Molecular Diagnostics and Targeted

Therapies in Solid Tumors

Gregory J. Tsongalis, Ph.D.

Professor of Pathology

Director, Molecular Pathology

Dartmouth Medical School

Dartmouth Hitchcock Medical Center

Norris Cotton Cancer Center

Lebanon, NH

1.4M New Cancers (US)

H&E for Morphology

Further Diagnostic Work-up

• Imaging (CT, MRI, PET)

• Immuno (IHC), molecular,

• Proteomic

Therapeutic

management

Current Patient Management Is Based on

Diagnosis

Hallmarks of Human Cancer

Evading

apoptosis

Self-sufficiency in

growth signals

Tissue invasion

and metastasis

Limitless replicative

potential

Sustained

angiogenesis

Insensitivity to anti-

growth signals

Gene Mutations Cause Cancer = New Biomarkers

Single gene = Single mutation = Cancer

Nothing Else Looks Like This!

Susan G. Komen Race for the Cure - Indianapolis

Traditional Processing of Tissues

ImmunoHistoChemistry

(IHC)

• Inexpensive

• Subjective scoring

• Semi-quantitative

• Protein target may be affected

by tissue processing

Traditional Technologies for Pathology Workup

• Fluorescent labeled DNA probes

hybridize to target

• Hybridized DNA probes fluoresce,

giving off brightly lit signals that are

easily viewed and analyzed

Fluorescence in situ

Hybridization (FISH)

EGFR Exon 21

Electropherogram

KRAS Gly13Asp BRAF V600E

Current Clinical Mutation Analysis Data

Evolving Molecular Technologies

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Cycle

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neg. control

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Fam (fluorescence)

Vic

(fl

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ce) SNP Genotyping Assay

Real Time PCR:

1. All specimen types

2. Increased sensitivity and specificity

3. Quantification possible

4. TAT = 2-3 hours

qBiomarker™ Somatic Mutation PCR Array: Human EGFR Pathway

Symbol COSMIC ID nt change AA change Known EGFR

Mutation Known KRAS

Mutation Known BRAF

Mutation BRAF 476 c.1799T>A p.V600E - - + EGFR 12366 c.2572C>A p.L858M + - - KRAS 532 c.38G>A p.G13D - + -

qBiomarker™ Somatic Mutation PCR Array Data

Genomic Approaches to Clinical Oncology

Tumor classification

Prognostic markers

Predictive indicators of drug response

New therapeutics

Monitoring disease

Susceptibility to cancer

Cystic Fibrosis

• Single gene disorder (CFTR - 1989)

• 27 exons, 23 kb

• F508

• >1,800 mutations

• Clinical heterogeneity

• Screening program – 2001

• Treatment of symptoms

• VX-770

– small molecule drug

– G551D specific (4%)

• VX-809 - F508 specific (90%)

• Challenges:

– Limited efficacy

– Emergence of resistance

– Unexpected toxicities

Therapies in Human Cancer

• The right treatment

for the right person

at the right time

Trial and Error

Personalized Medicine Current Practice

Personalized Medicine

One size fits all

• Trial and error

Personalized for the Individual Patient

Optimal drug response

Reduced adverse effects

Systemic response

Personalized Medicine Includes Predicting

Response to Targeted Therapies

Pharmacogenomics: Targeted Therapy

Most targeted therapies are directed against genes or gene products

found only in tumor cells – acquired or somatic genetic variants

O

Z

N

Tamoxifen

Targeted Therapy

HER History

1970s 2000s 1990s 1960s 1980s

EGF identified First HER-targeted MAb, trastuzumab (Herceptin®) approved for HER2+ MBC

Gefitinib (Iressa®) approved as

monotherapy for patients with

locally advanced or metastatic NSCLC

HER-targeted

therapies being explored in wide range of human

cancers

Overexpression of HER1/EGFR

reported in cancer cells

Monoclonals

identified

Small molecule

TK inhibitors identified

HER1/HER2

overexpression correlates with poor prognosis

Transmembrane structure of HER2 monomer

Extracellular domain

(632 amino acids)

Ligand-binding site

Intracellular domain

(580 amino acids)

Tyrosine kinase activity Cytoplasm

Plasma

membrane

Increased HER2 production

Normal Amplification/Overexpression

Cytoplasm

HER-2/neu receptor

protein

Cytoplasmic

membrane

Nucleus

HER-2/neu DNA

HER-2/neu

mRNA

Targeted Therapy

Anti-monoclonal Antibody and Anti-TKI Therapy Are Directed

Trastuzumab

(Herceptin) Lapatinib

(Tykerb)

Detecting HER-2

HER-2 Protein

HER-2 Genes Protein expression by IHC

Gene amplification by FISH

CML: The New Poster Child for Targeted

Therapy

• Diagnostic

• Therapeutic monitoring

• Resistance

THE BCR-ABL CHIMERIC ONCOPROTEIN

p185 (aa 426) BCR

BCR p210 (aa 902-927) NLS

Y Kinase SH3 SH2

NES

ABD DBD ABL

- Forms Dimers and Tetramers

- Constitutive Tyrosine Kinase Activity

- Exclusively Cytoplasmic

Therapeutic Options for CML

• ChemotherapyHydroxyurea, Busulfan

• Interferon-alpha

• Allogeneic Stem Cell Transplantation

• Gleevec® (Imatinib Mesylate)

Gleevec®-Tyrosine Kinase Inhibitor

Goldman JM. Lancet. 2000;355:1031-1032.

Bcr-Abl

ATP

Substrate

STI571

Y = Tyrosine

P = Phosphate

Bcr-Abl

Substrate

P P P

P

ATP in its specific binding site in the kinase domain of the protein is able to

phosphorylate tyrosine residues (Y) on selected substrates. The

phosphorylated substrate then binds with other molecules and activates

downstream pathways in leukaemogenesis. STI571 occupies the ATP pocket in

the BCR-ABL kinase domain and substrates cannot be phosphorylated.

Therapeutic Goals in CML

• Hematologic response: normal PB values and spleen size.

• Cytogenetic response: reduction of Ph+ cells in the blood or bone marrow. Complete = 0% Ph+ cells Partial CR = 1-35% Ph+ cells Minor CR = 36-95% Ph+ cells

• Molecular response: reduction or elimination of bcr-abl mRNA in marrow or PB.

Detection of BCR-ABL Positive for BCR-ABL

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Internal Control

BCR-ABL

>90% Imatinib resistance is due to reactivation of the bcr-abl

tyrosine kinase activity.

Persistance of bcr-abl kinase activity in the presence of imatinib

is mostly due to selected point mutations.

Imatinib Resistance

Kantarjian HM et al. Ann Intern Med 2006;145:913-923

Mutations That Induce Resistance to Imatinib

48 MUTATIONS

Other TKIs

KRAS & EGFR Testing:

predicting response to targeted therapy

in Colon and Lung Cancers

Prognostic vs Predictive Markers

• Prognostic marker- indicator of survival

independent of therapy; indicator of tumor

aggressiveness

• Predictive marker- indicator of response to

therapy, such as PFS or OS; (or in some

cases prediction of severe toxicity)

EGFR and KRAS

• EGFR- epidermal growth factor receptor, ErbB1

– Cell-surface receptor tyrosine kinase

– Activating mutations confer susceptibility to small

molecule TKI’s

– Resistance mutations also occur

• KRAS- Kirsten rat sarcoma virus (human homolog)

– GTP binding protein; signal transduction downstream of

cell surface receptor

– Activating mutations (reduced GTPase activity) negate

the requirement for upstream receptor activation

Mechanisms of EGFR Inhibition

Inhibition Strategies:

• EGFR-TK inhibitors

• Anti-EGFR mAb

inhibitors

mAb

Inhibitors

, Panitumumab

EGFR and Targeted Therapies

EGFR EGFR activating mutations confer susceptibility to small molecule TKI’s in

NSCLC.

Two common mutations account for >80% of EGFR mutant alleles. In frame del exon 19 and point mutation exon 21 (L858R)

Receptor L-domain

Receptor L-domain

Furin-like domain

Kinase domain

Transmembrane region E18

E19

E20

E21

Substitutions

In-frame deletions

Duplications/insertions

Substitutions

Substitutions

(L858R)

6%

6%

46%

42%

Lassus H, et al. J Mol Med. 2006;84:671-681. Lacroix L, et al. Int J Cancer. 2006;118:1068-1069. Stadlmann S, et al. Mod Path. 2006;19:607-610. Schilder RJ, et al. Clin Cancer Res. 2005;11:5539-5548.

KRAS Activating Mutations

• Found in 30-50% of CRCa’s

• Associated with smoking in NSCLC

• Occur most often in codons 12 and 13

• Missense mutations (change of amino acid)

• 7 common mutations, account for at least

95% of all identified

• A few mutations in other locations have

been reported ex: codon 61

KRAS Mutations

Lievre et al., J Clin Oncol 2008 (Jan);26:374

• Cetuximab – monoclonal Ab to EGFR

• KRAS mutations associated with resistance to

Cetuximab

Amado et al., J Clin Oncol 2008 (Mar);26:1

• Panitumamab – monoclonal Ab to EGFR

• Response associated with wild type KRAS

KRAS mutation testing

• DNA extracted from archival FFPE tumor sample (typically

primary tumor)

• Original kit (DxS, European) used allele-specific PCR

• Detection limit: 1% mutation in wild-type background

• Detects 7 mutations in codons 12 and 13

– Gly12Asp Gly12Cys

– Gly12Ala Gly12Arg

– Gly12Val Gly13Asp

– Gly12Ser

Allelic Discrimination for KRAS Mutations

GLY12ARG, 34G>C GLY12ASP, 35G>A

GLY12CYS, 34G>T GLY12ALA, 35G>C

KRAS Mutation Detected in FFPE Tumor

Codon

12

Codon

13

Gly Gly

G>A mutation

(Gly12Asp)

Correlating Molecular Findings with Pathology

Chen H. et al. Correlation of polypoid colorectal adenocarcinoma with

pre-existing adenomatous polyps and KRAS mutation.

Cancer Genet, Apr 204:245-251, 2011

Adenomatous polyp

with villous architecture

Pre-existing adenomatous

polyp with

villous architecture

Invasive adenocarcinoma

with ulceration

Correlating Molecular Findings with Pathology

A Changing Paradigm?

• Patient #1

– Poorly differentiated

– Aggressive

– Pos for therapeutic

targets

• Patient #2

– Well-Moderately

differentiated

– Less aggressive

– Neg for therapeutic

targets

Conclusions

• Targeted therapies offer a better way to

destroy cancer cells beyond the typical

chemotherapy

• Knowledge of tumor cell biology will lead to

more treatments against various pathway

constituents

• Our field is changing rapidly and so will our

roles

What if………..

DHMC Molecular Pathology Laboratory and

Translational Research Program

Samantha Allen

Claudine Bartels, Ph.D.

Heather Bentley

Betty Dokus

Susan Gallagher

Carol Hart

Arnold Hawk

Joel Lefferts, Ph.D.

Rebecca O’Meara

Elizabeth Reader

Mary Schwab

Laura Tafe, M.D.

Brian Ward

Brendan Wood

Eric York