The PD-1 expression balance between effector and regulatory T cells predicts the clinical efficacy of PD-1 blockade therapies.

Immune checkpoint blockade has provided a paradigm shift in cancer therapy, but the success of this approach is very variable; therefore, biomarkers predictive of clinical efficacy are urgently required. Here, we show that the frequency of PD-1+CD8+ T cells relative to that of PD-1+ regulatory T (Treg) cells in the tumor microenvironment can predict the clinical efficacy of programmed cell death protein 1 (PD-1) blockade therapies and is superior to other predictors, including PD ligand 1 (PD-L1) expression or tumor mutational burden. PD-1 expression by CD8+ T cells and Treg cells negatively impacts effector and immunosuppressive functions, respectively. PD-1 blockade induces both recovery of dysfunctional PD-1+CD8+ T cells and enhanced PD-1+ Treg cell-mediated immunosuppression. A profound reactivation of effector PD-1+CD8+ T cells rather than PD-1+ Treg cells by PD-1 blockade is necessary for tumor regression. These findings provide a promising predictive biomarker for PD-1 blockade therapies.

An Oncogenic Alteration Creates a Microenvironment that Promotes Tumor Progression by Conferring a Metabolic Advantage to Regulatory T Cells.

Only a small percentage of patients afflicted with gastric cancer (GC) respond to immune checkpoint blockade (ICB). To study the mechanisms underlying this resistance, we examined the immune landscape of GC. A subset of these tumors was characterized by high frequencies of regulatory T (Treg) cells and low numbers of effector T cells. Genomic analyses revealed that these tumors bore mutations in RHOA that are known to drive tumor progression. RHOA mutations in cancer cells activated the PI3K-AKT-mTOR signaling pathway, increasing production of free fatty acids that are more effectively consumed by Treg cells than effector T cells. RHOA mutant tumors were resistant to PD-1 blockade but responded to combination of PD-1 blockade with inhibitors of the PI3K pathway or therapies targeting Treg cells. We propose that the metabolic advantage conferred by RHOA mutations enables Treg cell accumulation within GC tumors, generating an immunosuppressive TME that underlies resistance to ICB.

Two novel high-risk adult B-cell acute lymphoblastic leukemia subtypes with high expression of CDX2 and IDH1/2 mutations.

The genetic basis of leukemogenesis in adults with B-cell acute lymphoblastic leukemia (B-ALL) is largely unclear, and its clinical outcome remains unsatisfactory. This study aimed to advance the understanding of biological characteristics, improve disease stratification, and identify molecular targets of adult B-ALL. Adolescents and young adults (AYA) (15 to 39 years old, n = 193) and adults (40 to 64 years old, n = 161) with Philadelphia chromosome-negative (Ph-) B-ALL were included in this study. Integrated transcriptomic and genetic analyses were used to classify the cohort into defined subtypes. Of the 323 cases included in the RNA sequencing analysis, 278 (86.1%) were classified into 18 subtypes. The ZNF384 subtype (22.6%) was the most prevalent, with 2 novel subtypes (CDX2-high and IDH1/2-mut) identified among cases not assigned to the established subtypes. The CDX2-high subtype (3.4%) was characterized by high expression of CDX2 and recurrent gain of chromosome 1q. The IDH1/2-mut subtype (1.9%) was defined by IDH1 R132C or IDH2 R140Q mutations with specific transcriptional and high-methylation profiles. Both subtypes showed poor prognosis and were considered inferior prognostic factors independent of clinical parameters. Comparison with a previously reported pediatric B-ALL cohort (n = 1003) showed that the frequencies of these subtypes were significantly higher in AYA/adults than in children. We delineated the genetic and transcriptomic landscape of adult B-ALL and identified 2 novel subtypes that predict poor disease outcomes. Our findings highlight the age-dependent distribution of subtypes, which partially accounts for the prognostic differences between adult and pediatric B-ALL.

The development of a custom RNA-sequencing panel for the identification of predictive and diagnostic biomarkers in glioma.

PURPOSE: Various molecular profiles are needed to classify malignant brain tumors, including gliomas, based on the latest classification criteria of the World Health Organization, and their poor prognosis necessitates new therapeutic targets. The Todai OncoPanel 2 RNA Panel (TOP2-RNA) is a custom-target RNA-sequencing (RNA-seq) using the junction capture method to maximize the sensitivity of detecting 455 fusion gene transcripts and analyze the expression profiles of 1,390 genes. This study aimed to classify gliomas and identify their molecular targets using TOP2-RNA. METHODS: A total of 124 frozen samples of malignant gliomas were subjected to TOP2-RNA for classification based on their molecular profiles and the identification of molecular targets. RESULTS: Among 55 glioblastoma cases, gene fusions were detected in 11 cases (20%), including novel MET fusions. Seven tyrosine kinase genes were found to be overexpressed in 15 cases (27.3%). In contrast to isocitrate dehydrogenase (IDH) wild-type glioblastoma, IDH-mutant tumors, including astrocytomas and oligodendrogliomas, barely harbor fusion genes or gene overexpression. Of the 34 overexpressed tyrosine kinase genes, MDM2 and CDK4 in glioblastoma, 22 copy number amplifications (64.7%) were observed. When comparing astrocytomas and oligodendrogliomas in gene set enrichment analysis, the gene sets related to 1p36 and 19q were highly enriched in astrocytomas, suggesting that regional genomic DNA copy number alterations can be evaluated by gene expression analysis. CONCLUSIONS: TOP2-RNA is a highly sensitive assay for detecting fusion genes, exon skipping, and aberrant gene expression. Alterations in targetable driver genes were identified in more than 50% of glioblastoma. Molecular profiling by TOP2-RNA provides ample predictive, prognostic, and diagnostic biomarkers that may not be identified by conventional assays and, therefore, is expected to increase treatment options for individual patients with glioma.

Dramatic response to entrectinib in a patient with malignant peripheral nerve sheath tumor harboring novel SNRNP70-NTRK3 fusion gene.

Neurotropic tropomyosin receptor kinase (NTRK) gene rearrangements have been reported in limited cases of sarcomas; however, to date, there has been only one report of such rearrangements in malignant peripheral nerve sheath tumors (MPNSTs). Herein, we describe a 51-year-old male patient with a buttock tumor arising from the sciatic nerve, which was diagnosed as MPNST with positive S-100 staining, negative SOX10 staining, and loss of trimethylation at lysine 27 of histone H3 (H3K27me3) confirmed by immunohistochemistry. Soon after the resection of the primary tumor, the patient was found to have pulmonary and lymph node metastases. Chemotherapy with eribulin and trabectedin showed limited effects. However, the patient responded rapidly to pazopanib, but severe side effects caused discontinuation of the treatment. RNA panel testing revealed a novel fusion gene between Small Nuclear Ribonucleoprotein U1 Subunit 70 (SNRNP70) gene and NTRK3 gene. Furthermore, loss of NF1, SUZ12, and CDKN2A genes was confirmed by DNA panel testing, which is compatible with a histological diagnosis of MPNST. SNRNP70 possesses a coiled-coiled domain and seems to induce constitutive activation of NTRK3 through dimerization. In fact, immunohistochemistry revealed diffuse staining of pan-TRK within tumor cells. Treatment with entrectinib, which is an NTRK inhibitor, showed a quick and durable response for 10 months. Although NTRK rearrangements are very rare in MPNST, this case highlights the importance of genetic testing in MPNST, especially using an RNA panel for the detection of rare fusion genes.

Genome Doubling Shapes High-Grade Transformation and Novel EWSR1::LARP4 Fusion Shows SOX10 Immunostaining in Hyalinizing Clear Cell Carcinoma of Salivary Gland.

Hyalinizing clear cell carcinoma (HCCC) is a rare indolent malignant tumor of minor salivary gland origin with EWSR1::ATF1 rearrangement. Pathologically, the tumor cells possess a clear cytoplasm in a background of hyalinized stroma. Generally, the tumor cells are positive for p63 and p40 and negative for s100 and α-smooth muscle actin, suggesting that they differentiate into squamous epithelium and not into myoepithelium. In this study, we performed a detailed histopathological and genomic analysis of 6 cases of HCCC, including 2 atypical subtypes-a case of "high-grade transformation" and 1 "possessing a novel partner gene for EWSR1." We performed a sequential analysis of the primary and recurrent tumor by whole-exome sequencing, RNA sequencing, Sanger sequencing, and fluorescence in situ hybridization to investigate the effect of genomic changes on histopathology and clinical prognosis. A fusion gene involving the EWSR1 gene was detected in all cases. Five cases, including the "high-grade transformation," harbored a known EWSR1::ATF1 fusion gene; however, 1 case harbored a novel EWSR1::LARP4 fusion gene. This novel EWSR1::LARP4-fused HCCC has a SOX10-positive staining, which is different from the EWSR1::ATF1-fused HCCC. According to whole-exome sequencing and fluorescence in situ hybridization analysis, the "whole-genome doubling" and focal deletion involving CDKN2A, CDKN2B, and PTEN were detected in HCCC with "high-grade transformation." Conclusively, we identified a novel partner gene for EWSR1, LARP4, in indolent HCCC. Importantly, "high-grade transformation" and poor prognosis were caused by whole-genome doubling and subsequent genomic aberrations.

Extensive analysis of 59 sarcoma-related fusion genes identified pazopanib as a potential inhibitor to COL1A1-PDGFB fusion gene.

Sarcomas are malignant mesenchymal tumors that are extremely rare and divergent. Fusion genes are involved in approximately 30% of sarcomas as driver oncogenes; however, their detailed functions are not fully understood. In this study, we determined the functional significance of 59 sarcoma-related fusion genes. The transforming potential and drug sensitivities of these fusion genes were evaluated using a focus formation assay (FFA) and the mixed-all-nominated-in-one (MANO) method, respectively. The transcriptome was also examined using RNA sequencing of 3T3 cells transduced with each fusion gene. Approximately half (28/59, 47%) of the fusion genes exhibited transformation in the FFA assay, which was classified into five types based on the resulting phenotype. The sensitivity to 12 drugs including multityrosine kinase inhibitors was assessed using the MANO method and pazopanib was found to be more effective against cells expressing the COL1A1-PDGFB fusion gene compared with the others. The downstream MAPK/AKT pathway was suppressed at the protein level following pazopanib treatment. The fusion genes were classified into four subgroups by cluster analysis of the gene expression data and gene set enrichment analysis. In summary, the oncogenicity and drug sensitivity of 59 fusion genes were simultaneously evaluated using a high-throughput strategy. Pazopanib was selected as a candidate drug for sarcomas harboring the COL1A1-PDGFB fusion gene. This assessment could be useful as a screening platform and provides a database to evaluate customized therapy for fusion gene-associated sarcomas.

Cells with stem-like properties are associated with the development of HPV18-positive cervical cancer.

The cellular origins of cervical cancer and the histological differentiation of human papillomavirus (HPV)-infected cells remain unexplained. To gain new insights into the carcinogenesis and histological differentiation of HPV-associated cervical cancer, we focused on cervical cancer with mixed histological types. We conducted genomic and transcriptomic analyses of cervical cancers with mixed histological types. The commonality of the cellular origins of these cancers was inferred using phylogenetic analysis and by assessing the HPV integration sites. Carcinogenesis was estimated by analyzing human gene expression profiles in different histological types. Among 42 cervical cancers with known HPV types, mixed histological types were detected in four cases, and three of them were HPV18-positive. Phylogenetic analysis of these three cases revealed that the different histological types had a common cell of origin. Moreover, the HPV-derived transcriptome and HPV integration sites were common among different histological types, suggesting that HPV integration could occur before differentiation into each histological type. Human gene expression profiles indicated that HPV18-positive cancer retained immunologically cold components with stem cell properties. Mixed cervical cancer has a common cellular origin among different histological types, and progenitor cells with stem-like properties may be associated with the development of HPV18-positive cervical cancer.

The pathogenic role of the BRCA2 c.7847C>T (p.Ser2616Phe) variant in breast and ovarian cancer predisposition.

Substantial numbers of variants of unknown significance (VUSs) have been identified in BRCA1/2 through genetic testing, which poses a significant clinical challenge because the contribution of these VUSs to cancer predisposition has not yet been determined. Here, we report 10 Japanese patients from seven families with breast or ovarian cancer harboring the BRCA2 c.7847C>T (p.Ser2616Phe) variant that was interpreted as a VUS. This variant recurs only in families from Japan and has not been reported in the global general population databases. A Japanese patient with Fanconi anemia with compound heterozygous variants c.7847C>T (p.Ser2616Phe) and c.475+1G>A in BRCA2 was reported. In silico predictions and quantitative cosegregation analysis suggest a high probability of pathogenicity. The clinical features of the variant carriers were not specific to, but were consistent with, those of patients with hereditary breast and ovarian cancer. A validated functional assay, called the mixed-all-nominated-in-one-BRCA (MANO-B) method and the accurate BRCA companion diagnostic (ABCD) test, demonstrated the deleterious effects of the variant. Altogether, following the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines, this variant satisfied the "PS3," "PM2," "PM3," and "PP3" criteria. We thus conclude that the BRCA2 c.7847C>T (p.Ser2616Phe) variant is a "likely pathogenic" variant that is specifically observed in the Japanese population, leading to a breast and ovarian cancer predisposition.

Molecular pathology quality control in Southeast Asia: Results of a multiregional quality assurance study from MASTER KEY Asia.

Tissue specimen quality assurance is a major issue of precision medicine for rare cancers. However, the laboratory standards and quality of pathological specimens prepared in Asian hospitals remain unknown. To understand the methods in Southeast Asian oncology hospitals and to clarify how pre-analytics affect the quality of formalin-fixed paraffin-embedded (FFPE) specimens, a questionnaire surveying pre-analytical procedures (Part I) was administered, quality assessment of immunohistochemistry (IHC) staining and DNA/RNA extracted from the representative FFPE specimens from each hospital (Part II) was conducted, and the quality of DNA/RNA extracted from FFPE of rare-cancer patients for genomic sequencing (Part III) was examined. Quality measurements for DNA/RNA included ΔΔCt, DV200, and cDNA yield. Six major cancer hospitals from Malaysia, Philippines, and Vietnam participated. One hospital showed unacceptable quality for the DNA/RNA assessment, but improved by revising laboratory procedures. Only 57% (n = 73) of the 128 rare-cancer patients' specimens met both DNA and RNA quality criteria for next-generation sequencing. Median DV200 was 80.7% and 64.3% for qualified and failed RNA, respectively. Median ΔΔCt was 1.25 for qualified and 4.89 for failed DNA. Longer storage period was significantly associated with poor DNA (fail to qualify ratio = 1579:321 days, p < 0.001) and RNA (fail to qualify ratio = 1070:280 days, p < 0.001). After improvement of pre-analytical factors, the qualification rate increased for hospitals A and E from 41.5% to 70.5% and 62.5% to 86%, respectively. This is the first report to elucidate the pre-analytical laboratory procedures of main Southeast Asian oncology hospitals. An external quality assessment program may improve factors associated with tumor FFPE specimen quality.

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).

Somatic mutations can induce a noninflamed tumour microenvironment via their original gene functions, despite deriving neoantigens.

BACKGROUND: Identifying biomarkers to predict immune checkpoint inhibitor (ICI) efficacy is warranted. Considering that somatic mutation-derived neoantigens induce strong immune responses, patients with a high tumour mutational burden reportedly tend to respond to ICIs. However, there are several conflicting data. Therefore, we focused on the original function of neoantigenic mutations and their impact on the tumour microenvironment (TME). METHODS: We evaluated 88 high-frequency microsatellite instability (MSI-H) colorectal cancers and analysed the function of the identified neoantigenic mutations and their influence on programmed cell death 1 (PD-1) blockade efficacy. The results were validated using The Cancer Genome Atlas (TCGA) datasets. RESULTS: We identified frameshift mutations in RNF43 as a common neoantigenic gene mutation in MSI-H tumours. However, loss-of-function RNF43 mutations induced noninflamed TME by activating the WNT/ß-catenin signalling pathway. In addition, loss of RNF43 function induced resistance to PD-1 blockade even in neoantigen-rich tumours. TCGA dataset analyses demonstrated that passenger rather than driver gene mutations were related to the inflamed TME in diverse cancer types. CONCLUSIONS: We propose a novel concept of "paradoxical neoantigenic mutations" that can induce noninflamed TME through their original gene functions, despite deriving neoantigens, suggesting the significance of qualities as well as quantities in neoantigenic mutations.

Subclonal accumulation of immune escape mechanisms in microsatellite instability-high colorectal cancers.

BACKGROUND: Intratumor heterogeneity (ITH) in microsatellite instability-high (MSI-H) colorectal cancer (CRC) has been poorly studied. We aimed to clarify how the ITH of MSI-H CRCs is generated in cancer evolution and how immune selective pressure affects ITH. METHODS: We reanalyzed public whole-exome sequencing data on 246 MSI-H CRCs. In addition, we performed a multi-region analysis from 6 MSI-H CRCs. To verify the process of subclonal immune escape accumulation, a novel computational model of cancer evolution under immune pressure was developed. RESULTS: Our analysis presented the enrichment of functional genomic alterations in antigen-presentation machinery (APM). Associative analysis of neoantigens indicated the generation of immune escape mechanisms via HLA alterations. Multiregion analysis revealed the clonal acquisition of driver mutations and subclonal accumulation of APM defects in MSI-H CRCs. Examination of variant allele frequencies demonstrated that subclonal mutations tend to be subjected to selective sweep. Computational simulations of tumour progression with the interaction of immune cells successfully verified the subclonal accumulation of immune escape mutations and suggested the efficacy of early initiation of an immune checkpoint inhibitor (ICI) -based treatment. CONCLUSIONS: Our results demonstrate the heterogeneous acquisition of immune escape mechanisms in MSI-H CRCs by Darwinian selection, providing novel insights into ICI-based treatment strategies.

HLA Class I Analysis Provides Insight Into the Genetic and Epigenetic Background of Immune Evasion in Colorectal Cancer With High Microsatellite Instability.

BACKGROUND & AIMS: A detailed understanding of antitumor immunity is essential for optimal cancer immune therapy. Although defective mutations in the B2M and HLA-ABC genes, which encode molecules essential for antigen presentation, have been reported in several studies, the effects of these defects on tumor immunity have not been quantitatively evaluated. METHODS: Mutations in HLA-ABC genes were analyzed in 114 microsatellite instability-high colorectal cancers using a long-read sequencer. The data were further analyzed in combination with whole-exome sequencing, transcriptome sequencing, DNA methylation array, and immunohistochemistry data. RESULTS: We detected 101 truncating mutations in 57 tumors (50%) and loss of 61 alleles in 21 tumors (18%). Based on the integrated analysis that enabled the immunologic subclassification of microsatellite instability-high colorectal cancers, we identified a subtype of tumors in which lymphocyte infiltration was reduced, partly due to reduced expression of HLA-ABC genes in the absence of apparent genetic alterations. Survival time of patients with such tumors was shorter than in patients with other tumor types. Paradoxically, tumor mutation burden was highest in the subtype, suggesting that the immunogenic effect of accumulating mutations was counterbalanced by mutations that weakened immunoreactivity. Various genetic and epigenetic alterations, including frameshift mutations in RFX5 and promoter methylation of PSMB8 and HLA-A, converged on reduced expression of HLA-ABC genes. CONCLUSIONS: Our detailed immunogenomic analysis provides information that will facilitate the improvement and development of cancer immunotherapy.

Optimization of microplate-based phenol-sulfuric acid method and application to the multi-sample measurements of cellulose nanofibers.

The phenol-sulfuric acid (PSA) method is a widely used colorimetric method for determining the total saccharides. Microplate-based PSA methods have been developed to handle a large number of samples and reduce the use of hazardous chemicals. However, the optimal procedures and measurement conditions for this method have not yet been fully established. To address this gap, we investigated the optimal procedure for microplate-based PSA. In addition to glucose (Glc), two types of cellulose nanofibers (CNFs) were also evaluated as they are a new type of nanomaterial, and a technique to quantify the concentration of CNFs is required in their safety assessment. The results showed that the thermal reaction with sulfuric acid before the addition of phenol resulted in a higher coloration than was shown after the addition of phenol. Furthermore, the longer the resting time after shaking with phenol, the greater the coloration and smaller the variation, with a resting time of 60 min or longer being optimal. This research provides valuable insights into improving the reliability and efficiency of the PSA method, which can facilitate the analysis of saccharides and other substances in a range of applications.

A new era of the Asian clinical research network: a report from the ATLAS international symposium.

This report summarizes the presentations and discussions in the first Asian Clinical Trials Network for Cancers (ATLAS) international symposium that was held on 24 April 2022, in Bangkok, Thailand, and hosted by the National Cancer Center Hospital (NCCH), co-hosted by the Pharmaceuticals and Medical Devices Agency (PMDA), Clinical Research Malaysia (CRM) and the Thai Society of Clinical Oncology (TSCO), and supported by Embassy of Japan in Thailand. Since 2020, the NCCH has conducted the ATLAS project to enhance research environments and infrastructures to facilitate international clinical research and cancer genomic medicine in the Asian region. The purpose of the symposium was to discuss what we can achieve under the ATLAS project, to share the latest topics and common issues in cancer research and to facilitate mutual understanding. Invitees included stakeholders from academic institutions, mainly at ATLAS collaborative sites, as well as Asian regulatory authorities. The invited speakers discussed ongoing collaborative research, regulatory perspectives to improve new drug access in Asia, the status of phase I trials in Asia, the introduction of research activities at the National Cancer Center (NCC) and the implementation of genomic medicine. As the next steps after this symposium, the ATLAS project will foster increased cooperation between investigators, regulatory authorities and other stakeholders relevant to cancer research, and establish a sustainable pan-Asian cancer research group to increase the number of clinical trials and deliver novel drugs to patients with cancer in Asia.

Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy.

Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase, a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking phosphoribosylformylglycinamidine synthase or phosphoribosyl pyrophosphate amidotransferase, another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mammalian target of rapamycin complex 1 (mTORC1) suppression and was profoundly suppressed in cells deficient for tuberous sclerosis complex 2, which negatively regulates mTORC1 through inhibition of Ras homolog enriched in brain, suggesting that purines regulate autophagy through the tuberous sclerosis complex-Ras homolog enriched in brain-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase and dihydroorotate dehydrogenase activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy.

Feasibility and clinical utility of comprehensive genomic profiling of hematological malignancies.

Identification of genetic alterations through next-generation sequencing (NGS) can guide treatment decision-making by providing information on diagnosis, therapy selection, and prognostic stratification in patients with hematological malignancies. Although the utility of NGS-based genomic profiling assays was investigated in hematological malignancies, no assays sufficiently cover driver mutations, including recently discovered ones, as well as fusions and/or pathogenic germline variants. To address these issues, here we have devised an integrated DNA/RNA profiling assay to detect various types of somatic alterations and germline variants at once. Particularly, our assay can successfully identify copy number alterations and structural variations, including immunoglobulin heavy chain translocations, IKZF1 intragenic deletions, and rare fusions. Using this assay, we conducted a prospective study to investigate the feasibility and clinical usefulness of comprehensive genomic profiling for 452 recurrently altered genes in hematological malignancies. In total, 176 patients (with 188 specimens) were analyzed, in which at least one alteration was detected in 171 (97%) patients, with a median number of total alterations of 7 (0-55). Among them, 145 (82%), 86 (49%), and 102 (58%) patients harbored at least one clinically relevant alteration for diagnosis, treatment, and prognosis, respectively. The proportion of patients with clinically relevant alterations was the highest in acute myeloid leukemia, whereas this assay was less informative in T/natural killer-cell lymphoma. These results suggest the clinical utility of NGS-based genomic profiling, particularly for their diagnosis and prognostic prediction, thereby highlighting the promise of precision medicine in hematological malignancies.

Tumor mutational burden measurement using comprehensive genomic profiling assay.

BACKGROUND: Tumors with a high number of mutations in the genome, or tumor mutational burden, are presumed to be more likely to respond to immune checkpoint inhibitors. However, the optimal method to calculate tumor mutational burden using comprehensive genomic profiling assays is unknown. METHODS: Todai OncoPanel is a dual panel of a deoxyribonucleic acid panel and a ribonucleic acid panel. Todai OncoPanel deoxyribonucleic acid panel version 6 is an improvement over version 3 with increased number of targeted genes and limited targeting of intronic regions. We calculated tumor mutational burden measured by Todai OncoPanel deoxyribonucleic acid panel versions 3 and 6 using three different calculation methods: all mutations within the targeted region (target tumor mutational burden), all mutations within the coding region (all coding tumor mutational burden) and non-synonymous mutations (non-synonymous coding tumor mutational burden). We then compared them with whole exosome sequencing tumor mutational burden. In addition, 16 lung cancer patients whose samples were analyzed using Todai OncoPanel deoxyribonucleic acid version 3 were treated with anti-PD-1 or PD-L1 antibody monotherapy. RESULTS: When compared with whole exosome sequencing tumor mutational burden as the standard, tumor mutational burden measured by Todai OncoPanel deoxyribonucleic acid version 3 resulted in accuracy of 71% for all three calculation methods. In version 6, accuracy was 96% for target tumor mutational burden and all coding tumor mutational burden and 91% for non-synonymous coding tumor mutational burden. Patients with either partial response or stable disease had higher non-synonymous coding tumor mutational burden (6.7/Mb vs. 1.6/Mb, P = 0.02) and higher PD-L1 expression (40% vs. 3%, P = 0.01) and a trend toward higher target tumor mutational burden (9.2/Mb vs. 2.4/Mb, P = 0.09) compared with patients with progressive disease. CONCLUSIONS: Increase in targeted gene number and limiting intronic regions improved tumor mutational burden measurement by Todai OncoPanel when compared with whole exosome sequencing tumor mutational burden. Target tumor mutational burden may be the method of choice to measure tumor mutational burden.

Development of an optimal protocol for molecular profiling of tumor cells in pleural effusions at single-cell level.

Liquid biopsy analyzes the current status of primary tumors and their metastatic regions. We aimed to develop an optimized protocol for single-cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test. FTCs were enriched using a negative selection of white blood cells by a magnetic-activated cell sorting system, and CD45-negative and cytokeratin-positive selection using a microfluidic cell separation system with a dielectrophoretic array. The enriched tumor cells were subjected to whole-genome amplification (WGA) followed by genome sequencing. The FTC analysis detected an EGFR exon 19 deletion in Case 1 (12/19 cells, 63.2%), and EML4-ALK fusion (17/20 cells, 85%) with an alectinib-resistant mutation of ALK (p.G1202R) in Case 2. To eliminate WGA-associated errors and increase the uniformity of the WGA product, the protocol was revised to sequence multiple single FTCs individually. An analytical pipeline, accurate single-cell mutation detector (ASMD), was developed to identify somatic mutations of FTCs. The large numbers of WGA-associated errors were cleaned up, and the somatic mutations detected in FTCs by ASMD were concordant with those found in tissue specimens. This protocol is applicable to circulating tumor cells analysis of peripheral blood and expands the possibility of utilizing molecular profiling of cancers.