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.

mTORC1 and muscle regeneration are regulated by the LINC00961-encoded SPAR polypeptide.

Although long non-coding RNAs (lncRNAs) are non-protein-coding transcripts by definition, recent studies have shown that a fraction of putative small open reading frames within lncRNAs are translated. However, the biological significance of these hidden polypeptides is still unclear. Here we identify and functionally characterize a novel polypeptide encoded by the lncRNA LINC00961. This polypeptide is conserved between human and mouse, is localized to the late endosome/lysosome and interacts with the lysosomal v-ATPase to negatively regulate mTORC1 activation. This regulation of mTORC1 is specific to activation of mTORC1 by amino acid stimulation, rather than by growth factors. Hence, we termed this polypeptide 'small regulatory polypeptide of amino acid response' (SPAR). We show that the SPAR-encoding lncRNA is highly expressed in a subset of tissues and use CRISPR/Cas9 engineering to develop a SPAR-polypeptide-specific knockout mouse while maintaining expression of the host lncRNA. We find that the SPAR-encoding lncRNA is downregulated in skeletal muscle upon acute injury, and using this in vivo model we establish that SPAR downregulation enables efficient activation of mTORC1 and promotes muscle regeneration. Our data provide a mechanism by which mTORC1 activation may be finely regulated in a tissue-specific manner in response to injury, and a paradigm by which lncRNAs encoding small polypeptides can modulate general biological pathways and processes to facilitate tissue-specific requirements, consistent with their restricted and highly regulated expression profile.

Impacts of the STING-IFNAR1-STAT1-IRF1 pathway on the cellular immune reaction induced by fractionated irradiation.

Radiotherapy (RT) combined with immune checkpoint inhibitors has recently produced outstanding results and is expected to be adaptable for various cancers. However, the precise molecular mechanism by which immune reactions are induced by fractionated RT is still controversial. We aimed to investigate the mechanism of the immune response regarding multifractionated, long-term radiation, which is most often combined with immunotherapy. Two human esophageal cancer cell lines, KYSE-450 and OE-21, were irradiated by fractionated irradiation (FIR) daily at a dose of 3 Gy in 5 d/wk for 2 weeks. Western blot analysis and RNA sequencing identified type I interferon (IFN) and the stimulator of IFN genes (STING) pathway as candidates that regulate immune response by FIR. We inhibited STING, IFNAR1, STAT1, and IFN regulatory factor 1 (IRF1) and investigated the effects on the immune response in cancer cells and the invasion of surrounding immune cells. We herein revealed type I IFN-dependent immune reactions and the positive feedback of STING, IRF1, and phosphorylated STAT1 induced by FIR. Knocking out STING, IFNAR1, STAT1, and IRF1 resulted in a poorer immunological response than that in WT cells. The STING-KO KYSE-450 cell line showed significantly less invasion of PBMCs than the WT cell line under FIR. In the analysis of STING-KO cells and migrated PBMCs, we confirmed the occurrence of STING-dependent immune activation under FIR. In conclusion, we identified that the STING-IFNAR1-STAT1-IRF1 axis regulates immune reactions in cancer cells triggered by FIR and that the STING pathway also contributes to immune cell invasion of cancer cells.

Comprehensive screening for drugs that modify radiation-induced immune responses.

BACKGROUND: Combination therapy based on radiotherapy and immune checkpoint inhibitors (ICIs) was recently reported as effective for various cancers. The radiation-induced immune response (RIIR) is an essential feature in ICI-combined radiotherapy; however, the effects of drugs used concomitantly with RIIR remain unclear. We screened for drugs that can modify RIIR to understand the mutual relationship between radiotherapy and combined drugs in ICI-combined radiotherapy. METHODS: We established a high-throughput system with reporter gene assays for evaluating RIIR, focusing on factors acting downstream of the STING-IRF pathway, which can stimulate cancer cells, T cells, and dendritic cells. We further quantified the effects of 2595 drugs, including those approved by the Food and Drug Administration, on RIIR in vitro. RESULTS: The reporter assay results correlated well with the expression of immune response proteins such as programmed death-ligand 1. This high-throughput system enabled the identification of drugs including cytotoxic agents, molecular-targeted agents, and other agents that activate or suppress RIIR. CONCLUSIONS: Our study provides an encyclopedic catalogue of clinically approved drugs based on their effect on RIIR. In ICIs combined radiotherapy, activation of STING-IFN may improve the therapeutic effect and our result could form a biological basis for further clinical trials combining radiotherapy with ICIs.

Comparative analysis of the immune responses in cancer cells irradiated with X-ray, proton and carbon-ion beams.

Radiotherapy (RT) is an effective treatment option for cancer; however, its efficacy remains less than optimal in locally advanced cancer. Immune checkpoint inhibitor-based therapy, including the administration of anti-PD-L1 antibodies, is a promising approach that works synergistically with RT. Proton beam therapy and carbon-ion therapy are common options for patients with cancer. Proton and carbon ions are reported to induce an immune reaction in cancer cells; however, the underlying mechanisms remain unclear. Here, we aimed to compare the immune responses after irradiation (IR) with X-ray, protons, and carbon ions in an oesophageal cancer cell line and the underlying mechanisms. An oesophageal cancer cell line, KYSE450, was irradiated with 1 fraction/15 GyE (Gy equivalent) of X-ray, proton, or carbon-ion beams, and then, the cells were harvested for RNA sequencing and gene enrichment analysis. We also knocked out STING and STAT1 in the quest for mechanistic insights. RNA sequencing data revealed that gene expression signatures and biological processes were different in KYSE450 irradiated with X-ray, proton, and carbon-ion beams 6-24 h after IR. However, after 3 days, a common gene expression signature was detected, associated with biological pathways involved in innate immune responses. Gene knock-out experiments revealed that the STING-STAT1 axis underlies the immune reactions after IR. X-Ray, proton, and carbon-ion IRs induced similar immune responses, regulated by the STING-STAT1 axis.

T cell receptor stimulation-induced epigenetic changes and Foxp3 expression are independent and complementary events required for Treg cell development.

The transcription factor Foxp3 is essential for the development of regulatory T (Treg) cells, yet its expression is insufficient for establishing the Treg cell lineage. Here we showed that Treg cell development was achieved by the combination of two independent processes, i.e., the expression of Foxp3 and the establishment of Treg cell-specific CpG hypomethylation pattern. Both events were induced by T cell receptor stimulation. The Treg cell-type CpG hypomethylation began in the thymus and continued to proceed in the periphery and could be fully established without Foxp3. The hypomethylation was required for Foxp3(+) T cells to acquire Treg cell-type gene expression, lineage stability, and full suppressive activity. Thus, those T cells in which the two events have concurrently occurred are developmentally set into the Treg cell lineage. This model explains how Treg cell fate and plasticity is controlled and can be exploited to generate functionally stable Treg cells.

Gastric mesenchymal tumor with smooth muscle differentiation and echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion.

Gastric mesenchymal tumors are relatively rare, and their molecular pathogeneses are poorly understood, except for gastrointestinal stromal tumor, desmoid, and inflammatory myofibroblastic tumors. We report a case of a gastric mesenchymal tumor with prominent smooth muscle cell differentiation and an echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion. On gross section, the tumor was 26 mm at the largest diameter, well-circumscribed, and located in the submucosal and muscular layers of the stomach wall. Histologically, the tumor comprised intersecting fascicles of spindle cells, non-atypical nuclei, and highly eosinophilic cytoplasm. Myxoid changes were observed focally, but inflammatory infiltrates were only evident in limited areas. Immunochemical staining revealed that the tumor was positive for α-smooth muscle actin and desmin. Diffuse positive staining for h-caldesmon was observed throughout the tumor, which suggested smooth muscle cell differentiation. Intracytoplasmic staining for ALK protein was also observed, and fluorescence in situ hybridization using ALK break-apart probes showed split chromosomal signals. RNA-sequencing analysis identified EML4-ALK fusion transcripts. We concluded that the tumor was a gastric mesenchymal tumor with smooth muscle differentiation based on its distinct differential smooth muscle properties, such as highly eosinophilic cytoplasm and diffuse expression of h-caldesmon. Furthermore, activated ALK may underly the tumor's pathogenesis.

Multifocal origin of occupational cholangiocarcinoma revealed by comparison of multilesion mutational profiles.

Recently identified occupational cholangiocarcinoma among printing workers is characterized by chronic bile duct injuries and precancerous or early cancerous lesions at multiple sites of the bile ducts. These observations suggested the potential multifocal carcinogenesis of the disease. We performed whole-exome analysis of multiple lesions, including the invasive carcinomas and precancerous lesions of four occupational cholangiocarcinoma cases. A much higher mutation burden was observed in both the invasive carcinomas (mean 76.3/Mb) and precancerous lesions (mean 71.8/Mb) than in non-occupational cholangiocarcinomas (mean 1.6/Mb). Most somatic mutations identified in 11 of 16 lesions did not overlap with each other. In contrast, a unique trinucleotide mutational signature of GpCpY to GpTpY was shared among the lesions. These results suggest that most of these lesions are multiclonal in origin and that common mutagenic processes, which may be induced by exposure to haloalkanes or their metabolites, generated somatic mutations at different sites of the bile ducts. A similarly high mutation rate had already been identified in the precancerous lesions, implying an increased potential for carcinogenesis throughout the biliary tree. These genomic features support the importance of ongoing close follow-up of the patients as a group at high risk of recurrence.

Comparative sequence analysis of patient-matched primary colorectal cancer, metastatic, and recurrent metastatic tumors after adjuvant FOLFOX chemotherapy.

BACKGROUND: In the era of genome-guided personalized cancer treatment, we must understand chemotherapy-induced genomic changes in tumors. This study evaluated whether adjuvant FOLFOX chemotherapy modifies the mutational profile of recurrent colorectal cancer (CRC). METHODS: Whole exome sequencing was performed on samples from primary CRC tumors, untreated metastatic tumors, and recurrent tumors following adjuvant FOLFOX chemotherapy. The samples were resected from four patients. RESULTS: The number of mutations or the mutation spectrum in individual patients was nearly identical. Copy number variants persisted regardless of FOLFOX therapy administration. The genomic signature of oxaliplatin exposure (G > T/C > A, T > A/A > T) was not enriched after FOLFOX chemotherapy. Overlapping single nucleotide variants (SNVs) and indels remained in 26-65% of the patient-matched tumor samples. One patient harbored an AKT1 E17K mutation in the recurrent tumor, whereas PIK3CA E542K and E88Q mutations were detected in the primary and untreated metastatic tumor samples. Genes related to intracellular Ca2+ homeostasis were enriched among the genes uniquely mutated after FOLFOX chemotherapy. CONCLUSIONS: We found that the mutation rates, mutation spectrum, and copy number variants were nearly identical regardless of the administration of FOLFOX therapy in the four CRC cases. The mutational discordance between the patient-matched tumor samples is likely caused by tumor heterogeneity and chemotherapy-induced clonal selection. These findings might be useful as pilot data for larger studies to clarify the changes in the mutational landscape induced by adjuvant FOLFOX chemotherapy.

Biobank Establishment and Sample Management in the Tohoku Medical Megabank Project.

The Tohoku Medical Megabank biobank (TMM biobank) is the first major population-based biobank established in Japan. The TMM biobank was established based on two population cohorts and is a reconstruction program from the Great East Japan Earthquake and Tsunami of 2011. The biobank stores more than 3.4 million tubes of biospecimens and associated health and analytic data obtained from approximately 150,000 TMM cohort participants between May 2013 and December 2018, and the TMM biobank currently shares high-quality specimens and data. Various biospecimens, including peripheral and cord blood mononuclear cells, buffy coat, plasma, serum, urine, breast milk and saliva have been collected in the TMM biobank. To minimize human error and maintain the quality of data and specimens, we have been utilizing laboratory information management system into various biobank procedures from registration to storage with various automation systems, such as liquid dispensing, DNA extraction and their storage. The biobank procedures for the quality management system (ISO 9001:2015) and information security management system (ISO 27001:2013) are certified by the International Organization for Standardization. The quality of our biobank samples fulfills the pre-analytical requirements for researchers conducting next-generation whole genome sequencing, DNA array analyses, proteomics, metabolomics, etc. We established analytical centers to conduct standard genomic and multiomic analyses in-house and share the generated data. Additionally, we generate thousands of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and proliferating T cells for functional studies. The TMM biobank serves as an indispensable infrastructure for academic, clinical and industrial research to actualize next-generation medicine in Japan.

Evaluation of reported pathogenic variants and their frequencies in a Japanese population based on a whole-genome reference panel of 2049 individuals.

Clarifying allele frequencies of disease-related genetic variants in a population is important in genomic medicine; however, such data is not yet available for the Japanese population. To estimate frequencies of actionable pathogenic variants in the Japanese population, we examined the reported pathological variants in genes recommended by the American College of Medical Genetics and Genomics (ACMG) in our reference panel of genomic variations, 2KJPN, which was created by whole-genome sequencing of 2049 individuals of the resident cohort of the Tohoku Medical Megabank Project. We searched for pathogenic variants in 2KJPN for 57 autosomal ACMG-recommended genes responsible for 26 diseases and then examined their frequencies. By referring to public databases of pathogenic variations, we identified 143 reported pathogenic variants in 2KJPN for the 57 ACMG recommended genes based on a classification system. At the individual level, 21% of the individuals were found to have at least one reported pathogenic allele. We then conducted a literature survey to review the variants and to check for evidence of pathogenicity. Our results suggest that a substantial number of people have reported pathogenic alleles for the ACMG genes, and reviewing variants is indispensable for constructing the information infrastructure of genomic medicine for the Japanese population.

Collaborative environmental DNA sampling from petal surfaces of flowering cherry Cerasus × yedoensis 'Somei-yoshino' across the Japanese archipelago.

Recent studies have shown that environmental DNA is found almost everywhere. Flower petal surfaces are an attractive tissue to use for investigation of the dispersal of environmental DNA in nature as they are isolated from the external environment until the bud opens and only then can the petal surface accumulate environmental DNA. Here, we performed a crowdsourced experiment, the "Ohanami Project", to obtain environmental DNA samples from petal surfaces of Cerasus × yedoensis 'Somei-yoshino' across the Japanese archipelago during spring 2015. C. × yedoensis is the most popular garden cherry species in Japan and clones of this cultivar bloom simultaneously every spring. Data collection spanned almost every prefecture and totaled 577 DNA samples from 149 collaborators. Preliminary amplicon-sequencing analysis showed the rapid attachment of environmental DNA onto the petal surfaces. Notably, we found DNA of other common plant species in samples obtained from a wide distribution; this DNA likely originated from the pollen of the Japanese cedar. Our analysis supports our belief that petal surfaces after blossoming are a promising target to reveal the dynamics of environmental DNA in nature. The success of our experiment also shows that crowdsourced environmental DNA analyses have considerable value in ecological studies.

DBTSS as an integrative platform for transcriptome, epigenome and genome sequence variation data.

DBTSS (http://dbtss.hgc.jp/) was originally constructed as a collection of uniquely determined transcriptional start sites (TSSs) in humans and some other species in 2002. Since then, it has been regularly updated and in recent updates epigenetic information has also been incorporated because such information is useful for characterizing the biological relevance of these TSSs/downstream genes. In the newest release, Release 9, we further integrated public and original single nucleotide variation (SNV) data into our database. For our original data, we generated SNV data from genomic analyses of various cancer types, including 97 lung adenocarcinomas and 57 lung small cell carcinomas from Japanese patients as well as 26 cell lines of lung cancer origin. In addition, we obtained publically available SNV data from other cancer types and germline variations in total of 11,322 individuals. With these updates, users can examine the association between sequence variation pattern in clinical lung cancers with its corresponding TSS-seq, RNA-seq, ChIP-seq and BS-seq data. Consequently, DBTSS is no longer a mere storage site for TSS information but has evolved into an integrative platform of a variety of genome activity data.

MicroRNA-126-mediated control of cell fate in B-cell myeloid progenitors as a potential alternative to transcriptional factors.

Lineage specification is thought to be largely regulated at the level of transcription, where lineage-specific transcription factors drive specific cell fates. MicroRNAs (miR), vital to many cell functions, act posttranscriptionally to decrease the expression of target mRNAs. MLL-AF4 acute lymphocytic leukemia exhibits both myeloid and B-cell surface markers, suggesting that the transformed cells are B-cell myeloid progenitor cells. Through gain- and loss-of-function experiments, we demonstrated that microRNA 126 (miR-126) drives B-cell myeloid biphenotypic leukemia differentiation toward B cells without changing expression of E2A immunoglobulin enhancer-binding factor E12/E47 (E2A), early B-cell factor 1 (EBF1), or paired box protein 5, which are critical transcription factors in B-lymphopoiesis. Similar induction of B-cell differentiation by miR-126 was observed in normal hematopoietic cells in vitro and in vivo in uncommitted murine c-Kit(+)Sca1(+)Lineage(-) cells, with insulin regulatory subunit-1 acting as a target of miR-126. Importantly, in EBF1-deficient hematopoietic progenitor cells, which fail to differentiate into B cells, miR-126 significantly up-regulated B220, and induced the expression of B-cell genes, including recombination activating genes-1/2 and CD79a/b. These data suggest that miR-126 can at least partly rescue B-cell development independently of EBF1. These experiments show that miR-126 regulates myeloid vs. B-cell fate through an alternative machinery, establishing the critical role of miRNAs in the lineage specification of multipotent mammalian cells.

The Babesia bovis gene and promoter model: an update from full-length EST analysis.

BACKGROUND: Babesia bovis is an apicomplexan parasite that causes babesiosis in infected cattle. Genomes of pathogens contain promising information that can facilitate the development of methods for controlling infections. Although the genome of B. bovis is publically available, annotated gene models are not highly reliable prior to experimental validation. Therefore, we validated a preproposed gene model of B. bovis and extended the associated annotations on the basis of experimentally obtained full-length expressed sequence tags (ESTs). RESULTS: From in vitro cultured merozoites, 12,286 clones harboring full-length cDNAs were sequenced from both ends using the Sanger method, and 6,787 full-length cDNAs were assembled. These were then clustered, and a nonredundant referential data set of 2,115 full-length cDNA sequences was constructed. The comparison of the preproposed gene model with our data set identified 310 identical genes, 342 almost identical genes, 1,054 genes with potential structural inconsistencies, and 409 novel genes. The median length of 5' untranslated regions (UTRs) was 152 nt. Subsequently, we identified 4,086 transcription start sites (TSSs) and 2,023 transcriptionally active regions (TARs) by examining 5' ESTs. We identified ATGGGG and CCCCAT sites as consensus motifs in TARs that were distributed around -50 bp from TSSs. In addition, we found ACACA, TGTGT, and TATAT sites, which were distributed periodically around TSSs in cycles of approximately 150 bp. Moreover, related periodical distributions were not observed in mammalian promoter regions. CONCLUSIONS: The observations in this study indicate the utility of integrated bioinformatics and experimental data for improving genome annotations. In particular, full-length cDNAs with one-base resolution for TSSs enabled the identification of consensus motifs in promoter sequences and demonstrated clear distributions of identified motifs. These observations allowed the illustration of a model promoter composition, which supports the differences in transcriptional regulation frameworks between apicomplexan parasites and mammals.

Genome-wide characterization of transcriptional start sites in humans by integrative transcriptome analysis.

We performed a genome-wide analysis of transcriptional start sites (TSSs) in human genes by multifaceted use of a massively parallel sequencer. By analyzing 800 million sequences that were obtained from various types of transcriptome analyses, we characterized 140 million TSS tags in 12 human cell types. Despite the large number of TSS clusters (TSCs), the number of TSCs was observed to decrease sharply with increasing expression levels. Highly expressed TSCs exhibited several characteristic features: Nucleosome-seq analysis revealed highly ordered nucleosome structures, ChIP-seq analysis detected clear RNA polymerase II binding signals in their surrounding regions, evaluations of previously sequenced and newly shotgun-sequenced complete cDNA sequences showed that they encode preferable transcripts for protein translation, and RNA-seq analysis of polysome-incorporated RNAs yielded direct evidence that those transcripts are actually translated into proteins. We also demonstrate that integrative interpretation of transcriptome data is essential for the selection of putative alternative promoter TSCs, two of which also have protein consequences. Furthermore, discriminative chromatin features that separate TSCs at different expression levels were found for both genic TSCs and intergenic TSCs. The collected integrative information should provide a useful basis for future biological characterization of TSCs.

DBTSS: DataBase of Transcriptional Start Sites progress report in 2012.

To support transcriptional regulation studies, we have constructed DBTSS (DataBase of Transcriptional Start Sites), which contains exact positions of transcriptional start sites (TSSs), determined with our own technique named TSS-seq, in the genomes of various species. In its latest version, DBTSS covers the data of the majority of human adult and embryonic tissues: it now contains 418 million TSS tag sequences from 28 tissues/cell cultures. Moreover, we integrated a series of our own transcriptomic data, such as the RNA-seq data of subcellular-fractionated RNAs as well as the ChIP-seq data of histone modifications and the binding of RNA polymerase II/several transcription factors in cultured cell lines into our original TSS information. We also included several external epigenomic data, such as the chromatin map of the ENCODE project. We further associated our TSS information with public or original single-nucleotide variation (SNV) data, in order to identify SNVs in the regulatory regions. These data can be browsed in our new viewer, which supports versatile search conditions of users. We believe that our new DBTSS will be an invaluable resource for interpreting the differential uses of TSSs and for identifying human genetic variations that are associated with disordered transcriptional regulation. DBTSS can be accessed at http://dbtss.hgc.jp.

Targeted therapy guided by circulating tumor DNA analysis in advanced gastrointestinal tumors.

Although comprehensive genomic profiling has become standard in oncology for advanced solid tumors, the full potential of circulating tumor DNA (ctDNA)-based profiling in capturing tumor heterogeneity and guiding therapy selection remains underexploited, marked by a scarcity of evidence on its clinical impact and the assessment of intratumoral heterogeneity. The GOZILA study, a nationwide, prospective observational ctDNA profiling study, previously demonstrated higher clinical trial enrollment rates using liquid biopsy compared with tissue screening. This updated analysis of 4,037 patients further delineates the clinical utility of ctDNA profiling in advanced solid tumors, showcasing a significant enhancement in patient outcomes with a 24% match rate for targeted therapy. Patients treated with matched targeted therapy based on ctDNA profiling demonstrated significantly improved overall survival compared with those receiving unmatched therapy (hazard ratio, 0.54). Notably, biomarker clonality and adjusted plasma copy number were identified as predictors of therapeutic efficacy, reinforcing the value of ctDNA in reflecting tumor heterogeneity for precise treatment decisions. These new insights into the relationship between ctDNA characteristics and treatment outcomes advance our understanding beyond the initial enrollment benefits. Our findings advocate for the broader adoption of ctDNA-guided treatment, signifying an advancement in precision oncology and improving survival outcomes in advanced solid tumors.

Gut microbiome associated with PARP inhibitor efficacy in patients with ovarian cancer.

OBJECTIVE: To investigate an association between the gut microbiome and efficacy of poly(ADP-ribose) polymerase inhibitors (PARPi) in ovarian cancer. METHODS: This study conducted fecal microbiome analysis (16S rRNA gene sequencing) and circulating tumor DNA (ctDNA) profiling for ovarian cancer patients who underwent PARPi maintenance therapy. Fecal and blood samples were collected at the baseline and the progressive disease (PD) or last follow-up. The relative abundance of gut microbes and progression-free survival (PFS) were analyzed using linear discriminant analysis of effect size and the Cox proportional hazard model according to BRCA1/2 mutation (BRCA1/2mut) status detected by ctDNA sequencing. RESULTS: Baseline samples were available from 23 BRCA1/2mut-positive patients and 33 BRCA1/2mut-negative patients. The microbes enriched in the baseline samples with long PFS were Bifidobacterium, Roseburia, Dialister, Butyricicoccus, and Bilophila for BRCA1/2mut-positive patients and Phascolarctobacterium for BRCA1/2mut-negative patients. In multivariate analyses dividing patients by the median values of relative abundances, no bacteria were associated with PFS in BRCA1/2mut-positive patients, whereas high Phascolarctobacterium abundances (≥1.11%) was significantly associated with longer PFS in BRCA1/2mut-negative patients (median 14.0 vs. 5.9 months, hazard ratio=0.28; 95% confidence interval=0.11-0.69; p=0.014). In the last samples, the relative abundances of Phascolarctobacterium were significantly higher in patients without PD (n=5) than those with PD (n=15) (median 1.25% vs. 0.06%; p=0.016). CONCLUSION: High fecal composition of Phascolarctobacterium was associated with prolonged PFS in patients with BRCA1/2mut-negative ovarian cancer receiving PARPi therapy. Our results would provide new insights for future research.

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 .