Investigation of Clinically Significant Molecular Aberrations in Patients with Prostate Cancer: Implications for Personalized Treatment, Prognosis and Genetic Testing.

The data on tumor molecular profiling of European patients with prostate cancer is limited. Our aim was to evaluate the prevalence and prognostic and predictive values of gene alterations in unselected patients with prostate cancer. The presence of gene alterations was assessed in patients with histologically confirmed prostate cancer using the ForeSENTIA® Prostate panel (Medicover Genetics), targeting 36 clinically relevant genes and microsatellite instability testing. The primary endpoint was the prevalence of gene alterations in homologous recombination repair (HRR) genes. Overall, 196 patients with prostate cancer were evaluated (median age 72.2 years, metastatic disease in 141 (71.9%) patients). Gene alterations were identified in 120 (61%) patients, while alteration in HRR genes were identified in 34 (17.3%) patients. The most commonly mutated HRR genes were ATM (17, 8.7%), BRCA2 (9, 4.6%) and BRCA1 (4, 2%). The presence of HRR gene alterations was not associated with advanced stage (p = 0.21), age at diagnosis (p = 0.28), Gleason score (p = 0.17) or overall survival (HR 0.72; 95% CI: 0.41-1.26; p = 0.251). We identified clinically relevant somatic gene alterations in European patients with prostate cancer. These molecular alterations have prognostic significance and therapeutic implications and/or may trigger genetic testing in selected patients. In the era of precision medicine, prospective research on the predictive role of these alterations for innovative treatments or their combinations is warranted.

Targeted pharmacologic inhibition of S-phase kinase-associated protein 2 (SKP2) mediated cell cycle regulation in lung and other RB-Related cancers: A brief review of current status and future prospects.

Small cell lung cancer (SCLC) often exhibits Rb deficiency, TRß and p130 deletion, and SKP2 amplification, suggesting TRß inactivation and SKP2 activation. It is reported that SKP2 targeted therapy is effective in some cancers in vitro and in vivo, but it is not reported for the treatment of SCLC and retinoblastoma. SKP2 is the synthetic lethal gene in SCLC and retinoblastoma, so SKP2 can be used for targeted therapy in SCLC and retinoblastoma. RB1 knockout mice develop several kinds of tumors, but Rb1 and SKP2 double knockout mice are healthy, suggesting that SKP2 targeted therapy may have significant effects on Rb deficient cancers with less side effects, and if successful in SCLC and retinoblastoma in vitro and in animal model, such compounds may be promising for the clinical treatment of SCLC, retinoblastoma, and variety of Rb deficient cancers. Previously our studies showed that retinoblastomas exhibit retinal cone precursor properties and depend on cone-specific thyroid hormone receptor ß2 (TRß2) and SKP2 signaling. In this study, we sought to suppress SCLC and retinoblastoma cell growth by SKP2 inhibitors as a prelude to targeted therapy in vitro and in vivo. We knocked down TRß2 and SKP2 or over-expressed p27 in SCLC and retinoblastoma cell lines to investigate SKP2 and p27 signaling alterations. The SCLC cell lines H209 as well as retinoblastoma cell lines Y79, WERI, and RB177 were treated with SKP2 inhibitor C1 at different concentrations, following which Western blotting, Immunostaining, and cell cycle kinetics studies were performed to study SKP2 and p27 expression ubiquitination, to determine impact on cell cycle regulation and growth inhibition. TRß2 knockdown in Y79, RB177 and H209 caused SKP2 downregulation and degradation, p27 up-regulation, and S phase arrest, whereas, SKP2 knockdown or p27 over-expression caused p27 accumulation and G1-S phase arrest. In the cell lines Y79, WERI, RB177, and H209 treatment with C1 caused SKP2 ubiquitination and degradation, p27 de-ubiquitination and accumulation, and cell growth arrest. SKP2 inhibitor C1 significantly suppressed retinoblastoma as well as SCLC cell growth by SKP2 degradation and p27 accumulation. In vivo study also showed inhibition of tumor growth with C1 treatment. Potential limitations of the success of such a therapeutic approach and its translational application in human primary tumors, and alternative approaches to overcome such limitations are briefly discussed for the treatment of retinoblastoma, SCLC and other RB-related cancers.

Molecular profile and clinical features of patients with gliomas using a broad targeted next generation-sequencing panel.

Gliomas are the most common malignant primary brain tumors characterized by poor prognosis. The genotyping of tumors using next generation sequencing (NGS) platforms enables the identification of genetic alterations that constitute diagnostic, prognostic and predictive biomarkers. The present study investigated the molecular profile of 32 tumor samples from 32 patients with high-grade gliomas by implementing a broad 80-gene targeted NGS panel while reporting their clinicopathological characteristics and outcomes. Subsequently, 14 of 32 tumor specimens were also genotyped using a 55-gene NGS panel to validate the diagnostic accuracy and clinical utility of the extended panel. The median follow-up was 19.2 months. In total, 129 genetic alterations including 33 structural variants were identified in 38 distinct genes. Among 96 variants (single nucleotide variants and insertions and deletions), 38 were pathogenic and 58 variants of unknown clinical significance. TP53 was the most frequently mutated gene, followed by PTEN and IDH1 genes. Glioma patients with IDH1 mutant tumors were younger and had significantly longer overall survival compared to patients with wild-type IDH1 tumors. Similarly, tumors with TP53 mutations were more likely observed in younger patients with glioma. Subsequently, a comparison of mutational profiles of samples analyzed by both panels was also performed. Implementation of the comprehensive pan-cancer and the MOL panels resulted in the identification of 37 and 15 variants, respectively. Of those, 13 were common. Comprehensive pan-cancer panel identified 24 additional variants, 22 of which were located in regions that were not targeted by the MOL panel. By contrast, the MOL panel identified two additional variants. Overall, the present study demonstrated that using an extended tumor profile assay instead of a glioma-specific tumor profile panel identified additional genetic changes that may be taken into consideration as potential therapeutic targets for glioma diagnosis and molecular classification.

Proof-of-Concept Pilot Study on Comprehensive Spatiotemporal Intra-Patient Heterogeneity for Colorectal Cancer With Liver Metastasis.

Introduction: The mechanisms underlying high drug resistance and relapse rates after multi-modal treatment in patients with colorectal cancer (CRC) and liver metastasis (LM) remain poorly understood. Objective: We evaluate the potential translational implications of intra-patient heterogeneity (IPH) comprising primary and matched metastatic intratumor heterogeneity (ITH) coupled with circulating tumor DNA (ctDNA) variability. Methods: A total of 122 multi-regional tumor and perioperative liquid biopsies from 18 patients were analyzed via targeted next-generation sequencing (NGS). Results: The proportion of patients with ITH were 53% and 56% in primary CRC and LM respectively, while 35% of patients harbored de novo mutations in LM indicating spatiotemporal tumor evolution and the necessity of multiregional analysis. Among the 56% of patients with alterations in liquid biopsies, de novo mutations in cfDNA were identified in 25% of patients, which were undetectable in both CRC and LM. All 17 patients with driver alterations harbored mutations targetable by molecularly targeted drugs, either approved or currently under evaluation. Conclusion: Our proof-of-concept prospective study provides initial evidence on potential clinical superiority of IPH and warrants the conduction of precision oncology trials to evaluate the clinical utility of I PH-driven matched therapy.

Clinical Significance of Germline Cancer Predisposing Variants in Unselected Patients with Pancreatic Adenocarcinoma.

Our aim was to determine the prevalence, prognostic and predictive role of germline pathogenic/likely pathogenic variants (P/LPVs) in cancer predisposing genes in patients with pancreatic ductal adenocarcinoma (PDAC). Germline testing of 62 cancer susceptibility genes was performed on unselected patients diagnosed from 02/2003 to 01/2020 with PDAC, treated at Hellenic Cooperative Oncology Group (HeCOG)-affiliated Centers. The main endpoints were prevalence of P/LPVs and overall survival (OS). P/LPVs in PDAC-associated and homologous recombination repair (HRR) genes were identified in 22 (4.0%) and 42 (7.7%) of 549 patients, respectively. P/LPVs were identified in 16 genes, including ATM (11, 2.0%) and BRCA2 (6, 1.1%), while 19 patients (3.5%) were heterozygotes for MUTYH P/LPVs and 9 (1.6%) carried the low-risk allele, CHEK2 p.(Ile157Thr). Patients carrying P/LPVs had improved OS compared to non-carriers (22.6 vs. 13.9 months, p = 0.006). In multivariate analysis, there was a trend for improved OS in P/LPV carriers (p = 0.063). The interaction term between platinum exposure and mutational status of HRR genes was not significant (p-value = 0.35). A significant proportion of patients with PDAC carries clinically relevant germline P/LPVs, irrespectively of age, family history or disease stage. The predictive role of these P/LPVs has yet to be defined. ClinicalTrials.gov Identifier: NCT03982446.

Whole-genome fetal and maternal DNA methylation analysis using MeDIP-NGS for the identification of differentially methylated regions.

DNA methylation is an epigenetic marker that has been shown to vary significantly across different tissues. Taking advantage of the methylation differences between placenta-derived cell-free DNA and maternal blood, several groups employed different approaches for the discovery of fetal-specific biomarkers. The aim of this study was to analyse whole-genome fetal and maternal methylomes in order to identify and confirm the presence of differentially methylated regions (DMRs). We have initially utilized methylated DNA immunoprecipitation (MeDIP) and next-generation sequencing (NGS) to identify genome-wide DMRs between chorionic villus sampling (CVS) and female non-pregnant plasma (PL) and peripheral blood (WBF) samples. Next, using specific criteria, 331 fetal-specific DMRs were selected and confirmed in eight CVS, eight WBF and eight PL samples by combining MeDIP and in-solution targeted enrichment followed by NGS. Results showed higher enrichment in CVS samples as compared to both WBF and PL samples, confirming the distinct methylation levels between fetal and maternal DNA for the selected DMRs. We have successfully implemented a novel approach for the discovery and confirmation of a significant number of fetal-specific DMRs by combining for the first time MeDIP and in-solution targeted enrichment followed by NGS. The implementation of this double-enrichment approach is highly efficient and enables the detailed analysis of multiple DMRs by targeted NGS. Also, this is, to our knowledge, the first reported application of MeDIP on plasma samples, which leverages the implementation of our enrichment methodology in the detection of fetal abnormalities in maternal plasma.

Inter-individual methylation variability in differentially methylated regions between maternal whole blood and first trimester CVS.

BACKGROUND: DNA methylation is the most studied form of epigenetic regulation, a process by which chromatin composition and transcription factor binding is altered to influence tissue specific gene expression and differentiation. Such tissue specific methylation patterns are investigated as biomarkers for cancer and cell-free fetal DNA using various methodologies. RESULTS: We have utilized methylation DNA immunoprecipitation (MeDIP) and real-time quantitative PCR to investigate the inter-individual methylation variability of differentially methylated regions (DMRs) on chromosomes 18 and 21. We have characterized 15 newly selected and seven previously validated DMRs in 50, 1(st) trimester Chorionic villus samplings (CVS) and 50 female non-pregnant peripheral blood (WBF) samples. qPCR results from MeDIP and genomic DNA (Input) assays were used to calculate fold enrichment values for each DMR. For all regions tested, enrichment was higher in CVS than in WBF samples with mean enrichments ranging from 0.22 to 6.4 and 0.017 to 1 respectively. Despite inter-individual variability, mean enrichment values for CVS were significantly different than those for WBF in all DMRs tested (p < 0.01). This observation is reinforced by the absence of overlap in CVS and WBF enrichment value distributions for 15 of 22 DMRs. CONCLUSIONS: Our work provides an expansion in the biomarker panel available for non-invasive prenatal diagnosis (NIPD) using the MeDIP-qPCR methology for Down syndrome and can eventually provide the starting point towards the development for assays towards the detection of Edwards syndrome. Furthermore, our data indicate that inter-experimental and inter-individual variation in methylation is apparent, yet the difference in methylation status across tissues is large enough to allow for robust tissue specific methylation identification.

The Epigenome View: An Effort towards Non-Invasive Prenatal Diagnosis.

Epigenetic modifications have proven to play a significant role in cancer development, as well as fetal development. Taking advantage of the knowledge acquired during the last decade, great interest has been shown worldwide in deciphering the fetal epigenome towards the development of methylation-based non-invasive prenatal tests (NIPT). In this review, we highlight the different approaches implemented, such as sodium bisulfite conversion, restriction enzyme digestion and methylated DNA immunoprecipitation, for the identification of differentially methylated regions (DMRs) between free fetal DNA found in maternal blood and DNA from maternal blood cells. Furthermore, we evaluate the use of selected DMRs identified towards the development of NIPT for fetal chromosomal aneuploidies. In addition, we perform a comparison analysis, evaluate the performance of each assay and provide a comprehensive discussion on the potential use of different methylation-based technologies in retrieving the fetal methylome, with the aim of further expanding the development of NIPT assays.

Apparent Homozygosity of p.Phe508del in CFTR due to a Large Gene Deletion of Exons 4-11.

We report a classic cystic fibrosis (CF) boy with a large deletion of exons 4-11 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene on one allele and p.Phe508del in exon 10 on the second allele. Both parents of Georgian and Ukrainian background had no personal or family history of the disease. The initial molecular diagnostic investigation identified the patient as homozygous for the p.Phe508del and not compatible with his parent's genetic status. The possibility of nonpaternity or uniparental disomy (UPD7) was investigated and excluded using microsatellite analysis of highly polymorphic markers on chromosome 7. Array-CGH was also performed on the patient and revealed a male profile with a subtle deletion within the CFTR gene on the long arm (q-arm) of chromosome 7 (7q31.2). The deletion was confirmed by MLPA extending from probe L02380 to probe L14978 (28.7 kb) and that was inherited from his father, while p.PheF508del was inherited from his mother. These data highlight the need for additional testing for large deletions in patients with apparent homozygosity for a mutated CFTR allele that do not match the carrier status of the parents. Not testing can lead to misdiagnosis and misinterpretation of mutation carrier status and the expected penetrance of the disorder.

21 Mb deletion in chromosome band 13q22.2q32.1 associated with mild/moderate psychomotor retardation, growth hormone insufficiency, short neck, micrognathia, hypotonia, dysplastic ears and other dysmorphic features.

We report on a 9-month old boy carrying a 21 Mb de novo 13q interstitial deletion. The imbalance was detected by chromosomal analysis and investigated by Fluorescence In Situ Hybridization (FISH) and Comparative Genomic Hybridization (array-CGH) using two different platforms: a BAC microarray with 516 kb resolution (Cytochip) and a 15 kb resolution oligonucleotide microarray (Agilent 244K). The deletion has been estimated to span 21.46 Mb on chromosomal bands 13q22.2-13q32.1. The patient has mild/moderate psychomotor retardation, growth hormone insufficiency, hypertelorism, short neck, micrognathia, hypotonia, dysplastic ears and other dysmorphic features. Further investigation revealed that the abnormality is de novo and causative of the patient's phenotype. The described patient is unique among similar rare cases with different deletion breakpoints. It is the first case of 13q22.2q32.1 deletion where the breakpoints are clearly defined, indicating the importance of detailed clinical description and high-resolution genomic analysis for characterization of rare genetic syndromes.

FoSTeS, MMBIR and NAHR at the human proximal Xp region and the mechanisms of human Xq isochromosome formation.

The recently described DNA replication-based mechanisms of fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR) were previously shown to catalyze complex exonic, genic and genomic rearrangements. By analyzing a large number of isochromosomes of the long arm of chromosome X (i(Xq)), using whole-genome tiling path array comparative genomic hybridization (aCGH), ultra-high resolution targeted aCGH and sequencing, we provide evidence that the FoSTeS and MMBIR mechanisms can generate large-scale gross chromosomal rearrangements leading to the deletion and duplication of entire chromosome arms, thus suggesting an important role for DNA replication-based mechanisms in both the development of genomic disorders and cancer. Furthermore, we elucidate the mechanisms of dicentric i(Xq) (idic(Xq)) formation and show that most idic(Xq) chromosomes result from non-allelic homologous recombination between palindromic low copy repeats and highly homologous palindromic LINE elements. We also show that non-recurrent-breakpoint idic(Xq) chromosomes have microhomology-associated breakpoint junctions and are likely catalyzed by microhomology-mediated replication-dependent recombination mechanisms such as FoSTeS and MMBIR. Finally, we stress the role of the proximal Xp region as a chromosomal rearrangement hotspot.

Differential DNA methylation as a tool for noninvasive prenatal diagnosis (NIPD) of X chromosome aneuploidies.

The demographic tendency in industrial countries to delay childbearing, coupled with the maternal age effect in common chromosomal aneuploidies and the risk to the fetus of invasive prenatal diagnosis, are potent drivers for the development of strategies for noninvasive prenatal diagnosis. One breakthrough has been the discovery of differentially methylated cell-free fetal DNA in the maternal circulation. We describe novel bisulfite conversion- and methylation-sensitive enzyme digestion DNA methylation-related approaches that we used to diagnose Turner syndrome from first trimester samples. We used an X-linked marker, EF3, and an autosomal marker, RASSF1A, to discriminate between placental and maternal blood cell DNA using real-time methylation-specific PCR after bisulfite conversion and real-time PCR after methylation-sensitive restriction digestion. By normalizing EF3 amplifications versus RASSF1A outputs, we were able to calculate sex chromosome/autosome ratios in chorionic villus samples, thus permitting us to correctly diagnose Turner syndrome. The identification of this new marker coupled with the strategy outlined here may be instrumental in the development of an efficient, noninvasive method of diagnosis of sex chromosome aneuploidies in plasma samples.

Multiplex Amplifiable Probe Hybridization (MAPH) methodology as an alternative to comparative genomic hybridization (CGH).

Genomic imbalances in locus copy-number are highly significant for the diagnosis and prognosis of cancer. Rapidly progressing DNA microarray technologies detect such pathogenic copy-number changes in the genome with high throughput, efficiency, and resolution. A variety of different microarray-based approaches have emerged, with array comparative genomic hybridization (array-CGH) being the method of choice in current clinical practice. Here we describe an alternative microarray-based technique called array-MAPH, derived from conventional Multiplex Amplifiable Probe Hybridization (MAPH).The main novelty of array-MAPH is the directed reduction of test DNA complexity prior to hybridization, yielding a mixture of specific probes, identical to target sequences on the microarray and thus increasing hybridization specificity. Unique amplifiable 400-600 bp fragments can be designed for any genomic region of interest, PCR-amplified, and spotted onto arrays as targets. The same sequences are combined into a probe mixture and hybridized to genomic DNA immobilized on a membrane. Bound probes are recovered by quantitative PCR and hybridized to the array. Array-MAPH can be used for the detection of small-scale copy-number changes, thereby providing new insights into the genetic basis of several diseases, including cancer.

Distal del(4) (q33) syndrome: detailed clinical presentation and molecular description with array-CGH.

The 4q deletion syndrome, comprising all microscopically visible deletions (interstitial and terminal) is a well-recognized distinctive malformation entity, with an estimated incidence of 1:10,000. Here we present the clinical and molecular findings in a 3-year-old male with a de novo distal deletion of 4q33 [46,XY,del(4) (q33)]. Clinical findings of the patient include: hypertelorism, broad nasal bridge, short nose with anteverted nares, long philtrum, thin upper lip, micro-retrognathia, low-set and protruding ears, pre-auricular tag unilaterally, low posterior hairline, clinodactyly of the 5th fingers, tapering fingers, hypospadias, and severe psychomotor retardation. Soon after birth he developed severe hypotonia and feeding difficulties. Echocardiography at 15 months documented aortic supravalvular membrane resulting in mild aortic stenosis and dysplasia of the pulmonary valve. Genome-wide screening using 1 Mb resolution array-CGH and subsequent FISH analyses defined a 18.9-22.9 Mb deletion located at the beginning of 4q33 and extending to the telomere. The description of additional cases with similar distal deletions of 4q33 will allow a more precise prognosis and is therefore of great value for genetic counsellors, while detailed molecular characterization in any well clinically characterized patient is expected to track down individual candidate genes for the specific features of the syndrome.