Elisa Schipper

Identification of differentially expressed microRNAs in human male breast cancer

BackgroundThe discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancer biology. Genetic and epigenetic data indicate oncogenic or tumor suppressor function of these pleiotropic regulators. Therefore, many studies analyzed the expression and function of microRNA in human breast cancer, the most frequent malignancy in females. However, nothing is known so far about microRNA expression in male breast cancer, accounting for approximately 1% of all breast cancer cases.MethodsThe expression of 319 microRNAs was analyzed in 9 primary human male breast tumors and in epithelial cells from 15 male gynecomastia specimens using fluorescence-labeled bead technology. For identification of differentially expressed microRNAs data were analyzed by cluster analysis and selected statistical methods.Expression levels were validated for the most up- or down-regulated microRNAs in this training cohort using real-time PCR methodology as well as in an independent test cohort comprising 12 cases of human male breast cancer.ResultsUnsupervised cluster analysis separated very well male breast cancer samples and control specimens according to their microRNA expression pattern indicating cancer-specific alterations of microRNA expression in human male breast cancer. miR-21, miR519d, miR-183, miR-197, and miR-493-5p were identified as most prominently up-regulated, miR-145 and miR-497 as most prominently down-regulated in male breast cancer.ConclusionsMale breast cancer displays several differentially expressed microRNAs. Not all of them are shared with breast cancer biopsies from female patients indicating male breast cancer specific alterations of microRNA expression.

Loss of Imprinting and Allelic Switching at the DLK1-MEG3 Locus in Human Hepatocellular Carcinoma

Deregulation of imprinted genes is an important molecular mechanism contributing to the development of cancer in humans. However, knowledge about imprinting defects in human hepatocellular carcinoma (HCC), the third leading cause of cancer mortality worldwide, is still limited. Therefore, a systematic meta-analysis of the expression of 223 imprinted loci in human HCC was initiated. This screen revealed that the DLK1-MEG3 locus is frequently deregulated in HCC. Deregulation of DLK1 and MEG3 expression accompanied by extensive aberrations in DNA methylation could be confirmed experimentally in an independent series of human HCC (n = 40) in more than 80% of cases. Loss of methylation at the DLK1-MEG3 locus correlates linearly with global loss of DNA methylation in HCC (r2 = 0.63, p<0.0001). Inhibition of DNMT1 in HCC cells using siRNA led to a reduction in MEG3-DMR methylation and concomitant increase in MEG3 RNA expression. Allele-specific expression analysis identified loss of imprinting in 10 out of 31 informative samples (32%), rendering it one of the most frequent molecular defects in human HCC. In 2 cases unequivocal gain of bi-allelic expression accompanied by substantial loss of methylation at the IG-DMR could be demonstrated. In 8 cases the tumour cells displayed allelic switching by mono-allelic expression of the normally imprinted allele. Allelic switching was accompanied by gains or losses of DNA methylation primarily at IG-DMR1. Analysis of 10 hepatocellular adenomas (HCA) and 5 cases of focal nodular hyperplasia (FNH) confirmed that this epigenetic instability is specifically associated with the process of malignant transformation and not linked to increased proliferation per se. This widespread imprint instability in human HCC has to be considered in order to minimize unwanted side-effects of therapeutic approaches targeting the DNA methylation machinery. It might also serve in the future as predictive biomarker and for monitoring response to epigenetic therapy.

Concordant hypermethylation of intergenic microRNA genes in human hepatocellular carcinoma as new diagnostic and prognostic marker

Epigenetic inactivation by aberrant DNA methylation has been reported for many microRNA genes in various human malignancies. However, relatively little is known about microRNA gene methylation in hepatocellular carcinoma (HCC). Therefore, a systematic screen for identification of aberrantly hypermethylated microRNA genes in HCC was initiated. The methylation status of 39 intergenic CpG island associated microRNA genes was analyzed in HCC cell lines (n = 7), immortalized hepatocytes (n = 2) and normal liver samples (n = 5). Subsequently, 13 differentially methylated microRNA genes were analyzed in primary human HCC samples (n = 40), benign liver tumors (n = 15) and the adjacent liver tissues employing pyrosequencing. Expression of microRNA genes was measured using quantitative real‐time polymerase chain reaction (RT‐PCR). In addition, DNA methylation and expression of microRNA genes were measured after DNMT1 knockdown or DNMT inhibition. Aberrant hypermethylation and concomitant reduction in expression of intergenic microRNA genes is a frequent event in human HCC: hsa‐mir‐9‐2 (23%), hsa‐mir‐9‐3 (50 %), hsa‐mir‐124‐1 (20%), hsa‐mir‐124‐2 (13%), hsa‐mir‐124‐3 (43%), hsa‐mir‐129‐2 (58%), hsa‐mir‐596 (28%) and hsa‐mir‐1247 (38%). Altogether, it affects 90% of the HCC specimens under study. MicroRNA gene methylation is not found in hepatocellular adenoma (n = 10) and focal nodular hyperplasia (n = 5). DNMT1 knockdown or DNMT inhibition reduced microRNA gene methylation and stimulated expression. In primary human HCC specimens hypermethylation and expression of microRNA genes showed an inverse correlation. Concordant hypermethylation of three or more microRNA genes is a highly specific marker for the detection of HCC and for poor prognosis.

Deregulation of RB1 expression by loss of imprinting in human hepatocellular carcinoma.

The tumour suppressor gene RB1 is frequently silenced in many different types of human cancer, including hepatocellular carcinoma (HCC). However, mutations of the RB1 gene are relatively rare in HCC. A systematic screen for the identification of imprinted genes deregulated in human HCC revealed that RB1 shows imprint abnormalities in a high proportion of primary patient samples. Altogether, 40% of the HCC specimens (16/40) showed hyper‐ or hypomethylation at the CpG island in intron 2 of the RB1 gene. Re‐analysis of publicly available genome‐wide DNA methylation data confirmed these findings in two independent HCC cohorts. Loss of correct DNA methylation patterns at the RB1 locus leads to the aberrant expression of an alternative RB1–E2B transcript, as measured by quantitative real‐time PCR. Demethylation at the intron 2 CpG island by DNMT1 knock‐down or aza‐deoxycytidine (DAC) treatment stimulated expression of the RB1–E2B transcript, accompanied by diminished RB1 main transcript expression. No aberrant DNA methylation was found at the RB1 locus in hepatocellular adenoma (HCA, n = 10), focal nodular hyperplasia (FNH, n = 5) and their corresponding adjacent liver tissue specimens. Deregulated RB1 expression due to hyper‐ or hypomethylation in intron 2 of the RB1 gene is found in tumours without loss of heterozygosity and is associated with a decrease in overall survival (p = 0.032) if caused by hypermethylation of CpG85. This unequivocally demonstrates that loss of imprinting represents an important additional mechanism for RB1 pathway inactivation in human HCC, complementing well‐described molecular defects. Copyright

Oncogenic PIK3CA mutations in lobular breast cancer progression

Infiltrating lobular breast cancer (ILBC) is a tumor‐biologically distinct breast cancer subtype. A high frequency of oncogenic PIK3CA mutations has been reported in ILBC, which may allow for targeted therapy with newly developed PI3K inhibitors. This is of particular clinical relevance for ILBC patients, who have failed to respond to current treatment regimes and suffer from tumor recurrence or dissemination. In anticipation of this therapeutic strategy, we investigated PIK3CA mutations in ILBC with special reference to late stage tumor progression. A total of 88 ILBCs from 73 patients, including primary tumors (PTs, n = 43), ipsilateral locally recurrent tumors (LRTs, n = 15), and distant organ metastases (DOMs, n = 30), were compiled on tissue microarrays. Established ILBC marker proteins were evaluated by immunohistochemistry and PIK3CA hot spot mutations in exons 9 and 20 by direct sequencing. Matched PT/LRT, PT/DOM, and DOM/DOM cases were characterized on a patient‐by‐patient basis. Following correction for redundant patient representations, mutation frequencies were compared in PTs versus LRTs or DOMs. Nearly all specimens were E‐cadherin‐negative (99%), estrogen receptor (ER)‐positive (91%), and lacked basal epithelial markers (100%), demonstrating correct ILBC classification. PIK3CA mutations were detected in 32/88 (36%) specimens. The mutation rate was similar in PTs (33%) and DOMs (26%, P = 0.769), but approximately two‐fold increased in LRTs (69%, P = 0.022). Consistently, matched PT/LRT and LRT/DOM cases showed additional PIK3CA mutations in LRTs. Intriguingly, these findings imply that PIK3CA mutations are positively selected for during ILBC progression to local recurrence but not distant metastasis, which may have clinical implications for PI3K inhibitor‐based therapy.

Routine clinical mutation profiling using next generation sequencing and a customized gene panel improves diagnostic precision in myeloid neoplasms

Microscopic examination of myelodysplastic syndromes (MDS) and myelodysplastic-myeloproliferative neoplasms (MDS/MPN) may be challenging because morphological features can overlap with those of reactive states. Demonstration of clonal hematopoiesis provides a diagnostic clue and has become possible by comprehensive mutation profiling of a number of frequently mutated genes, some of them with large coding regions. To emphasize the potential benefit of NGS in hematopathology we present sequencing results from routinely processed formalin-fixed and paraffin-embedded (FFPE) bone marrow trephines (n = 192). A customized amplicon-based gene panel including 23 genes frequently mutated in myeloid neoplasms was established and implemented. Thereby, 629,691 reads per sample (range 179,847–1,460,412) and a mean coverage of 2,702 (range 707–6,327) could be obtained, which are sufficient for comprehensive mutational profiling. Seven samples failed in sequencing (3.6%). In 185 samples we found in total 269 pathogenic variants (mean 1.4 variants per patient, range 0-5), 125 Patients exhibit at least one pathogenic mutation (67.6%). Variants show allele frequencies ranging from 6.7% up to 95.7%. Most frequently mutated genes were TET2 (28.7%), SRSF2 (19.5%), ASXL1 (8.6%) and U2AF1 (8.1%). The mutation profiling increases the diagnostic precision and adds prognostic information.

IPH-926 lobular breast cancer cells harbor a p53 mutant with temperature-sensitive functional activity and allow for profiling of p53-responsive genes

Profiling of p53-responsive genes has been carried out in different cellular models, most of which involved genetic modifications or cytotoxic stimulation. We report on the utilization of IPH-926 human lobular breast cancer cells for the profiling of p53-responsive genes using a novel approach without such modifications. We discovered that IPH-926 cells harbor a homozygous TP53 missense mutation encoding for a rare p53 mutant (E285K) with temperature-sensitive (ts) loss of function characteristics. This mutation had evolved as a late, secondary genetic event during the natural clonal evolution of the corresponding lobular carcinoma. In vitro temperature shifts reconstituted endogenous wild-type p53 activity in IPH-926, as evidenced by induction of p21Waf1. Transcriptional alterations associated with restored p53 function were profiled using Affymetrix microarrays and a new strategy to gate out non-specific temperature effects. At the P=0.0005 significance level, 60 genes were differentially expressed following reconstitution of p53 activity. These genes included CDKN1A, MDM2 and PHLDA3, a recently described p53-inducible inhibitor of AKT. Similar transcriptional alterations were observed upon reconstitution of p53 activity in BT-474 cells, which also harbor ts-p53 E285K, and in ASPC1 cells transduced with ts-p53 A138V. Consistent with these models, low PHLDA3 expression was associated with nuclear p53 accumulation, indicative of deleterious TP53 mutations, in primary breast cancers. From a molecular point of view, IPH-926 thus provides a new tool to study transcriptional programs controlled by p53. From a tumor pathology perspective, IPH-926 also provides the first direct evidence of a p53-related clonal evolutionary pathway in lobular breast cancer progression.

Comprehensive Molecular Profiling of Archival Bone Marrow Trephines Using a Commercially Available Leukemia Panel and Semiconductor-Based Targeted Resequencing

Comprehensive mutation profiling becomes more and more important in hematopathology complementing morphological and immunohistochemical evaluation of fixed, decalcified and embedded bone marrow biopsies for diagnostic, prognostic and also predictive purposes. However, the number and the size of relevant genes leave conventional Sanger sequencing impracticable in terms of costs, required input DNA, and turnaround time. Since most published protocols and commercially available reagents for targeted resequencing of gene panels are established and validated for the analysis of fresh bone marrow aspirate or peripheral blood it remains to be proven whether the available technology can be transferred to the analysis of archival trephines. Therefore, the performance of the recently available Ion AmpliSeq AML Research panel (LifeTechnologies) was evaluated for the analysis of fragmented DNA extracted from archival bone marrow trephines. Taking fresh aspirate as gold standard all clinically relevant mutations (n = 17) as well as 25 well-annotated SNPs could be identified reliably with high quality in the corresponding archival trephines of the training set (n = 10). Pre-treatment of the extracted DNA with Uracil-DNA-Glycosylase reduced the number of low level artificial sequence variants by more than 60%, vastly reducing time required for proper evaluation of the sequencing results. Subsequently, randomly picked FFPE samples (n = 41) were analyzed to evaluate sequencing performance under routine conditions. Thereby all known mutations (n = 43) could be verified and 36 additional mutations in genes not yet covered by the routine work-up (e.g., TET2, ASXL1, DNMT3A), demonstrating the feasibility of this approach and the gain of diagnostically relevant information. The dramatically reduced amount of input DNA, the increase in sensitivity as well as calculated cost-effectiveness, low hands on , and turn-around-time, necessary for the analysis of 237 amplicons strongly argue for replacing Sanger sequencing by this semiconductor-based targeted resequencing approach.

Is Upregulation of BCL2 a Determinant of Tumor Development Driven by Inactivation of CDH1/E- Cadherin?

Inactivation of CDH1, encoding E-cadherin, promotes cancer initiation and progression. According to a newly proposed molecular mechanism, loss of E-cadherin triggers an upregulation of the anti-apoptotic oncoprotein BCL2. Conversely, reconstitution of E-cadherin counteracts overexpression of BCL2. This reciprocal regulation is thought to be critical for early tumor development. We determined the relevance of this new concept in human infiltrating lobular breast cancer (ILBC), the prime tumor entity associated with CDH1 inactivation. BCL2 expression was examined in human ILBC cell lines (IPH-926, MDA-MB-134, SUM-44) harboring deleterious CDH1 mutations. To test for an intact regulatory axis between E-cadherin and BCL2, wild-type E-cadherin was reconstituted in ILBC cells by ectopic expression. Moreover, BCL2 and E-cadherin were evaluated in primary invasive breast cancers and in synchronous lobular carcinomas in situ (LCIS). MDA-MB-134 and IPH-926 showed little or no BCL2 expression, while SUM-44 ILBC cells were BCL2-positive. Reconstitution of E-cadherin failed to impact on BCL2 expression in all cell lines tested. Primary ILBCs were almost uniformly E-cadherin-negative (97%) and were frequently BCL2-negative (46%). When compared with an appropriate control group, ILBCs showed a trend towards an increased frequency of BCL2-negative cases (P = 0.064). In terminal duct-lobular units affected by LCIS, the E-cadherin-negative neoplastic component showed a similar or a reduced BCL2-immunoreactivity, when compared with the adjacent epithelium. In conclusion, upregulation of BCL2 is not involved in lobular breast carcinogenesis and is unlikely to represent an important determinant of tumor development driven by CDH1 inactivation.

Molecular Analysis of Circulating Cell-Free DNA from Lung Cancer Patients in Routine Laboratory Practice: A Cross-Platform Comparison of Three Different Molecular Methods for Mutation Detection

Circulating cell-free DNA (cfDNA), which is isolated from blood plasma, represents a noninvasive source for the detection of mutations conferring resistance against epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small-cell lung cancer patients. In advanced disease stages, performing regular biopsies is often not possible because of the general health condition of the patients. Furthermore, a biopsy of a single tumor lesion or metastasis may not reflect the heterogeneous genotype of the tumor and its metastases. Plasma cfDNA represents an alternative material for molecular monitoring of patients under therapy. Herein, we present a cross-platform comparison of three different molecular methods [digital PCR, next-generation sequencing (NGS), and quantitative PCR] to detect clinically relevant mutations in cfDNA. We validated our workflow with commercially available cfDNA reference material (5.0%, 1.0%, and 0.1% mutation frequency, respectively). Digital PCR and NGS detect reliably 0.1% allele frequency of the EGFR p.T790M mutation. Furthermore, we analyzed 55 cfDNA preparations from patients with lung cancer to compare reliability and sensitivity of the three methods under routine conditions and achieved 96.0% concordance of p.T790M results. A limit of detection for mutation calling using digital PCR (>0.1%) and NGS (>0.2%) was established. In total, 62.5% of known primary EGFR mutations were successfully detected in cfDNA. In 56.0% of the patients with detectable EGFR primary mutations, we identified a resistance conferring the EGFR p.T790M mutation.