Implications of Molecular and

Chemosensitivity Testing:

What Drug for What Marker–Are We

There?

NO!

Harvey I. Pass, MD

NYU Langone Medical Center

NO!

Disclosures• Research Funding from NCI/NIH, DOD, CDC, Covidien,

Mensanna, Rosetta Genomics, SomaLogic, Celera, SourceMDx,

Fujirebio, Pfizer, Response Genetics, Meso Scale Diagnostics,

Integrated ,and Fox, Stephen Banner Lung Foundation,

Simmons Mesothelioma Foundation, and a whole bunch of

patients.

• Medical Advisory Boards for Quest Diagnostics, Rosetta • Medical Advisory Boards for Quest Diagnostics, Rosetta

Genomics, Champions, Precision Therapeutics, Pinpoint

Genomics, GSK, and GE Healthcare

• CDAs with Avantra, Caris Life Sciences, Transgenomics,

Foundation Medicine

• Patents for use of osteopontin and microRNA for

diagnosis/prognosis of mesothelioma; osteopontin isoforms for

NSCLC; EFEMP1 for diagnosis of mesothelioma

Predictive Biomarkers

• “Hot off the press” Surgically Resected Prognostic Stratification

– WHO should get adjuvant therapy (as opposed to WHICH adjuvant therapy should be used)

• The Paucity of Validated Predictive Biomarkers

• Adjuvant Therapy and Predictive “tissue” • Adjuvant Therapy and Predictive “tissue” Biomarkers

• Next Generation Sequencing for Biomarker Discovery

• Chemosensitivity Testing: The Data, Please!

• In vivo Tumorgraft Personalized Treatment

Pro

gn

ost

ica

tio

n 14 genes

Non-Squamous

qPCR

Lancet 2012 March 3:379(918): 823-832

Pro

gn

ost

ica

tio

n

Predictive Markers

• Companion Diagnostics that hopefully will be able to discriminate populations which are sensitive to a cytotoxic or targeted therapy

• Great potential for personalized adjuvant therapy• Great potential for personalized adjuvant therapy

Which Stage IV Lung Cancer Treatments Have Companion Predictive Biomarkers?

� EGFR H-score (IHC) NO NO

TARGETCLINICALLY

VALIDATED

COMPANION

DX

� EGFR mutation YES NO

� ALK-Fusion YES YES

� EGFR H-score (IHC) NO NO

� C-MET NO NO

� ERCC1 NO NO

� RRM1 NO NO

� BRCA NO NO

� TS NO NO

ALK FUSION AND LUNG CANCEROnly 7% But a Predictive Assay and Treatment Selectivity

CRIZOTINIB

MET/ALK TKI

Novel fusion identified in Novel fusion identified in 20072007 in NSCLC; in NSCLC; EML4EML4--ALKALK

Soda et al., Nature 2007

Anaplastic Lymphoma Kinase (ALK)Anaplastic Lymphoma Kinase (ALK)

Echinoderm microtubule-associated protein-

like 4 (EML)

~250 kb ~300 kb

t(2;5) ALK gene

breakpoint region

2p23 regionTelomere Centromere

3’ 5’

FISH Assay for ALK Rearrangement*

Break-apart FISH assay

ALK 29.3

EML4 42.3

q14.1

q12.3q12.1

p12

p13.2

p14

p16.1

p16.3

p22.1

p23.2

p22.3

p24.1

p24.3

p25.2

q14.1

q12.3q12.1

p12

p13.2

p14

p16.1

p16.3

p22.1

p23.2

p22.3

p24.1

p24.3

p25.2

Break-apart FISH assay

for ALK-fusion genes1

ALK break-apart FISH assay

q36.1

q36.3

q37.2

q34

q32.1

q32.3

q33.2

q31.3

q24.3

q24.1

q23.2q22.2

q22.1

q21.2

q14.3

q36.1

q36.3

q37.2

q34

q32.1

q32.3

q33.2

q31.3

q24.3

q24.1

q23.2q22.2

q22.1

q21.2

q14.3

Split

signal

Non-split

signal

*Assay is positive if rearrangements can be detected in ≥15% of cells

FISH = fluorescence in situ hybridization Solomon, B et al:JTO

4:1450-1454,2009.

ALK-Positive Non-Small Cell Lung Cancer

Study A8081001:Crizotinib Response, PFS & OSApproved by the US FDA on 26 August 2011

Best Percent Change in Tumor Size (N*=106)

Median OS not reached, with 79% pts still in

follow-up. Survival probability at 6 months:

90.0%, at 12 months: 80.5% Median PFS = 10.0 mo

We Would Love To Have The EML4-ALK Story For Adjuvant Therapy……

� But we don’t because

– The biomarkers are not fully validated

– The biomarkers have various platforms for their

measurement without consensus in some cases of measurement without consensus in some cases of

which to use

• IHC vs mutation analysis vs FISH for EGFr

• rtPCR vs AQUA for ERCC1/RRM1

� Sooooo….what DO we have in order to

discover adjuvant predictive biomarkers?

Trial n Stage Chemotherapy 5-yr OS (%) HR (95% CI) p value

CT C

IALT * 1867 I–IIIA Cisplatin-based 44.5 40.4

0.86

(0.76–0.98) <0.03

JBR.10* 482 IB–II

Cis/

vinorelbine 69 54

0.69

(0.52–0.91) 0.009

Adjuvant Studies for Predictive Marker

Development and Validation

JBR.10* 482 IB–II vinorelbine 69 54 (0.52–0.91) 0.009

CALGB 344 IB

Carbo/

paclitaxel 59 57

0.80

(0.60–1.07) 0.1

LACE*

meta-

analysis 4584 I–IIIA Cisplatin-based 48.8 43.5

0.89

(0.82–0.96) 0.004

FFPE blocks are available from studies for IHC

P53, BCL2,

BAXp27

KRas, EGFr

Weinberg, Cell: 2000

βTubulins

RRM1

TS

VEGF, EGFr

Repair of DNA DamageERCC1

MSH2BRCA1

Biomarker Clinical Relevance Studies

ERCC1 Low ERCC1 =improved outcome after treatment

with platinum-based chemotherapy

IALT-Bio;

LACE-Bio Pending

MSH2 Low MSH2 = longer survival after treatment with

platinum-based chemotherapy

IALT-Bio;

alone and combined

BaxAssociation between high Bax levels and sensitivity Both IALT-Bio,

Predictive Biomarkers Validated Retrospectively in

Adjuvant Trials

BaxAssociation between high Bax levels and sensitivity

to chemotherapy

Both IALT-Bio,

and LACE-Bio

Class III

βtubulin

High expression of class III -tubulin = longer OS and

DFS survival (taxanes) JBR.10

15-gene

signatureHigh-risk patients benefit from adjuvant

chemotherapyJBR.10

Other Predictive Markers Moving to Prospective

Validation

BRCA1 RRM1 Thymidylate Synthase

• DNA repair, mitosis and cell

division

• High levels associated with

platinum resistance in vitro

• Important for DNA synthesis

• Enhanced expression is

associated with resistance to

gemcitabine in cell lines

• High TS expression is

associated with reduced

efficacy of pemetrexed in

vitroplatinum resistance in vitro gemcitabine in cell lines vitro

• High TS expression is

associated with squamous

cell carcinoma

Biomarker Trial

EGFrErlotonib vs placebo + adjuvant ChT (up to 4 cycles), IHC +

FISH

BRCA1Randomized Study of Customized Adjuvant Chemotherapy Based

on BRCA1 mRNA Levels in Completely Resected Stages II–IIIA in

Non-Small Cell Lung Cancer (GECP-SCAT)

ERCC1/RRM1 Gemcitabine and Cisplatin in Treating Patients With Stage I Non-

Ongoing Prospective Biomarker Studies Used in An

Adjuvant Setting

ERCC1/RRM1 Gemcitabine and Cisplatin in Treating Patients With Stage I Non-

Small Cell Lung Cancer That Was Removed by Surgery (SWOG

0720)

ERCC1/EGFR mutation

status TAilored Post-Surgical Therapy in Early Stage NSCLC (TASTE)

ERCC1/thymidylate

synthase

International Tailored Chemotherapy Adjuvant (ITACA) trial

Therapy

MAGE-A3 GSK1572932A Antigen-Specific Cancer Immunotherapeutic as

Adjuvant n Patients With Non-Small Cell Lung Cancer (MAGRIT)

15 gene signature Precision Therapeutics

GECP-SCAT

What is coming?

Search for Actionable Mutations with Next

Generation Sequencing

• Lung Cancer Mutation Consortium

• The Cancer Genome Atlas (TCGA)

– Squamous Cell Carcinoma

– Adenocarcinoma– Adenocarcinoma

• Industry-Based Mutation Analyses

– Foundation Medicine

• 200 most common mutations: $5000

Lung Cancer Mutation Consortium

Simultaneous Testing

for 8 mutations

by Snapshot Or Sequenome

and

MET amplification or

ALK Fusion by FISH

Panel testing is feasible but

many alterations are rare

Tyrosine Kinase Domains of Dominant “Driver” Mutations are

Established Therapeutic Targets

• EGFR

• HER2

• BRAF

• EML4/ALK

• MET Amplification

• EGFR Amplification

Mutation Testing

FISH Testing for Fusion

and/or Copy Number Testing

• BRAF

• PI3KCA

• AKT1

• MAP2K1

• KRAS

• NRAS

• EGFR Amplification

• HER2 Amplification

Drugs approved for NSCLC

Drugs approved for other cancers

Drugs targeting these genes in

clinical development

The majority of tested patients receive targeted drug

Chemoresponse Testing

Chemoresponse Assay Measure of Drug Effect

ATP tumor chemosensitivity assay(ATP-TCA)

Luciferase assay measures ATP content of tumor cells

Microculture kinetic (MiCK) assayContinuous measure of apoptosis induced by cytotoxic drugs

DISC , MTT, ATP, Caspase 3/7 assayCorrelative assays of cell death and

• Encouraging data in GYN malignancies (the only Medicare approval)

DISC , MTT, ATP, Caspase 3/7 assayCorrelative assays of cell death and apoptosis

ChemoFx® assayMeasures primary tumor cell survival in response to cytotoxic drugs

Relation to Lung Cancer Adjuvant Therapy?• Plate Prep: Automated, Robotic

• Cell Plating: Automated, Robotic

(384 wells, 24 hr incubation)

• Drug Solution Prep

• Serial Drug dilution Prep

(10 Concentrations)

• Add Drug to cells/wells

(72 hour incubation)

• Fix Cells

• Stain Cells to count cell nuclei

Schuchert, M.J., Cerfolio, R.J., et al. Ann Surg Onc. 2011; 18 (suppl 1; abstr 292)

Potential TumorGraft Attributes and Advantages

Maintain growth factor-

tumor interactions

Maintain cancer cell

heterogeneity

Maintain supporting

stroma

Maintain tumor-vascular

interactions

� Maintains fundamental genotype

� Maintains heterogeneity of original cancer

� Maintain supporting stroma & cancer stem cells as in the human tumor.

• At least through 9 generations (F9)

� Can be molecular categorized for targeted drug development

� Exploring means to shorten cycle times

Courtesy of Champions Oncology

SURGERY FIRST ROUND

PA

TIE

NT

RECOVERY

EXPANSION

4-6 months

TREATMENT

2-3 weeks

TU

MO

RG

RA

FT

RESECTIONENGRAFTMENT

24 hours

Courtesy of Champions Oncology

In Vitro vs In Vivo

Number Of

NSLC

Patients

Tested

Data

Turnover

Time

Cost Includes

Mutational

Analyses

Medicare

Approved

Published

Results For

Time To

Progression

ChemoFx Hundreds 1-2 weeks $5000 Soon Selectively YesChemoFx Hundreds 1-2 weeks $5000 Soon Selectively

in Ovarian

Yes

(ovarian)

Tumorgrafts < 20 6 months $5000-

25,000

Yes No Yes

(pancreatic)

Conclusions

• There are no validated companion tissue biomarkers at present which predict efficacy of adjuvant chemotherapy.

• However, prospective trials are ongoing which test the ability of biomarkers to predict the utility of companion biomarkers.companion biomarkers.

• Future targets for therapy may be defined by whole genome sequencing for targetable novel mutations in lung cancer.

• In vitro/in vivo testing for personalization of adjuvant therapy, although promising, awaits validation in well designed prospective trials.