Two Japanese families with familial pancreatic cancer with suspected pathogenic variants of CDKN2A: a case report.

BACKGROUND: Germline mutations in CDKN2A result in Familial Atypical Multiple Mole Melanoma Syndrome (FAMMM) (OMIM #155,601), which is associated with an increased risk of pancreatic ductal adenocarcinoma and melanoma. FAMMM has been reported globally, but it is quite rare in Japan. We report two families with familial pancreatic cancer with suspected pathogenic variants of CDKN2A that were incidentally identified through comprehensive genomic profiling. CASE PRESENTATION: The first case is a 74-year-old woman with a diagnosis of pancreatic carcinoma with multiple liver metastases. She had family histories of pancreatic cancer, but no personal or family history of malignant melanoma. Whole exon sequencing detected a germline CDKN2A variant evaluated as likely pathogenic. The results were disclosed to her daughters after she died, and the same CDKN2A variant was detected in one of the daughter. The daughter was referred to a nearby hospital for her clinical management. The second case is a 65-year-old man with pancreatic ductal adenocarcinoma. He had family histories of pancreatic cancer, but no personal or family history of malignant melanoma. He underwent a comprehensive genomic profiling test using pancreatic cancer tissue, and detected a presumed germline pathogenic variant of CDKN2A. Germline testing confirmed the same CDKN2A variant. Genetic analysis of his relatives produced negative results. Other blood relatives are scheduled for genetic analysis in the future. We report two families with familial pancreatic cancer with suspected pathogenic variants of CDKN2A that were incidentally identified through comprehensive genomic profiling. CONCLUSIONS: In current Japanese precision medicine, comprehensive genetic analysis can reveal rare genetic syndromes and offer us the opportunity to provide health management for patients and their relatives. However, gene-specific issues are raised in terms of the evaluation of a variant's pathogenicity and the extent of surveillance of the at-risk organs due to a lack of genetic and clinical data concerning CDKN2A variant carriers in Japan.

High levels of AXL expression in untreated EGFR-mutated non-small cell lung cancer negatively impacts the use of osimertinib.

For non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations, the initial therapeutic interventions will have crucial impacts on their clinical outcomes. Drug tolerant factors reportedly have an impact on EGFR-tyrosine kinase inhibitor sensitivity. This prospective study investigated the impacts of drug tolerant-related protein expression in tumors based on the efficacy of osimertinib in the first-setting of EGFR-mutated advanced NSCLC patients. A total of 92 patients with EGFR-mutated advanced or postoperative recurrent NSCLC were analyzed and treated with osimertinib at 14 institutions in Japan. AXL, p53, and programmed death-ligand 1 (PD-L1) expression in patient tumors was determined using immunohistochemistry. The AXL signaling pathway was investigated using a cell line-based assay and AXL-related gene expression in The Cancer Genome Atlas (TCGA) database. High levels of AXL and positive-p53 expression were detected in 26.1% and 53.3% of the pretreatment EGFR-mutated NSCLC tumors, respectively. High AXL expression levels were significantly associated with a shorter progression-free survival compared with low AXL expression levels, irrespective of the EGFR activating mutation status (p = 0.026). Cell line-based assays indicated that the overexpression of AXL protein accelerated PD-L1 expression, which induced insensitivity to osimertinib. In the TCGA database, AXL RNA levels were positively correlated with PD-L1 expression in the lung adenocarcinoma cohort. The results show that high AXL expression levels in tumors impact clinical predictions when using osimertinib to treat EGFR-mutated NSCLC patients. Trial Registration: UMIN000043942.

A Japanese case of ovarian mucinous adenocarcinoma with germline double variants of MSH2 and BRCA2.

Germline double heterozygosity (GDH) is rarely reported in cases of inherited cancer syndromes, and GDH of a mismatch repair gene and BRCA has never been reported in Japan. Nonetheless, the current report demonstrates a case of ovarian mucinous adenocarcinoma with initiated Lynch syndrome (LS)-related surveillance because of a known germline MSH2 variant. Six and a half years after oophorectomy, multiple tumors developed in the patient's lungs, bones, and lymph nodes, and histology results confirmed mucinous adenocarcinoma. Systemic chemotherapy including an anti-PD-L1 antibody was effective for >1 year, but brain metastases developed. Pathology of the brain tumors showed mucinous adenocarcinoma without expression of MSH2 and MSH6, while multi-gene panel testing demonstrated not only high microsatellite instability and a high tumor mutation burden, but also germline BRCA2 variants. Further, germline testing in relatives confirmed both variants were from the paternal line, from which many LS-related cancers develop, but not BRCA-related cancer.

The efficacy of a machine learning algorithm for assessing tumour components as a prognostic marker of surgically resected stage IA lung adenocarcinoma.

BACKGROUND: The importance of the stromal components in tumour progression has been discussed widely, but their prognostic role in small size tumours with lepidic components is not fully understood. Applying digital tissue image analysis to whole-slide imaging may enhance the accuracy and reproducibility of pathological assessment. This study aimed to evaluate the prognostic value of tumour components of lung adenocarcinoma by measuring the dimensions of the tumour consisting elements separately, using a machine learning algorithm. METHODS: Between September 2002 and December 2016, 317 patients with surgically resected, pathological stage IA adenocarcinoma with lepidic components were analysed. We assessed the whole tumour area, including the lepidic components, and measured the epithelium, collagen, elastin areas and alveolar air space. We analysed the prognostic impact of each tumour component. RESULTS: The dimensions of the epithelium and collagen areas were independent significant risk factors for recurrence-free survival (hazard ratio, 8.38; 95% confidence interval, 1.14-61.88; P = 0.037, and hazard ratio, 2.58; 95% confidence interval, 1.14-5.83; P = 0.022, respectively). According to the subgroup analysis when combining the epithelium and collagen areas as risk factors, patients with tumours consisting of both large epithelium and collagen areas showed significantly poor prognoses (P = 0.002). CONCLUSIONS: We assessed tumour components using a machine learning algorithm to stratify the post-operative prognosis of surgically resected stage IA adenocarcinomas. This method might guide the selection of patients with a high risk of recurrence.

Microsatellite instability is biased in Amsterdam II-defined Lynch-related cancer cases with family history but is rare in other cancers: a summary of 1000 analyses.

BACKGROUND: Microsatellite instability (MSI) is a key marker for predicting the response of immune checkpoint inhibitors (ICIs) and for screening Lynch syndrome (LS). AIM: This study aimed to see the characteristics of cancers with high level of MSI (MSI-H) in genetic medicine and precision medicine. METHODS: This study analyzed the incidence of MSI-H in 1000 cancers and compared according to several clinical and demographic factors. RESULTS: The incidence of MSI-H was highest in endometrial cancers (26.7%, 20/75), followed by small intestine (20%, 3/15) and colorectal cancers (CRCs)(13.7%, 64/466); the sum of these three cancers (15.6%) was significantly higher than that of other types (2.5%)(P < 0.0001). MSI-H was associated with LS-related cancers (P < 0.0001), younger age (P = 0.009), and family history, but not with smoking, drinking, or serum hepatitis virus markers. In CRC cases, MSI-H was significantly associated with a family history of LS-related cancer (P < 0.0001), Amsterdam II criteria [odds ratio (OR): 5.96], right side CRCs (OR: 4.89), and multiplicity (OR: 3.31). However, MSI-H was very rare in pancreatic (0.6%, 1/162) and biliary cancers (1.6%, 1/64) and was null in 25 familial pancreatic cancers. MSI-H was more recognized in cancers analyzed for genetic counseling (33.3%) than in those for ICI companion diagnostics (3.1%)(P < 0.0001). Even in CRCs, MSI-H was limited to 3.3% when analyzed for drug use. CONCLUSIONS: MSI-H was predominantly recognized in LS-related cancer cases with specific family histories and younger age. MSI-H was limited to a small proportion in precision medicine especially for non-LS-related cancer cases.

Present status of germline findings in precision medicine for Japanese cancer patients: issues in the current system.

OBJECTIVE: Since 2019, precision cancer medicine has been covered by national insurance in Japan; however, to date, germline findings have not been fully reported. The aim of this study was to evaluate the current status and raise a problem of germline finding analysis and disclosure in Japanese precision cancer medicine. METHODS: Germline findings of 52 genes were examined in 296 cases with advanced cancer by a case series study. RESULTS: Six (2.0%) cases were examined by the Oncoguide™ NCC Oncopanel with germline testing, but no germline findings were reported. The remaining 290 (98.0%) cases were analyzed by FoundationOne® CDx (tumor-only testing), which recognized 404 pathogenic variants; those of BRCA1/2 were recognized in 16 (5.5%) tumors. Our institutional algorithm suggested 39 candidate germline findings in 34 cases, while the public algorithm listed at least 91 candidate germline findings. Four germline findings had been previously identified (BRCA1: 3 and ATM: 1). Nine of 30 cases with candidate germline findings excluding these known germline findings refused or deferred germline testing. Only 4 of 16 cases that received counseling underwent germline testing, and those 4 revealed 3 germline findings (BRCA2, CDK4 and RAD51C); in total, 8 (2.7%) germline findings were revealed. Reasons for refusing genetic counseling and/or germline testing included extra hospital visits, added expense for germline testing due to limited national insurance coverage, poor patient physical condition and no known family members associated with the possible germline finding. CONCLUSIONS: In current Japanese precision cancer medicine, only a small fraction of the patients undergoes germline testing and demonstrated germline finding. The current results suggested a need for earlier indications for precision cancer medicine, broader insurance coverage and more efficient germline finding prediction algorithms, to increase the number of germline testings and to improve the following managements.

MAGEA10 expression is a predictive marker of early hepatic recurrence after curative gastrectomy for gastric and gastroesophageal junction cancer.

BACKGROUND: Resection for hepatic recurrence after gastrectomy in patients with gastric cancer may be curative; however, the prediction of hepatic recurrence remains intractable. Therefore, we aimed to explore predictive markers for hepatic recurrence in gastric and gastroesophageal junction cancer based on genetic information. METHODS: This study recruited 154 patients who underwent curative gastrectomy for pathological stage II or III primary gastric and gastroesophageal junction adenocarcinoma. Genes associated with hepatic recurrence were comprehensively analyzed using whole-exome sequencing and gene expression profiling (GEP), followed by immunohistochemistry analysis for MAGEA10. The cumulative incidences of hepatic recurrence, relapse-free survival, and overall survival were evaluated. RESULTS: A total of 12 patients with early hepatic recurrences were found within 2 years of surgery. Although there were no distinct gene mutations in recurrent patients, upregulation of MAGEA10 was identified in patients with early hepatic recurrence using GEP analysis. Immunostaining for MAGEA10 stained the cell nuclei in 29 (18.8%) of 154 samples. Furthermore, protein expression of MAGEA10 on immunohistochemistry was significantly related to a high MAGEA10 mRNA expression, high cumulative incidences of hepatic recurrence, and poor relapse-free survival. Overall survival did not differ significantly between positive and negative immunohistochemical staining for MAGEA10. The sensitivity and specificity of MAGEA10 staining for early hepatic recurrence were 58.3% and 84.5%, respectively. CONCLUSIONS: MAGEA10 represents a promising predictive marker for early hepatic recurrence after curative gastrectomy for gastric and gastroesophageal junction cancer.

Japanese version of The Cancer Genome Atlas, JCGA, established using fresh frozen tumors obtained from 5143 cancer patients.

This study aimed to establish the Japanese Cancer Genome Atlas (JCGA) using data from fresh frozen tumor tissues obtained from 5143 Japanese cancer patients, including those with colorectal cancer (31.6%), lung cancer (16.5%), gastric cancer (10.8%) and other cancers (41.1%). The results are part of a single-center study called "High-tech Omics-based Patient Evaluation" or "Project HOPE" conducted at the Shizuoka Cancer Center, Japan. All DNA samples and most RNA samples were analyzed using whole-exome sequencing, cancer gene panel sequencing, fusion gene panel sequencing and microarray gene expression profiling, and the results were annotated using an analysis pipeline termed "Shizuoka Multi-omics Analysis Protocol" developed in-house. Somatic driver alterations were identified in 72.2% of samples in 362 genes (average, 2.3 driver events per sample). Actionable information on drugs that is applicable in the current clinical setting was associated with 11.3% of samples. When including those drugs that are used for investigative purposes, actionable information was assigned to 55.0% of samples. Germline analysis revealed pathogenic mutations in hereditary cancer genes in 9.2% of samples, among which 12.2% were confirmed as pathogenic mutations by confirmatory test. Pathogenic mutations associated with non-cancerous hereditary diseases were detected in 0.4% of samples. Tumor mutation burden (TMB) analysis revealed 5.4% of samples as having the hypermutator phenotype (TMB ≥ 20). Clonal hematopoiesis was observed in 8.4% of samples. Thus, the JCGA dataset and the analytical procedures constitute a fundamental resource for genomic medicine for Japanese cancer patients.

Characterization of tumour mutation burden in patients with non-small cell lung cancer and interstitial lung disease.

BACKGROUND AND OBJECTIVE: The efficacy expectation of immune checkpoint inhibitors against NSCLC in patients with ILD seems to be high because these populations are supposed to have high TMB. However, information about the characterization of TMB in patients with NSCLC and ILD is limited. Therefore, this study aimed to evaluate TMB in samples of NSCLC with ILD and clarify factors that influence TMB values. METHODS: The medical records of patients with NSCLC who underwent thoracic surgery at our institution between January 2014 and January 2017 were retrospectively reviewed. Whole-exome sequencing with an Ion Proton system and gene expression profiling of fresh surgical specimens were performed. RESULTS: Among 367 patients with NSCLC, 62 (16.9%) were diagnosed with ILD. All samples were collected from primary tumours with a median TMB of approximately 2.1 (range: 0.1-64.4) mutation/Mb. Among 81 squamous cell carcinomas, we compared 27 tumours with concomitant ILD and 54 tumours without ILD. Univariate analyses revealed that tumours with concomitant ILD showed lower TMB values than those without ILD. Multivariate analysis revealed that concomitant ILD was significantly associated with low TMB values. Conversely, no difference was noted in the TMB value of adenocarcinoma between patients with and without ILD. CONCLUSION: Squamous cell carcinoma and adenocarcinoma with ILD do not have high TMB values. Therefore, considering the risk of severe pneumonitis, immune checkpoint inhibitors should not be used routinely against patients with NSCLC and ILD based on the expectation of high TMB values.

Mutational burden and signatures in 4000 Japanese cancers provide insights into tumorigenesis and response to therapy.

Tumor mutational burden (TMB) and mutational signatures reflect the process of mutation accumulation in cancer. However, the significance of these emerging characteristics remains unclear. In the present study, we used whole-exome sequencing to analyze the TMB and mutational signature in solid tumors of 4046 Japanese patients. Eight predominant signatures-microsatellite instability, smoking, POLE, APOBEC, UV, mismatch repair, double-strand break repair, and Signature 16-were observed in tumors with TMB higher than 1.0 mutation/Mb, whereas POLE and UV signatures only showed moderate correlation with TMB, suggesting the extensive accumulation of mutations due to defective POLE and UV exposure. The contribution ratio of Signature 16, which is associated with hepatocellular carcinoma in drinkers, was increased in hypopharynx cancer. Tumors with predominant microsatellite instability signature were potential candidates for treatment with immune checkpoint inhibitors such as pembrolizumab and were found in 2.8% of cases. Furthermore, based on microarray analysis, tumors with predominant signatures were classified into 2 subgroups depending on the expression of immune-related genes reflecting differences in the immune context of the tumor microenvironment. Tumor subpopulations differing in the content of infiltrating immune cells might respond differently to immunotherapeutics. An understanding of cancer characteristics based on TMB and mutational signatures could provide new insights into mutation-driven tumorigenesis.

Driver gene alterations and activated signaling pathways toward malignant progression of gastrointestinal stromal tumors.

Mutually exclusive KIT and PDGFRA mutations are considered to be the earliest events in gastrointestinal stromal tumors (GIST), but insufficient for their malignant progression. Herein, we aimed to identify driver genes and signaling pathways relevant to GIST progression. We investigated genetic profiles of 707 driver genes, including mutations, gene fusions, copy number gain or loss, and gene expression for 65 clinical specimens of surgically dissected GIST, consisting of six metastatic tumors and 59 primary tumors from stomach, small intestine, rectum, and esophagus. Genetic alterations included oncogenic mutations and amplification-dependent expression enhancement for oncogenes (OG), and loss of heterozygosity (LOH) and expression reduction for tumor suppressor genes (TSG). We assigned activated OG and inactivated TSG to 27 signaling pathways, the activation of which was compared between malignant GIST (metastasis and high-risk GIST) and less malignant GIST (low- and very low-risk GIST). Integrative molecular profiling indicated that a greater incidence of genetic alterations of driver genes was detected in malignant GIST (96%, 22 of 23) than in less malignant GIST (73%, 24 of 33). Malignant GIST samples groups showed mutations, LOH, and aberrant expression dominantly in driver genes associated with signaling pathways of PI3K (PIK3CA, AKT1, and PTEN) and the cell cycle (RB1, CDK4, and CDKN1B). Additionally, we identified potential PI3K-related genes, the expression of which was upregulated (SNAI1 and TPX2) or downregulated (BANK1) in malignant GIST. Based on our observations, we propose that inhibition of PI3K pathway signals might potentially be an effective therapeutic strategy against malignant progression of GIST.

PIK3CA mutation is a favorable prognostic factor in esophageal cancer: molecular profile by next-generation sequencing using surgically resected formalin-fixed, paraffin-embedded tissue.

BACKGROUND: Practical and reliable genotyping procedures with a considerable number of samples are required not only for risk-adapted therapeutic strategies, but also for stratifying patients into future clinical trials for molecular-targeting drugs. Recent advances in mutation testing, including next-generation sequencing, have led to the increased use of formalin-fixed paraffin-embedded tissue. We evaluated gene alteration profiles of cancer-related genes in esophageal cancer patients and correlated them with clinicopathological features, such as smoking status and survival outcomes. METHODS: Surgically resected formalin-fixed, paraffin-embedded tissue was collected from 135 consecutive patients with esophageal cancer who underwent esophagectomy. Based on the assessment of DNA quality with a quantitative PCR-based assay, uracil DNA glycosylase pretreatment was performed to ensure quality and accuracy of amplicon-based massively parallel sequencing. Amplicon-based massively parallel sequencing was performed using the Illumina TruSeq® Amplicon Cancer Panel. Gene amplification was detected by quantitative PCR-based assay. Protein expression was determined by automated quantitative fluorescent immunohistochemistry. RESULTS: Data on genetic alterations were available for 126 patients. The median follow-up time was 1570 days. Amplicon-based massively parallel sequencing identified frequent gene alterations in TP53 (66.7%), PIK3CA (13.5%), APC (10.3%), ERBB4 (7.9%), and FBXW7 (7.9%). There was no association between clinicopathological features or prognosis with smoking status. Multivariate analyses revealed that the PIK3CA mutation and clinical T stage were independent favorable prognostic factors (hazard ratio 0.34, 95% confidence interval: 0.12-0.96, p = 0.042). PIK3CA mutations were significantly associated with APC alterations (p = 0.0007) and BRAF mutations (p = 0.0090). CONCLUSIONS: Our study provided profiles of cancer-related genes in Japanese patients with esophageal cancer by next-generation sequencing using surgically resected formalin-fixed, paraffin-embedded tissue, and identified the PIK3CA mutation as a favorable prognosis biomarker.

Comprehensive characterization of genes associated with the TP53 signal transduction pathway in various tumors.

The TP53 signal transduction pathway is an attractive target for cancer treatments. In this study, we conducted a comprehensive molecular evaluation of 907 patients with cancer in Japan to identify genomic alterations in the TP53 pathway. TP53 mutations were frequently detected in many cancers, except melanoma, thymic tumors, gastrointestinal stromal tumors, and renal cancers. The frequencies of non-synonymous single nucleotide variants (SNVs) in the TP53 family members TP63 and TP73 were relatively low, although genes with increased frequencies of SNVs were as follows: PTEN (11.7%) in breast cancer, CDKN2A (11.1 and 9.6%) in pancreas and head and neck cancers, and ATM (18.0 and 11.1%) in liver and esophageal cancers. MDM2 expression was decreased or increased in patients with mutant or wild-type TP53, respectively. CDKN1A expression was increased with mutant TP53 in head and neck cancers. Moreover, TP63 overexpression was characteristically observed in squamous cell carcinomas of the lung, esophagus, and head and neck region. Additionally, overexpression of TP63 and TP73 was frequently observed in thymomas. Our results reveal a spectrum of genomic alterations in the TP53 pathway that is characteristic of many tumor types, and these data may be useful in the trials of targeted therapies.

Frequency of EGFR T790M mutation and multimutational profiles of rebiopsy samples from non-small cell lung cancer developing acquired resistance to EGFR tyrosine kinase inhibitors in Japanese patients.

BACKGROUND: The majority of non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation eventually develop resistance to EGFR tyrosine kinase inhibitors (TKIs). Minimal information exists regarding genetic alterations in rebiopsy samples from Asian NSCLC patients who develop acquired resistance to EGFR-TKIs. METHODS: We retrospectively reviewed the medical records of patients with NSCLC harboring EGFR mutations who had undergone rebiopsies after developing acquired resistance to EGFR-TKIs. We analyzed 27 practicable samples using a tumor genotyping panel to assess 23 hot-spot sites of genetic alterations in nine genes (EGFR, KRAS, BRAF, PIK3CA, NRAS, MEK1, AKT1, PTEN, and HER2), gene copy number of EGFR, MET, PIK3CA, FGFR1, and FGFR2, and ALK, ROS1, and RET fusions. Additionally, 34 samples were analyzed by commercially available EGFR mutation tests. RESULTS: Sixty-one patients underwent rebiopsy. Twenty-seven samples were analyzed using our tumor genotyping panel, and 34 samples were analyzed for EGFR mutations only by commercial clinical laboratories. Twenty-one patients (34 %) had EGFR T790M mutation. Using our tumor genotyping panel, MET gene copy number gain was observed in two of 27 (7 %) samples. Twenty patients received continuous treatment with EGFR-TKIs even after disease progression, and 11 of these patients had T790M mutation in rebiopsy samples. In contrast, only 10 of 41 patients who finished EGFR-TKI treatment at disease progression had T790M mutation. The frequency of T790M mutation in patients who received continuous treatment with EGFR-TKIs after disease progression was significantly higher than that in patients who finished EGFR-TKI treatment at disease progression (55 % versus 24 %, p = 0.018). CONCLUSIONS: The frequency of T790M mutation in this study was lower than that in previous reports examining western patients. These results suggest that continuous treatment with EGFR-TKI after disease progression may enhance the frequency of EGFR T790M mutation in rebiopsy samples.

Peroxisome proliferator-activated receptor γ agonist efatutazone impairs transforming growth factor ß2-induced motility of epidermal growth factor receptor tyrosine kinase inhibitor-resistant lung cancer cells.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) are effective for non-small cell lung cancers (NSCLC) with EGFR-activating mutations. However, most responders develop resistance. Efatutazone, a novel peroxisome proliferator-activated receptor gamma (PPARγ) agonist, is currently under clinical evaluation; it has antiproliferative effects and induces cellular morphological changes and differentiation. The present study investigated the effects of efatutazone in EGFR-TKI-resistant NSCLC cells, while focusing on cell motility. The PC-9-derived NSCLC cell lines PC-9ER and PC-9ZD, resistant to EGFR-TKI due to v-crk avian sarcoma virus CT10 oncogene homolog-like (CRKL) amplification-induced phosphatidylinositol 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (AKT) activation and an EGFR T790M mutation, respectively, were used. These cells exhibit enhanced cell motility due to transforming growth factor ß (TGF-ß)/Smad2 family member 2 (Smad2) pathway activation. Efatutazone had no growth-inhibitory effect on the tested cells but inhibited the motility of EGFR-TKI-resistant cells in wound closure and transwell assays. Efatutazone plus erlotinib treatment provided greater inhibition of PC-9ER cell migration than efatutazone or erlotinib alone. Efatutazone suppressed increased TGF-ß2 secretion from both cell lines (shown by ELISA) and downregulation of TGF-ß2 transcription (observed by quantitative RT-PCR). Immunoblot analysis and luciferase assays revealed that efatutazone suppressed Smad2 phosphorylation and its transcriptional activity. These results suggest that efatutazone inhibits cell motility by antagonizing the TGF-ß/Smad2 pathway and effectively prevents metastasis in NSCLC patients with acquired resistance to EGFR-TKI regardless of the resistance mechanism.

Assessment of mutational profile of Japanese lung adenocarcinoma patients by multitarget assays: a prospective, single-institute study.

BACKGROUND: Integration of mutational profiling to identify driver genetic alterations in a clinical setting is necessary to facilitate personalized lung cancer medicine. A tumor genotyping panel was developed and the Shizuoka Lung Cancer Mutation Study was initiated as a prospective tumor genotyping study. This study reports the frequency of driver genetic alterations in Japanese lung adenocarcinoma patients, and clinicopathologic correlations with each genotype. METHODS: Between July 2011 and January 2013, 411 lung adenocarcinoma patients admitted to the Shizuoka Cancer Center were included in this study with their written informed consent. Surgically resected tissues, tumor biopsies, and/or body cavity fluids were collected and tested for 23 hotspot sites of driver mutations in 9 genes (EGFR, KRAS, BRAF, PIK3CA, NRAS, MEK1, AKT1, PTEN, and HER2), gene amplifications in 5 genes (EGFR, MET, PIK3CA, FGFR1, and FGFR2), and ALK, ROS1, and RET fusions. RESULTS: Genetic alterations were detected in 54.3% (223 of 411) of all patients. The most common genetic alterations detected in this study were EGFR mutations (35.0%) followed by KRAS mutations (8.5%) and ALK fusions (5.0%). Concurrent genetic alterations were detected in 22 patients (5.4%), and EGFR mutations were observed in 16 patients as the most common partner for concurrent genetic alteration. Significantly more concurrent genetic alterations were observed in older patients. CONCLUSIONS: This is one of the largest reports of a prospective tumor genotyping study on Japanese patients with adenocarcinoma. These data suggest that mutational profiling data using a multimutational testing platform would be valuable for expanding the range of molecular-targeted therapeutics in lung cancer.

A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of rare tumor cells in blood.

BACKGROUND: Personalized cancer treatment relies on the accurate detection of actionable genomic aberrations in tumor cells. Circulating tumor cells (CTCs) could provide an alternative genetic resource for diagnosis; however, the technical difficulties in isolating and analyzing rare CTCs have limited progress to date. In this preclinical study, we aimed to develop an improved capture system for molecular characterization of CTCs based on a novel cell sorting technology. METHODS: We developed a cell capture platform using On-chip Sort (On-Chip Biotechnologies), a novel bench-top cell sorter equipped with a disposable microfluidic chip. Spike-in experiments comprising a series of lung cancer cell lines with varying epithelial cell adhesion molecule (EpCAM) expression levels were conducted to assess the capture and purification efficiency of the platform. Samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells, followed by sample fixation and labeling. The enriched and labeled samples were then sorted by On-chip Sort based on cytokeratin, vimentin, and CD45 expression. Captured cells were immediately subjected to whole genome amplification followed by mutation analysis using deep targeted sequencing, and copy number analysis using quantitative polymerase chain reaction (qPCR). RESULTS: Spike-in experiments revealed an excellent overall mean capture rate of 70.9%. A 100% success rate in the detection of EGFR, KRAS and BRAF mutations from captured cells was achieved using pyrosequencing and deep sequencing. The mutant variant detection rates were markedly higher than those obtained with the CellSearch profile kit. qPCR analysis of amplified DNA demonstrated reproducible detection of copy number changes of the EGFR in captured tumor cells. CONCLUSIONS: Using a novel cell sorter, we established an efficient and convenient platform for the capture of CTCs. Results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.

Prospective genetic profiling of squamous cell lung cancer and adenosquamous carcinoma in Japanese patients by multitarget assays.

BACKGROUND: Despite considerable recent progress in the treatment of lung adenocarcinoma, there has been little progress in the development of efficacious molecular targeted therapies for squamous cell lung cancer. In addition to the recent comprehensive genome-wide characterization of squamous cell lung cancer, it is also important to genotype this form of cancer. We therefore conducted the Shizuoka Lung Cancer Mutation Study to analyze driver mutations in patients with thoracic malignancies. Here we report the results of genotyping in patients with squamous cell lung cancer. METHODS: Based on the biobanking system, in conjunction with the clinic and pathology lab, we developed a genotyping panel designed to assess 24 mutations in 10 genes (EGFR, KRAS, BRAF, PIK3CA, NRAS, MEK1, AKT1, PTEN, HER2 and DDR2), EGFR, MET, PIK3CA, FGFR1 and FGFR2 copy numbers, and EML4-ALK and ROS1 translocations, using pyrosequencing plus capillary electrophoresis, quantitative polymerase chain reaction (PCR) and reverse-transcription PCR, respectively. RESULTS: A total of 129 patients with squamous cell lung cancer and adenosquamous carcinoma were enrolled in this study between July 2011 and November 2012. We detected genetic alterations in 40% of all cases. Gene alterations included: EGFR mutations, 6%; KRAS mutations, 4%; PIK3CA mutations, 13%; NRAS mutations, 1%; KIF5b-RET fusion gene, 1%; EGFR copy number gain, 5%; PIK3CA copy number gain, 15%; and FGFR1 copy number gain, 5%. Twelve patients (9%) harbored simultaneous genetic alterations. Genetic alterations were detected more frequently in surgically-resected, snap-frozen samples than in formalin-fixed, paraffin-embedded samples (50% vs. 29%). In addition, patients aged ≤70 years old and never-smokers showed high frequencies of genetic alterations. CONCLUSIONS: This study represents one of the largest prospective tumor-genotyping studies to be performed in Asian patients with squamous cell lung cancer. These results suggest that incorporation of genetic profiling into lung cancer clinical practice may facilitate the administration of personalized cancer treatments in patients with squamous cell lung cancer.

Biphenotypic sinonasal sarcoma diagnosed by detection of PAX3-MAML3 fusion gene using integrated whole-genome and transcriptome sequencing.

Biphenotypic sinonasal sarcoma (BSNS) is a double-phenotype sarcoma that shows differentiation in both the nervous and muscular systems. To date, whole-genome and transcriptome sequencing (WGTS) has not been used to analyze BSNS. We report a patient with BSNS who was diagnosed based on PAX3 rearrangement using WGTS. A 71-year-old Japanese male without remarkable symptoms showed a nasal tumor when undergoing computed tomography. Although pathological examination revealed a non-characteristic spindle cell tumor, a definitive diagnosis could not be made based on this examination. Endoscopic sinus surgery was performed for subsequent diagnosis, treatment, and WGTS. WGTS revealed a t(2; 4)(q35; q31.1) reciprocal translocation, resulting in a PAX3-MAML3 fusion gene, leading to a definitive diagnosis of BSNS. We also detected upregulation of the expression of PAX3, MAML3, and 11 known genes involved in neural and myogenic differentiation relevant to the BSNS phenotype. Hence, using WGTS in combination with conventional pathological diagnosis can contribute to a definitive diagnosis of rare cancers, including BSNS, by detecting chromosomal rearrangements or diagnostic markers.