Germline BRCA2 variant with low variant allele frequency detected in tumor-only comprehensive genomic profiling.

Germline BRCA1/2 variants in comprehensive genomic profiling (CGP) often exhibit variant allele frequency (VAF) exceeding 50%. However, when genomic loss occurs at the ipsilateral allele, including the germline variant in tumor cells, the VAF is low. This case report presents a patient with uterine sarcoma with a pathogenic BRCA2 mutation and low VAF in tumor-only CGP, which was later identified as a germline variant. When genomic alterations in BRCA1/2 are identified in tumor-only CGP, the possible germline origin of the variants should be considered, even if their VAF is very low.

BRCA1/2 reversion mutations in a pan-cancer cohort.

Tumor sensitivity to platinum (Pt)-based chemotherapy and poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors is increased by homologous recombination deficiency-causing mutations; in particular, reversion mutations cause drug resistance by restoring protein function. Treatment response is predicted by breast cancer susceptibility gene 1/2 (BRCA1/2) mutations; however, BRCA1/2 reversion mutations have not been comprehensively studied in pan-cancer cohorts. We aimed to characterize BRCA1/2 reversion mutations in a large pan-cancer cohort of Japanese patients by retrospectively analyzing sequencing data for BRCA1/2 pathogenic/likely pathogenic mutations in 3738 patients with 32 cancer types. We identified somatic mutations in tumors or circulating cell-free DNA that could restore the ORF of adverse alleles, including reversion mutations. We identified 12 (0.32%) patients with somatic BRCA1 (n = 3) and BRCA2 (n = 9) reversion mutations in breast (n = 4), ovarian/fallopian tube/peritoneal (n = 4), pancreatic (n = 2), prostate (n = 1), and gallbladder (n = 1) cancers. We identified 21 reversion events-BRCA1 (n = 3), BRCA2 (n = 18)-including eight pure deletions, one single-nucleotide variant, six multinucleotide variants, and six deletion-insertions. Seven (33.3%) reversion deletions showed a microhomology length greater than 1 bp, suggesting microhomology-mediated end-join repair. Disease course data were obtained for all patients with reversion events: four patients acquired mutations after PARP-inhibitor treatment failure, two showed somatic reversion mutations after disease progression, following Pt-based treatment, five showed mutations after both treatments, one patient with pancreatic cancer and BRCA1 reversion mutations had no history of either treatment. Although reversion mutations commonly occur in BRCA-associated cancers, our findings suggest that reversion mutations due to Pt-chemotherapy might be correlated with BRCA1/2-mediated tumorigenesis even in non-BRCA-associated histologies.

Clinical predominance of whole-exome sequencing to evaluate microsatellite instability status.

The microsatellite instability (MSI)/mismatch repair (MMR) status is one of the critical genomic biomarkers for predicting patient response to immune checkpoint inhibitors (ICIs). In this study, we aimed to investigate the concordance among the MSIsensor score obtained from whole-exome sequencing (WES), which could be a futuristic clinical cancer sequencing method, using only tumor tissues, MSI-PCR results, and immunohistochemistry (IHC) results to analyze various solid cancer types. We first endeavored to set the cut-off value of MSIsensor to determine functional deficient mismatch repair (f-dMMR) status. The MSI status of 1054 patients analyzed using WES was evaluated using MSIsensor. In addition, 87 of these patients were further analyzed using MSI-PCR and MMR IHC to calculate the sensitivity and specificity of the MSIsensor cut-off score. Our results showed that score 12.5 was an adequate cut-off score equivalent to PCR-confirmed MSS/MSI-low and MSI-high statuses, with sensitivity, specificity, and area under the curve values of 95.2%, 100%, and 0.998, respectively. Moreover, we identified false-positive cases of tumors with high mutational burden with an MSIsensor score <12.5, and optional IHC examination could rescue these cases. In conclusion, the MSIsensor score obtained using WES with tumor tissue showed a high clinical validity, with a cut-off value of 12.5 for f-dMMR detection, in combination with optional IHC analysis for MMR. Our novel algorithm will provide insights into the development of ICIs for cancer treatment, particularly when WES becomes a more common cancer genomic test in the near future.

Tumor Burden Monitoring with Circulating Tumor DNA During Treatment in Patients with Esophageal Squamous Cell Carcinoma.

BACKGROUND: To guide appropriate treatment strategy, an accurate tumor monitoring modality that reflects tumor burden during neoadjuvant treatment is required for esophageal squamous cell carcinoma (ESCC). We aimed to investigate the clinical utility of circulating tumor DNA (ctDNA) in plasma in patients who received neoadjuvant chemotherapy (NAC) followed by esophagectomy. PATIENTS AND METHODS: Longitudinally collected plasma samples for ctDNA combined with genomic DNA from primary lesions were obtained from patients with histologically confirmed ESCC who underwent NAC followed by subtotal esophagectomy. Next-generation sequencing was performed to identify mutations from the plasma and the primary tumor. The relationships between changes in ctDNA and the pathological response and recurrence were assessed in patients with locally advanced ESCC. RESULTS: In pretreatment samples from 13 patients, multiple concordant mutations in ctDNA and primary tumors were observed in 11 patients (85%), who were classified as ctDNA positive before treatment. The ctDNA positive rate after NAC correlated with the pathological response (responders, 25%; nonresponders, 100%; p = 0.007). The risk of recurrence increased significantly in patients with positive ctDNA after surgery in analysis of 16 patients; the 1-year recurrence-free survival rates were 90 and 0% in ctDNA-negative and ctDNA-positive groups, respectively (p = 0.0008). In two patients with postoperative recurrence, ctDNA was detected approximately 5.5 months earlier than the diagnosis using radiographical imaging. CONCLUSIONS: ctDNA is a promising biomarker for predicting pathological response and postoperative recurrence in ESCC. To demonstrate the external validity, we are currently preparing a multicenter prospective study.

The DNA integrity number and concentration are useful parameters for successful comprehensive genomic profiling test for cancer using formalin-fixed paraffin embedded tissue.

The acquisition of high-quality biospecimens and the appropriate handling of these materials are indispensable for successful clinical sequencing. We developed a cancer clinical sequencing system targeting 160 cancer genes: PleSSision-Rapid. Through the PleSSision-Rapid system, we have analyzed DNA quality evaluated by DIN (DNA integrity number) with 1329 formalin-fixed paraffin embedded (FFPE) samples including 477 prospectively collected tissues for genomic test (P) and 852 archival samples after routine pathological diagnosis (A1/A2). As a result, the samples with more than DIN 2.1 was 92.0% (439/477) in prospectively collected sample (P), while it was 85.6% (332/388) and 76.7% (356/464) in two types of archival samples (A1/A2). We performed the PleSSision-Rapid sequence using the samples with over DIN 2.1 and DNA concentration >10 ng/µL with which we were able to construct a DNA library, and the probability of sequence success was almost equivalent during all types of specimen processing, at 90.7% (398/439) in (P), 92.5% (307/332) in (A1) and 90.2% (321/356) in (A2), respectively. Our result indicated the clinical benefit to prepare the prospective collection of FFPE materials for indisputable clinical sequence, and that DIN ≥ 2.1 would be a solid parameter for sample preparation of comprehensive genomic profiling tests.

Hybridisation chain reaction-based visualisation and screening for lncRNA profiles in clear-cell renal-cell carcinoma.

BACKGROUND: Analysis of long noncoding RNA (lncRNA) localisation at both the tissue and subcellular levels can provide important insights into the cell types that are important for their function. METHODS: By applying new fluorescent in situ hybridisation technique called hybridisation chain reaction (HCR), we achieved a high-throughput lncRNA visualisation and evaluation of clinical samples. RESULTS: Assessing 1728 pairs of 16 lncRNAs and clear-cell renal-cell carcinoma (ccRCC) specimens, three lncRNAs (TUG1, HOTAIR and CDKN2B-AS1) were associated with ccRCC prognosis. Furthermore, we derived a new lncRNA risk group of ccRCC prognosis by combining the expression levels of these three lncRNAs. Examining genomic alterations underlying this classification revealed prominent features of tumours that could serve as potential biomarkers for targeting lncRNAs. We then derived combination of HCR with expansion microscopy and visualised nanoscale-resolution HCR signals in cell nuclei, uncovering intracellular colocalization of three lncRNA (TUG1, HOTAIR and CDKN2B-AS1) signals such as those located intra- or out of the nucleus or nucleolus in cancer cells. CONCLUSION: LncRNAs are expected to be desirable noncoding targets for cancer diagnosis or treatments. HCR involves plural probes consisting of small DNA oligonucleotides, clinically enabling us to detect cancerous lncRNA signals simply and rapidly at a lower cost.

Elevation of the Prognostic Factor Plasma Fibrinogen Reflects the Immunosuppressive Tumor Microenvironment in Esophageal Squamous Cell Carcinoma.

INTRODUCTION: Despite previous reports on the clinical significance of plasma fibrinogen (FNG) levels as a prognostic indicator of ESCC, its underlying mechanism remains unclear. This study aimed to validate the prognostic impact of plasma FNG levels and clarify its relationship with primary tumors in patients with esophageal squamous cell carcinoma (ESCC). METHODS: The prognostic impact of FNG was evaluated in patients with ESCC who underwent esophagectomy between 2000 and 2019. The RNA sequencing of the primary ESCC site, which was from pre-operative biopsy, was performed, followed by immune profile characterization using an immunogram. Those profiles were assessed via the immunohistochemical staining of tumor-associated macrophages (TAMs) and clinical response to nivolumab. RESULTS: Multivariate analysis identified FNG as a significant prognostic factor in ESCC. The immunogram suggested an immunosuppressive tumor environment in the high-FNG group. Immunostaining with the TAM markers CD163 and CD204, revealed that the high-FNG group had significantly higher number of TAMs compared with the low-FNG group. The immunosuppressive characteristics were clinically validated in patients with metastatic ESCC; those who had elevated FNG levels showed poor response to nivolumab. CONCLUSION: This study successfully validated the prognostic impact of plasma FNG levels in an expanded cohort with ESCC. Accordingly, our findings showed that increased plasma FNG reflects an immunosuppressive tumor microenvironment that facilitates tumor progression and poor responses to nivolumab.

Real-world application of next-generation sequencing-based test for surgically resectable colorectal cancer in clinical practice.

Aim: To evaluate the significance of next-generation sequencing-based gene panel testing in surgically resectable colorectal cancer by analyzing real-world data. Materials & methods: A total of 107 colorectal cancer patients who underwent curative surgery were included, and correlations between next-generation sequencing data and clinicopathological findings were evaluated. Results: More combination patterns in gene alteration were identified in advanced-stage tumors than in early-stage tumors. The copy number alteration count was significantly lower in right-sided colon tumors and early-stage tumors. Homologous recombination deficiency was more often identified in advanced-stage tumors, and high homologous recombination deficiency status was useful for identifying high-risk stage II tumors. Conclusion: Homologous recombination deficiency was identified as a useful result of gene panel testing with novel utility in clinical practice.

Landscape of prognostic signatures and immunogenomics of the AXL/GAS6 axis in renal cell carcinoma.

BACKGROUND: Cabozantinib is an oral tyrosine kinase inhibitor in renal cell carcinoma (RCC), whose targets include oncogenic AXL and unique ligand GAS6. Critical gaps in basic knowledge need to be addressed to devise an exclusive biomarker and candidate when targeting the AXL/GAS6 axis. METHODS: To clarify the effects of the AXL/GAS6 axis on RCC, we herein performed a large-scale immunogenomic analysis and single-cell counts including various metastatic organs and histological subtypes of RCC. We further applied genome-wide mutation analyses and methylation arrays. RESULTS: Varying patterns of AXL and GAS6 expression were observed throughout primary RCC tumours and metastases. Scoring individual AXL/GAS6 levels in the tumour centre and invasive margin, namely, the AXL/GAS6 score, showed a good ability to predict the prognosis of clear cell RCC. Metastasis- and histological subtype-specific differences in the AXL/GAS6 score existed since lung metastasis and the papillary subtype were weakly related to the AXL/GAS6 axis. Cell-by-cell immunohistological assessments clarified an immunosuppressive environment in tumours with high AXL/GAS6 scores. Genomic alterations in the PI3K-mTOR pathway and DNA methylation profiling revealed distinct differences with the AXL/GAS6 score in ccRCC. CONCLUSION: The AXL/GAS6 scoring system could predict the outcome of prognosis and work as a robust biomarker for the immunogenomic state in RCC.

ARID1A mutation/ARID1A loss is associated with a high immunogenic profile in clear cell ovarian cancer.

OBJECTIVES: ARID1A mutation is frequently found in clear cell ovarian cancer (CCC) and endometrioid ovarian cancer (EC). Anti-PD-1 monotherapy has been found to have limited efficacy in epithelial ovarian cancer; however, anti-PD-1 therapy showed significant clinical benefit in some CCC. We sought to define the relationship of ARID1A mutation/ARID1A expression to the immunogenic profile of different histologic subtypes of ovarian cancer. METHODS: We performed next-generation sequencing of 160 cancer-related genes. Also, we analyzed the immunohistochemical status of ARID1A, PD-L1, and CD8 with survival in different histologic subtypes of ovarian cancer in a total of 103 cases. RESULTS: ARID1A mutation was found in 0% of the high-grade serous ovarian cancer (HGSC) (n = 36), 41.5% of the CCC (n = 41), 45.0% of the EC (n = 20), and 33.3% of the mucinous ovarian cancer (MC) (n = 6) cases. ARID1A loss was found in 19.4% of the HGSC, 75.6% of the CCC, 60.0% of the EC and 0% of the MC cases. ARID1A mutation was found to be associated with high PD-L1 (p < 0.001) or CD8 levels (p < 0.001) in CCC but not in other histologic subtypes. Meanwhile, ARID1A loss was associated with high PD-L1 or CD8 levels in CCC (p < 0.001) and HGSC (p < 0.001) but not in EC and MC. In addition, ARID1A mutation was associated with high tumor mutation burden in CCC (p = 0.006). CONCLUSIONS: ARID1A mutation/ARID1A expression is associated with immune microenvironmental factors in CCC but not in EC. ARID1A status can be a biomarker for selecting candidates for immune checkpoint blockade in CCC.

The initial assessment of expert panel performance in core hospitals for cancer genomic medicine in Japan.

BACKGROUND: Since June 2019, cancer genomic profiling (CGP) tests have been reimbursed by the National Health Insurance system in Japan, with restrictions for government-designated hospitals with a molecular tumor board composed of multidisciplinary specialists, known as an expert panel (EP). The standardization of EPs is a critical challenge for implementing precision oncology in the clinical setting. METHODS: Data on consecutive cases who underwent the CGP tests at 11 core hospitals between June 2019 and January 2020 were collected. We evaluated the proportions of cases that received genomically matched treatments, including investigational new drugs (INDs) based on CGP results, and/or for which genetic counseling was recommended. Two simulated cases were annotated by each EP. The annotated reports were then centrally assessed. RESULTS: Each EP mainly discussed the applicability to genomically matched treatments and the necessity of performing genetic counseling. A pre-review of the report by key members in each EP reportedly made the EP conference more interactive and efficient, and thereby saved time. A total of 747 cases underwent CGP tests, 28 cases (3.7%) received genomically matched treatment, and 17 cases (2.3%) were referred for genetic counseling. Annotated reports for the simulated cases varied across the EPs, particularly the number of recommended IND trials, which seemed to be associated with the actual number of participants in IND trials. CONCLUSIONS: This investigation provides reference data for the application of precision oncology in a clinical setting. Further investigations on the standardization of clinical annotations are warranted.

Genetic Profiling of Malignant Melanoma Arising from an Ovarian Mature Cystic Teratoma: A Case Report.

Ovarian mature cystic teratomas comprise tissues derived from all three germ layers. In rare cases, malignant tumors arise from ovarian mature cystic teratoma. A variety of tumors can arise from mature cystic teratoma, among which primary malignant melanoma (MM), for which no molecular analyses such as genomic sequencing have been reported to date, is exceedingly rare, thereby limiting possible therapeutic options using precision medicine. We used targeted gene sequencing to analyze the status of 160 cancer-related genes in a patient with MM arising from an ovarian mature cystic teratoma (MM-MCT). KRAS amplification and homozygous deletion in PTEN and RB1 were detected in tumor samples collected from the patient. No KRAS amplification has been previously reported in cutaneous MM, indicating that the carcinogenesis of MM-MCT differs from that of primary cutaneous melanomas. A better understanding of the underlying genetic mechanisms will help clarify the carcinogenesis of MM-MCT. In turn, this will enable treatment with novel targeting agents as well as the initial exploration of gene-based precision oncological therapies, which aim to improve treatment outcomes for patients with this disease.

Germline Whole-Gene Deletion of FH Diagnosed from Tumor Profiling.

Hereditary leiomyomatosis and renal cell carcinoma (HL (RCC)) entails cutaneous and uterine leiomyomatosis with aggressive type 2 papillary RCC-like histology. HLRCC is caused by pathogenic variants in the FH gene, which encodes fumarate hydratase (FH). Here, we describe an episode of young-onset RCC caused by a genomic FH deletion that was diagnosed via clinical sequencing. A 35-year-old woman was diagnosed with RCC and multiple metastases: histopathological analyses supported a diagnosis of FH-deficient RCC. Although the patient had neither skin tumors nor a family history of HLRCC, an aggressive clinical course at her age and pathological diagnosis of FH-deficient RCC suggested a germline FH variant. After counseling, the patient provided written informed consent for germline genetic testing. She was simultaneously subjected to paired tumor profiling tests targeting the exome to identify a therapeutic target. Although conventional germline sequencing did not detect FH variants, exome sequencing revealed a heterozygous germline FH deletion. As such, paired tumor profiling, not conventional sequencing, was required to identify this genetic deletion. RCC caused by a germline FH deletion has hitherto not been described in Japan, and the FH deletion detected in this patient was presumed to be of maternal European origin. Although the genotype-phenotype correlation in HLRCC-related tumors is unclear, the patient's family was advised to undergo genetic counseling to consider additional RCC screening.

CDK12 and HER2 coamplification in two urothelial carcinomas with rapid and aggressive clinical progression.

Cyclin-dependent kinase 12 (CDK12), one of the key factors associated with DNA damage response pathways, is located on chromosome 17 proximal to Erb-B2 receptor tyrosine kinase 2 (ERBB2). In this report, CDK12 and ERBB2 coamplification was detected by targeted next-generation sequencing in two urothelial carcinomas. The staining intensity of the CDK12 and human epidermal growth factor receptor-2 proteins was associated with the prognosis of each urothelial carcinoma case. Our results suggest that CDK12 coamplification with ERBB2 might be associated with tumor aggressiveness and contribution to cancer pathogenesis. Therapies targeting CDK12 should be developed for these patients.

A first Japanese case of neuroendocrine prostate cancer accompanied by lung and brain metastasis with somatic and germline BRCA2 mutation.

Germline mutations and copy number changes in DNA damage repair (DDR) genes such as BRCA2 are associated with aggressive forms of prostate cancer (PCa). Although the prevalence of BRCA2 variants in PCa is increasing in Japan, the genomic and biological implications in Japanese patients are unclear. An 81-year-old male presented with prostatic adenocarcinoma with neuroendocrine differentiation accompanied by metastatic lung nodule and brain metastases. Platinum-based doublet chemotherapy combined with etoposide resulted in partial and complete remissions of brain and lung metastases, respectively. Next-generation sequencing of biopsy and peripheral blood samples demonstrated a somatic BRCA2 mutation at c.7008-2A>C and a germline mutation at p.E2877*. The patient's son had been diagnosed with breast cancer 2.5 years ago and was found to have the same germline BRCA2 mutation. BRCA2 mutation increases the risks of aggressive PCa and other cancer types in Japanese males. These forms may be highly responsive to platinum-based chemotherapy.

Salivary Duct Carcinoma Arising in the Submandibular Gland in a Patient with Neurofibromatosis Type 1.

A 71-year-old man with neurofibromatosis type 1 (NF1) presented to our department with a 1-week history of a painful mass in the left submandibular area. Computed tomography (CT) and magnetic resonance imaging revealed an irregular-shaped tumor with a diameter of 2.0 cm in the left submandibular gland and a metastatic lymph node with a diameter of 1.0 cm adjacent to the tumor. Fluorodeoxyglucose-positron emission tomography/CT revealed increased uptake in the tumor. Fine-needle aspiration cytology revealed atypical cells, suggesting salivary duct carcinoma (SDC). Left neck dissection with resection of the tumor and submandibular gland was performed under general anesthesia. Histologic examination revealed ductal formation with a solid, cystic, cribriform, and papillary structure with intraductal comedonecrosis, diagnosing as SDC originating in the submandibular gland (pT3N1M0 pStage III). Mutational analysis of 160 cancer-related genes by next-generation sequencing (NGS) revealed a germline and frameshift mutation in the NF1 gene (p.R2408Kfs*14) and a somatic and frameshift mutation in the TP53 gene (p.C176Wfs*22). The patient received postoperative radiotherapy to the left neck area at 66 Gy. No evidence of recurrence or metastasis has been observed as of 10 months postoperatively. This is the first reported case of SDC in the submandibular gland in a patient with NF1. The mutational data by NGS may contribute to a better understanding of the oncogenesis of SDC in patients with NF1.