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Supplementary Figures 1 - 9. Supp. Figure 1. Comparison of mutation rates between never-smokers and smokers. Supp. Figure 2. Druggable gene candidates. Supp. Figure 3. Prevalence of somatic mutations and the CNVs of the genes in the RB pathway. Supp. Figure 4. Association between RB pathway alterations and poor prognosis in early-stage LA patients who were not treated with adjuvant chemotherapy. Supp. Figure 5. Disease-free survival curve of randomly resampled early-stage LA patients included in the discovery cohort. Supp. Figure 6. Global landscape of significantly mutated genes and significantly altered CNV regions. Supp. Figure 7. Comparison of read depths for EGFR, KRAS, and BRAF. Supp. Figure 8. Mapped read depths of exome-captured regions of KEAP1 and STK11. Supp. Figure 9. Mutation rates between smokers and never-smokers.

Purpose:Genomic and transcriptomic alterations during metastasis are considered to affect clinical outcome of colorectal cancers, but detailed clinical implications of metastatic alterations are not fully uncovered. We aimed to investigate the effect of metastatic evolution on in vivo treatment outcome, and identify genomic and transcriptomic alterations associated with drug responsiveness.Experimental Design:We developed and analyzed patient-derived xenograft (PDX) models from 35 patients with colorectal cancer including 5 patients with multiple organ metastases (MOMs). We performed whole-exome, DNA methylation, and RNA sequencing for patient and PDX tumors. With samples from patients with MOMs, we conducted phylogenetic and subclonal analysis and in vivo drug efficacy test on the corresponding PDX models.Results:Phylogenetic analysis using mutation, expression, and DNA methylation data in patients with MOMs showed that mutational alterations were closely connected with transcriptomic and epigenomic changes during the tumor evolution. Subclonal analysis revealed that initial primary tumors with larger number of subclones exhibited more dynamic changes in subclonal architecture according to metastasis, and loco-regional and distant metastases occurred in a parallel or independent fashion. The PDX models from MOMs demonstrated therapeutic heterogeneity for targeted treatment, due to subclonal acquisition of additional mutations or transcriptomic activation of bypass signaling pathway during tumor evolution.Conclusions:This study demonstrated in vivo therapeutic heterogeneity of colorectal cancers using PDX models, and suggests that acquired subclonal alterations in mutations or gene expression profiles during tumor metastatic processes can be associated with the development of drug resistance and therapeutic heterogeneity of colorectal cancers.

Supplementary Figure from PET-Based Radiogenomics Supports mTOR Pathway Targeting for Hepatocellular Carcinoma

Purpose:This work aimed to explore in depth the genomic and molecular underpinnings of hepatocellular carcinoma (HCC) with increased 2[18F]fluoro-2-deoxy-d-glucose (FDG) uptake in PET and to identify therapeutic targets based on this imaging-genomic surrogate.Experimental Design:We used RNA sequencing and whole-exome sequencing data obtained from 117 patients with HCC who underwent hepatic resection with preoperative FDG-PET/CT imaging as a discovery cohort. The primary radiogenomic results were validated with transcriptomes from a second cohort of 81 patients with more advanced tumors. All patients were allocated to an FDG-avid or FDG–non-avid group according to the PET findings. We also screened potential drug candidates targeting FDG-avid HCCs in vitro and in vivo.Results:High FDG avidity conferred worse recurrence-free survival after HCC resection. Whole transcriptome analysis revealed upregulation of mTOR pathway signals in the FDG-avid tumors, together with higher abundance of associated mutations. These clinical and genomic findings were replicated in the validation set. A molecular signature of FDG-avid HCCs identified in the discovery set consistently predicted poor prognoses in the public-access datasets of two cohorts. Treatment with an mTOR inhibitor resulted in decreased FDG uptake followed by effective tumor control in both the hyperglycolytic HCC cell lines and xenograft mouse models.Conclusions:Our PET-based radiogenomic analysis indicates that mTOR pathway genes are markedly activated and altered in HCCs with high FDG retention. This nuclear imaging biomarker may stimulate umbrella trials and tailored treatments in precision care of patients with HCC.

Supplementary Tables 1 - 15. Supp. Table 1. Sequenced reads per tumor and normal sample. Supp. Table 2. Comparison of significantly mutated genes detected using various MuTect thresholds. Supp. Table 3. Comparison of the number and rate of substitutions between never-smokers and smokers. Supp. Table 4. Comparison of various methods used to compute the background mutation rate. Supp. Table 5. Significantly mutated genes detected in our discovery cohort. Supp. Table 6. Comparison of significantly mutated genes and previously reported cancer driver genes. Supp. Table 7. Thirteen significantly altered CNV regions detected in our discovery cohort. Supp. Table 8. Druggable target candidates in the 22 significantly mutated genes and the 60 genes in the significantly altered CNV regions detected in the discovery cohort. Supp. Table 9. Pathways significantly enriched in the 22 significantly mutated genes and the 60 genes included in the significantly altered CNV regions that were detected in the discovery cohort. Supp. Table 10. Cox multivariable regression analysis for RB pathway alterations. Supp. Table 11. Cox univariable regression analysis of RB pathway alterations. Supp. Table 12. Comparison of mutations detected using whole-exome sequencing and Sequenom in selected genes. Supp. Table 13. Prevalence of known driver candidates when using two different MuTect parameters. Supp. Table 14. Number of patients included in the overall, tumor, node, and metastasis stage categories. Supp. Table 15. List of potential cancer driver genes with somatic substitutions or small indels determined in previous studies.

Supplementary Methods, Supplementary Figure Legends, and Supplementary Table Legends.

Supplementary Data from PET-Based Radiogenomics Supports mTOR Pathway Targeting for Hepatocellular Carcinoma

Supplementary Materials and Methods, Supplementary Figures S1-S10 and Supplementary Tables S1-S6

Purpose: To better understand the complete genomic architecture of lung adenocarcinoma.Experimental Design: We used array experiments to determine copy number variations and sequenced the complete exomes of the 247 lung adenocarcinoma tumor samples along with matched normal cells obtained from the same patients. Fully annotated clinical data were also available, providing an unprecedented opportunity to assess the impact of genomic alterations on clinical outcomes.Results: We discovered that genomic alternations in the RB pathway are associated with significantly shorter disease-free survival in early-stage lung adenocarcinoma patients. This association was also observed in our independent validation cohort. The current treatment guidelines for early-stage lung adenocarcinoma patients recommend follow-up without adjuvant therapy after complete resection, except for high-risk patients. However, our findings raise the interesting possibility that additional clinical interventions might provide medical benefits to early-stage lung adenocarcinoma patients with genomic alterations in the RB pathway. When examining the association between genomic mutation and histologic subtype, we uncovered the characteristic genomic signatures of various histologic subtypes. Notably, the solid and the micropapillary subtypes demonstrated great diversity in the mutated genes, while the mucinous subtype exhibited the most unique landscape. This suggests that a more tailored therapeutic approach should be used to treat patients with lung adenocarcinoma.Conclusions: Our analysis of the genomic and clinical data for 247 lung adenocarcinomas should help provide a more comprehensive genomic portrait of lung adenocarcinoma, define molecular signatures of lung adenocarcinoma subtypes, and lead to the discovery of useful prognostic markers that could be used in personalized treatments for early-stage lung adenocarcinoma patients. Clin Cancer Res; 21(11); 2613–23. ©2014 AACR.See related commentary by Collisson, p. 2418

Supplementary Table 1. CXCL12 protein expression in epithelial cells of normal gastric epithelium, dysplasia (adenoma), and adenocarcinoma. Supplementary Table 2. The associations between CXCL12 expression in cancer cells and clinicopathologic variables. Supplementary Table 3. CXCL12 protein expression in stromal fibroblasts of normal gastric epithelium, dysplasia (adenoma), and adenocarcinoma. Supplementary Table 4. Relations between of CXCL12 expression in fibroblasts and clinicopathologic features in gastric adenocarcinoma.

Supplementary methods

Supplementary Figure 1. Acquired CAF phenotype by conditioned media of gastric cancer cell was maintained after removal of CM. Supplementary Figure 2. Densitometric analysis of western blots. Supplementary Figure 3. The IL-6 cytokine induces the generation of Twist1 positive CAFs in vivo. Supplementary Figure 4A. Overexpression of Twist1 induced the attributes of CAFs. Supplementary Figure 4B. H&E staining of xenograft of two gastric cancer cell lines (SNU668 and SNU638) to immune compromised mice. Supplementary Figure 5. Correlation between Twist1 and CXCL12 expression in various cancers using mRNA expression data from TCGA. Supplementary Figure 6. Survival of patients with CXCL12 expression in cancer cells in gastric cancer. Supplementary Figure 7. Suppression of CXCL12 attenuates ability of tumor progression during Twist1- induced activation in gastric normal fibroblasts.

Despite the epidemiological association between intrahepatic cholangiocarcinoma (iCCA) and HBV infection, little is known about the relevant oncogenic effects. We sought to identify the landscape and mechanism of HBV integration, along with the genomic architecture of HBV-infected iCCA (HBV-iCCA) tumors.We profiled a cohort of 108 HBV-iCCAs using whole-genome sequencing, deep sequencing, and RNA sequencing, together with preconstructed data sets of HBV-infected HCC (HBV-HCC; n = 167) and combined hepatocellular cholangiocarcinoma (HBV-cHCC/CCA; n = 59), and conventional (n = 154) and fluke-related iCCAs (n = 16). Platforms based on primary iCCA cell lines to evaluate the functional effects of chimeric transcripts were also used. We found that HBV had inserted at multiple sites in the iCCA genomes in 45 (41.7%) of the tumors. Recurrent viral integration breakpoints were found at nine different sites. The most common insertional hotspot (7 tumors) was in the TERT (telomerase reverse transcriptase) promoter, where insertions and mutations (11 tumors) were mutually exclusive, and were accompanied by promoter hyperactivity. Recurrent HBV integration events (5 tumors) were also detected in FAT2 (FAT atypical cadherin 2), and were associated with enrichment of epithelial-mesenchymal transition-related genes. A distinctive intergenic insertion (chr9p21.3), between DMRTA1 (DMRT like family A1) and LINC01239 (long intergenic non-protein coding RNA 1239), had oncogenic effects through activation of the mammalian target of rapamycin (mTOR)/4EBP/S6K pathway. Regarding the mutational profiles of primary liver cancers, the overall landscape of HBV-iCCA was closer to that of nonviral conventional iCCA, than to HBV-HCC and HBV-cHCC/CCA.Our findings provide insight into the behavior of iCCAs driven by various pathogenic mechanisms involving HBV integration events and associated genomic aberrations. This knowledge should be of use in managing HBV carriers.

We performed an in-depth examination of pathogenic germline variants (PGVs) and somatic variants in DNA damage response (DDR) genes in hepatocellular carcinoma (HCC) to explore their clinical and genomic impacts.We used a merged whole-exome or RNA sequencing data set derived from in-house (n = 230) and The Cancer Genome Atlas (n = 362) databases of multiethnic HCC samples. We also evaluated synthetic lethal approaches targeting mutations in homologous recombination (HR) genes using HCC cells selected from five genomic databases of cancer cell lines. A total of 110 PGVs in DDR pathways in 96 patients were selected. Of the PGV carriers, 44 were HR-altered and found to be independently associated with poorer disease-free survival after hepatectomy. The most frequently altered HR gene in both germline and somatic tissues was POLQ, and this variant was detected in 22.7% (10/44) and 23.8% (5/21) of all the corresponding carriers, respectively. PGVs in HR were significantly associated with upregulation of proliferation and replication-related genes and familial risk of HCC. Samples harboring PGVs in HR with loss of heterozygosity were most strongly correlated with the genomic footprints of deficient HR, such as mutation burden and denovoSig2 (analogous to Catalogue of Somatic Mutations in Cancer [COSMIC] 3), and poor outcome. Pharmacologic experiments with HCC cells defective in BRCA2 or POLQ suggested that tumors with this phenotype are synthetic lethal with poly(ADP-ribose) polymerase inhibitors.Our findings suggest that germline HR defects in HCC tend to confer a poor prognosis and result in distinctive genomic scarring. Tests of the clinical benefits of HR-directed treatments in the affected patients are needed.

Objective Adenosquamous carcinoma of the pancreas (ASCP) is a rare histologic subtype of pancreatic carcinoma. The clinicopathologic characteristics and surgical outcomes of ASCP are poorly understood due to the rarity of this disease. Recently, promising clinical responses in patients with pancreatic cancer have been obtained for antibodies against programmed death-1/programmed death-ligand 1 (PD-1/PD-L1). This study investigated the prevalence of PD-L1 expression and surgical outcomes of 56 ASCPs compared to 100 pancreatic ductal adenocarcinomas (PDACs). Methods A total of 56 resected cases of ASCPs were retrospectively reviewed; after matching for the T category, 100 PDACs were selected as a control group for comparison. Immunohistochemistry for p53, Smad4, and PD-L1 was performed in both groups. Results The ASCPs exhibited distinct clinicopathologic features, such as larger tumour, location in the distal pancreas, frequent vascular invasion and distant metastasis. In survival analysis, 1-and 2-year overall survival (OS) rates were 51.8% and 17.9%, respectively, with a median follow-up 13 months. According to multivariate analysis, vascular invasion and T category remained independent predictors of OS. Patients with ASCPs showed poorer survival than patients with PDACs after matching for the T category (p = 0.03). p53 and Smad4 were aberrantly expressed in 42 (75%) and 28 (50%) cases, respectively. Under the condition of a 10% cut-off value for PD-L1 positivity, approximately 11% of ASCPs were positive for PD-L1. Conclusions Approximately 11% of patients with ASCPs are assumed to be potential candidates for the application of antibodies against PD-1/PD-L1, as based on the immunohistochemical results for PD-L1.

Metastatic or recurrent colorectal cancer (CRC) patients require systemic chemotherapy, but the therapeutic options of targeted agents remain limited. CRC patients with KRAS or BRAF gene mutations exhibit a worse prognosis and are resistant to anti-EGFR treatment. Previous studies have shown that the expression of anti-apoptotic protein BCL-XL is increased in CRC patients with KRAS/BRAF mutations, suggesting BCL-XL as a therapeutic target for this subgroup. Here, we performed genome-wide CRISPR/Cas9 screens of cell lines with KRAS mutations to investigate the factors required for sensitivity to BCL-XL inhibitor ABT-263 using single-guide RNAs (sgRNAs) that induce loss-of-function mutations. In the presence of ABT-263, sgRNAs targeting negative regulators of WNT signaling (resulting in WNT activation) were enriched, whereas sgRNAs targeting positive regulators of WNT signaling (resulting in WNT inhibition) were depleted in ABT-263-resistant cells. The activation of WNT signaling was highly associated with an increased expression ratio of anti- to pro-apoptotic BCL-2 family genes in CRC samples. Genetic and pharmacologic inhibition of WNT signaling using β-catenin short hairpin RNA or TNIK inhibitor NCB-0846, respectively, augmented ABT-263-induced cell death in KRAS/BRAF-mutated cells. Inhibition of WNT signaling resulted in transcriptional repression of the anti-apoptotic BCL-2 family member, MCL1, via the functional inhibition of the β-catenin-containing complex at the MCL1 promoter. In addition, the combination of ABT-263 and NCB-0846 exhibited synergistic effects in in vivo patient-derived xenograft (PDX) models with KRAS mutations. Our data provide a novel targeted combination treatment strategy for the CRC patient subgroup with KRAS or BRAF mutations.

Although hepatocellular carcinomas (HCCs) often recur in patients undergoing hepatectomy, there are no reliable biomarkers of this undesirable event. Recent RNA-based efforts have developed valuable genetic indices prognostic of cancer outcomes. We aimed to identify molecular predictors of early recurrence after resection of HCC, and reveal the genomolecular structure of the resected tumors. Based on the transcriptomic and genomic datasets of 206 HCC samples surgically resected in the Asan Medical Center (AMC), we performed a differential gene expression analysis to identify quantitative markers associated with early recurrence and used the unsupervised clustering method to classify genomolecular subtypes. Differential gene expression profiling revealed that S100P was the highest-ranked overexpressed gene in HCCs that recurred within 2 years of surgery. This trend was reproduced in immunohistochemical studies of the original cohort and an independent AMC cohort. S100P expression also independently predicted HCC-specific mortality post-resection (adjusted hazard ratio 1.09, 95% confidence interval 1.01–1.19; p = 0.042). Validation in a Chinese cohort and in in vitro experiments confirmed the prognostic value of S100P in HCC. We further identified five discrete molecular subtypes of HCC; a subtype with stem cell features (‘AMC-C4’) was associated with the worst prognosis, both in our series and another two Asian datasets, and S100P was most strongly upregulated in that subtype. We identified a promising prognostic biomolecule, S100P, associated with early recurrence after HCC resection, and established the genomolecular architecture of tumors affecting clinical outcomes, particularly in Asian patients. These new insights into molecular mediators should contribute to effective care for affected patients.

Although a certain proportion of intramucosal carcinomas (IMCs) of the stomach does metastasize, the majority of patients are currently treated with endoscopic resection without lymph node dissection, and this potentially veils any existing metastasis and may put some patients in danger. In this regard, biological markers from the resected IMC that can predict metastasis are warranted. Here, we discovered unique miRNA expression profiles that consist of 21 distinct miRNAs that are specifically upregulated (miR-628-5p, miR-1587, miR-3175, miR-3620-5p, miR-4459, miR-4505, miR-4507, miR-4720-5p, miR-4742-5p, and miR-6779-5p) or downregulated (miR-106b-3p, miR-125a-5p, miR-151b, miR-181d-5p, miR-486-5p, miR-500a-3p, miR-502-3p, miR-1231, miR-3609, and miR-6831-5p) in metastatic (M)-IMC compared to nonmetastatic (N)-IMC, or nonneoplastic gastric mucosa. Intriguingly, most of these selected miRNAs showed stepwise increased or decreased expression from nonneoplastic tissue to N-IMC to M-IMC. This suggests that common oncogenic mechanisms are gradually intensified during the metastatic process. Using a machine-learning algorithm, we demonstrated that such miRNA signatures could distinguish M-IMC from N-IMC. Gene ontology and pathway analysis revealed that TGF-β signaling was enriched from upregulated miRNAs, whereas E2F targets, apoptosis-related, hypoxia-related, and PI3K/AKT/mTOR signaling pathways, were enriched from downregulated miRNAs. Immunohistochemical staining of samples from multiple institutions indicated that PI3K/AKT/mTOR pathway components, MAPK1, phospho-p44/42 MAPK, and pS6 were highly expressed and the expression of SMAD7, a TGF-β pathway component, was decreased in M-IMC, which could aid in distinguishing M-IMC from N-IMC. The miRNA signature discovered in this study is a valuable biological marker for identifying metastatic potential of IMCs, and provides novel insights regarding the metastatic progression of IMC.

The global incidence of early-onset colorectal cancer (EO-CRC) is rapidly rising. However, the reason for this rise in incidence as well as the genomic characteristics of EO-CRC remain largely unknown. We performed whole-exome sequencing in 47 cases of EO-CRC and targeted deep sequencing in 833 cases of CRC. Mutational profiles of EO-CRC were compared with previously published large-scale studies. EO-CRC and The Cancer Genome Atlas (TCGA) data were further investigated according to copy number profiles and mutation timing. We classified colorectal cancer into three subgroups: the hypermutated group consisted of mutations in POLE and mismatch repair genes; the whole-genome doubling group had early functional loss of TP53 that led to whole-genome doubling and focal oncogene amplification; the genome-stable group had mutations in APC and KRAS, similar to conventional colon cancer. Among non-hypermutated samples, whole-genome doubling was more prevalent in early-onset than in late-onset disease (54% vs 38%, Fisher’s exact P = 0.04). More than half of non-hypermutated EO-CRC cases involved early TP53 mutation and whole-genome doubling, which led to notable differences in mutation frequencies between age groups. Alternative carcinogenesis involving genomic instability via loss of TP53 may be related to the rise in EO-CRC., Colorectal cancer: tumor genetic classification could help guide treatment Researchers in South Korea have identified distinct types of genetic changes in colorectal cancer (CRC). Groups led by Tae Won Kim at the University of Ulsan, Seoul, and Jong-il Kim at Seoul National University looked for mutations in genes in tumors from 880 patients, including all active genes in 47 patients with early-onset colorectal cancer (EO-CRC). The cancers were classified into three subgroups. A “hypermutated” group had mutations in genes involved in DNA replication and repair. The entire genome was doubled in another group, which included the EO-CRC cases. In this group, TP53 gene function, which is known to be crucial in suppressing tumor formation, was lost. A third group had mutations in two other identified genes. Identifying which subgroups patients belong to may help determine the best treatment option.

Resistance to platinum-based chemotherapy is one of major causes of therapy failure and tumor recurrence in ovarian cancer patients. However, the molecular mechanism of platinum resistance is not fully understood. Here, we compared gene expression between cisplatin-sensitive and -resistant ovarian cancer cells using gene expression microarray analysis and found the significantly up-regulated expression of SERPINE1 encoding plasminogen activator inhibitor-1 (PAI-1), a serine protease regulating the fibrinolysis and extracelluar matrix, in cisplatin-resistant ovarian cancer cells. The correlation of high PAI-1 expression with low cisplatin sensitivity was confirmed in ovarian cancer cells. PAI-1 overexpression in ovarian cancer cells significantly decreased the sensitivity to cisplatin, whereas PAI-1 down-regulation by SERPINE1 siRNA or PAI-1 inhibitor increased cisplatin sensitivity. PAI-1 inhibited cisplatin-induced apoptosis in ovarian cancer cells by suppressing reactive oxygen species (ROS) production and DNA damage. PAI-1 also decreased cisplatin-induced ferroptosis through the upregulation of GPX4 levels. Furthermore, Kaplan-Meier survival analysis using TGGA data demonstrated that high PAI-1 expression was significantly associated with shorter platinum-free survival of patients with ovarian cancer. Collectively, these results illustrate that PAI-1 promotes cisplatin resistance in ovarian cancer cells by inhibiting ROS-mediated apoptosis and ferroptosis, suggesting that targeting PAI-1 increases the sensitivity to cisplatin in ovarian cancer. Citation Format: Mi Jeong Kwon, Ha Yeong Chae, So Young Lee, Shin Ung Kang, Tae Woong Jung, Soo Youn Cho, Jinil Han, Hyojin Jeong, Chang Ohk Sung, Young Kee Shin. Plasminogen activator inhibitor-1 (PAI-1) promotes cisplatin resistance in ovarian cancer cells by inhibiting ROS-mediated apoptosis and ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1684.

Early diagnosis of hepatocellular carcinoma (HCC) increases the chances of curative treatment and long-term survival.1 2 We read with interest the analytical study by Zhang et al presenting nomogram models predicting recurrence-free survival and guiding therapeutic decisions in a patient-tailored approach to treatment of early-stage HCC within Milan criteria.3 The refined Barcelona Clinic Liver Cancer (BCLC) guidelines no longer limit the size of solitary nodules in the definition of stage A HCC. However, there is continuing debate about the need to restrict the maximum diameter of single HCCs to inside the Milan limit (≤5 cm).4–6 In a survival-based confirmatory investigation, we primarily used a Korean nationwide cancer registry database of 10 510 new HCC cases. These cases were randomly sampled and registered in 54 hospitals, and investigated by the Korea Liver Cancer Study Group and the governmental organisation of the Korea Central Cancer Registry in 2008–2014. We included in the final analysis 1610 asymptomatic HCC subjects with BCLC stage A or stage B who had preserved liver function levels referred to as Child-Pugh class A without ascites and who initially received standard treatments as per BCLC stage (online supplemental figure S1).4 The population was divided into four groups: group 1, stage A disease fulfilling Milan criteria undergoing liver resection or transplantation, or local ablation; group 2, potential stage A with single nodules >5 cm undergoing resection; group 3, potential stage B with single nodules >5 cm undergoing transarterial chemoembolisation (TACE); and group 4, stage B undergoing TACE. Five-year overall survival (OS) rates were significantly different across the four groups (p

Mosaicisms caused by postzygotic mutational events are of increasing interest because of their potential association with various human diseases. Postzygotic somatic mutations have not been well characterized however in terms of their developmental lineage in humans. We conducted whole-genome sequencing (WGS) and targeted deep sequencing in 15 organs across three developmental lineages from a single male fetus with polycystic kidney disease (PKD) of 21 weeks gestational age. This fetus had no detectable neurological abnormalities at autopsy but germline mutations in the PKHD1 gene were identified that may have been associated with the PKD. Eight early embryonic mosaic variants with no alteration of protein function were detected. These variants were thought to have occurred at the two or four cell stages after fertilization with a mutational pattern involving frequent C>T and T>C transitions. In our current analyses, no tendency toward organ-specific mutation occurrences was found as the eight variants were detected in all 15 organs. However different allele fractions of these variants were found in different organs, suggesting a tissue-specific asymmetric growth of cells that reflected the developmental germ layer of each organ. This indicated that somatic mutation occurrences, even in early embryogenesis, can affect specific organ development or disease. Our current analyses demonstrate that multi-organ analysis is helpful for understanding genomic mosaicism. Our results also provide insights into the biological role of mosaicism in embryonic development and disease.

Neuroendocrine carcinoma (NEC) is an aggressive malignant tumor that rarely arises from the gallbladder. Here, we investigated the clinicopathologic and immunohistochemical characteristics of 34 NECs of the gallbladder. The patients were predominantly women (68%) with a median age of 63 years (range, 37 to 82 y). NECs frequently occurred in the fundus (44%) as mass-forming lesions (66%). Histologically, 17 tumors were of small cell type, and another 17 were of large cell type. Twenty-three cases (68%) were associated with biliary intraepithelial neoplasia (38%) and intracholecystic papillary neoplasm (29%). The majority of tumors exhibited a diffuse growth pattern (74%), followed by organoid (24%) or scirrhous (2%) growth patterns. Histologic features related to neuroendocrine differentiation, such as nuclear molding (56%), perilobular pseudopalisading (18%), and rosette formation (15%), were identified. Immunohistochemically, cytokeratin 7 and 20 were expressed in 19 (56%) and 8 (24%) cases, respectively. Loss of Rb1 expression and concomitant overexpression of p16 were observed in 25 (74%) cases. No BRAF mutations were identified in any of the 34 NECs. For survival analysis, the 1-, 3-, and 5-year overall survival rates were 64%, 35%, and 19%, respectively. In a multivariate analysis, the receipt of adjuvant chemoradiation therapy was identified as the only independent prognostic factor associated with the overall survival rate. The 1- and 3-year overall survival rates of patients with NECs were poorer for patients with poorly differentiated adenocarcinoma of the gallbladder (P

BackgroundSomatic mutations are a major driver of cancer development and many have now been identified in various cancer types, but the comprehensive somatic mutation status of the normal tissues matched to tumours has not been revealed.MethodWe analysed the somatic mutations of whole exome sequencing data in 392 patient tumour and normal tissue pairs based on the corresponding blood samples across 10 tumour types.ResultsMany of the mutations involved in oncogenic pathways such as PI3K, NOTCH and TP53, were identified in the normal tissues. The ageing-related mutational signature was the most prominent contributing signature found and the mutations in the normal tissues were frequently in genes involved in late replication time (pPIK3CA mutations were identified in 6 normal tissues and were harboured by all of the matched cancer tissues. Multiple types of PIK3CA mutations were found in normal breast and matched cancer tissues. The PIK3CA mutations exclusively present in normal tissue may indicate clonal expansions unrelated to the tumour. In addition, PIK3CA mutation was appeared that they arose before the occurrence of the allelic imbalance.ConclusionOur current results suggest that somatic mutant clones exist in normal tissues and that their clonal expansion could be linked to cancer development.

Tumor mutation burden (TMB) is an emerging biomarker, whose calculation requires targeted sequencing of many genes. We investigated if the measurement of mutation counts within a single gene is representative of TMB. Whole-exome sequencing (WES) data from the pan-cancer cohort (n = 10,224) of TCGA, and targeted sequencing (tNGS) and TTN gene sequencing from 24 colorectal cancer samples (AMC cohort) were analyzed. TTN was identified as the most frequently mutated gene within the pan-cancer cohort, and its mutation number best correlated with TMB assessed by WES (rho = 0.917, p TTN mutation frequencies observed in other FLAGS (FrequentLy mutAted GeneS) are associated with late replication time. Diagnostic accuracy for high TMB group did not differ between TTN-TMB and TMB assessed by tNGS. Classification modeling by machine learning using TTN-TMB for MSI-H diagnosis was constructed, and the diagnostic accuracy was 0.873 by area under the curve in external validation. TTN mutation was enriched in samples possessing high immunostimulatory signatures. We suggest that the mutation load within TTN represents high TMB status.

Purpose Recent clinical trials have reported response rates < 50% among patients treated with programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors for microsatellite instability‒high (MSI-H) colorectal cancer (CRC), and factors predicting treatment response have not been fully identified. This study aimed to identify potential biomarkers of PD-1/PD-L1 inhibitor treatment response among patients with MSI-H CRC. Materials and Methods MSI-H CRC patients enrolled in three clinical trials of PD-1/PD-L1 blockade at Asan Medical Center (Seoul, Republic of Korea) were screened and classified into two groups according to treatment response. Their histopathologic features and expression of 730 immune-related genes from the NanoString platform were evaluated, and a machine learning–based classification model was built to predict treatment response among MSI-H CRCs patients.Results A total of 27 patients (15 responders, 12 non-responders) were included. A high degree of lymphocytic/neutrophilic infiltration and an expansile tumor border were associated with treatment response and prolonged progression-free survival (PFS), while mucinous/signet-ring cell carcinoma was associated with a lack of treatment response and short PFS. Gene expression profiles revealed that the interferon-γ response pathway was enriched in the responder group. Of the top eight differentially expressed immune-related genes, PRAME had the highest fold change in the responder group. Higher expression of PRAME was independently associated with better PFS along with histologic subtypes in the multivariate analysis. The classification model using these genes showed good performance for predicting treatment response.Conclusion We identified histologic and immune-related gene expression characteristics associated with treatment response in MSI-H CRC, which may contribute to optimal patient stratification.

Immunoprofiling has an established impact on the prognosis of several cancers, however, its role and definition in high-grade serous ovarian cancer (HGSOC) are mostly unknown. This study is to investigate immunoprofiling which could be a prognostic factor in HGSOC. We produced tumor microarrays of 187 patients diagnosed with HGSOC. We performed a multiplexed immunofluorescence staining using Opal Multiplex IHC kit and quantitative analysis with Vectra-Inform system. The expression intensities of programmed death-ligand 1 (PD-L1), CD4, CD8, CD20, FoxP3, and CK in whole tumor tissues were evaluated. The enrolled patients showed general characteristics, mostly FIGO stage III/IV and responsive to chemotherapy. Each immune marker showed diverse positive densities, and each tumor sample represented its immune characteristics as an inflamed tumor or noninflamed tumor. No marker was associated with survival as a single one. Interestingly, high ratios of CD8 to FoxP3 and CD8 to PD-L1 were related to the favorable overall survival (77 vs. 39 months, 84 vs. 47 months, respectively), and CD8 to PD-L1 ratio was also a significant prognostic factor (HR 0.621, 95% CI 0.420–0.917, p = 0.017) along with well-known clinical prognostic factors. Additionally, CD8 to PD-L1 ratio was found to be higher in the chemosensitive group (p = 0.034). In conclusion, the relative expression levels of CD8, FoxP3, and PD-L1 were significantly related to the clinical outcome of patients with HGSOC, which could be a kind of significant immunoprofiling of ovarian cancer patients to apply for treatment.

Purpose: This work aimed to explore in depth the genomic and molecular underpinnings of hepatocellular carcinoma (HCC) with increased 2[18F]fluoro-2-deoxy-d-glucose (FDG) uptake in PET and to identify therapeutic targets based on this imaging-genomic surrogate. Experimental Design: We used RNA sequencing and whole-exome sequencing data obtained from 117 patients with HCC who underwent hepatic resection with preoperative FDG-PET/CT imaging as a discovery cohort. The primary radiogenomic results were validated with transcriptomes from a second cohort of 81 patients with more advanced tumors. All patients were allocated to an FDG-avid or FDG–non-avid group according to the PET findings. We also screened potential drug candidates targeting FDG-avid HCCs in vitro and in vivo. Results: High FDG avidity conferred worse recurrence-free survival after HCC resection. Whole transcriptome analysis revealed upregulation of mTOR pathway signals in the FDG-avid tumors, together with higher abundance of associated mutations. These clinical and genomic findings were replicated in the validation set. A molecular signature of FDG-avid HCCs identified in the discovery set consistently predicted poor prognoses in the public-access datasets of two cohorts. Treatment with an mTOR inhibitor resulted in decreased FDG uptake followed by effective tumor control in both the hyperglycolytic HCC cell lines and xenograft mouse models. Conclusions: Our PET-based radiogenomic analysis indicates that mTOR pathway genes are markedly activated and altered in HCCs with high FDG retention. This nuclear imaging biomarker may stimulate umbrella trials and tailored treatments in precision care of patients with HCC.

Matrix metalloproteinase 11 (MMP11), a member of the MMP family involved in the degradation of the extracellular matrix, has been implicated in cancer progression. Despite the stromal expression of MMP11 in breast cancer, the prognostic significance and role of MMP11 in immune or stromal cells of breast cancer remain unclear. Based on the immunohistochemical analysis of breast cancer tissues from 497 patients, we demonstrated that MMP11 expression in mononuclear inflammatory cells (predominantly macrophages) is an independent negative prognostic factor in breast cancer, whereas MMP11 expression in tumor cells and fibroblasts is not associated with patient survival. Enforced MMP11 expression in breast cancer cells did not promote cell proliferation and migration. However, MMP11-overexpressing macrophages enhanced the migration of HER2-positive (HER2+) breast cancer cells, recruitment of monocytes, and tube formation of endothelial cells. Furthermore, we found that the chemokine CCL2 secreted from MMP11-overexpressing macrophages activated the MAPK pathway via its receptor CCR2 in breast cancer cells, thereby promoting the migration of HER2+ breast cancer cells through MMP9 upregulation. We also found that MMP11 expression in macrophages was stimulated by MMP11-overepressing HER2+ breast cancer cells. Collectively, our findings provide evidence that MMP11 in macrophages may play a pro-tumoral role in HER2+ breast cancer through interaction with cancer cells, monocytes, and endothelial cells.

Notwithstanding remarkable treatment success with anti-PD-1 monoclonal antibody, oncogenic mechanism of PD-L1 regulation in gastric cancer (GC) remains poorly understood. We hypothesized that ARID1A might be related to tumor PD-L1 expression in GC. We found that tumor PD-L1 positivity was associated with loss of ARID1A and showed trend toward better survival of patients with various molecular subtypes of GC (experimental set, n = 273). Considering heterogeneous ARID1A expression, we validated this using whole tissue sections (n = 159) and found that loss of ARID1A was correlated with microsatellite instability-high (MSI-H), Epstein-Barr virus (EBV), and PD-L1 positivity. Furthermore, for patients with MSI-H tumors, the degree of PD-L1 expression was significantly higher in ARID1A-deficient tumors. After ARID1A knockdown in GC cell lines, total and membranous PD-L1 protein, and PD-L1 mRNA levels were increased based on Western blot, flow cytometry, and qRT-PCR, respectively. With IFN-γ treatment, PD-L1 expression was significantly increased both in ARID1A-deficient cancer cells and controls, but the increase was not more pronounced in the former. Loss of ARID1A increased PD-L1 via activating AKT signaling, while LY294002 (PI3K inhibitor) decreased PD-L1 levels. Furthermore, we found that 3 MSI-H tumors showing highest expression of PD-L1 had simultaneous KRAS mutation and loss of ARID1A, suggesting a possible synergistic role boosting PD-L1. Our results strongly indicate that loss of ARID1A is tightly associated with high PD-L1 expression in GC. These results would increase our understanding of the oncogenic mechanism of PD-L1 regulation in GC, and also help to find the optimal candidates for immunotherapy.