An Organoid Biobank of Neuroendocrine Neoplasms Enables Genotype-Phenotype Mapping.

Gastroenteropancreatic (GEP) neuroendocrine neoplasm (NEN) that consists of neuroendocrine tumor and neuroendocrine carcinoma (NEC) is a lethal but under-investigated disease owing to its rarity. To fill the scarcity of clinically relevant models of GEP-NEN, we here established 25 lines of NEN organoids and performed their comprehensive molecular characterization. GEP-NEN organoids recapitulated pathohistological and functional phenotypes of the original tumors. Whole-genome sequencing revealed frequent genetic alterations in TP53 and RB1 in GEP-NECs, and characteristic chromosome-wide loss of heterozygosity in GEP-NENs. Transcriptome analysis identified molecular subtypes that are distinguished by the expression of distinct transcription factors. GEP-NEN organoids gained independence from the stem cell niche irrespective of genetic mutations. Compound knockout of TP53 and RB1, together with overexpression of key transcription factors, conferred on the normal colonic epithelium phenotypes that are compatible with GEP-NEN biology. Altogether, our study not only provides genetic understanding of GEP-NEN, but also connects its genetics and biological phenotypes.

Deep whole-genome analysis of 494 hepatocellular carcinomas.

Over half of hepatocellular carcinoma (HCC) cases diagnosed worldwide are in China1-3. However, whole-genome analysis of hepatitis B virus (HBV)-associated HCC in Chinese individuals is limited4-8, with current analyses of HCC mainly from non-HBV-enriched populations9,10. Here we initiated the Chinese Liver Cancer Atlas (CLCA) project and performed deep whole-genome sequencing (average depth, 120×) of 494 HCC tumours. We identified 6 coding and 28 non-coding previously undescribed driver candidates. Five previously undescribed mutational signatures were found, including aristolochic-acid-associated indel and doublet base signatures, and a single-base-substitution signature that we termed SBS_H8. Pentanucleotide context analysis and experimental validation confirmed that SBS_H8 was distinct to the aristolochic-acid-associated SBS22. Notably, HBV integrations could take the form of extrachromosomal circular DNA, resulting in elevated copy numbers and gene expression. Our high-depth data also enabled us to characterize subclonal clustered alterations, including chromothripsis, chromoplexy and kataegis, suggesting that these catastrophic events could also occur in late stages of hepatocarcinogenesis. Pathway analysis of all classes of alterations further linked non-coding mutations to dysregulation of liver metabolism. Finally, we performed in vitro and in vivo assays to show that fibrinogen alpha chain (FGA), determined as both a candidate coding and non-coding driver, regulates HCC progression and metastasis. Our CLCA study depicts a detailed genomic landscape and evolutionary history of HCC in Chinese individuals, providing important clinical implications.

Helicobacter pylori, Homologous-Recombination Genes, and Gastric Cancer.

BACKGROUND: Helicobacter pylori infection is a well-known risk factor for gastric cancer. However, the contribution of germline pathogenic variants in cancer-predisposing genes and their effect, when combined with H. pylori infection, on the risk of gastric cancer has not been widely evaluated. METHODS: We evaluated the association between germline pathogenic variants in 27 cancer-predisposing genes and the risk of gastric cancer in a sample of 10,426 patients with gastric cancer and 38,153 controls from BioBank Japan. We also assessed the combined effect of pathogenic variants and H. pylori infection status on the risk of gastric cancer and calculated the cumulative risk in 1433 patients with gastric cancer and 5997 controls from the Hospital-based Epidemiologic Research Program at Aichi Cancer Center (HERPACC). RESULTS: Germline pathogenic variants in nine genes (APC, ATM, BRCA1, BRCA2, CDH1, MLH1, MSH2, MSH6, and PALB2) were associated with the risk of gastric cancer. We found an interaction between H. pylori infection and pathogenic variants in homologous-recombination genes with respect to the risk of gastric cancer in the sample from HERPACC (relative excess risk due to the interaction, 16.01; 95% confidence interval [CI], 2.22 to 29.81; P = 0.02). At 85 years of age, persons with H. pylori infection and a pathogenic variant had a higher cumulative risk of gastric cancer than noncarriers infected with H. pylori (45.5% [95% CI, 20.7 to 62.6] vs. 14.4% [95% CI, 12.2 to 16.6]). CONCLUSIONS: H. pylori infection modified the risk of gastric cancer associated with germline pathogenic variants in homologous-recombination genes. (Funded by the Japan Agency for Medical Research and Development and others.).

Genomic features of renal cell carcinoma developed during end-stage renal disease and dialysis.

Patients with end-stage renal disease (ESRD) or receiving dialysis have a much higher risk for renal cell carcinoma (RCC), but carcinogenic mechanisms and genomic features remain little explored and undefined. This study's goal was to identify the genomic features of ESRD RCC and characterize them for associations with tumor histology and dialysis exposure. In this study, we obtained 33 RCCs, with various histological subtypes, that developed in ESRD patients receiving dialysis and performed whole-genome sequencing and transcriptome analyses. Driver events, copy-number alteration (CNA) analysis and mutational signature profiling were performed using an analysis pipeline that integrated data from germline and somatic SNVs, Indels and structural variants as well as CNAs, while transcriptome data were analyzed for differentially expressed genes and through gene set enrichment analysis. ESRD related clear cell RCCs' driver genes and mutations mirrored those in sporadic ccRCCs. Longer dialysis periods significantly correlated with a rare mutational signature SBS23, whose etiology is unknown, and increased mitochondrial copy number. All acquired cystic disease (ACD)-RCCs, which developed specifically in ESRD patients, showed chromosome 16q amplification. Gene expression analysis suggests similarity between certain ACD-RCCs and papillary RCCs and in TCGA papillary RCCs with chromosome 16 gain identified enrichment for genes related to DNA repair, as well as pathways related to reactive oxygen species, oxidative phosphorylation and targets of Myc. This analysis suggests that ESRD or dialysis could induce types of cellular stress that impact some specific types of genomic damage leading to oncogenesis.

Variant spectrum of von Hippel-Lindau disease and its genomic heterogeneity in Japan.

Von Hippel-Lindau (VHL) disease is an autosomal dominant, inherited syndrome with variants in the VHL gene, causing predisposition to multi-organ neoplasms with vessel abnormality. Germline variants in VHL can be detected in 80-90% of patients clinically diagnosed with VHL disease. Here, we summarize the results of genetic tests for 206 Japanese VHL families, and elucidate the molecular mechanisms of VHL disease, especially in variant-negative unsolved cases. Of the 206 families, genetic diagnosis was positive in 175 families (85%), including 134 families (65%) diagnosed by exon sequencing (15 novel variants) and 41 (20%) diagnosed by multiplex ligation-dependent probe amplification (MLPA) (one novel variant). The deleterious variants were significantly enriched in VHL disease Type 1. Interestingly, five synonymous or non-synonymous variants within exon 2 caused exon 2 skipping, which is the first report of exon 2 skipping caused by several missense variants. Whole genome and target deep sequencing analysis were performed for 22 unsolved cases with no variant identified and found three cases with VHL mosaicism (variant allele frequency: 2.5-22%), one with mobile element insertion in the VHL promoter region, and two with a pathogenic variant of BAP1 or SDHB. The variants associated with VHL disease are heterogeneous, and for more accuracy of the genetic diagnosis of VHL disease, comprehensive genome and DNA/RNA analyses are required to detect VHL mosaicism, complicated structure variants and other related gene variants.

Comprehensive analysis of full-length transcripts reveals novel splicing abnormalities and oncogenic transcripts in liver cancer.

Genes generate transcripts of various functions by alternative splicing. However, in most transcriptome studies, short-reads sequencing technologies (next-generation sequencers) have been used, leaving full-length transcripts unobserved directly. Although long-reads sequencing technologies would enable the sequencing of full-length transcripts, the data analysis is difficult. In this study, we developed an analysis pipeline named SPLICE and analyzed cDNA sequences from 42 pairs of hepatocellular carcinoma (HCC) and matched non-cancerous livers with an Oxford Nanopore sequencer. Our analysis detected 46,663 transcripts from the protein-coding genes in the HCCs and the matched non-cancerous livers, of which 5,366 (11.5%) were novel. A comparison of expression levels identified 9,933 differentially expressed transcripts (DETs) in 4,744 genes. Interestingly, 746 genes with DETs, including the LINE1-MET transcript, were not found by a gene-level analysis. We also found that fusion transcripts of transposable elements and hepatitis B virus (HBV) were overexpressed in HCCs. In vitro experiments on DETs showed that LINE1-MET and HBV-human transposable elements promoted cell growth. Furthermore, fusion gene detection showed novel recurrent fusion events that were not detected in the short-reads. These results suggest the efficiency of full-length transcriptome studies and the importance of splicing variants in carcinogenesis.

Regulation of the innate immune response and gut microbiome by p53.

p53 is a key tumor suppressor mutated in half of human cancers. In recent years, p53 was shown to regulate a wide variety of functions. From the transcriptome analysis of 24 tissues of irradiated mice, we identified 553 genes markedly induced by p53. Gene Ontology (GO) enrichment analysis found that the most associated biological process was innate immunity. 16S rRNA-seq analysis revealed that Akkermansia, which has anti-inflammatory properties and is involved in the regulation of intestinal barrier integrity, was decreased in p53-knockout (p53-/- ) mice after radiation. p53-/- mice were susceptible to radiation-induced GI toxicity and had a significantly shorter survival time than p53-wild-type (p53+/+ ) mice following radiation. However, administration of antibiotics resulted in a significant improvement in survival and protection against GI toxicity. Mbl2 and Lcn2, which have antimicrobial activity, were identified to be directly transactivated by p53 and secreted by liver into the circulatory system. We also found the expression of MBL2 and LCN2 was decreased in liver cancer tissues with p53 mutations compared with those without p53 mutations. These results indicate that p53 is involved in shaping the gut microbiome through its downstream targets related to the innate immune system, thus protecting the intestinal barrier.

Postoperative recurrence detection using individualized circulating tumor DNA in upper tract urothelial carcinoma.

Biomarkers that could detect the postoperative recurrence of upper tract urothelial carcinoma (UTUC) have not been established. In this prospective study, we aim to evaluate the utility of individualized circulating tumor DNA (ctDNA) monitoring using digital PCR (dPCR) as a tumor recurrence biomarker for UTUC in the perioperative period. Twenty-three patients who underwent radical nephroureterectomy (RNU) were included. In each patient, whole exome sequencing by next-generation sequencing and TERT promoter sequencing of tumor DNA were carried out. Case-specific gene mutations were selected from sequencing analysis to examine ctDNA by dPCR analysis. We also prospectively collected plasma and urine ctDNA from each patient. The longitudinal variant allele frequencies of ctDNA during the perioperative period were plotted. Case-specific gene mutations were detected in 22 cases (96%) from ctDNA in the preoperative samples. Frequently detected genes were TERT (39%), FGFR3 (26%), TP53 (22%), and HRAS (13%). In all cases, we obtained plasma and urine samples for 241 time points and undertook individualized ctDNA monitoring for 2 years after RNU. Ten patients with intravesical recurrence had case-specific ctDNA detected in urine at the time of recurrence. The mean lead time of urinary ctDNA in intravesical recurrence was 60 days (range, 0-202 days). Two patients with distal metastasis had case-specific ctDNA in plasma at the time of metastasis. In UTUC, tumor-specific gene mutations can be monitored postoperatively as ctDNA in plasma and urine. Individualized ctDNA might be a minimally invasive biomarker for the early detection of postoperative recurrence.

Different risk genes contribute to clear cell and non-clear cell renal cell carcinoma in 1532 Japanese patients and 5996 controls.

Identifying causative genes via genetic testing is useful for screening, preventing and treating cancer. Several hereditary syndromes occur in patients with renal cell carcinoma (RCC). However, the evidence is from the European population; it remains unclear how the RCC-related genes and other cancer-predisposing genes contribute to RCC development in the Japanese population. A case-control study of 14 RCC-related genes and 26 cancer-predisposing genes was performed in 1563 Japanese patients with RCC and 6016 controls. The patients were stratified into clear cell RCC (ccRCC) or non-ccRCC (nccRCC). Gene-based analysis of germline pathogenic variants in patients with each subtype and cancer-free subjects was performed. Following quality control, 1532 patients with RCC and 5996 controls were analyzed. For ccRCC, 52 of 1283 (4.05%) patients carried pathogenic variants mainly in the cancer-predisposing genes such as TP53 (P = 1.73 × 10-4; OR, 5.8; 95% CI, 2.2-15.7). Approximately 80% of patients with pathogenic variants in TP53 had p.Ala189Val that was specific in East Asian population. For nccRCC, 14 of 249 (5.62%) patients carried pathogenic variants mainly in the RCC-related genes such as BAP1 and FH (P = 6.27 × 10-5; OR, Inf; 95% CI, 10.0-Inf). The patients with the pathogenic variants in the associated genes were diagnosed 15.8 years earlier and had a higher proportion of patients with a family history of RCC (OR, 20.0; 95% CI, 1.3-237.4) than the non-carriers. We showed different and population-specific contributions of risk genes between ccRCC and nccRCC in Japanese for improved personalized medicine.

Cutibacterium acnes invades prostate epithelial cells to induce BRCAness as a possible pathogen of prostate cancer.

BACKGROUND: Abundant evidence suggests that chronic inflammation is linked to prostate cancer and that infection is a possible cause of prostate cancer. METHODS: To identify microbiota or pathogens associated with prostate cancer, we investigated the transcriptomes of 20 human prostate cancer tissues. We performed de novo assembly of nonhuman sequences from RNA-seq data. RESULTS: We identified four bacteria as candidate microbiota in the prostate, including Moraxella osloensis, Uncultured chroococcidiopsis, Cutibacterium acnes, and Micrococcus luteus. Among these, C. acnes was detected in 19 of 20 prostate cancer tissue samples by immunohistochemistry. We then analyzed the gene expression profiles of prostate epithelial cells infected in vitro with C. acnes and found significant changes in homologous recombination (HR) and the Fanconi anemia pathway. Notably, electron microscopy demonstrated that C. acnes invaded prostate epithelial cells and localized in perinuclear vesicles, whereas analysis of γH2AX foci and HR assays demonstrated impaired HR repair. In particular, BRCA2 was significantly downregulated in C. acnes-infected cells. CONCLUSIONS: These findings suggest that C. acnes infection in the prostate could lead to HR deficiency (BRCAness) which promotes DNA double-strand breaks, thereby increasing the risk of cancer development.

Immunotherapies targeting neoantigens are effective in PD-1 blockade-resistant tumors.

Only a small fraction of tumor-infiltrating lymphocytes can specifically recognize and attack cancer cells in PD-1/PD-L1 blockade therapy. Here, we investigate approaches to expand the neoantigen-specific CD8+ T cells to overcome the difficulties in treating PD-1/PD-L1 blockade-resistant tumors. Mutation-associated neoepitopes of murine nonsmall cell lung cancer ASB-XIV were estimated by whole-exome and RNA sequencing and predicted by MHC-I binding affinity (FPKM >1) in silico. Using ASB-XIV-specific CD8+ T cells, we screened a panel of 257 neoepitope peptides derived from ASB-XIV missense and indel mutations. Mutated Phf3 peptide (mPhf3) was successfully identified as an immunogenic neoepitope. Prophylactic mPhf3-DC vaccination inhibited ASB-XIV tumor growth through CD8+ T cell-mediated antitumor immunity. Combining the mPhf3-DC vaccine and anti-PD-1 treatment elicited robust antitumor activity through the induction of mPhf3-specific CD8+ T cells in the tumor microenvironment. Furthermore, the adoptive transfer of mPhf3-specific CD8+ T cells eradicated ASB-XIV tumors. Likewise, the combination of mutated Cdt1 peptide (mCdt1)-DC vaccine and anti-PD-1 treatment or adoptive transfer of mCdt1-specific CD8+ T cells also led to significant regression of PD-1 blockade-resistant murine gastric YTN16 tumors. In conclusion, a novel immunogenic neoepitope of ASB-XIV was identified for immunotherapy targeting neoantigens. Identification of immunogenic neoantigens can extend the therapeutic strategies by increasing the frequency of neoantigen-specific T cells, even for PD-1/PD-L1 blockade-resistant tumors.

Hereditary cancer variants and homologous recombination deficiency in biliary tract cancer.

BACKGROUND & AIMS: The heritability and actionability of variants in homologous recombination-related genes in biliary tract cancers (BTCs) are uncertain. Although associations between BTC and BRCA germline variants have been reported, homologous recombination deficiency has not been investigated in BTCs. METHODS: We sequenced germline variants in 27 cancer-predisposing genes in 1,292 BTC cases and 37,583 controls without a personal nor family history of cancer. We compared pathogenic germline variant frequencies between cases and controls and documented the demographic and clinical characteristics of carriers. In addition, whole-genome sequencing of 45 BTC tissues was performed to evaluate homologous recombination deficiency status. RESULTS: Targeted sequencing identified 5,018 germline variants, which were classified into 317 pathogenic, 3,611 variants of uncertain significance, and 1,090 benign variants. Seventy-one BTC cases (5.5%) had at least one pathogenic variant among 27 cancer-predisposing genes. Pathogenic germline variants enriched in BTCs were present in BRCA1, BRCA2, APC, and MSH6 (p <0.00185). PALB2 variants were marginally associated with BTC (p = 0.01). APC variants were predominantly found in ampulla of Vater carcinomas. Whole-genome sequencing demonstrated that three BTCs with pathogenic germline variants in BRCA2 and PALB2, accompanied by loss of heterozygosity, displayed homologous recombination deficiency. Conversely, pathogenic germline variants without a second hit or variants of other homologous recombination-related genes such as ATM and BRIP1 showed homologous recombination-proficient phenotypes. CONCLUSIONS: In this study, we describe the heritability and actionability of variants in homologous recombination-related genes, which could be used to guide screening and therapeutic strategies for BTCs. IMPACT AND IMPLICATIONS: We found that 5.5% of biliary tract cancers (BTCs) in a Japanese population possessed hereditary cancer-predisposing gene alterations, including in BRCA and genes associated with colorectal cancer. Two hits in homologous recombination-related genes were required to confer a homologous recombination-deficient phenotype. PARP inhibitors and DNA-damaging regimens may be effective strategies against BTCs exhibiting homologous recombination deficiency. Hence, in this study, genome-wide sequencing has revealed a potential new therapeutic strategy that could be applied to a subset of BTCs.

Comprehensive analysis of indels in whole-genome microsatellite regions and microsatellite instability across 21 cancer types.

Microsatellites are repeats of 1- to 6-bp units, and approximately 10 million microsatellites have been identified across the human genome. Microsatellites are vulnerable to DNA mismatch errors and have thus been used to detect cancers with mismatch repair deficiency. To reveal the mutational landscape of microsatellite repeat regions at the genome level, we analyzed approximately 20.1 billion microsatellites in 2717 whole genomes of pan-cancer samples across 21 tissue types. First, we developed a new insertion and deletion caller (MIMcall) that takes into consideration the error patterns of different types of microsatellites. Among the 2717 pan-cancer samples, our analysis identified 31 samples, including colorectal, uterus, and stomach cancers, with a higher proportion of mutated microsatellite (≥0.03), which we defined as microsatellite instability (MSI) cancers of genome-wide level. Next, we found 20 highly mutated microsatellites that can be used to detect MSI cancers with high sensitivity. Third, we found that replication timing and DNA shape were significantly associated with mutation rates of microsatellites. Last, analysis of mutations in mismatch repair genes showed that somatic SNVs and short indels had larger functional impacts than germline mutations and structural variations. Our analysis provides a comprehensive picture of mutations in the microsatellite regions and reveals possible causes of mutations, as well as provides a useful marker set for MSI detection.

Endogenization and excision of human herpesvirus 6 in human genomes.

Sequences homologous to human herpesvirus 6 (HHV-6) are integrated within the nuclear genome of about 1% of humans, but it is not clear how this came about. It is also uncertain whether integrated HHV-6 can reactivate into an infectious virus. HHV-6 integrates into telomeres, and this has recently been associated with polymorphisms affecting MOV10L1. MOV10L1 is located on the subtelomere of chromosome 22q (chr22q) and is required to make PIWI-interacting RNAs (piRNAs). As piRNAs block germline integration of transposons, piRNA-mediated repression of HHV-6 integration has been proposed to explain this association. In vitro, recombination of the HHV-6 genome along its terminal direct repeats (DRs) leads to excision from the telomere and viral reactivation, but the expected "solo-DR scar" has not been described in vivo. Here we screened for integrated HHV-6 in 7,485 Japanese subjects using whole-genome sequencing (WGS). Integrated HHV-6 was associated with polymorphisms on chr22q. However, in contrast to prior work, we find that the reported MOV10L1 polymorphism is physically linked to an ancient endogenous HHV-6A variant integrated into the telomere of chr22q in East Asians. Unexpectedly, an HHV-6B variant has also endogenized in chr22q; two endogenous HHV-6 variants at this locus thus account for 72% of all integrated HHV-6 in Japan. We also report human genomes carrying only one portion of the HHV-6B genome, a solo-DR, supporting in vivo excision and possible viral reactivation. Together these results explain the recently-reported association between integrated HHV-6 and MOV10L1/piRNAs, suggest potential exaptation of HHV-6 in its coevolution with human chr22q, and clarify the evolution and risk of reactivation of the only intact (non-retro)viral genome known to be present in human germlines.

Mitochondrial DNA Variants at Low-Level Heteroplasmy and Decreased Copy Numbers in Chronic Kidney Disease (CKD) Tissues with Kidney Cancer.

The human mitochondrial genome (mtDNA) is a circular DNA molecule with a length of 16.6 kb, which contains a total of 37 genes. Somatic mtDNA mutations accumulate with age and environmental exposure, and some types of mtDNA variants may play a role in carcinogenesis. Recent studies observed mtDNA variants not only in kidney tumors but also in adjacent kidney tissues, and mtDNA dysfunction results in kidney injury, including chronic kidney disease (CKD). To investigate whether a relationship exists between heteroplasmic mtDNA variants and kidney function, we performed ultra-deep sequencing (30,000×) based on long-range PCR of DNA from 77 non-tumor kidney tissues of kidney cancer patients with CKD (stages G1 to G5). In total, this analysis detected 697 single-nucleotide variants (SNVs) and 504 indels as heteroplasmic (0.5% ≤ variant allele frequency (VAF) < 95%), and the total number of detected SNVs/indels did not differ between CKD stages. However, the number of deleterious low-level heteroplasmic variants (pathogenic missense, nonsense, frameshift and tRNA) significantly increased with CKD progression (p < 0.01). In addition, mtDNA copy numbers (mtDNA-CNs) decreased with CKD progression (p < 0.001). This study demonstrates that mtDNA damage, which affects mitochondrial genes, may be involved in reductions in mitochondrial mass and associated with CKD progression and kidney dysfunction.

The presence of vessels encapsulating tumor clusters is associated with an immunosuppressive tumor microenvironment in hepatocellular carcinoma.

Recently, a distinct vascular pattern in hepatocellular carcinoma (HCC) called vessels encapsulating tumor-forming clusters (VETC) has received attention because of its association with poor prognosis. However, little is known about the mechanism by which VETC promotes an aggressive phenotype at the molecular level. In our study, the association between differences in stepwise signal intensity in the HB phase and molecular subtypes and somatic mutations associated with the immune microenvironment were investigated using the International Cancer Genome Consortium (ICGC) cohort (66 patients). To our knowledge, this is the first study to analyze the molecular patterns of VETC using RNA-Seq data. The VETC+ HCC group showed significantly lower overall survival and higher cumulative incidence of extrahepatic metastasis after curative hepatic resection than the VETC- HCC group. The VETC+ group exhibited molecular features indicative of lower immune activation than the VETC- group, suggesting that tumor cells in the VETC+ group were more likely to escape from the immune response, which could lead to the shorter OS (Overall survival) and higher risk of metastasis. On the other hand, gene expression levels of fibroblast growth factor receptors were upregulated in VETC+ HCC, suggesting that VETC+ HCC might benefit from lenvatinib treatment. Our results demonstrate that VETC+ HCC was associated with the suppression of tumor immune responses at the molecular level.

Association between germline pathogenic variants in cancer-predisposing genes and lymphoma risk.

The application of advanced molecular technology has significantly expanded lymphoma classification, allowing risk stratification and treatment optimization. Limited evidence suggests the presence of a genetic predisposition in lymphoma, indicating the potential for better individualized clinical management based on a novel lymphoma classification. Herein, we examined the impact of germline pathogenic variants in 27 cancer-predisposing genes with lymphoma risk and explored the clinical characteristics of pathogenic variant carriers. This study included 2,066 lymphoma patients and 38,153 cancer-free controls from the Japanese population. Following quality control of sequencing data, samples from 1,982 lymphoma patients and 37,592 controls were further analyzed. We identified 309 pathogenic variants among 4,850 variants in the 27 cancer-predisposing genes. Pathogenic variants in the following four cancer-predisposing genes were associated with a high risk of lymphoma: ATM (odds ratio [OR], 2.63; 95% confidence interval [CI], 1.25-5.51; p = 1.06 × 10-2 ), BRCA1 (OR, 5.88; 95% CI, 2.65-13.02; p = 1.27 × 10-5 ), BRCA2 (OR, 2.94; 95% CI, 1.60-5.42; p = 5.25 × 10-4 ), and TP53 (OR, 5.22; 95% CI, 1.43-19.02; p = 1.23 × 10-2 ). The proportion of carriers of these genes was 1.6% of lymphoma patients. Furthermore, pathogenic variants in these genes were especially associated with a higher risk of mantle cell lymphoma (OR, 21.57; 95% CI, 7.59-61.26; p = 8.07 × 10-9 ). These results provide novel insights concerning monogenic form into lymphoma classification. Some lymphoma patients may benefit from surveillance and targeted treatment, such as other neoplasms.

Prognostic biomarker study in patients with clinical stage I esophageal squamous cell carcinoma: JCOG0502-A1.

We undertook genomic analyses of Japanese patients with stage I esophageal squamous cell carcinoma (ESCC) to investigate the frequency of genomic alterations and the association with survival outcomes. Biomarker analysis was carried out for patients with clinical stage T1bN0M0 ESCC enrolled in JCOG0502 (UMIN000000551). Whole-exome sequencing (WES) was performed using DNA extracted from formalin-fixed, paraffin-embedded tissue of ESCC and normal tissue or blood sample. Single nucleotide variants (SNVs), insertions/deletions (indels), and copy number alterations (CNAs) were identified. We then evaluated the associations between each gene alteration with a frequency of 10% or more and progression-free survival (PFS) using a Cox regression model. We controlled for family-wise errors at 0.05 using the Bonferroni method. Among the 379 patients who were enrolled in JCOG0502, 127 patients were successfully analyzed using WES. The median patient age was 63 years (interquartile range, 57-67 years), and 78.0% of the patients ultimately underwent surgery. The 3-year PFS probability was 76.3%. We detected 20 genes with SNVs, indels, or amplifications with a frequency of 10% or more. Genomic alterations in FGF19 showed the strongest association with PFS with a borderline level of statistical significance of P = .00252 (Bonferroni-adjusted significance level is .0025). Genomic alterations in FGF4, MYEOV, CTTN, and ORAOV1 showed a marginal association with PFS (P < .05). These genomic alterations were all CNAs at chromosome 11q13.3. We have identified new genomic alterations associated with the poor efficacy of ESCC (T1bN0M0). These findings open avenues for the development of new potential treatments for patients with ESCC.

Prognostic significance of pathogenic variants in BRCA1, BRCA2, ATM and PALB2 genes in men undergoing hormonal therapy for advanced prostate cancer.

BACKGROUND: The prognostic significance of germline variants in homologous recombination repair genes in advanced prostate cancer (PCa), especially with regard to hormonal therapy, remains controversial. METHODS: Germline DNA from 549 Japanese men with metastatic and/or castration-resistant PCa was sequenced for 27 cancer-predisposing genes. The associations between pathogenic variants and clinical outcomes were examined. Further, for comparison, DNA from prostate biopsy tissue samples from 80 independent patients with metastatic PCa were analysed. RESULTS: Forty-four (8%) patients carried germline pathogenic variants in one of the analysed genes. BRCA2 was most frequently altered (n = 19), followed by HOXB13 (n = 9), PALB2 (n = 5) and ATM (n = 5). Further, the BRCA1, BRCA2, PALB2 and ATM variants showed significant association with a short time to castration resistance and overall survival (hazard ratio = 1.99 and 2.36; 95% CI, 1.15-3.44 and 1.23-4.51, respectively), independent of other clinical variables. Based on log-rank tests, the time to castration resistance was also significantly short in patients with BRCA1, BRCA2, PALB2 or ATM somatic mutations and TP53 mutations. CONCLUSIONS: Germline variants in BRCA1, BRCA2, PALB2 or ATM are independent prognostic factors of the short duration of response to hormonal therapy in advanced PCa.

Population-based Screening for Hereditary Colorectal Cancer Variants in Japan.

BACKGROUND & AIMS: Colorectal cancer (CRC) is one of the most common cancers in the world. A small proportion of CRCs can be attributed to recognizable hereditary germline variants of known CRC susceptibility genes. To better understand cancer risk, it is necessary to explore the prevalence of hereditary CRC and pathogenic variants of multiple cancer-predisposing genes in non-European populations. METHODS: We analyzed the coding regions of 27 cancer-predisposing genes in 12,503 unselected Japanese CRC patients and 23,705 controls by target sequencing and genome-wide SNP chip. Their clinical significance was assessed using ClinVar and the guidelines by ACMG/AMP. RESULTS: We identified 4,804 variants in the 27 genes and annotated them as pathogenic in 397 and benign variants in 941, of which 43.6% were novel. In total, 3.3% of the unselected CRC patients and 1.5% of the controls had a pathogenic variant. The pathogenic variants of MSH2 (odds ratio (OR) = 18.1), MLH1 (OR = 8.6), MSH6 (OR = 4.9), APC (OR = 49.4), BRIP1 (OR=3.6), BRCA1 (OR = 2.6), BRCA2 (OR = 1.9), and TP53 (OR = 1.7) were significantly associated with CRC development in the Japanese population (P-values<0.01, FDR<0.05). These pathogenic variants were significantly associated with diagnosis age and personal/family history of cancer. In total, at least 3.5% of the Japanese CRC population had a pathogenic variant or CNV of the 27 cancer-predisposing genes, indicating hereditary cancers. CONCLUSIONS: This largest study of CRC heredity in Asia can contribute to the development of guidelines for genetic testing and variant interpretation for heritable CRCs.