Deciphering Genetic Alterations of Taiwanese Pancreatic Adenocarcinoma With Targeted Sequencing

Abstract Purpose Pancreatic adenocarcinoma (PAC) is the 8th leading cause of cancer-related death in Taiwan, and its incidence is increasing. The development of PAC involves successive accumulation of multiple genetic alternations. Understanding the molecular pathogenesis and heterogeneity of PAC may facilitate personalized treatment for PAC and identify therapeutic agents.Materials and Methods We performed next-generation sequencing (NGS) with targeted panels to explore the molecular changes underlying PAC in Taiwan. The Ion Torrent Oncomine Comprehensive Panel (OCP) was used for PAC metastatic lesions, and more PAC samples were sequenced with the Ion AmpliSeq Cancer Hot Spot (CHP) v2 panel.ResultsFive fresh-frozen paraffin-embedded (FFPE) specimens were successfully assayed with the OCP, and KRAS was the most prevalent alternation, which might contraindicate the use of anti-EGFR therapy. One PAC patient harbored a FGFR2 p.C382R mutation, who might derive benefit from FGFR tyrosine kinase inhibitor. Additional 38 samples assayed with CHP v2 showed 113 hotspot variants, corresponding to 54 COSMID IDs. The most frequently mutated genes were TP53, KRAS, HRAS, and PDGFRA, (29, 23, 14, 10 hotspot variants), impacting 11, 23, 14, and 10 PAC patients. Highly pathogenic variants including COSM22413 (PDGFRA, FATHMM predicted score: 0.88), COSM520, COSM521, COSM518 (KRAS, FATHMM predicted score: 0.98) were reported.ConclusionsBy using NGS with targeted panel, somatic mutations with therapeutic potential were identified. The combination of clinical and genetic information is useful for decision making and precise selection of targeted medicine.

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Next-Generation Sequencing Improves Diagnosis, Prognosis and Clinical Management of Myeloid Neoplasms

Cancers ◽

10.3390/cancers11091364 ◽

2019 ◽

Vol 11 (9) ◽

pp. 1364 ◽

Cited By ~ 6

Author(s):

Diego Carbonell ◽

Julia Suárez-González ◽

María Chicano ◽

Cristina Andrés-Zayas ◽

Juan Carlos Triviño ◽

...

Keyword(s):

Next Generation Sequencing ◽

Copy Number Variants ◽

Genetic Alterations ◽

Next Generation ◽

Pathogenic Variants ◽

Myeloid Neoplasms ◽

Diagnostic Samples ◽

Gene Panels ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Molecular diagnosis of myeloid neoplasms (MN) is based on the detection of multiple genetic alterations using various techniques. Next-generation sequencing (NGS) has been proved as a useful method for analyzing many genes simultaneously. In this context, we analyzed diagnostic samples from 121 patients affected by MN and ten relapse samples from a subset of acute myeloid leukemia patients using two enrichment-capture NGS gene panels. Pathogenicity classification of variants was enhanced by the development and application of a custom onco-hematology score. A total of 278 pathogenic variants were detected in 84% of patients. For structural alterations, 82% of those identified by cytogenetics were detected by NGS, 25 of 31 copy number variants and three out of three translocations. The detection of variants using NGS changed the diagnosis of seven patients and the prognosis of 15 patients and enabled us to identify 44 suitable candidates for clinical trials. Regarding AML, six of the ten relapsed patients lost or gained variants, comparing with diagnostic samples. In conclusion, the use of NGS panels in MN improves genetic characterization of the disease compared with conventional methods, thus demonstrating its potential clinical utility in routine clinical testing. This approach leads to better-adjusted treatments for each patient.

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A Novel Likely Pathogenic Variant in the BLOC1S5 Gene Associated with Hermansky-Pudlak Syndrome Type 11 and an Overview of Human BLOC-1 Deficiencies

Cells ◽

10.3390/cells10102630 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2630

Author(s):

Doris Boeckelmann ◽

Mira Wolter ◽

Barbara Käsmann-Kellner ◽

Udo Koehler ◽

Lea Schieber-Nakamura ◽

...

Keyword(s):

Genetic Defect ◽

Adaptor Protein ◽

Genetic Alterations ◽

Oculocutaneous Albinism ◽

Pathogenic Variants ◽

New Type ◽

Hermansky Pudlak Syndrome ◽

Lysosome Related Organelles ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Hermansky-Pudlak syndrome (HPS) is a heterogeneous disorder combining oculocutaneous albinism (OCA) and a platelet function disorder of varying severity as its most prominent features. The genes associated with HPS encode for different BLOC- (biogenesis of lysosome-related organelles complex) complexes and for the AP-3 (adaptor protein-3) complex, respectively. These proteins are involved in maturation, trafficking, and the function of lysosome-related organelles (LROs) such as melanosomes and platelet δ-granules. Some patients with different types of HPS can develop additional complications and symptoms like pulmonary fibrosis, granulomatous colitis, and immunodeficiency. A new type of HPS has recently been identified associated with genetic alterations in the BLOC1S5 gene, which encodes the subunit Muted of the BLOC-1 complex. Our aim was to unravel the genetic defect in two siblings with a suspected HPS diagnosis (because of OCA and bleeding symptoms) using next generation sequencing (NGS). Platelet functional analysis revealed reduced platelet aggregation after stimulation with ADP and a severe secretion defect in platelet δ-granules. NGS identified a novel homozygous essential splice site variant in the BLOC1S5 gene present in both affected siblings who are descendants of a consanguine marriage. The patients exhibited no additional symptoms. Our study confirms that pathogenic variants of BLOC1S5 cause the recently described HPS type 11.

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Peritoneal mesotheliomas characterized by less cell-cycle alterations and more TRAF7 alterations than malignant pleural mesotheliomas.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.9059 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. 9059-9059

Author(s):

Michael Offin ◽

Jacklynn V. Egger ◽

Andrea Cercek ◽

Garrett Michael Nash ◽

Marc Ladanyi ◽

...

Keyword(s):

Cell Cycle ◽

Genetic Alterations ◽

Time Interval ◽

Clinicopathologic Characteristics ◽

Targeted Next Generation Sequencing ◽

Molecular Alterations ◽

Pathogenic Variants ◽

Mutation Burden ◽

Well Differentiated ◽

Next Generation Sequencing Ngs

9059 Background: While peritoneal mesotheliomas (PM) are clinically distinct from malignant pleural mesotheliomas (MPM) it is unknown if genetic alterations reflect these differences. Here we report the molecular alterations and clinicopathologic characteristics of a prospectively collected PM cohort as compared to MPM. Methods: Patients with PM (n = 59) and targeted next generation sequencing (NGS; MSK-IMPACT) from January 2014 to January 2019 were assessed and followed through February 2020. Germline variants were analyzed in consented patients. NGS was compared to patients with MPM (n = 194) assessed in the same time interval. Results: Median age at diagnosis was 61 (range: 20-77), 56% were women (n = 33), and 92% had epithelioid histology (n = 54). 66% had ascites (n = 39) and 24% developed extra-abdominal metastases (n = 14; including lung, pleura, and mediastinum). 68% (n = 40) underwent surgical debulking and 80% (n = 47) had infusional therapy (median lines: 3) including platinum/pemetrexed (n = 38), EPIC (n = 22), HIPEC (n = 15), and immunotherapy (n = 16). The median overall survival (OS) from diagnosis was 5.4 years (median follow up 3.5 years). The median tumor mutation burden (TMB) was 1.8 mut/Mb (range: 0-14.9) in PM vs 2.0 mut/Mb (range: 0-31.5) in MPM (p = 0.049). More patients with PM had TRAF7 alterations than in MPM (5/59 vs 3/194; p = 0.02) while fewer had CDKN2A/ CDKN2B (4 vs 55; p = 0.0004). All patients with TRAF7 altered PM had well-differentiated papillary epithelioid histology. There was no difference in the prevalence of other common alterations such as BAP1 (32 vs 98; p = 0.66), NF2 (12 vs 55, p = 0.24), SETD2 (11 vs 24; p = 0.28), and TP53 (9 vs 28; p = 0.84) in PM vs MPM respectively. Patients with BAP1-altered PM had shorter OS (4.6 vs 9.8 years; HR 2.6, 95% CI 1.1-6.4; p = 0.04) while TRAF7-altered PM had improved OS (not reached vs 4.8 years; HR 0.3, 95% CI 0.1-0.9; p = 0.04) compared to wild type. 13% (4/30) of patients with PM had pathogenic variants on germline NGS ( POT1 I78T, MUTYH R109Y, BAP1 E402*, APC I1037K). Conclusions: NGS confirms the distinct biology of PM compared to MPM. Specifically, the former shows fewer cell cycle ( CDKN2) alterations compared to MPM. In contrast to MPM, BAP1 alteration was associated with shorter survival. As previously described, we found enrichment of TRAF7 in well differentiated papillary epithelioid PM associated with improved survival but notably some TRAF7 alterations were identified in poorly differentiated MPM. Consistent with other reports, the prevalence of germline alterations was 13%.

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Characteristics and Clinical Application of Extracellular Vesicle-Derived DNA

Cancers ◽

10.3390/cancers13153827 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3827

Author(s):

Jae Young Hur ◽

Kye Young Lee

Keyword(s):

Therapeutic Potential ◽

Extracellular Vesicle ◽

Clinical Settings ◽

Biomarker Detection ◽

Diagnosis And Prognosis ◽

Double Stranded Dna ◽

Fundamental Research ◽

The Stability ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Extracellular vesicles (EVs) carry RNA, proteins, lipids, and diverse biomolecules for intercellular communication. Recent studies have reported that EVs contain double-stranded DNA (dsDNA) and oncogenic mutant DNA. The advantage of EV-derived DNA (EV DNA) over cell-free DNA (cfDNA) is the stability achieved through the encapsulation in the lipid bilayer of EVs, which protects EV DNA from degradation by external factors. The existence of DNA and its stability make EVs a useful source of biomarkers. However, fundamental research on EV DNA remains limited, and many aspects of EV DNA are poorly understood. This review examines the known characteristics of EV DNA, biogenesis of DNA-containing EVs, methylation, and next-generation sequencing (NGS) analysis using EV DNA for biomarker detection. On the basis of this knowledge, this review explores how EV DNA can be incorporated into diagnosis and prognosis in clinical settings, as well as gene transfer of EV DNA and its therapeutic potential.

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One in seven pathogenic variants can be challenging to detect by NGS: an analysis of 450,000 patients with implications for clinical sensitivity and genetic test implementation

Genetics in Medicine ◽

10.1038/s41436-021-01187-w ◽

2021 ◽

Author(s):

Stephen E. Lincoln ◽

Tina Hambuch ◽

Justin M. Zook ◽

Sara L. Bristow ◽

Kathryn Hatchell ◽

...

Keyword(s):

Diagnostic Yield ◽

Copy Number Variants ◽

Low Complexity ◽

Clinical Genetic ◽

Clinical Sensitivity ◽

Pathogenic Variants ◽

Wide Range ◽

Large Indels ◽

Next Generation Sequencing Ngs ◽

The Impact

Abstract Purpose To evaluate the impact of technically challenging variants on the implementation, validation, and diagnostic yield of commonly used clinical genetic tests. Such variants include large indels, small copy-number variants (CNVs), complex alterations, and variants in low-complexity or segmentally duplicated regions. Methods An interlaboratory pilot study used synthetic specimens to assess detection of challenging variant types by various next-generation sequencing (NGS)–based workflows. One well-performing workflow was further validated and used in clinician-ordered testing of more than 450,000 patients. Results In the interlaboratory study, only 2 of 13 challenging variants were detected by all 10 workflows, and just 3 workflows detected all 13. Limitations were also observed among 11 less-challenging indels. In clinical testing, 21.6% of patients carried one or more pathogenic variants, of which 13.8% (17,561) were classified as technically challenging. These variants were of diverse types, affecting 556 of 1,217 genes across hereditary cancer, cardiovascular, neurological, pediatric, reproductive carrier screening, and other indicated tests. Conclusion The analytic and clinical sensitivity of NGS workflows can vary considerably, particularly for prevalent, technically challenging variants. This can have important implications for the design and validation of tests (by laboratories) and the selection of tests (by clinicians) for a wide range of clinical indications.

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One4Two®: An Integrated Molecular Approach to Optimize Infertile Couples’ Journey

Genes ◽

10.3390/genes12010060 ◽

2021 ◽

Vol 12 (1) ◽

pp. 60

Author(s):

Valeria D’Argenio ◽

Federica Cariati ◽

Rossella Tomaiuolo

Keyword(s):

Disease Genes ◽

Diagnostic Efficacy ◽

Whole Process ◽

Pathogenic Variants ◽

Limiting Step ◽

Number Of Genes ◽

Infertile Couples ◽

Next Generation Sequencing Ngs ◽

And Diffusion ◽

Generation Sequencing

The current diagnostic path of infertile couples is long lasting and often ineffective. Genetic tests, in particular, appear as a limiting step due to their jeopardized use on one side, and to the limited number of genes evaluated on the other. In this context, the development and diffusion, also in routine diagnostic settings, of next generation sequencing (NGS)-based methods for the analyses of several genes in multiple subjects at a time is improving the diagnostic sensitivity of molecular analyses. Thus, we developed One4Two®, a custom NGS panel to optimize the diagnostic journey of infertile couples. The panel validation was carried out in three steps analyzing a total of 83 subjects. Interestingly, all the previously identified variants were confirmed, assessing the analytic sensitivity of the method. Moreover, additional pathogenic variants have been identified underlying the diagnostic efficacy of the proposed method. One4Two® allows the simultaneous analysis of infertility-related genes, disease-genes of common inherited diseases, and of polymorphisms related to therapy outcome. Thus, One4Two® is able to improve the diagnostic journey of infertile couples by simplifying the whole process not only for patients, but also for laboratories and reproduction specialists moving toward an even more personalized medicine.

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A novel frequent BRCA1 recurrent variant c.5117G > A (p.Gly1206Glu) identified after 20 years of BRCA1/2 research in the Baltic region: cohort study and literature review

Hereditary Cancer in Clinical Practice ◽

10.1186/s13053-021-00168-z ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

P. Loza ◽

A. Irmejs ◽

Z. Daneberga ◽

E. Miklasevics ◽

E. Berga-Svitina ◽

...

Keyword(s):

Literature Review ◽

Local Spectrum ◽

Single Family ◽

Baltic Region ◽

Breast And Ovarian Cancer ◽

Pathogenic Variants ◽

Common Founder ◽

The Baltic ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Background Several recent studies in the Baltic region have found extended spectrum of pathogenic variants (PV) of the BRCA1/2 genes. The aim of current study is to analyze the spectrum of the BRCA1/2 PV in population of Latvia and to compare common PV between populations of the Baltic region. Methods We present a cohort of 9543 unrelated individuals including ones with cancer and unaffected individuals from population of Latvia, who were tested for three most common BRCA1 founder PV. In second line testing, 164 founder negative high-risk individuals were tested for PV of the BRCA1/2 using next generation sequencing (NGS). Local spectrum of the BRCA1/2 PV was compared with the Baltic region by performing a literature review. Results Founder PV c.5266dupC, c.4035delA or c.181 T > G was detected in 369/9543 (3.9%) cases. Other BRCA1/2 PV were found in 44/164 (26.8%) of NGS cases. Four recurrent BRCA1 variants c.5117G > A (p.Gly1706Glu), c.4675G > A (p.Glu1559Lys), c.5503C > T (p.Arg1835*) and c.1961delA (p.Lys654fs) were detected in 18/44 (41.0%), 5/44 (11.4%), 2/44 (4.5%) and 2/44 (4.5%) cases respectively. Additionally, 11 BRCA1 PV and six BRCA2 PV were each found in single family. Conclusions By combining three studies by our group of the same cohort in Latvia, frequency of the BRCA1/2 PV for unselected breast and ovarian cancer cases is 241/5060 (4.8%) and 162/1067 (15.2%) respectively. The frequency of three “historical” founder PV is up to 87.0% (369/424). Other non-founder PV contribute to at least 13.0% (55/424) and this proportion probably will rise by increasing numbers of the BRCA1/2 sequencing. In relative numbers, c.5117G > A is currently the third most frequent PV of the BRCA1 in population of Latvia, overcoming previously known third most common founder variant c.181 T > G. In addition to three BRCA1 founder PV, a total of five recurrent BRCA1 and two recurrent BRCA2 PV have been reported in population of Latvia so far. Many of the BRCA1/2 PV reported in Latvia are shared among other populations of the Baltic region.

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Repurposing cabozantinib with therapeutic potential in KIT-driven t(8;21) acute myeloid leukaemias

Cancer Gene Therapy ◽

10.1038/s41417-021-00329-1 ◽

2021 ◽

Author(s):

Kuan-Wei Su ◽

Da-Liang Ou ◽

Yu-Hsuan Fu ◽

Hwei-Fang Tien ◽

Hsin-An Hou ◽

...

Keyword(s):

Tyrosine Kinase ◽

Ribosome Biogenesis ◽

Kinase Inhibitor ◽

Therapeutic Potential ◽

Xenograft Model ◽

Sequencing Analysis ◽

Dependent Manner ◽

Leukocyte Activation ◽

Mouse Xenograft Model ◽

Acute Myeloid

AbstractCabozantinib is an orally available, multi-target tyrosine kinase inhibitor approved for the treatment of several solid tumours and known to inhibit KIT tyrosine kinase. In acute myeloid leukaemia (AML), aberrant KIT tyrosine kinase often coexists with t(8;21) to drive leukaemogenesis. Here we evaluated the potential therapeutic effect of cabozantinib on a selected AML subtype characterised by t(8;21) coupled with KIT mutation. Cabozantinib exerted substantial cytotoxicity in Kasumi-1 cells with an IC50 of 88.06 ± 4.32 nM, which was well within clinically achievable plasma levels. The suppression of KIT phosphorylation and its downstream signals, including AKT/mTOR, STAT3, and ERK1/2, was elicited by cabozantinib treatment and associated with subsequent alterations of cell cycle- and apoptosis-related molecules. Cabozantinib also disrupted the synthesis of an AML1-ETO fusion protein in a dose- and time-dependent manner. In a mouse xenograft model, cabozantinib suppressed tumourigenesis at 10 mg/kg and significantly prolonged survival of the mice. Further RNA-sequencing analysis revealed that mTOR-mediated signalling pathways were substantially inactivated by cabozantinib treatment, causing the downregulation of ribosome biogenesis and glycolysis, along with myeloid leukocyte activation. We suggest that cabozantinib may be effective in the treatment of AML with t(8;21) and KIT mutation. Relevant clinical trials are warranted.

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HGG-21. GERMLINE MUTATIONS IN MSH2 GENE IN PEDIATRIC PATIENTS WITH CONGENITAL AND SPORADIC GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa222.308 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii348-iii348

Author(s):

Maria Ejmont ◽

Małgorzata Rydzanicz ◽

Wiesława Grajkowska ◽

Marta Perek-Polnik ◽

Agnieszka Sowińska ◽

...

Keyword(s):

Germline Mutations ◽

Response To Therapy ◽

Msh2 Gene ◽

Illumina Hiseq ◽

Adult Type ◽

Targeted Next Generation Sequencing ◽

Pathogenic Variants ◽

New Treatment ◽

Next Generation Sequencing Ngs ◽

Ngs Data

Abstract INTRODUCTION Glioblastoma (GBM) remains one of the biggest therapeutic challenges in neuro-oncology. In spite of multimodal treatment approaches the prognosis of GBM is extremely poor, median survival is estimated about 12–16 months. Although GBM is one of the most common and malignant primary brain tumors, pediatric glioblastoma, including congenital is a very rare tumor, with an incidence of about 1.1–3.4 per million live births. Moreover, the mode of presentation, behavior, response to therapy and molecular background of pediatric glioblastomas differs from adult type of GBM. Until now, about ten patients with congenital glioblastoma have been described and in none of them germline markers were examined. Here we report two patients with GBM, one with congenital tumor with germline mutations in MSH2 gene. METHODS Targeted Next-Generation Sequencing (NGS) of the probands DNA extracted from leucocytes was performed using the TruSight One sequencing panel on an Illumina HiSeq 1500. Applied gene panel investigated the coding sequence and splice sites of 4813 genes associated with known disease phenotypes. The NGS data were analyzed using an in-house procedure. Identified variants were validated by Sanger sequencing. RESULTS NGS analysis of patients constitutional DNA revealed know, pathogenic variants c.940C>T and c.942 + 3A>T in MSH2 gene (NM_000251.3) associated with MMR-dependent hereditary cancer syndromes. CONCLUSION Molecular analysis are heavily needed for better understanding of pediatric GBM etiology and new treatment modality implementation. Identification of this oncogenic driver may provide insight into the pathogenesis of GBM, including congenital cases. Funded by National Science Centre, Poland (2016/23/B/NZ2/03064 and 2016/21/B/NZ2/01785).

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Genotype-Phenotype Correlations in Neurofibromatosis Type 1: A Single-Center Cohort Study

Cancers ◽

10.3390/cancers13081879 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1879

Author(s):

Marcello Scala ◽

Irene Schiavetti ◽

Francesca Madia ◽

Cristina Chelleri ◽

Gianluca Piccolo ◽

...

Keyword(s):

Neurofibromatosis Type 1 ◽

Neurofibromatosis Type ◽

Copy Number Variations ◽

Single Nucleotide Variants ◽

Multivariate Statistical ◽

Gene Deletions ◽

Pathogenic Variants ◽

Lisch Nodules ◽

Next Generation Sequencing Ngs

Neurofibromatosis type 1 (NF1) is a proteiform genetic condition caused by pathogenic variants in NF1 and characterized by a heterogeneous phenotypic presentation. Relevant genotype–phenotype correlations have recently emerged, but only few pertinent studies are available. We retrospectively reviewed clinical, instrumental, and genetic data from a cohort of 583 individuals meeting at least 1 diagnostic National Institutes of Health (NIH) criterion for NF1. Of these, 365 subjects fulfilled ≥2 NIH criteria, including 235 pediatric patients. Genetic testing was performed through cDNA-based sequencing, Next Generation Sequencing (NGS), and Multiplex Ligation-dependent Probe Amplification (MLPA). Uni- and multivariate statistical analysis was used to investigate genotype–phenotype correlations. Among patients fulfilling ≥ 2 NIH criteria, causative single nucleotide variants (SNVs) and copy number variations (CNVs) were detected in 267/365 (73.2%) and 20/365 (5.5%) cases. Missense variants negatively correlated with neurofibromas (p = 0.005). Skeletal abnormalities were associated with whole gene deletions (p = 0.05) and frameshift variants (p = 0.006). The c.3721C>T; p.(R1241*) variant positively correlated with structural brain alterations (p = 0.031), whereas Lisch nodules (p = 0.05) and endocrinological disorders (p = 0.043) were associated with the c.6855C>A; p.(Y2285*) variant. We identified novel NF1 genotype–phenotype correlations and provided an overview of known associations, supporting their potential relevance in the implementation of patient management.

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