Gene Expression and Gene Sequencing Using Cytology

Specimens

Jeffrey S. Ross, M.D.Albany Medical College

Albany, NYrossj@mail.amc.edu

24st Annual Advances in Cytology CytologyJune 13, 2012

Gene Expression Profiling in Cytology

• Can be performed on fresh FNA material• Difficult to perform on FFPE cell block

materials• Clinical utility limited to Oncology

– Cancer of unknown primary site– Prognosis assessment– Prediction of response to targeted therapy– Prediction of response to conventional therapy

Gene Expression Profiling Techniques

• RT-PCR– For individual genes – For genesets

• Oncotype DX• Others

• Genomic Micorarrays– For genesets

• Mammaprint• Others

• Mathematic Models and Algorithms– For results classification– For disease outcome prediction

Advantages of Cytology Specimens

• Relatively easy to obtain new specimens• Ease of repeated sampling for therapy response

information• Lower cost of sampling• FNA is Less invasive and better tolerated• Improved patient compliance• Tumor cell reduced cohesiveness enriches for

malignant cells vs stromal and inflammatory cells

Disadvantages of Cytology Specimens

• Small sample size– Lower mRNA yield– Reservoir of sample may be limited or absent

• May lack relevant histologic correlation(s)• Tumor cell reduced cohesiveness enriches for

malignant cells vs stromal and inflammatory cells

Expression Profiling for Disease Classification Examples

• Lymphoma and Leukemia

• Solid Tumors (Breast, Prostate, Colorectal and Lung Cancers)

• Carcinoma of Unknown Primary Site

• Microarray vs Multiplex RT-PCR

Microarray Classification of Non-Hodgkin’s Lymphoma

Staudt. Cancer Cell Vol. 2, No. 5, 11/02: 363 - 366

Site of Origin for Metastatic AdenocarcinomaDennis et al Cancer Res 2002;62:5999-6005

Expression Profiles of 61 Genes by SAGE

Established Tumor Markers by RT-PCR in Common Adenocarcinomas

Expression Profiling in Breast Cancer

• Molecular Portraits• Molecular Grading• ER/PR Testing• HER2 Testing• Oncotype DxTM Recurrence Score by RT-PCR

(Genomic Health)• MammaprintTM 70 Gene Recurrence Predictor

(Agendia)• Other Multigene Predictors

Feature IHC FISH CISH

RT-PCR Microarray

Starting Material FFPE FFPE FFPE or Fresh/Frozen

Fresh/Frozen

Slide Based/ Morphology

Driven

Yes Yes No No

Requires Microdissection

No No Yes Yes

Processing Impact

Yes Minimal Minimal No

Number of Genes Tested

Small Small Intermediate Large

Type of Measurement

Semi-quantitative

Semi-quantitativ

e

Quantitative Quantitative

Complexity of Statistical Algorithm

Straight-forward

Straight-forward

Complex Highly Complex

Comparison of Multigene Predictor Test Platforms (1)

Comparison of Multigene Predictor Test Platforms (2)

Potential for False Discovery

Low Low Intermediate High

Ability to assess multiple pathways

simultaneously

Low Low Intermediate High

Stand Alone Prognostic value

Established Established Established Established

Prediction of Response to

Hormonal Therapies

Established In current

routine practice

Not Established

EstablishedNot in current

routine practice

EstablishedNot in current

routine practice

Prediction of Response to HER2 Targeted Therapies

Established In current

routine practice

Established In current

routine practice

EstablishedNot in current

routine practice

EstablishedNot in current

routine practice

 

Platform Feature

IHC FISH CISH

RT-PCR Microarray

Comparison of Multigene Predictor Test Platforms (3)

Platform/Feature IHC FISH CISH

RT-PCR Microarray

Prediction of Response to

Cytotoxic Therapies

Not establishedNot in current routine clinical

practice

Not established

Not in routine practice

EstablishedIn current

routine clinical practice

EstablishedNot in current routine clinical

practice

Prediction of Therapy Toxicity

Not establishedNot in current routine clinical

practice

Not established

Not in routine practice

Not establishedNot in current routine clinical

practice

Not establishedNot in current routine clinical

practice

Cost Comparatively Low ($100-400)

Compara-tively Low ($300-600)

Very High($3,500 or

higher)

Very High($3,500 or

higher)

Amenable to standardization

Low Low Intermediate High

Amenable to Automated Processing

Intermediate Intermediate

High High

Molecular Portrait of Breast Cancers

Sørlie et al. Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10869-10874.

Basal–like

HER-2

“Normal

Luminal B

Luminal A

Molecular Grading of Breast Cancer

Sotiriou C, et al.. Gene expression profiling in breast cancer: understanding the molecular basis of histologic grade to improve prognosis. J Natl Cancer Inst. 2006 Feb 15;98(4):262-72.

Patterns of expression of grade-related genes and their association with histologic grade (HG) and relapse-free survival. GGI score of each tumor is plotted below the corresponding column. Relapse-free survival times in years are

indicated below the GGI scores.

• Gene expression profiling data indicates that there are 2 molecular grades of breast cancer

• Histologic Grade 2 cases redistribute into Molecular Grades 1 and 2

• Molecular grading has outperformed histologic grading in multivariate analysis of traditional prognostic factors including ER/PR and HER2 status

Microscopic Grading in the Molecular Era

• How can we reduce the number of grade II cases and increase the number of grade I and grade III tumors?

• Grade II (3+3+1 = 7/9 “Moderately Differentiated”)

– Architecture 3

– Nuclear grade 3

– Mitosis count 1

• Use Ki-67 labeling index to control for artifact low mitotic figure count in cases where tumors were left at room temperature for too long prior to fixation

Rela

tive E

R m

RN

A

Exp

ressio

n

ER Status by IHC on Core Needle Biopsies

+ + + + + + + + + + + + ++ + + + + + + + + + + + - - - - - - - - - - - - - - - - - - - + - + - -

25

20

15

10

5

0

ER Testing: Concordance Between IHC Status and mRNA Levels

HER-2 Testing: Is CISH the “Kish of Death” for FISH

and IHC?

• Will IHC continue to be the international method of choice for screening with

2+ cases triaged to FISH?

• Will primary FISH testing become the standard?

• Will mRNA detection gain in popularity?

• Will the recently approved CISH (SISH) assay become the preferred method?

• Will the ToGA Trial and FDA approval for trastuzumab in gastric/GEJ cancer

change how HER2 testing is done?

• Will pertuzimab and trastuzumab-DM1 require HER2+ testing prior to use?

IHC/FISH/CISH Concordance

Hanna WM, Kwok K. Chromogenic in-situ hybridization: a viable alternative to fluorescence in-situ hybridization in the HER2 testing algorithm. Mod Pathol. 2006 Apr;19(4):481-7.

Overall FISH/CISH Concordance = 98%

Multigene Classifiers and Predictors of Breast Cancer Clinical Outcome*

*Ross, JS, Hatzis C, Symmans WF, Pusztai L, Hortobagyi GN. Commericalized multi-gene predictors of clinical outcome in breast cancer. Oncologist. 2008;13:477-493.

Multi-Gene Prognostic Tests

• Pathways in common

– Proliferation

– Hormone Receptor

– HER2

• Challenges for Clinical Acceptance

– Associations are by chance only

• Overfitting the data

• Separate validation group must be used

– Bias

– Generalizability

Companion Diagnostics: Potential Uses in Cancer Drug Development

• Biomarkers for drug response and drug-induced toxicity

• Comparison of human response to pre-clinical animal models

• Identify genes with variants that may define patient populations

• Identify proteins as potential biomarkers

• Shorten duration and lower cost of clinical trials by selecting patients more likely to respond and less likely to suffer toxicity

• Improve patient compliance with “custom-designed” medicines

• Achieve “best in class” status and premium pricing to overcome market segmentation

Important Anti-Cancer Drugs Requiring Companion Diagnostic Testing for Use

• Tamoxifen and AI’s: ER/PR• Trastuzumab and Lapatinib: HER2• Imatinib and Dasatinib: BCR:ABL, CKIT, PDGF• Cetuximab and Panitumumab: KRAS, (BRAF)• Gefitinib and Erlotinib: EGFR• Vemurafenib: BRAF• Crizotinib: EML4:ALK

Fine Needle Aspirate vs Core Biopsy

FNA CBX

Less invasiveCell suspension enriched for

tumor cellsAverage 80-85% Tumor Cells

More invasiveIncludes stromal, benign epithelial

and inflammatory cellsAverage 55-60% Tumor Cells

Total RNA Yield From Single Pass FNA or Core Biopsy

FNA:Usable RNA in 46 / 63 samples (73%)

mean = 3.6 µgmedian = 2.2 µg

CBX:Usable RNA in 15 / 20 samples (75%)

mean = 2.8 µgmedian = 2.0 µg

0

5

10

Co

un

t

-5 0 5 10 15 20 25

RNA yield FNA

0

1

2

3

4

Co

un

t

-5 0 5 10 15 20 25

RNA yield CBXSymmans et al. Cancer 15;97(12):2960-2971

FNA vs Core Biopsy Conclusion• FNAs and CBxs are similarly suitable for

transcriptional profiling• FNAs have significant advantages

– More acceptable to patients, – Higher consent rate– Faster to perform– Less expensive– Provide A Higher Percentage Of Cancer Cells In The Sample

• The Stromal Transcriptional Component Will Probably NOT Influence The Predictive Power Of Most Genomic Studies For: – Prognosis – Biology – Therapy Response (?)

DNA Sequencing in Cytology• DNA yield from FNAs may not always be

satisfactory• Needle core biopsies now preferred• DNA can also be extracted from FFPE cellblocks

from fluids and FNAs• Sequencing NSCLC FNAs is especially important

– EGFR for gefitinib/erlotinib– EML4:ALK for crizotinib– KRAS for cetuximab (not approved in NSCLC)– EML4:ALK for crizotinib

Background (1)

• Next Generation DNA Sequencing (NGS) has recently been applied to FFPE cancer biopsies and major resections (Ross JS et al. J Clin Oncol 29: 2011)

• Current Hot-Spot Genotyping only detects:– Mutations restricted to specific exons and codons

• NGS detects:– Whole exome mutations in numerous cancer related genes– Insertions and deletions– Translocations and fusions– Copy number alterations (amplifications)

Background (2)• Recently, biomarker testing has emerged as a major driver of

the selection of therapy for non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and melanoma

• Currently, “hot-spot” DNA sequencing and FISH are used to select therapies for solid tumors:– EGFR genotyping in NSCLC for tyrosine kinase inhibitor (erlotinib)– EML4:ALK translocation testing in NSCLC for crizotinib– KRAS mutation testing in CRC for cetuximab– BRAF mutation testing in melanoma for vemurafenib

• The emergence of comprehensive genomic profiling by NGS has led investigators to question whether more thorough gene sequencing techniques could discover potential targets for the treatment of relapsed and metastatic NSCLC not currently searched for in current routine practice

Cancer Genome Profiling Workflow

<14-21 days

EGFR Activating Mutation – NSCLC

• Mutation: EGFR_c.2573T>G_p.L858R• Freq=32%, depth=53• 79 year old white female• FNA of lung mass: NSCLC

FNA sample cytocentrifuged and converted to an FFPE tissue block. Very small numbers of viable tumor

cells. Extensively necrotic.

KRAS Mutation – CRC• Mutation: KRAS_c.35G>T_p.G12V• Freq=30%, depth=283• 52 year old white male• KRASG12V mutation by “hot-spot” genotyping at

Commercial Laboratory • pT3 pN2 pMx CRC

Classic CRC with origin from mucosal surface at lower right

BRAF V600K Mutation – Metastatic MM• Mutation: BRAF_c.1798_1799GT>AA_p.V600K• Freq=10%, depth=416• 77 year old white male• Thick melanoma of back• Multiple posterior cervical lymph nodes positive for metastatic

melanoma

Metastatic Melanoma to a cervical lymph node

EML4-ALK Translocation in NSCLC• PF-02341066 (PF-1066) “Critzotinib”

– Oral ALK4 receptor kinase inhibitor– Phase I Trial on NSCLC patients with EML4-ALK

translocation• “echinoderm microtubule-associated protein-like 4”

– “anaplastic lymphoma kinase”– 10/19 (53%) had a partial response ASCO ‘09– 76 patients detected by break-apart FISH ASCO ‘10

• ORR (overall response rate) 64%• DCR (disease control rate) 90%

• Seen in 5-13% of adenocarcinomas– Papillary, solid and signet ring cell features– Mostly never or non-smokers

• All ELM4-ALK Positive NSCLC is Negative for EGFR mutation

• ELM4-ALK translocation can be detected by FISH or PCR

– IHC for ALK available, but no outcome data for crtizotinib treated patients

Shaw et al. J Clin Oncol. 27;2009:4247-4253.

NSCLC: JAK2 Mutation Detected by NGS• Sample: SM86• Mutation: JAK2_c.1849G>T_p.V617F• Freq=4%, depth=205• 77 year old white female• Lung adenocarcinoma diagnosed by pleural biopsy• Patient diagnosed with polycythemia vera

Low power of pleural biopsy positive for adenocarcinoma

High power view shows adenocarcinoma of the lung. Rare capillaries not blood filled. No nucleated RBC or

blasts seen.

G T A T G T G T C T G T G G A

Val Cys Val Cys Gly

c.1849G>T p.V617F

CONFIDENTIAL 35

ERBB2 RARA

HER2 Gene Copy Number Alteration Validation

Increased HER2 CNA detected by NGS

HER2 FISH Positive Breast Invasive Duct Carcinoma Demonstrating High HER2 CNA HER2 Protein 3+ Expression by IHC

CONFIDENTIAL 36

cMET Copy Number Alteration in Clear Cell Ovarian Carcinoma Validation

cMET gene CNA at FMI estimated at 6 copies in

40% purity

cMET average CNA by FISH at APS is 6.6 using Abbott-Vysis Probes for cMET and CEP 7

cMET IHC at APS shows H Score = 300

with 100% 3+ Membrane

Staining

Novel ALK Fusion in CRC Detected by NGS

Lipson et al. Nature Med, Feb, 2012

A 5,194,955-bp tandem duplication generates an in-frame C2orf44-ALK gene

fusion

The RNA sequence of the C2orf44-ALK gene fusion shows aberrant splicing

RNA sequencing shows an 89.8-fold increase in expression of ALK beginning

at exon 20 relative to exons 1–19.

pT4pN1pM1 Mucinous Adenocarcinoma associated with a serrated sessile polyp

RETKIF5B

ATG

ATG

32,316,377 bps 43,611,118 bps

KIF5B-RETRET-KIF5B

ATG

ATG

Break Break

ATG

Translation

KIF5B (exons 1—15) RET (exons 12—20)

Kinesin Coiled coil Tyrosine kinase

KIF5B-RET

Novel RET:KIF 5B Rearrangement in NSCLC (11.3Mb Pericentric Inversion)

Lipson et al. Nature Med, Feb, 2012

Novel gene fusion joining exons 1-15 of KIF5B to exons 12-20 of RET in lung adenocarcinoma

Percentage Of Samples With Actionable Mutations Across Major Tissue Types

N=94 N=76 N=31 N=29 N=24

39 CONFIDENTIAL

71% cases carried ≥1 plausibly actionable mutation32 % cases carried ≥2 plausibly actionable mutations

N = 111

Comparison of NGS with Traditional Hot-Spot Genotyping in NSCLC, CRC, Breast Cancer and Melanoma

Also Detected by Hot-Spot Genotyping

Targeted Therapies for Cancer

Molecular profiling is driving many new targeted cancer therapeutics

Subset of analyzed targets listed; data from BioCentury Online Intelligence Database

• ~500 compounds hitting ~140 targets in development

• Growing number of newly identified potential targets