Molecular CytologyMolecular Cytology

Teresa Alasio, MDJune 3, 2009

Teresa Alasio, MDJune 3, 2009

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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)

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

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

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

Non-GynNon-Gyn

EffusionsUrineWashings/brushings

EffusionsUrineWashings/brushings

Serous EffusionsSerous Effusions

FISH/CISHApplications:

MetastasisMesothelioma

Lymphomas involving serous membranesFlow cytometry

FISH/CISHApplications:

MetastasisMesothelioma

Lymphomas involving serous membranesFlow cytometry

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

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

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)

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

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

Normal urothelial cell with 2 copies of chr 3 (red), 7(green) 17 (aqua) and 9p21 locus (gold)

Urothelial carcinoma cell with 2 copies of chr 3 (red), increased chr 7 (green), chr 17 (aqua), and no 9p21 locus

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

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

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)

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

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.

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

FISH of breast aspirate

CEP17 = green

Her2/neu = orange

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

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

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

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)

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

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

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)

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.

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

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?

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.