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Introduction: Cutibacterium acnes (C. acnes) resides in various organs such as the skin, prostate, eye, nose, stomach, and intestine, indicating the possibility of extensive crosstalk between this bacterium and the human body. C. acnes strains are classified into three subspecies based on phylogenetics and distinguishable phenotypes. Among them, C. acnes subsp. defendens strains are characterized by anti-inflammatory features, raising expectations for their potential as future microbiome therapeutics. However, the heterogeneity of C. acnes subsp. defendens and its corresponding immunological functions have not been clearly addressed. Methods: The genetic diversity of the strains was assessed using single- and multi-locus sequence typing. Their immune-modulatory functions were evaluated in vitro using 2D and 3D assays with immune and epithelial cells. The anti-inflammatory effects were further confirmed in vivo using a rosacea-like mouse model. Comparative genomic and transcriptomic analyses were conducted to uncover mechanisms underlying the immunosuppressive activity of the strains. Results: We demonstrated that the newly isolated C. acnes subsp. defendens strains, exhibiting phenotypic heterogeneity, are distinctly clustered using single- and multi-locus sequence typing methods. These strains showed strong immune-regulatory functions in immune and epithelial cell-based 2D and 3D in vitro assays. Furthermore, their anti-inflammatory role was functionally confirmed in vivo using a rosacea-like mouse model, where they alleviated skin lesions characterized by hyperplasia and dermal inflammation. Comparative transcriptomics revealed that these strains may exert their immunosuppressive effects through the enhanced expression of acnecins and transcriptional variation in envelope stress regulators (specifically the two-component systems, CesSR homologs). Additionally, we propose that these C. acnes type II strains produce anti-inflammatory metabolites or peptides smaller than 3 kDa, which are associated with elevated pyrimidine and reduced L-arginine biosynthesis. Discussion: The newly isolated C. acnes subsp. defendens strains demonstrate significant anti-inflammatory properties both in vitro and in vivo , suggesting their potential as microbiome-based therapeutics. Their unique genomic and transcriptomic profiles, including the production of small bioactive compounds and specific transcriptomic patterns, underpin their immunosuppressive capabilities. These findings provide a foundation for developing novel treatments for inflammatory skin conditions, such as rosacea. [ABSTRACT FROM AUTHOR]

Background: N6-methyladenosine (m6A), is the most common modification found in mRNA and lncRNA in higher organisms and plays an important role in physiology and pathology. However, its role in pan-cancer has not been explored. Results: A total of 31 m6A modification regulators, including 12 writers, 2 erasers, and 17 readers are identified in the current study. The functional analysis of the regulators results in the enrichment of processes, primarily related to RNA modification and metabolism, and the PPI network reveals multiple interactions among the regulators. The mRNA expression analysis reveals a high expression for most of the regulators in pan-cancer. Most of the m6A regulators are found to be mutated across the cancers, with ZC3H13, VIRMA, and PRRC2A having a higher frequency rate. Significant correlations of the regulators with clinicopathological parameters, such as age, gender, tumor stage, and grade are identified in pan-cancer. The m6A regulators' expression is found to have significant positive correlations with the miRNAs in pan-cancer. The expression pattern of the m6A regulators is able to classify the tumors into different subclusters as well as into high- and low-risk groups. These tumor groups show differential patterns in terms of their immune cell infiltration, tumor stemness score, genomic heterogeneity score, expression of immune regulatory/checkpoint genes, and correlations between the regulators and the drugs. Conclusions: Our study provide a comprehensive overview of the functional roles, genetic and epigenetic alterations, and prognostic value of the RNA m6A regulators in pan-cancer. [ABSTRACT FROM AUTHOR]

Background: N6-methyladenosine (m6A), is the most common modification found in mRNA and lncRNA in higher organisms and plays an important role in physiology and pathology. However, its role in pan-cancer has not been explored. Results: A total of 31 m6A modification regulators, including 12 writers, 2 erasers, and 17 readers are identified in the current study. The functional analysis of the regulators results in the enrichment of processes, primarily related to RNA modification and metabolism, and the PPI network reveals multiple interactions among the regulators. The mRNA expression analysis reveals a high expression for most of the regulators in pan-cancer. Most of the m6A regulators are found to be mutated across the cancers, with ZC3H13, VIRMA, and PRRC2A having a higher frequency rate. Significant correlations of the regulators with clinicopathological parameters, such as age, gender, tumor stage, and grade are identified in pan-cancer. The m6A regulators’ expression is found to have significant positive correlations with the miRNAs in pan-cancer. The expression pattern of the m6A regulators is able to classify the tumors into different subclusters as well as into high- and low-risk groups. These tumor groups show differential patterns in terms of their immune cell infiltration, tumor stemness score, genomic heterogeneity score, expression of immune regulatory/checkpoint genes, and correlations between the regulators and the drugs. Conclusions: Our study provide a comprehensive overview of the functional roles, genetic and epigenetic alterations, and prognostic value of the RNA m6A regulators in pan-cancer.

Abstract Background Spatial chromatin structure is intricately linked with somatic aberrations, and somatic mutations of various cancer-related genes, termed co-mutations (CoMuts), occur in certain patterns during cancer initiation and progression. The functional mechanisms underlying these genetic events remain largely unclear in thyroid cancer (TC). With discrepant differentiation, papillary thyroid cancer (PTC) and anaplastic thyroid cancer (ATC) differ greatly in characteristics and prognosis. We aimed to reveal the spatial gene alterations and regulations between the two TC subtypes. Methods We systematically investigated and compared the spatial co-mutations between ATC (8305C), PTC (BCPAP and TPC-1), and normal thyroid cells (Nthy-ori-3–1). We constructed a framework integrating whole-genome sequencing (WGS), high-throughput chromosome conformation capture (Hi-C), and transcriptome sequencing, to systematically detect the associations between the somatic co-mutations of cancer-related genes, structural variations (SVs), copy number variations (CNVs), and high-order chromatin conformation. Results Spatial co-mutation hotspots were enriched around topologically associating domains (TADs) in TC. A common set of 227 boundaries were identified in both ATC and PTC, with significant overlaps between them. The spatial proximities of the co-mutated gene pairs in the two TC types were significantly greater than in the gene-level and overall backgrounds, and ATC cells had higher TAD contact frequency with CoMuts > 10 compared with PTC cells. Compared with normal thyroid cells, in ATC the number of the created novel three-dimensional chromatin structural domains increased by 10%, and the number of shifted TADs decreased by 7%. We found five TAD blocks with CoMut genes/events specific to ATC with certain mutations in genes including MAST-NSUN4, AM129B/TRUB2, COL5A1/PPP1R26, PPP1R26/GPSM1/CCDC183, and PRAC2/DLX4. For the majority of ATC and PTC cells, the HOXA10 and HIF2α signals close to the transcription start sites of CoMut genes within TADs were significantly stronger than those at the background. CNV breakpoints significantly overlapped with TAD boundaries in both TC subtypes. ATCs had more CNV losses overlapping with TAD boundaries, and noncoding SVs involved in intrachromosomal SVs, amplified inversions, and tandem duplication differed between ATC and PTC. TADs with short range were more abundant in ATC than PTC. More switches of A/B compartment types existed in ATC cells compared with PTC. Gene expression was significantly synchronized, and orchestrated by complex epigenetics and regulatory elements. Conclusion Chromatin interactions and gene alterations and regulations are largely heterogeneous in TC. CNVs and complex SVs may function in the TC genome by interplaying with TADs, and are largely different between ATC and PTC. Complexity of TC genomes, which are highly organized by 3D genome-wide interactions mediating mutational and structural variations and gene activation, may have been largely underappreciated. Our comprehensive analysis may provide key evidence and targets for more customized diagnosis and treatment of TC.

Background: Prostate cancer (PCa) ranks as the second most prevalent malignancy among males on a global scale. Accumulating evidence suggests that inflammation has an intricate relationship with tumorigenesis, tumor progression and tumor immune microenvironment. However, the overall impact of inflammation-related genes on the clinical prognosis and tumor immunity in PCa remains unclear. Methods: Machine learning methods were utilized to construct and validate a signature using The Cancer Genome Atlas (TCGA) for training, while the Memorial Sloan Kettering Cancer Center (MSKCC) and GSE70769 cohorts for independent validation. The efficacy of the signature in predicting outcomes and its clinical utility were assessed through a series of investigations encompassing in vitro experiments, survival analysis, and nomogram development. The association between the signature and precision medicine was explored via tumor immunity, genomic heterogeneity, therapeutic response, and molecular docking analyses, using bulk and single-cell RNA-sequencing data. Results: We identified 7 inflammation-related genes with prognostic significance and developed an inflammation-related prognostic signature (IRPS) with 6 genes. Furthermore, we demonstrated that both the IRPS and a nomogram integrating risk score and pathologic T stage exhibited excellent predictive ability for the survival outcomes in PCa patients. Moreover, the IRPS was found to be significantly associated with the tumor immune, genomic heterogeneity, therapeutic response, and drug selection. Conclusion: IRPS can serve as a reliable predictor for PCa patients. The signature may provide clinicians with valuable information on the efficacy of therapy and help personalize treatment for PCa patients.

Introduction: Macrophages are components of the innate immune system and can play an anti-tumor or pro-tumor role in the tumor microenvironment owing to their high heterogeneity and plasticity. Meanwhile, prostate cancer (PCa) is an immune-sensitive tumor, making it essential to investigate the value of macrophage-associated networks in its prognosis and treatment. Methods: Macrophage-related marker genes (MRMGs) were identified through the comprehensive analysis of single-cell sequencing data from GSE141445 and the impact of macrophages on PCa was evaluated using consensus clustering of MRMGs in the TCGA database. Subsequently, a macrophage-related marker gene prognostic signature (MRMGPS) was constructed by LASSO-Cox regression analysis and grouped based on the median risk score. The predictive ability of MRMGPS was verified by experiments, survival analysis, and nomogram in the TCGA cohort and GEO-Merged cohort. Additionally, immune landscape, genomic heterogeneity, tumor stemness, drug sensitivity, and molecular docking were conducted to explore the relationship between MRMGPS and the tumor immune microenvironment, therapeutic response, and drug selection. Results: We identified 307 MRMGs and verified that macrophages had a strong influence on the development and progression of PCa. Furthermore, we showed that the MRMGPS constructed with 9 genes and the predictive nomogram had excellent predictive ability in both the TCGA and GEO-Merged cohorts. More importantly, we also found the close relationship between MRMGPS and the tumor immune microenvironment, therapeutic response, and drug selection by multi-omics analysis. Discussion: Our study reveals the application value of MRMGPS in predicting the prognosis of PCa patients. It also provides a novel perspective and theoretical basis for immune research and drug choices for PCa. [ABSTRACT FROM AUTHOR]

Liver hepatocellular carcinoma (LIHC) is a prevalent and lethal malignancy with a complex molecular landscape. Fibrosis and ferroptosis are implicated in LIHC progression, yet their roles remain to be elucidated. The present study investigated the expression and prognostic significance of calcitonin receptor (CALCR), a gene that intersects the pathways of fibrosis and ferroptosis, across LIHC and other types of cancer. Data were obtained from The Cancer Genome Atlas and the Molecular Signatures Database. LIHC patients were classified into two clusters based on fibrosis-related gene expression using ConsensusClusterPlus. Single-sample gene set enrichment analysis was employed to quantify fibrosis and ferroptosis levels. Correlation, survival and nomogram analyses were performed to assess the prognostic value of CALCR. Additionally, single-cell RNA sequencing data from the Tumor Immune Single Cell Hub 2 (TISCH2) and pan-cancer analyses of genomic heterogeneity features were incorporated. The present study also identified a putative regulatory role for CALCR in LIHC cell migration, proliferation and apoptosis. CALCR was identified as a significant prognostic marker for LIHC. Patients with high CALCR expression exhibited shortened overall survival (OS) and disease-specific survival (DSS). Specifically, the hazard ratios (HRs) for OS and DSS were 1.76 [95% confidence interval (CI): 1.23=2.49) and 1.77 (95% CI: 1.13=2.78], respectively, with corresponding P-values of 0.002 for OS and 0.013 for DSS. Analyses of immune cell infiltration revealed a more complex immune environment in patients with low CALCR expression, suggesting differential responses to immunotherapy. Furthermore, in HepG-2 and HuH-7 cells, small interfering (si)-CALCR increased apoptosis while reducing proliferation and migration compared with si-negative control. CALCR serves as a significant prognostic biomarker for LIHC, influencing both molecular pathways and the immune landscape. Its expression is associated with improved survival outcomes and distinct genomic features, positioning it as a potential therapeutic target and predictor of immunotherapy efficacy. [ABSTRACT FROM AUTHOR]

Background: Spatial chromatin structure is intricately linked with somatic aberrations, and somatic mutations of various cancer-related genes, termed co-mutations (CoMuts), occur in certain patterns during cancer initiation and progression. The functional mechanisms underlying these genetic events remain largely unclear in thyroid cancer (TC). With discrepant differentiation, papillary thyroid cancer (PTC) and anaplastic thyroid cancer (ATC) differ greatly in characteristics and prognosis. We aimed to reveal the spatial gene alterations and regulations between the two TC subtypes. Methods: We systematically investigated and compared the spatial co-mutations between ATC (8305C), PTC (BCPAP and TPC-1), and normal thyroid cells (Nthy-ori-3–1). We constructed a framework integrating whole-genome sequencing (WGS), high-throughput chromosome conformation capture (Hi-C), and transcriptome sequencing, to systematically detect the associations between the somatic co-mutations of cancer-related genes, structural variations (SVs), copy number variations (CNVs), and high-order chromatin conformation. Results: Spatial co-mutation hotspots were enriched around topologically associating domains (TADs) in TC. A common set of 227 boundaries were identified in both ATC and PTC, with significant overlaps between them. The spatial proximities of the co-mutated gene pairs in the two TC types were significantly greater than in the gene-level and overall backgrounds, and ATC cells had higher TAD contact frequency with CoMuts > 10 compared with PTC cells. Compared with normal thyroid cells, in ATC the number of the created novel three-dimensional chromatin structural domains increased by 10%, and the number of shifted TADs decreased by 7%. We found five TAD blocks with CoMut genes/events specific to ATC with certain mutations in genes including MAST-NSUN4, AM129B/TRUB2, COL5A1/PPP1R26, PPP1R26/GPSM1/CCDC183, and PRAC2/DLX4. For the majority of ATC and PTC cells, the HOXA10 and HIF2α signals close to the transcription start sites of CoMut genes within TADs were significantly stronger than those at the background. CNV breakpoints significantly overlapped with TAD boundaries in both TC subtypes. ATCs had more CNV losses overlapping with TAD boundaries, and noncoding SVs involved in intrachromosomal SVs, amplified inversions, and tandem duplication differed between ATC and PTC. TADs with short range were more abundant in ATC than PTC. More switches of A/B compartment types existed in ATC cells compared with PTC. Gene expression was significantly synchronized, and orchestrated by complex epigenetics and regulatory elements. Conclusion: Chromatin interactions and gene alterations and regulations are largely heterogeneous in TC. CNVs and complex SVs may function in the TC genome by interplaying with TADs, and are largely different between ATC and PTC. Complexity of TC genomes, which are highly organized by 3D genome-wide interactions mediating mutational and structural variations and gene activation, may have been largely underappreciated. Our comprehensive analysis may provide key evidence and targets for more customized diagnosis and treatment of TC. [ABSTRACT FROM AUTHOR]

Liver metastases are common in pancreatic neuroendocrine tumors (PanNETs) patients and they are considered a poor prognostic marker. This study aims to analyze the spatiotemporal patterns of genomic variations between primary and metastatic tumors, and to identify the key related biomolecular pathways. We performed next-generation sequencing on paired tissue specimens of primary PanNETs (n = 11) and liver metastases (n = 12). Low genomic heterogeneity between primary PanNETs and liver metastases was observed. Genomic analysis provided evidence that polyclonal seeding is a prevalent event during metastatic progression, and may be associated with the progression-free survival. Besides this, copy number variations of BRCA1/BRCA2 seem to be associated with better prognosis. Pathways analysis showed that pathways in cancer, DNA repair, and cell cycle regulation-related pathways were significantly enriched in primary PanNETs and liver metastases. The study has shown a high concordance of gene mutations between the primary tumor and its metastases and the shared gene mutations may occur during oncogenesis and predates liver metastasis, suggesting an earlier onset of metastasis in patients with PanNETs, providing novel insight into genetic changes in metastatic tumors of PanNETs. [ABSTRACT FROM AUTHOR]

Reptin/RUVBL2 is involved in the remodeling of chromatin, DNA damage repair, and regulation of the cell cycle, all of which help to play essential roles in cancer. However, relevant pan-cancer analysis of Reptin is lacking. This study first investigated the potential oncogenic roles of Reptin and revealed a relationship between Reptin with clinicopathological characteristics and immune infiltration based on big data. Here, we showed that Reptin is overexpressed in many cancers. A significant association exists between the expression of Reptin and the prognosis of cancer cases. Reptin had a meaningful interaction with the immune infiltration of CD4+ Th1 cells and immune modulator genes in multiple cancer types. And negative correlation exists between Reptin and cancer-associated fibroblasts in BRCA, PRAD, TGCT, and THYM. A significant negative association exists between Reptin and regulatory T cells in TGCT and THCA. Moreover, Reptin is significantly associated with genomic heterogeneity, DNA mismatch repair genes, methyltransferase, and RNA modification genes in specific cancer types. Spliceosome, Hippo signaling pathway, DNA replication pathway, and acetyltransferase activity-associated functions were observed in the effect of Reptin on the tumor. This systematic analysis highlights Reptin as a vital cancer regulator among numerous genes and proved its potential prognosticator value and therapeutic target role for specific tumor types. [ABSTRACT FROM AUTHOR]

Reptin/RUVBL2 is involved in the remodeling of chromatin, DNA damage repair, and regulation of the cell cycle, all of which help to play essential roles in cancer. However, relevant pan-cancer analysis of Reptin is lacking. This study first investigated the potential oncogenic roles of Reptin and revealed a relationship between Reptin with clinicopathological characteristics and immune infiltration based on big data. Here, we showed that Reptin is overexpressed in many cancers. A significant association exists between the expression of Reptin and the prognosis of cancer cases. Reptin had a meaningful interaction with the immune infiltration of CD4+ Th1 cells and immune modulator genes in multiple cancer types. And negative correlation exists between Reptin and cancer-associated fibroblasts in BRCA, PRAD, TGCT, and THYM. A significant negative association exists between Reptin and regulatory T cells in TGCT and THCA. Moreover, Reptin is significantly associated with genomic heterogeneity, DNA mismatch repair genes, methyltransferase, and RNA modification genes in specific cancer types. Spliceosome, Hippo signaling pathway, DNA replication pathway, and acetyltransferase activity-associated functions were observed in the effect of Reptin on the tumor. This systematic analysis highlights Reptin as a vital cancer regulator among numerous genes and proved its potential prognosticator value and therapeutic target role for specific tumor types.

Abstract Bladder cancer (BC) is a heterogeneous disease that characterized by genomic instability and a high mutation rate. Heterogeneity in tumor may partially explain the diversity of responses to targeted therapies and the various clinical outcomes. A combination of cytology and cystoscopy is the standard methodology for BC diagnosis, prognosis, and disease surveillance. However, genomics analyses of single tumor‐biopsy specimens may underestimate the mutational burden of heterogeneous tumors. Liquid biopsy, as a promising technology, enables analysis of tumor components in the bodily fluids, such as blood and urine, at multiple time points and provides a minimally invasive approach that can track the evolutionary dynamics and monitor tumor heterogeneity. In this review, we describe the multiple faces of BC heterogeneity at the genomic and transcriptional levels and how they affect clinical care and outcomes. We also summarize the outcomes of liquid biopsy in BC, which plays a potential role in revealing tumor heterogeneity. Finally, we discuss the challenges that must be addressed before liquid biopsy can be widely used in clinical treatment.

Abstract Background The breast cancer genome dynamically evolves during malignant progression and recurrence. We investigated the genomic profiles of primary early-stage breast cancers and matched relapses to elucidate the molecular underpinnings of the metastatic process, focusing on potentially actionable alterations in the recurrences. Methods A mono-institutional cohort of 128 patients with breast cancers (n = 68 luminal B HER2, n = 6 luminal B HER2+, n = 1 HER2+ non-luminal, n = 56 triple negative) and at least one recurrence in a timeframe of 17 years was evaluated. Next-generation sequencing comprehensive genomic profiling was performed on 289 formalin-fixed paraffin-embedded (FFPE) samples, including primary tumors and matched relapses. Correlations of genomic aberrations with clinicopathologic factors and time to breast cancer relapse were analyzed. Results Genomic data were available for 188 of 289 FFPE samples that achieved the sequencing quality parameters (failure rate 34.9%), including 106 primary tumors and 82 relapses. All primary and relapse samples harbored at least one genomic alteration, with a median number of six alterations per sample (range 1–16). The most frequent somatic genomic alterations were mutations of TP53 (primary tumors = 49%, relapses = 49%) and PIK3CA (primary tumors = 33%, relapses = 30%). Distinctive genomic alterations of primary tumors were significantly associated with molecular subtypes. TP53, PIK3R1, and NF1 somatic alterations were more frequently detected in triple negative tumors (p value

Inferring the evolutionary dynamics at play during the process of speciation by analyzing the genomic landscape of divergence is a major pursuit in population genomics. However, empirical assessments of genomic landscapes under varying evolutionary scenarios that are known a priori are few, thereby limiting our ability to achieve this goal. Here we combine RAD-sequencing and individual-based simulations to evaluate the genomic landscape of divergence in the silvereye (Zosterops lateralis). Using pairwise comparisons that differ in divergence timeframe and the presence or absence of gene flow, we document how genomic patterns accumulate along the speciation continuum. In contrast to previous predictions, our results provide limited support for the idea that divergence accumulates around loci under divergent selection or that genomic islands widen with time. While a small number of genomic islands were found in populations diverging with and without gene flow, in few cases were SNPs putatively under selection tightly associated with genomic islands. The transition from localized to genome-wide levels of divergence was captured using individual-based simulations that considered only neutral processes. Our results challenge the ubiquity of existing verbal models that explain the accumulation of genomic differences across the speciation continuum and instead support the idea that divergence both within and outside of genomic islands is important during the speciation process.

Genetic diversity becomes structured among populations over time due to genetic drift and divergent selection. Although population structure is often treated as a uniform underlying factor, recent resequencing studies of wild populations have demonstrated that diversity in many regions of the genome may be structured quite dissimilar to the genome‐wide pattern. Here, we explored the adaptive and nonadaptive causes of such genomic heterogeneity using population‐level, whole genome resequencing data obtained from annual Mimulus guttatus individuals collected across a rugged environment landscape. We found substantial variation in how genetic differentiation is structured both within and between chromosomes, although, in contrast to other studies, known inversion polymorphisms appear to serve only minor roles in this heterogeneity. In addition, much of the genome can be clustered into eight among‐population genetic differentiation patterns, but only two of these clusters are particularly consistent with patterns of isolation by distance. By performing genotype‐environment association analysis, we also identified genomic intervals where local adaptation to specific climate factors has accentuated genetic differentiation among populations, and candidate genes in these windows indicate climate adaptation may proceed through changes affecting specialized metabolism, drought resistance, and development. Finally, by integrating our findings with previous studies, we show that multiple aspects of plant reproductive biology may be common targets of balancing selection and that variants historically involved in climate adaptation among populations have probably also fuelled rapid adaptation to microgeographic environmental variation within sites. [ABSTRACT FROM AUTHOR]

IntroductionCirculating tumor cells (CTCs) and cell-free tumor DNA (ctDNA) are tumor components present in circulation. Due to the limited access to both CTC enrichment platforms and ctDNA sequencing in most laboratories, they are rarely analyzed together.MethodsConcurrent isolation of ctDNA and single CTCs were isolated from lung cancer and breast cancer patients using the combination of size-based and CD45-negative selection method via DropCell platform. We performed targeted amplicon sequencing to evaluate the genomic heterogeneity of CTCs and ctDNA in lung cancer and breast cancer patients.ResultsHigher degrees of genomic heterogeneity were observed in CTCs as compared to ctDNA. Several shared alterations present in CTCs and ctDNA were undetected in the primary tumor, highlighting the intra-tumoral heterogeneity of tumor components that were shed into systemic circulation. Accordingly, CTCs and ctDNA displayed higher degree of concordance with the metastatic tumor than the primary tumor. The alterations detected in circulation correlated with worse survival outcome for both lung and breast cancer patients emphasizing the impact of the metastatic phenotype. Notably, evolving genetic signatures were detected in the CTCs and ctDNA samples during the course of treatment and disease progression.ConclusionsA standardized sample processing and data analysis workflow for concurrent analysis of CTCs and ctDNA successfully dissected the heterogeneity of metastatic tumor in circulation as well as the progressive genomic changes that may potentially guide the selection of appropriate therapy against evolving tumor clonality.

Background: Human papillomavirus–positive (HPV+) squamous cell carcinoma of the oropharynx (OPSCC) is the most prevalent HPV‐associated malignancy in the United States. Favorable treatment outcomes have led to increased interest in treatment de‐escalation to reduce treatment morbidity as well as the development of prognostic markers to identify appropriately low‐risk patients. Intratumoral genomic heterogeneity and copy number alteration burden have been demonstrated to be predictive of poor outcomes in many other cancers; therefore, we sought to determine whether intratumor heterogeneity and genomic instability are associated with poor outcomes in HPV+ OPSCC. Methods: Tumor heterogeneity estimates were made based on targeted exome sequencing of 45 patients with HPV+ OPSCC tumors. Analysis of an additional cohort of HPV+ OPSCC tumors lacking matched normal sequencing allowed copy number analysis of 99 patient tumors. Results: High intratumorally genomic heterogeneity and high numbers of copy number alterations were strongly associated with worse recurrence‐free survival. Tumors with higher heterogeneity and frequent copy number alterations were associated with loss of distal 11q, which encodes key genes related to double‐strand break repair, including ATM and MRE11A. Conclusions: Both intratumor genomic heterogeneity and high‐burden copy number alterations are strongly associated with poor recurrence‐free survival in patients with HPV+ OPSCC. The drivers of genomic instability and heterogeneity in these tumors remains to be elucidated. However, 11q loss and defective DNA double‐strand break repair have been associated with genomic instability in other solid tumors. Copy number alteration burden and intratumoral heterogeneity represent promising avenues for risk stratification of patients with HPV+OPSCC. Molecular markers are needed to determine which human papillomavirus (HPV)‐positive patients with squamous cell carcinoma of the oropharynx (OPSCC) are best served by reduced intensity treatment, which has the potential to reduce severe treatment‐related morbidity. We demonstrate that 11q‐related copy number variant burden and heterogeneity are highly associated with poor recurrence‐free survival in patients with HPV‐positive OPSCC and may have potential as translational biomarkers. [ABSTRACT FROM AUTHOR]

Introduction: Circulating tumor cells (CTCs) and cell-free tumor DNA (ctDNA) are tumor components present in circulation. Due to the limited access to both CTC enrichment platforms and ctDNA sequencing in most laboratories, they are rarely analyzed together. Methods: Concurrent isolation of ctDNA and single CTCs were isolated from lung cancer and breast cancer patients using the combination of size-based and CD45-negative selection method via DropCell platform. We performed targeted amplicon sequencing to evaluate the genomic heterogeneity of CTCs and ctDNA in lung cancer and breast cancer patients. Results: Higher degrees of genomic heterogeneity were observed in CTCs as compared to ctDNA. Several shared alterations present in CTCs and ctDNA were undetected in the primary tumor, highlighting the intra-tumoral heterogeneity of tumor components that were shed into systemic circulation. Accordingly, CTCs and ctDNA displayed higher degree of concordance with the metastatic tumor than the primary tumor. The alterations detected in circulation correlated with worse survival outcome for both lung and breast cancer patients emphasizing the impact of the metastatic phenotype. Notably, evolving genetic signatures were detected in the CTCs and ctDNA samples during the course of treatment and disease progression. Conclusions: A standardized sample processing and data analysis workflow for concurrent analysis of CTCs and ctDNA successfully dissected the heterogeneity of metastatic tumor in circulation as well as the progressive genomic changes that may potentially guide the selection of appropriate therapy against evolving tumor clonality. [ABSTRACT FROM AUTHOR]

Gastroesophageal junction (GEJ) cancer is a tumor that occurs at the junction of stomach and esophagus anatomically. GEJ cancer frequently metastasizes to lymph nodes, however the heterogeneity and clonal evolution process are unclear. This study is the first of this kind to use single cell DNA sequencing to determine genomic variations and clonal evolution related to lymph node metastasis. Multiple Annealing and Looping Based Amplification Cycles (MALBAC) and bulk exome sequencing were performed to detect single cell copy number variations (CNVs) and single nucleotide variations (SNVs) respectively. Four GEJ cancer patients were enrolled with two (Pt.3, Pt.4) having metastatic lymph nodes. The most common mutation we found happened in the TTN gene, which was reported to be related with the tumor mutation burden in cancers. Significant intra-patient heterogeneity in SNVs and CNVs were found. We identified the SNV subclonal architecture in each tumor. To study the heterogeneity of CNVs, the single cells were sequenced. The number of subclones in the primary tumor was larger than that in lymph nodes, indicating the heterogeneity of primary site was higher. We observed two patterns of multi-station lymph node metastasis: one was skip metastasis and the other was to follow the lymphatic drainage. Taken together, our single cell genomic analysis has revealed the heterogeneity and clonal evolution in GEJ cancer.

Gastroesophageal junction (GEJ) cancer is a tumor that occurs at the junction of stomach and esophagus anatomically. GEJ cancer frequently metastasizes to lymph nodes, however the heterogeneity and clonal evolution process are unclear. This study is the first of this kind to use single cell DNA sequencing to determine genomic variations and clonal evolution related to lymph node metastasis. Multiple Annealing and Looping Based Amplification Cycles (MALBAC) and bulk exome sequencing were performed to detect single cell copy number variations (CNVs) and single nucleotide variations (SNVs) respectively. Four GEJ cancer patients were enrolled with two (Pt.3, Pt.4) having metastatic lymph nodes. The most common mutation we found happened in the TTN gene, which was reported to be related with the tumor mutation burden in cancers. Significant intra-patient heterogeneity in SNVs and CNVs were found. We identified the SNV subclonal architecture in each tumor. To study the heterogeneity of CNVs, the single cells were sequenced. The number of subclones in the primary tumor was larger than that in lymph nodes, indicating the heterogeneity of primary site was higher. We observed two patterns of multi-station lymph node metastasis: one was skip metastasis and the other was to follow the lymphatic drainage. Taken together, our single cell genomic analysis has revealed the heterogeneity and clonal evolution in GEJ cancer. [ABSTRACT FROM AUTHOR]

Background: High tumor mutational burden (TMB) is one of the widely researched predictive biomarkers of immune checkpoint inhibitors and has been shown to be closely related with response to immunotherapy in multiple cancer types. However, for patients who have failed conventional therapy and are about to undergo immunotherapy, there is no consensus recommendation on the timing of tumor sampling for TMB analysis, and the effects of different therapies on TMB have not been clarified. This retrospective observational study aimed to investigate the heterogeneity of TMB and genomic mutation under the treatment pressure., Patients and Methods: We retrospectively collected the available genomic and therapeutic information from 8051 samples across 15 tumor types (>50 samples/tumor) found in 30 published studies and investigated the distribution and heterogeneity of TMB under treatment across diverse cohorts., Results: This integrated analysis has shown anticancer treatments increased TMB. Significant effects of treatment on TMB were more frequently observed in tumor types with lower treatment-naïve TMB, including breast, prostate, and pediatric cancers. For different cancer therapies, chemotherapy was prone to be correlated with an increased TMB in most cancer types. Meanwhile, the fraction of the TMB-high category of breast, prostate, and bladder cancers and glioma increased significantly after chemotherapy. Several actionable genes including ERS1 and NF1 in breast cancer, as well as some prognostic markers including TERT in bladder cancer and IDH1 in glioma, were significantly changed in post-chemotherapy tumors compared to treatment-naïve tumors., Conclusion: Our study reveals the heterogeneity of TMB under treatment across diverse cancer types and provides evidences that chemotherapy was associated with increases in TMB as well as the fraction of TMB-high category, suggesting that resampling tumor tissues for calculating post-chemotherapy TMB could be a better option for predicting the response to immunotherapy, especially for tumors with initially low TMB., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Abstract Background In lung cancer, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor sensitizing mutations co-existing with rare minor EGFR mutations are known as compound mutations. These minor EGFR mutations can lead to acquired resistance after EGFR tyrosine kinase inhibitor treatment, so determining the mutation status of patients is important. However, using amplicon-based targeted deep sequencing based on next-generation sequencing to characterize mutations is prone to sequencing error. We therefore assessed the benefit of incorporating molecular barcoding with high-throughput sequencing to investigate genomic heterogeneity in treatment-naïve patients who have undergone resection of their non-small cell lung cancer (NSCLC) EGFR mutations. Methods We performed amplicon-based targeted sequencing with the molecular barcoding system (MBS) to detect major common EGFR mutations and uncommon minor mutations at a 0.5% allele frequency in fresh–frozen lung cancer samples. Results Profiles of the common mutations of EGFR identified by MBS corresponded with the results of clinical testing in 63 (98.4%) out of 64 cases. Uncommon mutations of EGFR were detected in seven cases (10.9%). Among the three types of major EGFR mutations, patients with the G719X mutation had a significantly higher incidence of compound mutations than those with the L858R mutation or exon 19 deletion (p = 0.0052). This was validated in an independent cohort from the Cancer Genome Atlas dataset (p = 0.018). Conclusions Our findings demonstrate the feasibility of using the MBS to establish an accurate NSCLC patient genotype. This work will help understand the molecular basis of EGFR compound mutations in NSCLC, and could aid the development of new treatment modalities.

The lack of clinical biomarkers for head and neck cancer subtypes limits early diagnosis and monitoring of disease progression. This study investigates genetic alterations in clinically identical tumor, tumor-adjacent dysplastic epithelium (TADE) and normal epithelium (NE) in five oral cancer patients to identify differences and commonalities between oral cancer, TADE and NE. A VELscope®Vx device was used to identify TADE and NE surrounding a clinical tumor for analysis of genetic alterations using the OncoScan® assay. One of the tumor samples examined was an “M” class tumor with a high confidence BRAF:p.G469A:c.1406G>C somatic mutation, which is the first to be reported in oral cancer. Another tumor showed mosaicism in genetic alterations, indicating the presence of multiple clones. Overall, each patient's tumor, TADE and NE showed a distinct genetic profile which indicates intertumoral clonal/genetic diversity. Interestingly, four tumors showed gain of 3q26.2, 5q14.3, 8q24.3, 8q22.3, 14q32.33 and loss/LOH in 9p21.3 while all TADE had LOH on 22q11.23. In addition, some genetic alterations progressed from NE through TADE into tumor in individual patients. Furthermore, no molecular event was identified that is common to all NE and/or TADE that progressed into tumor. This pilot study demonstrates the presence of genetic heterogeneity in oral tumorigenesis, and suggests that there might exist some common genetic alterations between tumors and TADE. However, this observation would need to be further investigated and validated in a larger cohort of oral cancer patients for its potential role in oral tumorigenesis.

The lack of clinical biomarkers for head and neck cancer subtypes limits early diagnosis and monitoring of disease progression. This study investigates genetic alterations in clinically identical tumor, tumor-adjacent dysplastic epithelium (TADE) and normal epithelium (NE) in five oral cancer patients to identify differences and commonalities between oral cancer, TADE and NE. A VELscope®Vx device was used to identify TADE and NE surrounding a clinical tumor for analysis of genetic alterations using the OncoScan® assay. One of the tumor samples examined was an "M" class tumor with a high confidence BRAF :p.G469A:c.1406G>C somatic mutation, which is the first to be reported in oral cancer. Another tumor showed mosaicism in genetic alterations, indicating the presence of multiple clones. Overall, each patient's tumor, TADE and NE showed a distinct genetic profile which indicates intertumoral clonal/genetic diversity. Interestingly, four tumors showed gain of 3q26.2, 5q14.3, 8q24.3, 8q22.3, 14q32.33 and loss/LOH in 9p21.3 while all TADE had LOH on 22q11.23. In addition, some genetic alterations progressed from NE through TADE into tumor in individual patients. Furthermore, no molecular event was identified that is common to all NE and/or TADE that progressed into tumor. This pilot study demonstrates the presence of genetic heterogeneity in oral tumorigenesis, and suggests that there might exist some common genetic alterations between tumors and TADE. However, this observation would need to be further investigated and validated in a larger cohort of oral cancer patients for its potential role in oral tumorigenesis. [ABSTRACT FROM AUTHOR]

Genomic heterogeneity constitutes one of the most distinctive features of cancer diseases, limiting the efficacy and availability of medical treatments. Tumorigenesis emerges as a strongly stochastic process, producing a variable landscape of genomic configurations. In this context, matrix factorisation techniques represent a suitable approach for modelling such complex patterns of variability. In this work, we present a hierarchical factorisation model conceived from a systems biology point of view. The model integrates the topology of molecular pathways, allowing to simultaneously factorise genes and pathways activity matrices. The protocol was evaluated by using simulations, showing a high degree of accuracy. Furthermore, the analysis with a real cohort of breast cancer patients depicted the internal composition of some of the most relevant altered biological processes in the disease, describing gene and pathway level strategies and their observed combinations in the population of patients. We envision that this kind of approaches will be essential to better understand the hallmarks of cancer.

Small ruminant lentiviruses (SRLV) are viruses that retro-transcribe RNA to DNA and show high rates of genetic variability. SRLV affect animals with strains specific for each host species (sheep or goats), resulting in a series of clinical manifestations depending on the virulence of the strain, the host’s genetic background and farm production system. The aim of this work was to present an up-to-date overview of the genomic epidemiology and genetic diversity of SRLV in Italy over time (1998–2019). In this study, we investigated 219 SRLV samples collected from 17 different Italian regions in 178 geographically distinct herds by CEREL. Our genetic study was based on partial sequencing of the gag-pol gene (800 bp) and phylogenetic analysis. We identified new subtypes with high heterogeneity, new clusters and recombinant forms. The genetic diversity of Italian SRLV strains may have diagnostic and immunological implications that affect the performance of diagnostic tools. Therefore, it is extremely important to increase the control of genomic variants to improve the control measures.

Bacillus cereus is a human pathogenic bacterium found in foods with the potential to cause emesis and diarrhea. This study estimated the presence, toxigenic and genomic diversity of B. cereus s.l. obtained from cassava starch samples collected in bakeries and powdered food companies in Medellín (Colombia). Bacillus cereuss.l. was found in 43 of 75 (57%) cassava starch samples and 98 isolates were obtained. The nheABC, hblCDAB, cytK2, entFM and cesB toxin genes were detected by multiplex PCR and the most frequent operon was nheABC, whereas cesB gene was not found. Twelve toxigenic profiles were determined by the detection of toxin genes, and the most frequent profiles harbored all enterotoxin genes. A broad genomic diversity was detected according to GTG5-PCR fingerprinting results with 76 B. cereus s.l. grouped in sixteen clusters and the 22 isolates clustering separately. No relationship was observed between genomic background and toxigenic profiles. In general, the results showed a high genomic and enterotoxigenic diversity in B. cereus s.l. found in cassava starch. These results should incentive future studies to understand the distribution of B. cereus s.l. isolated on raw materials in comparison with finished products.

Osteosarcoma is the most common primary bone sarcoma and is often diagnosed in the 2nd–3rd decades of life. Response to the aggressive and highly toxic neoadjuvant methotrexate-doxorubicin-cisplatin (MAP) chemotherapy schedule is strongly predictive of outcome. Outcomes for patients with osteosarcoma have not significantly changed for over thirty years. There is a need for more effective treatment for patients with high risk features but also reduced treatment-related toxicity for all patients. Predictive biomarkers are needed to help inform clinicians to de-escalate or add therapy, including immune therapies, and to contribute to future clinical trial designs. Here, we review a variety of approaches to improve outcomes and quality of life for patients with osteosarcoma with a focus on incorporating toxicity reduction, immune therapy and molecular analysis to provide the most effective and least toxic osteosarcoma therapy.

Precision medicine, which includes comprehensive genome sequencing, is a potential therapeutic option for treating high-grade serous carcinoma (HGSC). However, HGSC is a heterogeneous tumor at the architectural, cellular, and molecular levels. Intratumoral molecular heterogeneity currently limits the precision of medical strategies based on the gene mutation status. This study was carried out to analyze the presence of 160 cancer-related genetic alterations in three tissue regions with different pathological features in a patient with HGSC. The patient exhibited histological heterogeneous features with different degrees of large atypical cells and desmoplastic reactions. TP53 mutation, ERBB2 and KRAS amplification, and WT1, CDH1, and KDM6A loss were detected as actionable gene alterations. Interestingly, the ERBB2 and KRAS amplification status gradually changed according to the region examined. The difference was consistent with the differences in pathological features. Our results demonstrate the need for sampling of the appropriate tissue region showing progression of pathological features for molecular analysis to solve issues related to tumor heterogeneity prior to developing precision oncology strategies.

Background The biofilm of a methanol-fed, fluidized denitrification system treating a marine effluent is composed of multi-species microorganisms, among which Hyphomicrobium nitrativorans NL23 and Methylophaga nitratireducenticrescens JAM1 are the principal bacteria involved in the denitrifying activities. Strain NL23 can carry complete nitrate (NO ${}_{3}^{-}$ 3 − ) reduction to N2, whereas strain JAM1 can perform 3 out of the 4 reduction steps. A small proportion of other denitrifiers exists in the biofilm, suggesting the potential plasticity of the biofilm in adapting to environmental changes. Here, we report the acclimation of the denitrifying biofilm from continuous operating mode to batch operating mode, and the isolation and characterization from the acclimated biofilm of a new denitrifying bacterial strain, named GP59. Methods The denitrifying biofilm was batch-cultured under anoxic conditions. The acclimated biofilm was plated on Methylophaga specific medium to isolate denitrifying Methylophaga isolates. Planktonic cultures of strains GP59 and JAM1 were performed, and the growth and the dynamics of NO ${}_{3}^{-}$ 3 − , nitrite (NO ${}_{2}^{-}$ 2 − ) and N2O were determined. The genomes of strains GP59 and JAM1 were sequenced and compared. The transcriptomes of strains GP59 and JAM1 were derived from anoxic cultures. Results During batch cultures of the biofilm, we observed the disappearance of H. nitrativorans NL23 without affecting the denitrification performance. From the acclimated biofilm, we isolated strain GP59 that can perform, like H. nitrativorans NL23, the complete denitrification pathway. The GP59 cell concentration in the acclimated biofilm was 2–3 orders of magnitude higher than M. nitratireducenticrescens JAM1 and H. nitrativorans NL23. Genome analyses revealed that strain GP59 belongs to the species M. nitratireducenticrescens. The GP59 genome shares more than 85% of its coding sequences with those of strain JAM1. Based on transcriptomic analyses of anoxic cultures, most of these common genes in strain GP59 were expressed at similar level than their counterparts in strain JAM1. In contrast to strain JAM1, strain GP59 cannot reduce NO ${}_{3}^{-}$ 3 − under oxic culture conditions, and has a 24-h lag time before growth and NO ${}_{3}^{-}$ 3 − reduction start to occur in anoxic cultures, suggesting that both strains regulate differently the expression of their denitrification genes. Strain GP59 has the ability to reduce NO ${}_{2}^{-}$ 2 − as it carries a gene encoding a NirK-type NO ${}_{2}^{-}$ 2 − reductase. Based on the CRISPR sequences, strain GP59 did not emerge from strain JAM1 during the biofilm batch cultures but rather was present in the original biofilm and was enriched during this process. Discussion These results reinforce the unique trait of the species M. nitratireducenticrescens among the Methylophaga genus as facultative anaerobic bacterium. These findings also showed the plasticity of denitrifying population of the biofilm in adapting to anoxic marine environments of the bioreactor.

Background: In lung cancer, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor sensitizing mutations co-existing with rare minor EGFR mutations are known as compound mutations. These minor EGFR mutations can lead to acquired resistance after EGFR tyrosine kinase inhibitor treatment, so determining the mutation status of patients is important. However, using amplicon-based targeted deep sequencing based on next-generation sequencing to characterize mutations is prone to sequencing error. We therefore assessed the benefit of incorporating molecular barcoding with high-throughput sequencing to investigate genomic heterogeneity in treatment-naïve patients who have undergone resection of their non-small cell lung cancer (NSCLC) EGFR mutations.Methods: We performed amplicon-based targeted sequencing with the molecular barcoding system (MBS) to detect major common EGFR mutations and uncommon minor mutations at a 0.5% allele frequency in fresh-frozen lung cancer samples.Results: Profiles of the common mutations of EGFR identified by MBS corresponded with the results of clinical testing in 63 (98.4%) out of 64 cases. Uncommon mutations of EGFR were detected in seven cases (10.9%). Among the three types of major EGFR mutations, patients with the G719X mutation had a significantly higher incidence of compound mutations than those with the L858R mutation or exon 19 deletion (p = 0.0052). This was validated in an independent cohort from the Cancer Genome Atlas dataset (p = 0.018).Conclusions: Our findings demonstrate the feasibility of using the MBS to establish an accurate NSCLC patient genotype. This work will help understand the molecular basis of EGFR compound mutations in NSCLC, and could aid the development of new treatment modalities. [ABSTRACT FROM AUTHOR]

Abstract Background Tissue genomic heterogeneity (t-HET) in patients with epithelial ovarian cancer (OVCA) is related to tissue plasticity, i.e., flexibility to adapt to adverse molecular environments. Here, we interrogated the presence and clinical relevance of OVCA t-HET. Methods We applied high-depth (>2000×) sequencing on 297 paraffin tissue samples (fallopian tubes, ovaries, intra-abdominal metastases) from 71 treatment-naïve patients who subsequently received first-line platinum-based chemotherapy. Based on tissue mutation patterns, we distinguished tissue genotypes into: no mutation (33/297 samples; 11.1%), stable (173; 58.2%) and unstable (91; 30.7%). We profiled genotypes per patient and assessed t-HET in 69 patients. Predicted pathogenic mutations refer to germline and/or tissues. Results Among all 71 patients, 46 (64.8%) had pathogenic BRCA1 mutations and 15 (21.7%) had BRCA1/2 disruption (i.e., pathogenic mutations with position-LOH). We classified 29 patients with t-HET (42%), all with pathogenic BRCA1; t-HET was observed in 64% with such mutations (p < 0.001). As opposed to non-t-HET, matched tissues in t-HET shared pathogenic BRCA1 (p < 0.001) but not BRCA2 and TP53. Germline BRCA1 mutations in tissues exhibited position-LOH; heterozygous status; or, partial loss of the inherited allele accompanied by additional clonal mutations. Patients with t-HET had worse outcome (log-rank p = 0.048 [progression-free]; p = 0.037 [overall survival]), including 12/15 patients with disrupted BRCA1/2 and 3 BRCA1 carriers with partial germline loss in tissues. Conclusions Pathogenic BRCA1 mutations appear necessary but may not be sufficient for the establishment of t-HET. t-HET may be associated with worse outcome, including in patients with disrupted BRCA1/2, which is usually considered as a favourable marker. OVCA t-HET may need to be addressed for treatment decisions. Highlights • Intra-patient tissue genomic heterogeneity (t-HET) is common in ovarian cancer (OVCA). • t-HET coexists with pathogenic BRCA1 & TP53 mutations in tissues; such mutations do not necessarily coexist with t-HET. • Pathogenic BRCA1 mutations are shared in t-HET; BRCA2 and TP53 are not. • Disrupted BRCA1 & 2 are positively associated with t-HET; disrupted TP53 is not. • t-HET may reflect tissue plasticity against BRCA1 and affect OVCA patient outcome. [ABSTRACT FROM AUTHOR]

Summary Endometriosis is characterized by ectopic endometrial-like epithelium and stroma, of which molecular characteristics remain to be fully elucidated. We sequenced 107 ovarian endometriotic and 82 normal uterine endometrial epithelium samples isolated by laser microdissection. In both endometriotic and normal epithelium samples, numerous somatic mutations were identified within genes frequently mutated in endometriosis-associated ovarian cancers. KRAS is frequently mutated in endometriotic epithelium, with a higher mutant allele frequency (MAF) accompanied by arm-level allelic imbalances. Analyses of MAF, combined with multiregional sequencing, illuminated spatiotemporal evolution of the endometriosis and uterine endometrium genomes. We sequenced 109 single endometrial glands and found that each gland carried distinct cancer-associated mutations, demonstrating the heterogeneity of the genomic architecture of endometrial epithelium. Remarkable increases in MAF of mutations in cancer-associated genes in endometriotic epithelium suggest retrograde flow of endometrial cells already harboring cancer-associated mutations, with selective advantages at ectopic sites, leading to the development of endometriosis. [ABSTRACT FROM AUTHOR]

Background: The biofilm of a methanol-fed, fluidized denitrification system treating a marine effluent is composed of multi-species microorganisms, among which Hyphomi- crobium nitrativorans NL23 and Methylophaga nitratireducenticrescens JAM1 are the principal bacteria involved in the denitrifying activities. Strain NL23 can carry complete nitrate (NO-3) reduction to N2, whereas strain JAM1 can perform 3 out of the 4 reduction steps. A small proportion of other denitrifiers exists in the biofilm, suggesting the potential plasticity of the biofilm in adapting to environmental changes. Here, we report the acclimation of the denitrifying biofilm from continuous operating mode to batch operating mode, and the isolation and characterization from the acclimated biofilm of a new denitrifying bacterial strain, named GP59. Methods: The denitrifying biofilm was batch-cultured under anoxic conditions. The acclimated biofilm was plated on Methylophaga specific medium to isolate denitrifying Methylophaga isolates. Planktonic cultures of strains GP59 and JAM1 were performed, and the growth and the dynamics of NO-3, nitrite (NO-2) and N2O were determined. The genomes of strains GP59 and JAM1 were sequenced and compared. The transcriptomes of strains GP59 and JAM1 were derived from anoxic cultures. Results: During batch cultures of the biofilm, we observed the disappearance of H. nitrativorans NL23 without affecting the denitrification performance. From the acclimated biofilm, we isolated strain GP59 that can perform, like H. nitrativorans NL23, the complete denitrification pathway. The GP59 cell concentration in the acclimated biofilm was 2-3 orders of magnitude higher than M. nitratireducenticrescens JAM1 and H. nitrativorans NL23. Genome analyses revealed that strain GP59 belongs to the species M. nitratireducenticrescens. The GP59 genome shares more than 85% of its coding sequences with those of strain JAM1. Based on transcriptomic analyses of anoxic cultures, most of these common genes in strain GP59 were expressed at similar level than their counterparts in strain JAM1. In contrast to strain JAM1, strain GP59 cannot reduce NO-3 under oxic culture conditions, and has a 24-h lag time before growth and NO-3 reduction start to occur in anoxic cultures, suggesting that both strains regulate differently the expression of their denitrification genes. Strain GP59 has the ability to reduce NO-2 as it carries a gene encoding a NirK-type NO-2 reductase. Based on the CRISPR sequences, strain GP59 did not emerge from strain JAM1 during the biofilm batch cultures but rather was present in the original biofilm and was enriched during this process. Discussion. These results reinforce the unique trait of the species M. nitratireducen- ticrescens among the Methylophaga genus as facultative anaerobic bacterium. These findings also showed the plasticity of denitrifying population of the biofilm in adapting to anoxic marine environments of the bioreactor. [ABSTRACT FROM AUTHOR]

Sixty strains (n = 60) of Vibrio vulnificus were examined for their multiple antibiotic resistance (MAR) index, plasmid profiles, and DNA polymorphisms. Thirty-seven strains (n = 37) were isolated from cockles (Anadara granosa) in Malaysia, while 23 (n = 23) isolates were isolated from clams (Mercenaria mercenaria) in Qatar. All isolates were resistant to two or more of the antibiotics tested, with the most common resistances were demonstrated towards penicillin (93%), ampicillin (70%), cephalothin (65%), clindamycin (66%), vancomycin (64%), and erythromycin (51%). The antibiotic that experienced the least resistance was kanamycin (6%), and all isolates were susceptible to cefoperazone, streptomycin, and tetracycline. The MAR index for the V. vulnificus isolated from Malaysia and Qatar, possessed similar values which ranged from 0.2 to 0.7, respectively. Plasmid analysis demonstrated that 65% of V. vulnificus strains harbored plasmids, while 35% were not. Nineteen (P1−P19) plasmids profiles were observed. No specific cluster or group was observed although they were isolated from different sample sources and locations by phylogenetic analysis using GelCompar II software at an 80% similarity level. Results demonstrated the high MAR index and genomic heterogeneity of V. vulnificus, which are of great concern to the human health of those who have consumed cockles and clams from the study area.

Prostate cancer is one of the most common types of cancer in males. Heterogeneous genomic aberrations may lead to prostate cancer onset, progression and metastasis. This heterogeneity also contributes to the variety in cancer risk and outcomes, different drug responses and progression, observed between individual patients. Classical prognostic factors, including prostate-specific antigen, Gleason Score and clinical tumor staging, are not sufficient to portray the complexity of a clinically relevant cancer diagnosis, risk prognosis, treatment choice and therapy monitoring. There is a requirement for novel genetic biomarkers in order to understand the oncogenic heterogeneity in a patient-personalized clinical setting and to improve the efficacy of risk prognosis and treatment choice. A number of biomarkers and gene panels have been established from patient sample cohort studies. These previous studies have provided distinct information to the investigation of heterogeneous malignancy in prostate cancer, which aids in clinical decision-making. Biomarker-guided therapies may facilitate the effective selection of drugs during early treatment; therefore, are beneficial to the individual patient. A non-invasive approach allows for convenient and repeated sampling to screen for cancer and monitor treatment response without the requirement for invasive tissue biopsies. With the current availability of numerous advanced technologies, reliable detection of the minimal tumor residues present following treatment may become clinical practice and, therefore, inform further in the field of personalized medicine. [ABSTRACT FROM AUTHOR]

Background: Glioma is the most frequent primary tumor of the central nervous system. The high heterogeneity of glioma tumors enables them to adapt to challenging environments, leading to resistance to treatment. Therefore, to detect the driving factors and improve the prognosis of glioma, it is essential to have a comprehensive understanding of the genomic heterogeneity, stemness, and immune microenvironment of glioma., Methods: We classified gliomas into various subtypes based on stemness, genomic heterogeneity, and immune microenvironment consensus clustering analysis. We identified risk hub genes linked to heterogeneous characteristics using WGCNA, LASSO, and multivariate Cox regression analysis and utilized them to create an effective risk model., Results: We thoroughly investigated the genomic heterogeneity, stemness, and immune microenvironment of glioma and identified the risk hub genes RAB42, SH2D4A, and GDF15 based on the TCGA dataset. We developed a risk model utilizing these genes that can reliably predict the prognosis of glioma patients. The risk signature showed a positive correlation with T cell exhaustion and increased infiltration of immunosuppressive cells, and a negative correlation with the response to immunotherapy. Moreover, we discovered that SH2D4A, one of the risk hub genes, could stimulate the migration and proliferation of glioma cells., Conclusions: This study identified risk hub genes and established a risk model by analyzing the genomic heterogeneity, stemness, and immune microenvironment of glioma. Our findings will facilitate the diagnosis and prediction of glioma prognosis and may lead to potential treatment strategies for glioma.

To test the traditional model of tumor progression, Darwinian-type evolution, against the more recent Big Bang model, we selected 6 microsatellite-stable colorectal standard-type adenocarcinomas and their synchronous lymph node and liver metastases. Somatic genomic variants were identified by whole-exome sequencing (WES) of large tumor fragments from the primaries and 1 liver metastasis each, and used to design targeted resequencing next-generation sequencing (NGS) panels, 1 per case. Targeted deep resequencing (mean coverage, 2725; median, 2222) was performed with DNA from punch samples (1-mm tissue microarrayer needles) obtained from different regions of the primaries and their metastases. In total, 255 genomic variants were interrogated in 108 punch samples. Clonal heterogeneity was infrequent: a pattern of clonal heterogeneity consistent with a role in metastasis formation was observed only in 1 case in a single gene (p. Asp604Tyr of the PTPRT gene). However, when comparing variant allele frequencies (VAFs) of genomic variants in adjacent positions on chromosomes ("matched genomic variant loci") across punch samples, differences that exceeded 2 SD of the NGS assay variations (ad hoc dubbed VAF dysbalance) were observed in 7.1% of the punch samples (2.6%-12.0% per case), which indicates an intricate intermixing of mutated and nonmutated tumor cells ("intrinsic heterogeneity"). Additional OncoScan array analyses on a subset of the punch samples (31 in total) showed gross genomic aberrations as a possible explanation in only some (39.2%) of the matched genomic variant loci with VAF dysbalance. Our study provides a fairly direct (statistical model-free) view of the genomic states of microsatellite-stable colorectal carcinomas and their metastases, and suggests that Darwinian-type tumor evolution is not the key pathway of the metastasizing disease; instead, we recorded an "intrinsic" genomic heterogeneity, which may echo an initial Big Bang-like event., (Copyright © 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Precision oncology relies on the accurate molecular characterization of individual patients with cancer at the time of treatment initiation. However, tumor molecular profiles are not static, and cancers continually evolve because of ongoing mutagenesis and clonal selection. Here, we performed genomic analyses of primary tumors, metastases, and plasma collected from individual patients to define the concordance of actionable genomic alterations and to identify drivers of metastatic disease progression. We observed a high degree of discordance of actionable genomic alterations, with 23% discordant between primary and metastatic disease sites. Among chromatin-modifying genes, ARID1A mutations, when discordant, were exclusive to the metastatic tumor samples. Our findings indicate that the high degree of lesion-to-lesion genomic heterogeneity may be a barrier to precision oncology approaches for bladder cancer and that circulating tumor DNA profiling may be preferred to tumor sequencing for a subset of patients. [Display omitted] • Mutations of chromatin-modifying genes vary between grade/stage in bladder cancer • Characterized by early branched evolution and lesion-to-lesion genomic heterogeneity • Primary and metastatic sites have 23% discordance in actionable genomic alterations • Plasma cfDNA identifies targetable genes not detected in tumor specimens Clinton et al. define the concordance of genomic alterations in urothelial carcinoma from a localized to metastatic state to identify drivers of progression. Within individual patients, there is significant discordance between primary and metastatic sites. Additionally, plasma cell-free DNA (cfDNA) can identify alterations not detected by tumor sequencing. [ABSTRACT FROM AUTHOR]

Introduction: Diffuse intrinsic pontine glioma (DIPG) and midline high-grade glioma (mHGG) are lethal childhood brain tumors. Spatial genomic heterogeneity has been well-described in adult HGG but has not been comprehensively characterized in pediatric HGG. We performed whole exome sequencing on 38-matched primary, contiguous, and metastatic tumor sites from eight children with DIPG (n = 7) ormHGG (n = 1) collected using a unique MRI-guided autopsy protocol. Validation was performed using Sanger sequencing, Droplet Digital polymerase-chain reaction, immunohistochemistry, and fluorescent in-situ hybridization. Results: Median age at diagnosis was 6.1 years (range: 2.9-23.3 years). Median overall survival was 13.2 months (range: 11.2-32.2 months). Contiguous tumor infiltration and distant metastases were observed in seven and six patients, respectively, including leptomeningeal dissemination in three DIPGs. Histopathological heterogeneity was evident in seven patients, including intra-pontine heterogeneity in two DIPGs, ranging from World Health Organization grade II to IV astrocytoma. We found conservation of heterozygous K27M mutations in H3F3A (n = 4) or HIST1H3B (n = 3) across all primary, contiguous, and metastatic tumor sites in all DIPGs. ACVR1 (n = 2), PIK3CA (n = 2), FGFR1 (n = 2), and MET (n = 1) were also intra-tumorally conserved. ACVR1 was co-mutated with HIST1H3B (n = 2). In contrast, PDGFRA amplification and mutation were spatially heterogeneous, as were mutations in BCOR (n = 1), ATRX (n = 2), and MYC (n = 1). TP53 aberrations (n = 3 patients) varied by type and location between primary and metastatic tumors sites but were intra-tumorally conserved. Conclusion: Spatial conservation of prognostically-relevant and therapeutically-targetable somatic mutations in DIPG and mHGG contrasts the significant heterogeneity of driver mutations seen in adult HGG and supports uniform implementation of diagnostic biopsy in DIPG and mHGG to classify molecular risk groups and guide therapeutic strategy. [ABSTRACT FROM AUTHOR]

Multifocal breast cancer ( MFBC), defined as multiple synchronous unilateral lesions of invasive breast cancer, is relatively frequent and has been associated with more aggressive features than unifocal cancer. Here, we aimed to investigate the genomic heterogeneity between MFBC lesions sharing similar histopathological parameters. Characterization of different lesions from 36 patients with ductal MFBC involved the identification of non-silent coding mutations in 360 protein-coding genes (171 tumour and 36 matched normal samples). We selected only patients with lesions presenting the same grade, ER, and HER2 status. Mutations were classified as 'oncogenic' in the case of recurrent substitutions reported in COSMIC or truncating mutations affecting tumour suppressor genes. All mutations identified in a given patient were further interrogated in all samples from that patient through deep resequencing using an orthogonal platform. Whole-genome rearrangement screen was further conducted in 8/36 patients. Twenty-four patients (67%) had substitutions/indels shared by all their lesions, of which 11 carried the same mutations in all lesions, and 13 had lesions with both common and private mutations. Three-quarters of those 24 patients shared oncogenic variants. The remaining 12 patients (33%) did not share any substitution/indels, with inter-lesion heterogeneity observed for oncogenic mutation(s) in genes such as PIK3CA, TP53, GATA3, and PTEN. Genomically heterogeneous lesions tended to be further apart in the mammary gland than homogeneous lesions. Genome-wide analyses of a limited number of patients identified a common somatic background in all studied MFBCs, including those with no mutation in common between the lesions. To conclude, as the number of molecular targeted therapies increases and trials driven by genomic screening are ongoing, our findings highlight the presence of genomic inter-lesion heterogeneity in one-third, despite similar pathological features. This implies that deeper molecular characterization of all MFBC lesions is warranted for the adequate management of those cancers. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]