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Tumor mutation burden (TMB) is evaluated as a biomarker of response to immunotherapy. We present the efforts of the Onconetwork Immuno-Oncology Consortium to validate a commercial targeted sequencing test for TMB calculation. A three-phase study was designed to validate the Oncomine Tumor Mutational Load (OTML) assay at nine European laboratories. Phase 1 evaluated reproducibility and accuracy on seven control samples. In phase 2, six formalin-fixed, paraffin-embedded samples tested with FoundationOne were reanalyzed with the OTML panel to evaluate concordance and reproducibility. Phase 3 involved analysis of 90 colorectal cancer samples with known microsatellite instability (MSI) status to evaluate TMB and MSI association. High reproducibility of TMB was demonstrated among the sites in the first and second phases. Strong correlation was also detected between mean and expected TMB in phase 1 (r 2  = 0.998) and phase 2 (r 2  = 0.96). Detection of actionable mutations was also confirmed. In colorectal cancer samples, the expected pattern of MSI-high/high-TMB and microsatellite stability/low-TMB was present, and gene signatures produced by the panel suggested the presence of a POLE mutation in two samples. The OTML panel demonstrated robustness and reproducibility for TMB evaluation. Results also suggest the possibility of using the panel for mutational signatures and variant detection. Collaborative efforts between academia and companies are crucial to accelerate the translation of new biomarkers into clinical research., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: β-catenin activation plays a crucial role for tumourigenesis in the large intestine but except for Lynch syndrome (LS) associated cancers stabilizing mutations of β-catenin gene (CTNNB1) are rare in colorectal cancer (CRC). Previous animal studies provide an explanation for this observation. They showed that CTNNB1 mutations induced transformation in the colon only when CTNNB1 was homozygously mutated or when membranous β-catenin binding was hampered by E-cadherin haploinsufficiency. We were interested, if these mechanisms are also found in human CTNNB1 mutated CRCs., Results: Among 869 CRCs stabilizing CTNNB1 mutations were found in 27 cases. Homo- or hemizygous CTNNB1 mutations were detected in 74% of CTNNB1 mutated CRCs (13 microsatellite instabile (MSI-H), 7 microsatellite stabile (MSS)) but only in 3% (1/33) of extracolonic CTNNB1 mutated cancers. In contrast to MSS CRC, CTNNB1 mutations at codon 41 or 45 were highly selected in MSI-H CRC. Of the examined three CRC cell lines, β-catenin and E-cadherin expression was similar in cell lines without or with hetereozygous CTNNB1 mutations (DLD1 and HCT116), while a reduced E-cadherin expression combined with cytoplasmic accumulation of β-catenin was found in a cell line with homozygous CTNNB1 mutation (LS180). Reduced expression of E-cadherin in human MSI-H CRC tissue was identified in 60% of investigated cancers, but no association with the CTNNB1 mutational status was found., Conclusions: In conclusion, this study shows that in contrast to extracolonic cancers stabilizing CTNNB1 mutations in CRC are commonly homo- or hemizygous indicating a higher threshold of β-catenin stabilization to be required for transformation in the colon as compared to extracolonic sites. Moreover, we found different mutational hotspots in CTNNB1 for MSI-H and MSS CRCs suggesting a selection of different effects on β-catenin stabilization according to the molecular pathway of tumourigenesis. Reduced E-cadherin expression in CRC may further contribute to higher levels of transcriptionally active β-catenin, but it is not directly linked to the CTNNB1 mutational status.

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., (© 2018 UICC.)

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.)

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.

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.)

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.)

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., (© 2015 Wiley Periodicals, Inc.)

A multistep model of disease progression and genomic landscape has been firmly established for colorectal cancer (CRC) primaries, but the genetic makeup of related metastases and the dynamics of genetic changes during metastatic progression are scarcely known. To address these issues, we used multigene high-coverage next-generation sequencing of 24 microsatellite-stable CRC primaries, matched normal tissue, and related multiple metastases to nodes, liver, lung, and brain with a CRC-specific gene panel to infer the degree of clonal evolution during metastatic progression of the disease. Somatic mutations were detected in 40% of CRC-related genes, and we observed a striking 100% genetic concordance between primary and multiple secondary sites for APC, KRAS, FBXW7, PIK3CA, BRAF, SMAD4, and ACVR2A. Except for true de novo mutations in 4 cases (affecting SYNE1, CTNNB1, TP53, and PTEN), all remaining cases (84.4%) shared the genetic lesions of the primary tumors with all investigated metastases irrespective of the site of metastasis or time lapse between primary tumor resection and the occurrence of metastatic spread. Putative biomarkers and druggable targets were identified in 25% of the cases. Our data proves that genetic alterations occurring early in CRC carcinogenesis are remarkably stable during metastatic progression, indicating (i) a very low degree of genetic heterogeneity between primary and multiple secondary sites with respect to CRC driver mutations and (ii) that genetic interrogation of archived primary tumor samples appears to be sufficient for the application of cancer precision medicine in the metastatic setting.

Background: According to current clinical guidelines mutational analysis for KRAS and NRAS is recommended prior to EGFR-directed therapy of colorectal cancer (CRC) in the metastatic setting. Therefore, reliable, fast, sensitive and cost-effective methods for routine tissue based molecular diagnostics are required that allow the assessment of the CRC mutational status in a high throughput fashion., Methods: We have developed a custom designed assay for routine mass-spectrometric (MS) (MassARRAY, Agena Bioscience) analysis to test the presence/absence of 18 KRAS, 14 NRAS and 4 BRAF mutations. We have applied this assay to 93 samples from patients with CRC and have compared the results with Sanger sequencing and a chip hybridization assay (KRAS LCD-array Kit, Chipron). In cases with discordant results, next-generation sequencing (NGS) was performed., Results: MS detected a KRAS mutation in 46/93 (49%), a NRAS mutation in 2/93 (2%) and a BRAF mutation in 1/93 (1%) of the cases. MS results were in agreement with results obtained by combination of the two other methods in 92 (99%) of 93 cases. In 1/93 (1%) of the cases a G12V mutation has been detected by Sanger sequencing and MS, but not by the chip assay. In this case, NGS has confirmed the G12V mutation in KRAS., Conclusions: Mutational analysis by MS is a reliable method for routine diagnostic use, which can be easily extended for testing of additional mutations.

Recent molecular profiling studies reported a new class of ultramutated colorectal cancers (CRCs), which are caused by exonuclease domain mutations (EDMs) in DNA polymerase ϵ (POLE). Data on the clinical implications of these findings as to whether these mutations define a unique CRC entity with distinct clinical outcome are lacking. We performed Sanger sequencing of the POLE exonuclease domain in 431 well-characterized patients with microsatellite stable (MSS) CRCs of a population-based patient cohort. Mutation data were analyzed for associations with major epidemiological, clinical, genetic, and pathological parameters including overall survival (OS) and disease-specific survival (DSS). In 373 of 431 MSS CRC, all exons of the exonuclease domain were analyzable. Fifty-four mutations were identified in 46 of these samples (12.3%). Besides already reported EDMs, we detected many new mutations in exons 13 and 14 (corresponding to amino acids 410-491) as well as in exon 9 and exon 11 (corresponding to aa 268-303 and aa 341-369). However, we did not see any significant associations of EDMs with clinicopathological parameters, including sex, age, tumor location and tumor stage, CIMP, KRAS, and BRAF mutations. While with a median follow-up time of 5.0 years, survival analysis of the whole cohort revealed nonsignificantly different adjusted hazard ratios (HRs) of 1.35 (95% CI: 0.82-2.25) and 1.44 (0.81-2.58) for OS and DSS indicating slightly impaired survival of patients with EDMs, subgroup analysis for patients with stage III/IV disease receiving chemotherapy revealed a statistically significantly increased adjusted HR (1.87; 95%CI: 1.02-3.44). In conclusion, POLE EDMs do not appear to define an entirely new clinically distinct disease entity in CRC but may have prognostic or predictive implications in CRC subgroups, whose significance remains to be investigated in future studies., (© 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)