Baseline ctDNA gene alterations as a biomarker of survival after panitumumab and chemotherapy in metastatic colorectal cancer.

Certain genetic alterations and right-sided primary tumor location are associated with resistance to anti-epidermal growth factor (EGFR) treatment in metastatic colorectal cancer (mCRC). The phase 3 PARADIGM trial (n = 802) demonstrated longer overall survival with first-line anti-EGFR (panitumumab) versus antivascular endothelial growth factor (bevacizumab) plus modified FOLFOX6 in patients with RAS wild-type mCRC with left-sided primary tumors. This prespecified exploratory biomarker analysis of PARADIGM (n = 733) evaluated the association between circulating tumor DNA (ctDNA) gene alterations and efficacy outcomes, focusing on a broad panel of gene alterations associated with resistance to EGFR inhibition, including KRAS, NRAS, PTEN and extracellular domain EGFR mutations, HER2 and MET amplifications, and ALK, RET and NTRK1 fusions. Overall survival was prolonged with panitumumab plus modified FOLFOX6 versus bevacizumab plus modified FOLFOX6 in patients with ctDNA that lacked gene alterations in the panel (that is, negative hyperselected; median in the overall population: 40.7 versus 34.4 months; hazard ratio, 0.76; 95% confidence interval, 0.62-0.92) but was similar or inferior with panitumumab in patients with ctDNA that contained any gene alteration in the panel (19.2 versus 22.2 months; hazard ratio, 1.13; 95% confidence interval, 0.83-1.53), regardless of tumor sidedness. Negative hyperselection using ctDNA may guide optimal treatment selection in patients with mCRC. ClinicalTrials.gov registrations: NCT02394834 and NCT02394795 .

Patient survey on cancer genomic medicine in Japan under the national health insurance system.

In Japan, comprehensive genomic profiling (CGP) tests have been reimbursed under the national health care system for solid cancer patients who have finished standard treatment. More than 50,000 patients have taken the test since June 2019. We performed a nation-wide questionnaire survey between March 2021 and July 2022. Questionnaires were sent to 80 designated Cancer Genomic Medicine Hospitals. Of the 933 responses received, 370 (39.7%) were web based and 563 (60.3%) were paper based. Most patients (784, 84%) first learned about CGP tests from healthcare professionals, and 775 (83.1%) gave informed consent to their treating physician. At the time of informed consent, they were most worried about test results not leading to novel treatment (536, 57.4%). On a scale of 0-10, 702 respondents (75.2%) felt that the explanations of the test result were easy to understand (7 or higher). Ninety-one patients (9.8%) started their recommended treatment. Many patients could not receive recommended treatment because no approved drugs or clinical trials were available (102/177, 57.6%). Ninety-eight patients (10.5%) did not wish their findings to be disclosed. Overall satisfaction with the CGP test process was high, with 602 respondents (64.5%) giving a score of 7-10. The major reason for choosing 0-6 was that the CGP test result did not lead to new treatment (217/277, 78.3%). In conclusion, satisfaction with the CGP test process was high. Patients and family members need better access to information. More patients need to be treated with genomically matched therapy.

Metabolic Hallmarks for Purine Nucleotide Biosynthesis in Small Cell Lung Carcinoma.

Small cell lung cancer (SCLC) has a poor prognosis, emphasizing the necessity for developing new therapies. The de novo synthesis pathway of purine nucleotides, which is involved in the malignant growth of SCLC, has emerged as a novel therapeutic target. Purine nucleotides are supplied by two pathways: de novo and salvage. However, the role of the salvage pathway in SCLC and the differences in utilization and crosstalk between the two pathways remain largely unclear. Here, we found that deletion of the HPRT1 gene, which codes for the rate-limiting enzyme of the purine salvage pathway, significantly suppressed tumor growth in vivo in several SCLC cells. We also demonstrated that HPRT1 expression confers resistance to lemetrexol (LMX), an inhibitor of the purine de novo pathway. Interestingly, HPRT1-knockout had less effect on SCLC SBC-5 cells, which are more sensitive to LMX than other SCLC cell lines, suggesting that a preference for either the purine de novo or salvage pathway occurs in SCLC. Furthermore, metabolome analysis of HPRT1-knockout cells revealed increased intermediates in the pentose phosphate pathway and elevated metabolic flux in the purine de novo pathway, indicating compensated metabolism between the de novo and salvage pathways in purine nucleotide biosynthesis. These results suggest that HPRT1 has therapeutic implications in SCLC and provide fundamental insights into the regulation of purine nucleotide biosynthesis. IMPLICATIONS: SCLC tumors preferentially utilize either the de novo or salvage pathway in purine nucleotide biosynthesis, and HPRT1 has therapeutic implications in SCLC.

A Learning Program for Treatment Recommendations by Molecular Tumor Boards and Artificial Intelligence.

Importance: Substantial heterogeneity exists in treatment recommendations across molecular tumor boards (MTBs), especially for biomarkers with low evidence levels; therefore, the learning program is essential. Objective: To determine whether a learning program sharing treatment recommendations for biomarkers with low evidence levels contributes to the standardization of MTBs and to investigate the efficacy of an artificial intelligence (AI)-based annotation system. Design, Setting, and Participants: This prospective quality improvement study used 50 simulated cases to assess concordance of treatment recommendations between a central committee and participants. Forty-seven participants applied from April 7 to May 13, 2021. Fifty simulated cases were randomly divided into prelearning and postlearning evaluation groups to assess similar concordance based on previous investigations. Participants included MTBs at hub hospitals, treating physicians at core hospitals, and AI systems. Each participant made treatment recommendations for each prelearning case from registration to June 30, 2021; participated in the learning program on July 18, 2021; and made treatment recommendations for each postlearning case from August 3 to September 30, 2021. Data were analyzed from September 2 to December 10, 2021. Exposures: The learning program shared the methodology of making appropriate treatment recommendations, especially for biomarkers with low evidence levels. Main Outcomes and Measures: The primary end point was the proportion of MTBs that met prespecified accreditation criteria for postlearning evaluations (approximately 90% concordance with high evidence levels and approximately 40% with low evidence levels). Key secondary end points were chronological enhancements in the concordance of treatment recommendations on postlearning evaluations from prelearning evaluations. Concordance of treatment recommendations by an AI system was an exploratory end point. Results: Of the 47 participants who applied, 42 were eligible. The accreditation rate of the MTBs was 55.6% (95% CI, 35.3%-74.5%; P < .001). Concordance in MTBs increased from 58.7% (95% CI, 52.8%-64.4%) to 67.9% (95% CI, 61.0%-74.1%) (odds ratio, 1.40 [95% CI, 1.06-1.86]; P = .02). In postlearning evaluations, the concordance of treatment recommendations by the AI system was significantly higher than that of MTBs (88.0% [95% CI, 68.7%-96.1%]; P = .03). Conclusions and Relevance: The findings of this quality improvement study suggest that use of a learning program improved the concordance of treatment recommendations provided by MTBs to central ones. Treatment recommendations made by an AI system showed higher concordance than that for MTBs, indicating the potential clinical utility of the AI system.

Comprehensive single-cell analysis demonstrates radiotherapy-induced infiltration of macrophages expressing immunosuppressive genes into tumor in esophageal squamous cell carcinoma.

Radiotherapy (RT) combined with immunotherapy is promising; however, the immune response signature in the clinical setting after RT remains unclear. Here, by integrative spatial and single-cell analyses using multiplex immunostaining (CODEX), spatial transcriptome (VISIUM), and single-cell RNA sequencing, we substantiated the infiltration of immune cells into tumors with dynamic changes in immunostimulatory and immunosuppressive gene expression after RT. In addition, our comprehensive analysis uncovered time- and cell type-dependent alterations in the gene expression profile after RT. Furthermore, myeloid cells showed prominent up-regulation of immune response-associated genes after RT. Notably, a subset of infiltrating tumor-associated myeloid cells showing PD-L1 positivity exhibited significant up-regulation of immunostimulatory (HMGB1 and ISG15), immunosuppressive (SIRPA and IDO1), and protumor genes (CXCL8, CCL3, IL-6, and IL-1AB), which can be targets of immunotherapy in combination with PD-L1. These datasets will provide information on the RT-induced gene signature to seek an appropriate target for personalized immunotherapy combined with RT and guide the timing of combination therapy.

Japanese Society of Medical Oncology/Japan Society of Clinical Oncology/Japanese Society of Pediatric Hematology/Oncology-led clinical recommendations on the diagnosis and use of immunotherapy in patients with DNA mismatch repair deficient (dMMR) tumors, third edition.

BACKGROUND: Clinical trials have reported the efficacy of immune checkpoint inhibitors in the treatment of mismatch repair-deficient (dMMR) advanced solid tumors. The accumulated evidence of tumor agnostic agent has been made since PD-1 inhibitor was approved and used in clinical practice. Therefore, we have revised the guideline "Japan Society of Clinical Oncology provisional clinical opinion for the diagnosis and use of immunotherapy in patients with deficient DNA mismatch repair tumors, cooperated by Japanese Society of Medical Oncology, First Edition". METHODS: Clinical questions regarding medical care were formulated for patients with dMMR advanced solid tumors. Relevant publications were searched by PubMed and Cochrane Database. Critical publications and conference reports were added manually. Systematic reviews were performed for each clinical question for the purpose of developing clinical recommendations. The committee members identified by Japan Society of Clinical Oncology (JSCO), Japanese Society of Medical Oncology (JSMO), and Japanese society of pediatric hematology/oncology (JSPHO) voted to determine the level of each recommendation considering the strength of evidence, expected risks and benefits to patients, and other related factors. Thereafter, a peer review by experts nominated from JSCO, JSMO, and JSPHO and the public comments among all societies' members were done. RESULTS: The current guideline describes two clinical questions and eight recommendations for whom, when, and how MMR status should be tested. CONCLUSION: In this guideline, the committee proposed eight recommendations for performing MMR testing properly to select patients who are likely to benefit from immunotherapy.

The prognostic impact of a high number of peritumoral alveolar macrophages in neuroendocrine carcinoma in the lung.

Alveolar macrophages (AMs) are resident macrophages in the lungs; however, whether the number of AMs plays a role in the lung neuroendocrine tumor (NET) prognosis remains unclear. We counted the number of AMs located around the tumor (peritumoral alveolar macrophages [pAMs]) and the number of AMs located apart from the tumor (distant macrophages; dAMs). In 73 cases of neuroendocrine carcinoma (NEC: small cell lung carcinoma and large cell neuroendocrine carcinoma), the group that contained higher pAMs (≥86/µm2 ) revealed shorter recurrent-free survival (RFS) than those with lower pAMs (<86/µm2 ) (p = 0.005). Bivariate analysis showed that the number of pAMs was an independent predictor of a poor RFS. In contrast, in the carcinoid tumor cohort (n = 29), there was no statistically significant correlation between the two groups with high and low numbers of pAMs in RFS (p = 0.113). Furthermore, we examined the correlation between genomic alterations and the number of pAMs in NEC, but no significant correlation was observed. In conclusion, the number of pAMs is a prognostic factor for NEC in the lung and pAMs may contribute to tumor progression within the peritumoral microenvironment.

Transposable elements potentiate radiotherapy-induced cellular immune reactions via RIG-I-mediated virus-sensing pathways.

Radiotherapy (RT) plus immunotherapy is a promising modality; however, the therapeutic effects are insufficient, and the molecular mechanism requires clarification to further develop combination therapies. Here, we found that the RNA virus sensor pathway dominantly regulates the cellular immune response in NSCLC and ESCC cell lines. Notably, transposable elements (TEs), especially long terminal repeats (LTRs), functioned as key ligands for the RNA virus sensor RIG-I, and the mTOR-LTR-RIG-I axis induced the cellular immune response and dendritic cell and macrophage infiltration after irradiation. Moreover, RIG-I-dependent immune activation was observed in ESCC patient tissue. scRNA sequencing and spatial transcriptome analysis revealed that radiotherapy induced the expression of LTRs, and the RNA virus sensor pathway in immune and cancer cells; this pathway was also found to mediate tumour conversion to an immunological hot state. Here, we report the upstream and ligand of the RNA virus sensor pathway functions in irradiated cancer tissues.

Japanese Society of Medical Oncology/Japan Society of Clinical Oncology/Japanese Society of Pediatric Hematology/Oncology-led clinical recommendations on the diagnosis and use of immunotherapy in patients with high tumor mutational burden tumors.

The development of novel antitumor agents and accompanying biomarkers has improved survival across several tumor types. Previously, we developed recommendations for tumor-agnostic treatments in patients with solid tumors with DNA mismatch repair deficient or neurotrophic receptor tyrosine kinase fusions. Recently, immune checkpoint inhibitors have shown efficacy in patient with tumor mutation burden-high (TMB-H) solid tumors and have been established as a third tumor-agnostic agent, making it necessary to develop the guideline prioritized for these patients. Clinical questions regarding medical care were formulated for patients with TMB-H advanced solid tumors. Relevant publications were searched by PubMed and Cochrane Database. Critical publications and conference reports were added manually. Systematic reviews were performed for each clinical question for the purpose of developing clinical recommendations. The committee members identified by Japan Society of Clinical Oncology (JSCO), Japanese Society of Medical Oncology (JSMO), and Japanese society of pediatric hematology/oncology (JSPHO) voted to determine the level of each recommendation considering the strength of evidence, expected risks and benefits to patients, and other related factors. Thereafter, a peer review by experts nominated from JSCO, JSMO, and JSPHO, and the public comments among all societies' members was done. The current guideline describes three clinical questions and seven recommendations for whom, when, and how TMB should be tested, and what is recommended for patients with TMB-H advanced solid tumors. In this guideline, the committee proposed seven recommendations for performing TMB testing properly to select patients who are likely to benefit from immunotherapy.

Human resources for administrative work to carry out a comprehensive genomic profiling test in Japan.

Comprehensive genomic profiling (CGP) tests have been nationally reimbursed in Japan since June 2019 under strict restrictions, and over 46,000 patients have taken the test. Core Hospitals and Designated Hospitals host molecular tumor boards, which is more time-consuming than simply participating in them. We sent a questionnaire to government-designated Cancer Genomic Medicine Hospitals, including all 12 Core Hospitals, all 33 Designated Hospitals, and 117 of 188 Cooperative Hospitals. The questionnaire asked how much time physicians and nonphysicians spent on administrative work for cancer genomic medicine. For every CGP test, 7.6 h of administrative work was needed. Physicians spent 2.7 h/patient, while nonphysicians spent 4.9 h/patient. Time spent preparing for molecular tumor boards, called Expert Panels, was the longest, followed by time spent participating in Expert Panels. Assuming an hourly wage of ¥24,000/h for physicians and ¥2800/h for nonphysicians, mean labor cost was ¥78,071/patient. On a monthly basis, more time was spent on administrative work at Core Hospitals compared with Designated Hospitals and Cooperative Hospitals (385 vs. 166 vs. 51 h/month, respectively, p < 0.001). Consequently, labor cost per month was higher at Core Hospitals than at Designated Hospitals and Cooperative Hospitals (¥3,951,854 vs. ¥1,687,167 vs. ¥487,279/month, respectively, p < 0.001). Completing a CGP test for a cancer patient in Japan is associated with significant labor at each hospital, especially at Core Hospitals. Streamlining the exchange of information and simplifying Expert Panels will likely alleviate this burden.

Japanese Society of Medical Oncology/Japan Society of Clinical Oncology/Japanese Society of Pediatric Hematology/Oncology-led clinical recommendations on the diagnosis and use of tropomyosin receptor kinase inhibitors in adult and pediatric patients with neurotrophic receptor tyrosine kinase fusion-positive advanced solid tumors.

BACKGROUND: Clinical trials have reported the efficacy of tropomyosin receptor kinase (TRK) inhibitors against neurotrophic receptor tyrosine kinase (NTRK) fusion gene-positive advanced solid tumors. The accumulated evidence of tumor-agnostic agent has made since TRK inhibitors were approved and used in clinical practice. Therefore, we have revised the 'Japan Society of Clinical Oncology (JSCO)/Japanese Society of Medical Oncology (JSMO)-led clinical recommendations on the diagnosis and use of tropomyosin receptor kinase inhibitors in adult and pediatric patients with neurotrophic receptor tyrosine kinase fusion-positive advanced solid tumors, cooperated by the Japanese Society of Pediatric Hematology/Oncology (JSPHO)'. METHODS: Clinical questions regarding medical care were formulated for patients with NTRK fusion-positive advanced solid tumors. Relevant publications were searched by PubMed and Cochrane Database. Critical publications and conference reports were added manually. Systematic reviews were performed for each clinical question for the purpose of developing clinical recommendations. The committee members identified by JSCO, JSMO, and JSPHO voted to determine the level of each recommendation considering the strength of evidence, expected risks and benefits to patients, and other related factors. Thereafter, a peer review by experts nominated from JSCO, JSMO, and JSPHO, and the public comments among all societies' members was done. RESULTS: The current guideline describes 3 clinical questions and 14 recommendations for whom, when, and how NTRK fusion should be tested, and what is recommended for patients with NTRK fusion-positive advanced solid tumors. CONCLUSION: The committee proposed 14 recommendations for performing NTRK testing properly to select patients who are likely to benefit from TRK inhibitors.

Panitumumab vs Bevacizumab Added to Standard First-line Chemotherapy and Overall Survival Among Patients With RAS Wild-type, Left-Sided Metastatic Colorectal Cancer: A Randomized Clinical Trial.

Importance: For patients with RAS wild-type metastatic colorectal cancer, adding anti-epidermal growth factor receptor (anti-EGFR) or anti-vascular endothelial growth factor (anti-VEGF) monoclonal antibodies to first-line doublet chemotherapy is routine, but the optimal targeted therapy has not been defined. Objective: To evaluate the effect of adding panitumumab (an anti-EGFR monoclonal antibody) vs bevacizumab (an anti-VEGF monoclonal antibody) to standard first-line chemotherapy for treatment of RAS wild-type, left-sided, metastatic colorectal cancer. Design, Setting, and Participants: Randomized, open-label, phase 3 clinical trial at 197 sites in Japan in May 2015-January 2022 among 823 patients with chemotherapy-naive RAS wild-type, unresectable metastatic colorectal cancer (final follow-up, January 14, 2022). Interventions: Panitumumab (n = 411) or bevacizumab (n = 412) plus modified fluorouracil, l-leucovorin, and oxaliplatin (mFOLFOX6) every 14 days. Main Outcomes and Measures: The primary end point, overall survival, was tested first in participants with left-sided tumors, then in the overall population. Secondary end points were progression-free survival, response rate, duration of response, and curative (defined as R0 status) resection rate. Results: In the as-treated population (n = 802; median age, 66 years; 282 [35.2%] women), 604 (75.3%) had left-sided tumors. Median follow-up was 61 months. Median overall survival was 37.9 months with panitumumab vs 34.3 months with bevacizumab in participants with left-sided tumors (hazard ratio [HR] for death, 0.82; 95.798% CI, 0.68-0.99; P = .03) and 36.2 vs 31.3 months, respectively, in the overall population (HR, 0.84; 95% CI, 0.72-0.98; P = .03). Median progression-free survival for panitumumab vs bevacizumab was 13.1 vs 11.9 months, respectively, for those with left-sided tumors (HR, 1.00; 95% CI, 0.83-1.20) and 12.2 vs 11.4 months overall (HR, 1.05; 95% CI, 0.90-1.24). Response rates with panitumumab vs bevacizumab were 80.2% vs 68.6%, respectively, for left-sided tumors (difference, 11.2%; 95% CI, 4.4%-17.9%) and 74.9% vs 67.3% overall (difference, 7.7%; 95% CI, 1.5%-13.8%). Median duration of response with panitumumab vs bevacizumab was 13.1 vs 11.2 months for left-sided tumors (HR, 0.86; 95% CI, 0.70-1.10) and 11.9 vs 10.7 months overall (HR, 0.89; 95% CI, 0.74-1.06). Curative resection rates with panitumumab vs bevacizumab were 18.3% vs 11.6% for left-sided tumors; (difference, 6.6%; 95% CI, 1.0%-12.3%) and 16.5% vs 10.9% overall (difference, 5.6%; 95% CI, 1.0%-10.3%). Common treatment-emergent adverse events were acneiform rash (panitumumab: 74.8%; bevacizumab: 3.2%), peripheral sensory neuropathy (panitumumab: 70.8%; bevacizumab: 73.7%), and stomatitis (panitumumab: 61.6%; bevacizumab: 40.5%). Conclusions and Relevance: Among patients with RAS wild-type metastatic colorectal cancer, adding panitumumab, compared with bevacizumab, to standard first-line chemotherapy significantly improved overall survival in those with left-sided tumors and in the overall population. Trial Registration: ClinicalTrials.gov Identifier: NCT02394795.

Regulation of MEK inhibitor selumetinib sensitivity by AKT phosphorylation in the novel BRAF L525R mutant.

BACKGROUND: Oncogenic mutations in BRAF genes are found in approximately 5-10% of colorectal cancers. The majority of BRAF mutations are located within exons 11-15 of the catalytic kinase domains, with BRAF V600E accounting for more than 80% of the observed BRAF mutations. Sensitivity to BRAF- and mitogen-activated protein kinase (MEK) inhibitors varies depending on BRAF mutations and tumor cell types. Previously, we newly identified, BRAF L525R-mutation, in the activation segment of the kinase in colorectal cancer patient. Here, we characterized the function of the BRAF L525R mutation. METHODS: HEK293 cells harboring a BRAF mutation (V600E or L525R) were first characterized and then treated with cetuximab, dabrafenib, and selumetinib. Cell viability was measured using WST-1 assay and the expression of proteins involved in the extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signaling pathways was evaluated using western blot analysis. RESULTS: The MEK inhibitor selumetinib effectively inhibited cell proliferation and ERK phosphorylation in BRAF L525R cells but not in BRAF V600E cells. Further studies revealed that AKT phosphorylation was reduced by selumetinib in BRAF L525R cells but not in BRAF V600E cells or selumetinib-resistant BRAF L525R cells. Moreover, the AKT inhibitor overcame the selumetinib resistance. CONCLUSIONS: We established a model system harboring BRAF L525R using HEK293 cells. BRAF L525R constitutively activated ERK. AKT phosphorylation caused sensitivity and resistance to selumetinib. Our results suggest that a comprehensive network analysis may provide insights to identify effective therapies.

Efficacy of Targeted Trials and Signaling Pathway Landscape in Advanced Gastrointestinal Cancers From SCRUM-Japan GI-SCREEN: A Nationwide Genomic Profiling Program.

PURPOSE: Genomic profiling programs have been implemented to apply next-generation sequencing (NGS) for facilitating trial enrollment. SCRUM-Japan GI-SCREEN is a large-scale genomic profiling program in advanced gastrointestinal cancers using a validated genomic assay with the goal of facilitating enrollment in targeted clinical trials, generating real-world data, and performing clinicogenomic analysis for biomarker discovery. PATIENTS AND METHODS: Genotyping of tumor tissue samples from 5,743 patients with advanced gastrointestinal cancers enrolled in GI-SCREEN was centrally performed with NGS. Patients were enrolled in matched trials of targeted agents affiliated with GI-SCREEN on the basis of genotyping results. RESULTS: A total of 11 gastrointestinal cancers were included, with colorectal cancer being the most common. The median age ranged from 59 to 70.5 years across cancer types. Patients enrolled after initiation of first-line treatment had significantly longer overall survival (OS) than that before treatment initiation with a median survival time difference of 8.9 months and a hazard ratio (HR) ranging from 0.25 to 0.73 across cancer types, demonstrating an immortal time bias. One hundred and forty-nine patients received matched therapies in clinical trials on the basis of their identified alterations. Among patients with colorectal cancer harboring actionable alterations, the median OS was significantly longer in patients who received matched therapies in trials than in those who did not (HR, 0.52; 95% CI, 0.26 to 1.01; P = .049). Cancer-specific pathway alterations were significantly associated with shorter survival and related to primary resistance to matched trial therapies. CONCLUSION: Our genomic profiling program led to patient enrollment in targeted clinical trials and improved survival of patients with colorectal cancer who received matched therapies in clinical trials. To avoid immortal time bias, precautions are needed when using data from patients who have undergone NGS testing after initiation of the evaluated treatment line.

Classification and characterization of alternative promoters in 26 lung adenocarcinoma cell lines.

BACKGROUND: Genome-wide landscape of alternative promoter use remains unknown. We determined expression profiles of promoters in 26 lung adenocarcinoma cell lines using the transcriptional start site-sequencing data and proposed an index 'canonical promoter usage' to quantify the diversity of alternative promoter usage. METHODS: Transcriptional start site-sequencing and other datasets were obtained from the DataBase of Transcriptional Start Sites. Transcriptional start site-sequencing read clusters were mapped onto RefGene to determine the promoters. Commonly used promoters were designated as canonical promoters. The sequence logos, CpG islands, DNA methylation and histone modifications of canonical and non-canonical promoters were examined. Canonical promoter usage was calculated by dividing 'read counts of a canonical promoter' by 'read counts of all the units of promoters' on each gene. The expressed genes were subjected to hierarchical clustering according to their canonical promoter usage. RESULTS: Among 104 455 promoters for 14 297 genes, 8659 canonical and 68 197 non-canonical promoters were identified. Corresponding to higher expression, canonical promoters showed core promoter sequences, higher CpG island positivity, less DNA methylation and higher transcription-promoting histone modifications. Gene ontology enrichment analysis revealed that the clusters with lower canonical promoter usage were related to signalling pathways, whereas clusters of tightly regulated genes with higher canonical promoter usage were related to housekeeping genes. CONCLUSION: Canonical promoters were regulated by conventional transcriptional machinery, while non-canonical promoters would be targets of 'leaky' expression. Further investigation is warranted to analyse the correlation between alternative promoter usage and biological characteristics contributing to carcinogenesis.

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.

The impact of tertiary lymphoid structures on clinicopathological, genetic and gene expression characteristics in lung adenocarcinoma.

INTRODUCTION: Tertiary lymphoid structures (TLS) are observed in several cancers and are associated with favorable prognosis. This study aimed to examine the clinicopathological, genetic, and gene expression profiles of lung adenocarcinoma patients with TLS. METHODS: A total of 112 patients with pathological stage IB lung adenocarcinoma who underwent complete resection between 2011 and 2015 were enrolled in this study. We investigated whether TLS correlated with prognosis and programmed death-ligand 1 (PD-L1) expression. Furthermore, the correlation of TLS with tumor mutation burden (TMB) and genetic mutations was evaluated in patients for whom whole-exon sequencing data were available. In addition, using the Cancer Genome Atlas Lung Adenocarcinoma (TCGA-LUAD) dataset, gene expression analysis according to the TLS status was performed. RESULTS: Among the 112 patients, 49 were TLS-positive (TLS+). TLS+ correlated with longer recurrence-free survival (RFS) than TLS-negative cases (TLS-) (hazard ratio [HR], 0.47; 95 % confidence interval [CI]: 0.23-0.88, p = 0.02). In the multivariate analysis, TLS was a better independent prognostic factor for RFS (HR 0.37, 95 %CI 0.18-0.72, p < 0.01). PD-L1 expression was not significantly different between TLS+ and TLS- patients (p = 0.54). TMB in TLS+ was similar to that in TLS- patients (p = 0.39); however, it tended to be lower than that in TLS- especially among smokers (p = 0.07). In gene expression analysis, RNA expression of chemokines related to lymph node formation, such as CXCL13, CCL19 and CCL21, was significantly higher, and biological processes such as positive regulation of humoral immune response and regulation of antigen receptor-mediated signaling pathway were enhanced in TLS+. CONCLUSIONS: TLS was a favorable prognostic factor and was not associated with PD-L1 expression in patients with lung adenocarcainoma. Moreover, gene expression analysis indicated that TLS is a site for the generation and regulation of antitumor immune responses.

Fecal microbiota in patients with a stoma decreases anaerobic bacteria and alters taxonomic and functional diversities.

Colorectal cancer (CRC) is one of the most common malignant diseases. Generally, stoma construction is performed following surgery for the resection of the primary tumor in patients with CRC. The association of CRC with the gut microbiota has been widely reported, and the gut microbiota is known to play an important role in the carcinogenesis, progression, and treatment of CRC. In this study, we compared the microbiota of patients with CRC between with and without a stoma using fecal metagenomic sequencing data from SCRUM-Japan MONSTAR-SCREEN, a joint industry-academia cancer research project in Japan. We found that the composition of anaerobes was reduced in patients with a stoma. In particular, the abundance of Alistipes, Akkermansia, Intestinimonas, and methane-producing archaea decreased. We also compared gene function (e.g., KEGG Orthology and KEGG pathway) and found that gene function for methane and short-chain fatty acids (SCFAs) production was underrepresented in patients with a stoma. Furthermore, a stoma decreased Shannon diversity based on taxonomic composition but increased that of the KEGG pathway. These results suggest that the feces of patients with a stoma have a reduced abundance of favorable microbes for cancer immunotherapy. In conclusion, we showed that a stoma alters the taxonomic and functional profiles in feces and may be a confounding factor in fecal microbiota analysis.