The Diagnostic and Prognostic Potentials of Non-Coding RNA in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a rare biliary tract tumor with high malignancy. CCA is the second most common primary hepatobiliary cancer after hepatocarcinoma. Despite its rarity, the incidence of CCA is steadily increasing globally. Most patients with CCA are asymptomatic in the early stages, resulting in a late-stage diagnosis and poor prognosis. Finding reliable biomarkers is essential to improve CCA's early diagnosis and survival rate. Non-coding RNAs (ncRNAs) are non-protein coding RNAs produced by genomic transcription. This includes microRNAs, long non-coding RNAs, and circular RNAs. ncRNAs have multiple functions in regulating gene expression and are crucial for maintaining normal cell function and developing diseases. Many studies have shown that aberrantly expressed ncRNAs can regulate the occurrence and development of CCA. ncRNAs can be easily extracted and detected through tumor tissue and liquid biopsies, representing a potential tool for diagnosing and prognosis CCA. This review will provide a detailed update on the diagnostic and prognostic potentials of lncRNAs and cirRNAs as biomarkers in CCA.

Principles in the Management of Glioblastoma.

Glioblastoma, the most aggressive and common malignant primary brain tumour, is characterized by infiltrative growth, abundant vascularization, and aggressive clinical evolution. Patients with glioblastoma often face poor prognoses, with a median survival of approximately 15 months. Technological progress and the subsequent improvement in understanding the pathophysiology of these tumours have not translated into significant achievements in therapies or survival outcomes for patients. Progress in molecular profiling has yielded new omics data for a more refined classification of glioblastoma. Several typical genetic and epigenetic alterations in glioblastoma include mutations in genes regulating receptor tyrosine kinase (RTK)/rat sarcoma (RAS)/phosphoinositide 3-kinase (PI3K), p53, and retinoblastoma protein (RB) signalling, as well as mutation of isocitrate dehydrogenase (IDH), methylation of O6-methylguanine-DNA methyltransferase (MGMT), amplification of epidermal growth factor receptor vIII, and codeletion of 1p/19q. Certain microRNAs, such as miR-10b and miR-21, have also been identified as prognostic biomarkers. Effective treatment options for glioblastoma are limited. Surgery, radiotherapy, and alkylating agent chemotherapy remain the primary pillars of treatment. Only promoter methylation of the gene MGMT predicts the benefit from alkylating chemotherapy with temozolomide and it guides the choice of first-line treatment in elderly patients. Several targeted strategies based on tumour-intrinsic dominant signalling pathways and antigenic tumour profiles are under investigation in clinical trials. This review explores the potential genetic and epigenetic biomarkers that could be deployed as analytical tools in the diagnosis and prognostication of glioblastoma. Recent clinical advancements in treating glioblastoma are also discussed, along with the potential of liquid biopsies to advance personalized medicine in the field of glioblastoma, highlighting the challenges and promises for the future.

Identification of Novel Molecular and Clinical Biomarkers of Survival in Glioblastoma Multiforme Patients: A Study Based on The Cancer Genome Atlas Data.

Glioblastoma multiforme (GBM) is the most prevalent type of brain tumour; although advancements in treatment have been made, the median survival time for GBM patients has persisted at 15 months. This study is aimed at investigating the genetic alterations and clinical features of GBM patients to find predictors of survival. GBM patients' methylation and gene expression data along with clinical information from TCGA were retrieved. The most overrepresented pathways were identified independently for each omics dataset. From the genes found in at least 30% of these pathways, one gene that was identified in both sets was further examined using the Kaplan-Meier method for survival analysis. Additionally, three groups of patients who started radio and chemotherapy at different times were identified, and the influence of these variations in treatment modality on patient survival was evaluated. Four pathways that seemed to negatively impact survival and two with the opposite effect were identified. The methylation status of PRKCB was highlighted as a potential novel biomarker for patient survival. The study also found that treatment with chemotherapy prior to radiotherapy can have a significant impact on patient survival, which could lead to improvements in clinical management and therapeutic approaches for GBM patients.

Current Applications and Challenges of Next-Generation Sequencing in Plasma Circulating Tumour DNA of Ovarian Cancer.

Circulating tumour DNA (ctDNA) facilitates longitudinal study of the tumour genome, which, unlike tumour tissue biopsies, globally reflects intratumor and intermetastatis heterogeneity. Despite its costs, next-generation sequencing (NGS) has revolutionised the study of ctDNA, ensuring a more comprehensive and multimodal approach, increasing data collection, and introducing new variables that can be correlated with clinical outcomes. Current NGS strategies can comprise a tumour-informed set of genes or the entire genome and detect a tumour fraction as low as 10-5. Despite some conflicting studies, there is evidence that ctDNA levels can predict the worse outcomes of ovarian cancer (OC) in both early and advanced disease. Changes in those levels can also be informative regarding treatment efficacy and tumour recurrence, capable of outperforming CA-125, currently the only universally utilised plasma biomarker in high-grade serous OC (HGSOC). Qualitative evaluation of sequencing shows that increasing copy number alterations and gene variants during treatment may correlate with a worse prognosis in HGSOC. However, following tumour clonality and emerging variants during treatment poses a more unique opportunity to define treatment response, select patients based on their emerging resistance mechanisms, like BRCA secondary mutations, and discover potential targetable variants. Sequencing of tumour biopsies and ctDNA is not always concordant, likely as a result of clonal heterogeneity, which is better captured in the plasma samples than it is in a large number of biopsies. These incoherences may reflect tumour clonality and reveal the acquired alterations that cause treatment resistance. Cell-free DNA methylation profiles can be used to distinguish OC from healthy individuals, and NGS methylation panels have been shown to have excellent diagnostic capabilities. Also, methylation signatures showed promise in explaining treatment responses, including BRCA dysfunction. ctDNA is evolving as a promising new biomarker to track tumour evolution and clonality through the treatment of early and advanced ovarian cancer, with potential applicability in prognostic prediction and treatment selection. While its role in HGSOC paves the way to clinical applicability, its potential interest in other histological subtypes of OC remains unknown.

Cx43 can form functional channels at the nuclear envelope and modulate gene expression in cardiac cells.

Classically associated with gap junction-mediated intercellular communication, connexin43 (Cx43) is increasingly recognized to possess non-canonical biological functions, including gene expression regulation. However, the mechanisms governing the localization and role played by Cx43 in the nucleus, namely in transcription modulation, remain unknown. Using comprehensive and complementary approaches encompassing biochemical assays, super-resolution and immunogold transmission electron microscopy, we demonstrate that Cx43 localizes to the nuclear envelope of different cell types and in cardiac tissue. We show that translocation of Cx43 to the nucleus relies on Importin-ß, and that Cx43 significantly impacts the cellular transcriptome, likely by interacting with transcriptional regulators. In vitro patch-clamp recordings from HEK293 and adult primary cardiomyocytes demonstrate that Cx43 forms active channels at the nuclear envelope, providing evidence that Cx43 can participate in nucleocytoplasmic shuttling of small molecules. The accumulation of nuclear Cx43 during myogenic differentiation of cardiomyoblasts is suggested to modulate expression of genes implicated in this process. Altogether, our study provides new evidence for further defining the biological roles of nuclear Cx43, namely in cardiac pathophysiology.

AZD-7648, a DNA-PK Inhibitor, Induces DNA Damage, Apoptosis, and Cell Cycle Arrest in Chronic and Acute Myeloid Leukemia Cells.

The non-homologous end joining pathway is vital for repairing DNA double-strand breaks (DSB), with DNA-dependent protein kinase (DNA-PK) playing a critical role. Altered DNA damage response (DDR) in chronic (CML) and acute myeloid leukemia (AML) offers potential therapeutic opportunities. We studied the therapeutic potential of AZD-7648 (DNA-PK inhibitor) in CML and AML cell lines. This study used two CML (K-562 and LAMA-84) and five AML (HEL, HL-60, KG-1, NB-4, and THP-1) cell lines. DDR gene mutations were obtained from the COSMIC database. The copy number and methylation profile were evaluated using MS-MLPA and DDR genes, and telomere length using qPCR. p53 protein expression was assessed using Western Blot, chromosomal damage through cytokinesis-block micronucleus assay, and γH2AX levels and DSB repair kinetics using flow cytometry. Cell density and viability were analyzed using trypan blue assay after treatment with AZD-7648 in concentrations ranging from 10 to 200 µM. Cell death, cell cycle distribution, and cell proliferation rate were assessed using flow cytometry. The cells displayed different DNA baseline damage, DDR gene expressions, mutations, genetic/epigenetic changes, and p53 expression. Only HEL cells displayed inefficient DSB repair. The LAMA-84, HEL, and KG-1 cells were the most sensitive to AZD-7648, whereas HL-60 and K-562 showed a lower effect on density and viability. Besides the reduction in cell proliferation, AZD-7648 induced apoptosis, cell cycle arrest, and DNA damage. In conclusion, these results suggest that AZD-7648 holds promise as a potential therapy for myeloid leukemias, however, with variations in drug sensitivity among tested cell lines, thus supporting further investigation to identify the specific factors influencing sensitivity to this DNA-PK inhibitor.

Basal cell carcinomas of the scalp after radiotherapy for tinea capitis: Clinicopathological study in a case series of 96 patients with analysis of 427 tumours.

BACKGROUND/OBJECTIVES: Low-dose X-ray radiotherapy to treat tinea capitis during childhood is a well-known risk factor for scalp basal cell carcinomas (BCCs). Post-radiotherapy BCCs are often multiple, and it has been suggested that they display more aggressive features. Our main objective was to study the clinicopathological aspects of post-radiotherapy BCCs to evaluate their biological behaviour and identify features that may differ from other BCCs. METHODS: We performed an observational, retrospective study assessing multiple clinical and pathological characteristics of patients with post-radiotherapy BCCs. RESULTS: We studied 96 patients with 427 post-radiotherapy scalp BCCs. Post-radiotherapy BCCs were often multiple (median of 4 lesions/patient, ranging from 1 to 54). Significant comorbidities included a high incidence of thyroid disease and meningiomas. Recurrences were observed in 23% of patients, but there may be confounding factors, such as referral bias, heterogenous treatment modalities and occurrence of new tumours due to field effect. We found a high incidence of infundibulocystic BCCs (in 14.6% of patients and corresponding to 5.4% of the total number of tumours), trichoblastomas (5.2%) and neurofibromas of the scalp (10%). CONCLUSIONS: This study is consistent with the occurrence of multiple lesions (sometimes numerous) and a relatively high tendency for recurrence in post-radiotherapy BCCs, as suggested by previous studies. We also found a high incidence of the infundibulocystic variant and a higher risk of follicular tumours and neurofibromas, which suggests that radiotherapy may influence the type of differentiation of BCCs and contribute to induce neoplasms of different cell lines.

Small supernumerary marker chromosomes derived from human chromosome 11.

Introduction: With only 39 reported cases in the literature, carriers of a small supernumerary marker chromosome (sSMC) derived from chromosome 11 represent an extremely rare cytogenomic condition. Methods: Herein, we present a review of reported sSMC(11), add 18 previously unpublished cases, and closely review eight cases classified as 'centromere-near partial trisomy 11' and a further four suited cases from DECIPHER. Results and discussion: Based on these data, we deduced the borders of the pericentric regions associated with clinical symptoms into a range of 2.63 and 0.96 Mb for chromosome 11 short (p) and long (q) arms, respectively. In addition, the minimal pericentric region of chromosome 11 without triplo-sensitive genes was narrowed to positions 47.68 and 60.52 Mb (GRCh37). Furthermore, there are apparent differences in the presentation of signs and symptoms in carriers of larger sSMCs derived from chromosome 11 when the partial trisomy is derived from different chromosome arms. However, the number of informative sSMC(11) cases remains low, with overlapping presentation between p- and q-arm-imbalances. In addition, uniparental disomy (UPD) of 'normal' chromosome 11 needs to be considered in the evaluation of sSMC(11) carriers, as imprinting may be an influencing factor, although no such cases have been reported. Comprehensively, prenatal sSMC(11) cases remain a diagnostic and prognostic challenge.

Basal Cell Carcinomas After Radiotherapy Show More Frequent Follicular Differentiation Than Tumors From Sun-Exposed Areas: Immunohistochemical Study With a Special Focus on Infundibulocystic Basal Cell Carcinoma.

ABSTRACT: Patients submitted to radiotherapy for tinea capitis in childhood have an increased incidence of scalp basal cell carcinomas (BCCs) but also of other neoplasms, namely, follicular tumors. In a cohort of such patients, we also found a high incidence of infundibulocystic BCCs, an otherwise rare variant. We thus hypothesized that postradiotherapy BCCs could be more prone to display follicular differentiation. We compared the histological and immunohistochemical features of postradiotherapy BCCs [both conventional (16 cases) and infundibulocystic (16 cases)] with those of BCCs arising in sun-exposed areas (16 cases), using markers of follicular differentiation (PHLDA-1, CK15, CD34, ß-catenin, and calretinin). Postradiotherapy BCCs showed slightly higher tendency for infundibular and/or trichilemmal differentiation than BCCs from sun-exposed areas (37.5% vs. 18.8%), but this difference was not statistically significant. Nevertheless, infundibulocystic BCCs showed more frequent expression of PHLDA-1 and stronger cytoplasmic expression of CK15 compared with the other lesions. In addition, CD34 highlighted a characteristic meshwork of stromal cells surrounding the epithelial component in all infundibulocystic BCCs, in contrast to the other postradiotherapy BCCs and UV-related BCCs, in which 78.1% were negative or only focally positive. In conclusion, our study suggests a tendency for more frequent follicular differentiation in postradiotherapy BCCs compared with BCCs from sun-exposed areas. In addition, the immunohistochemical study confirms previous data from the literature regarding infundibulocystic BCCs (higher CK15 and PHLDA-1 expression) and shows a distinctive stromal positivity for CD34 that has not been previously acknowledged in these tumors.

Integrated Multi-Omics Signature Predicts Survival in Head and Neck Cancer.

Head and Neck Cancer (HNC) is characterized by phenotypic, biological, and clinical heterogeneity. Despite treatment modalities, approximately half of all patients will die of the disease. Several molecular biomarkers have been investigated, but until now, without clinical translation. Here, we identified an integrative nine-gene multi-omics signature correlated with HNC patients' survival independently of relapses or metastasis development. This prognosis multi-omic signature comprises genes mapped in the chromosomes 1q, 3p, 8q, 17q, 19p, and 19q and encompasses alterations at copy number, gene expression, and methylation. Copy number alterations in LMCD1-A1S and GRM7, the methylation status of CEACAM19, KRT17, and ST18, and the expression profile of RPL29, UBA7, FCGR2C, and RPSAP58 can predict the HNC patients' survival. The difference higher than two years observed in the survival of HNC patients that harbor this nine-gene multi-omics signature can represent a significant step forward to improve patients' management and guide new therapeutic targets development.

Recommendations for reporting results of diagnostic genomic testing.

Results of clinical genomic testing must be reported in a clear, concise format to ensure they are understandable and interpretable. It is important laboratories are aware of the information which is essential to make sure the results are not open to misinterpretation. As genomic testing has continued to evolve over the past decade, the European Society of Human Genetics (ESHG) recommendations for reporting results of diagnostic genetic testing (biochemical, cytogenetic and molecular genetic) published in 2014 have been reviewed and updated to provide the genomic community with guidance on reporting unambiguous results.

Zinc Prevents DNA Damage in Normal Cells but Shows Genotoxic and Cytotoxic Effects in Acute Myeloid Leukemia Cells.

Genomic instability is prevented by the DNA damage response (DDR). Micronutrients, like zinc (Zn), are cofactors of DDR proteins, and micronutrient deficiencies have been related to increased cancer risk. Acute myeloid leukemia (AML) patients commonly present Zn deficiency. Moreover, reports point to DDR defects in AML. We studied the effects of Zn in DDR modulation in AML. Cell lines of AML (HEL) and normal human lymphocytes (IMC) were cultured in standard culture, Zn depletion, and supplementation (40 µM ZnSO4) conditions and exposed to hydrogen peroxide (H2O2) or ultraviolet (UV) radiation. Chromosomal damage, cell death, and nuclear division indexes (NDI) were assessed through cytokinesis-block micronucleus assay. The phosphorylated histone H2AX (yH2AX) expression was monitored at 0 h, 1 h, and 24 h after exposure. Expression of DDR genes was evaluated by quantitative real time polymerase chain reaction (qPCR). Zn supplementation increased the genotoxicity of H2O2 and UV radiation in AML cells, induced cytotoxic and antiproliferative effects, and led to persistent yH2AX activation. In contrast, in normal lymphocytes, supplementation decreased damage rates, while Zn depletion favored damage accumulation and impaired repair kinetics. Gene expression was not affected by Zn depletion or supplementation. Zn presented a dual role in the modulation of genome damage, preventing damage accumulation in normal cells and increasing genotoxicity and cytotoxicity in AML cells.

Development of a genomic predictive model for cholangiocarcinoma using copy number alteration data.

AIMS: Cholangiocarcinoma (CC) is a rare tumour arising from the biliary tract epithelium. The aim of this study was to perform a genomic characterisation of CC tumours and to implement a model to differentiate extrahepatic (ECC) and intrahepatic (ICC) cholangiocarcinoma. METHODS: DNA extracted from tumour samples of 23 patients with CC, namely 10 patients with ECC and 13 patients with ICC, was analysed by array comparative genomic hybridisation. A support vector machine algorithm for classification was applied to the genomic data to distinguish between ICC and ECC. A survival analysis comparing both groups of patients was also performed. RESULTS: With these whole genome results, we observed several common alterations between tumour samples of the same CC anatomical type, namely gain of Xp and loss of 3p, 11q11, 14q, 16q, Yp and Yq in ICC tumours, and gain of 16p25.3 and loss of 3q26.1, 6p25.3-22.3, 12p13.31, 17p, 18q and Yp in ECC tumours. Gain of 2q37.3 was observed in the samples of both tumour subtypes, ICC and ECC. The developed genomic model comprised four chromosomal regions that seem to enable the distinction between ICC and ECC, with an accuracy of 71.43% (95% CI 43% to 100%). Survival analysis revealed that in our cohort, patients with ECC survived on average 8 months less than patients with ICC. CONCLUSIONS: This genomic characterisation and the introduction of genomic models to clinical practice could be important for patient management and for the development of targeted therapies. The power of this genomic model should be evaluated in other CC populations.

Genomic characterisation of multiple myeloma: study of a Portuguese cohort.

Multiple myeloma (MM) genomic complexity reflects in the variable patients' clinical presentation. Genome-wide studies seem to be a reasonable alternative to identify critical genomic lesions. In the current study, we have performed the genomic characterisation of a Portuguese cohort of patients with MM by array comparative genomic hybridisation. Overall, the most frequently detected alterations were 13q deletions, gains of 1q, 19p, 15q, 5p and 7p and trisomy 9. Even though some identified genomic alterations were previously associated with a prognostic value, other abnormalities remain with unknown, but putative significance for patients' clinical practice. These genomic alterations should be further assessed as possible biomarkers.

Erratum zu: Über die Notwendigkeit der Anerkennung von sog. Kernberufsgruppen innerhalb der genetischen Gesundheitsversorgung in Europa.

[This corrects the article DOI: 10.1515/medgen-2022-2116.].

Genomic-Metabolomic Associations Support the Role of LIPC and Glycerophospholipids in Age-Related Macular Degeneration.

PURPOSE: Large-scale genome-wide association studies (GWAS) have reported important single nucleotide polymorphisms (SNPs) with significant associations with age-related macular degeneration (AMD). However, their role in disease development remains elusive. This study aimed to assess SNP-metabolite associations (i.e., metabolite quantitative trait loci [met-QTL]) and to provide insights into the biological mechanisms of AMD risk SNPs. DESIGN: Cross-sectional multicenter study (Boston, Massachusetts, and Coimbra, Portugal). PARTICIPANTS: Patients with AMD (n = 388) and control participants (n = 98) without any vitreoretinal disease (> 50 years). METHODS: Age-related macular degeneration grading was performed using color fundus photographs according to the Age-Related Eye Disease Study classification scheme. Fasting blood samples were collected and evaluated with mass spectrometry for metabolomic profiling and Illumina OmniExpress for SNPs profiling. Analyses of met-QTL of endogenous metabolites were conducted using linear regression models adjusted for age, gender, smoking, 10 metabolite principal components (PCs), and 10 SNP PCs. Additionally, we analyzed the cumulative effect of AMD risk SNPs on plasma metabolites by generating genetic risk scores and assessing their associations with metabolites using linear regression models, accounting for the same covariates. Modeling was performed first for each cohort, and then combined by meta-analysis. Multiple comparisons were accounted for using the false discovery rate (FDR). MAIN OUTCOME MEASURES: Plasma metabolite levels associated with AMD risk SNPs. RESULTS: After quality control, data for 544 plasma metabolites were included. Meta-analysis of data from all individuals (AMD patients and control participants) identified 28 significant met-QTL (ß = 0.016-0.083; FDR q-value < 1.14 × 10-2), which corresponded to 5 metabolites and 2 genes: ASPM and LIPC. Polymorphisms in the LIPC gene were associated with phosphatidylethanolamine metabolites, which are glycerophospholipids, and polymorphisms in the ASPM gene with branched-chain amino acids. Similar results were observed when considering only patients with AMD. Genetic risk score-metabolite associations further supported a global impact of AMD risk SNPs on the plasma metabolome. CONCLUSIONS: This study demonstrated that genomic-metabolomic associations can provide insights into the biological relevance of AMD risk SNPs. In particular, our results support that the LIPC gene and the glycerophospholipid metabolic pathway may play an important role in AMD, thus offering new potential therapeutic targets for this disease.

A seven-gene signature to predict the prognosis of oral squamous cell carcinoma.

The prognosis of oral squamous cell carcinoma (OSCC) patients remains poor without implemented biomarkers in the clinical routine practice to help in the patient's management. With this study we aimed to identify specific prognostic biomarkers for OSCC using a whole genome technology as well as to verify the clinical utility of a head and neck cancer-specific multiplex ligation-dependent probe amplification (MLPA) panel. A genomic characterization of tumor samples from 62 OSCC patients was performed using array comparative genomic hybridization (aCGH) and a more straightforward and cost-effective molecular technology, MLPA. The identification of a genomic signature and prognosis biomarkers was carried out by applying several statistical methods. With aCGH we observed that the chromosomes most commonly altered were 3p, 3q, 5q, 6p, 7q, 8p, 8q, 11q, 15q, 17q, and 18q. The MLPA results showed that the chromosomes with a higher frequency of alterations were 3p, 3q, 8p, 8q, and 11q. We identified a genomic signature with seven genes OCLN (3p21.31), CLDN16 (3q29), SCRIB (3q29), IKBKB (3q22.3), PAK2 (8q22.3), PIK3CB (3q28), and YWHAZ (8q24.3) that together allow to differentiate the patients that developed metastases or relapses after primary tumor treatment, with an overall accuracy of 79%. Amplification of PIK3CB as a predictor of metastases or relapses development was validated using TCGA data. This amplified gene showed a reduction in more than 5 years in the median survival of the patients. The identified biomarkers might have a significant impact in the patients' management and could leverage the OSCC precision medicine.