A Multi-Disciplinary Approach

Targeted Therapies in the Management of Non-Small Cell Lung Cancer

Course Faculty

Medical Oncologists:

Dr. Barb Melosky – British Columbia Cancer Agency, Vancouver, BC

Dr. Jeff Rothenstein – Lakeridge Health Oshawa, Oshawa, ON

Dr. Sunil Verma – Odette Cancer Center, Toronto, ON

Radiation Oncologist:

Dr. Patrick Cheung – Odette Cancer Center, Toronto, ON

Pathologists:

Dr. Ming Tsao – Princess Margaret Hospital, Toronto, ON

Disclosures

• Dr. Barb Meloskyo Advisory Board – Astra Zeneca, Roche, Boehringer Ingelheim, Pfizer, Lilly

• Dr. Jeff Rothensteino Advisory Board – Lilly, BMS; Grants/honorarium – Novartis; Clinical trials –

Roche, BMS, AstraZeneca, Boehringer Ingelheim, Novartis

• Dr. Sunil Vermao Advisory Board – AstraZeneca, Roche, Boehringer Ingelheim, Novartis, Lilly

• Dr. Patrick Cheungo None to declare

• Dr. Ming Tsaoo Grants/honorarium – Pfizer, Merck, Roche, AstraZeneca, Boehringer

Ingelheim

Course Objectives

1. To review the evidence for use of biomarkers to help make treatment decisions for patients with advanced non small cell lung cancer

2. To discuss the current opportunities and challenges in integrating biomarkers in clinical care of lung cancer patients

3. To review the latest evidence on targeted therapies incorporating biomarkers

4. To discuss the emerging roles of radiotherapy in the management of metastatic NSCLC.

Course Outline

• Module 1: Case Study & NSCLC Treatment Overview

• Module 2: Molecular Testing in NSCLC

• Module 3: EGFR/TKI: Evidence

• Module 4: ALK Inhibitors: Evidence

Module 2: Molecular Testing in NSCLC

Targeted Therapies in the Management of Non-Small Cell Lung Cancer: A Multi-

Disciplinary Approach

Dr. Ming TsaoPathologist, Princess Margaret Hospital, Toronto, ON

EGFR and ALK Testing in Non-Small Cell Lung Cancer

Module 2 Objectives

• Assess the guidelines and molecular testing procedures for selecting EGFR and/or ALK lung cancer patients.

• Describe the diagnostic procedures for molecular testing in NSCLC patients including sample selection, techniques and other requirements.

• Review Canadian algorithm and resources for molecular testing in NSCLC patients.

Current Biomarkers for FDA-Approved Therapies for NSCLC Patients

ALK = anaplastic lymphoma kinase; EGFR = epidermal growth factor receptorNSCLC = non-small-cell lung cancer; TKI = tyrosine kinase inhibitor

Agent Patient selection Tumour type

Bevacizumab

Histology Non-squamous

Pemetrexed

EGFR TKIsEGFR mutations

(first-line)Predominantly in adenocarcinoma Crizotinib

CeritinibALK (& ROS1)rearrangements

Nivolumab None Non-Small Cell

Pembrolizumab PD-L1 (22C3) Non-Small Cell

EGFR Mutation Rates: Low in Squamous Cell Carcinoma

Number of studies

Number of patients

MutationPositive

% Mutation Prevalence

Sex

Female 46 4858 1886 38.8

Male 46 3574 853 23.8

Smoking

Never 40 2913 1509 51.8

Ever 40 4755 834 17.5

Ethnicity

East Asian 27 3178 1483 46.6

Caucasian 19 5254 1256 23.9

Histology

Adeno ca 44 7718 2544 33

Squamous ca 17 278 14 5

Adenosquamous ca 4 14 5 36

Large cell ca 10 21 3 14

Adapted from Lindeman et al, J Thorac Oncol 2013;8:823-59; Mascaux et al, Thoracic Oncology: IASLC Multidisciplinary Approach, Chapter 18

EGFR Mutation Rates in East Asian Patients: Highest Among Female/Never-

SmokersMale Female Ever-smoker Never-

smoker

Hsieh (Taiwan) 23% 72% 21% 66%

Chou (Taiwan) 52% 72% 44% 69%

Yang (Taiwan) 55% 64% 37% 62%

Wu (Taiwan) 57% 65% 54% 65%

Kosaka (Japan) 36% 62% 30% 68%

Tokumo (Japan) 33% 67% 27% 73%

Takano (Japan) 53% 69% 51% 68%

Han (S. Korea) 9% 33% 13% 26%

Shigematsu (USA) 18% 54% 13% 56%

IPASS (many countries) 49% 63% - -

ALK Re-arranged Lung Cancer:Also Low in Squamous Cell Carcinoma

5.2 5.7

11.1

1.3

9

4.8

NSCLC ADC(unselected)

ADC(selected)

SCC ADSC NSCLC-Others

NSCLC (N=4025)

ADC (N=6775)

SCC (n=1411)

ADSC (n=78)

NSCLC-Others (n=376)

Number of Tumors Tested (n=12,665)

Estimated Prevalence Rate (% tested)

NSCLC: non-small cell lung cancer; ADC: adenocarcinoma; SCC: squamous cell carcinoma; ADSC: adenosquamous carcinoma

Adapted from IASLC ATLAS of ALK Testing in Lung Cancer , Chapter 2, 2013

Guideline for Molecular Testing in Lung Cancer from CAP/IASLC/AMP

• Aim: To establish evidence-based recommendations for the molecular analysis of lung cancers

• Systematic review conducted by: the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP)

Lindeman NI, et al. J Thorac Oncol. 2013;8:823–59; Lindeman NI, et al. Arch Pathol Lab Med. 2013;137:828–60; Lindeman NI, et al. J Mol Diagn. 2013;15:415–53

ASCO Endorsement

Patient Selection

Clinical factors: None

Histology:

• Adenocarcinoma or NSCLC with adenocarcinoma

component

• Small samples for which an adenocarcinoma component

cannot be excluded

• Pure Squamous and neuroendocrine carcinomas be tested

only in never smokers and young patients

1st Step in Mutation/FISH Testing

Shiau CJ, et al. J Thorac Oncol 2014 (7):9:947-56

Pathologist must review HE slide and mark areas for analysis

What (samples) to Test?

• Primary or metastatic lesions are suitable

• In most instances, available paraffin embedded diagnostic tumor samples:

o Resection specimens: patients with recurrence after previous surgery

o Needle core or aspiration (including EBUS) biopsy: for newly diagnosed advanced cancer patients

o Re-biopsy/effusion: for patients without readily available samples

• Cytology specimens

o Smear (air dried > alcohol fixed)

o Cytospin slide

o Cell block / gel block (FFPE)

Primary: 62%

Lymph node: 4%

Metastatic: 27%

Pleural fluid: 5%

Others: 2%

ONTARIO PROVINCE-WIDE EXPERIENCE

Shiau CJ, et al. J Thorac Oncol 2014 (7):9:947-56

Excision: 37%

Small biopsy: 40%

Cytology: 23%

ONTARIO PROVINCE-WIDE EXPERIENCE

Shiau CJ, et al. J Thorac Oncol 2014 (7):9:947-56

What are the Sample Requirements?

• Molecular Testing:

o At least 5 to 10 unstained slides at 4 microns plus an H and E with tissue being at least 0.25cm2 and at minimum 30% tumor cellularity

o Will work on paraffin embedded cytological material such as FNAs, as long as the tumor cellularity is sufficient

o Decalcified samples have lower rate of success for molecular testing and should be avoided.

• FISH

o At least 2 to 3 unstained sections on charged slides at 4 micron thickness

• IHC

o 2-3 unstained 4 micron tissue sections on charged slides for IHC plus an H&E

o Should not be more than 3 weeks after cutting

2293 cases analyzed1780 histology (77.6%)513 cytology (22.4%)

124 inconclusive cases (5.4%)101 histology (5.7%)23 cytology (5.6%)

239 Exon 19 deletions (53.6%)166 histology (52.2%)73 cytology (57.0%)

1723 EGFR Wild-type (79.4%)1361 histology (81.1%)362 cytology (73.9%)

207 Exon 21 L858R (46.4%)152 histology (47.8%)55 cytology (43.0%)

446 with EGFR mutation (20.6%)318 histology (18.9%)128 cytology (26.1%)

2169 successful cases (94.6%)1679 histology (94.3%)490 cytology (95.5%)

Ontario (Canada) Province-wide Mutation Testing Program : 18 months period

(2010-11)

Shiau CJ, et al. J Thorac Oncol 2014 (7):9:947-56

Testing in Cytology Specimens

Variable n (%) Successful test (%)Abundant 143 (27.8) 140 (97.9)Moderate 128 (25.0) 127 (99.2)

Small-clusters 118 (23.0) 112 (94.9)Minimal 83 (16.2) 73 (88.0)

Shiau CJ, et al. J Thorac Oncol 2014 (7):9:947-56

EGFR Mutation: PCR/Direct Sequencing

Minimum >30% tumor cellularityMay potentially miss up to 30% of mutations

Mutation Tests with Increased Sensitivity

Method% Tumor DNA

required

Targeted or screening

method

EGFR mutations

detected

Detection of

deletions and

insertions

Sanger direct sequencing 25 screening Known and new yes

Real time/Taqman PCR 10 targeted Known only no

High Resolution Melting Analysis 5-10 screening Known and new yes

COBAS (Roche) 5-10 targeted Known yes

Pyrosequencing 5-10 screening Known only yes

SNaPshot PCR 1-10 targeted Known only yes

Cycleave PCR 5 targeted Known only yes

Fragment length and RFLP analysis 5 Screening/targeted Known only yes

Allelic specific PCR/Scorpions ARMS 1 targeted Known only no

MassARRAY 1 targeted Known only yes

PNA-LNA PCR clamp 1 targeted Known only no

Denaturing HPLC 1 screening Known and new yes

Massively parallel/NGS 0.1 screening Known and new yes

Mascaux et al, Thoracic Oncology: IASLC Multidisciplinary Approach, Chapter 18

Must Test EGFR Mutations

Sharma SV, et al. Nat Rev Cancer 2007;7:169-81

90%

Desirable to Test EGFR Mutations

Sharma SV, et al. Nat Rev Cancer 2007;7:169-81

Lesscommonmutationstodetect

V1E13;A20E20;A20

E20ins18;A20E6;A20

E6ins33;A20E14;ins11del49A20

T3;A20

KI24;A20

KI15;A20

E6;A19

E2;A20E2;ins117A20E3;ins69A20

E14;del12A20E14;del36A20

E17;ins30A20E17ins61;ins34A20

E15del19;del20A20E18;A20

V2

V3aV3bV4

V5bV5a

V7V6

V8aV8b

“V5”“V4”

E17ins68;A20

EML4–ALK

KIF5B–ALK

TFG–ALK

KI17;A20

PTPN3-ALK

KLC1–ALK KL9;A20

ALKkinasedomain

Ou SH, et al. Oncologist 2012;17:1351–75

ALK – Multiple Variants Detectable by FISH

ALK Gene Rearrangement:FISH with Break Apart Probes

~12 MbALK EML4

e20

Optimized IHC to Detect ALK Fusion Protein Requires Signal Amplification

• Advantage: routinely used in pathology practice, fast and cost-effective

IASLC ATLAS of ALK Testing in Lung Cancer , Chapter 4, 2013

Canada Algorithm for Routine ALK Testing

Cutz et al. J Thorac Oncol 2014;9(9):1255-63

AntibodyTotal

cases studied

IHC negative IHC positive IHC doubtful (equivocal)

FISH - FISH + FISH - FISH + FISH - FISH +

Rodig (2009) ALK1 243 233 2 0 8 - -

Yi (2011) ALK1 101 69 0 22 10 - -

Paik (2011) 5A4 640 602 0 10 28 - -

Park (2012) 5A4 262 234 0 3 25 - -McLeer-Florin (2012)

5A4 81 59 0 1 19 0 2

Zhang (2013) SP8 130 110 0 0 15 - -

Sholl (2013) 5A4 176 162 1 0 13 - -

Minca (2013) D5F3 249 217 0 0 32 - -

Selinger (2013) ALK1/D5F3 587 581 0 6 7 - -

Martinez (2013) D5F3 79 73 0 1 5 - -

Takamochi (2013) 5A4 360 347 0 3 10 - -

TOTAL - 2908 2687 3 73 172 0 2

CALK (2014) 5A4 373 326 0 0 18 29 0FISH, fluorescence in situ hybridization; IHC, immunohistochemistry.

Concordance Between ALK IHC and FISH Testing

Current EGFR Testing Program

Physician

Request Testing for First line Rx with

gefitinib Holding Lab

Testing Lab

Prepare sample and ship to testing lab

Complete EGFR test and send results to

physician

Sample delivery to testing centre

Pathologist to QC the HE slides

Lab performance of the assay

Reporting of results

Turn Around Time

Test requested by Oncologist

1-2 days

7-10 days

Total: 10-21 days(average 14 days)

1-7 days

1-2 daysPrepare HE &

unstained section

Macro-dissectionDNA isolation

Mutation assay

Sample delivery to testing centre

Pathologist to QC the HE slides

Lab performance of the assay

Reporting of results

Ideally, at Diagnosis

Test requested by Oncologist

1-2 days

7-10 days

Total: 10-21 days(average 14 days)

1-7 days

Reflex Testing by Pathologist at time of diagnosis

Result available at consultation

1-2 daysPrepare HE &

unstained section

Macro-dissectionDNA isolation

Mutation assay

Next Generation Testing:High Throughput and Multiplex Platforms

• Mass spectrometry

(Sequenom)

• SNaPShot multiplex PCR (Applied Biosystems)

• Next Gen Sequencing: MiSeq, HiSeq, Ion Torrent, etc.

Next Generation Testing: Plasma circulating tumour cells and ctDNA

CTC = circulating tumour cell Haber & Velculescu. Cancer Disc June 2014

Where to Test?

Accredited clinical laboratory using validated assays!