Molecular Molecular CytopathologyCytopathology

Robert A. Edwards, M.D., Robert A. Edwards, M.D., Ph.D.Ph.D.

University of California, University of California, IrvineIrvine

August 22, 2006August 22, 2006

IntroductionIntroduction

The presentation will discuss recent The presentation will discuss recent progress in molecular diagnostics which progress in molecular diagnostics which have application in cytopathology. Topics have application in cytopathology. Topics will include techniques applicable to will include techniques applicable to adjunctive HPV testing, including hybrid adjunctive HPV testing, including hybrid capture, linear array PCR, and capture, linear array PCR, and microarray techniques. Examples of microarray techniques. Examples of molecular techniques for the diagnosis of molecular techniques for the diagnosis of neoplasia in fine needle aspirates from neoplasia in fine needle aspirates from solid organ tumors will also be presented.solid organ tumors will also be presented.

Learning ObjectivesLearning Objectives

Identify the principles of nucleic acid Identify the principles of nucleic acid –based testing in the cytopathology –based testing in the cytopathology laboratory. laboratory.

Apply the current guidelines and Apply the current guidelines and techniques for adjunct HPV testing. techniques for adjunct HPV testing.

Recognize the role of molecular Recognize the role of molecular diagnostics in non-gynecologic diagnostics in non-gynecologic cytology cytology

OutlineOutline

What’s Driving Molecular What’s Driving Molecular Cytopathology ?Cytopathology ?

Gynecologic CytopathologyGynecologic Cytopathology HPV Testing MethodologiesHPV Testing Methodologies HPV vaccination and HPV vaccination and

Non-Gynecologic CytopathologyNon-Gynecologic Cytopathology Applications in Solid TumorsApplications in Solid Tumors Applications in Effusion CytologyApplications in Effusion Cytology

What is Driving Advances inWhat is Driving Advances in Molecular Cytopathology? Molecular Cytopathology?

The Era of Personalized Medicine:The Era of Personalized Medicine: Advances in Molecular EpidemiologyAdvances in Molecular Epidemiology

Cancer Susceptibility Polymorphisms Cancer Susceptibility Polymorphisms (BRCA, MSI/MMP’s, XME’s)(BRCA, MSI/MMP’s, XME’s)

Heart Disease (LDL-R mutations)Heart Disease (LDL-R mutations) Therapeutic Responses Therapeutic Responses

(Pharmacogenomics)(Pharmacogenomics) Thrombotic Disease (Factor V Leiden, Thrombotic Disease (Factor V Leiden,

Prothrombin)Prothrombin)

Prognostic significance of specific Prognostic significance of specific mutation/translocation variantsmutation/translocation variants

What is Driving Advances inWhat is Driving Advances in Molecular Cytopathology? Molecular Cytopathology?

Advances in Human Molecular GenomicsAdvances in Human Molecular Genomics Solid tumor databases of genome-wide Solid tumor databases of genome-wide

expression patternsexpression patterns Identifies a tumor’s “signature”Identifies a tumor’s “signature”

SAGE (SAGE (http://www.ncbi.nlm.nih.gov/SAGE/http://www.ncbi.nlm.nih.gov/SAGE/))

CGAP (CGAP (http://cgap.nci.nih.gov/http://cgap.nci.nih.gov/))

What is Driving Advances inWhat is Driving Advances in Molecular Cytopathology? Molecular Cytopathology?

From Lab to Hospital : CP Leading the Way From Lab to Hospital : CP Leading the Way

Molecular diagnostics:- 7% of current CP testing: 20% by Molecular diagnostics:- 7% of current CP testing: 20% by 2010*2010* Factor V Leiden G1691A PCR/RFLP Factor V Leiden G1691A PCR/RFLP Factor II (Prothrombin) G20210A Polymorphism Factor II (Prothrombin) G20210A Polymorphism

PCR/RFLPPCR/RFLP Hemochromatosis- HFE Cys282Tyr Mutation Detection- Hemochromatosis- HFE Cys282Tyr Mutation Detection-

PCRPCR Mycobacterial Detection via rRNA PCR/ Line Probe AssayMycobacterial Detection via rRNA PCR/ Line Probe Assay Apo E Genotyping - PCRApo E Genotyping - PCR

Advances in High-Throughput Molecular Diagnostics/Sample Advances in High-Throughput Molecular Diagnostics/Sample PrepPrep Instrumentation/QC/BioinformaticsInstrumentation/QC/Bioinformatics

*Medical Laboratory Observer, 1/04: 36(1):11

What is Driving Advances inWhat is Driving Advances in Molecular Cytopathology? Molecular Cytopathology?

Minimally Invasive Sampling/MiniaturizationMinimally Invasive Sampling/Miniaturization Large scale epidemiology now done with gDNA Large scale epidemiology now done with gDNA

from buccal smears- 20 from buccal smears- 20 g gDNA from 2 swabs.g gDNA from 2 swabs. Hybrid Capture and PCR-based diagnostics require Hybrid Capture and PCR-based diagnostics require

low-microgram quantities gDNA or RNAlow-microgram quantities gDNA or RNA RNALater enables preservation of RNA in aspirates, RNALater enables preservation of RNA in aspirates,

effusions, urine sediments (Yield ~1 effusions, urine sediments (Yield ~1 g/100 ml)g/100 ml) Harvesting pure tumor DNA/RNA samples:Harvesting pure tumor DNA/RNA samples:

Laser Capture MicroscopyLaser Capture Microscopy Can capture live single cells for study/ tumor cell lines.Can capture live single cells for study/ tumor cell lines.

Mol. Cytol. Already in Mol. Cytol. Already in useuse

HBV genotypingHBV genotyping Forensics/patient identificationForensics/patient identification Genetic analysis (CVS +/- FISH)Genetic analysis (CVS +/- FISH) Pharmacogenomics for tailoring Pharmacogenomics for tailoring

drug therapy?drug therapy?

Molecular Testing in Molecular Testing in Gynecologic CytopathologyGynecologic Cytopathology

HPV: Molecular VirologyHPV: Molecular Virology

>90 HPV genotypes cloned, characterized high-risk: 16, 18, 31, 33, 35, 39, etc potentially high-risk : 26, 53, 66 low-risk: 6, 11, 40, 42, 43, 44, 54, 61, etc.

Many patients infected with high risk HPV, only 5-10% develop persistent infection

* NEJM 2003; 348:518-527* NEJM 2003; 348:518-527

HPV and Cervical HPV and Cervical Cancer: ScopeCancer: Scope

Long-term high-risk HPV infection Long-term high-risk HPV infection predisposes to progression to cancerpredisposes to progression to cancer

99.7% HPV prevalence in Cervical CA99.7% HPV prevalence in Cervical CA 2 million ASCUS diagnoses in US/year2 million ASCUS diagnoses in US/year Conventional Pap*:Conventional Pap*:

Sensitivity- 30% - 87% Sensitivity- 30% - 87% Specificity- 86% to 100% percent for Specificity- 86% to 100% percent for

detection of cervical cancer and its detection of cervical cancer and its precursors precursors

Possible role for adjunctive HPV DNA Possible role for adjunctive HPV DNA testingtesting

* Nanda et al., Ann Intern Med 2000 May 16;132(10):810-9

The ALTS TrialThe ALTS Trial Large prospective RCT to assess optimal

management of ASCUS and LSIL, 1996-2000#

Triage following a Dx of ASCUS or LSIL: 1). Immediate colposcopy 2). Accelerated repeat cytology 3) High-risk HPV testing

RESULT: With RESULT: With ASCUS dx, one HPV test ID’s 92.4% of CIN III

Established Role for Adjunctive HPV testing

* Am J Obstet Gynecol 2003; 188:1383-9

# http://www.cancer.gov/PREVENTION/alts/index.html

Indications for HPV Indications for HPV testingtesting

Primary screeningPrimary screening #1: Women with an ASCUS diagnosis#1: Women with an ASCUS diagnosis

If ASCUS and (+) for high risk HPV- colposcopy If ASCUS and (+) for high risk HPV- colposcopy IF ASCUS and (-) for high risk HPV - Repeat IF ASCUS and (-) for high risk HPV - Repeat

smear in twelve monthssmear in twelve months

#2: Combined (HPV and cytology) testing #2: Combined (HPV and cytology) testing — — For ‘low risk’ women over age 30 who are For ‘low risk’ women over age 30 who are

being screened for cervical cancer is another being screened for cervical cancer is another option for primary screening. option for primary screening.

FDA-approved in 2003, done not more than every FDA-approved in 2003, done not more than every 3 years3 years

Normal result: < 1 in 2000 chance of HSIL or worse in Normal result: < 1 in 2000 chance of HSIL or worse in subsequent 5 years subsequent 5 years

ASCCP/ACS/ACOG Interim ASCCP/ACS/ACOG Interim Guidelines for Adjunct HPV Guidelines for Adjunct HPV

Testing:Testing:

Wright, T.C. et al. Obstetrics & Gynecology 2004;103:304-309

Cytology HPV Follow-up

Neg Neg Screen in 3 yrs

Neg Pos Repeat combined test 6-12 mos

ASCUS Neg Repeat cytology 12 months

ASCUS Pos Colposcopy

>ASCUS Pos or Neg Colposcopy

ASCCP Consensus ASCCP Consensus Conference, 2006 Conference, 2006

http://www.asccp.org/consensus/indhttp://www.asccp.org/consensus/index.shtmlex.shtml

Will revise 2001 Guidelines for Will revise 2001 Guidelines for managing Cytological Abnormalities managing Cytological Abnormalities and CIN 9/18+19/06and CIN 9/18+19/06

See above URL for draft See above URL for draft recommendationsrecommendations

Considerations for HPV Considerations for HPV Testing:Testing:

Hybrid Capture II (Digene) Hybrid Capture II (Digene) vs PCRvs PCR

Requires liquid based samplesRequires liquid based samples Cost effectiveness depends on pt Cost effectiveness depends on pt

population, F/U population, F/U Must be high-throughput compatible Must be high-throughput compatible

with high sensitivity and specificity.with high sensitivity and specificity. Neither HC-II nor Amplicor specifically Neither HC-II nor Amplicor specifically

ID HPV serotype, while Roche Linear ID HPV serotype, while Roche Linear Array PCR does- (Clinical utility is Array PCR does- (Clinical utility is unclear)unclear)

Considerations for HPV Considerations for HPV Testing:Testing:

Hybrid Capture vs PCRHybrid Capture vs PCR Reimbursement/ Medicolegal:Reimbursement/ Medicolegal:

Only HCII FDA-Approved (3/2000)Only HCII FDA-Approved (3/2000) US Roche Amplicor HPV test US Roche Amplicor HPV test

announced 1/30/04, launched in announced 1/30/04, launched in EU in 4/04 EU in 4/04 Currently ‘Home-Brew” UseCurrently ‘Home-Brew” Use FDA approval?FDA approval?

Lysis of Sample with Base Lysis of Sample with Base Requires ~4 ml of residual sample, Requires ~4 ml of residual sample,

adequate in >95% of casesadequate in >95% of cases Denatures Viral (or bacterial) DNADenatures Viral (or bacterial) DNA Low Risk and High-Risk HPV Low Risk and High-Risk HPV

standards lysed in parallelstandards lysed in parallel Incubate 65Incubate 65ooC for 30 minutesC for 30 minutes ** Proper lysis prevents non-** Proper lysis prevents non-

specific DNA-RNA hybridsspecific DNA-RNA hybrids

Add pooled HPV RNA probes to Add pooled HPV RNA probes to specimensspecimens High-risk HPV RNA Cocktail: High-risk HPV RNA Cocktail:

(16/18/31/33/35/39/45/51/52/56/58/59/68)

Incubate 65Incubate 65ooC for 60 minutesC for 60 minutes

Digene Hybrid Capture II-Procedure

http://www.digene.com/clinician_4.html

Capture of Hybrids to Solid PhaseCapture of Hybrids to Solid Phase 96-well plate coated with anti-DNA-96-well plate coated with anti-DNA-

RNA hybrid Ab’s, incubated with RNA hybrid Ab’s, incubated with sample/probe mix @ RT for 60 sample/probe mix @ RT for 60 minutesminutes

Hc3: hybrids are captured with Hc3: hybrids are captured with immobilized pooled HPV-specific immobilized pooled HPV-specific biotinylated oligonucleotides biotinylated oligonucleotides (specific for HPV RNA probes). (specific for HPV RNA probes). Greater Specificity**, < FP’sGreater Specificity**, < FP’s

Label for Detection Label for Detection -Captured RNA:DNA hybrids incubated -Captured RNA:DNA hybrids incubated with alkaline phosphatase-labelled anti-with alkaline phosphatase-labelled anti-DNA-RNA antibody. (Major step of DNA-RNA antibody. (Major step of signal amplication)signal amplication)

** Castle PE, et al; J Clin Microbiol. 2003 Sep;41(9):4022-30

The bound alkaline phosphatase The bound alkaline phosphatase cleaves the chemiluminescent cleaves the chemiluminescent dioxetane substrate, producing dioxetane substrate, producing light measured via luminometerlight measured via luminometer

Limitations/IssuesLimitations/Issues Specificity: High-risk probe cocktail Specificity: High-risk probe cocktail

cross-reactivity with at least 15 cross-reactivity with at least 15 additional HPV genotypes* additional HPV genotypes* Poljak et al., J ClinVirol 2002; 25: S89-97

BUT- minimal effect on BUT- minimal effect on Sensitivity/Specificity for Sensitivity/Specificity for CIN 2 CIN 2 lesions lesions (Cancer Epidemiol Biomarkers Prev. 2002 Nov;11(11):1394-9) (Cancer Epidemiol Biomarkers Prev. 2002 Nov;11(11):1394-9)

Controversy over defining lower limit Controversy over defining lower limit of normalof normal

*.

Roche AmplicorRoche Amplicor

Amplicor HPV Microwell plate (MWP) Amplicor HPV Microwell plate (MWP) assayassay13 high risk anogential HPV types only13 high risk anogential HPV types onlyGreater genotype specificity than Greater genotype specificity than current testscurrent tests

Linear Array HPVLinear Array HPVGenotypes 37 anogenital HPV typesGenotypes 37 anogenital HPV types

Based on amplification of HPV L1 Based on amplification of HPV L1 allele variable regionsallele variable regions

Amplicor HPV TestAmplicor HPV Test

http://www.cytopathology.org/Assets/ASCCoolNewStuff.pdf

Amplicor vs HCIIAmplicor vs HCII

Poljak et al: Amplicor more sensitive, 85% Poljak et al: Amplicor more sensitive, 85% concordance (using multiplex PCR as gold concordance (using multiplex PCR as gold standard)standard) Acta Dermatovenerol Alp Panonica Adriat. 2005 Dec;14(4):147-Acta Dermatovenerol Alp Panonica Adriat. 2005 Dec;14(4):147-

5252. . Sandri et al: Amplicor = HCII, 83% concordance, Sandri et al: Amplicor = HCII, 83% concordance,

more FN with HCIImore FN with HCII, more FP with , more FP with Amplicor Amplicor J Clin Microbiol. 2006 Jun;44(6):2141-6.J Clin Microbiol. 2006 Jun;44(6):2141-6.

““Real Time” Real Time” (quantitative) PCR(quantitative) PCR

‘‘Regular” PCR: non-linear Regular” PCR: non-linear amplificationamplification

qPCR: Enables [product] qPCR: Enables [product] measurements each cycle. measurements each cycle. Exponential phase is prop to initial Exponential phase is prop to initial mRNA abundance. mRNA abundance.

From Janet Kornegay, http://www.cytopathology.org/Assets/ASCCoolNewStuff.pdf

Microarray- PrincipleMicroarray- Principle

HPV Testing- FutureHPV Testing- Future

• Hc3- Improved HPV typing; Rapid Capture Instrument for automated HPV testing

• Combination of HPV testing with other biomarkers to further stratify HPV+ women into different screening intervals

• Digene vs Roche competition for market• 108 patients’ insurance will cover HPV testing

HPV vaccinesHPV vaccines

Preventative vaccination:Preventative vaccination: Composed of VLP’s of recombinant self Composed of VLP’s of recombinant self

assembled L1 capsid and L2 peptide- assembled L1 capsid and L2 peptide- Mimics infectious virionMimics infectious virion Zhou et al., Zhou et al., Virology 1991; 185:251-7

Induces high-titer neutralizing humoral Induces high-titer neutralizing humoral (Ab) response against L1 capsid protein(Ab) response against L1 capsid protein

Two vaccines:Two vaccines:

Gardisil (Merck)Gardisil (Merck) -Tetravalent (HPV-6,11,16,18)-Tetravalent (HPV-6,11,16,18) 3 doses, 0, 1, and 6 months3 doses, 0, 1, and 6 months $120 per dose$120 per dose FDA approved for women 9-26, 6/8/06FDA approved for women 9-26, 6/8/06

Cervarix (Glaxo-SK)Cervarix (Glaxo-SK) Bivalent (16,18)Bivalent (16,18) FDA approval pending 12/06FDA approval pending 12/06

Issues:Issues: Immunize boys as well? (Cost vs. Immunize boys as well? (Cost vs.

efficacy)efficacy) Need for booster shots? Need for booster shots?

Only have 4 year data (so far)Only have 4 year data (so far) In HBV, memory T cells present even when In HBV, memory T cells present even when

anti-HBs is gone)anti-HBs is gone) Effect on current screening?Effect on current screening?

Decades before vaccinated cohort will enable Decades before vaccinated cohort will enable decreased HBV screening effortsdecreased HBV screening efforts

Cost- most needed in developing Cost- most needed in developing countriescountries

Non-Gynecologic Non-Gynecologic CytologyCytology

Applications of Molecular Techniques Applications of Molecular Techniques to Solid to Solid

Tumor Cytopathology: Tumor Cytopathology: BreastBreast PancreasPancreas ThyroidThyroid

Effusion/Fluids CytologyEffusion/Fluids Cytology Peritoneal EffusionsPeritoneal Effusions UrineUrine

Breast Cancer: SAGE Breast Cancer: SAGE Identification of Markers of Identification of Markers of

Invasion Invasion Cytology: Difficult to DDX DCIS from IDCACytology: Difficult to DDX DCIS from IDCA SAGE: 103 genes specific for IDCA vs DCIS or Nl SAGE: 103 genes specific for IDCA vs DCIS or Nl

breast; 68 ‘known’ genesbreast; 68 ‘known’ genes ISH reveals specific expression patterns among the 68ISH reveals specific expression patterns among the 68

Neoplastic epitheliumNeoplastic epithelium EndotheliumEndothelium Inflammatory responseInflammatory response Pan-StromaPan-Stroma Juxtatumoral-Stroma – differ between Juxtatumoral-Stroma – differ between

breast/pancreas, suggesting tumor-specific ID of breast/pancreas, suggesting tumor-specific ID of desmoplastic response. desmoplastic response.

Identified marker genes for infiltration, and possible Identified marker genes for infiltration, and possible DDX of in-situ vs. infiltrative CA. DDX of in-situ vs. infiltrative CA.

Iacobuzio-Donahue CA Cancer Res. 2002 Sep 15;62(18):5351-7

Microarray of Breast CA by Microarray of Breast CA by FNAFNA

38 breast FNA’s used to generate cDNA 38 breast FNA’s used to generate cDNA probes for high-density microarray probes for high-density microarray transcriptional profiling (TP)transcriptional profiling (TP) Average RNA yield from FNA material = 2 Average RNA yield from FNA material = 2 g g

(range 1-25 (range 1-25 g)g) 18 cases - established expression ratio cutoffs 18 cases - established expression ratio cutoffs

for ER and HER-2,:for ER and HER-2,: 20/20 prospective ID of HER2 status, 19/20 for ER 20/20 prospective ID of HER2 status, 19/20 for ER

status.status. SO:SO:

Transcriptional Profiling can be done from FNATranscriptional Profiling can be done from FNA TP reliably measures single-gene prognostic TP reliably measures single-gene prognostic

markersmarkers Complex transcriptional patterns can also identify Complex transcriptional patterns can also identify

clinical ER status. clinical ER status. Pusztai L, et al. Pusztai L, et al. Clin Cancer Res. 2003 Jul;9(7):2406-15.Clin Cancer Res. 2003 Jul;9(7):2406-15.

Other Applications to Other Applications to Breast Cancer?Breast Cancer?

Detection of Axillary LN Mets via real-time Detection of Axillary LN Mets via real-time qPCR?:qPCR?: H&E: 1 CA cell/ 10H&E: 1 CA cell/ 1044; IHC 1 in 10; IHC 1 in 1055. . RT-qPCR using anti-CK 19: 1 in 10RT-qPCR using anti-CK 19: 1 in 1077 cells, obviates the cells, obviates the

high false positive rate by enabling quantitation above high false positive rate by enabling quantitation above a threshold. a threshold.

Pilot Study: Pilot Study: (Baker M., et al. Amer J Surg 186(4): October 2003, Pages 351-(Baker M., et al. Amer J Surg 186(4): October 2003, Pages 351-358 D)358 D)

Preoperative ID of Micrometastases in non-palpable Preoperative ID of Micrometastases in non-palpable sentinel LN by U/S- FNA- 183 patientssentinel LN by U/S- FNA- 183 patients

57% sensitivity, 96% specificity in node (+) pts57% sensitivity, 96% specificity in node (+) pts AND:AND:

Schroder CPSchroder CP Int J Cancer. 2003 Sep 10;106(4):611-8. Int J Cancer. 2003 Sep 10;106(4):611-8. 75 sentinel nodes compared by H&E, IHC, and CK19 qRT-PCR:75 sentinel nodes compared by H&E, IHC, and CK19 qRT-PCR: IHC more sensitive and specific than qRT-PCRIHC more sensitive and specific than qRT-PCR

Pancreas: Genomics-Based Pancreas: Genomics-Based Marker Farming for Marker Farming for

Pancreatic FNA/ ERCPPancreatic FNA/ ERCP Pancreatic FNA interpretation limited by Pancreatic FNA interpretation limited by

morphologic overlap of neoplastic/reactive morphologic overlap of neoplastic/reactive processesprocesses

By SAGE, mesothelin is expressed in By SAGE, mesothelin is expressed in pancreatic CA but not normal pancreas pancreatic CA but not normal pancreas ((Argani PArgani P et et

al Clin Cancer Res. 2001 Dec;7(12):3862-8)al Clin Cancer Res. 2001 Dec;7(12):3862-8)

30 pancreatic FNA’s (19 CA, 9 suspicious, 11 30 pancreatic FNA’s (19 CA, 9 suspicious, 11 benign) stained for mesothelin. IHC for benign) stained for mesothelin. IHC for mesothelin shows 68% sens/91% specificitymesothelin shows 68% sens/91% specificity

6/9 suspicious> CA: 5/6 mesothelin+, 6/9 suspicious> CA: 5/6 mesothelin+, 3/9 suspicious> Neg: 3/3 mesothelin -.3/9 suspicious> Neg: 3/3 mesothelin -.

(McCarthy DM et al Appl Immunohistochem Mol Morphol. 2003 Sep;11(3):238-43)(McCarthy DM et al Appl Immunohistochem Mol Morphol. 2003 Sep;11(3):238-43)

ID of THYROID CA-ID of THYROID CA-SPECIFIC GENES for use SPECIFIC GENES for use

with future FNAwith future FNA Lifetime risk of developing clinically significant Lifetime risk of developing clinically significant

thyroid nodule = 10% thyroid nodule = 10% Problem- up to 30% of thyroid FNA = Problem- up to 30% of thyroid FNA =

Follicular Neoplasm NOS, lack of single Follicular Neoplasm NOS, lack of single markers for malignancymarkers for malignancy

Microarray Analysis of 17 follicular tumors Microarray Analysis of 17 follicular tumors performed, 105 genes differentially expressed performed, 105 genes differentially expressed (adenoma vs CA) . (adenoma vs CA) .

Generated a clustering analysis training setGenerated a clustering analysis training set Tested five follicular adenomas and two known Tested five follicular adenomas and two known

cancers, all correctly identified. cancers, all correctly identified. Barden CB, et al. Barden CB, et al. Clin Cancer Res. 2003 May;9(5):1792-800Clin Cancer Res. 2003 May;9(5):1792-800

Effusion CytologyEffusion Cytology Genetic-based testing for malignancy NOS:Genetic-based testing for malignancy NOS:

Telomerase overactivityTelomerase overactivity AneuploidyAneuploidy

Telomerase: Ribonucleoprotein involved in Telomerase: Ribonucleoprotein involved in maintaining chromosome ends. maintaining chromosome ends.

Immortalized (i.e.- cancer) cells overexpress Immortalized (i.e.- cancer) cells overexpress hTERT, can quantitate by qPCRhTERT, can quantitate by qPCR

Aneuploidy:Aneuploidy: Comparative Genomic Hybridization enables a Comparative Genomic Hybridization enables a

genome-wide snapshot of chromosomal genome-wide snapshot of chromosomal imbalances. Identifies losses or gains as small imbalances. Identifies losses or gains as small as 3-5 MB. as 3-5 MB.

hTERT RT-PCR for Effusion hTERT RT-PCR for Effusion CytologyCytology

Hiroi et al: Evaluated hTERT in 20 Hiroi et al: Evaluated hTERT in 20 malignant and 16 benign effusions by RT-malignant and 16 benign effusions by RT-PCRPCR

hTERT expressed in 19/20 malignant hTERT expressed in 19/20 malignant cytology specimens; negative in 16/16 cytology specimens; negative in 16/16 reactive specimensreactive specimens

Kovacs et al: Kovacs et al: 35 cytologic specimens: 92.3% specificity 35 cytologic specimens: 92.3% specificity

and 60% sensitivity and 60% sensitivity Hiroi, S, et al . Diagn Cytopathol. 2003 Oct;29(4):212-6.

Kovacs RB Int J Mol Med. 2004 Feb;13(2):303-9

Effusion Cytology and Effusion Cytology and CGHCGH

Nagel et al: Performed CGH on 15 malignant effusions:Nagel et al: Performed CGH on 15 malignant effusions: 8 ovarian adenocarcinomas8 ovarian adenocarcinomas 3 breast adenocarcinomas3 breast adenocarcinomas 1 colon adenocarcinoma1 colon adenocarcinoma 3 mesotheliomas3 mesotheliomas Benign Control: normal diploid human cell lines. Benign Control: normal diploid human cell lines. 14/15 yielded high-qaulity gDNA. Requires 10^5 to 10^6 cells 14/15 yielded high-qaulity gDNA. Requires 10^5 to 10^6 cells

SO: CGH on cytologic samples can provide primary dx SO: CGH on cytologic samples can provide primary dx of malignancy. of malignancy. Pattern of CGH may be informative about the primary Pattern of CGH may be informative about the primary Cons- labor intensive, requires gDNA isolation, sample and Cons- labor intensive, requires gDNA isolation, sample and

control gDNA labeling, hybridization (48-72hr), image analysis control gDNA labeling, hybridization (48-72hr), image analysis and interpretation. and interpretation.

Nagel H et al.Mod Pathol. 2002 Aug;15(8):818-25

Urine Cytology & Molecular Urine Cytology & Molecular DiagnosticsDiagnostics

The problem: ~60% sensitivity for urothelial The problem: ~60% sensitivity for urothelial Ca in conventional urine cytology specimensCa in conventional urine cytology specimens

Many diagnostic markers have been studiedMany diagnostic markers have been studied Some have higher sensitivity, esp. for Low Some have higher sensitivity, esp. for Low

GradeGrade BUT-BUT- Lower specificity, more false positives, esp. w/ Lower specificity, more false positives, esp. w/

concurrent inflammatory diseaseconcurrent inflammatory disease See Konety, B.R., See Konety, B.R., Urol. Onc.: Seminars and Urol. Onc.: Seminars and

Original InvestigationsOriginal Investigations 24(4): 326-337, 2006 24(4): 326-337, 2006

Current FDA approved tests Current FDA approved tests & Mean & Mean

Sensitivity/Specificity Sensitivity/Specificity CytologyCytology (48/96)(48/96) Hb dipstick testHb dipstick test (71/67)(71/67) NMP-22NMP-22 (ELISA) (ELISA) (67/74)(67/74) BTA-TRAK, BTA-Stat (Latex Agg.)BTA-TRAK, BTA-Stat (Latex Agg.) (62/73, (62/73,

69/74)69/74) ImmunoCyt (IF microscopy) ImmunoCyt (IF microscopy) (58/79)(58/79) Urovysion (FISH- 3,7,17,16) Urovysion (FISH- 3,7,17,16) (81/96)(81/96)

hTERT RT-PCR for hTERT RT-PCR for Urothelial CaUrothelial Ca

146 voided urines- pts with UroCa, 146 voided urines- pts with UroCa, 128 ctrls.128 ctrls.

Samples for cytology, RT-PCR for Samples for cytology, RT-PCR for hTERT. hTERT.

92% Cancers PCR+, 44% detected by 92% Cancers PCR+, 44% detected by cytologycytology

Specificity for both hTERT and Specificity for both hTERT and cytology = 96%cytology = 96%

BUT- not FDA approved BUT- not FDA approved Melissourgos N, Urology. 2003 Aug;62(2):362-7

Vysis Urovysion by FISHVysis Urovysion by FISH Low Grade Papillary Urothelial CA: Low Grade Papillary Urothelial CA:

Chr 9q32-4Chr 9q32-4 High Grade Papillary Urothelial CA: High Grade Papillary Urothelial CA:

p53, Chr 17p53, Chr 17 Urovysion detects aneuploidy of Urovysion detects aneuploidy of

3,7,17,9p 3,7,17,9p FDA approved for recurrence FDA approved for recurrence

surveillance in pts with Ca Dx.surveillance in pts with Ca Dx.

Mayo: UroVysion FISH vs. Mayo: UroVysion FISH vs. OthersOthers

280 voided urines tested for BTA, Hemoglobin 280 voided urines tested for BTA, Hemoglobin dipstick, Telomerase PCR, and UroVysiondipstick, Telomerase PCR, and UroVysion

75 pts shown to have UroCa:75 pts shown to have UroCa: Sensitivity: Urovysion (81%), BTA Stat (78%), Hb Sensitivity: Urovysion (81%), BTA Stat (78%), Hb

dipstick (74%) and Telomerase (46%)dipstick (74%) and Telomerase (46%) Specificity: Urovysion (96%), Telomerase (91%), Specificity: Urovysion (96%), Telomerase (91%),

BTA Stat (74%), Hb dipstick (51%) BTA Stat (74%), Hb dipstick (51%)

SO: UroVysion most sensitive and specific for Dx SO: UroVysion most sensitive and specific for Dx UroCaUroCa

Telomerase: Good specificity, poor sensitivity.Telomerase: Good specificity, poor sensitivity. BTA stat/hemoglobin dipstick: Good sensitivity, poor BTA stat/hemoglobin dipstick: Good sensitivity, poor

specificityspecificity

Halling KC J Urol. 2002 May;167(5):2001-6

ConclusionsConclusions

Molecular Medicine has come to Molecular Medicine has come to cytologycytology

Challenges:Challenges: Prospective studies/EfficacyProspective studies/Efficacy Costs/ReimbursementCosts/Reimbursement

A useful adjunct to some difficult A useful adjunct to some difficult morphologic diagnosesmorphologic diagnoses