via humoral factors, we exposed healthy BM-derived CD34+ HSPC to cell-free supernatants derived from AML cell lines as well as from 24 newly diagnosed AML patients. Exposure to AML-derived supernatants significantly inhibited proliferation, cell cycling, colony formation, and differentiation of healthy CD34+ HSPC. RNA sequencing of healthy CD34+ HSPC after exposure to leukemic conditions revealed a specific signature of genes related to proliferation, cell-cycle regulation, and differentiation, thereby reflecting their functional inhibition on a molecular level. Experiments with paired patient samples showed that these inhibitory effects are markedly related to the immunomagnetically enriched CD34+ leukemic cell population. Using PCR, ELISA, and RNA sequencing, we detected overexpression of TGFβ1 in leukemic cells on the transcriptional and protein level and, correspondingly, a molecular signature related to TGFβ1 signaling in healthy CD34+ HSPC. This inhibitory effect of TGFβ1 on healthy hematopoiesis was functionally corrobated and could be pharmacologically reverted by SD208, an inhibitor of TGFβ receptor 1 signaling. Overall, these data indicate that leukemic cells induce functional inhibition of healthy CD34+ HSPC, at least in part, through TGFβ1, suggesting that blockage of this pathway may improve hematopoiesis in AML.

Findings 5585 families with Lynch syndrome from 22 countries were eligible for the analysis. Of these, there were insufficient numbers to estimate penetrance for Asia and South America, and for those with EPCAM variants. Therefore, we used data (collected between July 11, 2014, and Dec 31, 2018) from 5255 families (1829 MLH1, 2179 MSH2, 798 MSH6, and 449 PMS2), comprising 79 809 relatives, recruited in 15 countries in North America, Europe, and Australasia. There was strong evidence of the existence of unknown familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers (p 0 center dot 0001 for each of the three three continents). These familial risk factors resulted in a wide within-gene variation in the risk of colorectal cancer for men and women from each continent who all carried pathogenic variants in the same gene or the MSH2 c.942+3A T variant. The variation was especially prominent for MLH1 and MSH2 variant carriers, depending on gene, sex and continent, with 7-56% of carriers having a colorectal cancer penetrance of less than 20%, 9-44% having a penetrance of more than 80%, and onlyBackground Existing clinical practice guidelines for carriers of pathogenic variants of DNA mismatch repair genes (Lynch syndrome) are based on the mean age-specific cumulative risk (penetrance) of colorectal cancer for all carriers of pathogenic variants in the same gene. We aimed to estimate the variation in the penetrance of colorectal cancer between carriers of pathogenic variants in the same gene by sex and continent of residence. Methods In this retrospective cohort study, we sourced data from the International Mismatch Repair Consortium, which comprises 273 members from 122 research centres or clinics in 32 countries from six continents who are involved in Lynch syndrome research. Families with at least three members and at least one confirmed carrier of a pathogenic or likely pathogenic variant in a DNA mismatch repair gene (MLH1, MSH2, MSH6, or PMS2) were included. The families of probands with known de-novo pathogenic variants were excluded. Data were collected on the method of ascertainment of the family, sex, carrier status, cancer diagnoses, and ages at the time of pedigree collection and at last contact or death. We used a segregation analysis conditioned on ascertainment to estimate the mean penetrance of colorectal cancer and modelled unmeasured polygenic factors to estimate the variation in penetrance. The existence of unknown familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers was tested by use of a Wald p value for the null hypothesis that the polygenic SD is zero. Findings 5585 families with Lynch syndrome from 22 countries were eligible for the analysis. Of these, there were insufficient numbers to estimate penetrance for Asia and South America, and for those with EPCAM variants. Therefore, we used data (collected between July 11, 2014, and Dec 31, 2018) from 5255 families (1829 MLH1, 2179 MSH2, 798 MSH6, and 449 PMS2), comprising 79 809 relatives, recruited in 15 countries in North America, Europe, and Australasia. There was strong evidence of the existence of unknown familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers (pT variant. The variation was especially prominent for MLH1 and MSH2 variant carriers, depending on gene, sex and continent, with 7-56% of carriers having a colorectal cancer penetrance of less than 20%, 9-44% having a penetrance of more than 80%, and only 10-19% having a penetrance of 40-60%. Interpretation Our study findings highlight the important role of risk modifiers, which could lead to personalised risk assessments for precision prevention and early detection of colorectal cancer for people with Lynch syndrome. Funding National Health and Medical Research Council, Australia. Copyright (c) 2021 Elsevier Ltd. All rights reserved.Methods In this retrospective cohort study, we sourced data from the International Mismatch Repair Consortium, which comprises 273 members from 122 research centres or clinics in 32 countries from six continents who are involved in Lynch syndrome research. Families with at least three members and at least one confirmed carrier of a pathogenic or likely pathogenic variant in a DNA mismatch repair gene (MLH1, MSH2, MSH6, or PMS2) were included. The families of probands with known de-novo pathogenic variants were excluded. Data were collected on the method of ascertainment of the family, sex, carrier status, cancer diagnoses, and ages at the time of pedigree collection and at last contact or death. We used a segregation analysis conditioned on ascertainment to estimate the mean penetrance of colorectal cancer and modelled unmeasured polygenic factors to estimate the variation in penetrance. The existence of unknown familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers was tested by use of a Wald p value for the null hypothesis that the polygenic SD is zero.

Azacitidine is the first drug to demonstrate a survival benefit for patients with MDS. However, only half of patients respond and almost all patients eventually relapse. Limited and conflicting data are available on predictive factors influencing response. We analyzed 128 patients from two institutions with MDS or AML treated with azacitidine to identify prognostic indicators. Genetic mutations in ASXL1, RUNX1, DNMT3A, IDH1, IDH2, TET2, TP53, NRAS, KRAS, FLT3, KMT2A-PTD, EZH2, SF3B1, and SRSF2 were assessed by next-generation sequencing. With a median follow up of 5.6 years median survival was 1.3 years with a response rate of 49%. The only variable with significant influence on response was del(20q). All 6 patients responded (p = 0.012) but survival was not improved. No other clinical, cytogenetic or molecular marker for response or survival was identified. Interestingly, patients from poor-risk groups as high-risk cytogenetics (55%), t-MDS/AML (54%), TP53 mutated (48%) or relapsed after chemotherapy (60%) showed a high response rate. Factors associated with shorter survival were low platelets, AML vs. MDS, therapy-related disease, TP53 and KMT2A-PTD. In multivariate analysis anemia, platelets, FLT3-ITD, and therapy-related disease remained in the model. Poor-risk factors such as del(7q)/-7, complex karyotype, ASXL1, RUNX1, EZH2, and TP53 did not show an independent impact. Thus, no clear biomarker for response and survival can be identified. Although a number of publications on predictive markers for response to AZA exist, results are inconsistent and improved response rates did not translate to improved survival. Here, we provide a comprehensive overview comparing the studies published to date.

About 40% of patients with myelodysplastic syndromes (MDSs) present with a normal karyotype, and they are facing different courses of disease. To advance the biological understanding and to find molecular prognostic markers, we performed a high-resolution oligonucleotide array study of 107 MDS patients (French American British) with a normal karyotype and clinical follow-up through the Duesseldorf MDS registry. Recurrent hidden deletions overlapping with known cytogenetic aberrations or sites of known tumor-associated genes were identified in 4q24 (TET2, 2x), 5q31.2 (2x), 7q22.1 (3x) and 21q22.12 (RUNX1, 2x). One patient with a 7q22.1 deletion had an additional 5q31.2 deletion of the acute myeloid leukemia/MDS region, the smallest deletion identified so far and including the putative tumor suppressor (ts) genes, EGR1 and CTNNA1. One TET2 deletion was homozygous and one heterozygous, with a missense mutation in the remaining allele, further supporting its role as a ts gene. Besides these recurrent alterations, additional individual imbalances were found in 34 cases; in total, 42/107 (39%) cases had genomic imbalances. These patients had an inferior survival as compared with the rest of the patients (P=0.002). This study emphasizes the heterogeneity of MDS, but points to interesting genes that may have diagnostic and prognostic impact.

Hereditary non-polyposis colorectal cancer, an autosomal dominant predisposition to colorectal cancer and other malignancies, is caused by inactivating mutations of DNA mismatch repair genes, mainly MLH1 and MSH2. Missense mutations affect protein structure or function, but may also cause aberrant splicing, if located within splice sites (ss) or cis-acting sequences of splicing regulatory proteins, i.e., exonic splicing enhancers or exonic splicing silencers. Despite significant progress of ss scoring algorithms, the prediction for the impact of mutations on splicing is still unsatisfactory. For this study, we assessed ten ss and nine missense mutations outside ss in MLH1 and MSH2, including eleven newly identified mutations, and experimentally analyzed their effect at the RNA level. We additionally tested and compared the reliability of several web-based programs for the prediction of splicing outcome for these mutations.

EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) was recently described as an antagonist of angiogenesis. Motivated by a strong dependence of tumor growth and metastasis on angiogenesis, we investigated the role of EFEMP1 in human breast cancer. We applied RNA microarray expression analysis and quantitative real-time PCR (QRT) in a total of 45 sporadic breast cancer tissues and found EFEMP1 down-regulation in 59% and 61% of the analyzed tissues, respectively. This down-regulation was confirmed on protein level. Immunohistochemistry in 211 breast cancer tissues resulted in reduced or even abolished EFEMP1 expression in 57-62.5% of the tumors. Bisulphite genomic sequencing in breast cancer cell lines and primary breast cancer tissues revealed promoter methylation as the major cause of this down-regulation. Furthermore, analysis of 203 clinically well characterized primary breast cancers displayed a significant correlation of reduced EFEMP1 protein expression with poor disease-free (p = 0.037) and overall survival (p = 0.032), particularly in those node-positive patients who received adjuvant anthracycline-based chemotherapy, but not in those treated by either cyclophosphamide-methotrexate-5-fluorouracil (CMF) or Tamoxifen. In summary, the presented data demonstrate for the first time the reduced EFEMP1 expression on RNA and protein level in a substantial number of sporadic breast carcinomas and its correlation with epigenetic alterations. Furthermore, these data point towards a possible predictive impact of EFEMP1 expression in primary breast cancer.

Extensive hypermethylation and consecutive transcriptional silencing of tumorsuppressor genes have been documented in multiple tumor entities including breast cancer. In a microarray based genome-wide methylation analysis of five sporadic breast carcinomas we identified a hypermethylated CpG island within the first intron of the prospero related homeobox gene 1 (PROX1). We, therefore, investigated CpG island methylation of PROX1 in a series of 33 pairs of primary breast cancer and corresponding normal tissue samples by bisulfite sequencing and COBRA analyses. Seventeen of these (52%) breast cancer samples revealed a significant accumulation of methylated CpG sites along with a significant reduction of PROX1 transcription compared to normal breast tissues of the same patients. Frequent methylation was also observed in brain metastases from primary breast cancer (21/37 = 57% of cases). Secondary, we analysed 38 brain metastases of primary breast carcinomas and detected a significantly reduced expression of PROX1 compared to normal breast tissue (p < 0.001) and primary breast carcinomas (p < 0.05), respectively. Additionally, treatment of breast cancer cell lines with demethylating agents could reactivate PROX1 transcription. In summary, we have identified PROX1 as a novel target gene that is hypermethylated and transcriptionally silenced in primary and metastatic breast cancer.

The canonical Wnt signalling pathway plays a key role during embryogenesis and defects in this pathway have been implicated in the pathogenesis of various types of tumours, including breast cancer. The gene for secreted frizzled-related protein 1 (SFRP1) encodes a soluble Wnt antagonist and is located in a chromosomal region (8p22–p12) that is often deleted in breast cancer. In colon, lung, bladder and ovarian cancer SFRP1 expression is frequently inactivated by promoter methylation. We have previously shown that loss of SFRP1 protein expression is a common event in breast tumours that is associated with poor overall survival in patients with early breast cancer. To investigate the cause of SFRP1 loss in breast cancer, we performed mutation, methylation and expression analysis in human primary breast tumours and breast cell lines. No SFRP1 gene mutations were detected. However, promoter methylation of SFRP1 was frequently observed in both primary breast cancer (61%, n=130) and cell lines analysed by methylation-specific polymerase chain reaction (MSP). We found a tight correlation (P

To establish the importance of CHEK2 mutations for familial breast cancer incidence in the German population, we have screened all 14 of the coding exons in 516 families negative for mutations in both the BRCA1 and BRCA2 genes. We found 12 distinct variants in 30 unrelated patients (5.81%), including 5 that are novel and an additional 4 found for the first time in breast cancer. These aberrations were evaluated in 500 healthy women aged over 50 years and in the case of the 2 exon 10 mutations, 1100delC and 1214del4bp, in 1315 randomized healthy controls. According to our results, a statistically significant association for the exon 10 mutations was observed (p = 0.006). The prevalence of the 1100delC mutation in the German population, however, is significantly lower than those reported for other Caucasian populations both in familial breast cancer patients (1.6%) and controls (0.5%), and shows independent segregation with breast cancer in 2 of 4 families analyzed. The remaining 10 variants were more abundant in patients (21) compared to the controls (12) although the difference was not statistically significant. Interestingly, we found no increased breast cancer risk associated with the splice site mutation IVS2+1G-->A or the most common missense mutation I157T, which account for more than half (12/21) of the variants observed in patients. The low prevalence and penetrance of the exon 10 deletion mutations together with no, or an uncertain elevation in risk for other CHEK2 mutations suggests a limited relevance for CHEK2 mutations in familial breast cancer. Further evaluation of the unique variants observed in breast cancer is required to determine if they may play a role in a polygenic model of familial breast cancer. Nevertheless, it seems premature to include CHEK2 screening in genetic testing.

Introduction An "ideal" marker to monitor MRD after allo-SCT should be informative in the majority of pts and facilitate the use of a method with high sensitivity and specificity in a standardized manner. In addition, to allow repeated monitoring in timely tight intervals but also ensuring patients' comfort such marker should ideally be measurable in peripheral blood (PB). Despite recent identification of several molecular aberrations AML and MDS, many of these do not fulfil the above-mentioned requirements, as they are present only in small patient groups, their potential instability during disease course, the absence of standardized assays and the need for BM as optimal sample source. A molecular marker which might provide these properties is the WT1 gene, as it is overexpressed in the majority of AML pts and in about 50% MDS pts and is measurable in PB by a standardized assay. To estimate its value after allo-SCT we compared serial WT1 measurement with other methods used to monitor MRD in a real-life situation. Patients and Methods For this retrospective analysis all AML and MDS pts who underwent allo-SCT at our center between 2012 and 2016 were screened for PB WT1 mRNA overexpression using the ELN certified Ipsogen® WT1 ProfileQuant® Kit. Pts with WT1 overexpression, as defined by an validated cut-off level of 50 copies/104 ABL copies, were routinely monitored after transplant. In addition, in all pts STR-based chimerism analysis was performed. In pts with chromosomal aberrations existing prior allo-SCT metaphase and FISH analysis was performed, while XY FISH was additionally conducted in pts with sex-mismatched donor-recipient constellation. Furthermore, pts with molecular markers were regularly monitored by NGS or qPCR. Results of WT1 monitoring were correlated with clinical course and compared with results obtained from the other methods. Results Of 104 screened pts 59 (57%) showed an WT1 overexpression at diagnosis and underwent an allo-SCT. This included 40 AML pts (WT1 overexpressed in 66%) and 19 MDS pts (WT1 overexpressed in 44%). Chimerism analysis was accessible in all 59 pts (100%), while 20 pts (34%) could also be monitored by XY-FISH. Additionally, in 40 pts (68%) cytogenetics and FISH were applicable, while 22 pts (37%) could be investigated by NGS or qPCR. Overall, in 5 pts MRD could be monitored by WT1 and chimerism only, while in 29 pts MRD could be monitored by 1, in 22 pts by 2 and in 3 pts by 3 additional methods. With a median follow up of 13 months (2 - 51) we analyzed a total of 472 WT1 samples reflecting a median of 8 samples per patient (2 - 19). One month after allo-SCT 57 pts (97%) showed complete remission and a rapid decrease of WT1 expression below threshold. Only 2 pts had persistant hematological disease with sustained WT1 overexpression. Twenty-four pts (41%) experienced hematologic (62%) or molecular (38%) relapse at a median of 126 d (28 - 938 ) after allo-SCT. In 20 (83%) of these at least one WT1 value wasabove the cut-off before relapse. Median time from first elevated WT1 to relapse was 2 weeks (0 - 27). Only 4 pts (17%) with molecular relapse showed WT1 expression below cut-off. In contrast, known molecular aberrations were found again in 63% and loss of complete donor-chimerism or a positive signal in XY-FISH analyses were only seen in 46% and 57% before relapse. Furthermore, cytogenetics or FISH detected known or new aberrations in 39% before relapse. From 35 pts remaining in CR for a median of 13 months, only 1 (3%) had a transient increase of WT1 expression above the cut-off, whereas WT1 levels of the other 34 pts persisted below. Three pts (9%) with ongoing remission showed a transient decrease of donor-chimerism, whereas even 31% of pts accessible for XY-FISH showed temporary conspicuous results. Conventional cytogenetics and FISH in pts with CR showed transient abnormalities in 18%, whereas in 14% with molecular aberrations these were temporary detectable. Conclusion Measurement of PB WT1 overexpression is an easy accessible method to monitor MRD after allo-SCT that can be employed by a standardized assay in the majority of AML and MDS pts independent from their individual leukemic genotype. Besides these advantages, the results from our real-life experience also suggest that WT1 overexpression allows sensitive detection of imminent relapse after allo-SCT. Taking into account the methodical restrictions of this retrospective analysis, our data requires confirmation in a prospective trial. Disclosures Gattermann: Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: travel, accomodation expenses, Research Funding. Kobbe:Jansen: Honoraria, Other: travel support; Celgene: Honoraria, Other: travel support, Research Funding.

Molecular sensing in the lingual mucosa and in the gastro-intestinal tract play a role in the detection of ingested harmful drugs and toxins. Therefore, genetic polymorphisms affecting the capability of initiating these responses may be critical for the subsequent efficiency of avoiding and/or eliminating possible threats to the organism. By using a tagging approach in the region of Taste Receptor 2R38 (TAS2R38) gene, we investigated all the common genetic variation of this gene region in relation to colorectal cancer risk with a case-control study in a German population (709 controls and 602 cases) and in a Czech population (623 controls and 601 cases). We found that there were no significant associations between individual SNPs of the TAS2R38 gene and colorectal cancer in the Czech or in the German population, nor in the joint analysis. However, when we analyzed the diplotypes and the phenotypes we found that the non-taster group had an increased risk of colorectal cancer in comparison to the taster group. This association was borderline significant in the Czech population, (OR = 1.28, 95% CI 0.99-1.67; P(value) = 0.058) and statistically significant in the German population (OR = 1.36, 95% CI 1.06-1.75; P(value) = 0.016) and in the joint analysis (OR = 1.34, 95% CI 1.12-1.61; P(value) = 0.001). In conclusion, we found a suggestive association between the human bitter tasting phenotype and the risk of CRC in two different populations of Caucasian origin.

Contains fulltext : 96266.pdf (Publisher’s version ) (Closed access) Recently, we identified 3' end deletions in the EPCAM gene as a novel cause of Lynch syndrome. These truncating EPCAM deletions cause allele-specific epigenetic silencing of the neighboring DNA mismatch repair gene MSH2 in tissues expressing EPCAM. Here we screened a cohort of unexplained Lynch-like families for the presence of EPCAM deletions. We identified 27 novel independent MSH2-deficient families from multiple geographical origins with varying deletions all encompassing the 3' end of EPCAM, but leaving the MSH2 gene intact. Within The Netherlands and Germany, EPCAM deletions appeared to represent at least 2.8% and 1.1% of the confirmed Lynch syndrome families, respectively. MSH2 promoter methylation was observed in epithelial tissues of all deletion carriers tested, thus confirming silencing of MSH2 as the causative defect. In a total of 45 families, 19 different deletions were found, all including the last two exons and the transcription termination signal of EPCAM. All deletions appeared to originate from Alu-repeat mediated recombination events. In 17 cases regions of microhomology around the breakpoints were found, suggesting nonallelic homologous recombination as the most likely mechanism. We conclude that 3' end EPCAM deletions are a recurrent cause of Lynch syndrome, which should be implemented in routine Lynch syndrome diagnostics.

Germline mutations in mismatch repair (MMR) genes, tumours with high microsatellite instability (MSI-H) and loss of MMR protein expression are the hallmarks of HNPCC (Lynch syndrome). While somatic MLH1 promoter hypermethylation is generally accepted in the tumorigenesis of sporadic tumours, abnormal MLH1 promoter methylation in normal body cells is controversially discussed as a mechanism predisposing patients to HNPCC. In all 94 patients suspected of HNPCC-syndrome with a mean age of onset of 45.5 years, MLH1-deficiency in their tumours but no germline mutation, underwent methylation-specific PCR-screening for MLH1 promoter methylation. In peripheral blood cells of 12 patients an MLH1 promoter methylation, in seven informative cases allele-specific, was found. Normal colonic tissue, buccal mucosa, and tumour tissue available from three patients also presented abnormal methylation in the MLH1 promoter. The heredity of aberrant methylation is questionable. Pro: MLH1 promoter methylation was found in a patient and his mother giving evidence for a familial predisposition for an epimutation in MLH1. Contra: a de novo set-up of methylation in one patient, a mosaic or incomplete methylation pattern in six patients, and no evidence for inheritance of MLH1 promoter methylation in the remaining families. Our findings provide strong evidence that MLH1 promoter methylation in normal body cells mimics HNPCC and constitutes a pathogenic pre-lesion in MLH1. The identification of hypermethylation as an epigenetic defect has important implications for surveillance recommendations, as these patients should be treated like Lynch syndrome patients, whereas the heritability of methylation is still under investigation.

Fanconi anaemia (FA) is a rare recessive DNA repair disorder clinically characterised by congenital malformations, progressive bone marrow failure and a high propensity for developing malignancies at an early age, predominantly acute myeloid leukaemia (AML) and squamous cell carcinoma. It is conceivable that a number of patients with hypomorphic mutations are not diagnosed as FA until severe complications in the treatment of a malignancy occur. Here, we report on a patient with FA-A, diagnosed only at the age of 49 years due to persistent pancytopenia and myelodysplastic syndrome/AML induced by a first cycle of chemotherapy for bilateral metachronic breast cancer. This exceptional case clearly demonstrates that, in instances of long-lasting mild pancytopenia or development of malignancies, especially at an unusually young age, FA should be ruled out, irrespective of the patient's age and features, especially before inflicting severe genotoxic stress.

Resistance to CD95 (Apo-1/Fas)-mediated apoptosis is a typical feature of breast cancer cells. Recent studies identified deleterious mutations of the CD95 gene not only in a variety of B cell lymphomas but also in a number of solid tumor entities. Therefore, we amplified and sequenced selected regions of the CD95 gene from 48 breast cancer cases and 10 cell lines but no mutation was found. In the presence of both polymorphic alleles, loss of heterozygosity was excluded in 27 informative cases. We conclude, that relevant somatic mutations of the CD95 gene occur, if at all, at a low frequency and are not the primary cause for resistance to CD95-mediated apoptosis in breast cancer. © 2001 Wiley-Liss, Inc.

Introduction Myelodysplastic syndromes (MDS) are heterogeneous regarding clinical characteristics, as well as prognosis and treatment approaches. Therapeutic decision making relies greatly on prognostic scoring systems. Recently, the gold-standard IPSS (International prognostic scoring system [Greenberg Blood 1997]) has been revised. The new IPSS-R still uses cell counts, marrow blast count, and karyotypic abnormalities to stratify patients (pts) into risk groups [Greenberg Blood 2012]. However, more than 50% of all MDS pts present with a normal karyotype and even in pts with identical chromosomal abnormalities outcome may vary. Somatic mutations are more common than cytogenetic abnormalities and can be identified in over 70% of pts including pts with normal karyotype [Bejar JCO 2012]. Therefore, the addition of such mutations to common prognostic markers might help to refine prognostication in MDS pts and improve therapeutic procedures by individualizing MDS treatment. Patients and Methods We analyzed 182 pts with different subtypes of MDS: RCUD, RARS, MDS-U, 5q- (n=28), RCMD +/-RS (n=85), RAEB 1 (n=16), RAEB 2 (n=25), MDS/MPD (n=24), AML Various molecular assays were performed including sensitive next-generation sequencing for mutations in ASXL1, DNMT3A, EZH2, FLT3-ITD, IDH1, KRAS, MLL-PTD, NRAS, RUNX1, SF3B1, SFRS2, TET2, and TP53. Most of the data was collected prospectively within the clinical routine diagnostic procedures. During that time the marker panel was adjusted, when new analyses became available. Thus, not all markers are currently available for all pts (see table). To assess the impact of the biomarkers, Kaplan-Meier curves were estimated starting at the day of diagnosis. Results The most frequent mutation was TET2 (30.2%), followed by ASXL1 (25.4%), SF3B1 (22.9%), SFRS2 (22.2%), RUNX1 (20.4%), DNMT3A (13.7%), TP53 (9.9%), EZH2 (9.0%), NRAS (4.7%), MLL-PTD (3.8), IDH1 (3.5%), FLT3-ITD (3.3%), KRAS (2.8%), IDH2 (2.8%), and CBL (2.2%). Three pts with normal karyotype, who would have been classified as ICUS (idiopathic cytopenia of undetermined significance) due to only mild dysplasia, were reclassified as MDS-U as they exhibited typical somatic mutations (RUNX1 plus TET2; TET2, and DNMT3A). Median follow up was 3.8 years (95% confidence interval [CI] 3.1-4.5). During this time 74 pts died and 108 pts were censored at the last date they were known to be alive. Median survival was 4.9 years (95% CI 2.7-7.1). A significant influence on survival in univariate analysis could be demonstrated for TP53 (p Conclusion We could confirm mutations in TP53, EZH2, RUNX1, and ASXL1 to be predictors of poor overall survival, while SF3B1 mutations conferred a favorable prognosis in pts with MDS. Integrating mutation assessment into the clinical routine might improve diagnostic procedures as well as prognostication, and individualize treatment approaches. Further analyses are ongoing. Disclosures: Kuendgen: Celgene: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Gattermann:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Götze:Celgene Corp: Honoraria. Germing:Celgene: Honoraria, Research Funding.

Background Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHSR), has two major functions: the stimulation of the growth hormone production and the stimulation of food intake. Accumulating evidence also indicates a role of ghrelin in cancer development. Methods We conducted a case-control study to examine the association of common genetic variants in the genes coding for ghrelin (GHRL) and its receptor (GHSR) with colorectal cancer risk. Pairwise tagging was used to select the 11 polymorphisms included in the study. The selected polymorphisms were genotyped in 680 cases and 593 controls from the Czech Republic. Results We found two SNPs associated with lower risk of colorectal cancer, namely SNPs rs27647 and rs35683. We replicated the two hits, in additional 569 cases and 726 controls from Germany. Conclusion A joint analysis of the two populations indicated that the T allele of rs27647 SNP exerted a protective borderline effect (Ptrend = 0.004).