Distinct copy number signatures between residual benign and transformed areas of carcinoma ex pleomorphic adenoma.

The mechanisms involved with the pathogenesis of carcinoma ex pleomorphic adenoma (CXPA) seem to be associated with the accumulation of molecular alterations in the pleomorphic adenoma (PA). In this sense, using array-based comparative genomic hybridization (aCGH) a rare series of 27 cases of CXPA and 14 residual PA (rPA) adjacent to the transformation area, we investigated the profile of the copy number alterations (CNAs) comparing benign residual and transformed areas. The main findings were correlated with the histopathological classification by histologic subtype and degree of invasion. The distribution of losses (p = 0.187) and amplifications (p = 0.172) was not statistically different between rPA and CXPA. The number of gains was increased in the transformed areas compared to the benign residual areas (p = 0.005). PLAG1 gain was maintained along the malignant transformation, as it was observed in both residual PA and CXPA samples, likely being an earlier event during transformation. The amplification of GRB7 and ERBB2 may also be an initial step in the malignant transformation of PA to CXPA (salivary duct carcinoma subtype). Furthermore, the amplification of HMGA2 and RPSAP52 were the most prevalent alterations among the studied samples. It was noteworthy that amplified genes in the transformed areas of the tumors were enriched for biological processes related to immune signaling. In conclusion, our results underscored for the first-time crucial CNAs in CXPA, some of them shared with the residual benign area adjacent to the transformation site. These CNAs included PLAG1 gain, as well as amplification of GRB7, ERBB2, HMGA2, and RPSAP52.

Efficiency of Non-Invasive Prenatal Testing in Detecting Fetal Copy Number Variation: A Retrospective Cohort Study.

Purpose: Screening of pathological copy number variations (CNVs) is important for early-diagnosis of hereditary disease. This study was designed to investigate the efficiency of non-invasive prenatal testing (NIPT) in detecting fetal CNVs. Methods: This retrospective analysis included fetuses with CNVs between January 2018 and December 2020. Karyotype analysis and CNV sequencing (CNV-seq) were performed. We then analyzed the positive predictive values of the subchromosomal microdeletions and microduplications. Results: Fifty-eight subjects with aberrant CNVs were screened after NIPT, among which 44 finally underwent amniocentesis. CNV-seq confirmed the presence of CNVs in 24 cases. This indicated that false positivity rate of NIPT was 45.5%. Among 24 cases with CNVs after CNV-seq, only 4 showed consistent findings with karyotype analysis, which showed that karyotyping analysis yielded a missed diagnosis rate of 83.3% for the genome CNV. Positive predictive value (PPV) was 50.0% for CNVs with a length of <5 Mb after NIPT screening. PPV for CNVs with a length of 5 Mb-10 Mb was 33.3%, while that for CNVs with a length of ≥10Mb was 60%. For CNVs duplication after NIPT, the PPV was 65.2%, while that for deletion was 36.4%. Conclusion: For CNVs detected after NIPT, it should be combined with ultrasonographic findings, karyotype analysis, CNV-seq or CMA to determine the pregnancy outcome. Expanding NIPT may increase the risk of unnecessary invasive surgery and unintended selective termination of pregnancy.

Genomic alterations in retinoblastoma tumors of Argentine patients.

INTRODUCTION: Retinoblastoma is initiated by inactivation of RB1 gene, but additional alterations may be required for tumor progression. Substitution and INDEL variants in different genes, aside RB1, are infrequent, while large copy number variants (CNVs) like gains on 1q, 2p, 6p and loss on 16q are common, they include oncogenes or tumor suppressors and are typical of retinoblastoma. AIM: To provide the molecular profile that is useful for prognosis and understanding of retinoblastoma development. METHODS: To identify genomic variants in six retinoblastoma tumors whole exome sequencing and informatic analysis were performed. RESULTS: RB1 was the only gene with nonsense or frameshift mutations. SNVs in other 11 genes were missense and at non-canonical splice-sites, all nonpathogenic. CNVs, similar to those reported, were identified in all retinoblastoma tumors. The most frequent were 1q gain and 16q loss. Additionally, deletions were identified on 13q, including RB1 gene, and on the X chromosome, including BCOR gene, the most frequently mutated, after RB1, in retinoblastoma. The number of CNVs detected in each tumor was between 1 and 7, depending on the age at diagnosis. CONCLUSION: The analysis of genomic alterations in retinoblastoma is useful to understand the severity of tumor progression and to apply appropriate treatments.

Identification of genome-wide copy number variation-driven subtypes for the treatment and prognostic prediction of esophageal carcinoma.

Background: Esophageal carcinoma (ESCA) is a frequently detected gastrointestinal cancer. Copy number variants (CNVs) have a dramatic impact on the screening, diagnosis and prognostic prediction of cancers. However, the mechanism of action of CNVs on ESCA occurrence and progression remains unclear. Methods: ESCA samples from The Cancer Genome Atlas (TCGA) were typed by consensus clustering using CNV-associated genes. Weighted Gene Co-Expression Network Analysis (WGCNA) was used to section gene modules closely related to the two clusters, and sub-networks were constructed as hub genes. In addition, seven prognosis-correlated genes were further screened and retained by multivariate Cox regression analysis to develop a prognostic assessment model. The ssGSEA algorithm assessed energy metabolism levels in patients from different clusters and risk groups. Finally, quantitative real-time PCR (qRT-PCR) and live-dead cell staining verified the expression of genes associated with CNV risk scores. Results: ESCA was classified into two subtypes based on CNV values. Compared with cluster 1, cluster 2 had significantly higher level of immune score and tumor-associated immune cell infiltration as well as a noticeably better overall survival. The three modules most associated with the two clusters were identified by WGCNA, and a prognostic model with a strong prediction performance was constructed with their genes. Glycolysis, lactate metabolism, fatty acid synthesis, glutathione, methionine, and tryptophan metabolic pathway enrichment scores were remarkably higher in patients in cluster 1 and the high-risk group than in cluster 2 and the low-risk group. Knockdown PIK3C2A promoted ESCA cells apoptosis and inhibited cell vibiality. Conclusion: The current research maybe provides new understanding for the pathogenesis of ESCA based on CNV, providing an effective guidance for its clinical diagnosis and prognostic evaluation.

FANCD2 genome binding is nonrandom and is enriched at large transcriptionally active neural genes prone to copy number variation.

Fanconi anemia (FA) is a rare genetic disease characterized by congenital abnormalities and increased risk for bone marrow failure and cancer. Central nervous system defects, including acute and irreversible loss of neurological function and white matter lesions with calcifications, have become increasingly recognized among FA patients, and are collectively referred to as Fanconi Anemia Neurological Syndrome or FANS. The molecular etiology of FANS is poorly understood. In this study, we have used a functional integrative genomics approach to further define the function of the FANCD2 protein and FA pathway. Combined analysis of new and existing FANCD2 ChIP-seq datasets demonstrates that FANCD2 binds nonrandomly throughout the genome with binding enriched at transcription start sites and in broad regions spanning protein-coding gene bodies. FANCD2 demonstrates a strong preference for large neural genes involved in neuronal differentiation, synapse function, and cell adhesion, with many of these genes implicated in neurodevelopmental and neuropsychiatric disorders. Furthermore, FANCD2 binds to regions of the genome that replicate late, undergo mitotic DNA synthesis (MiDAS) under conditions of replication stress, and are hotspots for copy number variation. Our analysis describes an important targeted role for FANCD2 and the FA pathway in the maintenance of large neural gene stability.

Predicting the survival of patients with painful tumours treated with palliative radiotherapy: a secondary analysis using the 3-variable number-of-risk-factors model.

BACKGROUND: The 3-variable number-of-risk-factors (NRF) model is a prognostic tool for patients undergoing palliative radiotherapy (PRT). However, there is little research on the NRF model for patients with painful non-bone-metastasis tumours treated with PRT, and the efficacy of the NRF model in predicting survival is unclear to date. Therefore, we aimed to assess the prognostic accuracy of a 3-variable NRF model in patients undergoing PRT for bone and non- bone-metastasis tumours. METHODS: This was a secondary analysis of studies on PRT for bone-metastasis (BM) and PRT for miscellaneous painful tumours (MPTs), including non-BM tumours. Patients were grouped in the NRF model and survival was compared between groups. Discrimination was evaluated using a time-independent C-index and a time-dependent area under the receiver operating characteristic curve (AUROC). A calibration curve was used to assess the agreement between predicted and observed survival. RESULTS: We analysed 485 patients in the BM group and 302 patients in the MPT group. The median survival times in the BM group for groups I, II, and III were 35.1, 10.1, and 3.3 months, respectively (P < 0.001), while in the MPT group, they were 22.1, 9.5, and 4.6 months, respectively (P < 0.001). The C-index was 0.689 in the BM group and 0.625 in the MPT group. In the BM group, time-dependent AUROCs over 2 to 24 months ranged from 0.738 to 0.765, while in the MPT group, they ranged from 0.650 to 0.689, with both groups showing consistent accuracy over time. The calibration curve showed a reasonable agreement between the predicted and observed survival. CONCLUSIONS: The NRF model predicted survival moderately well in both the BM and MPT groups.

Postmenopausal Shrinkage of Uterine Myomas: A Retrospective Study of 97 Cases Monitored Annually for 10 Years.

Introduction and aim Both patients and gynecologists are concerned about how much and how quickly myomas shrink after menopause. This study aimed to elucidate clinical findings that may be associated with postmenopausal shrinkage of uterine myomas. Materials and methods This study included 97 patients who underwent menopause by August 2012, had myoma nodules with the longest diameter between 50 mm and 160 mm, and visited our specialized myoma clinic annually for at least 10 years after menopause. They underwent transabdominal ultrasonography at least once per year. An experienced gynecologist measured the longest diameter of myoma nodules with a maximum diameter between 50 mm and 160 mm. The shrinkage rate of myoma diameters after menopause compared to premenopausal diameters was calculated each year for 10 years. The shrinkage rate of the longest diameter of the largest nodule 10 years after menopause (10-year shrinkage rate) and its relationship with clinical findings (the age at menopause, parity, body mass index {BMI}, number of nodules, MRI findings on T2-weighted image, location of the nodule, and longest diameter of the largest nodule before menopause) were analyzed. Additionally, we examined annual changes in shrinkage rate of myomas over a 10-year period after menopause (annual trend), and the relationship between annual trends and factors such as BMI and the number of nodules. Results In this examination of 10-year shrinkage rate, the group with a BMI of less than 25 showed a significantly greater shrinkage rate compared to the group with a BMI of 25 or more (25.0% vs 15.7%, p=0.023). Additionally, the group with a single nodule showed a significantly greater 10-year shrinkage rate compared to the group with four or more nodules (26.3% vs 15.2%, p=0.036). For annual trends, the rate of change in the first two years after menopause was significantly faster compared to the trend from the third to the 10th year (difference in slope: 3.888 points per year, p<0.001). When divided into two groups based on the number of nodules (one or two nodules group and three or more nodules group), the group with one or two nodules showed a significant difference in the shrinkage rate between up to two years after menopause and from the period from the third to the 10th year (difference in slope: 4.590 points per year, p<0.001). However, for the group with three or more nodules, there was no significant difference in the annual trend between the first two years after menopause and the rate from the third to the 10th year (difference in slope: 1.626 points per year, p=0.107). Conclusion BMI and the number of myoma nodules were significantly related to the 10-year shrinkage rate. Although myomas shrank significantly faster within the first two years after menopause compared to the later period, the early annual trend did not differ significantly from the trend in the later period when there were multiple nodules with a maximum diameter of 50 mm or more.

Deciphering genetic and nongenetic factors underlying tumour dormancy: insights from multiomics analysis of two syngeneic MRD models of melanoma and leukemia.

BACKGROUND: Tumour dormancy, a resistance mechanism employed by cancer cells, is a significant challenge in cancer treatment, contributing to minimal residual disease (MRD) and potential relapse. Despite its clinical importance, the mechanisms underlying tumour dormancy and MRD remain unclear. In this study, we employed two syngeneic murine models of myeloid leukemia and melanoma to investigate the genetic, epigenetic, transcriptomic and protein signatures associated with tumour dormancy. We used a multiomics approach to elucidate the molecular mechanisms driving MRD and identify potential therapeutic targets. RESULTS: We conducted an in-depth omics analysis encompassing whole-exome sequencing (WES), copy number variation (CNV) analysis, chromatin immunoprecipitation followed by sequencing (ChIP-seq), transcriptome and proteome investigations. WES analysis revealed a modest overlap of gene mutations between melanoma and leukemia dormancy models, with a significant number of mutated genes found exclusively in dormant cells. These exclusive genetic signatures suggest selective pressure during MRD, potentially conferring resistance to the microenvironment or therapies. CNV, histone marks and transcriptomic gene expression signatures combined with Gene Ontology (GO) enrichment analysis highlighted the potential functional roles of the mutated genes, providing insights into the pathways associated with MRD. In addition, we compared "murine MRD genes" profiles to the corresponding human disease through public datasets and highlighted common features according to disease progression. Proteomic analysis combined with multi-omics genetic investigations, revealed a dysregulated proteins signature in dormant cells with minimal genetic mechanism involvement. Pathway enrichment analysis revealed the metabolic, differentiation and cytoskeletal remodeling processes involved in MRD. Finally, we identified 11 common proteins differentially expressed in dormant cells from both pathologies. CONCLUSIONS: Our study underscores the complexity of tumour dormancy, implicating both genetic and nongenetic factors. By comparing genomic, transcriptomic, proteomic, and epigenomic datasets, our study provides a comprehensive understanding of the molecular landscape of minimal residual disease. These results provide a robust foundation for forthcoming investigations and offer potential avenues for the advancement of targeted MRD therapies in leukemia and melanoma patients, emphasizing the importance of considering both genetic and nongenetic factors in treatment strategies.

Molecular substratification of endometrial carcinomas with no special molecular profile (NSMP) by using a limited NGS custom panel may facilitate effective patient selection for the PIK3CA-targeted therapy.

Endometrial carcinomas (EC) of no special molecular profile (NSMP) represent the largest molecular category of EC, comprising a mixture of tumors with different histology and molecular profiles. These facts likely point to different tumor biology, clinical outcomes, and targeted therapy responses within this molecular category. The PIK3CA is currently the only targetable kinase oncoprotein directly implicated in EC carcinogenesis. Investigating a unique single-institution cohort, we attempted to stratify NSMP ECs based on the presence of the PIK3CA pathogenic mutation. Those cases were further analyzed for other well-established-associated oncogenic driver gene mutations. Histological and clinical variables were also correlated in each case. Altogether, 175 ECs were prospectively tested by a limited custom NGS panel containing ARID1A, BCOR, BRCA1, BRCA2, CTNNB1, KRAS, MLH1, MSH2, MSH6, NRAS, PIK3CA, PMS2, POLD1, POLE, PTEN,and TP53 genes. We identified 24 PIK3CA mutated cases in the group of 80 NSMP ECs, with another co-occurring mutation in at least one oncogenic driver gene (CTNNB1, PTEN, ARID1A, KRAS, BCOR, PMS2) in 19 cases. In conclusion, a limited NGS panel can effectively test EC tissue for specific pathogenetically relevant oncogene mutations. The NSMP EC category contains 30% of the PIK3CA mutated cases. Of those, 21% contain the PIK3CA mutation as a sole EC-associated oncogene mutation, while 79% harbor at least one more mutated gene. These findings may inform future healthcare planning and improve the effectiveness of EC patient selection for the PIK3CA-targeted therapy.

Low-pass whole genome sequencing of circulating tumor cells to evaluate chromosomal instability in triple-negative breast cancer.

Chromosomal Instability (CIN) is a common and evolving feature in breast cancer. Large-scale Transitions (LSTs), defined as chromosomal breakages leading to gains or losses of at least 10 Mb, have recently emerged as a metric of CIN due to their standardized definition across platforms. Herein, we report the feasibility of using low-pass Whole Genome Sequencing to assess LSTs, copy number alterations (CNAs) and their relationship in individual circulating tumor cells (CTCs) of triple-negative breast cancer (TNBC) patients. Initial assessment of LSTs in breast cancer cell lines consistently showed wide-ranging values (median 22, range 4-33, mean 21), indicating heterogeneous CIN. Subsequent analysis of CTCs revealed LST values (median 3, range 0-18, mean 5), particularly low during treatment, suggesting temporal changes in CIN levels. CNAs averaged 30 (range 5-49), with loss being predominant. As expected, CTCs with higher LSTs values exhibited increased CNAs. A CNA-based classifier of individual patient-derived CTCs, developed using machine learning, identified genes associated with both DNA proliferation and repair, such as RB1, MYC, and EXO1, as significant predictors of CIN. The model demonstrated a high predictive accuracy with an Area Under the Curve (AUC) of 0.89. Overall, these findings suggest that sequencing CTCs holds the potential to facilitate CIN evaluation and provide insights into its dynamic nature over time, with potential implications for monitoring TNBC progression through iterative assessments.

Mitochondrial genetic variations in leukemia: a comprehensive overview.

Leukemias are a group of heterogeneous hematological malignancies driven by diverse genetic variations, and the advent of genomic sequencing technologies facilitates the investigation of genetic abnormalities in leukemia. However, these sequencing-based studies mainly focus on nuclear DNAs. Increasing evidence indicates that mitochondrial dysfunction is an important mechanism of leukemia pathogenesis, which is closely related to the mitochondrial genome variations. Here, we provide an overview of current research progress concerning mitochondrial genetic variations in leukemia, encompassing gene mutations and copy number variations. We also summarize currently accessible mitochondrial DNA (mtDNA) sequencing methods. Notably, somatic mtDNA mutations may serve as natural genetic barcodes for lineage tracing and longitudinal assessment of clonal dynamics. Collectively, these findings enhance our understanding of leukemia pathogenesis and foster the identification of novel therapeutic targets and interventions.

In-host density-dependent model of high-risk HPV virions, basal cells, lymphocytes t-cells incorporating functional responses.

Cervical cancer is one of the most common types of cancer and it is caused mostly by high-risk Human Papillomavirus (HPV) and continues to spread at an alarming rate. While HPV impacts have been investigated before, there are currently only a scanty number of mathematical models that account for HPV's dynamic role in cervical cancer. The objectives were to develop an in-host density-dependent deterministic model for the dynamics implications of basal cells, virions, and lymphocytes incorporating immunity and functional responses. Analyze the model using techniques of epidemiological models such as basic reproduction number and simulate the model using Matlab ODE solver. Six compartments are considered in the model that is; Susceptible cells (S), Infected cells (I), Precancerous cells (P), Cancerous cells (C), Virions (V), and Lymphocytes (L). Next generation matrix (NGM), survival function, and characteristic polynomial method were used to determine the basic reproduction number denoted as R 0 . R 0 was obtained using three methods because NGM has some weaknesses hence the need for the other two methods. The findings from this research indicated that Disease-Free Equilibrium point is locally asymptotically stable whenever R 0 * < 1 and globally asymptotically stable if R 0 * ≤ 1 and the Endemic Equilibrium is globally asymptotically stable if R 0 * > 1 . The results obtained shows that the progression rate of precancerous cells to cancerous cells ( θ ) has the most direct impact on the model. The model was able to estimate the longevity of a patient as 10 days when ( θ ) increases by 0.08 . The findings of this research will help healthcare providers, public health authorities, and non-governmental health groups in creating effective prevention strategies to slow the development of cervical cancer. More research should be done to determine the exact number of cancerous cells that can lead to the death of a cervical cancer patient since this paper estimated a proportion of 75 % .

Safety evaluation of curdlan as a food additive.

The EFSA Panel on Food Additives and Flavourings (FAF) provides a scientific opinion on the safety of curdlan as a new food additive used as firming and gelling agent, stabiliser, thickener. Curdlan is a high molecular weight polysaccharide consisting of ß-1,3-linked glucose units, produced by fermentation from Rhizobium radiobacter biovar 1 strain NTK-u. The toxicological dataset consisted of sub-chronic, chronic and carcinogenicity, reproductive and developmental toxicity studies as well as genotoxicity. In vivo data showed that curdlan is not absorbed as such but is extensively metabolised by the gut microbiota into CO2 and other innocuous compounds. Curdlan was not genotoxic and was well-tolerated with no overt organ-specific toxicity. Effects observed at very high doses of curdlan, such as decreased growth and increased cecum weight, are common for indigestible bulking compounds and therefore considered physiological responses. In a combined three-generation reproductive and developmental toxicity study, decreased pup weight was observed during lactation at 7500 mg curdlan/kg body weight (bw) per day, the highest dose tested. The Panel considered the observed effects as treatment-related and adverse, although likely secondary to nutritional imbalance and identified a conservative no observed adverse effect level (NOAEL) of 2500 mg/kg bw per day. Despite the limitations noted in the dataset, the Panel was able to conclude applying the margin of exposure (MOE) approach. Given that curdlan and its break-down products are not absorbed and that the identified adverse effect is neither systemic nor local, no adjustment factor was deemed necessary. Thus, an MOE of at least 1 was considered sufficient. The highest exposure estimate was 1441 mg/kg bw per day in toddlers at the 95th percentile of the proposed maximum use level exposure assessment scenario. The Panel concluded that there is no safety concern for the use of curdlan as a food additive at the proposed uses and use levels.

Molecular Profiling Defines Three Subtypes of Synovial Sarcoma.

Synovial Sarcomas (SS) are characterized by the presence of the SS18::SSX fusion gene, which protein product induce chromatin changes through remodeling of the BAF complex. To elucidate the genomic events that drive phenotypic diversity in SS, we performed RNA and targeted DNA sequencing on 91 tumors from 55 patients. Our results were verified by proteomic analysis, public gene expression cohorts and single-cell RNA sequencing. Transcriptome profiling identified three distinct SS subtypes resembling the known histological subtypes: SS subtype I and was characterized by hyperproliferation, evasion of immune detection and a poor prognosis. SS subtype II and was dominated by a vascular-stromal component and had a significantly better outcome. SS Subtype III was characterized by biphasic differentiation, increased genomic complexity and immune suppression mediated by checkpoint inhibition, and poor prognosis despite good responses to neoadjuvant therapy. Chromosomal abnormalities were an independent significant risk factor for metastasis. KRT8 was identified as a key component for epithelial differentiation in biphasic tumors, potentially controlled by OVOL1 regulation. Our findings explain the histological grounds for SS classification and indicate that a significantly larger proportion of patients have high risk tumors (corresponding to SS subtype I) than previously believed.

The prognostic effect of blast count in TP53 mutant myeloid neoplasms -the Minnesota experience.

In 2022, the World Health Organization (WHO) and International Consensus Classification (ICC) recognized TP53 as an entity-defining alteration in myeloid neoplasms, yet with differing criteria that could lead to discrepant diagnoses and affect clinical trial eligibility. We studied 67 patients with TP53 mutant myeloid neoplasms, reclassifying them using both criteria. While most cases fulfill the criteria for TP53 mutant defined entities, most discrepancies were found in cases with ≥20% blasts. Patients were stratified into three groups based on blast count (<10%, 10-19%, and ≥20%) which revealed comparable clinicopathologic features, genetic characteristics, and outcomes. Notably, patients with ≥10% blasts had shorter overall survival compared to those with <10% blasts (8.1 vs. 12.4 months; p = 0.03). This study is among the few to examine TP53 mutant myeloid neoplasms as a single entity and suggests that the 10% blast count threshold could serve as a gateway to a more harmonized classification for these patients.

Comprehensive Analyses of Somatic Copy Number Alterations and Mutations Based on the Adenoma-Carcinoma Sequence.

AIMS: Identifying molecular alterations in the adenoma and carcinoma components within the same tumor would greatly contribute to understanding the neoplastic progression of early colorectal cancer. METHODS AND RESULTS: We examined somatic copy number alterations (SCNAs) and mutations involved in the adenoma and carcinoma components obtained from the same tumor in 46 cases of microsatellite-stable carcinoma in adenoma, using a genome-wide SNP array and gene mutation panel. In addition, we also performed hierarchical clustering to determine the SCNA frequencies in the tumors, resulting in stratification of the samples into two subgroups according to SCNA frequency. Subgroup 1 was characterized by multiple SCNAs and carcinoma components exclusively, while Subgroup 2 was characterized by a low frequency of SCNAs and both the adenoma and carcinoma components. The numbers of total genes and genes with gains were higher in the carcinoma than adenoma components. The three most frequent gains in both components were located at 1p36.33-1q44, 2p25.3-2q37.3, and 3p26.3-3q29. However, no candidate genes mapped to these regions. APC and KRAS mutations were common in both components, whereas the frequency of TP53 mutations was statistically higher in the carcinoma than adenoma component. However, TP53 mutations were not correlated with SCNA frequency. CONCLUSIONS: We suggest that considerable SCNAs and TP53 mutations are required for progression from adenoma to carcinoma within the same intramucosal neoplastic lesion.

Exploring the impact of intra-tumoural heterogeneity on liquid biopsy cell-free DNA methylation and copy number in head and neck squamous cell carcinoma.

Liquid biopsy profiling is gaining increasing promise towards biomarker-led identification and disease stratification of tumours, particularly for tumours displaying significant intra-tumoural heterogeneity (ITH). For head and neck squamous cell carcinoma (HNSCC), which display high levels of genetic ITH, identification of epigenetic modifications and methylation signatures has shown multiple uses in stratification of HNSCC for prognosis, treatment, and HPV status. In this study, we investigated the potential of liquid biopsy methylomics and genomic copy number to profile HNSCC. We conducted multi-region sampling of tumour core, tumour margin and normal adjacent mucosa, as well as plasma cell-free DNA (cfDNA) across 9 HNSCC patients. Collectively, our work highlights the prevalence of methylomic ITH in HNSCC, and demonstrates the potential of cfDNA methylation as a tool for ITH assessment and serial sampling.

Age-related differences in the number of chronic diseases in association with trajectories of depressive symptoms: a population-based cohort study.

BACKGROUND: The number of chronic diseases has been associated with changes in depressive symptoms over time among middle-aged and older adults. This study aimed to explore the association between the number of chronic diseases and trajectories of depressive symptoms and the role of age in this association. METHODS: A total of 12,974 middle-aged and older Chinese adults (≥ 45 years) participated in the China Health and Retirement Longitudinal Study (CHARLS) in waves 2011, 2013, 2015, 2018, and 2020. The number of chronic diseases was determined by self-reported hospital diagnosis of hypertension, dyslipidemia, diabetes, cancer, chronic lung diseases, liver disease, heart diseases, stroke, kidney diseases, digestive diseases, emotional, nervous, or psychiatric problems, memory-related disease, arthritis or rheumatism, asthma, and then obtaining the total number of chronic diseases. Depressive symptoms were measured by the 10-item Center for Epidemiologic Studies Depression Scale (CESD-10). Group-based trajectory modeling (GBTM) was adopted to capture the trajectories of depressive symptoms over time. Multinomial logistic regressions were conducted to examine the association between the number of chronic diseases and trajectories of depressive symptoms and the role of age in this association. RESULTS: Four distinct trajectories of depressive symptoms were observed in 34.68% individuals in mild, 40.76% in moderate, 19.41% in increasing, and 5.15% in severe group. Compared to participants without chronic diseases, those with one, two, three or more chronic diseases had a 1.81, 3, and 7.49-fold higher risk of developing severe depressive symptom trajectory, respectively. Moreover, the association between the number of chronic diseases and severe depressive symptoms trajectory differed by age (45-59 and ≥ 60 years) (P for interaction < 0.05). CONCLUSION: Participants with middle age may play a promoting role in the association between the number of chronic disease and severe depressive symptoms. The severe depressive symptoms intervention may be more beneficial for middle-aged adults.

Resolution of Optimal Mitochondrial and Nuclear DNA Enrichment in Target-Panel Sequencing and Physiological Mitochondrial DNA Copy Number Estimation in Liver Cancer and Non-Liver Cancer Subjects.

Mitochondria generate energy to support cells. They are important organelles that engage in key biological pathways. The dysfunction of mitochondria can be linked to hepatocarcinogenesis, which has been actively explored in recent years. To investigate the mitochondrial dysfunction caused by genetic variations, target-panel sequencing is a flexible and promising strategy. However, the copy number of mitochondria generally exceeds nuclear DNA, which raises a concern that uneven target enrichment of mitochondrial DNA (mtDNA) and nuclear DNA (ncDNA) in target-panel sequencing would lead to an undesirably biased representation of them. To resolve this issue, we evaluated the optimal pooling of mtDNA probes and ncDNA probes by a series of dilutions of mtDNA probes in both genomic DNA (gDNA) and cell-free DNA (cfDNA) samples. The evaluation was based on read count, average sequencing depth and coverage of targeted regions. We determined that an mtDNA:ncDNA probe ratio of around 1:10 would offer a good balance of sequencing performance and cost effectiveness. Moreover, we estimated the median physiological mtDNA:ncDNA copy ratio as 38.1 and 2.9 in cfDNA and gDNA samples of non-liver cancer subjects, respectively, whereas they were 20.0 and 2.1 in the liver cancer patients. Taken together, this study revealed the appropriate pooling strategy of mtDNA probes and ncDNA probes in target-panel sequencing and suggested the normal range of physiological variation of the mtDNA:ncDNA copy ratio in non-liver cancer individuals. This can serve as a useful reference for future target-panel sequencing investigations of the mitochondrial genome in liver cancer.

A Pro-Inflammatory Stimulus versus Extensive Passaging of DITNC1 Astrocyte Cultures as Models to Study Astrogliosis.

Astrogliosis is a process by which astrocytes, when exposed to inflammation, exhibit hypertrophy, motility, and elevated expression of reactivity markers such as Glial Fibrillar Acidic Protein, Vimentin, and Connexin43. Since 1999, our laboratory in Chile has been studying molecular signaling pathways associated with "gliosis" and has reported that reactive astrocytes upregulate Syndecan 4 and αVß3 Integrin, which are receptors for the neuronal glycoprotein Thy-1. Thy-1 engagement stimulates adhesion and migration of reactive astrocytes and induces neurons to retract neurites, thus hindering neuronal network repair. Reportedly, we have used DITNC1 astrocytes and neuron-like CAD cells to study signaling mechanisms activated by the Syndecan 4-αVß3 Integrin/Thy-1 interaction. Importantly, the sole overexpression of ß3 Integrin in non-reactive astrocytes turns them into reactive cells. In vitro, extensive passaging is a simile for "aging", and aged fibroblasts have shown ß3 Integrin upregulation. However, it is not known if astrocytes upregulate ß3 Integrin after successive cell passages. Here, we hypothesized that astrocytes undergoing long-term passaging increase ß3 Integrin expression levels and behave as reactive astrocytes without needing pro-inflammatory stimuli. We used DITNC1 cells with different passage numbers to study reactivity markers using immunoblots, immunofluorescence, and astrocyte adhesion/migration assays. We also evaluated ß3 Integrin levels by immunoblot and flow cytometry, as well as the neurotoxic effects of reactive astrocytes. Serial cell passaging mimicked the effects of inflammatory stimuli, inducing astrocyte reactivity. Indeed, in response to Thy-1, ß3 Integrin levels, as well as cell adhesion and migration, gradually increased with multiple passages. Importantly, these long-lived astrocytes expressed and secreted factors that inhibited neurite outgrowth and caused neuronal death, just like reactive astrocytes in culture. Therefore, we describe two DITNC1 cell types: a non-reactive type that can be activated with Tumor Necrosis Factor (TNF) and another one that exhibits reactive astrocyte features even in the absence of TNF treatment. Our results emphasize the importance of passage numbers in cell behavior. Likewise, we compare the pro-inflammatory stimulus versus long-term in-plate passaging of cell cultures and introduce them as astrocyte models to study the reactivity process.