Search Results (56)

Acute lymphoblastic leukaemia is the most common childhood malignancy that remains a leading cause of death in childhood. It may be characterised by multiple known recurrent genetic aberrations that inform prognosis, the most common being hyperdiploidy and t(12;21) ETV6::RUNX1. We aimed to assess the applicability of a new imaging flow cytometry methodology that incorporates cell morphology, immunophenotype, and fluorescence in situ hybridisation (FISH) to identify aneuploidy of chromosomes 4 and 21 and the translocation ETV6::RUNX1. We evaluated this new “immuno-flowFISH” platform on 39 cases of paediatric ALL of B-lineage known to have aneuploidy of chromosomes 4 and 21 and the translocation ETV6::RUNX1. After identifying the leukaemic population based on immunophenotype (i.e., expression of CD34, CD10, and CD19 antigens), we assessed for copy numbers of loci for the centromeres of chromosomes 4 and 21 and the ETV6 and RUNX1 regions using fluorophore-labelled DNA probes in more than 1000 cells per sample. Trisomy 4 and 21, tetrasomy 21, and translocations of ETV6::RUNX1, as well as gains and losses of ETV6 and RUNX1, could all be identified based on FISH spot counts and digital imagery. There was variability in clonal makeup in individual cases, suggesting the presence of sub-clones. Copy number alterations and translocations could be detected even when the cell population comprised less than 1% of cells and included cells with a mature B-cell phenotype, i.e., CD19-positive, lacking CD34 and CD10. In this proof-of-principle study of 39 cases, this sensitive and specific semi-automated high-throughput imaging flow cytometric immuno-flowFISH method has been able to show that alterations in ploidy and ETV6::RUNX1 could be detected in the 39 cases of paediatric ALL. This imaging flow cytometric FISH method has potential applications for diagnosis and monitoring disease and marrow regeneration (i.e., distinguishing residual ALL from regenerating haematogones) following chemotherapy. Full article

Introduction: Invasive prenatal testing with chromosomal microarray analysis after first-trimester screening is a relevant option but there is still debate regarding the indications. Therefore, we evaluated the prevalence of numerical chromosomal aberrations detected by classic karyotype and clinically relevant copy number variants (CNVs) in prenatal samples using array comparative genomic hybridization (aCGH) stratified to NT thickness: <the 95th percentile, the 95th percentile–2.9 mm, 3.0–3.4 mm, 3.5–3.9 mm, 4.0–4.5 mm, and >4.5 mm, and by the presence/absence of associated structural anomalies detected by ultrasonography. Materials and Methods: Retrospective cohort study carried out at two tertiary Polish centers for prenatal diagnosis (national healthcare system) in central and south regions from January 2018 to December 2021. A total of 1746 prenatal samples were received. Indications for invasive prenatal testing included high risk of Down syndrome in the first-trimester combined test (n = 1484) and advanced maternal age (n = 69), and, in 193 cases, other reasons, such as parental request, family history of congenital defects, and genetic mutation carrier, were given. DNA was extracted directly from amniotic fluid (n = 1582) cells and chorionic villus samples (n = 164), and examined with classic karyotype and aCGH. Results: Of the entire cohort of 1746 fetuses, classical karyotype revealed numerical chromosomal aberrations in 334 fetuses (19.1%), and aCGH detected CNV in 5% (n = 87). The frequency of numerical chromosomal aberrations increased with NT thickness from 5.9% for fetuses with NT < p95th to 43.3% for those with NT > 4.5 mm. The highest rate of numerical aberrations was observed in fetuses with NT > 4.5 mm having at least one structural anomaly (50.2%). CNVs stratified by NT thickness were detected in 2.9%, 2.9%, 3.5%, 4.3%, 12.2%, and 9.0% of fetuses with NT < 95th percentile, 95th percentile–2.9 mm, 3.0–3.4 mm, 3.5–3.9 mm, 4.0–4.5 mm, and >4.5 mm, respectively. After exclusion of fetuses with structural anomalies and numerical aberrations, aCGH revealed CNVs in 2.0% of fetuses with NT < 95th percentile, 1.5% with NTp95–2.9 mm, 1.3% with NT 3.0–3.4 mm, 5.4% with NT 3.5–3.9 mm, 19.0% with NT 4.0–4.5 mm, and 14.8% with NT > 4.5 mm. Conclusions: In conclusion, our study indicates that performing aCGH in samples referred to invasive prenatal testing after first-trimester screening provides additional clinically valuable information over conventional karyotyping, even in cases with normal NT and anatomy. Full article

JARID2 (Jumonji, AT-rich interactive domain 2) haploinsufficiency is associated with a clinically distinct neurodevelopmental syndrome. It is characterized by intellectual disability, developmental delay, autistic features, behavior abnormalities, cognitive impairment, hypotonia, and dysmorphic features. JARID2 acts as a transcriptional repressor protein that is involved in the regulation of histone methyltransferase complexes. JARID2 plays a role in the epigenetic machinery, and the associated syndrome has an identified DNA methylation episignature derived from sequence variants and intragenic deletions involving JARID2. For this study, our aim was to determine whether patients with larger deletions spanning beyond JARID2 present a similar DNA methylation episignature and to define the critical region involved in aberrant DNA methylation in 6p22–p24 microdeletions. We examined the DNA methylation profiles of peripheral blood from 56 control subjects, 13 patients with (likely) pathogenic JARID2 variants or patients carrying copy number variants, and three patients with JARID2 VUS variants. The analysis showed a distinct and strong differentiation between patients with (likely) pathogenic variants, both sequence and copy number, and controls. Using the identified episignature, we developed a binary model to classify patients with the JARID2-neurodevelopmental syndrome. DNA methylation analysis indicated that JARID2 is the driver gene for aberrant DNA methylation observed in 6p22–p24 microdeletions. In addition, we performed analysis of functional correlation of the JARID2 genome-wide methylation profile with the DNA methylation profiles of 56 additional neurodevelopmental disorders. To conclude, we refined the critical region for the presence of the JARID2 episignature in 6p22–p24 microdeletions and provide insight into the functional changes in the epigenome observed when regulation by JARID2 is lost. Full article

Sarcoma classification is challenging and can lead to treatment delays. Previous studies used DNA aberrations and machine-learning classifiers based on methylation profiles for diagnosis. We aimed to classify sarcomas by analyzing methylation signatures obtained from low-coverage whole-genome sequencing, which also identifies copy-number alterations. DNA was extracted from 23 suspected sarcoma samples and sequenced on an Oxford Nanopore sequencer. The methylation-based classifier, applied in the nanoDx pipeline, was customized using a reference set based on processed Illumina-based methylation data. Classification analysis utilized the Random Forest algorithm and t-distributed stochastic neighbor embedding, while copy-number alterations were detected using a designated R package. Out of the 23 samples encompassing a restricted range of sarcoma types, 20 were successfully sequenced, but two did not contain tumor tissue, according to the pathologist. Among the 18 tumor samples, 14 were classified as reported in the pathology results. Four classifications were discordant with the pathological report, with one compatible and three showing discrepancies. Improving tissue handling, DNA extraction methods, and detecting point mutations and translocations could enhance accuracy. We envision that rapid, accurate, point-of-care sarcoma classification using nanopore sequencing could be achieved through additional validation in a diverse tumor cohort and the integration of methylation-based classification and other DNA aberrations. Full article

It has been shown that the loss of function of the BRCA1, BRCA2, and PALB2 genes due to a number of hereditary mutations or chromosomal aberrations can affect the effectiveness of chemotherapy treatment and disease prognosis in patients with various types of cancer, and in particular in breast cancer. Thus, the aim of the work was to evaluate the predictive and prognostic potential of DNA copy number aberrations and mutations in the BRCA1, BRCA2, and PALB2 genes in breast tumors. Materials and Methods: The study included 66 patients with breast cancer. DNA copy number aberrations (CNA) were assessed by high-density CytoScanHD™ Array micro matrix analysis. Gene mutations were assessed by sequencing on the MiSeq™ Sequencing System using the Accel-Amplicon BRCA1, BRCA2, and PALB2 Panel. Results: It has been established that the presence of a normal copy number of PALB2 is associated with a lack of response to chemotherapy in Taxotere-containing treatment regimens (p = 0.05). In addition, the presence of a PALB2 deletion is associated with 100% metastatic survival rates (log-rank test p = 0.04). As a result of sequencing, 25 mutations were found in the BRCA1 gene, 42 mutations in BRCA2, and 27 mutations in the PALB2 gene. The effect of mutations on the effectiveness of treatment is controversial, but an effect on the survival of patients with breast cancer has been shown. So, in the presence of pathogenic mutations in the BRCA2 gene, 100% metastatic survival is observed (log-rank test p = 0.05), as well as in the elimination of PALB2 mutations during treatment (log-rank test p = 0.07). Conclusion: Currently, there is little data on the effect of chromosomal aberrations and mutations in the BRCA1/2 and PALB2 genes on the effectiveness of treatment and prognosis of the disease. At the same time, the study of these genes has great potential for testing focused on a personalized approach to the treatment of patients with breast cancer. Full article

Background: Liquid biopsy is widely recognized as an efficient diagnostic method in oncology for disease detection and monitoring. Though the examination of circulating tumor cells (CTC) is mostly implemented for the assessment of genomic aberrations, the need of complex methodologies for their detection has impeded its acceptance in low-resource settings. We evaluated cell-free DNA (cfDNA) as a liquid biopsy tool and investigated its utility in breast cancer patients. Methods: Total cell-free DNA was extracted from the plasma of breast cancer patients (n = 167) with a median follow-up of more than 5 years, at various stages of the disease. Quantitative PCR was performed to estimate the copy numbers of two fractions of ALU repetitive elements (ALU 115 and ALU 247), and DNA integrity (DI) was calculated as the ratio of ALU 247/115. Mutations in TP53 and PIK3CA in the cfDNA were estimated by next-gen sequencing (NGS) in a subset of samples. Associations of the levels of both the ALU fragments with various clinico-pathological factors and disease-free survival at various stages were examined. Nomogram models were constructed with clinical variables and ALU 247 levels to predict disease-free survival and the best performing model was evaluated by decision curve analysis. Results: DI and ALU 247 levels were significantly lower (p < 0.0001) in the post-operative plasma when compared to their pre-surgery levels. DI and ALU 247 were found to be significantly higher in patients with metastasis (p < 0.05). Patients with higher levels of ALU 247 in their post-operative plasma had significant poor disease-free survival (p = 0.005). Higher levels of ALU 247 in the circulation also correlated with low tumor-infiltrating lymphocytes (TIL) within their primary tumors in the ER-negative breast cancer subtype (p = 0.01). Cox proportional hazard analysis confirmed ALU 247 as an independent variable of disease-free survival both in univariate and multivariate analysis [HR 1.3 (95% CI 1.047 to 1.613, p = 0.017)]. The nomogram model showed that the addition of ALU 247 with other variables significantly improved (C-index 0.823) the predictive ability of the model. Conclusion: Our results confirm the utility of cfDNA as an evolving liquid biopsy tool for molecular analysis. Evaluation of larger fragments of cfDNA estimated through ALU 247 can provide vital information concurrent with the pathological process of disease evolution in breast cancer and warrants expansion to other cancer types. Full article

Background: Progress in the diagnosis and treatment of clear cell renal cell carcinoma (ccRCC) has significantly prolonged patient survival. However, ccRCC displays an extreme heterogenous characteristic and metastatic tendency, which limit the benefit of targeted or immune therapy. Thus, identifying novel biomarkers and therapeutic targets for ccRCC is of great importance. Method: Pan cancer datasets, including the expression profile, DNA methylation, copy number variation, and single nucleic variation, were introduced to decode the aberrance of copper death regulators (CDRs). Then, FDX1 was systematically analyzed in ccRCC to evaluate its impact on clinical characteristics, prognosis, biological function, immune infiltration, and therapy response. Finally, in vivo experiments were utilized to decipher FDX1 in ccRCC malignancy and its role in tumor immunity. Result: Copper death regulators were identified at the pancancer level, especially in ccRCC. FDX1 played a protective role in ccRCC, and its expression level was significantly decreased in tumor tissues, which might be regulated via CNV events. At the molecular mechanism level, FDX1 positively regulated fatty acid metabolism and oxidative phosphorylation. In addition, FDX1 overexpression restrained ccRCC cell line malignancy and enhanced tumor immunity by increasing the secretion levels of IL2 and TNFγ. Conclusions: Our research illustrated the role of FDX1 in ccRCC patients’ clinical outcomes and its impact on tumor immunity, which could be treated as a promising target for ccRCC patients. Full article

The concept of BRCAness was developed because of similarities between sporadic and hereditary breast cancer. BRCAness defines the pathogenesis and treatment sensitivity of many types of cancer, as well as the presence of a defect in the homologous recombination repair of tumor cells simulating the loss of BRCA1 or BRCA2, as in the presence of germline mutations. The question of treatment effectiveness for BRCA-like tumors is controversial and open. Thus, the aim of this work was to study the effectiveness of neoadjuvant chemotherapy (NAC) in BRCA-deficient breast cancer patients without germline mutations. The study involved 130 patients with breast cancer in stages IIA–IIIB. The treatment regimen included neoadjuvant chemotherapy, surgery, and adjuvant chemotherapy. The materials used were tumor samples from before and after chemotherapy. DNA and RNA were isolated from the tumor material. RNA was used to assess the expression level of BRCA1, while DNA was used for methyl-sensitive PCR. A microarray analysis was performed on high-density DNA chips from an Affymetrix CytoScan^TM HD Array to assess DNA copy number aberration (CNA status) and loss of heterozygosity. A statistical analysis was performed using the Statistica 8.0 application package. It was noted that the existence of copy number aberrations in genes was statistically significantly associated with tumor treatment response and disease prognosis. Patients with partial regression had a statistically significantly higher amount of deletion than patients without an objective response (5/25 patients; 16%), as shown in the general sample of patients (52.9% versus 27.1%, respectively) at p = 0.0001 and in patients treated with anthracycline-containing regimen (p = 0.0001). In addition, it was shown that patients with BRCA1 deletion had higher rates of metastatic-free survival (log rank test, p = 0.009). BRCAness patients had a higher rate of 5-year metastatic survival, but not of treatment efficacy. The prospective study showed the positive effect of assessing the BRCAness phenotype of a tumor before treatment and of prescribing personalized NAC regimens. The objective response rate was statistically significantly more often observed in the group of patients with personalized chemotherapy (85.0% (34/40 patients) versus 62.3% (56/90 patients); p = 0.007). Despite the controversial effectiveness of BRCA-like tumor treatment, our data showed high predictive and prognostic significance of the BRCAness phenotype for the personalization of platinum and taxane regimens. Full article

Molecular biomarkers, such as IDH1/IDH2 mutations and 1p19q co-deletion, are included in the histopathological and clinical criteria currently used to diagnose and classify gliomas. IDH1/IDH2 mutation is a common feature of gliomas and is associated with a glioma-CpG island methylator phenotype (CIMP). Aberrant genomic methylation patterns can also be used to extrapolate information about copy number variation in a tumor. This project’s goal was to assess the feasibility of DNA methylation array for the simultaneous detection of glioma biomarkers as a more effective testing strategy compared to existing single analyte tests. Methods: Whole-genome methylation array (WGMA) testing was performed using 48 glioma DNA samples to detect methylation aberrations and chromosomal gains and losses. The analyzed samples include 39 tumors in the discovery cohort and 9 tumors in the replication cohort. Methylation profiles for each sample were correlated with IDH1 p.R132G mutation, immunohistochemistry (IHC), and previous 1p19q clinical testing to assess the sensitivity and specificity of the WGMA assay for the detection of these variants. Results: We developed a DNA methylation signature to specifically distinguish a IDH1/IDH2 mutant tumor from normal samples. This signature is composed of 11 CpG sites that were significantly hypermethylated in the IDH1/IDH2 mutant group. Copy number analysis using WGMA data was able to identify five of five positive samples for 1p19q co-deletion and was concordant for all negative samples. Conclusions: The DNA methylation signature presented here has the potential to refine the utility of WGMA to predict IDH1/IDH2 mutation status of gliomas, thus improving diagnostic yield and efficiency of laboratory testing compared to single analyte IDH1/IDH2 or 1p19q tests. Full article

Medulloblastoma is a pediatric brain malignancy that consists of four transcriptional subgroups. Structural and numerical aneuploidy are common in all subgroups, although they are particularly profound in Group 3 and Group 4 medulloblastoma and in a subtype of SHH medulloblastoma termed SHHα. This suggests that chromosomal instability (CIN), the process leading to aneuploidy, is an important player in medulloblastoma pathophysiology. However, it is not known if there is ongoing CIN in medulloblastoma or if CIN affects the developing cerebellum and promotes tumor formation. To investigate this, we performed karyotyping of single medulloblastoma cells and demonstrated the presence of distinct tumor cell clones harboring unique copy number alterations, which is suggestive of ongoing CIN. We also found enrichment for processes related to DNA replication, repair, and mitosis in both SHH medulloblastoma and in the highly proliferative compartment of the presumed tumor cell lineage-of-origin, the latter also being sensitive to genotoxic stress. However, when challenging these tumor cells-of-origin with genetic lesions inducing CIN using transgenic mouse modeling, we found no evidence for large chromosomal aberrations in the cerebellum or for medulloblastoma formation. We therefore conclude that without a background of specific genetic mutations, CIN is not tolerated in the developing cerebellum in vivo and, thus, by itself is not sufficient to initiate medulloblastoma. Full article

Introduction: Salivary gland cancer (SGC) is a rare cancer for which systemic treatment options are limited. Therefore, it is important to characterize its genetic landscape in search for actionable aberrations, such as NTRK gene fusions. This research aimed to identify these actionable aberrations by combining NGS-based analysis of RNA (gene fusions) and DNA (single and multiple nucleotide variants, copy number variants, microsatellite instability and tumor mutational burden) in a large cohort of SGC patients. Methods: RNA and DNA were extracted from archival tissue of 121 patients with various SGC subtypes. Gene fusion analysis was performed using a customized RNA-based targeted NGS panel. DNA was sequenced using a targeted NGS panel encompassing 523 cancer-related genes. Cross-validation of NGS-based NTRK fusion detection and pan-TRK immunohistochemistry (IHC) was performed. Results: Fusion transcripts were detected in 50% of the cases and included both known (MYB-NFIB, MYBL1-NFIB, CRTC1-MAML2) and previously unknown fusions (including transcripts involving RET, BRAF or RAD51B). Only one NTRK fusion transcript was detected, in a secretory carcinoma case. Pan-TRK IHC (clone EPR17341) was false positive in 74% of cases. The proportion of patients with targets for genetically matched therapies differed among subtypes (salivary duct carcinoma: 82%, adenoid cystic carcinoma 28%, mucoepidermoid carcinoma 50%, acinic cell carcinoma 33%). Actionable aberrations were most often located in PIK3CA (n = 18, 15%), ERBB2 (n = 15, 12%), HRAS and NOTCH1 (both n = 9, 7%). Conclusions: Actionable genetic aberrations were seen in 53.7% of all SGC cases on the RNA and DNA level, with varying percentages between subtypes. Full article

Neuroblastoma (NB) is a tumor of the developing sympathetic nervous system. Despite recent advances in understanding the complexity of NB, the mechanisms that determine its regression or progression are still largely unknown. Stage 4S NB is characterized by a favorable course of disease and often by spontaneous regression, while progression to true stage 4 is a very rare event. Here, we focused on genomic analysis of an NB case that progressed from stage 4S to stage 4 with a very poor outcome. Array-comparative genomic hybridization (a-CGH) on tumor-tissue DNA, and whole-exome sequencing (WES) on exosomes DNA derived from plasma collected at the onset and at the tumor progression, pointed out relevant genetic changes that can explain this clinical worsening. The combination of a-CGH and WES data allowed for the identification iof somatic copy number aberrations and single-nucleotide variants in genes known to be responsible for aggressive NB. KLRB1, MAPK3 and FANCA genes, which were lost at the time of progression, were studied for their possible role in this event by analyzing in silico the impact of their expression on the outcome of 786 NB patients. Full article

Alveolar soft part sarcoma (ASPS) is a rare subtype of soft tissue sarcoma characterized by an unbalanced translocation, resulting in ASPSCR1-TFE3 fusion that transcriptionally upregulates MET expression. The European Organization for Research and Treatment of Cancer (EORTC) 90101 “CREATE” phase II trial evaluated the MET inhibitor crizotinib in ASPS patients, achieving only limited antitumor activity. We performed a comprehensive molecular analysis of ASPS tissue samples collected in this trial to identify potential biomarkers correlating with treatment outcome. A tissue microarray containing 47 ASPS cases was used for the characterization of the tumor microenvironment using multiplex immunofluorescence. DNA isolated from 34 available tumor samples was analyzed to detect recurrent gene copy number alterations (CNAs) and mutations by low-coverage whole-genome sequencing and whole-exome sequencing. Pathway enrichment analysis was used to identify diseased-associated pathways in ASPS sarcomagenesis. Kaplan–Meier estimates, Cox regression, and the Fisher’s exact test were used to correlate histopathological and molecular findings with clinical data related to crizotinib treatment, aiming to identify potential factors associated with patient outcome. Tumor microenvironment characterization showed the presence of PD-L1 and CTLA-4 in 10 and 2 tumors, respectively, and the absence of PD-1 in all specimens. Apart from CD68, other immunological markers were rarely expressed, suggesting a low level of tumor-infiltrating lymphocytes in ASPS. By CNA analysis, we detected a number of broad and focal alterations. The most common alteration was the loss of chromosomal region 1p36.32 in 44% of cases. The loss of chromosomal regions 1p36.32, 1p33, 1p22.2, and 8p was associated with shorter progression-free survival. Using whole-exome sequencing, 13 cancer-associated genes were found to be mutated in at least three cases. Pathway enrichment analysis identified genetic alterations in NOTCH signaling, chromatin organization, and SUMOylation pathways. NOTCH4 intracellular domain dysregulation was associated with poor outcome, while inactivation of the beta-catenin/TCF complex correlated with improved outcome in patients receiving crizotinib. ASPS is characterized by molecular heterogeneity. We identify genetic aberrations potentially predictive of treatment outcome during crizotinib therapy and provide additional insights into the biology of ASPS, paving the way to improve treatment approaches for this extremely rare malignancy. Full article

In colorectal cancer, somatic mutations have played an important role as prognostic and predictive biomarkers, with some also functioning as therapeutic targets. Another genetic aberration that has shown significance in colorectal cancer is copy number alterations (CNAs). CNAs occur when a change to the DNA structure propagates gain/amplification or loss/deletion in sections of DNA, which can often lead to changes in protein expression. Multiple techniques have been developed to detect CNAs, including comparative genomic hybridization with microarray, low pass whole genome sequencing, and digital droplet PCR. In this review, we summarize key findings in the literature regarding the role of CNAs in the pathogenesis of colorectal cancer, from adenoma to carcinoma to distant metastasis, and discuss the roles of CNAs as prognostic and predictive biomarkers in colorectal cancer. Full article

One of the important reasons for the ineffectiveness of chemotherapy in breast cancer (BC) is considered to be the formation of a multidrug resistance phenotype in tumour cells, which is caused by the expression of energy-dependent ABC transporters. The aim of this work was to assess chromosomal aberrations and the level of transcripts of all 49 known ABC transporter genes in breast tumours. Materials and Methods. The study included 129 patients with breast cancer. A microarray study of all tumour samples was carried out on microchips. Results. This study established that the presence of a deletion in genes ABCB1, ABCB4, ABCB8, ABCC7, ABCC11, ABCC12, ABCF2, and ABCG4 is associated with an objective response to treatment (p ≤ 0.05). A decrease in the expression of genes was associated with a good response to chemotherapy, whereas an increase in expression caused the progression and stabilization of the tumour. Analysis of metastatic-free survival rates showed that the presence of ABCB1/4 and ABCC1/6 deletions was associated with 100% survival (log-rank test p = 0.01 and p = 0.03). Conclusions. The study showed that the aberrant state of ABC transporter genes, as well as a decrease in the expression of these genes, is a predictor of the effectiveness of therapeutic treatment and a potential prognostic marker of metastatic survival. Full article