APC

CTNNB1

EGFR

IDH1

KRAS

NOTCH1

PIK3CA

PTEN

Rapid Tumor Genotyping in Solid Tumors

AKT1

BRAF

KIT

MAP2K1

TP53

NRAS

Growth Factor

Receptortyrosine kinase

RAS

MAP2K1(MEK1)

RAF

ERK

Proliferation Survival

PI3K

AKT

mTOR PTEN

P P

Mitogen Activated Protein Kinase (MAPK) Pathway

Tumor Genotyping

www.genpathdiagnostics.com/oncology/OnkoMatch

Personalized Medicine Requires Personalized DiagnosticsBiological discoveries utilizing advanced sequencing techniques are unraveling the key drivers of cancer. These discoveries are now entering the clinic by personalizing treatment based on molecular profile. For example, conventional testing in lung cancer relies on 1 or 2 mutational events (EGFR

and ALK) but research groups such as the Lung Cancer Consortium1 are profiling patients across several oncogenes implicated in oncogenesis. This multidimensional view of cancer is taking hold in various solid tumors.

1. The Lung Cancer Mutation Consortium (LCMC) is an NCI sponsored initiative made up of 14 leading cancer centers across the country.

Major pathways have been identified in cancer proliferation including:

Mitogen Activated Protein Kinase (MAPK) signaling: EGFR, BRAF, KRAS, NRAS, MAP2K1, KIT

mTOR: PIK3CA, AKT, PTEN

Tumor Suppressor and DNA Repair: TP53, PTEN, APC

Cell Signaling: NOTCH1, CTNNB1

The molecular profiling of cancer patients is becoming the standard for disease management in top medical centers.

www.genpathdiagnostics.com/oncology/OnkoMatch

Same Histology but Different Genotypes = Different Targeted Therapies?

The Paradigm Shift in Tumor GenotypingRapid Detection of 68 Mutations Across 14 Oncogenes from 1 Tumor Specimen

GenPath introduces OnkoMatch, a proprietary assay utilizing technology pioneered at Mass General Hospital. OnkoMatch is a reliable and robust tumor genotyping platform for detecting 68 mutations across 14 oncogenes from one specimen.

Tumor 1 Tumor 2 Tumor 3

Treatment naive Refractory to cisplatin regimens

Refractory to cisplatin regimens

EGFR mutated PIK3CA mutated MEK mutated

Erlotinib XL147 – PIK3CA inhibitor MEK-162 inhibitor

FDA approved Investigational Agent Investigational Agent

Example: 3 patients with stage IV adenocarcinoma

Therapies Matched to the Patient’s Specific Molecular Profile

OnkoMatch Methodology: PCR amplification followed by single base extension detection of hotspot mutations that have been identified as key driver mutations.

Past Conventional Next Generation

Histology Histology Histology

Single Gene Test Tumor Genotyping (ex KRAS) AKT1 - APC - BRAF - CTNNB1 EGFR - IDH1 - KIT - KRAS MAP2K1 - NOTCH1 - NRAS PIK3CA - PTEN - TP53

OnkoMatch provides fast

results with a 7 day turnaround.

APCCTNNB1

EGFR

IDH1

KRAS

NOTCH1 PIK3CA

PTEN

BRAF

MAP2K1

TP53

NRAS

AKT1 KIT

Tumor Genotyping

EGFR

KRAS

Single Gene Test

OnkoMatch represents the new paradigm in cancer diagnostics enabling clinicians to see a more complete picture of cancer drivers and not mutations confined to one or more genes.

Tumor Genotyping

www.genpathdiagnostics.com/oncology/OnkoMatch

Solid Tumor Cancer: Lung, Breast, Colon, GI, Pancreas, Melanoma, Head and Neck, Ovarian, Thyroid

ADVANCED STAGE CANCER – Patients who have become refractory (treatment resistant) to prior therapies and are searching

for investigational targeted therapies offered in clinical trials.

NON-METASTATIC CANCER – Patients who do not have immediate requirement for investigational therapy

but would like to know their mutational profile for potential future clinical trials or future approvals of targeted therapies.

When is OnkoMatch Genotyping Recommended?

Mutational Incidence Breakdown for Lung

TP53: 5%

PTEN: 4%PIK3CA: 3%

NRAS: 1%MET: 5%KRAS: 25%

HER2: 2%

EGFR: 15%

CTNNB1: 3%

BRAF: 2%

APC: 3%

ALK: 5%

AKT1: 1%

BRAF HER2

EGFR ALK

MEK PIK3

KRASAKT1

FDA APROVED

THE FUTURE

HER2: 15%CTNNB1: 2%

EGFR: 2%AKT1: 4%

APC: 3%

BRAF: 3%

KRAS: 4%

MAP2K1: 2%

NRAS: 2%

PIK3CA: 25%

PTEN: 6%

TP53: 23%

Mutational Incidence Breakdown for Breast

www.genpathdiagnostics.com/oncology/OnkoMatch

The Age of Targeted Therapy Based on Molecular ProfileMedicine has moved past the age of broad based toxic therapies whose efficacy in select populations is unknown. Cancer genetics has revealed extensive

tumor heterogeneity within the same disease state. This diversity requires tailored therapies based on the patient’s tumor genotype.

Response Rate

Crizotinib – ALK 61%

Vemurafenib – BRAF 48%

FDA 2011 ApprovalsRecent FDA approvals for ALK and BRAF inhibitors have proven that MATCHING genetic profiling with a targeted therapy is both efficacious and minimally toxic to the patient.

Investigational therapies, currently in clinical trials, are now targeting oncogenes such as AKT1, KRAS, MEK and PIK3CA. Inhibitors of these pathways could be the next success in cancer treatment.

Source: COSMIC database, Wellcome Trust Sanger Institute

Source: FDA Package Insert

Tumor Genotyping

www.genpathdiagnostics.com/oncology/OnkoMatch

Source: Nature Medicine Vol 17 • Number 3 • March 2011 • Page 298

Biomarker Discovery → Drug Development Has Accelerated

41 Years

13 Years

9 Years

4 Years

1960 1973 1999

1996

2002 2011

2007 2011

1998

20011993–1995

1985–1987

Discovery of ‘Philadelphiachromosome’

Mechanism of action:translocation of the

ABL oncogene

BCR-ABL inhibitors(patent �led)

Hematological responses in CML

(53 of 54 patients)

ERBB2 cloning &ID ampli�cation

ERBB2 expression ispredictive of response

ID of BRAF mutationsin cell lines and

malignant melanoma

Responses in BRAF

mutant tumors

ALK fusionspredict

response

Drug repositioning based on EML4-ALK

translocation in NSCLC

BCR-ABL inhibition(Gleevec)

ERBB2 inhibition(Herceptin)

BRAF inhibition(Vemurafenib)

ALK inhibition(Crizotinib)

Years for FDA Approval of Personalized Therapies

The time between biological marker discovery and drug development has significantly shortened. Gleevec was developed 41 years after the discovery of the Philadelphia chromosome whereas Vemurafenib took only 9 years from BRAF identification in cell lines. The discovery of oncogenes that drive cancer progression has spurred on a dramatic drug discovery race as shown in the more than 100 clinical trials (see Investigational Agents sheet) currently underway.

www.genpathdiagnostics.com/oncology/OnkoMatch

Tumor Genotyping from MGH to your practice Technology used routinely at Mass General Hospital is now available nationally in community oncology practices.

OnkoMatch tumor genotyping technology has been licensed exclusively from Massachusetts General Hospital’s (MGH) Division of Translational Medicine.1 Dr. John Iafrate, MD, PhD (Director of Molecular Diagnostics at MGH and Associate Professor of Pathology, Harvard Medical School) and his team at MGH designed a multiplex genotyping assay for solid tumors based on SNaPshot2 technology. GenPath leveraged MGH’s sophisticated DNA extraction method and mutational analysis for detecting multiple mutations across several oncogenes in one test.

Tumor genotyping of solid tumors such as lung are part of MGH’s normal protocol for patient management. “Analysis by SNaPshot Multiplex System is now part of the routine pathological assessment of lung cancers at Mass General Hospital”3, John Iafrate, MD, PhD.

Patients at MGH with detected mutations may be placed in appropriate clinical trials that target the oncogenic pathway. More than 1,500 genotypes have been reported into MGH’s medical records reflecting the wide diversity of tumor profiles.

Annals of Oncology 22: 2616–2624, 2011

doi:10.1093/annonc/mdr489

Published online 9 November 2011original article

Implementing multiplexed genotyping of non-small-celllung cancers into routine clinical practice

L. V. Sequist1,2*, R. S. Heist1,2, A. T. Shaw1,2, P. Fidias1,2, R. Rosovsky1,2,3, J. S. Temel1,2,I. T. Lennes1,2, S. Digumarthy2,4, B. A. Waltman2, E. Bast1, S. Tammireddy1, L. Morrissey1,A. Muzikansky2,5, S. B. Goldberg1,2, J. Gainor2,6, C. L. Channick2,7, J. C. Wain2,8,H. Gaissert2,8, D. M. Donahue2,8, A. Muniappan2,8, C. Wright2,8, H. Willers2,9,D. J. Mathisen2,8, N. C. Choi2,9, J. Baselga1,2, T. J. Lynch10, L. W. Ellisen1,2, M. Mino-Kenudson2,11,M. Lanuti2,8, D. R. Borger1,2, A. J. Iafrate2,11, J. A. Engelman1,2 & D. Dias-Santagata2,11

1Massachusetts General Hospital Cancer Center, Boston; 2Harvard Medical School, Boston; 3The Mass General/North Shore Cancer Center, Danvers; 4Department of

Radiology; 5Department of Biostatistics; 6Department of Medicine; 7Division of Pulmonary and Critical Care Medicine; 8Division of Thoracic Surgery; 9Department of

Radiation Oncology, Massachusetts General Hospital, Boston; 10Yale University School of Medicine and Yale Cancer Center, New Haven; 11Department of Pathology,

Massachusetts General Hospital, Boston, USA

Received 14 August 2011; revised 17 September 2011; accepted 26 September 2011

Background: Personalizing non-small-cell lung cancer (NSCLC) therapy toward oncogene addicted pathway

inhibition is effective. Hence, the ability to determine a more comprehensive genotype for each case is becoming

essential to optimal cancer care.

Methods: We developed a multiplexed PCR-based assay (SNaPshot) to simultaneously identify >50 mutations in several

key NSCLC genes. SNaPshot and FISH for ALK translocations were integrated into routine practice as Clinical Laboratory

Improvement Amendments-certified tests. Here, we present analyses of the first 589 patients referred for genotyping.

Results: Pathologic prescreening identified 552 (95%) tumors with sufficient tissue for SNaPshot; 51% had ‡1mutation identified, most commonly in KRAS (24%), EGFR (13%), PIK3CA (4%) and translocations involving ALK (5%).

Unanticipated mutations were observed at lower frequencies in IDH and b-catenin. We observed several associations

between genotypes and clinical characteristics, including increased PIK3CA mutations in squamous cell cancers.

Genotyping distinguished multiple primary cancers from metastatic disease and steered 78 (22%) of the 353 patients

with advanced disease toward a genotype-directed targeted therapy.

Conclusions: Broad genotyping can be efficiently incorporated into an NSCLC clinic and has great utility in

influencing treatment decisions and directing patients toward relevant clinical trials. As more targeted therapies are

developed, such multiplexed molecular testing will become a standard part of practice.

Key words: carcinoma, non-small cell, genotype, molecular targeted therapy

introduction

Certain genetically defined cancers are ‘oncogene addicted’ toactivated kinases and are thereby highly sensitive to drugs thatselectively inhibit the corresponding kinase. Employinggenotype-based therapy has been highly successful in chronicmyelogenous leukemia, gastrointestinal stromal tumors, non-small-cell lung cancer (NSCLC) and melanoma, and in manyinstances, the targeted agent is far more effective thantraditional chemotherapy [1–9]. This shifting paradigm hasdramatically impacted lung cancer treatments. Until recently,therapeutic options for advanced NSCLC were limited to

chemotherapies that were ‘personalized’ only by considering

the side-effect profiles of a number of similar modestly effective

regimens. Response rates were typically 20%–30% and

progression-free survival (PFS) was 3–5 months [10–13]. But

now, we know that determining NSCLC genotype can inform

the most effective personalized therapies. Patients with

mutations in the epidermal growth factor receptor (EGFR) gene

benefit from EGFR tyrosine kinase inhibitors (TKIs) with

a response rate of �75%, PFS of 9–13 months and improved

quality of life compared with chemotherapy [8, 14–16].

Similarly, patients with EML4-ALK translocations have a 60%

response rate, 9-month PFS and a low degree of toxicity when

treated with crizotinib, an ALK TKI [6].Although these landmark studies have focused on a single or

small number of genetic mutations, there is an increasing

motivation to develop technologies that can simultaneously

determine the mutational status of many genes. Responding to

original

article

*Correspondence to: Dr L. V. Sequist, Massachusetts General Hospital Cancer Center,

55 Fruit Street, Professional Office Building room 212, Boston, MA 02114, USA. Tel: +1-617-726-7812; Fax: +1-617-724-3166; E-mail: lvsequist@partners.org and Dr Dora

Dias-Santagata, Translational Research Laboratory, Massachusetts General Hospital,

55 Fruit Street, Jackson 1028, Boston, MA 02114, USA. Tel: +1-617-724-1261; Fax:+1-617-726-6974; E-mail: ddiassantagata@partners.org

ª The Author 2011. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

All rights reserved. For permissions, please email: journals.permissions@oup.com

at New

York M

edical College H

ealth Sciences Library on Novem

ber 29, 2011http://annonc.oxfordjournals.org/

Dow

nloaded from

Annals of Oncology 22: 2616–2624, 2011

doi:10.1093/annonc/mdr489

Published online 9 November 2011original article

Implementing multiplexed genotyping of non-small-celllung cancers into routine clinical practice

L. V. Sequist1,2*, R. S. Heist1,2, A. T. Shaw1,2, P. Fidias1,2, R. Rosovsky1,2,3, J. S. Temel1,2,I. T. Lennes1,2, S. Digumarthy2,4, B. A. Waltman2, E. Bast1, S. Tammireddy1, L. Morrissey1,A. Muzikansky2,5, S. B. Goldberg1,2, J. Gainor2,6, C. L. Channick2,7, J. C. Wain2,8,H. Gaissert2,8, D. M. Donahue2,8, A. Muniappan2,8, C. Wright2,8, H. Willers2,9,D. J. Mathisen2,8, N. C. Choi2,9, J. Baselga1,2, T. J. Lynch10, L. W. Ellisen1,2, M. Mino-Kenudson2,11,M. Lanuti2,8, D. R. Borger1,2, A. J. Iafrate2,11, J. A. Engelman1,2 & D. Dias-Santagata2,11

1Massachusetts General Hospital Cancer Center, Boston; 2Harvard Medical School, Boston; 3The Mass General/North Shore Cancer Center, Danvers; 4Department of

Radiology; 5Department of Biostatistics; 6Department of Medicine; 7Division of Pulmonary and Critical Care Medicine; 8Division of Thoracic Surgery; 9Department of

Radiation Oncology, Massachusetts General Hospital, Boston; 10Yale University School of Medicine and Yale Cancer Center, New Haven; 11Department of Pathology,

Massachusetts General Hospital, Boston, USA

Received 14 August 2011; revised 17 September 2011; accepted 26 September 2011

Background: Personalizing non-small-cell lung cancer (NSCLC) therapy toward oncogene addicted pathway

inhibition is effective. Hence, the ability to determine a more comprehensive genotype for each case is becoming

essential to optimal cancer care.

Methods: We developed a multiplexed PCR-based assay (SNaPshot) to simultaneously identify >50 mutations in several

key NSCLC genes. SNaPshot and FISH for ALK translocations were integrated into routine practice as Clinical Laboratory

Improvement Amendments-certified tests. Here, we present analyses of the first 589 patients referred for genotyping.

Results: Pathologic prescreening identified 552 (95%) tumors with sufficient tissue for SNaPshot; 51% had ‡1mutation identified, most commonly in KRAS (24%), EGFR (13%), PIK3CA (4%) and translocations involving ALK (5%).

Unanticipated mutations were observed at lower frequencies in IDH and b-catenin. We observed several associations

between genotypes and clinical characteristics, including increased PIK3CA mutations in squamous cell cancers.

Genotyping distinguished multiple primary cancers from metastatic disease and steered 78 (22%) of the 353 patients

with advanced disease toward a genotype-directed targeted therapy.

Conclusions: Broad genotyping can be efficiently incorporated into an NSCLC clinic and has great utility in

influencing treatment decisions and directing patients toward relevant clinical trials. As more targeted therapies are

developed, such multiplexed molecular testing will become a standard part of practice.

Key words: carcinoma, non-small cell, genotype, molecular targeted therapy

introduction

Certain genetically defined cancers are ‘oncogene addicted’ toactivated kinases and are thereby highly sensitive to drugs thatselectively inhibit the corresponding kinase. Employinggenotype-based therapy has been highly successful in chronicmyelogenous leukemia, gastrointestinal stromal tumors, non-small-cell lung cancer (NSCLC) and melanoma, and in manyinstances, the targeted agent is far more effective thantraditional chemotherapy [1–9]. This shifting paradigm hasdramatically impacted lung cancer treatments. Until recently,therapeutic options for advanced NSCLC were limited to

chemotherapies that were ‘personalized’ only by considering

the side-effect profiles of a number of similar modestly effective

regimens. Response rates were typically 20%–30% and

progression-free survival (PFS) was 3–5 months [10–13]. But

now, we know that determining NSCLC genotype can inform

the most effective personalized therapies. Patients with

mutations in the epidermal growth factor receptor (EGFR) gene

benefit from EGFR tyrosine kinase inhibitors (TKIs) with

a response rate of �75%, PFS of 9–13 months and improved

quality of life compared with chemotherapy [8, 14–16].

Similarly, patients with EML4-ALK translocations have a 60%

response rate, 9-month PFS and a low degree of toxicity when

treated with crizotinib, an ALK TKI [6].Although these landmark studies have focused on a single or

small number of genetic mutations, there is an increasing

motivation to develop technologies that can simultaneously

determine the mutational status of many genes. Responding to

original

article

*Correspondence to: Dr L. V. Sequist, Massachusetts General Hospital Cancer Center,

55 Fruit Street, Professional Office Building room 212, Boston, MA 02114, USA. Tel: +1-617-726-7812; Fax: +1-617-724-3166; E-mail: lvsequist@partners.org and Dr Dora

Dias-Santagata, Translational Research Laboratory, Massachusetts General Hospital,

55 Fruit Street, Jackson 1028, Boston, MA 02114, USA. Tel: +1-617-724-1261; Fax:+1-617-726-6974; E-mail: ddiassantagata@partners.org

ª The Author 2011. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

All rights reserved. For permissions, please email: journals.permissions@oup.com

at New

York M

edical College H

ealth Sciences Library on Novem

ber 29, 2011http://annonc.oxfordjournals.org/

Dow

nloaded from

Annals of Oncology 22: 2616–2624, 2011doi:10.1093/annonc/mdr489Published online 9 November 2011

B Y E R I K A C H E C K H A Y D E N

After Van VanderMeer was diagnosed

with advanced lung cancer, the results

of a genetic test offered some hope. Last

year, the 64-year-old lawyer learned that his

cancer featured a genetic rearrangement that

might render it vulnerable to a drug being tested

in clinical trials. But because the experimental

drug, crizotinib, was being given only to patients

who had failed chemotherapy, VanderMeer had

to wait for more than a year to gain access to the

drug. Even though VanderMeer’s tumours had

by then spread to both of his lungs, crizotinib

vaporized them within two weeks.

VanderMeer is now doing well and hoping to

continue beating the disease: more than half of

patients who take the drug, made by Pfizer of

New York, seem to have a better prognosis than

do those who didn’t receive treatment. But what

if VanderMeer had started taking it sooner?

Now oncologists, pathologists and geneti-

cists are hoping to answer that question with

a study that will test whether genetically tar-

geted treatments, applied soon enough, can

cure patients of lung cancer rather than buying

them a few extra months of life.

Targeted therapies have now been approved

for many cancers, and it has become routine

for major cancer centres to genotype patients’

tumours to determine whether they might

benefit from targeted drugs, in case standard

treatments fail. But the clinical trial, which

will be conducted by the Alliance for Clinical

Trials in Oncology, a nationwide group

funded by the US National Cancer Institute in

Bethesda, Maryland, will test whether using

targeted treatments earlier can prevent patients

with lung cancer from ever reaching that point.

In the trial, tumours will be genotyped after

surgery to determine whether mutations are

present in a gene encoding epidermal growth

factor receptor (EGFR). Mutations in this gene

are targeted by many molecular therapies,

including erlotinib and gefitinib, which are

approved for the treatment of advanced lung

cancer. Some of the patients who have EGFR

mutations will begin taking erlotinib after sur-

gery, instead of waiting to see whether their

cancer recurs.

Although similar approaches have been

tested in smaller trials, yielding mixed results,

organizers say that a larger, better-defined

study is needed to provide a clear answer.

“We have never tested these drugs in the

right population,” says oncologist Ramaswamy

Govindan of Washington University in St Louis,

leader of the trial. “We have never tested a group

of patients who have mutations in EGFR and

then asked the question, ‘could these patients

be cured by gefitinib or erlotinib?’”

He hopes to expand the analysis to include

crizotinib, which targets a different genetic

rearrangement and was approved by the US

Food and Drug Administration in August.

Other targeted therapies are in the pipeline.

In a 9 November paper, for instance, a con-

sortium of researchers from Massachusetts

General Hospital and Harvard Medical

School, both in Boston, and Yale University in

New Haven, Connecticut, describe the results

of a study that tested more than 500 patients

with non-small cell lung cancer (L. V. Sequist

et al. Ann. Oncol. http://dx.doi.org/10.1093/

annonc/mdr489; 2011). The authors examined

mutations in several genes relevant to therapies

that have been approved or are in development

(see ‘Identifying targets’). Of the 353 patients

with the most advanced lung cancers, 22%

were matched to clinical trials appropriate for

their cancer type.

The Alliance trial will be logistically diffi-

cult. Only 10–20% of patients with non-small

cell lung cancer have mutations in the EGFR

gene; only 20% of patients are diagnosed early

enough to benefit from surgery; and only a

fraction of patients with the appropriate muta-

tions will actually gain any advantage from

targeted treatments. To reach their target of

400 participants, Govindan and his colleagues

may need to screen as many as 1,500 people.

VanderMeer, for one, hopes that the efforts

pay off — and spare other patients from what

he calls the “blunderbuss” of chemo therapy.

“I’d hate for anyone to have to go through

the blunderbuss before they get to the stiletto,”

he says. ■

M E D I C I N E

Targeted treatment tested

as potential cancer cure

Trial will deploy genetically targeted therapy early, rather than as last resort.

MORE ONLINE

E X P L A I N E R

The science

behind

Australia’s war

on tobacco

advertising

go.nature.com/

zjrfci

M O R E N E W S

● Sickle-cell mystery solved go.nature.

com/dxx61h ● Ancient adaptations to parasites

drove human genetic variation

go.nature.com/d4es4b

● Proof found for unifying quantum

principle go.nature.com/dt8syh

F R O M T H E B L O G

Japan

funds

Fukushima

clean-up

projects

go.nature.com/wdfscp

No mutation

49%

KRAS

24%EGFR

13%

TP53

5%CTTNB1

2%NRAS

1%

HER2 ~1%

IDH1 ~1% or less

ALK

5%

PIK3CA

4%BRAF

2%IDENTIFYING TARGETS

Genotyping of lung tumours from more than 500 patients revealed genetic changes

that could be targeted by drugs. Some patients had more than one mutation.

TEP

CO

AU

STR

ALI

AN

CA

NC

ER C

OU

NC

IL

SO

UR

CE:

L. V

. SEQ

UIS

T ET

AL.

AN

N. O

NC

OL.

(2011)

1 7 N O V E M B E R 2 0 1 1 | V O L 4 7 9 | N A T U R E | 2 8 1

IN FOCUS NEWS

© 2011 Macmillan Publishers Limited. All rights reserved

17 NOVEMBER 2011 | VOL 479 |NATURE

nature

MGH’s tumor genotyping test, SNaPshot, has recently been included in prestigious journals, explaining the assay’s novel findings.

1 MGH is a registered trademark of Massachusetts General Hospital 2 SNaPshot is a registered trademark of Applied Biosystems, Inc. 3 Shaw AT, Iafrate JA, et. al. Case 21-2011: A 31-Year-Old Man with ALK-Positive Adenocarcinoma of the Lung. NEJM 2011; 365:163.

481 Edward H Ross Drive · Elmwood Park, NJ 07407 ·1 800 627 1479 tel · 1 201 791 8760 fax · www.genpathdiagnostics.com GenPath is a business unit of BioReference Laboratories, Inc. © 2012 BioReference Laboratories, Inc. All rights reserved. 91031 1/12

Tumor Genotyping

Reporting of open clinical trials related to mutation detected

• Results consolidated in 1 simple table• Mutation identified in pathway

Tumor Genotyping

Oncogenes TestedGene DNA Variant Protein VariantAKT1 None Detected

APC None Detected

BRAF c.1798_1799GT>AA p.V600K

CTNNB1 None Detected

EGFR None Detected

IDH1 None Detected

KIT None Detected

KRAS None Detected

MAP2K1 None Detected

NOTCH1 None Detected

NRAS None Detected

PIK3CA None Detected

PTEN None Detected

TP53 None Detected

Additional MarkersEGFR exon 19 deletion Not Detected

Final Report

®

John Smith, M. D.

ONCOLOGY CENTER

123 MAIN ST

Elmwood Park, NJ 12345

X1111 M1

DOCTOR

PATIENT

SAMPLE

Name: Barbara Jones

Addr: 123 Smith Street

DOB: 1/12/1952 Age: 60

SEX: F

ID No:

Date of Report: 1/22/2012

Date Collected: 1/15/2012

Date Received: 1/15/2012

Specimen ID: 30000000

Source: Skin

Clinical Info: Metastatic melanoma

Genpath is a business unit of BioReference Laboratories Inc.481 Edward H. Ross DriveElmwood Park, NJ 07407(800)633-4522

James Weisberger, M.D.Laboratory Director

Page 1 of 3Created 1/23/12 9:57 AM

TP2011-202778295

BRAF c.1798_1799GT>AA,p.V600K

Variant Description:The BRAF V600K mutation arises from a complex nucleotide change (c.1798_1799GT>AA) and results in an amino acid substitution of the valine (V) at position 600 by a lysine (K).

Prognostic Relevance: In one study of a consecutive series of patients with metastatic melanoma, the presence of a BRAF muta-tion was associated with a more aggressive clinical course and shorter survival for patients that were not treated with a BRAF inhibitor. (Long, Menzies, 2011).

Therapeutic Relevance:A phase III clinical trial showed that vemurafenib (PLX4032), a potent and orally-available BRAF kinase inhibitor, improved the rates of overall and progression-free survival in patients with previously untreated metastatic

OnkoMatch

Interpretative Information

HER1(EGFR)

RAS

RAF

MAP2K1

MAPK

PIK3CA

AKT1

Cytoplasm

Cell Membrane

Nucleus Cell Proliferation, Cell Survival, Invasion & Metastasis

Tumor-Induced Neoangiogenesis

Tyrosine Kinase DomainsP P

PTEN

CT

See report section on open clinical trialsCT

Results Primary Tumor Type MELANOMA

Genpath is a business unit of BioReference Laboratories Inc.481 Edward H. Ross DriveElmwood Park, NJ 07407(800)633-4522

James Weisberger, M.D.Laboratory Director

Page 2 of 3Created 1/23/12 9:57 AM

TP2011-202778295

Methodology

Clinical Trials

Methodology text

Sponsor: Novartis Pharmaceuticals)

ClinicalTrials.gov Identifier: NCT01221077

1. A Study of RO5212054 (PLX3603) in Patients With BRAF V600-mutated Advanced Solid Tumours

2. A Phase I Study of Oral LGX818 in Adult Patients With Advanced or Metastatic BRAF Mutant Melanoma

melanoma with the BRAF V600E or V600K mutations, when compared to chemotherapy (dacarbazine) (Chapman, 2011). Preliminary results from early phase clinical studies using other targeted agents are encouraging and include the BRAF inhibitor GSK2118436, and the RAF kinase inhibitor XL281 (Shepherd, 2011). Multiple MEK inhibitors are currently being evaluated for the treatment of advanced melanoma and other solid tumors, and include AZD6244, PD0325901 and GSK1120212 (Flaherty, CurrOpinOnc 2010).

Interpretative Information (continued)

Failed Probes

Comments

EGFR, c.2156G>C (p.G719A)

Final Report

®

John Smith, M. D.

ONCOLOGY CENTER

123 MAIN ST

Elmwood Park, NJ 12345

X1111 M1

DOCTOR

PATIENT

SAMPLE

Name: Barbara Jones

Addr: 123 Smith Street

DOB: 1/12/1952 Age: 60

SEX: F

ID No:

Date of Report: 1/22/2012

Date Collected: 1/15/2012

Date Received: 1/15/2012

Specimen ID: 30000000

Source: Skin

Clinical Info: Metastatic melanoma

Sponsor: Hoffmann-La Roche

ClinicalTrials.gov Identifier: NCT01143753

Clear Reporting of Results