Assigning evidence to actionability: An introduction to variant interpretation in precision cancer medicine.

Modern concepts in precision cancer medicine are based on increasingly complex genomic analyses and require standardized criteria for the functional evaluation and reporting of detected genomic alterations in order to assess their clinical relevance. In this article, we propose and address the necessary steps in systematic variant evaluation consisting of bioinformatic analysis, functional annotation and clinical interpretation, focusing on the latter two aspects. We discuss the role and clinical application of current variant classification systems and point out their scope and limitations. Finally, we highlight the significance of the molecular tumor board as a platform for clinical decision-making based on genomic analyses.

KRAS/GNAS-testing by highly sensitive deep targeted next generation sequencing improves the endoscopic ultrasound-guided workup of suspected mucinous neoplasms of the pancreas.

Pancreatic cysts or dilated pancreatic ducts are often found by cross-sectional imaging, but only mucinous lesions can become malignant. Therefore, distinction between mucinous and non-mucinous lesions is crucial for adequate patient management. We performed a prospective study including targeted next generation sequencing (NGS) of cell-free DNA in the diagnostic endoscopic ultrasound (EUS)-guided workup. Pancreatic cyst(s) or main duct fluid obtained by EUS-guided FNA was analysed by carcinoembryonic antigen (CEA), cytology and deep targeted NGS of 14 known gastrointestinal cancer genes (AKT1, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53, APC) with a limit of detection down to variant allele frequency of 0.01%. Results were correlated to histopathology and clinical follow-up. One hundred and thirteen patients with pancreatic cyst(s) and/or a dilated pancreatic main duct (≥5 mm) were screened. Sixty-six patients had to be excluded, mainly due to inoperability or small cyst size (≤10 mm). Forty-seven patients were enrolled for further analysis. A final diagnosis was available in 27 cases including 8 negative controls. In 43/47 (91.5%) of patients a KRAS- and/or GNAS-mutation was diagnosed by NGS. 27.0% of the KRAS-mutated and 10.0% of the GNAS-mutated lesions harbored multiple mutations. KRAS/GNAS-testing by NGS, cytology, and CEA had a sensitivity and specificity of 94.7/100%, 38.1/100%, and 42.1/75.0%, respectively. KRAS/GNAS-testing was significantly superior to CEA (P = .0209) and cytology (P = .0016). In conclusion, KRAS/GNAS-testing by deep targeted NGS is a suitable method to distinguish mucinous from non-mucinous pancreatic lesions, suggesting its usage as a single diagnostic test. Results must be confirmed in a larger cohort.

Next generation sequencing of the cellular and liquid fraction of pancreatic cyst fluid supports discrimination of IPMN from pseudocysts and reveals cases with multiple mutated driver clones: First findings from the prospective ZYSTEUS biomarker study.

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.

Size matters: Dissecting key parameters for panel-based tumor mutational burden analysis.

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.

Measurement of tumor mutational burden (TMB) in routine molecular diagnostics: in silico and real-life analysis of three larger gene panels.

Assessment of Tumor Mutational Burden (TMB) for response stratification of cancer patients treated with immune checkpoint inhibitors is emerging as a new biomarker. Commonly defined as the total number of exonic somatic mutations, TMB approximates the amount of neoantigens that potentially are recognized by the immune system. While whole exome sequencing (WES) is an unbiased approach to quantify TMB, implementation in diagnostics is hampered by tissue availability as well as time and cost constrains. Conversely, panel-based targeted sequencing is nowadays widely used in routine molecular diagnostics, but only very limited data are available on its performance for TMB estimation. Here, we evaluated three commercially available larger gene panels with covered genomic regions of 0.39 Megabase pairs (Mbp), 0.53 Mbp and 1.7 Mbp using i) in silico analysis of TCGA (The Cancer Genome Atlas) data and ii) wet-lab sequencing of a total of 92 formalin-fixed and paraffin-embedded (FFPE) cancer samples grouped in three independent cohorts (non-small cell lung cancer, NSCLC; colorectal cancer, CRC; and mixed cancer types) for which matching WES data were available. We observed a strong correlation of the panel data with WES mutation counts especially for the gene panel >1Mbp. Sensitivity and specificity related to TMB cutpoints for checkpoint inhibitor response in NSCLC determined by wet-lab experiments well reflected the in silico data. Additionally, we highlight potential pitfalls in bioinformatics pipelines and provide recommendations for variant filtering. In summary, our study is a valuable data source for researchers working in the field of immuno-oncology as well as for diagnostic laboratories planning TMB testing.

Combined targeted DNA and RNA sequencing of advanced NSCLC in routine molecular diagnostics: Analysis of the first 3,000 Heidelberg cases.

Tyrosine kinase inhibitors currently confer the greatest survival gain for nonsmall cell lung cancer (NSCLC) patients with actionable genetic alterations. Simultaneously, the increasing number of targets and compounds poses the challenge of reliable, broad and timely molecular assays for the identification of patients likely to benefit from novel treatments. Here, we demonstrate the feasibility and clinical utility of comprehensive, NGS-based genetic profiling for routine workup of advanced NSCLC based on the first 3,000 patients analyzed in our department. Following automated extraction of DNA and RNA from formalin-fixed, paraffin-embedded tissue samples, parallel sequencing of DNA and RNA for detection of mutations and gene fusions, respectively, was performed using PCR-based enrichment with an ion semiconductor sequencing platform. Overall, 807 patients (27%) were eligible for currently approved, EGFR-/BRAF-/ALK- and ROS1-directed therapies, while 218 additional cases (7%) with MET, ERBB2 (HER2) and RET alterations could potentially benefit from experimental targeted compounds. In addition, routine capturing of comutations, e.g. TP53 (55%), KEAP1 (11%) and STK11 (11%), as well as the precise typing of fusion partners and involved exons in case of actionable translocations including ALK and ROS1, are prognostic and predictive tools currently gaining importance for further refinement of therapeutic and surveillance strategies. The reliability, low dropout rates (<5%), minimal tissue requirements, fast turnaround times (6 days on average) and lower costs of the diagnostic approach presented here compared to sequential single-gene testing, highlight its practicability in order to support individualized decisions in routine patient care, enrollment in molecularly stratified clinical trials, as well as translational research.

Variant classification in precision oncology.

Next-generation sequencing has become a cornerstone of therapy guidance in cancer precision medicine and an indispensable research tool in translational oncology. Its rapidly increasing use during the last decade has expanded the options for targeted tumor therapies, and molecular tumor boards have grown accordingly. However, with increasing detection of genetic alterations, their interpretation has become more complex and error-prone, potentially introducing biases and reducing benefits in clinical practice. To facilitate interdisciplinary discussions of genetic alterations for treatment stratification between pathologists, oncologists, bioinformaticians, genetic counselors and medical scientists in specialized molecular tumor boards, several systems for the classification of variants detected by large-scale sequencing have been proposed. We review three recent and commonly applied classifications and discuss their individual strengths and weaknesses. Comparison of the classifications underlines the need for a clinically useful and universally applicable variant reporting system, which will be instrumental for efficient decision making based on sequencing analysis in oncology. Integrating these data, we propose a generalizable classification concept featuring a conservative and a more progressive scheme, which can be readily applied in a clinical setting.

Targeted deep sequencing of effusion cytology samples is feasible, informs spatiotemporal tumor evolution, and has clinical and diagnostic utility.

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.

EML4-ALK fusion variant V3 is a high-risk feature conferring accelerated metastatic spread, early treatment failure and worse overall survival in ALK+ non-small cell lung cancer.

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.

Genome-wide analysis of pediatric-type follicular lymphoma reveals low genetic complexity and recurrent alterations of TNFRSF14 gene.

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.

Mutational profiles of Brenner tumors show distinctive features uncoupling urothelial carcinomas and ovarian carcinoma with transitional cell histology.

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.

Synonymous EGFR variant p.Q787Q is neither prognostic nor predictive in patients with lung adenocarcinoma.

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.

Targeted next-generation sequencing enables reliable detection of HER2 (ERBB2) status in breast cancer and provides ancillary information of clinical relevance.

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.

Tubular, lactating, and ductal adenomas are devoid of MED12 Exon2 mutations, and ductal adenomas show recurrent mutations in GNAS and the PI3K-AKT pathway.

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.

Precision oncology based on omics data: The NCT Heidelberg experience.

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.

Prevalence of somatic mitochondrial mutations and spatial distribution of mitochondria in non-small cell lung cancer.

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.

Colorectal mixed adenoneuroendocrine carcinomas and neuroendocrine carcinomas are genetically closely related to colorectal adenocarcinomas.

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.

High-throughput diagnostic profiling of clinically actionable gene fusions in lung cancer.

Molecular profiling of non-small cell lung cancers (NSCLC) has a strong impact on clinical decision making and current oncological therapies. Besides detection of activating mutations in EGFR, analysis of ALK and ROS1 gene rearrangements has come into focus for targeted therapies. Targeted massive parallel sequencing (MPS) has been established for routine diagnostic profiling of the most prevalent oncogenic mutations in NSCLC, but not for the detection of gene rearrangements yet. Here, we present and evaluate an MPS-based panel sequencing approach which simultaneously detects ALK, ROS1, and RET fusions as well as somatic mutations in a single multiplex assay using formalin-fixed paraffin-embedded (FFPE) tissue. To this end, we first evaluated sensitivity and specificity of the fusion assay retrospectively by employing it to a set of 50 NSCLC with known gene fusions (n = 35) and with no gene fusions (n = 15). The sensitivity and specificity of the MPS assay for the detection of known fusions was 100%. In a second prospective phase, we implemented the approach of parallel mutation and gene fusion detection in our routine diagnostic workflow to assess performance of the test in a diagnostic outreach setting. Our prospective screening of 109 NSCLC samples revealed four gene fusions all of which were confirmed by FISH. In conclusion, our approach facilitates simultaneous high-throughput detection of gene fusions and somatic mutations in NSCLC samples and is able to replace conventional methods.

Genetic heterogeneity in synchronous colorectal cancers impacts genotyping approaches and therapeutic strategies.

Synchronous colorectal carcinomas (sCRC) are clinically challenging neoplasms. Although the epidemiological characteristics are quite well established, their biological basis is still poorly understood. Hence, we performed comprehensive molecular profiling of 23 sCRC cases comprising 50 synchronous primary tumors, 5 metastases, and corresponding normal tissue by targeted deep sequencing of 30 CRC-related genes, microsatellite analysis and analysis for methylated MLH1. We identified a striking inter- and intratumoral genetic heterogeneity of sCRC. Twenty (87%) cases showed genetic heterogeneity leaving only three cases with tumors that had an identical genetic make-up. Intertumoral heterogeneity was frequently observed for clinically actionable genes, including KRAS. Specifically, 44% of the cases harbored tumors of which at least one was KRAS mutated and the other KRAS wildtype. Moreover, 48% of the cases had at least double, sometimes even triple or quadruple mutations in KRAS, APC, TP53, PIK3CA, and TGFBR2, most of them being subclonal events. Lastly, we detected four cases (17%) with microsatellite instable (MSI) tumors with one case harboring one MSI- and a distinct microsatellite stable carcinoma. Our data demonstrate a striking genetic heterogeneity not only between different sCRC of a single case but also within a single tumor. These results contribute to the biological understanding of sCRC and directly impact genotyping strategies and oncological decision making. Testing one tumor or a single metastasis may not suffice in the sCRC setting as clinically relevant and tumor-specific genetic information may be left undetected compromising optimal oncological therapy. © 2015 Wiley Periodicals, Inc.

Genotyping of colorectal cancer for cancer precision medicine: Results from the IPH Center for Molecular Pathology.

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.