Clinical utility of Todai OncoPanel in the setting of approved comprehensive cancer genomic profiling tests in Japan.

Comprehensive cancer genome profiling (CGP) has been nationally reimbursed in Japan since June 2019. Less than 10% of the patients have been reported to undergo recommended treatment. Todai OncoPanel (TOP) is a dual DNA-RNA panel as well as a paired tumor-normal matched test. Two hundred patients underwent TOP as part of Advanced Medical Care B with approval from the Ministry of Health, Labour and Welfare between September 2018 and December 2019. Tests were carried out in patients with cancers without standard treatment or when patients had already undergone standard treatment. Data from DNA and RNA panels were analyzed in 198 and 191 patients, respectively. The percentage of patients who were given therapeutic or diagnostic recommendations was 61% (120/198). One hundred and four samples (53%) harbored gene alterations that were detected with the DNA panel and had potential treatment implications, and 14 samples (7%) had a high tumor mutational burden. Twenty-two samples (11.1%) harbored 30 fusion transcripts or MET exon 14 skipping that were detected by the RNA panel. Of those 30 transcripts, 6 had treatment implications and 4 had diagnostic implications. Thirteen patients (7%) were found to have pathogenic or likely pathogenic germline variants and genetic counseling was recommended. Overall, 12 patients (6%) received recommended treatment. In summary, patients benefited from both TOP DNA and RNA panels while following the same indication as the approved CGP tests. (UMIN000033647).

Integrin alpha 2 is associated with tumor progression and postoperative recurrence in non-small cell lung cancer.

BACKGROUND: Integrins are transmembrane proteins that mediate cell adhesion to extracellular matrix. Whereas expression of integrin alpha 2 is associated with motility, invasiveness and cellular differentiation in various tumors, the role of integrin alpha 2 in lung cancer has not been studied in detail. The aim of this study was to determine whether and how aberrant integrin alpha 2 expression in non-small cell lung cancer leads to different outcomes. METHODS: We measured expression of integrin alpha 2 by quantitative polymerase chain reaction in 100 samples collected from non-small cell lung cancer patients who had undergone surgical resection. We assigned patients to high and low expression groups and analyzed survival. Cellular morphology, adhesion, proliferation, migration and invasion were examined in human lung cancer cell lines. RESULTS: Among 100 cases, 41 were female, with a median age of 71 years. High expression of integrin alpha 2 in non-small cell lung cancer was associated with lower recurrence-free survival (P = 0.004). Overexpression of integrin alpha 2 in cell lines had no effect on cell proliferation or invasion but resulted in increased cell size (1416 µm2 versus 470 µm2 in H522 cells, P < 0.001; 1822 µm2 versus 1029 µm2 in H661 cells, P = 0.02), adhesion (P < 0.001 in H522 and H661 cells) and migration (gap area filled was 71% versus 36% in H522 cells, P < 0.001; 57% versus 26% in H661 cells, P = 0.001). These changes were suppressed by E7820, an inhibitor of integrin alpha 2. CONCLUSIONS: Integrin alpha 2 may play a significant role in lung cancer adhesion and migration, and may lead to a higher risk of recurrence.

Integrin α11 in non-small cell lung cancer is associated with tumor progression and postoperative recurrence.

Integrins are transmembrane proteins that mediate cell adhesion to the extracellular matrix. Integrin α11 (ITGA11) is not expressed in normal alveolar epithelial cells and is a known receptor for collagen. While integrin α11ß1 overexpression in the tumor stroma has been associated with tumor growth and metastatic potential of non-small cell lung cancer (NSCLC), little is known about the role of ITGA11 in tumor cells. Thus, we examined the RNA expression of ITGA11 by quantitative RT-PCR in 80 samples collected from NSCLC patients who had undergone surgical resection and analyzed the clinical outcomes. We found that high expression of ITGA11 was associated with lower recurrence-free survival in all NSCLC patients (P = 0.043) and in stage I NSCLC patients (P = 0.049). These results were consistent with in silico analyses of the Cancer Genome Atlas database. We also analyzed cell proliferation, migration and invasion capacity in lung cancer cell lines after overexpression of ITGA11. Overexpression of ITGA11 in lung cancer cell lines had little effect on cell proliferation but resulted in increased migration and invasion capacity. Our findings suggest that ITGA11 plays a significant role in cancer migration and invasion, leading to higher recurrence. ITGA11 expression may be a predictor of poor prognosis in patients with surgically resected NSCLC.

Identification of a metastatic lung adenocarcinoma of the palate mucosa through genetic and histopathological analysis: a rare case report and literature review.

BACKGROUND: Cancers of unknown primary origin (CUPs) are reported to be the 3-4th most common causes of cancer death. Recent years have seen advances in mutational analysis and genomics profiling. These advances could improve accuracy of diagnosis of CUPs and might improve the prognosis of patients with CUPs. CASE PRESENTATION: A 76-year old male with an adenocarcinoma of unknown primary origin in the lung presented with another tumor of the palate mucosa. The tumor cells in the pleural effusion were all negative for immunohistochemical markers (TTF-1 and Napsin A) and lung-specific oncogenic driver alterations (EGFR mutation and ALK translocation). The tumor of the palate mucosa was likewise identified as an adenocarcinoma, and the cells showed cytological similarities with the tumor cells in the pleural effusion; TTF-1, Napsin A, EGFR mutation and ALK translocation were all negative. This result suggested that origins of the tumors of the palate mucosa and in the lung were the same, even though the origin had not yet been determined. Next, we addressed whether the tumor of the palate mucosa was a primary tumor or not. Secretory carcinoma (SC), which is a common type of minor salivary gland tumor (MSGT), was suspected; however, mammaglobin was negative and ETV6-NTRK3 (EN) fusion was not observed. Other MSGTs were excluded based on histological and immunohistochemical findings. Furthermore, an additional examination demonstrated an oncogenic KRAS mutation at codon 12 (p.G12D) in both palate tumor and in pleural effusion. KRAS mutation is known to exist in one-third of lung adenocarcinomas (LUADs), but quite rare in MSGTs. The possibility of metastasis from other organs was considered unlikely from the results of endoscopic and imaging studies. This result indicated that the primary site of the CUP was indeed the lung, and that the tumor of the palate mucosa was a metastasis of the LUAD. CONCLUSIONS: A tumor of the palate mucosa that showed diagnostic difficulties was determined to be a metastatic LUAD by genomic alterations and histopathological findings.

Altered editing level of microRNAs is a potential biomarker in lung adenocarcinoma.

Adenosine-to-inosine (A-to-I) microRNA editing is associated with tumor phenotypes in various cancer types. Recent analyses of The Cancer Genome Atlas (TCGA) dataset have shown several microRNAs that undergo A-to-I editing in human cancers, some of which have been reported to be associated with prognosis. Herein, we examined published small RNA deep sequencing data of 74 cases of lung adenocarcinoma (AD) and the corresponding normal counterpart (NC) specimen in silico in order to identify A-to-I microRNA editing events. Editing levels of miR-379-5p, miR-99a-5p, and miR-497-5p were lower in AD than in NC and, in a large number of cases, the editing level of miR-200b-3p was higher in AD than in NC. Difference in the editing level between AD and NC was largest for miR-99a-5p. Then, we examined the editing level of miR-99a-5p in 50 surgically resected lung adenocarcinoma cases at our institution by a conventional sequence-based method, and its association with clinical outcomes. The editing level of miR-99a-5p was significantly lower in 19 cases of AD (38%) than in corresponding NC. These cases showed a shorter overall survival as assessed using the log-rank test (P = .047). This trend was consistent with previous analyses of TCGA dataset. The altered editing level of microRNAs in lung adenocarcinoma could serve as a potential biomarker.

Glutamate-cysteine ligase catalytic subunit is associated with cisplatin resistance in lung adenocarcinoma.

BACKGROUND: Cisplatin is a key drug for treating lung adenocarcinoma, and its sensitivity to cisplatin is directly related to prognosis. We aimed to reveal the roles of genes related to glutathione synthesis (glutamate-cysteine ligase catalytic subunit, GCLC) and cystine uptake (cystine/glutamate transporter, xCT and CD44v8-10) in cisplatin resistance and prognosis in lung adenocarcinoma. METHODS: We established cell lines stably expressing GCLC, xCT, standard isoform of CD44, and CD44v8-10, and investigated their sensitivities to cisplatin. We also measured mRNA expression levels of these genes in the tumor tissues from 92 lung adenocarcinoma patients. Patients were divided into high-expression (upper quartile, N = 23) and low-expression groups (remaining patients, N = 69). Recurrence-free survival, overall survival (N = 92), and post-recurrence survival (N = 22) were selected as endpoints. RESULTS: Compared with the control green fluorescent protein-expressing cell line (inhibitory concentration 50:6.9 µM), all the stable cell lines were more resistant to cisplatin (12.9 µM, P = 0.025; 13.9 µM, P = 0.028; 26.7 µM, P = 0.001; 17.7 µM, P = 0.008, respectively). In contrast, there was no significant difference in recurrence-free or overall survival between the high- and low-expression groups for any of the genes. However, high expression of GCLC was a risk factor for poorer post-recurrence survival (hazard ratio, 6.26; 95% confidence interval, 1.37-28.7; P = 0.018). CONCLUSION: High expression levels of genes related to glutathione synthesis and cystine uptake promote cisplatin resistance in lung adenocarcinoma cell lines. High expression of GCLC in tumor tissue may be a potential predictor of treatment failure.

High expression of IRE1 in lung adenocarcinoma is associated with a lower rate of recurrence.

OBJECTIVE: Recent reports have shown that endoplasmic reticulum stress is associated with cancer. However, the impacts of endoplasmic reticulum stress on the prognosis of lung cancer are unknown. Therefore, in this study, we sought to reveal the relationship between the expression of endoplasmic reticulum stress-related genes (endoplasmic reticulum oxidoreductase 1L, protein kinase RNA-like endoplasmic reticulum kinase, activating transcription factor 6 and inositol-requiring kinase 1) and the outcome of lung adenocarcinoma. METHODS: One hundred and twenty-six patients with surgically resected lung adenocarcinomas were subjected to an endoplasmic reticulum stress-related mRNA expression analysis using quantitative RT-PCR. The following parameters were analyzed for all the study patients: age, sex, disease stage, smoking status, lymph node invasion (ly), vascular invasion (v) and EGFR mutation status. We assigned patients to either a high-expression group or a low-expression group according to the expression levels of endoplasmic reticulum stress-related genes. RESULTS: High expressions of endoplasmic reticulum stress-related genes were observed in patients with lower stages of lung adenocarcinoma and minimal vascular invasion. A Kaplan-Meier analysis showed significant differences in recurrence-free survival and overall survival between high-expression group and low-expression group. High inositol-requiring kinase 1 expression was an independent predictor of recurrence-free survival among patients with lung adenocarcinoma (hazard ratio, 0.396; 95% confidence interval, 0.188-0.834; P = 0.015). CONCLUSIONS: Inositol-requiring kinase 1 may be a useful biomarker to predict recurrence in surgically resected lung adenocarcinoma patients.

The chimeric transcript RUNX1-GLRX5: a biomarker for good postoperative prognosis in Stage IA non-small-cell lung cancer.

Stage IA non-small-cell lung cancer cases have been recognized as having a low risk of relapse; however, occasionally, relapse may occur. To predict clinical outcome in Stage IA non-small-cell lung cancer patients, we searched for chimeric transcripts that can be used as biomarkers and identified a novel chimeric transcript, RUNX1-GLRX5, comprising RUNX1, a transcription factor, and GLRX5. This chimera was detected in approximately half of the investigated Stage IA non-small-cell lung cancer patients (44/104 cases, 42.3%). Although there was no significant difference in the overall survival rate between RUNX1-GLRX5-positive and -negative cases (P = 0.088), a significantly lower relapse rate was observed in the RUNX1-GLRX5-positive cases (P = 0.039), indicating that this chimera can be used as a biomarker for good prognosis in Stage IA patients. Detection of the RUNX1-GLRX5 chimeric transcript may therefore be useful for the determination of a postoperative treatment plan for Stage IA non-small-cell lung cancer patients.

Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3.

Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation.

Alternative polyadenylation is associated with lower expression of PABPN1 and poor prognosis in non-small cell lung cancer.

Alternative polyadenylation (APA), which induces shortening of the 3'UTR, is emerging as an important phenomenon in gene regulation. APA is involved in development, cancer and cell proliferation. APA may lead to disruption of microRNA-mediated gene silencing in cancer cells via detachment of microRNA binding sites. We studied the correlation between the APA profile and the tumor aggressiveness in cases of lung cancer. We selected the top 10 genes showing significant 3'UTR shortening in lung cancer, using the package of the Bioconductor for probe-level analyses of expression microarrays. We established and evaluated the APA score by quantitative RT-PCR in 147 clinical specimens of non-small cell lung cancer and compared the results with the clinical outcomes and expression levels of APA-related genes, including PABPN1, CPEB1, E2F1 and proliferation markers (MKI67, TOP2A and MCM2). High APA scores were correlated with an advanced tumor stage and a poor prognosis (P < 0.001). Multivariate analysis identified the APA score as an independent prognostic factor (hazard ratio, 3.0; P = 0.03). Both lower expression of PABPN1 and higher expression of the proliferation markers were correlated with high APA scores and a poor prognosis, with suppression of PABPN1 exerting its influence independent of gain of the proliferation markers. Moreover, the APA score was correlated with the maximum standardized uptake value of the tumors on positron emission tomography (r = 0.53; P < 0.001). Our results indicate that the loss of PABPN1, a suppressor of APA, might promote tumor aggressiveness by releasing the cancer cells from microRNA-mediated gene regulation.

Correlation between variant call accuracy and quality parameters in comprehensive cancer genomic profiling tests.

Background: Comprehensive genomic profiling (CGP) tests have been widely utilized in clinical practice. In this test, the variant list automatically output from the data analysis pipeline often contains false-positive variants, although the correlation between the quality parameters and prevalence of false-positive variants remains unclear. Methods: We analyzed 125 CGP tests performed in our laboratory. False-positive variants were manually detected via visual inspection. The quality parameters of both wet and dry processes were also analyzed. Results: Among the 125 tests, 52 (41.6%) required more than one correction of the called variants, and 21 (16.8%) required multiple corrections. A significant correlation was detected between somatic false-positive variants and quality parameters in the wet (ΔΔCq, pre-capture library peak size, pre-capture library DNA amount, capture library peak size, and capture library concentration) and dry processes (total reads, mapping rates, duplication rates, mean depth, and depth coverage). Capture library concentration and mean depth were strong independent predictors of somatic false-positive variants. Conclusions: We demonstrated a correlation between somatic false-positive variants and quality parameters in the CGP test. This study facilitates gaining a better understanding of CGP test quality management.

Analysis of quality metrics in comprehensive cancer genomic profiling using a dual DNA-RNA panel.

Background: The nucleic acid quality from formalin-fixed paraffin-embedded (FFPE) tumor vary among samples, resulting in substantial variability in the quality of comprehensive cancer genomic profiling tests. The objective of the study is to investigate how nucleic acid quality affects sequencing quality. We also examined the variations in nucleic acid quality among different hospitals or cancer types. Methods: Three nucleic acid quality metrics (ddCq, Q-value, and DV200) and five sequencing quality metrics (on-target rate, mean depth, coverage uniformity, target exon coverage, and coverage of the housekeeping gene) were examined using 585 samples from the Todai OncoPanel, a dual DNA-RNA panel. Results: In the DNA panel, ddCq served as an indicator of sequencing depth and Q-value reflected the uniformity of sequencing across different regions. It was essential to have favorable values not only for ddCq but also for Q-value to obtain ideal sequencing results. For the RNA panel, DV200 proved to be a valuable metric for assessing the coverage of the housekeeping genes. Significant inter-hospital differences were observed for DNA quality (ddCq and Q-value), but not for RNA quality (DV200). Differences were also observed among cancer types, with Q-value being the lowest in lung and the highest in cervix, while DV200 was the highest in lung and the lowest in bowel. Conclusions: We demonstrated distinct characteristics and high predictive performances of ddCq, Q-value, and DV200. Variations were observed in the nucleic acid quality across hospitals and cancer types. Further study is warranted on preanalytical factors in comprehensive cancer genomic profiling tests.

NECTIN2 is a prognostic biomarker and potential therapeutic target in lung adenocarcinoma.

INTRODUCTION: NECTINs are transmembrane proteins mediating cell-to-cell adhesion. NECTINs interact with integrins or other membrane receptors to trigger multiple cellular functions. Aberrant NECTIN expression is associated with cancer progression and poor outcomes. While NECTIN2 is overexpressed in various cancer types, its role in lung cancer is not well understood. MATERIAL AND METHODS: We investigated the function of NECTIN2 in lung adenocarcinoma (LUAD) using the Cancer Genome Atlas (TCGA) dataset and clinical samples of 105 LUAD patients who had undergone surgical resection. Cell proliferation, apoptosis, migration and invasion were investigated using human lung adenocarcinoma cell lines. RESULTS: We found that high NECTIN2 expression correlated with reduced overall survival in LUAD in TCGA database. In clinical samples, high NECTIN2 expression was associated with lower recurrence-free survival in all patients (P < 0.001) and in stage I patients (P = 0.001). Functional analyses demonstrated that NECTIN2 knockout promoted cell apoptosis and diminished cell proliferation and migration capacity. NECTIN2 overexpression did not significantly affect cellular functions. DISCUSSION: Our results suggest that NECTIN2 plays a significant role in cell apoptosis and cancer cell migration, leading to increased postoperative recurrence. Furthermore, NECTIN2 serves as a prognostic indicator and a potential therapeutic target in LUAD. CONCLUSIONS: High NECTIN2 expression in LUAD was found to be associated with postoperative recurrence, and was observed to play an important role in cell apoptosis and migration.

Detection of novel paraja ring finger 2-fer tyrosine kinase mRNA chimeras is associated with poor postoperative prognosis in non-small cell lung cancer.

Previously, we reported that the overexpression of fer tyrosine kinase (FER), a non-receptor tyrosine kinase, is correlated with poor postoperative prognosis and cancer-cell survival in non-small cell lung cancer (NSCLC). In the present study, we further analyzed FER-overexpressed NSCLC cases and identified various patterns of chimeric mRNAs, composed of paraja ring finger 2 (PJA2) and FER. We detected no genomic rearrangements between PJA2 and FER and attributed these chimeric mRNAs to alterations at the transcriptome level: i.e., trans-splicing. Several chimeric patterns were detected concurrently in each patient, and the pattern sets varied among patients, although the pattern in which PJA2 exon 1 was fused to FER exon 3 (designated as Pe1-Fe3 mRNA) was detected constantly. Therefore, in a wide screening for PJA2-FER mRNAs in NSCLC, we focused on this chimeric pattern as a representative chimera. In analyses of 167 NSCLC samples, Pe1-Fe3 mRNA was identified in about 10% of the patients, and the presence of chimeric mRNA was significantly correlated with a high expression level of parental FER mRNA. Furthermore, we found that the detection of Pe1-Fe3 mRNA was correlated with poor postoperative survival periods in NSCLC, consistent with a previous finding in which FER overexpression was correlated with poor postoperative prognosis in NSCLC. This report is the first to suggest a correlation between chimeric mRNA and the expression level of parental mRNA. Furthermore, our findings may be clinically beneficial, suggesting that PJA2-FER mRNAs might serve as a novel prognostic biomarker in NSCLC.

[Long-term results of open stent-grafting applied with a Matsui-Kitamura( MK) stent in treatment of arch aneurysm].

We report the initial and long-term results of open stent-grafting (OSG) applied with a Matsui-Kitamura (MK) stent in the treatment of thoracic aortic aneurysm (TAA). From August 2005 to March 2013, OSG for TAA was applied in 46 cases( male/female, 36/10, 54-86 years old, mean age 71). During deep hypothermic circulatory arrest with antegrade selective cerebral perfusion, stent graft was delivered through the transected proximal aortic arch, followed by arch replacement with a 4-branched prosthesis. Operative mortality within 30 days was 6.5%(respiratory failure in 1, multiple organ failure in 1and ischemic enteritis in 1) and there was 1 in hospital death due to brain stem infarction. Perioperative morbidity included 2( 4.3%) stroke, 5( 10.9%) spinal cord injuries( paraplegia in 1, paraparesis in 1 and transient paraparesis in 3). In long-term follow-up, survival was 86.8%, 77.2%, and 72.0% for 1, 3 and 5 years. Freedom from aortic event was 97.3%, 93.8% and 89.1% for 1, 3 and 5 years. Theseresults suggested the OSG method applied with a MK stent is a useful surgical procedure for the treatment of TAA.

Genome structure-based screening identified epigenetically silenced microRNA associated with invasiveness in non-small-cell lung cancer.

MicroRNA (miRNA) expression is frequently altered in human cancers. To search for epigenetically silenced miRNAs in non-small-cell lung cancer (NSCLC), we mapped human miRNAs on autosomal chromosomes and selected 55 miRNAs in silico. We treated six NSCLC cell lines with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) and determined the expressions of the 55 miRNAs. Fourteen miRNAs were decreased in the cancer cell lines and were induced after 5-aza-CdR treatment. After a detailed DNA methylation analysis, we found that mir-34b and mir-126 were silenced by DNA methylation. Mir-34b was silenced by the DNA methylation of its own promoter, whereas mir-126 was silenced by the DNA methylation of its host gene, EGFL7. A chromatin immunoprecipitation assay revealed H3K9me2 and H3K9me3 in mir-34b and EGFL7, and H3K27me3 in EGFL7. The overexpression of mir-34b and mir-126 decreased the expression of c-Met and Crk, respectively. The 5-aza-CdR treatment of lung cancer cell line resulted in increased mir-34b expression and decreased c-Met protein. We next analyzed the DNA methylation status of these miRNAs using 99 primary NSCLCs. Mir-34b and mir-126 were methylated in 41 and 7% of all the cases, respectively. The DNA methylation of mir-34b was not associated with c-Met expression determined by immunohistochemistry, but both mir-34b methylation (p = 0.007) and c-Met expression (p = 0.005) were significantly associated with lymphatic invasion in a multivariate analysis. The DNA methylation of mir-34b can be used as a biomarker for an invasive phenotype of lung cancer.

Brief, manualised and semistructured individual psychotherapy programme for patients with advanced cancer in Japan: study protocol for Managing Cancer and Living Meaningfully (CALM) phase 2 trial.

INTRODUCTION: Managing Cancer and Living Meaningfully (CALM) is a novel, brief and manualised psychotherapeutic intervention intended to treat and prevent depression and end-of-life distress in patients with advanced cancer. This phase 2 trial aims to assess the feasibility and preliminary efficacy of CALM in Japanese patients with cancer. METHODS AND ANALYSIS: This study is a single-arm clinical trial. All patients involved in the study are ≥18 years of age, have been diagnosed with advanced or metastatic solid-tumour cancer, and their expected survival is at least 6 months. CALM comprises three to six individual therapy sessions, each lasting approximately 45-60 min, provided over 3- 6 months. The participants will be asked to complete questionnaires at baseline (t0), 3 months (t1) and 6 months (t2). The primary outcomes are rates of completion of the intervention and of the outcome measures and improvement of depressive symptoms measured using the Patient Health Questionnaire-9 between t0 and t2. The criteria for the successful rate of completion is that at least 70% participants who participate in at least three sessions will complete measures at t2. The secondary outcomes are the improvement in scores on: (1) the Quality of Life at the End of Life-Cancer Scale, (2) the Experiences in Close Relationships scale, (3) the Death and Dying Distress Scale and (4) the Clinical Evaluation Questionnaire. ETHICS AND DISSEMINATION: This study was approved by the Research Ethics Committee of The University of Tokyo, Cancer Institute Hospital of Japanese Foundation for Cancer Research and Yamaguchi University. We will conduct the study in accordance with the Declaration of Helsinki and the Ethical Guidelines for Medical and Health Research Involving Human Subjects. The results of this study will be submitted for peer-reviewed publication and presentation at local, national and international scientific meetings and conferences. TRAIL REGISTRATION NUMBER: UMIN000040032; Pre-results.

Concordance Between Recommendations From Multidisciplinary Molecular Tumor Boards and Central Consensus for Cancer Treatment in Japan.

Importance: Quality assurance of molecular tumor boards (MTBs) is crucial in cancer genome medicine. Objective: To evaluate the concordance of recommendations by MTBs and centrally developed consensus treatment recommendations at all 12 leading institutions for cancer genomic medicine in Japan using 50 simulated cases. Design, Setting, and Participants: This was a prospective quality improvement study of 50 simulated cancer cases. Molecular tumor boards from 12 core hospitals independently recommended treatment for 50 cases blinded to the centrally developed consensus treatment recommendations. The study's central committee consisted of representatives from all 12 core hospitals in Japan who selected the 50 simulated cases from The Cancer Genome Atlas database, including frequently observed genomic alterations. The central committee recommended centrally developed consensus treatment. The concordance rate for genomically matched treatments between MTBs and centrally developed consensus treatment recommendations was evaluated. Data analysis was conducted from January 22 to March 3, 2021. Exposures: Simulated cases of cancer. Main Outcomes and Measures: The primary outcome was concordance, defined as the proportion of recommendations by MTBs concordant with centrally developed consensus treatment recommendations. A mixed-effects logistic regression model, adjusted for institutes as a random intercept, was applied. High evidence levels were defined as established biomarkers for which the treatment was ready for routine use in clinical practice, and low evidence levels were defined as biomarkers for genomically matched treatment that were under investigation. Results: The Clinical Practice Guidance for Next-Generation Sequencing in Cancer Diagnosis and Treatment (edition 2.1) was used for evidence-level definition. The mean concordance between MTBs and centrally developed consensus treatment recommendations was 62% (95% CI, 57%-65%). Each MTB concordance varied from 48% to 86%. The concordance rate was higher in the subset of patients with colorectal cancer (100%; 95% CI, 94.0%-100%), ROS1 fusion (100%; 95% CI, 85.5%-100%), and high evidence level A/R (A: 88%; 95% CI, 81.8%-93.0%; R:100%; 95% CI, 92.6%-100%). Conversely, the concordance rate was lower in cases of cervical cancer (11%; 95% CI, 3.1%-26.1%), TP53 mutation (16%; 95% CI, 12.5%-19.9%), and low evidence level C/D/E (C: 30%; 95% CI, 24.7%-35.9%; D: 25%; 95% CI, 5.5%-57.2%; and E: 18%; 95% CI, 13.8%-23.0%). Multivariate analysis showed that evidence level (high [A/R] vs low [C/D/E]: odds ratio, 4.4; 95% CI, 1.8-10.8) and TP53 alteration (yes vs no: odds ratio, 0.06; 95% CI, 0.03-0.10) were significantly associated with concordance. Conclusions and Relevance: The findings of this study suggest that genomically matched treatment recommendations differ among MTBs, particularly in genomic alterations with low evidence levels wherein treatment is being investigated. Sharing information on matched therapy for low evidence levels may be needed to improve the quality of MTBs.

CpG island methylation of microRNAs is associated with tumor size and recurrence of non-small-cell lung cancer.

We investigated whether the CpG island methylation of certain microRNAs was associated with the clinicopathological features and the prognosis of non-small-cell lung cancer. The methylation of mir-152, -9-3, -124-1, -124-2, and -124-3 was analyzed in 96 non-small-cell lung cancer specimens using a combined bisulfite restriction analysis. The median observation period was 49.5 months. The methylation of mir-9-3, -124-2, and -124-3 was individually associated with an advanced T factor independent of age, sex, and smoking habit. Moreover, the methylation of multiple microRNA loci was associated with a poorer progression-free survival in a univariate analysis. Our result enlightens the accumulation of aberrant DNA methylation which occurs in concordance with the tumor progression.

Polymerase reaction without primers throughout for the reconstruction of full-length cDNA from products of rapid amplification of cDNA ends (RACE).

Rapid amplification of cDNA ends (RACE) has widely been used to determine both ends of the cDNA from its partial sequence. Conventionally, 5'- and 3'-RACE products were ligated at a restriction site in the overlap region to reconstruct the full-length cDNA; however, reconstruction is difficult if no appropriate restriction enzymes are available. Here, we report a novel method to reconstruct full-length cDNA with DNA polymerase. Instead of usual PCR, chain reactions were avoided and the elongation time was shortened, which enables non-specific products or undesired point mutations to be minimized. We successfully reconstructed and TA-cloned a full-length cDNA of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene variant 2 from RACE products obtained from a surgically resected lung adenocarcinoma sample. We also evaluated some parameters to provide recommendations for this new method.