GENE-42. EXOME SEQUENCING ANALYSIS TO IDENTIFY POSSIBLE GENOMIC DRIVERS OF METASTATIC INVASION INTO THE SPINE

Abstract The spine is a common site of metastatic spread of many cancers, causing debilitating pain and suffering. Treatment of spinal metastases is limited by resistance to radiation, chemotherapy, and proximity to the spinal cord. It is currently not known what causes the high incidence of spinal metastases, yet theories have been proposed, like the venous metastatic spread theory (Batson, 1940) and the “seed and soil” hypothesis (Paget, 1889), postulating that factors intrinsic to the tumor cells or to the microenvironment determine the location of cancer dissemination. Recent advances in high throughput sequencing allow for in depth analyses of the molecular signatures of tumors. To identify if there are intrinsic genetic alterations common to cancer cells that disseminate into the spine, we sequenced the exome of metastatic tumor cells harvested from the vertebrae of 9 patients with different primary tumors: carcinomas and sarcomas. Exome sequencing was performed using the HiSeq 4000 (Illumina) platform on DNA from tumor cell lines and control blood or bone marrow. Data was analyzed at the University of Michigan Bioinformatics Core and variants were called using the VarScan method (http://varscan.sourceforge.net/) in somatic mode. This analysis identified a total of 2366 genes with high impact mutations (888–1026 per sample); of these, 232 genes are common to all patients analyzed. Ninety-six of the identified genes (4%) are included in the Catalogue of Somatic Mutations in Cancer, and of these, seven: ACSL6, ACVR1, ALK, FGFR2, HSP90AA1, PTPN6 and PTPRB, have high impact mutations in all 9 patients with spinal metastatic disease. Pathway analysis of genes mutated in 5 or more patients shows significant overrepresentation of 41 KEGG pathways including TGFb, HIF1-a, VEGF, Wnt and Estrogen signaling pathways. Ongoing experiments are performed to validate the sequencing analysis and characterize functional consequences of the common mutations identified.

Download Full-text

BCR-ABL1 p190 in CML: A Minor Breakpoint with a Major Impact

Blood ◽

10.1182/blood-2019-126584 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 190-190

Author(s):

Shady Adnan Awad ◽

Helena Hohtari ◽

Komal Kumar Javarappa ◽

Tania Brandstoetter ◽

Daehong Kim ◽

...

Keyword(s):

Cell Cycle ◽

Exome Sequencing ◽

Rna Sequencing ◽

Cell Lines ◽

Research Funding ◽

Drug Sensitivity ◽

Genetic Alterations ◽

Resistance Testing ◽

Sequencing Analysis ◽

Distinct Features

Introduction: The oncoprotein Bcr-Abl has two major isoforms, depending on the breakpoint in BCR gene, p190 and p210. While p210 is the hallmark of chronic myeloid leukemia (CML), p190 occurs in the majority of Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients. p190 occurs as a sole transcript in 1-2% CML patients, associated with distinct features like monocytosis and frequent additional cytogenetic abnormalities (ACA) at diagnosis. It also confers a risk of treatment failure and progression in chronic phase (CP) CML patients. However, the underlying mechanisms are largely unknown. Here we explore the characteristics of p190 and p210 in CML and ALL patients using next generation sequencing, phospho-flowcytometry and high throughput drug testing. Patients and methods: Peripheral blood mononuclear cells (PMNC) were collected at diagnosis from four CP-CML patients harboring p190 isoform from Helsinki University Hospital. Genetic alterations were identified by whole exome sequencing. RNA sequencing was employed to analyze transcriptional profiles of p190 CML (n=3) in contrast to p210 CML patients (n=4). A thorough transcriptional, phosphorylation and drug sensitivity profiling were applied to five p190- and three p210-expressing Ph+ALL patients. Expression alterations were further characterized in two cell line models mimicking BCR-ABL positive leukemia (Ba/F3 and HPCLSK). Phosphorylation profiles were analyzed by flowcytometry and phospho-array (Tyrosine Phosphorylation ProArray, Full Moon Biosystems). For drug sensitivity and resistance testing (DSRT), a custom plate set comprising 75 approved and investigational oncology drugs was used for patient samples and more extensive 528-drugs plates were used for the cell lines. Results: CML patients with p190 had a median age of 72.5 years at the diagnosis (range: 50-80) and all received imatinib as a frontline treatment. Only one patient achieved a fluctuating major molecular response (MMR) by 12 months while the rest of the patients showed primary resistance to treatment and were shifted to a 2nd line TKI, nilotinib (n=2) or proceeded to HSCT (n=1). By exome sequencing we identified 26 variants in p190-CML samples (median per patient=7, range: 2-10), including variants in ASXL1, DNMT3A and KDM4D genes. RNA-sequencing analysis identified 19 and 97 dysregulated genes (Q <0.05) between p190- and p210 in CML and Ph+ ALL cells respectively. In CML, enrichment analysis revealed upregulation of TNF, interferon (IFN), IL1-R and Toll-like receptor (TLR) signaling, TP53-related, cell cycle and apoptosis pathways. Among Ph+ ALL samples, many CML-related genes were upregulated in samples encompassing p210 while IFN-, TP53- and cell cycle-related molecules were upregulated in p190 samples. p190 samples exhibited hyper-phosphorylation of Src kinase compared to p210 samples. DSRT results also revealed increased sensitivity of primary Ph+ ALL-p190 cells to Src-inhibitors (dasatinib and saracatinib), glucocorticoids and MDM2 inhibitors/TP53 activators (SAR405838 and idasanutlin). Regarding cell lines, Ba/F3-p190 showed the upregulation of interferon signaling pathways compared to p210. Src was also hyperphosphorylated in both Ba/F3 and HPCLSK p190 models. In addition to glucocorticoids and Src-inhibitors, compounds blocking the activity of the inhibitors of apoptosis protein (IAP) family were highly effective at reducing the viability of p190 compared to p210 cells in both cell lines. Conclusions: In CML, p190 isoform of BCR-ABL1 is associated with distinct features and should be considered as a high-risk group. Combining clinical, genomic, phosphorylation and drug sensitivity data, we demonstrated that p190 activates specific cancer pathways, notably Src signaling and interferon pathways. Data also suggests that CML patients with p190 could benefit from broad spectrum TKI with Src inhibiting activity or combination of TKI with MDM2- or IAP-inhibitors. Disclosures Heckman: Orion Pharma: Research Funding; Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding. Porkka:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding. Mustjoki:Novartis: Research Funding; Pfizer: Research Funding; BMS: Honoraria, Research Funding.

Download Full-text

Using single-cell sequencing technology to detect circulating tumor cells in solid tumors

Molecular Cancer ◽

10.1186/s12943-021-01392-w ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Jiasheng Xu ◽

Kaili Liao ◽

Xi Yang ◽

Chengfeng Wu ◽

Wei Wu ◽

...

Keyword(s):

Single Cell ◽

Solid Tumors ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

High Throughput Sequencing ◽

Metastatic Potential ◽

Sequencing Analysis ◽

Sequencing Technology ◽

Single Cell Sequencing

AbstractCirculating tumor cells are tumor cells with high vitality and high metastatic potential that invade and shed into the peripheral blood from primary solid tumors or metastatic foci. Due to the heterogeneity of tumors, it is difficult for high-throughput sequencing analysis of tumor tissues to find the genomic characteristics of low-abundance tumor stem cells. Single-cell sequencing of circulating tumor cells avoids interference from tumor heterogeneity by comparing the differences between single-cell genomes, transcriptomes, and epigenetic groups among circulating tumor cells, primary and metastatic tumors, and metastatic lymph nodes in patients' peripheral blood, providing a new perspective for understanding the biological process of tumors. This article describes the identification, biological characteristics, and single-cell genome-wide variation in circulating tumor cells and summarizes the application of single-cell sequencing technology to tumor typing, metastasis analysis, progression detection, and adjuvant therapy.

Download Full-text

Germline mutations in MSH2 and ATM gene in patients with GIST (gastrointestinal stromal tumor) and second epitelial tumors.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e23520 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e23520-e23520

Author(s):

Silvia Gasperoni ◽

Laura Papi ◽

Francesca Castiglione ◽

Francesca Gensini ◽

Roberta Sestini ◽

...

Keyword(s):

Genetic Counseling ◽

Lynch Syndrome ◽

Colon Adenocarcinoma ◽

Germline Mutations ◽

Genetic Alterations ◽

Second Primary ◽

Sequencing Analysis ◽

Msh2 Gene ◽

Primary Tumors ◽

Therapeutic Implications

e23520 Background: In adult GISTs are frequently sporadic, while rarely GISTs are linked to Carney Triad and Carney-Stratakis Syndrome and NF1. GISTs with second primary tumors are reported in 4-33% of patients in literature and genetic counseling is suggested to explore an underlying germline mutations pathway. Methods: In our Academic Hospital Centre (EURACAN member) in Florence, Italy, we are following patients with GIST and multiple primary tumors with genetic counseling (72 GISTs with second tumors/185 patients with GIST) and germline analysis of the following genetic panel is performed as clinically indicated: BRCA1, BRCA2, MUTYH, MLH1, MSH2, MSH6, CDH1, ATM, TP53, PTEN, CHECK2, PALB2, BARD1, BRIP1, BLM, RAD51C, RAD51D, XRCC2, PMS2, MRE11A, RAD50, NBN, FAM175A, EPKAM, TSK1, MEN1 by sequencing analysis with Illumina MiSeq by kit multiplicom BRCA Hereditary cancer Mastr plus, and bioinformatic analysis by software SOPHIADDM (Sophia genetics) for point genetic alterations of BRCA1 NM_007294.3, BRCA2 NM_000059.3, MUTYH NM_000249, MSH2 NM_000251, MSH6 NM_000179, CDH1 NM_00444360, ATM NM_000051, TP53 NM_000546, PTEN NM_000314, CHEK2 NM_001005735, PALB2 NM_024675, BARD1 NM_000465, BRIP1 NM_032043, BLM NM_000057, RAD51C NM_002876, RAD51D NM_001142571, XRCC2 NM_005431, PMS2 NM_000535, MRE11A NM_005590, RAD50 NM_006732, NBN NM_002485, FAM175A NM_139076, EPCAM NM_002354, STK1 NM_000455, MEN1 NM_000244 and MLPA (Multiplex Ligation-dependent Probe Amplification) test analysis for patients with kit P087-BRCA1,P045-BRCA2(CHEK2, P248-MLH1-MSH2, P003-MLH1/MSH2, P072-MSH6-MUTYH (MRC-Holland). Results: In 3 patients germline mutations have been observed: 1 patient showed the c.1192dupG, p.(Ala398Glyfs*19) pathogenic mutation in exon 7 of MSH2 gene, confirmed by Sanger Sequencing, 1 patient showed c.565-?_1130+?del mutation consisting in heterozygous 3-4-5-6 exons deletion of MSH2 gene, confirmed by MLPA analysis, and in 1 patient the following ATM alteration has been identified in heterozygosis: ATM c.5319+2T > C, p.(?). In the 2 patients with Lynch syndrome with colon adenocarcinoma (MSI-H), synchronous GISTs (1 patient quadruple WT and 1 patient kit ex 11 mutated ) were diagnosed; in the patient with ATM mutation, the diagnosis of GIST (kit ex 11 mutated) occurred after prostate adenocarcinoma and before colon adenocarcinoma (MSI-H). Conclusions: Our analysis suggests that GIST diagnosis could be tumor-related to multiple hereditary tumor syndromes as Lynch Syndrome and Ataxia-Teleangectasia syndrome, the latter being linked in eterozygosis to tumor susceptibility to breast in female. This report represents a high value in terms of genetic counseling for relatives and in terms of therapeutic implications for the patients.

Download Full-text

Identification of Somatic Genetic Alterations Using Whole-Exome Sequencing of Uterine Leiomyosarcoma Tumors

Frontiers in Oncology ◽

10.3389/fonc.2021.687899 ◽

2021 ◽

Vol 11 ◽

Author(s):

Lihua Chen ◽

Jiajia Li ◽

Xiaohua Wu ◽

Zhong Zheng

Keyword(s):

Exome Sequencing ◽

Colony Formation ◽

Direct Sequencing ◽

Genetic Alterations ◽

Uterine Sarcoma ◽

Driver Mutations ◽

Exome Capture ◽

Uterine Leiomyosarcoma ◽

Sequencing Analysis ◽

Whole Exome

BackgroundThe genomic abnormalities associated with uterine leiomyosarcoma (uLMS) have not been fully elucidated to date.ObjectiveTo understand the pathogenesis of uLMS and to identify driver mutations and potential therapeutic targets in uLMS.MethodsThree matched tumor-constitutional DNA pairs from patients with recurrent uLMS were subjected to whole-exome capture and next-generation sequencing. The role of the selected gene SHARPIN in uLMS was analyzed by the CCK-8 assay and colony formation assay after specific siRNA knockdown.ResultsWe identified four genes with somatic SNVs, namely, SLC39A7, GPR19, ZNF717, and TP53, that could be driver mutations. We observed that 30.7% (4/13) of patients with uLMS had TP53 mutations as analyzed by direct sequencing. Analysis of somatic copy number variants (CNVs) showed regions of chromosomal gain at 1q21-23, 19p13, 17q21, and 17q25, whereas regions of chromosomal loss were observed at 2q35, 2q37, 1p36, 10q26, 6p22, 8q24, 11p15, 11q12, and 9p21. The SHARPIN gene was amplified in two patients and mutated in another (SHARPIN: NM_030974: exon2: c.G264C, p.E88D). Amplification of the SHARPIN gene was associated with shorter PFS and OS in soft tissue sarcoma, as shown by TCGA database analysis. Knockdown of SHARPIN expression was observed to decrease cell growth and colony formation in uterine sarcoma cell lines.ConclusionsExome sequencing revealed mutational heterogeneity of uLMS. The SHARPIN gene was amplified in uLMS and could be a candidate oncogene.

Download Full-text

Whole-Exome Sequencing Of Adult Philadephia-Negative Acute Lymphoblastic Leukemia From Diagnosis To Relapse After Allogeneic Hematopoietic Stem Cell Transplantation Reveals Clonal Evolution and Relapse-Associated Mutated Genes

Blood ◽

10.1182/blood.v122.21.156.156 ◽

2013 ◽

Vol 122 (21) ◽

pp. 156-156

Author(s):

Haowen Xiao ◽

Yi Luo ◽

Xiaoyu Lai ◽

Jimin Shi ◽

Yamin Tan ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Somatic Mutations ◽

Lymphoblastic Leukemia ◽

Gene Mutations ◽

Genetic Alterations ◽

Sequencing Analysis ◽

Hematopoietic Stem ◽

Whole Exome ◽

Matched Samples

Abstract Introduction Although steady progress of effective chemotherapy in childhood acute lymphoblastic leukemia (ALL) carried with exceeding 80% of individuals now cured, the majority of adult patients with ALL are not cured by chemotherapy, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative option. However, relapse remains the most leading cause of death after allo-HSCT. Adverse genetic alterations are generally accepted to be responsible for treatment failure and relapse. Several structural chromosomal alterations including rearrangement of the myeloid-lymphoid or mixed-lineage leukemia gene (MLL) and Philadelphia chromosome (Ph), have been mostly found in relapsed ALL. However, many Ph-negative (Ph-) ALL patients with normal karyotype , lacking known risk factors, also experienced relapse. The underlying pathologic determinants leading to relapse and prognostic markers in these cases remain poorly understood. More importantly, allo-HSCT is a distinct treatment option from tradtional chemotherapy and has 2 important forms to eliminate and select on malignant cells. The malignant cells that go on to causing relapse must initially survive ablation of chemotherapy before allo-HSCT and conditioning regimen in allo-HSCT. Then, after allo-HSCT, they must survive the effect of graft-versus- leukemia (GVL) reaction. Following this rationale, we hypothesized that there may be pivotal genetic causes confer leukemic cells a fitness advantage to undergo huge selective pressures and expand after allo-HSCT. To elucidate the genomic basis underlying relapse after allo-HSCT to aid to discover novel predictive biomarkers and identify therapeutic targets, we carried out the first whole-exome sequencing analysis in longitudinal matched samples from diagnosis to relapse after allo-HSCT in adult patients with the most common subtype of ALL, Ph- B-cell ALL (B-ALL). Methods Whole-exome sequencing was conducted for 9 genomic DNA samples from 3 relapsed cases with Ph- B-ALL (discovery cohort) at 3 specific time points including: diagnosis, complete remission (CR) after induction chemotherapy before allo-HSCT, relapse after allo-HSCT to discover candidate relapse-associated mutated genes. We identified putative somatic mutations by comparing each tumor ( diagnostic samples or relapsed samples) to normal (CR samples) from the same patient. To confirm candidate somatic gene mutations, screen relapse-associated gene mutations and define the frequency of somatic mutations identified by whole-exome sequencing analysis, we further carried out target genes whole coding regions sequencing in an ALL extended validation cohort including 58 adult Ph- B-ALL cases, where 27 patients experienced relapse at a median time of 6.5 (range 2-33) months after allo-HSCT and 31 patients did not relapse after allo-HSCT at a median follow-up for 34 (range 12–56) months. Results (1) We discovered novel associations of recurrently mutated genes (CREBBP, KRAS, PTPN21) with the pathogenesis of adult Ph- B-ALL relapse after allo-HSCT, which were mutated in at least two relapsed cases, but were not mutated in non- relapsed patients. (2) The generation of high-depth whole-exome sequencing data in longitudinal matched samples from diagnosis to relapse after allo-HSCT in initial 3 patients allowed us to directly assessed the evolution of somatic mutations. Our data suggested that in the progression of leukemia relapse after allo-HSCT, the relapse clone had a clear relationship to the diagnosis clone, either arising from a subclone already exsiting in the diagnostic tumor, or originating from a common preleukemic progenitor with the diagnosis clone. In the latter pattern, the relapse clone acquires new genetic alterations while retaining some but not all of the alterations found in the diagnostic tumor. In contrast, in some cases, leukemia recurrences afer allo-HSCT may be composed of second malignancies with completely distinct sets of mutations from the primary tumor. Conclusions Our study is the first to explore genetic basis of adult Ph- B-ALL from diagnosis to relapse after allo-HSCT over time, which will provide novel genetic biomarkers on risk “index” to improve individualized treatment intensification and intervention strategies, and potential therapeutic targets for Ph--ALL relapse after allo-HSCT. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Gene expression differences between disseminated tumor cells and tumor cells from overt bone metastases in patients with metastatic breast cancer

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.1040 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. 1040-1040

Author(s):

R. J. Broom ◽

E. Amir ◽

T. Cawthorn ◽

O. Freedman ◽

D. Gianfelice ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Bone Marrow ◽

Bone Metastases ◽

Tumor Cells ◽

Disseminated Tumor Cells ◽

Genetic Alterations ◽

Differentially Expressed ◽

Primary Tumors ◽

Ct Guided

1040 Background: Despite extensive work evaluating molecular differences between primary tumors, circulating tumor cells, disseminated tumor cells (DTCs) and established metastases, it is not apparent which genetic alterations are required to form viable, independent bone metastases (BM). A major limitation in exploring the genetic differences between DTCs and established BM is the paucity of fresh BM tissue available. Methods: Ten breast cancer patients with BM underwent a CT-guided BM biopsy and a bone marrow aspiration (for DTCs). Tumor cells were enriched by immunomagnetic separation and RNA was extracted from each sample. Gene expression profiling was conducted using Illumina Human Ref-8 bead arrays. Microarray data was analyzed using BeadStudio software to identify differentially expressed genes. Ingenuity Pathway Analysis software was used to identify genes integral to specific pathways involved in tumor dissemination. Results: The yield of analyzable malignant cells from BM and bone marrow aspirates was 60% and 80%, respectively. A signature of 133 genes was identified that was differentially expressed between the two sample types. Paired analysis of samples from the same patients identified a subset of 161 genes, of which 52 overlapped with the initial unmatched signature. Several genes relevant to breast cancer metastasis to bone (i.e., osteopontin, CTGF, parathyroid hormone receptor, EGFR) were significantly over-expressed in the BM compared to the DTCs. Conclusions: Results suggest that there are specific subsets of genes, which are required for DTCs in the bone marrow to form overt BM. A number of genes identified are already known to participate in osteolytic BM formation. This signature may allow identification of patients at increased risk for developing BM. No significant financial relationships to disclose.

Download Full-text

EYA2 suppresses the progression of hepatocellular carcinoma via SOCS3-mediated blockade of JAK/STAT signaling

Molecular Cancer ◽

10.1186/s12943-021-01377-9 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Ze-Kun Liu ◽

Can Li ◽

Ren-Yu Zhang ◽

Ding Wei ◽

Yu-Kui Shang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Exome Sequencing ◽

Methylation Status ◽

Sequencing Analysis ◽

Loss Of Function ◽

Gain Of Function ◽

Primary Tumors ◽

Stat Signaling ◽

Specific Deletion

Abstract Background Somatic mutations are involved in hepatocellular carcinoma (HCC) progression, but the genetic mechanism associated to hepatocarcinogenesis remains poorly understood. We report that Eyes absent homolog 2 (EYA2) suppresses the HCC progression, while EYA2(A510E) mutation identified by exome sequencing attenuates the tumor-inhibiting effect of EYA2. Methods Whole-exome sequencing was performed on six pairs of human HCC primary tumors and matched adjacent tissues. Focusing on EYA2, expression level of EYA2 in human HCC samples was evaluated by quantitative real-time PCR, western blot and immunohistochemistry. Loss- and gain-of-function studies, hepatocyte-specific deletion of EYA2 (Eya2−/−) in mice and RNA sequencing analysis were used to explore the functional effect and mechanism of EYA2 on HCC cell growth and metastasis. EYA2 methylation status was evaluated using Sequenom MassARRAY and publicly available data analysis. Results A new somatic mutation p.Ala510Glu of EYA2 was identified in HCC tissues. The expression of EYA2 was down-regulated in HCC and associated with tumor size (P = 0.001), Barcelona Clinic Liver Cancer stage (P = 0.016) and tumor differentiation (P = 0.048). High level of EYA2 was correlated with a favorable prognosis in HCC patients (P = 0.003). Results from loss-of-function and gain-of-function experiments suggested that knockdown of EYA2 enhanced, while overexpression of EYA2 attenuated, the proliferation, clone formation, invasion, and migration of HCC cells in vitro. Delivery of EYA2 gene had a therapeutic effect on inhibition of orthotopic liver tumor in nude mice. However, EYA2(A510E) mutation led to protein degradation by unfolded protein response, thus weakening the inhibitory function of EYA2. Hepatocyte-specific deletion of EYA2 in mice dramatically promoted diethylnitrosamine-induced HCC development. EYA2 was also down-regulated in HCC by aberrant CpG methylation. Mechanically, EYA2 combined with DACH1 to transcriptionally regulate SOCS3 expression, thus suppressing the progression of HCC via SOCS3-mediated blockade of the JAK/STAT signaling pathway. Conclusions In our study, we identified and validated EYA2 as a tumor suppressor gene in HCC, providing a new insight into HCC pathogenesis.

Download Full-text

JS1.1 Specific genetic alterations of breast tumors lead to Yo paraneoplastic cerebellar syndromes

Neuro-Oncology ◽

10.1093/neuonc/noz126.010 ◽

2019 ◽

Vol 21 (Supplement_3) ◽

pp. iii4-iii4

Author(s):

V Desestret ◽

D Pissaloux ◽

I Treilleux ◽

M Small ◽

M Robert ◽

...

Keyword(s):

Immune Response ◽

Tumor Cells ◽

Hormone Receptors ◽

Breast Tumors ◽

Genetic Alterations ◽

Copy Number Variations ◽

Sequencing Analysis ◽

Breast Cancers ◽

Genetic Characteristics ◽

Ovarian Cancers

Abstract BACKGROUND Paraneoplastic cerebellar degenerations with anti-Yo antibodies (Yo-PCD) are rare syndromes associated with ovarian or breast cancers and caused by an auto-immune response against neuronal antigens expressed by tumor cells. We previously demonstrated in Yo-PCD ovarian cancers an association between massive infiltration of ovarian tumors by activated immune effector cells and recurrent gains and/or mutations of onconeural Yo genes (CDR2L and CDR2), suggesting that such genetic alterations in ovarian tumor cells may trigger immune tolerance breakdown and initiation of the auto-immune reaction against Purkinje cells. MATERIAL AND METHODS We pursued the characterization of Yo-PCD tumors and specifically studied breast cancer by IHC, FISH, CGH array and RNA sequencing analysis of 17 breast Yo-PCD tumors and by comparing their genetic characteristics with 10 sporadic breast tumors and public databases. RESULTS We confirmed that specific genetic alterations were also present in breast cancers associated with Yo-PCD. Moreover, this study provides additional evidence for a role of tumor cell specificities in PCD immunopathology by revealing peculiarities in Yo-PCD breast tumors compared to Yo-PCD ovarian cancer. Indeed, not only the CDR2L Yo gene was amplified in 8/9 breast Yo-PCD cancers but also the Erb2/Her2 gene in 15/16 (both genes are on chromosome 17q). In addition to this original Her2 and Yo antigen amplification confirmed by FISH, we observed an overexpression of these proteins by IHC analysis. These Yo-PCD breast cancers are also all negative for hormone receptors (HR). Thus, Yo-PCD breast tumors seem to belong to the molecularly and clinically distinct class of HR-negative and Her2-enriched breast cancers, which represents less than 10 % of breast cancers in the general population. Transcriptomic analysis confirmed that breast Yo-PCD tumors differ by their expression programs from classical breast cancers molecular subtypes. CONCLUSION Understanding the tumor genetic features leading to the immune breakdown and anti-tumor immune response as well as nervous tissue attack remains challenging and seems to be specific according to the tumor subtypes. Herein, our results suggest that, despite sharing common genetic alterations (copy number variations and mutations affecting Yo genes), the Yo-PCD immunopathogenesis of breast and ovarian cancers differ by involving different tumor-specific molecular pathways.

Download Full-text

Whole Exome Sequencing Analysis in AML with Normal Karyotype Not Harboring FLT3/ITD Mutation Reveals Novel Genetic Alterations.

Blood ◽

10.1182/blood.v120.21.2593.2593 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2593-2593

Author(s):

Il-Kwon Lee ◽

Namshin Kim ◽

Yeo-Kyeoung Kim ◽

Dennis Dong Hwan Kim ◽

Quang Trinh ◽

...

Keyword(s):

Complete Remission ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Conflicts Of Interest ◽

Genetic Alterations ◽

Sequencing Analysis ◽

Novel Mutations ◽

Whole Exome ◽

Recurrent Mutations ◽

Custom Made

Abstract Abstract 2593 Background: NK-AML represents genetically heterogeneous group of disease. However genetic lesions affecting treatment outcome in patients with NK-AML are relatively unknown. Methods: The discovery cohort consists of 67 NK-AML patients in complete remission (median age: 49.2, ranges: 19–70) without FLT-3 mutations. Genomic DNA was extracted from enriched AML cells at diagnosis or control specimens obtained after complete remission. Whole exomes were captured using Agilent SureSelect and sequencing were performed by HiSeq2000 with 41∼89× coverage. Bioinformatics analysis and identification of somatic mutation has been done by series of software such as BWA, Picard, GATK, VarScan 2, and custom-made scripts. All the data has been re-checked by manual inspection. Validation has been done independent set of cohort (358 NK-AML patients, median age: 51, ranges: 15–85) with Sanger sequencing on highly mutated target sites. Results: Filtering against dbSNP and COSMIC database generated 485 genes with somatic and structural variations. Among them, 41 genes were detected in more than two patients. In addition to well-known 28 mutations, 13 novel mutations with different frequencies were identified including genes responsible for structural maintenance of chromosome (SMC1A, 6.0%) and tumor suppressor function (FAT1, 6.0%). Most common type of mutation was missense mutation (70.8%), and substantial fraction of mutation was splicing site mutations (3.8%). The hematological system development and hematologic function were most highly enriched by the Ingenuity Pathway Analysis (IPA) as expected. CIRCOS plot analysis showed similar co-occurring pattern of recurrent mutations with previous reports. Hierarchical clustering analysis divided into four different groups according to the number of harboring mutations. In network analysis four distinct subgroups were observed ranging 21 to 3 gene network. Conclusion: Using whole exome sequencing approach, a catalog of recurrent mutations was successfully defined in the patients with NK-AML without FLT3/ITD mutation. This candidate list of novel mutations should be tested further for therapeutic target and prognostic marker in the patients with NK-AML. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Whole Exome Sequencing Analysis of ABCC8 and ABCD2 Genes Associating With Clinical Course of Breast Carcinoma

Physiological Research ◽

10.33549/physiolres.933212 ◽

2015 ◽

pp. S549-S557 ◽

Cited By ~ 3

Author(s):

P. SOUCEK ◽

V. HLAVAC ◽

K. ELSNEROVA ◽

R. VACLAVIKOVA ◽

R. KOZEVNIKOVOVA ◽

...

Keyword(s):

Breast Cancer ◽

Exome Sequencing ◽

Cancer Progression ◽

Clinical Data ◽

In Silico ◽

Genetic Alterations ◽

Sequencing Analysis ◽

Whole Exome ◽

Generation Sequencing

The aim of the present study was to introduce methods for exome sequencing of two ATP-binding cassette (ABC) transporters ABCC8 and ABCD2 recently suggested to play a putative role in breast cancer progression and prognosis of patients. We performed next generation sequencing targeted at analysis of all exons in ABCC8 and ABCD2 genes and surrounding noncoding sequences in blood DNA samples from 24 patients with breast cancer. The revealed alterations were characterized by in silico tools. We then compared the most frequent functionally relevant polymorphism rs757110 in ABCC8 with clinical data of patients. In total, the study identified 113 genetic alterations (>70 % novel ones) in both genes. Of these alterations, 83 were noncoding, 13 synonymous, 10 frameshifts and 7 were missense alterations. Four in silico programs predicted pathogenicity of two polymorphisms and four newly identified alterations. Rs757110 polymorphism in ABCC8 did not significantly associate with clinical data of the patients. In conclusion, exome sequencing identified several functionally relevant alterations in ABCC8 and ABCD2 genes that may further be used for a larger follow-up study aiming to assess their clinical significance.

Download Full-text