molecular cytology2
DESCRIPTION
The intended audience is pathologists, pathology residents, cytopathology fellows and medical students.TRANSCRIPT
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Molecular CytologyMolecular Cytology
Teresa Alasio, MDJune 3, 2009
Teresa Alasio, MDJune 3, 2009
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OverviewOverview
Molecular cytology is defined as molecular studies applied on all types of cytologic specimens Gyn Non-gyn FNA
Liquid based preparatory techniques have made application of techniques feasible
Molecular cytology is defined as molecular studies applied on all types of cytologic specimens Gyn Non-gyn FNA
Liquid based preparatory techniques have made application of techniques feasible
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Influence of Molecular Technologies to Cytopathology
Influence of Molecular Technologies to Cytopathology
Molecular techniques as adjunct to morphology for diagnosis
Molecular results are meaningful when interpreted with proper morphologic correlation
Reshaping clinical cytology and training programs
Molecular techniques as adjunct to morphology for diagnosis
Molecular results are meaningful when interpreted with proper morphologic correlation
Reshaping clinical cytology and training programs
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Advantages to use of Cytology Specimens for
Molecular Analysis
Advantages to use of Cytology Specimens for
Molecular Analysis
DNA and RNA are better preserved in cytology specimens Samples in liquid based preservative offer
high quality molecular material Ease of obtaining fresh, whole cells Assessment can be immediately done A small quantity of material is needed
DNA and RNA are better preserved in cytology specimens Samples in liquid based preservative offer
high quality molecular material Ease of obtaining fresh, whole cells Assessment can be immediately done A small quantity of material is needed
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Types of SpecimensTypes of Specimens
Gyn Non-Gyn
Urine Effusions Washings and
brushings
Gyn Non-Gyn
Urine Effusions Washings and
brushings
FNA Sarcomas Thyroid Breast Pancreas Lymph nodes
FNA Sarcomas Thyroid Breast Pancreas Lymph nodes
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Molecular TechniquesMolecular Techniques
Comparative genomic hybridization Hybrid capture
FISH/CISH (chromogenic in situ hybridization)
LOH Telomerase assay RT-PCR Southern blotting
Comparative genomic hybridization Hybrid capture
FISH/CISH (chromogenic in situ hybridization)
LOH Telomerase assay RT-PCR Southern blotting
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Gyn CytologyGyn Cytology
Application of molecular techniques is logical progression in the evolution of gynecologic cytology
Liquid based cytology not only reduces number of unsat samples, also provides perfect media for molecular analysis
Application of molecular techniques is logical progression in the evolution of gynecologic cytology
Liquid based cytology not only reduces number of unsat samples, also provides perfect media for molecular analysis
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Prevalence – General Population
Prevalence – General Population
Human papilloma virus (HPV) is the most common sexually transmitted infection in the United States
20 million Americans 5.5 million new cases diagnosed annually
75% of all sexually active people will become infected with HPV during their lifetimes
Human papilloma virus (HPV) is the most common sexually transmitted infection in the United States
20 million Americans 5.5 million new cases diagnosed annually
75% of all sexually active people will become infected with HPV during their lifetimes
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Estimates of HPV Associated Disease in the US
Estimates of HPV Associated Disease in the US
Genital warts: 1%, 1.4 million Colposcopic (sub-clinical) changes: 4%,
5 million DNA positive, no lesions: 10%, 14
million HPV antibody positive but no DNA or
lesions: 60%, 81 million OVERALL 75% of population exposed
Genital warts: 1%, 1.4 million Colposcopic (sub-clinical) changes: 4%,
5 million DNA positive, no lesions: 10%, 14
million HPV antibody positive but no DNA or
lesions: 60%, 81 million OVERALL 75% of population exposed
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Question:Question:
What is the percentage of cervical carcinoma associated with HPV?
High Risk HPV types have been identified in 99.7% of all cervical cancers
What is the percentage of cervical carcinoma associated with HPV?
High Risk HPV types have been identified in 99.7% of all cervical cancers
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Human PapillomavirusesHuman Papillomaviruses
More than 100 types, fully sequenced
Typing based on nucleic acid sequence
Types assigned sequential number based on order of discovery No relation to phylogeny
More than 100 types, fully sequenced
Typing based on nucleic acid sequence
Types assigned sequential number based on order of discovery No relation to phylogeny
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HPV TypesHPV Types
Two major phylogenetic branches, differing affinities for site of infection
Cutaneous: Keratinized squamous epithelium
Mucosal: Non-keratinized squamous epithelium
Two major phylogenetic branches, differing affinities for site of infection
Cutaneous: Keratinized squamous epithelium
Mucosal: Non-keratinized squamous epithelium
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HPV Mucosal Types and Variants
HPV Mucosal Types and Variants
More than 30 types found in anogenital tract “Low risk” types: rarely found in cancers “High risk” types: frequently found in
cancers or similar to types found in cancer Variants best characterized for HPV 16
E6/E7 polymorphisms could modify oncogenicity
Cross-reactive in ELISA assays
More than 30 types found in anogenital tract “Low risk” types: rarely found in cancers “High risk” types: frequently found in
cancers or similar to types found in cancer Variants best characterized for HPV 16
E6/E7 polymorphisms could modify oncogenicity
Cross-reactive in ELISA assays
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Low Risk HPVLow Risk HPV
Types 6 and 11 are examples97% of condyloma cases
Cause benign lesionsGenital warts (condyloma acuminata)Low grade SIL
Not found in cervical cancers
Types 6 and 11 are examples97% of condyloma cases
Cause benign lesionsGenital warts (condyloma acuminata)Low grade SIL
Not found in cervical cancers
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LSILsLSILs
Majority of LSILs will regress spontaneously 92-94% of young women Older adult women – 60% regress and 20-
30% of LSILs progress to HSIL Treatment of LSIL includes observation
or HPV DNA testing, rather than immediate colposcopy in young women
Majority of LSILs will regress spontaneously 92-94% of young women Older adult women – 60% regress and 20-
30% of LSILs progress to HSIL Treatment of LSIL includes observation
or HPV DNA testing, rather than immediate colposcopy in young women
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HSILsHSILs
Persistent infection required for development of HSIL
Infection with multiple types predictive of persistence
Subjective diagnosis Less than 40% of HSILs will regress Treatment recommended for diagnosis
of HSIL
Persistent infection required for development of HSIL
Infection with multiple types predictive of persistence
Subjective diagnosis Less than 40% of HSILs will regress Treatment recommended for diagnosis
of HSIL
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High Risk HPVHigh Risk HPV
Types 16, 18, 31, 33, 45 are high risk
Type 16 most likely to persist40-60% of invasive cancer
Incubation period from initial infection to progression to carcinoma is 7-12 years
Types 16, 18, 31, 33, 45 are high risk
Type 16 most likely to persist40-60% of invasive cancer
Incubation period from initial infection to progression to carcinoma is 7-12 years
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Genome OrganizationGenome Organization
Similar for all papillomaviruses Only one strand transcribed Open reading frames (ORFs) named in
relation to bovine papillomavirus genes “Early” genes E1-E7 (but no E3 in HPV) “Late” genes L1 and L2, coding for major
and minor capsid proteins
Similar for all papillomaviruses Only one strand transcribed Open reading frames (ORFs) named in
relation to bovine papillomavirus genes “Early” genes E1-E7 (but no E3 in HPV) “Late” genes L1 and L2, coding for major
and minor capsid proteins
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Upstream regulatory region
Also called long control region (LCR) or non-coding region (NCR)
Contains transcriptional and replication regulatory elements
E1: Viral replication; maintains episome E2: Transcriptional regulation, co-factor for viral replication E4: Disrupts cytokeratins E5: Interacts with growth factor receptors E6: Transforming protein; p53 degradation E7: Transforming protein; Rb binding
L1 is major capsid proteinCapsid is 72 pentamers of L1 Expressed L1 assembles into viral conformation, viral-like particles (VLPs) L2 is minor capsid protein Required for encapsidation of viral genome
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Viral ReplicationViral Replication
Replication and assembly in nucleus Infection initiated in basal epithelial
cells Steady-state viral replication, some early-
region transcription Presumed site of latent infection
High-copy viral replication, late gene transcription and virion production limited to differentiating cells
Replication and assembly in nucleus Infection initiated in basal epithelial
cells Steady-state viral replication, some early-
region transcription Presumed site of latent infection
High-copy viral replication, late gene transcription and virion production limited to differentiating cells
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HPV Hybrid Capture AssayHPV Hybrid Capture Assay
Current FDA approved test 1995 tube format; 1999 micro-titer format
Liquid hybridization technique Chemiluminescent detection
Semi-quantitative signal, but no control for amount of input DNA
RNA probes react with DNA targets RNA-DNA hybrids captured and detected
with monoclonal antibody to hybrids
Current FDA approved test 1995 tube format; 1999 micro-titer format
Liquid hybridization technique Chemiluminescent detection
Semi-quantitative signal, but no control for amount of input DNA
RNA probes react with DNA targets RNA-DNA hybrids captured and detected
with monoclonal antibody to hybrids
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Hybrid Capture AssayHybrid Capture Assay
Low risk probe mix HPV types 6, 11, 42, 43, 44
High risk probe mix HPV types 16, 18, 31, 33, 35, 39, 45,
51, 52, 56, 58, 59 and 68 Good inter-laboratory comparison Results not type-specific
Low risk probe mix HPV types 6, 11, 42, 43, 44
High risk probe mix HPV types 16, 18, 31, 33, 35, 39, 45,
51, 52, 56, 58, 59 and 68 Good inter-laboratory comparison Results not type-specific
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Hybrid Capture II AssayHybrid Capture II Assay
Designed to work with exfoliated cervical sample
Recommended collection kit includes brush and sample transport media Collects endo- and ectocervical cells
5% of total specimen assayed for each probe group
Designed to work with exfoliated cervical sample
Recommended collection kit includes brush and sample transport media Collects endo- and ectocervical cells
5% of total specimen assayed for each probe group
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HPV PCR AssaysHPV PCR Assays
Small portion of genome targeted Allows testing of samples with poor quality
DNA Small changes in virus (variants or
integration) may give false negative results Amount of DNA assayed varies (limits
number of cells sampled)
Small portion of genome targeted Allows testing of samples with poor quality
DNA Small changes in virus (variants or
integration) may give false negative results Amount of DNA assayed varies (limits
number of cells sampled)
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HPV PCR AssaysHPV PCR Assays
Type specific assays Generally target E6/E7 region
Consensus assays Generally target L1 region Type(s) determined by type specific
hybridization, restriction digestion or sequencing
Type specific assays Generally target E6/E7 region
Consensus assays Generally target L1 region Type(s) determined by type specific
hybridization, restriction digestion or sequencing
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HPV QuantitationHPV Quantitation
“ Viral load” difficult to estimate because of uneven tissue distribution and variation in sampling
Requires some measure of number of cells in assay (denominator)
Quantitative PCR assays, usually type-specific
“ Viral load” difficult to estimate because of uneven tissue distribution and variation in sampling
Requires some measure of number of cells in assay (denominator)
Quantitative PCR assays, usually type-specific
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HPV In Situ HybridizationHPV In Situ Hybridization
Only method permitting direct visualization of virus in morphologic context
Applicable to formalin-fixed paraffin-embedded tissues
Type specificity is good, but cross-hybridization cannot be totally avoided
Results are very technique dependent Integration status can be determined
Only method permitting direct visualization of virus in morphologic context
Applicable to formalin-fixed paraffin-embedded tissues
Type specificity is good, but cross-hybridization cannot be totally avoided
Results are very technique dependent Integration status can be determined
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Non-GynNon-Gyn
EffusionsUrineWashings/brushings
EffusionsUrineWashings/brushings
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Serous EffusionsSerous Effusions
FISH/CISHApplications:
MetastasisMesothelioma
Lymphomas involving serous membranesFlow cytometry
FISH/CISHApplications:
MetastasisMesothelioma
Lymphomas involving serous membranesFlow cytometry
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Chromosomal Alterations in Effusions
Chromosomal Alterations in Effusions
Mesotheliomas Chromosome 6 (loss of long arm)
Adenocarcinomas Chromosome 8 (gains or inversions (8q) -
pulmonary adenocarcinomas Numerical and structural alterations on
chromosomes 1,3,6,7,8,9,11,12,17 have been seen in metastatic breast and ovarian cancer
Mesotheliomas Chromosome 6 (loss of long arm)
Adenocarcinomas Chromosome 8 (gains or inversions (8q) -
pulmonary adenocarcinomas Numerical and structural alterations on
chromosomes 1,3,6,7,8,9,11,12,17 have been seen in metastatic breast and ovarian cancer
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Comparative Genomic Hybridization (CGH)
Comparative Genomic Hybridization (CGH)
Promising application for cytology Permits outlining of DNA sequences and
copy number alteration losses, gains, amplifications are identified May provide information about site of
primary tumors and distinguish cytogenetic alterations affecting oncogenes and tumor suppressor genes involved in neoplastic progression and metastasis
Promising application for cytology Permits outlining of DNA sequences and
copy number alteration losses, gains, amplifications are identified May provide information about site of
primary tumors and distinguish cytogenetic alterations affecting oncogenes and tumor suppressor genes involved in neoplastic progression and metastasis
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Telomerase AssayTelomerase Assay
Telomerase is prevents incomplete chromosomal replication, nuclease degradations and end-to-end fusions, inactivated, leads to senescence in somatic cells
Telomerase is reactivated in neoplasia Telomerase repeat amplification protocol
(TRAP) applied to cytology with high spec/sens in malignant cytology samples (effusions, breast fnas)
Telomerase is prevents incomplete chromosomal replication, nuclease degradations and end-to-end fusions, inactivated, leads to senescence in somatic cells
Telomerase is reactivated in neoplasia Telomerase repeat amplification protocol
(TRAP) applied to cytology with high spec/sens in malignant cytology samples (effusions, breast fnas)
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UrineUrine
Diagnose primary bladder cancerMonitor patients for recurrenceLow grade urothelial tumors“Atypical” urothelial cellsReflex testing can be performed
and reimbursed
Diagnose primary bladder cancerMonitor patients for recurrenceLow grade urothelial tumors“Atypical” urothelial cellsReflex testing can be performed
and reimbursed
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UroVysionUroVysion Multi-targeted FISH Four single stranded fluorescently
labeled nucleic acid probes Chromosome enumeration probes (CEPs) to
chromosomes 3, 7 and 17 Frequently accumulated in urothelial tumors,
reflects aneuploidy Single locus specific identifier (LSI) probe
9p21 Present in early, noninvasive tumors
Multi-targeted FISH Four single stranded fluorescently
labeled nucleic acid probes Chromosome enumeration probes (CEPs) to
chromosomes 3, 7 and 17 Frequently accumulated in urothelial tumors,
reflects aneuploidy Single locus specific identifier (LSI) probe
9p21 Present in early, noninvasive tumors
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Normal urothelial cell with 2 copies of chr 3 (red), 7(green) 17 (aqua) and 9p21 locus (gold)
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Urothelial carcinoma cell with 2 copies of chr 3 (red), increased chr 7 (green), chr 17 (aqua), and no 9p21 locus
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Bronchial SpecimensBronchial Specimens
Interphase multicolor FISH (IM-FISH) containing locus specific probes to Chromosome 6 (gains) 5p15 8q24 (c-myc) 7p12 (EGFR) gene
More sensitive than conventional cytology for detecting lung cancer in these specimens Detects tumors at earlier stages and higher
proportion of peripheral tumors
Interphase multicolor FISH (IM-FISH) containing locus specific probes to Chromosome 6 (gains) 5p15 8q24 (c-myc) 7p12 (EGFR) gene
More sensitive than conventional cytology for detecting lung cancer in these specimens Detects tumors at earlier stages and higher
proportion of peripheral tumors
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FISH/CISH Sarcomas Lymphomas Breast Thyroid
FISH/CISH Sarcomas Lymphomas Breast Thyroid
PCR-based assays Lymphomas
(monoclonal rearrangements)
PCR/RT-PCR Soft tissue Pancreas
Southern blotting
PCR-based assays Lymphomas
(monoclonal rearrangements)
PCR/RT-PCR Soft tissue Pancreas
Southern blotting
Molecular Techniques used on Fine Needle Aspiration Specimens
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SarcomasSarcomas
FISH probes are commercially available for specific chromosomal translocations Can be applied to conventional cytologic
preparations FISH used for
t(11;22)(q24;q12) t(X;18)(q11;q11) t(2;13), t(1;13) t(12;16)
FISH probes are commercially available for specific chromosomal translocations Can be applied to conventional cytologic
preparations FISH used for
t(11;22)(q24;q12) t(X;18)(q11;q11) t(2;13), t(1;13) t(12;16)
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ThyroidThyroid
Molecular alterations in PTC include RET TRK rearrangements BRAF and RAS mutations
Follicular carcinomas PAX8-PPAR gamma translocations RAS mutations
Mutations in PTC are not present in all PTCs, may only be able to help in cases of follicular variant
More work needed in this field
Molecular alterations in PTC include RET TRK rearrangements BRAF and RAS mutations
Follicular carcinomas PAX8-PPAR gamma translocations RAS mutations
Mutations in PTC are not present in all PTCs, may only be able to help in cases of follicular variant
More work needed in this field
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BreastBreast
Her2/neu is important prognostic and predictive factor in breast cancer Proto-oncogene on chromosome 17
FISH and IHC are both used for Her2/neu Her2/neu status is usually concordant
between primary tumor and locoregional and distant mets, so FNA specimens of mets are used Important clinical application because
treatments are based on overexpression of Her2/neu in metastases.
Her2/neu is important prognostic and predictive factor in breast cancer Proto-oncogene on chromosome 17
FISH and IHC are both used for Her2/neu Her2/neu status is usually concordant
between primary tumor and locoregional and distant mets, so FNA specimens of mets are used Important clinical application because
treatments are based on overexpression of Her2/neu in metastases.
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FISH on FNA vs. TissueFISH on FNA vs. Tissue
It has been reported that FISH for Her2/neu performed on direct FNA smears has comparable results with tissue sections
Because FNA smears provide whole nuclei, these are ideal specimens for evaluation of Her2/neu by FISH
It has been reported that FISH for Her2/neu performed on direct FNA smears has comparable results with tissue sections
Because FNA smears provide whole nuclei, these are ideal specimens for evaluation of Her2/neu by FISH
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FISH of breast aspirate
CEP17 = green
Her2/neu = orange
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Other Molecular StudiesOther Molecular Studies
FISH for chromosomal aneusomy Chromosomes 1,8,11,17
PCR for allelotyping Determination of methylation status
Establish benign vs. malignant RAR2, RASSF1A, cyclin D2
Clonality assays LOH analysis
Transcription profiling cDNA microarrays
FISH for chromosomal aneusomy Chromosomes 1,8,11,17
PCR for allelotyping Determination of methylation status
Establish benign vs. malignant RAR2, RASSF1A, cyclin D2
Clonality assays LOH analysis
Transcription profiling cDNA microarrays
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ThinPrep of atypical breast epithelial cells.
Clusters and individual epithelial cells hybridized with centromeric probes for chromosomes 1, 811 and 17, showing polysomy for all chromosomes
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PancreasPancreas
EUS FNA Analysis of cyst fluid for CEA and DNA
Cystic neoplasms which lack cellularity cannot make diagnosis on cytology
Molecular studies help identify potential for malignancy where cytology cannot make diagnosis
EUS FNA Analysis of cyst fluid for CEA and DNA
Cystic neoplasms which lack cellularity cannot make diagnosis on cytology
Molecular studies help identify potential for malignancy where cytology cannot make diagnosis
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Indications for Molecular Analysis of Cyst Fluid
Indications for Molecular Analysis of Cyst Fluid
Pancreatic cystic lesion (unknown type) Mucinous or non-mucinous? Benign or malignant? IPMN of unknown risk for malignancy Non-diagnostic FNA of pancreatic mass
Pancreatic or biliary stricture of unknown etiology
Reactive or neoplastic? Pancreatic endocrine tumors (PET)
Pancreatic cystic lesion (unknown type) Mucinous or non-mucinous? Benign or malignant? IPMN of unknown risk for malignancy Non-diagnostic FNA of pancreatic mass
Pancreatic or biliary stricture of unknown etiology
Reactive or neoplastic? Pancreatic endocrine tumors (PET)
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Mucinous Cysts Mucinous Cysts
Molecular events seen in the development of pancreatic cancer, including
K-ras mutationp53 overexpressionLoss of p16SMAD4
Molecular events seen in the development of pancreatic cancer, including
K-ras mutationp53 overexpressionLoss of p16SMAD4
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PANDA StudyPANDA Study
Pancreas Cyst DNA Analysis Multi-center prospective study
124 patients pancreatic cyst/surgical histology correlates MGH, UPMC, UPenn, MUSC, Wash U,
Indiana U
Pancreas Cyst DNA Analysis Multi-center prospective study
124 patients pancreatic cyst/surgical histology correlates MGH, UPMC, UPenn, MUSC, Wash U,
Indiana U
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PANDA Study: ResultsPANDA Study: Results
Presence of K-ras point mutation and multiple LOH mutations each independently predicted mucinous vs. non-mucinous etiology (p<.0001)
Combination of K-ras and multiple LOH mutations showed >95% specificity for the presence of malignancy
High amount, good quality DNA and cumulative amount of high amplitude LOH mutations associated with presence of high grade dysplasia/malignancy (p<.0001)
Presence of K-ras point mutation and multiple LOH mutations each independently predicted mucinous vs. non-mucinous etiology (p<.0001)
Combination of K-ras and multiple LOH mutations showed >95% specificity for the presence of malignancy
High amount, good quality DNA and cumulative amount of high amplitude LOH mutations associated with presence of high grade dysplasia/malignancy (p<.0001)
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PANDA Study: ConclusionsPANDA Study: Conclusions Combination of tests need to be performed to
maximize the diagnostic yield of pancreatic cyst FNAs CEA level Cytology DNA analysis
DNA analysis should be considered when cyst cytology is negative
In presence of large amounts of DNA, high-amplitude mutations or mutational sequency of k-ras mutation followed by allelic loss, presence of malignancy should be suspected.
Combination of tests need to be performed to maximize the diagnostic yield of pancreatic cyst FNAs CEA level Cytology DNA analysis
DNA analysis should be considered when cyst cytology is negative
In presence of large amounts of DNA, high-amplitude mutations or mutational sequency of k-ras mutation followed by allelic loss, presence of malignancy should be suspected.
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ConclusionsConclusions
Molecular techniques can be applied to cytology specimens in most if not all specimen types Gyn Non-gyn FNA
Early detection of cancers as well as pre-malignant lesions are identified using molecular techniques on cytology specimens
Molecular techniques can be applied to cytology specimens in most if not all specimen types Gyn Non-gyn FNA
Early detection of cancers as well as pre-malignant lesions are identified using molecular techniques on cytology specimens
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ConclusionsConclusions
Information obtained from use of molecular analysis should be correlated with cytologic findings to obtain the best diagnosis
Implications for prognosis and treatment are evident
Is this the future of cytology?
Information obtained from use of molecular analysis should be correlated with cytologic findings to obtain the best diagnosis
Implications for prognosis and treatment are evident
Is this the future of cytology?
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ReferencesReferences Schmitt FC, Longatto-Filho A, Valent A, Vielh P. Molecular
techniques in cytopathology practice. Journal of Clinical Pathology 2008; 61:258-267.
Krishnamurthy S. Applications of Molecular Techniques to Fine-Needle Aspiration Biopsy. Cancer Cytopathology 2007: 111(2), 106-122.
Bubendorf L, Grilli B. Multiprobe FISH for Enhanced Detection of Bladder Cancer in Voided Urine Specimens and Bladder Washings. AJCP 2001; 115(5).
Sneige N, Liu B, Yin G, et al. Correlation of cytologic findings and chromosomal instability detected by fluorescence in situ hybridization in breast fine-needle aspiration specimens from women at high risk for breast cancer. Modern Pathology (2006) 19: 622-629.
Beaty BG, Bryant R, Wang W, et al. HER2/neu Detection in Fine Needle Aspirates of Breast Cancer, 2004; 122(2).
Khalid A, et al. Pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts: a report of the PANDA study. 2009. Gastrointestinal Endoscopy.
Schmitt FC, Longatto-Filho A, Valent A, Vielh P. Molecular techniques in cytopathology practice. Journal of Clinical Pathology 2008; 61:258-267.
Krishnamurthy S. Applications of Molecular Techniques to Fine-Needle Aspiration Biopsy. Cancer Cytopathology 2007: 111(2), 106-122.
Bubendorf L, Grilli B. Multiprobe FISH for Enhanced Detection of Bladder Cancer in Voided Urine Specimens and Bladder Washings. AJCP 2001; 115(5).
Sneige N, Liu B, Yin G, et al. Correlation of cytologic findings and chromosomal instability detected by fluorescence in situ hybridization in breast fine-needle aspiration specimens from women at high risk for breast cancer. Modern Pathology (2006) 19: 622-629.
Beaty BG, Bryant R, Wang W, et al. HER2/neu Detection in Fine Needle Aspirates of Breast Cancer, 2004; 122(2).
Khalid A, et al. Pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts: a report of the PANDA study. 2009. Gastrointestinal Endoscopy.