Anatomic Molecular Pathology:Anatomic Molecular Pathology:An Emerging FieldAn Emerging Field
Antonia R. Sepulveda M.D., Ph.D.Antonia R. Sepulveda M.D., Ph.D.University of PennsylvaniaUniversity of Pennsylvania
[email protected]@mail.med.upenn.edu
2008 ASIP Annual Meeting2008 ASIP Annual Meeting
Anatomic pathology (U.S.) is a medical specialtythat is concerned with the diagnosis of diseasebased on the gross, microscopic, and molecularexamination of organs, tissues, and cells.
Molecular pathology
Molecular pathology is an emerging discipline within anatomicalpathology which is focused on the use of nucleic acidbasedtechniques such as insitu hybridization, reversetranscription,PCR, and nucleic acid microarrays for specialized studies ofdisease in tissues and cells.
Molecular pathology shares some aspects of practice with bothanatomic and clinical pathology, and is sometimes considered a"crossover" discipline.
Anatomic PathologyAnatomic Pathologynn Surgical PathologySurgical Pathologynn Surgical resection specimensSurgical resection specimensnnDiagnosis, stagingDiagnosis, stagingnnPrognostic/predictive markersPrognostic/predictive markers
nn BiopsiesBiopsiesnndiagnostic, stagingdiagnostic, stagingnn Prognostic/predictive markersPrognostic/predictive markers
nn CytopathologyCytopathologynn Cells in biological fluidsCells in biological fluidsnn Fine needle aspirationFine needle aspiration
nn Autopsy PathologyAutopsy Pathology
Surgical PathologySurgical Pathology
nn Surgical specimenSurgical specimennn SamplingSampling
nn Paraffin embedded tissueParaffin embedded tissue
Surgical PathologySurgical Pathology
Diagnosis:Diagnosis:Moderately differentiated adenocarcinomaModerately differentiated adenocarcinoma
SR086XX
Molecular Tests in AnatomicMolecular Tests in AnatomicPathologyPathology
nn Tumor tissueTumor tissueüü Mutational analysis: EGFR, BRAF,Mutational analysis: EGFR, BRAF, KrasKras, LOH, LOHüü Microsatellite Instability & DNA MMR IHCMicrosatellite Instability & DNA MMR IHCüü CpG methylation: hMLH1CpG methylation: hMLH1üü Chromosomal TranslocationsChromosomal Translocationsüü Gene expression profilesGene expression profilespredictive markerspredictive markersnn Microarray gene expression profilesMicroarray gene expression profilesnn MicroRNAMicroRNA profilesprofilesnn Proteomic testingProteomic testing
nn NonNonneoplasticneoplasticüü Infectious agentsInfectious agents
nn OtherOtherüü Identity tests (STR panels)Identity tests (STR panels)
Tissue Based Molecular Tests forTissue Based Molecular Tests forHereditary Nonpolyposis colorectal
Cancer (HNPCC) WorkupHNPCC) Workup
HNPCC and DNA Mismatch Repair Proteins
nn MutS:MutS: MSH2MSH2,, MSH6MSH6, MSH3, MSH3nn MutL:MutL: MLH1MLH1,, PMS2PMS2, MLH3, MLH3
MMR proteins function as heterodimersMMR proteins function as heterodimersThe heterodimers are required for protein stabilityThe heterodimers are required for protein stability
Hereditary Nonpolyposis Colorectal Cancer (HNPCC):1 Loss of function/expression of DNA mismatch
repair protein in tumors (germline mutation)2 Mutator Phenotype: Microsatellite Instability (MSI)
Algorithm forLaboratoryTestingfor HNPCC
Lindor, NM et al. JAMA,296 (12), 2006
Algorithm forAlgorithm forLaboratoryLaboratoryTestingTestingfor HNPCCfor HNPCC
Lindor, NM et al. JAMA,296 (12), 2006
MSI
MLH1 methyland/orBRAF mut
TESTING MSI IN TUMORSTESTING MSI IN TUMORSPROTOCOLPROTOCOL
nn Unstained sections from formalin fixedUnstained sections from formalin fixedparaffin embedded tissueparaffin embedded tissue
nn Scrape selected areas for DNA extractionScrape selected areas for DNA extractionnn PCR amplification with MSI panelPCR amplification with MSI panelnn DNA fragment characterization byDNA fragment characterization by
electrophoresiselectrophoresis
BAT25 BAT26 D5S346 D17S250
N N N N
T T T T
COLORECTAL ADENOCARCINOMA:
ØHigh Level MicrosatelliteInstability (MSIH)ØLoss of hMLH1 Protein
Expression
Gologan & Sepulveda et al.Arch Pathol Lab Med 2005;129: 13901397
Incidence and functional consequences ofIncidence and functional consequences ofhMLH1hMLH1 promoter hypermethylation in colorectalpromoter hypermethylation in colorectal
carcinomacarcinoma
11 of 13 (84%) MSIpositive cancersexhibited prominent methylation,compared with 2 of 21 MSInegative cancers (P =0.0001).
Methylation specific PCR (MSP)
James G. Herman et al. and Stephen B.James G. Herman et al. and Stephen B. BaylinBaylinPNAS, Vol. 95, Issue 12, 6870PNAS, Vol. 95, Issue 12, 68706875, 19986875, 1998
Distinction of HNPCC and Sporadic MicrosatelliteDistinction of HNPCC and Sporadic MicrosatelliteUnstable CRC through Quantification ofUnstable CRC through Quantification of
MLH1 Methylation by RealMLH1 Methylation by Realtime PCRtime PCR
MM BettstetterBettstetter et al and Wet al and W DietmaierDietmaier.. Clinical Cancer ResearchClinical Cancer Research 13, 322113, 32213228, 20073228, 2007
BRAF Mutation is Frequently Present in SporadicCRC with Methylated hMLH1, but not in HNPCC
nn BRAF:BRAF: cytoplasmic serine/threonine kinasecytoplasmic serine/threonine kinasenn Function:Function: mitogenmitogenactivated protein kinaseactivated protein kinase
signaling pathwaysignaling pathwaynn Most of the mutations inMost of the mutations in BRAFBRAF : thymine to: thymine to
adenineadenine transversiontransversion at nucleotide 1796at nucleotide 1796nn Substitution ofSubstitution of valinevaline for glutamate (V600E)for glutamate (V600E)nn Results inResults in BRAFBRAF activating mutationactivating mutation
nn BRAFBRAF V600E mutationsV600E mutations occur in:occur in:nn Sporadic MSISporadic MSIH CRC (40H CRC (4074%)74%)nn Sporadic MSS CRC 4% to 12%Sporadic MSS CRC 4% to 12%nn BRAF V600E mutations not detectedBRAF V600E mutations not detected
in HNPCC patientsin HNPCC patientsG Deng et al. and YS Kim Clin Cancer Res, 10, 191195, 2004
EPIDERMAL GROWTH FACTOREPIDERMAL GROWTH FACTORRECEPTOR (EGFR) TESTING INRECEPTOR (EGFR) TESTING IN
LUNG CANCERLUNG CANCER
Molecular Diagnosis in Lung CancerMolecular Diagnosis in Lung CancerNCCN Practice Guidelines (v.2.2008)NCCN Practice Guidelines (v.2.2008)
nn EGFR mutations define a subgroup ofEGFR mutations define a subgroup ofNSCLC patients who will benefit fromNSCLC patients who will benefit fromEGFR tyrosine kinase inhibitor (TKI)EGFR tyrosine kinase inhibitor (TKI)treatmenttreatment
Distribution of Mutations andDistribution of Mutations andResponse Rate to TKIResponse Rate to TKI
Cancer Science (OnlineEarly Articles).doi:10.1111/j.13497006.2007.00607.x
~90%
EX19 DEL
L858R
Not all EGRF mutationsassociated with response
Detection of EGFR Exon 19Detection of EGFR Exon 19DeletionsDeletions
Exon 19Inframe deletionsusually 9 –24 bp
208 bp –190 bp = 18 bp
Positive fordeletion
Normal
Oncotype DX is validated for use inbreast cancer patients
MultigeneMultigene Expression Assay (RTExpression Assay (RTPCR)PCR)to Predict Recurrence ofto Predict Recurrence of TamoxifenTamoxifenTreated, NodeTreated, NodeNegative Breast CancerNegative Breast CancerSelection of Patients for ChemotherapySelection of Patients for Chemotherapy
S.PaikS.Paik et al.et al. NEJM. 351:2817NEJM. 351:28172826, 20042826, 2004
nn Gene expressionGene expressionmicroarraymicroarraybased testbased test
nnHelps identify tissue ofHelps identify tissue oforigin of poorly &origin of poorly &undifferentiated cancersundifferentiated cancers
nn15 tissue types15 tissue types
PathworkPathwork™™ TissueTissueof Origin Testof Origin Test
CancerTYPEMolecularClassification Testn92gene realtime
quantitative PCR assay
nMolecular classificationof human cancers
nn39 tumor classes39 tumor classes
Molecular Diagnosis of InfectiousMolecular Diagnosis of InfectiousAgentsAgents
nn Whipple’s disease:Whipple’s disease: TropherymaTropheryma whipelliiwhipelliinn Test: PCR forTest: PCR for rRNArRNA DNA sequenceDNA sequencenn Confirmation of diagnosis and followConfirmation of diagnosis and followupup
The Molecular Anatomic PathologyThe Molecular Anatomic PathologyLaboratoryLaboratory
yeinjee.com/.../2007/08/parislouvrepyramid.jpg
Anatomic PathologyAnatomic Pathology
Molecular DiagnosticsMolecular Diagnostics
Career Opportunities in MolecularCareer Opportunities in MolecularAnatomic PathologyAnatomic Pathology
Environment:Environment:ØØAcademia, Hospitals, ReferenceAcademia, Hospitals, Reference
Laboratories and IndustryLaboratories and IndustryPositionsPositionsØØScientific DirectorsScientific DirectorsØØResearch and Clinical TechniciansResearch and Clinical Technicians
and Technologistsand TechnologistsRoles:Roles:ØØTranslational researchTranslational researchØØTest developmentTest developmentØØValidation and clinical testingValidation and clinical testing
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