The genomic landscape of breast and non-breast cancers from individuals with germline CHEK2 deficiency.

CHEK2 is considered to be involved in homologous recombination repair (HRR). Individuals who have germline pathogenic variants (gPVs) in CHEK2 are at increased risk to develop breast cancer and likely other primary cancers. PARP inhibitors (PARPi) have been shown to be effective in the treatment of cancers that present with HRR deficiency-for example, caused by inactivation of BRCA1/2. However, clinical trials have shown little to no efficacy of PARPi in patients with CHEK2 gPVs. Here, we show that both breast and non-breast cancers from individuals who have biallelic gPVs in CHEK2 (germline CHEK2 deficiency) do not present with molecular profiles that fit with HRR deficiency. This finding provides a likely explanation why PARPi therapy is not successful in the treatment of CHEK2-deficient cancers.

Clinical Validity of Tumor-Informed Circulating Tumor DNA Analysis in Patients Undergoing Surgery of Colorectal Metastases.

BACKGROUND: Accurate biomarkers to monitor tumor load and response in metastatic colorectal cancer patients undergoing surgery could optimize treatment regimens. OBJECTIVE: This study aimed to explore the clinical validity of tumor-informed quantification of circulating tumor DNA in blood using ultradeep sequencing. DESIGN: Resection specimens from 53 colorectal cancer patients were analyzed for tumor-specific mutations in 15 genes. These mutations were used to measure the presence of circulating tumor DNA in preoperatively collected plasma samples using hybrid capture-based sequencing. Additional postoperative measurements were performed 1 week after surgery in 16 patients. SETTINGS: The study was conducted at the Radboud University Medical Center. PATIENTS: A total of 53 colorectal cancer patients undergoing surgery of metastases were included. MAIN OUTCOME MEASURES: The detection of circulating tumor DNA. RESULTS: At least 1 tumor-specific mutation was detected in all tumor samples. In preoperative plasma samples, circulating tumor DNA was detected in 88% (37/42) of systemic treatment-naïve patients and in 55% (6/11) of patients who received preoperative chemotherapy. More specifically, circulating tumor DNA was detected in 0% (0/3) of cases with a subtotal or partial pathologic response and in 75% (6/8) of cases without a pathologic response in the resection specimen ( p = 0.06). In postoperative plasma samples, circulating tumor DNA was detected in 80% (4/5) of patients with an incomplete resection and in 0% (0/11) of those with a complete resection ( p = 0.003). LIMITATIONS: The study was limited by the heterogeneity of the cohort and the small number of postoperative plasma samples. CONCLUSIONS: These data indicate that tumor-informed circulating tumor DNA detection in the plasma of patients undergoing surgery for metastatic colorectal cancer is feasible and may have clinical value in response monitoring and predicting residual disease. Prospective studies are needed to establish the clinical utility of circulating tumor DNA analysis to guide treatment decisions in these patients. See Video Abstract at http://links.lww.com/DCR/B990 . VALIDEZ CLNICA DEL ANLISIS DE ADN DEL TUMOR CIRCULANTE INFORMADO POR EL TUMOR EN PACIENTES SOMETIDOS A CIRUGA DE METSTASIS COLORRECTALES: ANTECEDENTES:Los biomarcadores precisos para monitorear la carga tumoral y la respuesta en pacientes con cáncer colorrectal metastásico que se someten a cirugía podrían optimizar los regímenes de tratamiento.OBJETIVO:Este estudio explora la validez clínica de la cuantificación informada por el tumor del ADN tumoral circulante en sangre mediante secuenciación ultraprofunda.DISEÑO:Se analizaron muestras de resección de 53 pacientes con cáncer colorrectal en busca de mutaciones específicas del tumor en quince genes. Estas mutaciones se usaron para medir la presencia de ADN tumoral circulante en muestras de plasma recolectadas antes de la operación usando secuenciación basada en captura híbrida. Se realizaron mediciones postoperatorias adicionales una semana después de la cirugía en dieciséis pacientes.AJUSTES:El estudio se realizó en el centro médico de la universidad de Radboud.PACIENTES:Se incluyeron un total de 53 pacientes con cáncer colorrectal sometidos a cirugía de metástasis.PRINCIPALES MEDIDAS DE RESULTADO:La detección de ADN tumoral circulante.RESULTADOS:Se detectó al menos una mutación específica de tumor en todas las muestras de tumor. En muestras de plasma preoperatorias, se detectó ADN tumoral circulante en el 88% (37/42) de los pacientes sin tratamiento sistémico previo y en el 55% (6/11) de los pacientes que recibieron quimioterapia preoperatoria. Más concretamente, en el 0% (0/3) de los casos con respuesta patológica subtotal o parcial y en el 75% (6/8) de los casos sin respuesta patológica en la pieza de resección ( p = 0,06). En muestras de plasma postoperatorio se detectó ADN tumoral circulante en el 80% (4/5) de los pacientes con una resección incompleta y en el 0% (0/11) de los que tenían resección completa ( p = 0,003).LIMITACIONES:El estudio estuvo limitado por la heterogeneidad de la cohorte y el pequeño número de muestras de plasma postoperatorias.CONCLUSIONES:Estos datos indican que la detección de ADN tumoral circulante informado por el tumor en el plasma de pacientes sometidos a cirugía por cáncer colorrectal metastásico es factible y puede tener valor clínico en el control de la respuesta y la predicción de la enfermedad residual. Se necesitan estudios prospectivos para establecer la utilidad clínica del análisis de ADN tumoral circulante para guiar las decisiones de tratamiento en estos pacientes. Consulte Video Resumen en http://links.lww.com/DCR/B990 . (Traducción-Dr. Mauricio Santamaria ).

Comprehensive clinicopathological and genomic profiling of gallbladder cancer reveals actionable targets in half of patients.

Gallbladder cancer (GBC) is a rare, highly aggressive malignancy with a 5-year survival rate of 5-10% in advanced cases, highlighting the need for more effective therapies. The aim of this study was to identify potentially actionable therapeutic targets for GBC. Specimens and clinicopathological data of 642 GBC patients, diagnosed between 2000 and 2019 were collected using the Dutch Pathology Registry (PALGA) and the Netherlands Cancer Registry. All cases were histologically reviewed and a subset was subjected to a comprehensive next generation sequencing panel. We assessed mutations and gene amplifications in a panel of 54 actionable genes, tumor-mutational burden (TMB), and microsatellite instability (MSI). Additionally, the entire cohort was screened for HER2, PD-L1, pan-TRK, and p53 expression with immunohistochemistry. Histopathological subtypes comprised biliary-type adenocarcinoma (AC, 69.6%), intestinal-type AC (20.1%) and other subtypes (10.3%). The median total TMB was 5.5 mutations/Mb (range: 0-161.1) and 17.7% of evaluable cases had a TMB of >10 mutations/Mb. MSI was observed in two cases. Apart from mutations in TP53 (64%), tumors were molecularly highly heterogeneous. Half of the tumors (50%) carried at least one molecular alteration that is targetable in other tumor types, including alterations in CDKN2A (6.0% biallelically inactivated), ERBB2 (9.3%) and PIK3CA (10%). Immunohistochemistry results correlated well with NGS results for HER2 and p53: Pearson r = 0.82 and 0.83, respectively. As half of GBC patients carry at least one potentially actionable molecular alteration, molecular testing may open the way to explore targeted therapy options for GBC patients.

Identification of Fusion Genes and Targets for Genetically Matched Therapies in a Large Cohort of Salivary Gland Cancer Patients.

Introduction: Salivary gland cancer (SGC) is a rare cancer for which systemic treatment options are limited. Therefore, it is important to characterize its genetic landscape in search for actionable aberrations, such as NTRK gene fusions. This research aimed to identify these actionable aberrations by combining NGS-based analysis of RNA (gene fusions) and DNA (single and multiple nucleotide variants, copy number variants, microsatellite instability and tumor mutational burden) in a large cohort of SGC patients. Methods: RNA and DNA were extracted from archival tissue of 121 patients with various SGC subtypes. Gene fusion analysis was performed using a customized RNA-based targeted NGS panel. DNA was sequenced using a targeted NGS panel encompassing 523 cancer-related genes. Cross-validation of NGS-based NTRK fusion detection and pan-TRK immunohistochemistry (IHC) was performed. Results: Fusion transcripts were detected in 50% of the cases and included both known (MYB-NFIB, MYBL1-NFIB, CRTC1-MAML2) and previously unknown fusions (including transcripts involving RET, BRAF or RAD51B). Only one NTRK fusion transcript was detected, in a secretory carcinoma case. Pan-TRK IHC (clone EPR17341) was false positive in 74% of cases. The proportion of patients with targets for genetically matched therapies differed among subtypes (salivary duct carcinoma: 82%, adenoid cystic carcinoma 28%, mucoepidermoid carcinoma 50%, acinic cell carcinoma 33%). Actionable aberrations were most often located in PIK3CA (n = 18, 15%), ERBB2 (n = 15, 12%), HRAS and NOTCH1 (both n = 9, 7%). Conclusions: Actionable genetic aberrations were seen in 53.7% of all SGC cases on the RNA and DNA level, with varying percentages between subtypes.

Germline MBD4 deficiency causes a multi-tumor predisposition syndrome.

We report an autosomal recessive, multi-organ tumor predisposition syndrome, caused by bi-allelic loss-of-function germline variants in the base excision repair (BER) gene MBD4. We identified five individuals with bi-allelic MBD4 variants within four families and these individuals had a personal and/or family history of adenomatous colorectal polyposis, acute myeloid leukemia, and uveal melanoma. MBD4 encodes a glycosylase involved in repair of G:T mismatches resulting from deamination of 5'-methylcytosine. The colorectal adenomas from MBD4-deficient individuals showed a mutator phenotype attributable to mutational signature SBS1, consistent with the function of MBD4. MBD4-deficient polyps harbored somatic mutations in similar driver genes to sporadic colorectal tumors, although AMER1 mutations were more common and KRAS mutations less frequent. Our findings expand the role of BER deficiencies in tumor predisposition. Inclusion of MBD4 in genetic testing for polyposis and multi-tumor phenotypes is warranted to improve disease management.

Mutational signature analysis in non-small cell lung cancer patients with a high tumor mutational burden.

BACKGROUND: Lung cancer is the leading cause of cancer death worldwide. With the growing number of targeted therapies and the introduction of immuno-oncology (IO), personalized medicine has become standard of care in patients with metastatic disease. The development of predictive and prognostic biomarkers is of great importance. Mutational signatures harbor potential clinical value as predictors of therapy response in cancer. Here we set out to investigate particular mutational processes by assessing mutational signatures and associations with clinical features, tumor mutational burden (TMB) and targetable mutations. METHODS: In this retrospective study, we studied tumor DNA from patients with non-small cell lung cancer (NSCLC) irrespective of stage. The samples were sequenced using a 2 megabase (Mb) gene panel. On each sample TMB was determined and defined as the total number of single nucleotide mutations per Mb (mut/Mb) including non-synonymous mutations. Mutational signature profiling was performed on tumor samples in which at least 30 somatic single base substitutions (SBS) were detected. RESULTS: In total 195 samples were sequenced. Median total TMB was 10.3 mut/Mb (range 0-109.3). Mutational signatures were evaluated in 76 tumor samples (39%; median TMB 15.2 mut/Mb). SBS signature 4 (SBS4), associated with tobacco smoking, was prominently present in 25 of 76 samples (33%). SBS2 and/or SBS13, both associated with activity of the AID/APOBEC family of cytidine deaminases, were observed in 11 of 76 samples (14%). SBS4 was significantly more present in early stages (I and II) versus advanced stages (III and IV; P = .005). CONCLUSION: In a large proportion of NSCLC patients tissue panel sequencing with a 2 Mb panel can be used to determine the mutational signatures. In general, mutational signature SBS4 was more often found in early versus advanced stages of NSCLC. Further studies are needed to determine the clinical utility of mutational signature analyses.

Unraveling Neuroendocrine Gallbladder Cancer: Comprehensive Clinicopathologic and Molecular Characterization.

PURPOSE: Neuroendocrine carcinomas and mixed neuroendocrine non-neuroendocrine neoplasms of the gallbladder (NE GBC) are rare and highly aggressive entities. The cell of origin of NE GBC has been a matter of controversy. Here, we performed a comparative histopathologic and molecular analysis of NE GBC cases and, if present, associated precancerous lesions. PATIENTS AND METHODS: We selected cases diagnosed between 2000 and 2019 in the Netherlands. Precursors and carcinomas were immunohistochemically compared and analyzed for mutations, gene amplifications, microsatellite instability, and tumor mutational burden using an next-generation sequencing panel containing 523 cancer-related genes. In addition, presence of fusion genes was analyzed using a panel of 55 genes. RESULTS: Sixty percent of neuroendocrine cases (6/10) presented with a precursor lesion, either intracholecystic papillary neoplasm (n = 3) or biliary intraepithelial neoplasia (n = 3). Immunohistochemically, neuroendocrine components were different from the epithelial precursor lesions. Molecular profiling, however, revealed TP53 mutations shared between different components in five of six cases, indicating a clonal relation. Furthermore, 40% of cases (4/10) harbored at least one potentially actionable alteration. This included (likely) pathogenic mutations in RAD54L, ATM, and BRCA2; amplifications of ERBB2 and MDM2; and a gene fusion involving FGFR3-TACC3. All cases were microsatellite-stable and had a tumor mutational burden of < 10 mutations/Mb. CONCLUSION: Our data provide insight into the development of NE GBC and suggest a common origin of precancerous epithelial lesions and invasive neuroendocrine components, favoring the hypothesis of lineage transformation. Moreover, nearly half of the NE GBCs carried at least one potentially actionable molecular alteration, highlighting the importance of molecular testing in this highly lethal cancer.

Comprehensive routine diagnostic screening to identify predictive mutations, gene amplifications, and microsatellite instability in FFPE tumor material.

BACKGROUND: Sensitive and reliable molecular diagnostics is needed to guide therapeutic decisions for cancer patients. Although less material becomes available for testing, genetic markers are rapidly expanding. Simultaneous detection of predictive markers, including mutations, gene amplifications and MSI, will save valuable material, time and costs. METHODS: Using a single-molecule molecular inversion probe (smMIP)-based targeted next-generation sequencing (NGS) approach, we developed an NGS panel allowing detection of predictive mutations in 33 genes, gene amplifications of 13 genes and microsatellite instability (MSI) by the evaluation of 55 microsatellite markers. The panel was designed to target all clinically relevant single and multiple nucleotide mutations in routinely available lung cancer, colorectal cancer, melanoma, and gastro-intestinal stromal tumor samples, but is useful for a broader set of tumor types. RESULTS: The smMIP-based NGS panel was successfully validated and cut-off values were established for reliable gene amplification analysis (i.e. relative coverage ≥3) and MSI detection (≥30% unstable loci). After validation, 728 routine diagnostic tumor samples including a broad range of tumor types were sequenced with sufficient sensitivity (2.4% drop-out), including samples with low DNA input (< 10 ng; 88% successful), low tumor purity (5-10%; 77% successful), and cytological material (90% successful). 75% of these tumor samples showed ≥1 (likely) pathogenic mutation, including targetable mutations (e.g. EGFR, BRAF, MET, ERBB2, KIT, PDGFRA). Amplifications were observed in 5.5% of the samples, comprising clinically relevant amplifications (e.g. MET, ERBB2, FGFR1). 1.5% of the tumor samples were classified as MSI-high, including both MSI-prone and non-MSI-prone tumors. CONCLUSIONS: We developed a comprehensive workflow for predictive analysis of diagnostic tumor samples. The smMIP-based NGS analysis was shown suitable for limited amounts of histological and cytological material. As smMIP technology allows easy adaptation of panels, this approach can comply with the rapidly expanding molecular markers.

Evaluation of a Hybrid Capture-Based Pan-Cancer Panel for Analysis of Treatment Stratifying Oncogenic Aberrations and Processes.

Stratification of patients for targeted and immune-based therapies requires extensive genomic profiling that enables sensitive detection of clinically relevant variants and interrogation of biomarkers, such as tumor mutational burden (TMB) and microsatellite instability (MSI). Detection of single and multiple nucleotide variants, copy number variants, MSI, and TMB was evaluated using a commercially available next-generation sequencing panel containing 523 cancer-related genes (1.94 megabases). Analysis of formalin-fixed, paraffin-embedded tissue sections and cytologic material from 45 tumor samples showed that all previously known MSI-positive samples (n = 7), amplifications (n = 9), and pathogenic variants (n = 59) could be detected. TMB and MSI scores showed high intralaboratory and interlaboratory reproducibility (eight samples tested in 11 laboratories). For reliable TMB analysis, 20 ng DNA was shown to be sufficient, even for relatively poor-quality samples. A minimum of 20% neoplastic cells was required to minimize variations in TMB values induced by chromosomal instability or tumor heterogeneity. Subsequent analysis of 58 consecutive lung cancer samples in a diagnostic setting was successful and revealed sufficient somatic mutations to generate mutational signatures in 14 cases. In conclusion, the 523-gene assay can be applied for evaluation of multiple DNA-based biomarkers relevant for treatment selection.

Mutational Signature Analysis Reveals NTHL1 Deficiency to Cause a Multi-tumor Phenotype.

Biallelic germline mutations affecting NTHL1 predispose carriers to adenomatous polyposis and colorectal cancer, but the complete phenotype is unknown. We describe 29 individuals carrying biallelic germline NTHL1 mutations from 17 families, of which 26 developed one (n = 10) or multiple (n = 16) malignancies in 14 different tissues. An unexpected high breast cancer incidence was observed in female carriers (60%). Mutational signature analysis of 14 tumors from 7 organs revealed that NTHL1 deficiency underlies the main mutational process in all but one of the tumors (93%). These results reveal NTHL1 as a multi-tumor predisposition gene with a high lifetime risk for extracolonic cancers and a typical mutational signature observed across tumor types, which can assist in the recognition of this syndrome.