1-s2.0-S2210776215001027-main
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Brazil. The use of the array CGH (aCGH) has aided signifi- cantly the genetic diagnosis in recent years. In our laboratory aCGH combined with conventional karyotyping allowed an improvement of 20% to 30% in the diagnostic rate of patients with intellectual disabillity, dysmorphisms, autism and devel- opmental delay. We used the Affymetrix chip Cytoscan HD and 750 K Cytoscan arrays (Santa Clara, CA) and analysis through the respective software system and for conventional karyotype (G Banding). We selected 2 cases to report: Case 1: Two siblings with distinct developmental disturbance phenotypes. Sibling one is a 7-year-old female with severe developmental delay phenotype, microcephaly and consanguineous parents. She presented an interstitial deletion of 12,1 Mb on chromo- some 5, arr [hg19] 5p14.3- p15.31 (6,801,589-18,992,827) 1, featuring the Cri-du-chat syndrome. Her brother, a 12-year-old male with mild intellectual disability (ID) a duplication of the exact same size and region that was deleted in the sister was found arr [hg19] 5p14.3-p15.31 (6,801,589-18,992,827) 3. Parental aCGH was normal. However, conventional karyotype of the father revealed 2 reciprocal translocation between chromosomes 1 and 2, 5 and 7: (46, XY, t(1, 2) (q44;w p23- pter) t(5; 7)(p14.3-p15.31; p22). Case 2: A 16-year-old female with consanguineous parents and normal conventional kar- yotyping. She presents significant developmental delay, dys- lalia, severe ID, autistic traits, irritability, hyperactivity and autoagression, without obvious dysmorphias and with normal: MRI, electroencephalogram and CT scan results. Microarray analysis revealed a homozygous microdeletion of 197 Kbp on chromosome 8:arr [hg19] 8p22 (15,451,748-15,649,733) 1, spanning almost the entire TUSC3 gene, except promoter and first exon (601385*).This is the seventh family with a reported TUSC3 mutation with mostly non syndromic ID and the second with the same deletion (Khan et al, 2011). Three of the families reported in the literature carried a deletion which encompassed only the TUSC3 promoter region and the first exon, 2 families presented distinct point mutations and 1 an intragenic dupli- cation. All 7 families presented homozygous mutations, only 1 was not consanguineous however they were from the same village. Conclusion: These cases illustrate the importance of inte- grating conventional karyotyping techniques and microarray in genetic diagnosis. Both cases were referred for genetic coun- selling and family research. Unusual ROS1 Translocation Pattern in a 61 Year-old Woman with Metastatic Adenocarcinoma of Lung Hui Chen a , Rajyalakshmi Luthra b , Neda Kalhor a , John Heymach c , Ronald Abraham b , Meenakshi Mehrotra b , Bal Mukund Mishra b , Keyur P. Patel b , Rajesh R. Singh b , Xinyan Lu b a Department of Pathology, MD Anderson Cancer Center, Houston, TX, USA; b Department of Hematopathology, MD Anderson Cancer Center, Houston, TX, USA; c Department of Thoracic/Head &Neck Medical Oncology, MD Anderson Cancer Center, Houston, TX, USA FISH assay has enabled us to detect gene amplification, deletion and translocation in solid tumors. However, FISH is limited to focused genes of interest. Currently the alternative microarray based genome-wide analysis for copy number ab- erration and allelic imbalances of oncogenes remains under- characterized. In this study, we explored genomic copy number analysis on a 61-year-old woman with metastatic adenocarci- noma of lung and refractory to chemo- and adjuvant therapies. Primary tumor from fine needle aspiration of lung and meta- static adenocarcinoma from core biopsy of lymph node were used for immunohistochemistry (IHC), FISH assay and next generation sequencing analysis to detect overexpression/ amplification, translocation, and somatic mutations. Addition- ally, genomic DNA from metastatic tumor was subjected to molecular inversion probe array (MIP) by OncoScan FFPE Assay kit (Affymetrix). Genomic copy number and allelic imbalance calls were performed by OncoScan Console and data reviewed by OncoScan Nexus Express (BioDiscovery). Both primary and metastatic tumors were pan negative for EGFR, KRAS and BRAF mutations and ALK translocation. FISH analysis demonstrated ROS1 translocation with unusual double splitting signals suggestive of allelic imbalance in lymph node metastasis but not in primary tumor. MIP study on lymph node metastasis showed loss of heterozygosity involving 4 chromosomes/arms including chromosome 6 where ROS1 located. In addition, MIP array detected copy number gains involving 9 chromosomes/arms. Genome instability with numerous copy number gain and allelic imbalances, and ROS1 translocation might be one of contributing factors to patient poor clinical outcome. A Novel Mutation in Calreticulin (CALR) was Identified in a Patient of African American Origin with Thrombocytosis D.P. Dash a , Sherine Joseph Thomas b a Blood Center of Wisconsin, Milwaukee, WI, USA; b Georgia Cancer Specialists and Northside Hospital Cancer Institute, Atlanta, GA, USA Recent studies show that Calreticulin (CALR) somatic mu- tations provide a diagnostic marker in JAK2/MPL wild type essential thrombocythemia (ET) and primary myelofibrosis (PMF) with a mutation frequency of 67% to 71% and 56% to 88%, respectively (Tefferi A and Pardanani A, Nat Rev Clin Oncol, 2014). In general, CALR mutations are not seen in Polycythemia Vera (PV), post-PV myelofibrosis or JAK2 V617F or MPL mutated ET or PMF. Studies showed that CALR muta- tions may also provide prognostic information and therefore is a promising molecular marker for diagnosis and prognosis for patients with myeloproliferative neoplasm (MPN). The exact mechanism by which CALR mutations produce the myeloprolif- erative disease phenotype is unknown, but multiple studies have shown that CALR mutations disrupt the C-terminal endoplasmic reticulum -retention sequence (KDEL), generate a novel C-ter- minus, and activate the STAT5 pathway. A 40-year-old female with persistent moderate thrombocytosis was referred for eval- uation. Workup was negative for pseudo-thrombocytosis. She was noted to have mild iron deficiency. Reactive thrombocytosis and essential thrombocytosis were considered in the differential diagnosis. She was referred for clinical testing of CALR mutation analysis after she was found negative for JAK2. Mutation screening of CALR Exon 09 by PCR based Sanger sequencing revealed a novel mutation c.1191_1199del (p.E398_D400del) in this patient with African American ethnicity. Mutation status in CALR will aid the diagnosis of ET and PMF patients and risk stratification. Abstracts 363
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Brazil. Theuseof thearrayCGH(aCGH) hasaidedsigni-cantlythegeneticdiagnosisinrecent years. InourlaboratoryaCGH combined with conventional karyotyping allowed animprovement of 20%to30%inthediagnosticrateof patientswithintellectual disabillity, dysmorphisms, autismanddevel-opmental delay. We used the Affymetrix chip Cytoscan HD and750 K Cytoscan arrays (Santa Clara, CA) and analysis throughtherespectivesoftwaresystemandforconventionalkaryotype(GBanding). We selected 2 cases to report: Case 1: Twosiblings withdistinct developmental disturbancephenotypes.Siblingoneisa7-year-oldfemalewithseveredevelopmentaldelayphenotype, microcephalyandconsanguineousparents.Shepresentedaninterstitial deletionof 12,1Mbonchromo-some 5, arr [hg19] 5p14.3- p15.31 (6,801,589-18,992,827) 1,featuringtheCri-du-chatsyndrome.Herbrother,a12-year-oldmalewithmildintellectual disability(ID) aduplicationof theexactsamesizeandregionthatwasdeletedinthesisterwasfound arr [hg19] 5p14.3-p15.31 (6,801,589-18,992,827) 3.Parental aCGHwasnormal.However,conventional karyotypeof the father revealed 2 reciprocal translocation betweenchromosomes1and2, 5and7: (46, XY, t(1, 2)(q44;w p23-pter)t(5;7)(p14.3-p15.31;p22).Case2:A16-year-oldfemalewith consanguineous parents and normal conventional kar-yotyping. Shepresentssignicant developmental delay, dys-lalia, severe ID, autistic traits, irritability, hyperactivity andautoagression, without obviousdysmorphiasandwithnormal:MRI, electroencephalogramandCTscanresults. Microarrayanalysisrevealedahomozygousmicrodeletionof 197Kbponchromosome8:arr [hg19] 8p22(15,451,748-15,649,733) 1,spanningalmosttheentireTUSC3gene,exceptpromoterandrst exon(601385*).ThisistheseventhfamilywithareportedTUSC3 mutation with mostly non syndromic ID and the secondwith the same deletion (Khan et al, 2011). Three of the familiesreported in the literature carried a deletion which encompassedonlytheTUSC3promoterregionandtherst exon, 2familiespresenteddistinct point mutationsand1anintragenicdupli-cation.All 7familiespresentedhomozygousmutations,only1wasnot consanguineoushowever theywerefromthesamevillage.Conclusion: Thesecasesillustratetheimportanceof inte-gratingconventional karyotypingtechniquesandmicroarrayingeneticdiagnosis. Bothcaseswerereferredfor geneticcoun-sellingandfamilyresearch.UnusualROS1TranslocationPatternina61Year-oldWomanwithMetastaticAdenocarcinomaofLungHui Chena,Rajyalakshmi Luthrab,NedaKalhora,JohnHeymachc,RonaldAbrahamb,Meenakshi Mehrotrab,Bal MukundMishrab,KeyurP.Patelb,RajeshR.Singhb,XinyanLubaDepartmentofPathology,MDAndersonCancerCenter,Houston,TX,USA;bDepartmentofHematopathology,MDAndersonCancerCenter,Houston,TX,USA;cDepartmentofThoracic/Head&NeckMedical Oncology, MD Anderson Cancer Center, Houston, TX, USAFISHassayhasenabledustodetect geneamplication,deletionandtranslocationinsolidtumors. However, FISHislimitedtofocusedgenesof interest. Currentlythealternativemicroarraybasedgenome-wideanalysisfor copynumber ab-errationandallelicimbalancesof oncogenesremainsunder-characterized. In this study, we explored genomic copy numberanalysisona61-year-oldwomanwithmetastaticadenocarci-nomaof lungandrefractorytochemo-andadjuvant therapies.Primarytumor fromneneedleaspirationof lungandmeta-staticadenocarcinomafromcorebiopsyof lymphnodewereusedfor immunohistochemistry (IHC), FISHassay andnextgeneration sequencing analysis to detect overexpression/amplication, translocation, andsomaticmutations. Addition-ally, genomic DNAfrommetastatic tumor was subjected tomolecular inversion probe array (MIP) by OncoScan FFPEAssay kit (Affymetrix). Genomic copy number and allelicimbalancecalls wereperformedby OncoScanConsoleanddatareviewedby OncoScanNexus Express (BioDiscovery).Both primary and metastatic tumors were pan negative forEGFR, KRASand BRAF mutations and ALKtranslocation.FISHanalysisdemonstratedROS1translocationwithunusualdouble splitting signals suggestive of allelic imbalance in lymphnodemetastasisbutnotinprimarytumor.MIPstudyonlymphnode metastasis showed loss of heterozygosity involving 4chromosomes/arms including chromosome 6 where ROS1located. Inaddition, MIParray detectedcopy number gainsinvolving 9 chromosomes/arms. Genome instability withnumerous copy number gain and allelic imbalances, and ROS1translocationmight beoneof contributingfactors topatientpoorclinical outcome.ANovelMutationinCalreticulin(CALR)wasIdentiedinaPatientofAfricanAmericanOriginwithThrombocytosisD.P.Dasha,SherineJosephThomasbaBlood Center of Wisconsin, Milwaukee, WI, USA;bGeorgia CancerSpecialistsandNorthsideHospital CancerInstitute,Atlanta,GA,USARecent studiesshowthat Calreticulin(CALR) somaticmu-tations provide a diagnostic marker in JAK2/MPL wild typeessential thrombocythemia (ET) and primary myelobrosis(PMF) withamutationfrequencyof 67%to71%and56%to88%, respectively (Tefferi AandPardanani A, Nat Rev ClinOncol, 2014). In general, CALRmutations are not seen inPolycythemiaVera(PV),post-PVmyelobrosisorJAK2V617ForMPLmutatedETorPMF.StudiesshowedthatCALRmuta-tions may also provide prognostic informationand therefore is apromising molecular marker for diagnosis and prognosis forpatients with myeloproliferative neoplasm(MPN). The exactmechanismbywhichCALRmutationsproducethemyeloprolif-erative disease phenotype is unknown, but multiple studies haveshown thatCALRmutationsdisruptthe C-terminalendoplasmicreticulum-retentionsequence(KDEL), generateanovel C-ter-minus, andactivatetheSTAT5pathway. A40-year-oldfemalewithpersistent moderatethrombocytosiswasreferredforeval-uation. Workupwasnegativefor pseudo-thrombocytosis. Shewas noted to have mild iron deciency. Reactive thrombocytosisandessential thrombocytosis were consideredinthe differentialdiagnosis. She was referred for clinical testing of CALR mutationanalysis after she was found negative for JAK2. MutationscreeningofCALRExon09byPCRbasedSangersequencingrevealed a novel mutation c.1191_1199del (p.E398_D400del) inthispatient withAfricanAmericanethnicity. MutationstatusinCALRwill aidthediagnosisof ETandPMFpatientsandriskstratication.Abstracts 363
