Mate-pair Next Generation Sequencing Research Articles

Stable transmission of complex chromosomal rearrangements involving chromosome 1q derived from constitutional chromoanagenesis

BackgroundChromoanagenesis events encompassing chromoanasynthesis, chromoplexy, and chromothripsis are described in cancers and can result in highly complex chromosomal rearrangements derived from ‘all-at-once’ catastrophic cellular events. The complexity of these rearrangements and the original descriptions in cancer cells initially led to the assumption that it was an acquired anomaly. While rare, these phenomena involving chromosome 1 have been reported a few individuals in a constitutional setting.Case presentationHere, we describe a newborn baby who was initially referred for cytogenetic testing for multiple congenital anomalies including cystic encephalomalacia, patent ductus arteriosus, inguinal hernia, and bilateral undescended testicles. Chromosome analysis was performed and revealed a derivative chromosome 1 with an 1q24-q31 segment inserted into 1q42.13 resulting in gain of 1q24-q31. Whole genome SNP microarray analysis showed a complex pattern of copy number variants with four gains and one loss involving 1q24-q31. Mate pair next-generation sequencing analysis revealed 18 chromosome breakpoints, six gains along an 1q24-q31 segment, one deletion of 1q31.3 segment and one deletion of 1q42.13 segment, which is strongly evocative of a chromoanasynthesis event for developing this complex rearrangement. Parental chromosome analyses were performed and showed the same derivative chromosome 1 in the mother.ConclusionsTo our knowledge, our case is the first case with familial constitutional chromoanagenesis involving chromosome 1q24-q42. This report emphasizes the value of performing microarray and mate pair next-generation sequencing analysis for individuals with germline abnormal or complex chromosome rearrangements.

Different whole genome sequencing strategies for the clinical management of patients with oropharyngeal squamous cell carcinoma

Oropharyngeal squamous cell carcinoma (OPSCC) is a cancer of the base of the tongue and tonsils. This cancer was previously found in individuals with a smoking and drinking history but is increasingly being promoted by the presence of high risk types of human papillomavirus (HPV). In an attempt to determine the exact role that HPV plays in the development of this cancer we have been using mate-pair next generation sequencing (MP-Seq). Using MP-Seq we have found that HPV is only integrated into the human genome in 30% of HPV-positive OPSCCs, which is dramatically different than what is found in cervical cancer. MP-Seq not only can determine the physical status of HPV in a given sample, but can also ascertain genome-wide changes in any cancer. We describe how MP-Seq could be used in the clinical management of HPV-positive OPSCC patients. We have also explored another whole genome sequencing strategy that has been developed by the Beijing Genomics Institute. They can currently do whole genome sequencing for a total cost (and this includes analysis) of $600 and are developing methodologies to reduce this to $100. I will describe how these types of whole genome sequencing strategies could be used to direct therapies for these patients including facilitating the use of the liquid biopsy to monitor patients during their clinical treatment.

Heterogeneity of Programmed Cell Death Ligand 1 Expression in Multifocal Lung Cancer.

The expression of programmed cell death ligand 1 (PD-L1) provides limited predictive value in identifying patients most likely to respond to immunotherapy. As the heterogeneity of PD-L1 expression may lead to sampling error and the misclassification of PD-L1 status, we assessed the distribution of PD-L1 expression in paired, resected multifocal lung cancers. PD-L1 was assessed by IHC. Paired lesions were defined as independent primaries or related lesions using mate pair next-generation sequencing. Agreement statistics were used for analysis. Sixty-seven multifocal lung cancers from 32 patients were sequenced and stained for PD-L1. There was agreement of PD-L1 expression by the tumor cells in paired lesions of 20 patients and disagreement of PD-L1 expression by the tumor cells in paired lesions of 12 patients (κ = 0.01). Sequencing identified that 23 patients had independent primary lung cancers and that 9 patients had related cancers. In paired lesions of patients with independent cancers, there was agreement of PD-L1 expression by the tumor cells in 12 patients and disagreement in 11 patients (κ = 0.31). In paired lesions of patients with related lung cancers, there was agreement of PD-L1 expression by the tumor cells in 8 patients and disagreement in 1 patient (κ = 0.73). The expression of PD-L1 is heterogeneous among paired independent lung cancers, but there are high levels of agreement in intrapulmonary metastasis. Clin Cancer Res; 22(9); 2177-82. ©2015 AACR.

Highlights of This Issue

Preclinical evaluation of targeted therapies does not follow simple criteria and often heavily relies on the use of patient-derived xenograft (PDX), which present major disadvantages: potential failure of tumor transplantation, long waiting-time to response evaluation, and extensive laboratory cost. Alternative clinical decision-making tools, therefore, are needed. This study compares PDX-based treatment response of targeted therapies in lung cancer patients with the result of a specific in silico response prediction (ISRP) tool and provides evidence for a substantial agreement between an ISPR and PDX response prediction. The ISPR allows fast and cheap individual-targeted response prediction in parallel for several substances.The heterogeneity of programmed cell death ligand 1 (PD-L1) expression may not be accurately represented by analysis of a single tumor. Mansfield and colleagues assessed the distribution of PD-L1 expression in paired, resected multifocal lung cancers and used mate pair next-generation sequencing to define the lineage relationship of these lesions. The expression of PD-L1 was heterogeneous among independent primary lung cancers, but there were high levels of agreement among related, intrapulmonary metastases. These data highlight that a single biopsy may not accurately capture PD-L1 expression status and emphasize the need for novel methods of patient selection for immunotherapy.PD-L1 status and tumor-infiltrating lymphocyte (TIL) recruitment have been suggested to be the most theoretical biomarker to predict the good responder of anti-PD-1/PD-L1 inhibitors. To prove this concept, Ock and colleagues analyzed the immunogenomic properties in The Cancer Genome Atlas (N = 9,677) according to the classification of tumor into four groups based on PD-L1 expression and TIL status, assessed by CD8A expression. Tumor microenvironment immune types (TMIT) I, high PD-L1, and CD8A expressions were significantly associated with a high number of somatic mutations as well as neoantigen, PD-L1 amplification, and infection with Epstein-Barr virus or human papillomavirus.The increased expression of phosphodiesterase (PDE) 5 in various human malignancies, coupled with the success of PDE5 inhibitors in different diseases, has led to interest in investigating the role of this enzyme in cancer. Catalano and colleagues showed that PDE5 overexpression increased motile and invasive properties of breast cancer cells. In patients, PDE5 is differentially expressed according to molecular subtypes, with a positive correlation with tumor grading, and high levels predict a worse prognosis. This study holds promise for PDE5 as a valuable candidate with prognostic significance and an attractive target for future therapy in breast cancers.

Mate pair sequencing for the detection of chromosomal aberrations in patients with intellectual disability and congenital malformations

Recently, microarrays have replaced karyotyping as a first tier test in patients with idiopathic intellectual disability and/or multiple congenital abnormalities (ID/MCA) in many laboratories. Although in about 14-18% of such patients, DNA copy-number variants (CNVs) with clinical significance can be detected, microarrays have the disadvantage of missing balanced rearrangements, as well as providing no information about the genomic architecture of structural variants (SVs) like duplications and complex rearrangements. Such information could possibly lead to a better interpretation of the clinical significance of the SV. In this study, the clinical use of mate pair next-generation sequencing was evaluated for the detection and further characterization of structural variants within the genomes of 50 ID/MCA patients. Thirty of these patients carried a chromosomal aberration that was previously detected by array CGH or karyotyping and suspected to be pathogenic. In the remaining 20 patients no causal SVs were found and only benign aberrations were detected by conventional techniques. Combined cluster and coverage analysis of the mate pair data allowed precise breakpoint detection and further refinement of previously identified balanced and (complex) unbalanced aberrations, pinpointing the causal gene for some patients. We conclude that mate pair sequencing is a powerful technology that can provide rapid and unequivocal characterization of unbalanced and balanced SVs in patient genomes and can be essential for the clinical interpretation of some SVs.

978 PTEN DELETION AND MUTATION IN CLINICALLY INSIGNIFICANT AND SIGNIFICANT PROSTATE CANCER: AN ANALYSIS BY MATE-PAIR NEXT GENERATION AND SANGER SEQUENCING

You have accessJournal of UrologyProstate Cancer: Basic Research (V)1 Apr 2013978 PTEN DELETION AND MUTATION IN CLINICALLY INSIGNIFICANT AND SIGNIFICANT PROSTATE CANCER: AN ANALYSIS BY MATE-PAIR NEXT GENERATION AND SANGER SEQUENCING R. Jeffrey Karnes, john cheville, george vasmatzis, and william sukov R. Jeffrey KarnesR. Jeffrey Karnes Rochester, MN More articles by this author , john chevillejohn cheville Rochester, MN More articles by this author , george vasmatzisgeorge vasmatzis Rochester, MN More articles by this author , and william sukovwilliam sukov Rochester, MN More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.560AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Deletion of PTEN is associated with worse prognosis in prostate cancer. However, little is known about the impact of PTEN mutations, and there is limited data regarding the frequency and clinical impact of deletion and mutation in clinically insignificant tumors compared to significant tumors. METHODS We identified 40 prostatectomy cases with adenocarcinoma consisting of 9 clinically insignificant tumors (defined as GS6 and tumor volume <0.5 cm3), 7 cases of large volume (>1.0 cm3) GS6, 23 cases of GS7, and 1 case of GS8) and performed massively parallel mate-pair next generation sequencing (NGS) on whole genome-amplified DNA extracted from laser capture microdissected tumor tissue. Sequence analysis of exonic regions was performed using standard Sanger methodology. Follow-up information was available for 31 patients. Tumor-free survival from date of prostatectomy was assessed using the Kaplan-Meier method and the log-rank test. RESULTS PTEN deletion detected by mate-pair NGS occurred in 10 cases and three of these cases showed additional deleterious mutations (2 nonsense and 1 frameshift) in the remaining PTEN allele by Sanger sequencing. Although not mutually exclusive, alterations in PTEN occurred more frequently in the setting of ERG rearrangements (70%). PTEN abnormalities were absent in clinically insignificant GS6, and large volume GS6 but were common in GS7 or higher tumors (10 of 23 cases; 43%)(p=0.032). Radiographic or biochemical recurrence occurred in 5 patients with PTEN abnormalities (HR=4.65, p=0.036), including metastatic recurrences in 2 of the 3 patients with confirmed bi-allelic loss of PTEN. The overall 5-year tumor-free survival rate among patients with Gleason 7-8 tumors was 57% with PTEN abnormalities and 79% in the remaining cases (p=0.039). CONCLUSIONS PTEN abnormalities were absent in clinically insignificant and large volume GS6 tumors, but occurred in a significant number of GS7 cases, Although our series is small, patients that had PTEN deletion and mutation has a significantly worse outcome.. These results suggest that PTEN abnormalities identify a subset of prostatic adenocarciomas at higher risk for progression, for which molecular and cytogenetic testing for PTEN abnormalities may provide significant prognostic information. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e402 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information R. Jeffrey Karnes Rochester, MN More articles by this author john cheville Rochester, MN More articles by this author george vasmatzis Rochester, MN More articles by this author william sukov Rochester, MN More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

SVDetect: a tool to identify genomic structural variations from paired-end and mate-pair sequencing data

Summary: We present SVDetect, a program designed to identify genomic structural variations from paired-end and mate-pair next-generation sequencing data produced by the Illumina GA and ABI SOLiD platforms. Applying both sliding-window and clustering strategies, we use anomalously mapped read pairs provided by current short read aligners to localize genomic rearrangements and classify them according to their type, e.g. large insertions–deletions, inversions, duplications and balanced or unbalanced inter-chromosomal translocations. SVDetect outputs predicted structural variants in various file formats for appropriate graphical visualization.Availability: Source code and sample data are available at http://svdetect.sourceforge.net/Contact: svdetect@curie.frSupplementary information: Supplementary data are available at Bioinformatics online.