Search

Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer. It is defined by cholangiocytic differentiation and has poor prognosis. Recently, epigenetic processes have been shown to play an important role in cholangiocarcinogenesis. We performed an integrative analysis on 52 iCCAs using both genetic and epigenetic data with a specific focus on DNA methylation components. We found recurrent isocitrate dehydrogenase 1 (IDH1) and IDH2 (28%) gene mutations, recurrent arm-length copy number alterations (CNAs), and focal alterations such as deletion of 3p21 or amplification of 12q15, which affect BRCA1 Associated Protein 1, polybromo 1, and mouse double minute 2 homolog. DNA methylome analysis revealed excessive hypermethylation of iCCA, affecting primarily the bivalent genomic regions marked with both active and repressive histone modifications. Integrative clustering of genetic and epigenetic data identified four iCCA subgroups with prognostic relevance further designated as IDH, high (H), medium (M), and low (L) alteration groups. The IDH group consisted of all samples with IDH1 or IDH2 mutations and showed, together with the H group, a highly disrupted genome, characterized by frequent deletions of chromosome arms 3p and 6q. Both groups showed excessive hypermethylation with distinct patterns. The M group showed intermediate characteristics regarding both genetic and epigenetic marks, whereas the L group exhibited few methylation changes and mutations and a lack of CNAs. Methylation-based latent component analysis of cell-type composition identified differences among these four groups. Prognosis of the H and M groups was significantly worse than that of the L group. Conclusion: Using an integrative genomic and epigenomic analysis approach, we identified four major iCCA subgroups with widespread genomic and epigenomic differences and prognostic implications. Furthermore, our data suggest differences in the cell-of-origin of the iCCA subtypes., (© 2019 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of American Association for the Study of Liver Diseases.)

Introduction: Circulating tumor cells (CTCs) may be used to improve cancer diagnosis, prognosis, and treatment. However, because knowledge regarding CTC biology is limited and the numbers of CTCs and CTC-positive cancer patients are low, progress in this field is slow. We addressed this limitation by combining diagnostic leukapheresis (DLA) and microfluidic enrichment to obtain large numbers of viable CTCs from metastasized breast cancer patients., Methods: DLA was applied to 9 patients, and 7.5 mL of peripheral blood was drawn. CTCs were enriched with the Parsortix™ system. The quality of CTCs from fresh and cryopreserved DLA products was tested, and CTCs were cultured in vitro. Single uncultured and cultured CTCs were isolated by micromanipulation to determine different parameters, such as genomic aberrations and mutation profiles of selected tumor-associated genes. Expression levels of estrogen receptor and HER2/neu were monitored during in vitro culture., Results: Viable CTCs from peripheral blood and fresh or frozen DLA products could be enriched. DLA increased the likelihood of successful CTC culture. Cryopreserved DLA products could be stored with minimal CTC loss and no overt reduction in the tumor cell quality and viability during an observation period of up to 3 years. The analyzed parameters did not change during in vitro culture. DLA samples with high CTC numbers and lower ratios of apoptotic CTCs were more likely to grow in culture., Conclusions: The increased CTC numbers from fresh or cryopreserved DLA products facilitate multiple functional and molecular analyses and, thus, could improve our knowledge of their biology., (© 2019 American Association for Clinical Chemistry.)

Lung adenocarcinoma (ADC) is the most prevalent subtype of lung cancer and characterized by considerable morphological and mutational heterogeneity. However, little is known about the epigenomic intratumor variability between spatially separated histological growth patterns of ADC. In order to reconstruct the clonal evolution of histomorphological patterns, we performed global DNA methylation profiling of 27 primary tumor regions, seven matched normal tissues and six lymph node metastases from seven ADC cases. Additionally, we investigated the methylation data from 369 samples of the TCGA ADC cohort. All regions showed varying degrees of methylation changes between segments of different, but also of the same growth patterns. Similarly, copy number variations were seen between spatially distinct segments of each patient. Hierarchical clustering of promoter methylation revealed extensive heterogeneity within and between the cases. Intratumor DNA methylation heterogeneity demonstrated a branched clonal evolution of ADC regions driven by genomic instability with subclonal copy number changes. Notably, methylation profiles within tumors were not more similar to each other than to those from other individuals. In two cases, different tumor regions of the same individuals were represented in distant clusters of the TCGA cohort, illustrating the extensive epigenomic intratumor heterogeneity of ADCs. We found no evidence for the lymph node metastases to be derived from a common growth pattern. Instead, they had evolved early and separately from a particular pattern in each primary tumor. Our results suggest that extensive variation of epigenomic features contributes to the molecular and phenotypic heterogeneity of primary ADCs and lymph node metastases., (© 2018 UICC.)

Tumor mutational burden (TMB) represents a new determinant of clinical benefit from immune checkpoint blockade that identifies responders independent of PD-L1 expression levels and is currently being explored in clinical trials. Although TMB can be measured directly by comprehensive genomic approaches such as whole-genome and exome sequencing, broad availability, short turnaround times, costs and amenability to formalin-fixed and paraffin-embedded tissue support the use of gene panel sequencing for approximating TMB in routine diagnostics. However, data on the parameters influencing panel-based TMB estimation are limited. Here, we report an extensive in silico analysis of the TCGA data set that simulates various panel sizes and compositions. We demonstrate that panel size is a critical parameter that influences confidence intervals (CIs) and cutoff values as well as important test parameters including sensitivity, specificity, and positive predictive value. Moreover, we evaluate the Illumina TSO500 panel, which will be made available for TMB estimation, and propose dynamic, entity-specific cutoff values based on current clinical trial data. Optimizing the cost-benefit ratio, our data suggest that panels between 1.5 and 3 Mbp are ideally suited to estimate TMB with small CIs, whereas smaller panels tend to deliver imprecise TMB estimates for low to moderate TMB (0-30 muts/Mbp), connected with insufficient separation of hypermutated tumors from non-hypermutated tumors., (© 2018 UICC.)

Anaplastic lymphoma kinase (ALK) sequencing can identify resistance mechanisms and guide next-line therapy in ALK+ non-small-cell lung cancer (NSCLC), but the clinical significance of other rebiopsy findings remains unclear. We analysed all stage-IV ALK+ NSCLC patients with longitudinally assessable TP53 status treated in our institutions (n = 62). Patients with TP53 mutations at baseline (TP53mutbas, n = 23) had worse overall survival (OS) than patients with initially wild-type tumours (TP53wtbas, n = 39, 44 vs. 62 months in median, p = 0.018). Within the generally favourable TP53wtbas group, detection of TP53 mutations at progression defined a "converted" subgroup (TP53mutconv, n = 9) with inferior OS, similar to that of TP53mutbas and shorter than that of patients remaining TP53 wild-type (TP53wtprogr, 45 vs. 94 months, p = 0.043). Progression-free survival (PFS) under treatment with tyrosine kinase inhibitors (TKI) for TP53mutconv was comparable to that of TP53mutbas and also shorter than that of TP53wtprogr cases (5 and 8 vs. 13 months, p = 0.0039). Fewer TP53wtprogr than TP53mutbas or TP53mutconv cases presented with metastatic disease at diagnosis (67% vs. 91% or 100%, p < 0.05). Thus, acquisition of TP53 mutations at progression is associated with more aggressive disease, shorter TKI responses and inferior OS in ALK+ NSCLC, comparable to primary TP53 mutated cases., Competing Interests: n/a

Tyrosine kinase inhibitors (TKI) have improved prognosis in metastatic anaplastic lymphoma kinase (ALK)-driven lung adenocarcinoma, but patient outcomes vary widely. We retrospectively analyzed the clinical course of all cases with assessable baseline TP53 status and/or ALK fusion variant treated at our institutions (n = 102). TP53 mutations were present in 17/87 (20%) and the echinoderm microtubule-associated protein-like 4 (EML4)-ALK variant 3 (V3) in 41/92 (45%) patients. The number of metastatic sites at diagnosis was affected more by the presence of V3 than by TP53 mutations, and highest with both factors (mean 5.3, p < 0.001). Under treatment with ALK TKI, progression-free survival (PFS) was shorter with either TP53 mutations or V3, while double positive cases appeared to have an even higher risk (hazard ratio [HR] = 2.9, p = 0.015). The negative effect of V3 on PFS of TKI-treated patients was strong already in the first line (HR = 2.5, p = 0.037) and decreased subsequently, whereas a trend for PFS impairment under first-line TKI by TP53 mutations became stronger and statistically significant only when considering all treatment lines together. Overall survival was impaired more by TP53 mutations (HR = 4.9, p = 0.003) than by V3 (HR = 2.4, p = 0.018), while patients with TP53 mutated V3-driven tumors carried the highest risk of death (HR = 9.1, p = 0.02). Thus, TP53 mutations and V3 are independently associated with enhanced metastatic spread, shorter TKI responses and inferior overall survival in ALK + lung adenocarcinoma. Both markers could assist selection of cases for more aggressive management and guide development of novel therapeutic strategies. In combination, they define a patient subset with very poor outcome., (© 2018 UICC.)

Approximately half of all pancreatic cysts are neoplastic, mainly comprising intraductal papillary mucinous neoplasms (IPMN), which can progress to invasive carcinoma. Current Fukuoka guidelines have limited sensitivity and specificity in predicting progression of asymptomatic pancreatic cysts. We present first results of the prospective ZYSTEUS biomarker study investigating (i) whether detection of driver mutations in IPMN by liquid biopsy is technically feasible, (ii) which compartment of IPMN is most suitable for analysis, and (iii) implications for clinical diagnostics. Twenty-two patients with clinical inclusion criteria were enrolled in ZYSTEUS. Fifteen cases underwent endoscopic ultrasound (EUS)-guided fine-needle aspiration and cytological diagnostics. Cellular and liquid fraction of the cysts of each case were separated and subjected to deep targeted next generation sequencing (NGS). Clinical parameters, imaging findings (EUS and MRI), and follow-up data were collected continuously. All IPMN cases (n = 12) showed at least one mutation in either KRAS (n = 11) or GNAS (n = 4). Three cases showed both KRAS and GNAS mutations. Six cases harbored multiple KRAS/GNAS mutations. In the three cases with pseudocysts, no KRAS or GNAS mutations were detected. DNA yields were higher and showed higher mutation diversity in the cellular fraction. In conclusion, mutation detection in pancreatic cyst fluid is technically feasible with more robust results in the cellular than in the liquid fraction. Current results suggest that, together with imaging, targeted sequencing supports discrimination of IPMN from pseudocysts. The prospective design of ZYSTEUS will provide insight into diagnostic value of NGS in preoperative risk stratification. Our data provide evidence for an oligoclonal nature of IPMN., (© 2018 Wiley Periodicals, Inc.)

Objectives: The potential role of cancer associated somatic mutations of the mitochondrial genome (mtDNA) is controversial and still poorly understood. Our group and others recently challenged a direct tumorigenic impact and suggested a passenger-like character. In combination with the known increased mutation rate, somatic mtDNA mutations account for an interesting tool to delineate tumor evolution. Here, we comprehensively analyzed the spatial distribution of somatic mtDNA mutations throughout whole tumor sections of pulmonary adenocarcinoma (ADC)., Materials and Methods: Central sections of 19 ADC were analyzed in a segmented manner (11-34 segments/tumor) together with non-neoplastic tissue samples and lymph node metastasis, if present. We performed whole mtDNA sequencing and real-time PCR based quantification of mtDNA copy numbers for all samples. Further, histological growth patterns were determined on H&E sections and the tumor cell content was quantified by digital pathology analyses., Results: Somatic mtDNA mutations were present in 96% (18/19) of the analyzed tumors, either ubiquitously or restricted to specific tumor regions. Spatial and histological mapping of the mutations enabled the identification of subclonal structures and phylogenetic relations within a tumor section indicating different progression levels. In this regard, lymph node metastases seem to be related to early events in ADC development. There was no concurrence between histological and mtDNA mutation based clusters. However, micropapillary patterns occurred only in tumors with ubiquitous mutations. ADC with more than two ubiquitous mutations were associated with shorter disease-free survival (p < 0.01)., Conclusion: Cancer related mtDNA mutations are interesting candidates for the understanding of subclonal ADC evolution and perspectively for monitoring tumor progression. Our data reveal a potential prognostic relevance of somatic mtDNA mutations., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Tumor mutational burden (TMB) is a new biomarker for prediction of response to PD-(L)1 treatment. Comprehensive sequencing approaches (i.e., whole exome and whole genome sequencing) are ideally suited to measure TMB directly. However, as their applicability in routine diagnostics is currently limited by high costs, long turnaround times and poor availability of fresh tissue, targeted next-generation sequencing (NGS) of formalin-fixed and paraffin-embedded (FFPE) samples appears to be a more feasible and straight-forward approach for TMB approximation, which can be seamlessly integrated in already existing diagnostic workflows and pipelines. In this work, we provide an overview of the clinical implications of TMB testing and highlight key parameters including pre-analysis, analysis and post-analytical steps that influence and shape TMB approximation by panel sequencing. Collectively, the data will not only serve as a field guide and state of the art knowledge source for molecular pathologists who consider implementation of TMB measurement in their lab, but also enable clinicians in understanding the specific parameters influencing TMB test results and reporting., Competing Interests: Conflicts of Interest: V Endris: advisory board and lecture fees from AstraZeneca and ThermoFisher. J Leichsenring: consultancy contract with AstraZeneca. S Fröhling: speaker’s honoraria from Amgen, Lilly, PharmaMar and Roche; research funding from AstraZeneca, Pfizer and PharmaMar. M Thomas: advisory board honoraria from Novartis, Lilly, BMS, MSD, Roche, Celgene, Takeda, AbbVie, Boehringer, speaker’s honoraria from Lilly, MSD, Takeda, research funding from AstraZeneca, BMS, Celgene, Novartis, Roche and travel grants from BMS, MSD, Novartis, Boehringer. P Schirmacher: advisory board honoraria from Pfizer, Roche, Novartis, AstraZeneca as well as speaker’s honoraria and research funding from Roche, AstraZeneca and Novartis. A Stenzinger: advisory board honoraria from BMS, AstraZeneca, Novartis, ThermoFisher, speaker’s honoraria from BMS, Illumina, AstraZeneca, MSD, Novartis, Roche, ThermoFisher, and research funding from Chugai. The other authors have no conflicts of interest to declare.

A deeper understanding of melanoma biology has opened up new avenues for mechanistically informed therapies. However, data on the prevalence of druggable genetic lesions in melanoma are still conflicting and real-world performance data on high-throughput genetic profiling of melanoma cases using formalin fixed, paraffin embedded (FFPE) tissue with variable tumour cellularity and quality are lacking. We retrospectively analysed targeted next-generation sequencing data of 274 consecutive melanoma samples obtained for routine diagnostics between December 2013 and July 2017. Actionable mutations were detected in 197 cases (71.9%), of which activating BRAF (mostly p.V600E/K) and RAS (mostly p.Q61R/K) mutations occurred in 40.5% (n = 111) and 30.3% (n = 83) of cases, respectively. We identified driver mutations of the Triple-WT subgroup in 10.6% of cases (n = 29; 10 with activating KIT mutations). Median turnaround time was 5 working days with no dropouts. Tumour cellularity ranged from 5% to 95% and successful sequencing was possible at DNA concentrations as low as 0.03 ng/μL (median 10.58 ng/μL; range 0.03-209.05 ng/μL). Fast, quality-controlled high-throughput genetic profiling of FFPE melanoma samples is feasible and provides a landscape of genetic aberrations in melanoma that is currently relevant in clinical practice and approximates TCGA subtypes., (Copyright © 2018 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Purpose: Rapidly evolving genomics technologies, in particular comprehensive next-generation sequencing (NGS), have led to exponential growth in the understanding of cancer biology, shifting oncology toward personalized treatment strategies. However, comprehensive NGS approaches, such as whole-exome sequencing, have limitations that are related to the technology itself as well as to the input source. Hence, clinical implementation of comprehensive NGS in a quality-controlled diagnostic workflow requires both the standardization of sequencing procedures and continuous validation of sequencing results by orthogonal methods in an ongoing program to enable the determination of key test parameters and continuous improvement of NGS and bioinformatics pipelines., Patients and Methods: We present validation data on 220 patients who were enrolled between 2013 and 2016 in a multi-institutional, genomics-guided precision oncology program (Molecularly Aided Stratification for Tumor Eradication Research) of the National Center for Tumor Diseases Heidelberg and the German Cancer Consortium., Results: More than 90% of clinically actionable genomic alterations identified by combined whole-exome sequencing and transcriptome sequencing were successfully validated, with varying frequencies of discordant results across different types of alterations (fusions, 3.7%; single-nucleotide variants, 2.6%; amplifications, 1.1%; overexpression, 0.9%; deletions, 0.6%). The implementation of new computational methods for NGS data analysis led to a substantial improvement of gene fusion calling over time., Conclusion: Collectively, these data demonstrate the value of a rigorous validation program that partners with comprehensive NGS to successfully implement and continuously improve cancer precision medicine in a clinical setting.

Harnessing the immune system by checkpoint blockade has greatly expanded the therapeutic options for advanced cancer. Since the efficacy of immunotherapies is influenced by the molecular make-up of the tumor and its crosstalk with the immune system, comprehensive analysis of genetic and immunologic tumor characteristics is essential to gain insight into mechanisms of therapy response and resistance. We investigated the association of immune cell contexture and tumor genetics including tumor mutational burden (TMB), copy number alteration (CNA) load, mutant allele heterogeneity (MATH) and specific mutational signatures (MutSigs) using TCGA data of 5722 tumor samples from 21 cancer types. Among all genetic variables, MutSigs associated with DNA repair deficiency and AID/APOBEC gene activity showed the strongest positive correlations with immune parameters. For smoking-related and UV-light-exposure associated MutSigs a few positive correlations were identified, while MutSig 1 (clock-like process) correlated non-significantly or negatively with the major immune parameters in most cancer types. High TMB was associated with high immune cell infiltrates in some but not all cancer types, in contrast, high CNA load and high MATH were mostly associated with low immune cell infiltrates. While a bi- or multimodal distribution of TMB was observed in colorectal, stomach and endometrial cancer where its levels were associated with POLE / POLD1 mutations and MSI status, TMB was unimodal distributed in the most other cancer types including NSCLC and melanoma. In summary, this study uncovered specific genetic-immunology associations in major cancer types and suggests that mutational signatures should be further investigated as interesting candidates for response prediction beyond TMB.

We used whole-genome and transcriptome sequencing to identify clinically actionable genomic alterations in young adults with pancreatic ductal adenocarcinoma (PDAC). Molecular characterization of 17 patients with PDAC enrolled in a precision oncology program revealed gene fusions amenable to pharmacologic inhibition by small-molecule tyrosine kinase inhibitors in all patients with KRAS wild-type ( KRAS WT ) tumors (4 of 17). These alterations included recurrent NRG1 rearrangements predicted to drive PDAC development through aberrant ERBB receptor-mediated signaling, and pharmacologic ERBB inhibition resulted in clinical improvement and remission of liver metastases in 2 patients with NRG1 -rearranged tumors that had proved resistant to standard treatment. Our findings demonstrate that systematic screening of KRAS WT tumors for oncogenic fusion genes will substantially improve the therapeutic prospects for a sizeable fraction of patients with PDAC. Significance: Advanced PDAC is a malignancy with few treatment options that lacks molecular mechanism-based therapies. Our study uncovers recurrent gene rearrangements such as NRG1 fusions as disease-driving events in KRAS wt tumors, thereby providing novel insights into oncogenic signaling and new therapeutic options in this entity. Cancer Discov; 8(9); 1087-95. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1047 ., (©2018 American Association for Cancer Research.)

The aim of this study was to subcategorise large cell carcinoma (LCC) with null immunophenotype according to the World Health Organization (WHO) Classification of 2015 into the existing groups of adenocarcinoma and squamous cell carcinoma by further molecular genetic analysis. Lineage-specific molecular alterations of these tumours could depict additional therapeutic approaches. We analysed a cohort of 35 LCC diagnosed according to the 2004 WHO classification and reclassified them according to the criteria of the 2015 WHO classification. Subsequently, tumours with a null immunophenotype were analysed by targeted next generation sequencing (42 marker genes including TP53, EGFR, KRAS, STK11 and SMARC4A) and fluorescence in situ hybridisation (ROS1, ALK). By applying the criteria of the 2015 WHO classification and subsequent molecular subtyping we could show that out of 35 previously diagnosed LCC, 16 cases could be reclassified into specific NSCLC subtypes using immunohistochemistry. Additionally, based on their mutational pattern, eight of the remaining 19 cases with null immunophenotype could be assigned as 'favour adenocarcinoma'. We demonstrate that molecular subtyping is helpful to further categorise LCC with null immunophenotype. Our findings argue for an algorithm including stratified molecular analysis of all respective cases., (Copyright © 2018 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Lynch syndrome is caused by germline mutations of DNA mismatch repair (MMR) genes. MMR deficiency has long been regarded as a secondary event in the pathogenesis of Lynch syndrome colorectal cancers. Recently, this concept has been challenged by the discovery of MMR-deficient crypt foci in the normal mucosa. We aimed to reconstruct colorectal carcinogenesis in Lynch syndrome by collecting molecular and histology evidence from Lynch syndrome adenomas and carcinomas. We determined the frequency of MMR deficiency in adenomas from Lynch syndrome mutation carriers by immunohistochemistry and by systematic literature analysis. To trace back the pathways of pathogenesis, histological growth patterns and mutational signatures were analyzed in Lynch syndrome colorectal cancers. Literature and immunohistochemistry analysis demonstrated MMR deficiency in 491 (76.7%) out of 640 adenomas (95% CI: 73.3% to 79.8%) from Lynch syndrome mutation carriers. Histologically normal MMR-deficient crypts were found directly adjacent to dysplastic adenoma tissue, proving their role as tumor precursors in Lynch syndrome. Accordingly, mutation signature analysis in Lynch colorectal cancers revealed that KRAS and APC mutations commonly occur after the onset of MMR deficiency. Tumors lacking evidence of polypous growth frequently presented with CTNNB1 and TP53 mutations. Our findings demonstrate that Lynch syndrome colorectal cancers can develop through three pathways, with MMR deficiency commonly representing an early and possibly initiating event. This underlines that targeting MMR-deficient cells by chemoprevention or vaccines against MMR deficiency-induced frameshift peptide neoantigens holds promise for tumor prevention in Lynch syndrome., (© 2018 UICC.)

In order to identify anaplastic lymphoma kinase-driven non-small cell lung cancer (ALK + NSCLC) patients with a worse outcome, who might require alternative therapeutic approaches, we retrospectively analyzed all stage IV cases treated at our institutions with one of the main echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion variants V1, V2 and V3 as detected by next-generation sequencing or reverse transcription-polymerase chain reaction (n = 67). Progression under tyrosine kinase inhibitor (TKI) treatment was evaluated both according to Response Evaluation Criteria in Solid Tumors (RECIST) and by the need to change systemic therapy. EML4-ALK fusion variants V1, V2 and V3 were found in 39%, 10% and 51% of cases, respectively. Patients with V3-driven tumors had more metastatic sites at diagnosis than cases with the V1 and V2 variants (mean 3.3 vs. 1.9 and 1.6, p = 0.005), which suggests increased disease aggressiveness. Furthermore, V3-positive status was associated with earlier failure after treatment with first and second-generation ALK TKI (median progression-free survival [PFS] by RECIST in the first line 7.3 vs. 39.3 months, p = 0.01), platinum-based combination chemotherapy (median PFS 5.4 vs. 15.2 months for the first line, p = 0.008) and cerebral radiotherapy (median brain PFS 6.1 months vs. not reached for cerebral radiotherapy during first-line treatment, p = 0.028), and with inferior overall survival (39.8 vs. 59.6 months in median, p = 0.017). Thus, EML4-ALK fusion variant V3 is a high-risk feature for ALK + NSCLC. Determination of V3 status should be considered as part of the initial workup for this entity in order to select patients for more aggressive surveillance and treatment strategies., (© 2018 UICC.)

This case report presents a male patient with epithelioid mesothelioma that was initially misdiagnosed as cancer of unknown primary (CUP) and correctly identified using molecular panel sequencing. The patient had a prior history of colon and breast cancer. To assess the enlarged mediastinal lymph nodes, retrosternal lymphadenectomy was performed in 2016. The lymph nodes were histologically deemed unrelated to the known breast cancer by the reference pathologist, thus leading to the diagnosis of a CUP syndrome. When the patient presented to our center, targeted deep sequencing of both breast cancer and presumed CUP was performed to address the clonal relationship between both malignancies. A missense mutation in BAP1 was revealed in both samples, with coverage data indicating a germline event. The patient was subsequently counseled by a human geneticist and underwent genetic testing, which confirmed the germline nature of this mutation. Collectively, these data led to the diagnosis of BAP1 ( BRCA1 -associated protein-1) tumor predisposition syndrome (TPDS). With the knowledge of an underlying BAP1 mutation and its known frequent association with epithelioid mesothelioma, the histology was reassessed and the diagnosis was revised to epithelioid mesothelioma. At this point, peritoneal involvement of mesothelioma could be diagnosed and histologically confirmed. This case illustrates the potential of integrated histopathologic and molecular diagnostics in helping to decipher CUP syndromes and establish the correct diagnosis. Additionally, this case highlights typical features of BAP1 TPDS with its general susceptibility to cancers, with pleural and peritoneal mesotheliomas as most prevalent clinical entities and the typically more benign course of these epithelioid mesotheliomas compared with BAP1 -unrelated cases of mesotheliomas., (Copyright © 2018 by the National Comprehensive Cancer Network.)

Background: Somatic copy number alterations (CNAs) contribute to the clinically targetable aberrations in the tumor genome. For both routine diagnostics and biomarkers research, CNA analysis in a single assay together with somatic mutations is highly desirable., Results: Ioncopy is a validated method and easy-to-use software for CNA calling from targeted NGS data. Copy number and significance of CNA are estimated for each gene in each sample. Copy number gains and losses are called after multiple testing corrections controlling FWER or FDR., Conclusions: Ioncopy facilitates calling of CNAs in a cohort of tumors tissues with or without using normal (germline) DNA controls.

The interpatient variability of tumor proteomes has been investigated on a large scale but many tumors display also intratumoral heterogeneity regarding morphological and genetic features. It remains largely unknown to what extent the local proteome of tumors intrinsically differs. Here, we used hepatocellular carcinoma as a model system to quantify both inter- and intratumor heterogeneity across human patient specimens with spatial resolution. We defined proteomic features that distinguish neoplastic from the directly adjacent nonneoplastic tissue, such as decreased abundance of NADH dehydrogenase complex I. We then demonstrated the existence of intratumoral variations in protein abundance that re-occur across different patient samples, and affect clinically relevant proteins, even in the absence of obvious morphological differences or genetic alterations. Our work demonstrates the suitability and the benefits of using mass spectrometry-based proteomics to analyze diagnostic tumor specimens with spatial resolution. Data are available via ProteomeXchange with identifier PXD007052., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The genetic landscape of rare benign tumours and their malignant counterparts is still largely unexplored. While recent work showed that mutant HRAS is present in subsets of poromas and porocarcinomas, a more comprehensive genetic view on these rare adnexal neoplasms is lacking. Using high-coverage next generation sequencing, we investigated the mutational profile of 50 cancer-related genes in 12 cases (six poromas and six porocarcinomas). Non-synonymous mutations were found in two-thirds of both poromas and porocarcinomas. Hotspot HRAS mutations were identified in two poromas (p.G13R and p.Q61R) and one porocarcinoma (p.G13C). While in poromas only few cases showed single mutated genes, porocarcinomas showed greater genetic heterogeneity with up to six mutated genes per case. Recurrent TP53 mutations were found in all porocarcinomas that harboured mutated genes. Non-recurrent mutations in porocarcinomas were found in several additional tumour suppressors (RB1, APC, CDKN2A, and PTEN), and genes implicated in PI3K-AKT and MAPK signalling pathways (ABL1, PDGFRA, PIK3CA, HRAS, and RET). UV-associated mutations were found in TP53, APC, CDKN2A, PTEN, and RET. In conclusion, our study confirms and extends the spectrum of genetic lesions in poromas and porocarcinomas. While poromas exhibited only few mutations, which did not involve TP53, the majority of porocarcinomas harboured UV-mediated mutations in TP53 with some of these cases showing considerable genetic heterogeneity that may be clinically exploitable., (Copyright © 2017 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Background: Combination chemotherapy has shown benefit in the treatment of biliary cancer and further improvements might be achieved by the addition of a biological agent. We report here the effect of chemotherapy with the monoclonal EGFR antibody panitumumab as therapy for KRAS wild-type biliary cancer., Patients and Methods: Patients with advanced biliary tract cancer were randomised (2:1) to receive cisplatin 25 mg/m 2 and gemcitabine 1000 mg/m 2 on day 1 and day 8/q3w with (arm A) or without panitumumab (arm B; 9 mg/kg BW, i.v q3w). The primary end-point was the evaluation of progression-free survival (PFS) at 6 months. Secondary end-points included objective response rate (ORR), overall survival (OS), and toxicity. In addition, a post hoc assessment of genetic alterations was performed. Finally, we performed a meta-analysis of trials with chemotherapy with and without EGFR antibodies., Results: Sixty-two patients were randomised in arm A and 28 patients in arm B. Patients received 7 treatment cycles in median (1-35) with a median treatment duration of 4.7 months (141 days, 8-765). PFS rate at 6 months was 54% in patients treated with cisplatin/gemcitabine and panitumumab but was 73% in patients treated with cisplatin/gemcitabine without antibody, respectively. Secondary end-points were an ORR of 45% in treatment arm A compared with 39% receiving treatment B and a median OS of 12.8 months (arm A) and of 20.1 months (arm B), respectively. In contrast to the p53-status, genetic alterations in IDH1/2 were linked to a high response after chemotherapy and prolonged survival. In accordance with our results, the meta-analysis of 12 trials did not reveal a survival advantage for patients treated with EGFR antibodies compared with chemotherapy alone., Conclusions: Panitumumab in combination with chemotherapy does not improve ORR, PFS and OS in patients with KRAS wild-type, advanced biliary cancer. Genetic profiling should be included in CCA trials to identify and validate predictive and prognostic biomarkers., Clinical Trials Number: The trial was registered with NCT01320254., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Recently, many genome-wide profiling studies provided insights into the molecular make-up of major cancer types. The deeper understanding of these genetic alterations and their functional consequences led to the discovery of novel therapeutic opportunities improving clinical management of cancer patients. While tissue-based molecular patient stratification is the gold standard for precision medicine, it has certain limitations: Tissue biopsies are invasive sampling procedures carrying the risk of complications and may not represent the entire tumor due to underlying genetic heterogeneity. In this context, complementary characterization of genetic information in the blood of cancer patients can serve as minimal-invasive 'liquid biopsy'. Fragments of circulating cell-free DNA (cfDNA) are released from tissues of healthy individuals as well as cancer patients. The fraction of cfDNA that is released from primary tumors or metastases (i.e. circulating tumor DNA, ctDNA) represents genetic aberrations in cancer cells, which are a potential source for diagnostic, prognostic, and predictive biomarkers. Recent studies have demonstrated technical feasibility and clinical applications including detection of drug targets and resistance mutations as well as longitudinal monitoring of tumors under therapy. To this end, a variety of pre-analytical procedures for blood processing, isolation and quantification of cfDNA are being employed and several analytical methods and technologies ranging from PCR-based single locus assays to genome-wide approaches are available, which considerably differ in sensitivity, specificity, and throughput. However, broad implementation of ctDNA analysis in daily clinical practice requires a thorough understanding of theoretical, technical, and biological concepts and necessitates standardization and validation of pre-analytical and analytical procedures across different technologies. Here, we review the pertinent literature and discuss the advantages and limitations of available methodologies and their potential applications in molecular diagnostics., (© 2017 Wiley Periodicals, Inc.)

During the course of disease, many cancer patients eventually present with metastatic disease including peritoneal or pleural spread. In this context, cytology specimens derived from ascites or pleural effusion may help to differentiate malignant from benign conditions and sometimes yield diagnosis of a malignancy. However, even when supported by immunohistochemistry, cytological interpretation can be challenging, especially if tumor cellularity is low. Here, we investigated whether targeted deep sequencing of formalin-fixed and paraffin embedded (FFPE) cytology specimens of cancer patients is feasible, and has diagnostic and clinical impact. To this end, a cohort of 20 matched pairs was compiled, each comprising a cytology sample (FFPE cell block) and at least one biopsy/surgical resection specimen serving as benchmark. In addition, 5 non-malignant effusions were sequenced serving as negative-controls. All samples yielded sufficient libraries and were successfully subjected to targeted sequencing employing a semiconductor based next-generation sequencing platform. Using gene panels of different size and composition, including the Oncomine Comprehensive Assay, for targeted sequencing, somatic mutations were detected in the tissue of all 20 cases. Of these, 15 (75%) harbored mutations that were also detected in the corresponding cytology samples. In four of these cases (20%), additional private mutations were detected in either cytology or tissue samples, reflecting spatiotemporal tumor evolution. Of the five remaining cases, three (15%) showed wild type alleles in cytology material whereas tumor tissue had mutations in interrogated genes. Two cases were discordant, showing different private mutations in the cytology and in the tissue sample, respectively. In summary, sequencing of cytology specimens (FFPE cell block) reflecting spatiotemporal tumor evolution is feasible and yields adjunct genetic information that may be exploitable for diagnostics and therapy., (© 2017 Wiley Periodicals, Inc.)

Multiregional analysis provided first indications for morphological and molecular heterogeneity in lung adenocarcinomas (ADCs), but comprehensive morpho-molecular comparisons are still lacking. The purpose of our study was to investigate the spatial distribution of EGFR and KRAS alterations systematically throughout whole tumor cross-sections in correlation with the tumor cell content and the histopathological patterns. Central sections of 19 ADCs were subdivided into 467 segments of 5 mm × 5 mm. We determined the predominant histological growth pattern and the allele frequencies of driver gene mutations by digital PCR in every segment. We further quantified the absolute cell counts and proportions of tumor and non-neoplastic cells in all segments to normalize the mutant allele frequencies. Driver gene mutations could be detected in >99% of the tumor containing segments, with high levels of inter- and intratumor heterogeneity regarding the mutant allele frequency (range: 0.04-19.36). Different patterns for the distribution of the variant allele frequency within a tumor were recognizable. While some cases showed ubiquitously low or high levels, others revealed regions with focally elevated frequencies. Differences between KRAS and EGFR alterations were not significant. The great majority of the analyzed tumor sections (16/19) exhibited two or more morphological growth patterns. Mutant allele frequencies were significantly higher in segments with a predominant solid pattern compared to all other histologies (p < 0.01). Our data indicate that driver gene mutations are present with high levels of inter- and intratumor heterogeneity throughout the whole tumor, with a correlation between the allele frequencies and histological growth patterns., (© 2017 UICC.)

Brenner tumors (BT) are rare ovarian tumors encompassing benign, borderline, and malignant variants. While the histopathology of BTs and their clinical course is well described, little is known about the underlying genetic defects. We employed targeted next generation sequencing to analyze the mutational landscape in a cohort of 23 BT cases (17 benign, 2 borderline, and 4 malignant) and 3 ovarian carcinomas with transitional cell histology (TCC). Copy number variations (CNV) were validated by fluorescence in-situ hybridization (FISH) and quantitative PCR-based copy number assays. Additionally, we analyzed the TERT promotor region by conventional Sanger sequencing. We identified 25 different point mutations in 23 of the analyzed genes in BTs and 10 mutations in 8 genes in TCCs. About 57% percent of mutations occurred in genes involved in cell cycle control, DNA repair, and epigenetic regulation processes. All TCC cases harbored TP53 mutations whereas all BTs were negative and none of the mutations observed in BTs were present in TCCs. CNV analysis revealed recurrent MDM2 amplifications in 3 out of 4 of the malignant BT cases with one case harboring a concomitant amplification of CCND1. No mutations were observed in the TERT promoter region in BTs and TCCs, which is mutated in about 50%-75% of urothelial carcinoma and in 16% of ovarian clear-cell carcinomas. In conclusion, our study highlights distinct genetic features of BTs, and detection of the triplet phenotype MDM2 amplification/TP53 wt/TERT wt may aid diagnosis of malignant BT in difficult cases. Moreover, selected genetic lesions may be clinically exploitable in a metastatic setting., (© 2017 Wiley Periodicals, Inc.)

The European Commision (EC) recently approved osimertinib for the treatment of adult patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC) harboring EGFR T790M mutations. Besides tissue-based testing, blood samples containing cell-free circulating tumor DNA (ctDNA) can be used to interrogate T790M status. Herein, we describe the conditions and results of a round robin trial (RRT) for T790M mutation testing in NSCLC tissue specimens and peripheral blood samples spiked with cell line DNA mimicking tumor-derived ctDNA. The underlying objectives of this two-staged external quality assessment (EQA) approach were (a) to evaluate the accuracy of T790M mutations testing across multiple centers and (b) to investigate if a liquid biopsy-based testing for T790M mutations in spiked blood samples is feasible in routine diagnostic. Based on a successfully completed internal phase I RRT, an open RRT for EGFR T790M mutation testing in tumor tissue and blood samples was initiated. In total, 48 pathology centers participated in the EQA. Of these, 47 (97.9%) centers submitted their analyses within the pre-defined time frame and 44 (tissue), respectively, 40 (plasma) successfully passed the test. The overall success rates in the RRT phase II were 91.7% (tissue) and 83.3% (blood), respectively. Thirty-eight out of 48 participants (79.2%) successfully passed both parts of the RRT. The RRT for blood-based EGFR testing initiated in Germany is, to the best of our knowledge, the first of his kind in Europe. In summary, our results demonstrate that blood-based genotyping for EGFR resistance mutations can be successfully integrated in routine molecular diagnostics complementing the array of molecular methods already available at pathology centers in Germany.

Precision oncology implies the ability to predict which patients will likely respond to specific cancer therapies based on increasingly accurate, high-resolution molecular diagnostics as well as the functional and mechanistic understanding of individual tumors. While molecular stratification of patients can be achieved through different means, a promising approach is next-generation sequencing of tumor DNA and RNA, which can reveal genomic alterations that have immediate clinical implications. Furthermore, certain genetic alterations are shared across multiple histologic entities, raising the fundamental question of whether tumors should be treated by molecular profile and not tissue of origin. We here describe MASTER (Molecularly Aided Stratification for Tumor Eradication Research), a clinically applicable platform for prospective, biology-driven stratification of younger adults with advanced-stage cancer across all histologies and patients with rare tumors. We illustrate how a standardized workflow for selection and consenting of patients, sample processing, whole-exome/genome and RNA sequencing, bioinformatic analysis, rigorous validation of potentially actionable findings, and data evaluation by a dedicated molecular tumor board enables categorization of patients into different intervention baskets and formulation of evidence-based recommendations for clinical management. Critical next steps will be to increase the number of patients that can be offered comprehensive molecular analysis through collaborations and partnering, to explore ways in which additional technologies can aid in patient stratification and individualization of treatment, to stimulate clinically guided exploratory research projects, and to gradually move away from assessing the therapeutic activity of targeted interventions on a case-by-case basis toward controlled clinical trials of genomics-guided treatments., (© 2017 UICC.)

Blockade of immune checkpoints has become a powerful tool in cancer medicine, which is effective across various solid cancer types and hematologic malignancies. While immunohistochemical detection of PD-L1 expression in tumor cells, immune cells, or both has been introduced as predictive biomarker in several clinical trials, shortcomings and limitations of this approach were quickly recognized. As a single biomarker is unlikely to adequately reflect the complex interplay between immune cells and cancer, various genetic determinants of therapy success, including microsatellite instability, mutational burden, and PD-L1 amplification, are being investigated. Very recent work indicates that mutations in B2M, JAK1, and JAK2 render melanoma resistant to immune checkpoint blockade, thus serving as negative response predictors. Using the TCGA dataset, we performed a pan-cancer analysis of potentially damaging mutations in key genes implicated in antigen presentation and interferon-gamma signaling and investigated associations with transcript levels of immune checkpoint genes, cytolytic activity, and mutational burden. For B2M, JAK1, and JAK2, we observed overall mutation frequencies of 1.8%, 2%, and 2.6%, respectively, and found significant associations with mutational burden. On pathway level, melanoma as well as bladder, gastric, and lung cancer were most frequently affected by putative resistance mutations with mutation rates of 27%-50% in the antigen presentation pathway and of 16%-21% in the interferon signaling pathway. Our analysis suggests that a significant number of tumors harbor mutations that may negatively interfere with immune checkpoint inhibition, or confer a higher likelihood of resistance for which a second hit is ultimately required. Since these mutations are prevalent in treatment-naïve tumors, genetic screening prior to therapy might complement current approaches at predicting response to immune checkpoint blockade., (© 2017 Wiley Periodicals, Inc.)

Background: Mitochondria are considered relevant players in many tumour entities and first data indicate beneficial effects of mitochondria-targeted antioxidants in both cancer prevention and anticancer therapies. To further dissect the potential roles of mitochondria in NSCLC we comprehensively analysed somatic mitochondrial mutations, determined the spatial distribution of mitochondrial DNA within complete tumour sections and investigated the mitochondrial load in a large-scale approach., Methods: Whole mitochondrial genome sequencing of 26 matched tumour and non-neoplastic tissue samples extended by reviewing published data of 326 cases. Systematical stepwise real-time PCR quantification of mitochondrial DNA covering 16 whole surgical tumour sections. Immunohistochemical determination of the mitochondrial load in 171 adenocarcinoma and 145 squamous cell carcinoma., Results: Our results demonstrate very low recurrences (max. 1.7%) and a broad distribution of 456 different somatic mitochondrial mutations. Large inter- and intra-tumour heterogeneity were seen for mitochondrial DNA copy numbers in conjunction with a correlation to the predominant histological growth pattern. Furthermore, tumour cells had significantly higher mitochondrial level compared to adjacent stroma, whereas differences between tumour entities were negligible., Conclusions: Non-evident somatic mitochondrial mutations and highly varying mitochondrial DNA level delineate challenges for the approach of mitochondria-targeted anticancer therapies in NSCLC.

Mutations in BRCA1 or BRCA2 define a subset of prostate cancer patients. Herein, we address the question whether BRCA1/2 mutations have a predictive impact on chemotherapy with docetaxel, a widely used drug in patients with metastatic castration resistant prostate cancer (mCRPC). Fifty-three men treated with docetaxel for mCRPC were tested for somatic BRCA1/2 mutations of the primary tumor. In a subgroup of patients, BRCA1/2 protein expression was tested as a potential surrogate marker for BRCA1/2 inactivation. Eight of 53 patients (15.1%) harbored a deleterious BRCA2 mutation. No BRCA1 mutation was found. Patients with a BRCA2 mutation showed a response rate of 25% to docetaxel in comparison to 71.1% in men with wildtype BRCA2 (p = 0.019). While the time to develop castration resistance was similar in both subgroups, the overall survival was significantly shorter in patients harboring a BRCA2 mutation. No correlation between the BRCA1/2 protein expression and the response to docetaxel was found. While the presence of a BRCA2 mutation does not preclude a response to docetaxel, there is overall a significant correlation between BRCA2 inactivation and a poor response rate. Our results suggest that a close oncological monitoring of patients with BRCA2 mutations for taxane resistance is warranted.

The functional relationship between bile acid (BA) and autophagy has not been evaluated in the context of pancreatitis. Here we investigated whether BA and their nuclear farnesoid X receptor (FXR) modulate autophagy and the development of pancreatitis. FXR expression, autophagy, apoptosis and necroptosis were determined in human chronic pancreatitis (CP) tissue in vivo and in pancreatic cells lines in vitro by means of real-time PCR, immunoblots and immunofluorescence. Pancreatic cell lines exposed to the most abundant BAs glycochenodeoxycholate (GCDC) and taurocholic acid (TCA) increased the expression of nuclear FXR and diminished that of the essential autophagy-related protein ATG7. BA was also elevated in pancreatic tissues from CP patients, correlating with elevated FXR and curtailed ATG7 expression with locally reduced autophagic activity. This was accompanied by an increased manifestation of CP hallmarks including apoptosis, necroptosis, inflammation and fibrosis. The present results suggest a cascade of events in which local accumulation of BA signals via FXR to suppress autophagy in pancreatic acinar cells, thereby unleashing acinar cell apoptosis and necroptosis. Thus, BA may cause CP by suppressing autophagy and exacerbating acinar cell apoptosis and necroptosis., Competing Interests: The authors declare no conflict of interest.

The approval, in 2015, of the first poly (adenosine diphosphate-ribose) polymerase inhibitor (PARPi; olaparib, Lynparza) for platinum-sensitive relapsed high-grade ovarian cancer with either germline or somatic BRCA1/2 deleterious mutations is changing the way that BRCA1/2 testing services are offered to patients with ovarian cancer. Ovarian cancer patients are now being referred for BRCA1/2 genetic testing for treatment decisions, in addition to familial risk estimation, and irrespective of a family history of breast or ovarian cancer. Furthermore, testing of tumor samples to identify the estimated 3%-9% of patients with somatic BRCA1/2 mutations who, in addition to germline carriers, could benefit from PARPi therapy is also now being considered. This new testing paradigm poses some challenges, in particular the technical and analytical difficulties of analyzing chemically challenged DNA derived from formalin-fixed, paraffin-embedded specimens. The current manuscript reviews some of these challenges and technical recommendations to consider when undertaking BRCA1/2 testing in tumor tissue samples to detect both germline and somatic BRCA1/2 mutations. Also provided are considerations for incorporating genetic analysis of ovarian tumor samples into the patient pathway and ethical requirements., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Colorectal mixed adenoneuroendocrine carcinomas are rare and clinically aggressive neoplasms with considerable morphological heterogeneity. Data on their genomic characteristics and molecular associations to either conventional colorectal adenocarcinomas or poorly differentiated neuroendocrine neoplasms is still scarce, hampering optimized patient treatment and care. Tissue from 19 colorectal mixed adenoneuroendocrine carcinomas and eight colorectal poorly differentiated neuroendocrine neoplasms (neuroendocrine carcinomas) was microdissected and subjected to next-generation sequencing using a colorectal adenocarcinoma-specific panel comprising 196 amplicons covering 32 genes linked to colorectal adenocarcinoma, and poorly differentiated neuroendocrine neoplasm tumorigenesis. Mixed adenoneuroendocrine carcinomas were also examined for microsatellite instability and MLH-1 promoter methylation status. In three mixed adenoneuroendocrine carcinomas, exocrine and endocrine components were analyzed separately. Genetic testing of colorectal mixed adenoneuroendocrine carcinomas identified 43 somatic mutations clustering in 13/32 genes. Sixteen (84%) tumors harbored at least one somatic mutation, two tumors (11%) displayed high microsatellite instability. Compared with colorectal adenocarcinomas, mixed adenoneuroendocrine carcinomas were more frequently BRAF (37%; P=0.006), and less frequently KRAS (21%; P=0.043) and APC (16%; P=0.001) mutated. Point mutations in neuroendocrine neoplasm-related genes like RB1 or RET were not detected, but one tumor harbored a heterozygous RB1 deletion. Separately analyzed adenocarcinoma and neuroendocrine carcinoma components revealed a shared mutational trunk of driver genes involved in colorectal adenocarcinoma carcinogenesis. Colorectal neuroendocrine carcinomas were similar in their mutation profile to colorectal adenocarcinomas, but compared with mixed adenoneuroendocrine carcinomas, had a higher rate of APC mutations (P=0.027). Our data indicate that colorectal mixed adenoneuroendocrine carcinomas and neuroendocrine carcinomas are genetically closely related to colorectal adenocarcinomas, suggesting that the cells giving rise to these tumors primarily have an intestinal coinage. The identification of BRAF mutations and the frequently present KRAS wild-type status principally render some mixed adenoneuroendocrine carcinomas eligible to targeted treatment strategies used for colorectal adenocarcinomas.

HER2-positive breast cancers are a heterogeneous group of tumors, which share amplification and overexpression of HER2. In routine diagnostics, the HER2 (ERBB2) status is currently assessed by immunohistochemistry (IHC) and in situ hybridization (ISH). Data on targeted next-generation sequencing (NGS) approaches that could be used to determine the HER2 status are sparse. Employing two breast cancer-related gene panels, we performed targeted NGS of 41 FFPE breast cancers for which full pathological work-up including ISH and IHC results was available. Selected cases were analyzed by qPCR. Of the 41 cases, the HER2 status of the 4 HER2-positive and 6 HER2-negative tumors was independently detected by our NGS approach achieving a concordance rate of 100%. The remaining 31 cases were equivocal HER2 cases by IHC of which 5 showed amplification of HER2 by ISH. Our NGS approach classified all non-amplified cases correctly as HER2 negative and corroborated all but one of the 5 cases with amplified HER2 as detected by ISH. For the overall cohort, concordance between the gold standard and NGS was 97.6% (sensitivity 88.9% and specificity 100%). Additionally, we observed mutations in PIK3CA (44%), HER2 (8%), and CDH1 (6%) among others. Amplifications were found in CCND1 (12%), followed by MYC (10%) and EGFR (2%) and deletions in CDKN2A (10%), MAP2K4 and PIK3R1 (2% each). We here show that targeted NGS data can be used to interrogate the HER2 status with high specificity and high concordance with gold standard methods. Moreover, this approach identifies additional genetic events that may be clinically exploitable. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Metaphase karyotyping is an established diagnostic standard in acute myeloid leukemia (AML) for risk stratification. One of the cytogenetic findings in AML is structurally highly abnormal marker chromosomes. In this study, we have assessed frequency, cytogenetic characteristics, prognostic impact, and underlying biological origin of marker chromosomes. Given their inherent gross structural chromosomal damage, we speculated that they may arise from chromothripsis, a recently described phenomenon of chromosome fragmentation in a single catastrophic event. In 2 large consecutive prospective, randomized, multicenter, intensive chemotherapy trials (AML96, AML2003) from the Study Alliance Leukemia, marker chromosomes were detectable in 165/1026 (16.1%) of aberrant non-core-binding-factor (CBF) karyotype patients. Adverse-risk karyotypes displayed a higher frequency of marker chromosomes (26.5% in adverse-risk, 40.3% in complex aberrant, and 41.2% in abnormality(17p) karyotypes, P < .0001 each). Marker chromosomes were associated with a poorer prognosis compared with other non-CBF aberrant karyotypes and led to lower remission rates (complete remission + complete remission with incomplete recovery), inferior event-free survival as well as overall survival in both trials. In multivariate analysis, marker chromosomes independently predicted poor prognosis in the AML96 trial ≤60 years. As detected by array comparative genomic hybridization, about one-third of marker chromosomes (18/49) had arisen from chromothripsis, whereas this phenomenon was virtually undetectable in a control group of marker chromosome-negative complex aberrant karyotypes (1/34). The chromothripsis-positive cases were characterized by a particularly high degree of karyotype complexity, TP53 mutations, and dismal prognosis. In conclusion, marker chromosomes are indicative of chromothripsis and associated with poor prognosis per se and not merely by association with other adverse cytogenetic features., (© 2017 by The American Society of Hematology.)

Patients with non-small cell lung cancer (NSCLC) harboring activating mutations in the Epidermal Growth Factor Receptor (EGFR) benefit from targeted therapies. A synonymous polymorphism (rs1050171, p.Q787Q) was shown to be associated with improved overall survival (OS) in colorectal cancer patients. As data in NSCLC are limited, we retrospectively analyzed associations of p.Q787Q with clinicopathological parameters including clinical response and outcome in patients with lung adenocarcinoma (ADC) who received tyrosine kinase inhibitor (TKI) therapy. Of 642 ADC patients whose tumors were profiled by next generation sequencing, 102 (15.9%) carried EGFR mutations targetable by TKIs (30.4% male patients, median age 65.1 y, 19.6% smokers with 12.8 median pack years). Seventy-nine patients (77.5%) received TKI therapy either as a first- or second-line therapy. Of the 102 EGFR-mutant tumors, 72 (70.6%) exhibited the p.Q787Q polymorphism and another 12 (11.8%) cases with p.Q787Q harbored an additional TKI insensitive mutation (p.T790M). The polymorphism was neither associated with classic clinicopathological parameters nor with overall survival (21.1 months vs. 20.1 months; P-value = 0.91) or clinical response (P-value = 0.122). The patients with p.T790M had worse survival compared to EGFR activating mutation carriers with and without p.Q787Q when analyzed as a separate group (27.5 months, P-value = 0.02). In conclusion, p.Q787Q is neither a suitable prognostic nor predictive biomarker for ADC patients receiving anti-EGFR therapy in first- or second-line of therapy. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Pancreatic ductal adenocarcinoma (PDAC) has generally a poor prognosis, but recent data suggest that there are molecular subtypes differing in clinical outcome. This study examines the association between histopathologic heterogeneity, genetic profile, and survival. Tumor histology from 177 resected PDAC patients with follow-up data was subclassified according to predominant growth pattern, and four key genes were analyzed. PDACs were classified as conventional (51%), combined with a predominant component (41%), variants and special carcinomas (8%). Patients with combined PDACs and a dominant cribriform component survived longer than patients with conventional or other combined PDACs. Genetic alterations in at least two out of four genes were found in 95% of the patients (KRAS 93%, TP53 79%, CDKN2A/p16 75%, SMAD4 37%). Patients with less than four mutations survived significantly longer (p = 0.04) than those with alterations in all four genes. Patients with either wildtype KRAS or CDKN2A/p16 lived significantly longer than those with alterations in these genes (p = 0.018 and p = 0.006, respectively). Our data suggest that the number of altered genes, the mutational status of KRAS and certain morphological subtypes correlate with the outcome of patients with PDAC. Future pathology reporting of PDAC should therefore include the KRAS status and a detailed morphological description., Competing Interests: The authors declare no competing financial interests.

Soft-tissue sarcomas (STS) are rare malignancies that account for 1% of adult cancers and comprise more than 50 entities. Current therapeutic options for advanced-stage STS are limited. Immune checkpoint inhibitors targeting the PD-1/PD-L1 signaling axis are being explored as new treatment modality in STS; however, the determinants of response to these agents are largely unknown. Using the sarcoma data set of The Cancer Genome Altas (TCGA) and an independent cohort of untreated high-grade STS, we analyzed DNA copy number status and mRNA expression of PD-L1 in a total of 335 STS cases. Copy number gains (CNG) were detected in 54 TCGA cases (21.1%), of which 21 (8.2%) harbored focal PD-L1 CNG and that were most prevalent in myxofibrosarcoma (35%) and undifferentiated pleomorphic sarcoma (34%). In the untreated high-grade STS cohort, we detected CNG in six cases (7.6%). Analysis of co-amplified genes identified a 5.6-Mb core region comprising 27 genes, including JAK2 . Patients with PD-L1 CNG had higher PD-L1 expression compared with STS without CNG (fold change, 1.8; p = 0.02), an effect that was most pronounced in the setting of focal PD-L1 CNG (fold change, 3.0; p = 0.0027). STS with PD-L1 CNG showed a significantly higher mutational load compared with tumors with a diploid PD-L1 locus (median number of mutated genes; 58 vs. 40; p = 3.6E-06), and PD-L1 CNG were associated with inferior survival (HR = 1.82; p = 0.025). In contrast, T-cell infiltrates quantified by mRNA expression of CD3Z were associated with improved survival (HR = 0.88; p = 0.024) and consequently influenced the prognostic power of PD-L1 CNG, with low CD3Z levels conferring poor survival in cases with PD-L1 CNG (HR = 1.8; p = 0.049). These data demonstrate that PD-L1 GNG and elevated expression of PD-L1 occur in a substantial proportion of STS, have prognostic impact that is modulated by T-cell infiltrates, and thus warrant investigation as response predictors for immune checkpoint inhibition.

Multiple targeted therapy for advanced clear-cell renal cell carcinoma (RCC) has substantially improved patient outcome, but complete remission is uncommon and many tumors eventually develop resistance. Mechanistic, preclinical, and early clinical data highlight c-Met / hepatocyte growth factor receptor as a promising target for RCC therapeutic agents.We have examined MET expression, frequency of MET gene copy gains and MET gene mutation in a large, hospital-based series of renal cell carcinomas with long-term follow-up information.Out of a total of 572 clear-cell RCC, only 17% were negative for MET expression whereas 32% showed high protein levels. High MET expression and MET copy number gains were associated with an aggressive phenotype and an unfavorable patient outcome. Elevated protein levels in absence of gene amplification were not attributed to mutations, based on results of targeted next-generation sequencing.Our data reveal that clear-cell RCC with MET upregulation show an aggressive behavior and MET copy number increase is evident in a substantial percentage of patients with high-grade carcinomas and metastatic disease. Diagnostic assessment of MET expression and amplification may be of predictive value to guide targeted therapy against MET signaling in patients with clear-cell RCC.

Adenomas of the breast are rare benign tumors although single cases with malignant behavior have been reported. However, the genetic basis of these tumors is unknown. Employing targeted next generation sequencing of 50 cancer-related genes as well as Sanger sequencing, we profiled a cohort of 18 mammary adenomas comprising 9 ductal, 6 tubular, and 3 lactating adenoma. Missense mutations were detected in 8 of the 18 cases (44%). Specifically, five (56%) ductal adenomas and three (50%) tubular adenomas harbored mutated genes. No mutations were detected in lactating adenomas. Three of the nine ductal adenomas showed mutant AKT1 (p.E17K) with two of them harboring an additional GNAS mutation (p.R201C). One case had mutant PIK3CA (p.H1047R) and another case a mutation in GNAS (p.R201C). The three cases of mutated tubular adenomas showed mutations in either MET or FGFR3. Of note, we did not detect copy number changes and none of the cases including tubular adenomas had mutations in exon 2 of MED12. Our results suggest that ductal adenomas are related to papillomas of the breast and screening for mutations in exon 2 of MED12 might help to facilitate differential diagnosis between tubular adenoma and fibroadenoma in difficult cases. Lastly, our data exemplarily demonstrate that mutations in cancer-related genes per se do not indicate malignancy but occur in benign tumors. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Circulating tumor cells (CTC) are rare cells which have left the primary tumor to enter the blood stream. Although only a small CTC subgroup is capable of extravasating, the presence of CTCs is associated with an increased risk of metastasis and a shorter overall survival. Understanding the heterogeneous CTC biology will optimize treatment decisions and will thereby improve patient outcome. For this, robust workflows for detection and isolation of CTCs are urgently required. Here, we present a workflow to characterize CTCs by combining the advantages of both the CellSearch ® and the CellCelector™ micromanipulation system. CTCs were isolated from CellSearch ® cartridges using the CellCelector™ system and were deposited into PCR tubes for subsequent molecular analysis (whole genome amplification (WGA) and massive parallel multigene sequencing). By a CellCelector™ screen we reidentified 97% of CellSearch ® SKBR-3 cells. Furthermore, we isolated 97% of CellSearch ® -proven patient CTCs using the CellCelector™ system. Therein, we found an almost perfect correlation of R 2  = 0.98 (Spearman's rho correlation, n = 20, p < 0.00001) between the CellSearch ® CTC count (n = 271) and the CellCelector™ detected CTCs (n = 252). Isolated CTCs were analyzed by WGA and massive parallel multigene sequencing. In total, single nucleotide polymorphisms (SNPs) could be detected in 50 genes in seven CTCs, 12 MCF-7, and 3 T47D cells, respectively. Taken together, CTC quantification via the CellCelector™ system ensures a comprehensive detection of CTCs preidentified by the CellSearch ® system. Moreover, the isolation of CTCs after CellSearch ® using the CellCelector™ system guarantees for CTC enrichment without any contaminants enabling subsequent high throughput genomic analyses on single cell level. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:125-132, 2017., (© 2016 American Institute of Chemical Engineers.)

Background: Inhibition of the oncogenic fusion-gene EML4-ALK is a current first-line approach for patients with stage IV non-small cell lung cancer. While FISH was established as the gold standard for identifying these patients, there is accumulating evidence that other methods of detection, i.e., immunohistochemistry and next-generation sequencing (NGS), exist that may be equally successful. However, the concordance of these methods is under investigation., Case Presentation: Adding to the current literature, we here report a 56 year old female never-smoker with stage IV lung adenocarcinoma whose biopsy was IHC and FISH inconclusive but positive in NGS. Retroactive profiling of the resection specimen corroborated fusion reads obtained by NGS, FISH-positivity and showed weak ALK-positivity by IHC. Consequently, we diagnosed the case as ALK-positive rendering the patient eligible to crizotinib treatment., Conclusions: With IHC on biopsy material only, this case would have been overlooked withholding effective therapy.

Targeted deep massive parallel sequencing has been implemented in routine molecular diagnostics for high-throughput genetic profiling of formalin-fixed paraffin-embedded (FFPE) cancer samples. This approach is widely used to interrogate simple somatic mutations but experience with the analysis of copy number variations (CNV) is limited. Here, we retrospectively analyzed CNV in 822 cancer cases (135 melanoma, 468 non-small cell lung cancers (NSCLC), 219 colorectal cancers (CRC)). We observed a decreasing frequency of CNV in clinically actionable genes from melanoma to NSCLC to CRC. The overall cohort displayed 168 (20%) amplifications in 17 druggable targets. The majority of BRAF mutant melanomas (54%) showed co-occurring CNV in other genes, mainly affecting CDKN2A. Subsets showed clustered deletions in ABL1, NOTCH1, RET or STK11, GNA11, and JAK3. Most NRAS mutant melanomas (49%) harbored CNVs in other genes with CDKN2A and FGFR3 being most frequently affected. Five BRAF/NRASwt tumors had co-amplifications of KDR, KIT, PDGFRA and another six mutated KIT. Among all NSCLC, we identified 14 EGFRamp (with ten EGFRmut) and eight KRASamp (with seven KRASmut). KRASmut tumors displayed frequent amplifications of MYC (n = 10) and MDM2 (n = 5). Fifteen KRAS/EGFR/BRAFwt tumors had MET mutations/amplifications. In CRC, amplified IGF2 was most prevalent (n = 13) followed by MYC (n = 9). Two cases showed amplified KRAS wildtype alleles. Two of the KRASmut cases harbored amplifications of NRAS and three KRASwt cases amplification of EGFR. In conclusion, we demonstrate that our approach i) facilitates detection of CNV, ii) enables detection of known CNV patterns, and iii) uncovers new CNV of clinically actionable genes in FFPE tissue samples across cancers. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: TP53 mutations are frequent in breast cancer, however their clinical relevance in terms of response to chemotherapy is controversial., Methods: 450 pre-therapeutic, formalin-fixed, paraffin-embedded core biopsies from the phase II neoadjuvant GeparSixto trial that included HER2-positive and triple negative breast cancer (TNBC) were subjected to Sanger sequencing of exons 5-8 of the TP53 gene. TP53 status was correlated to response to neoadjuvant anthracycline/taxane-based chemotherapy with or without carboplatin and trastuzumab/lapatinib in HER2-positive and bevacizumab in TNBC. p53 protein expression was evaluated by immunohistochemistry in the TNBC subgroup., Results: Of 450 breast cancer samples 297 (66.0%) were TP53 mutant. Mutations were significantly more frequent in TNBC (74.8%) compared to HER2-positive cancers (55.4%, P < 0.0001). Neither mutations nor different mutation types and effects were associated with pCR neither in the whole study group nor in molecular subtypes (P > 0.05 each). Missense mutations tended to be associated with a better survival compared to all other types of mutations in TNBC (P = 0.093) and in HER2-positive cancers (P = 0.071). In TNBC, missense mutations were also linked to higher numbers of tumor-infiltrating lymphocytes (TILs, P = 0.028). p53 protein overexpression was also linked with imporved survival (P = 0.019)., Conclusions: Our study confirms high TP53 mutation rates in TNBC and HER2-positive breast cancer. Mutations did not predict the response to an intense neoadjuvant chemotherapy in these two molecular breast cancer subtypes.

Here, we report the case of an acute promyelocytic leukemia (APL) patient who-although negative for FLT3 mutations at diagnosis-developed isolated FLT3 tyrosine kinase II domain (FLT3-TKD)-positive meningeal relapse, which, in retrospect, could be traced back to a minute bone marrow subclone present at first diagnosis. Initially, the 48-yr-old female diagnosed with high-risk APL had achieved complete molecular remission after standard treatment with all-trans retinoic acid (ATRA) and chemotherapy according to the AIDA (ATRA plus idarubicin) protocol. Thirteen months after the start of ATRA maintenance, the patient suffered clinically overt meningeal relapse along with minute molecular traces of PML/RARA (promyelocytic leukemia/retinoic acid receptor alpha) in the bone marrow. Following treatment with arsenic trioxide and ATRA in combination with intrathecal cytarabine and methotrexate, the patient achieved a complete molecular remission in both cerebrospinal fluid (CSF) and bone marrow, which currently lasts for 2 yr after completion of therapy. Whole-exome sequencing and subsequent ultradeep targeted resequencing revealed a heterozygous FLT3-TKD mutation in CSF leukemic cells (p.D835Y, c.2503G>T, 1000/1961 reads [51%]), which was undetectable in the concurrent bone marrow sample. Interestingly, the FLT3-TKD mutated meningeal clone originated from a small bone marrow subclone present in a variant allele frequency of 0.4% (6/1553 reads) at initial diagnosis. This case highlights the concept of clonal evolution with a subclone harboring an additional mutation being selected as the "fittest" and leading to meningeal relapse. It also further supports earlier suggestions that FLT3 mutations may play a role for migration and clonal expansion in the CSF sanctuary site.

Pediatric-type follicular lymphoma (PTFL) is a variant of follicular lymphoma (FL) with distinctive clinicopathological features. Patients are predominantly young males presenting with localized lymphadenopathy; the tumor shows high-grade cytology and lacks both BCL2 expression and t(14;18) translocation. The genetic alterations involved in the pathogenesis of PTFL are unknown. Therefore, 42 PTFL (40 males and 2 females; mean age, 16 years; range, 5-31) were genetically characterized. For comparison, 11 cases of conventional t(14:18)(-) FL in adults were investigated. Morphologically, PTFL cases had follicular growth pattern without diffuse areas and characteristic immunophenotype. All cases showed monoclonal immunoglobulin (IG) rearrangement. PTFL displays low genomic complexity when compared with t(14;18)(-) FL (mean, 0.77 vs 9 copy number alterations per case; P <001). Both groups presented 1p36 alterations including TNFRSF14, but copy-number neutral loss of heterozygosity (CNN-LOH) of this locus was more frequently observed in PTFL (40% vs 9%; P =075). TNFRSF14 was the most frequently affected gene in PTFL (21 mutations and 2 deletions), identified in 54% of cases, followed by KMT2D mutations in 16%. Other histone-modifying genes were rarely affected. In contrast, t(14;18)(-) FL displayed a mutational profile similar to t(14;18)(+) FL. In 8 PTFL cases (19%), no genetic alterations were identified beyond IG monoclonal rearrangement. The genetic landscape of PTFL suggests that TNFRSF14 mutations accompanied by CNN-LOH of the 1p36 locus in over 70% of mutated cases, as additional selection mechanism, might play a key role in the pathogenesis of this disease. The genetic profiles of PTFL and t(14;18)(-) FL in adults indicate that these are two different disorders.

Cancer of unknown primary (CUP) is defined as metastatic solid malignancy where no primary tumor is detected despite appropriate staging. About 90% of CUP represent adenocarcinoma or undifferentiated carcinoma. Since therapy regimens are only modestly effective, identification of the molecular landscape of these neoplasms might be a promising approach to direct CUP therapy and aid in tumor classification. We screened a cohort of 128 patients with adenocarcinoma or undifferentiated carcinoma meeting the definition of CUP. Massive parallel multigene sequencing of 50 genes, which had been selected due to their relevance as oncogenic drivers or druggable molecular targets could ultimately be performed on samples from 55 patients for whom complete clinical datasets were also available. Overall, 60 tumor-specific mutations and 29 amplifications/deletions, as revealed by coverage analysis, were detected in 46 cases (84%). The most frequently mutated genes were TP53 (30 cases, 55%), KRAS (9 cases, 16%), CDKN2A (5 cases, 9%), and SMAD4 (5 cases, 9%). The most frequently deleted gene was CDKN2A (8 cases, 15%). KRAS and CDKN2A mutations significantly correlated with poor progression-free survival (PFS) and, in case of KRAS, overall survival (OS). WIldtype TP53 and female sex defined a relatively favorable category, with favorable PFS and OS. 8 cases (15%) harbored mutations that may be targetable by currently approved drugs. Taken together, Mutations of relevant driver genes are present in the vast majority of CUP tumors. Some of them impact on prognosis and a subset is putatively druggable., Competing Interests: The authors declare no conflicts of interest.

The activating mutation of the BRAF serine/threonine protein kinase (BRAF V600E) is the key driver mutation in hairy cell leukemia (HCL), suggesting opportunities for therapeutic targeting. We analyzed the course of 21 HCL patients treated with vemurafenib outside of trials with individual dosing regimens (240-1920 mg/d; median treatment duration, 90 days). Vemurafenib treatment improved blood counts in all patients, with platelets, neutrophils, and hemoglobin recovering within 28, 43, and 55 days (median), respectively. Complete remission was achieved in 40% (6/15 of evaluable patients) and median event-free survival was 17 months. Response rate and kinetics of response were independent of vemurafenib dosing. Retreatment with vemurafenib led to similar response patterns (n = 6). Pharmacodynamic analysis of BRAF V600E downstream targets showed that vemurafenib (480 mg/d) completely abrogated extracellular signal-regulated kinase phosphorylation of hairy cells in vivo. Typical side effects also occurred at low dosing regimens. We observed the development of acute myeloid lymphoma (AML) subtype M6 in 1 patient, and the course suggested disease acceleration triggered by vemurafenib. The phosphatidylinositol 3-kinase hotspot mutation (E545K) was identified in the AML clone, providing a potential novel mechanism for paradoxical BRAF activation. These data provide proof of dependence of HCL on active BRAF signaling. We provide evidence that antitumor and side effects are observed with 480 mg vemurafenib, suggesting that dosing regimens in BRAF-driven cancers could warrant reassessment in trials with implications for cost of cancer care., (© 2016 by The American Society of Hematology.)

Cancer precision medicine has opened up new avenues for the treatment of colorectal cancer (CRC). To fully realize its potential, high-throughput sequencing platforms that allow genotyping beyond KRAS need to be implemented and require performance assessment. We comprehensively analyzed first-year data of 202 consecutive formalin-fixed paraffin embedded (FFPE) CRC samples for which prospective genotyping at our institution was requested. Deep targeted genotyping was done using a semiconductor-based sequencing platform and a self-designed panel of 30 CRC-related genes. Additionally, microsatellite status (MS) was determined. Ninety-seven percent of tumor samples were suitable for sequencing and in 88% MS could be assessed. The minimal drop-out rates of 6 and 25 cases, respectively were due to too low amounts or heavy degradation of DNA. Of 557 nonsynonymous mutations, 90 (16%) have not been described in COSMIC at the time of data query. Forty-three cases (22%) had double- or triple mutations affecting a single gene. Sixty-four percent had genetic alterations influencing oncological therapy. Eight percent of patients (MSI phenotype: 6%; mutated POLE: 2%) were potentially eligible for treatment with immune checkpoint inhibitors. Of 56% of KRASwt CRC that potentially qualified for anti-EGFR treatment, 30% presented with mutations in BRAF/NRAS. Mutated PIK3CA was detected in 21%. In conclusion, we here present real-life routine diagnostics data that not only demonstrate the robustness and feasibility of deep targeted sequencing and MS-analysis of FFPE CRC samples but also contribute to the understanding of CRC genetics. Most importantly, in more than half of the patients our approach enabled the selection of the best treatment currently available. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)