Ming Tsao, MD, FRCPCProfessor of Laboratory Medicine and Pathobiology, University of TorontoConsultant Pathologist and Senior Scientist, University Health Network

Disclaimer

• The information in this presentation is not a substitute for clinical judgement in the care of a particular patient. CAP is not liable for any damages arising from the use or misuse of any information contained in or implied by the information in this presentation.

Made possible through an educational grant from Eli Lilly Canada.

Copyright © 2010

Disclosure Related to this Lecture• I have been a Consultant for and received

Honoraria from:– Lilly Canada (Lung cancer histopathology)– AstraZeneca Canada (EGFR mutation testing)– Roche Oncology (EGFR TKI biomarkers)– Pfizer (Targeted therapy and ALK testing)

• I will not discuss off label use and/or investigational use in my presentation.

Learning Objectives • Participation at this Royal College MOC (Maintenance of Certification)

Section 1 Accredited Group Learning event will enable participants to:

– Learn about the latest advances in lung cancer targeted therapy and recognize the importance of histology in targeted therapy for NSCLC

– Recognize predictive biomarkers currently used in clinical practice in patients with lung cancer

– Learn about the accuracy and reliability of NSCLC subclassification in small tissue samples

– Know the commonly used immunohistochemical stains in the diagnosis and sub-classification of NSCLC

– Appreciate the value of prioritizing the use of small tissue samples from patients with advanced lung cancer

– Understand the increasingly important role of the Anatomical Pathologist in targeted therapy for lung cancer

Leading Causes of Deaths (WHO)In 2002:1. Ischaemic heart disease2. Cerebrovascular disease3. Lower respiratory infection4. HIV/AIDS5. COPD6. Perinatal conditions 7. Diarrheal diseases8. Tuberculosis9. Lung cancer10.Road traffic accidents

In 2030:1. Ischemic heart disease2. Cerebrovascular disease3. HIV/AIDS4. COPD5. Lower respiratory infection6. Lung cancer7. Diabetes Mellitus8. Road traffic accidents9. Perinatal conditions10. Stomach cancer

Mathers CD, Loncar D, PLosMed 2006;3:e442

Cancer Survival Rates (1975 – 2003)5-

year

sur

viva

l rat

e (%

)

Cancer Facts & Figures 2008

Canadian Cancer Statistics 2009Estimated New cases

Est. Deaths

Death/Case R

5-yr survival

Lung 23400 20500 0.876 15%

Breast 22900

18900

0.246 87%

Prostate 25500 0.172 95%

Colorectal 22000 0.414 62%

Canadian Cancer Statistics 2009: www.cancer.ca

HIGH DEATH RATES: 70% of Lung Cancer Patients are Diagnosed at Advanced Stage

NSCLC

SCLC

SEER STATISTICS(US NCI Surveillance Epidemiology

and End Results)

local

regional

distant

Lung cancer survival

2004 WHO Classification of Malignant Lung Cancer

• Squamous cell carcinoma • Small cell carcinoma

– Combined• Adenocarcinoma

– Mixed type (>80%)

– Acinar type– Papillary type– Bronchioloalveol

ar carcinoma– Solid type

• Large cell carcinoma– LCNEC

(neuroendocrine)

– Etc.• Adenosquamous carcinoma• Sarcomatoid carcinoma

• Carcinoid tumour– Typical– Atypical

• Salivary gland tumors– Mucoepidermoid– Adenoid cystic– Epithelial-myoepithelial

• Mesenchymal tumours– Epithelioid

hemangioendothelioma– Etc.

Small cell lung cancer: 20% “Non-small cell lung cancer” (NSCLC): 80%

Practical Classification for Treatment Decision

Others (15%)

Adeno(~ 50%)

Squamous(~ 25%)

Before 2004

• Histological classification underwent minor revisions (1982, 91, 97, 2004)

• Most important to distinguish small cell from non-small cell carcinoma

• Distinction between major subtypes of NSCLC, i.e. adeno, squamous, large cell are not crucial

• Use of non-specific term such as “Non-small cell NOS” has been acceptable

Standard Therapy for NSCLC

• EARLY STAGE NSCLC – surgical resection• Adjuvant chemotherapy

• LOCALLY ADVANCED NSCLC – combined radiation and chemotherapy – Sometimes surgery

• ADVANCED STAGE NSCLC – palliative chemotherapy and/or radiation– Combinations of chemotherapy agents

60-70%

Landmark Discoveries or Studies Pacing for Paradigm Shift in Lung Cancer Diagnosis

1. Activating (oncogenic) mutations in the EGFR gene in NSCLC, mainly adenocarcinoma

2. Mutations may confer “oncogene addiction” to tumor cells, which sensitize them to drugs targeting protein encoded by the gene

3. New targeted anti-cancer drugs may have tumor specific efficacy or toxicity, necessitating more accurate markers to select patients for therapy

N Engl J Med 2009;361:947-57.

No. at RiskPlacebo 132 108 71 31 11 3 0Carboplatin 129 103 37 7 2 1 0+paclitaxel

0.0

0.2

0.4

0.6

0.8

1.0

0 4 12 16 24Prob

abili

ty o

f Pro

gres

sion

-fre

e Su

rviv

al

Months Since Randomization

P<0.001Hazard ratio, 0.48 (95% CI, 0.36-0.64)Events: gefitinib, 97 (73.5%); carboplatin + paclitaxel, 111 (86.0%)

Carboplatin+ paclitaxel

Gefitinib

EGFR-Mutation: Positive

208

No. at RiskPlacebo 91 21 4 2 1 0 0Carboplatin 85 58 14 1 0 0 0+paclitaxel

0.0

0.2

0.4

0.6

0.8

1.0

0 4 12 16 24Prob

abili

ty o

f Pro

gres

sion

-fre

e Su

rviv

al

Months Since Randomization

P<0.001Hazard ratio, 2.85 (95% CI, 0.36-0.64)Events: gefitinib, 97 (73.5%); carboplatin + paclitaxel, 111 (86.0%)

Carboplatin+ paclitaxel

Gefitinib

EGFR-Mutation: Negative

208

Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.

Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, Sunpaweravong P, Han B, Margono B, Ichinose Y, Nishiwaki Y, Ohe Y, Yang JJ, Chewaskulyong B,

Jiang H, Duffield EL, Watkins CL, Armour AA, Fukuoka M.

N Engl J Med. 2009 Sep 3;361(10):947.

Gefitinib Labelling in Canada (also in Europe)

Phase III studies of EGFR-TKI vs. Platinum doublet

in EGFR Mutant PatientsGroup EGFR mutation Primary

endpointN

(TKI vs. CT) TKI Control

WJOG 3405 EX19, L858R PFS172

(HR=0.49)G CDDP+DOC

NEJ 002 EX19, L858R, G719X, L861Q PFS

320

(HR=0.69)G CBDCA+PAC

EURTARC EX19, L858R PFS174

(HR=0.37)E Pt doublet

Optimal EX19, L858R PFS165

(HR=0.16)E CBDCA+GEM

Adapted from Mitsudomi, 2011

Epidermal Growth Factor Receptor (EGFR/HER/ErbB)

Burgess, A et al (2003) Mol Cell 12(3): 541.

Roskoski, R (2004) Biochem Biophys Res Commun 319(1): 1.

Activation Follows Dimerization Induced by Ligand Binding

Kumar A, et al. J Clin Oncol 26:1742-1751

Phosphorylated tyrosine

ras

raf

erk

mek

EGFRGPR

PI3K

aktrac Ral-GDS

SURVIVAL PROLIFERATION

INVASION

DIFFERENTIATION

ANGIOGENESIS

EGFR Signaling

Reported EGFR TK Domain Mutations

Sharma, S et al (2007) Nat Rev Cancer 7(3): 169.

Standard Testing Method: PCR/Direct Sequencing

Exon 18 19 20 21 22 23 24

EXON 21 (L858R)EXON Deletion (E746_A750)

Santos, G and Tsao, MS (2010)

Limitations of Standard Direct Sequencing Method

• Contamination of mutant sequences (from tumour cells) with wild type sequences (from normal cells) may decrease sensitivity of assay

Minimum requirement 25-40% tumour cells, but usually over-estimated

Assay Methods with Increased Sensitivity

Adapted from: Pao, W and Ladanyi , M (2007) Clin Cancer Res 13(17): 4954.

Method Sensitivity Mutations identifiedDirect Sequencing 25% Known and new

PCR-SSCP 10% Known and new

TaqMan PCR 10% Known only

Loop-hybrid mobility shift assay 7.5% Known only

Cycleave PCR 5% Known only

PCR-RLFP (fragment length analysis) 5% Known only

MassARRAY genotyping 5% Known only

LNA -PCR clamp 1% Known only

Scorpion ARMS (DxS) 1% Known only

dHPLC 1% Known and new

COLD-TaqMan PCR 0.05% Known only

All assays—other than direct sequencing—claim to be able to detect mutations in samples containing ≤10% mutant alleles

Assa

ys u

sed

in C

anad

ian

Labo

rato

ries

Potential Laboratory Generated Errors

• False Positive– Specimen contamination– “Formalin mutation”– PCR artifacts

• False Negative– Poor quality specimen– Lack of sensitivity of assay

IMPORTANT THAT TESTING BE DONE IN ACCREDITED CLINICAL DIAGNOSTIC LABORATORIES

Effect of Various Fixatives on Quality of Nucleic Acids

Fixative DNA Quality

RNA Quality

Unbuffered formalin

Poor Poor

Buffered formalin Fair Fair

Methacarn Good Good

Ethanol Good Good

CytoLyt Fair Unknown

Glutaraldehyde (Karnovsky)

Good Unknown

Histochoice Good Unknown

HOPE Good Good

Zenker Poor Poor

Reprinted from Hunt (2008) Arch Pathol Lab Med 132(2): 248-260. Copyright 2008. College of American Pathologists; Boldrini et al (2007). J Thorac Oncol 2(12): 1086.

Fixative DNA Quality

RNA Quality

Carnoy Good Good

UMFIX (universal molecular fixative)

Good Good

Prefer Poor Poor

Decalcifying acids Poor Poor

Mercury-containing solutions (B-5)

Poor Poor

Bouin Poor Poor

Hollande Fair Fair

Zinc buffered formalin

Good Good

EGFR Mutation Testing Samples• Testing may be attempted on any tumour

samples in paraffin block (or freshly obtained)

– Cytology (BW, BB, EBUS, FNA)– Biopsies (core or open) – Effusions– Resections

• Testing should start with histologic evaluation of the samples by a pathologist to determine adequacy of sample and select tumour-rich areas

Scant samples: May be extra-challenging but doable

Macro-dissection to Enrich for Tumour Cells

Fine Needle Aspiration (FNA) Cell Blocks

• Potentially inadequate or excellent materials• Need the pathologist to evaluate section

EGFR Mutation Analysis on FNA/Fluid Materials

BB: Bronchial Brushing; BL: Bronchial Lavage; BiSeq: Bidirectionally sequencing; BrB: Bronchoscopic Biopsy, BW: Bronchial Washing; CB: Core Biopsy; Cseq: Cyclic Sequencing; DSeq: direct sequencing; DxS: Scorpion ARMS; Ebx: endobronchial biopsy; Eff: Effusion; FNA: fine needle aspiration ; HRMA: high resolution melting analysis; SP: Sputum; WGA: whole genome amplification. *Results reported for Scorpions test; ** Other samples were either not

analyzed or results were inconclusive; †Tissue processed immediately

Reference Sample Fixative NSCLC(N)

Analysis Method

Mutant(N, %)

WT /Negative(N, %)

Nomoto et al (2006) FNA/BW/BB/Eff Ethanol 29 HRMA/DSeq 17 (59%) 12 (41)

Smith et al (2008) FNA Air-dried 11 HRMA/BiSeq 3 (27%) 8 (73%)

Lim et al (2009) BrB/FNA/CB RNAlater 88 DSeq/WGA 21 (24%) 67 (76%)

Fassina et al (2009) FNA FineFix 77 HRMA/DSeq 2 (3%) 75 (%)

Wu et al (2008) Effusion -80oC 136 DSeq 93 (68%) 43 (32%)

Kimura et al (2006) Effusion -80oC 43 DSeq 11 (26%) 32 (74%)

Kimura et al (2006) Effusion -80oC 24 DxS/DSeq* 8 (33%) 16 (67%)

Boldrini et al (2007) FNA/SP/BW/BB Cytolyt 23 CSeq 3 (13%) 20 (87%)

Horiike et al (2007) TBNA -80oC 94 DxS/DSeq* 27 (29%) 67 (71%)

Smouse et al (2009) FNA, Eff/BW/BL Formalin 18 BiSeq 7 (39%) 4 (22%)**

Otani et al (2008) Wash Fluid None† 34 DSeq 17 (50%) 17 (50%)

EGFR Mutation Analysis on EBUS-TBNA

and CT-guided Biopsy Samples

CT: computed tomography ; DSeq: direct sequencing; DxS: EGFR29 Mutation Kit; EBUS-TBNA: endobronchial ultrasound-guided transbronchial needle aspiration; EUS: endoscopic ultrasound guided aspiration; USG: ultrasonography

Reference Sample Fixative NSCLC(N)

Analysis EGFR Mutant

Wild-type/Negative

Garcia-Olive´et al . (2010) Eur Respir J 35:391.

EBUS-TBNAOther biopsy/cytology

Ethanol 26 25

Seq 2 (8%)3 (12%)

24 (92%)22 (88%)

Nicholson et al. (2010) J Thorac Oncol 5:436.

Blind-,EBUS-, EUS-TBNA Endobronchial biopsyEffusion

Formalin 622

DxS 000

6 (100%)2 (100%)2 (100%)

Nakajima et al. (2007) Chest132:597.

EBUS-TBNA FFPE 43 DSeq 11 (26%) 32 (74%)

Chen et al. (2008) Acta Radiol 49:991.

CT-guided biopsy Frozen 17 Not specified

12 (71%) 5 (29%)

Shih et al. (2006) Int. J. Cancer 118:963.

CT-guided core biopsyUSG-guided core biopsyEndoscopic biopsyEffusion cell blocks

FFPE 2018169

DSeq 12 (60%)8 (44%)5 (31%)4 (44%)

Not reported

Cancer (Cancer Cytopathol) 2011;119:80-91.

Which Mutations Need to be Tested?

Sharma S et al (2007) Nat Rev Cancer 7(3): 169.

At minimum must test for: • Exon 19 deletions

• Exon 21 L858R mutation

EGFR Mutation Rates among Different Histologies of NSCLC (Multi-

institutional)

Low among SQC but significant among LCC or NSCLC NOS patients

Reference Study ADC , % (total pts)

SQC , %(total pts)

LCC/others , % (total pts)

Non-ADC , % (total pts)

Zhu et al (2008) BR.21 20%(107) - - 13%(97)Tamura et al (2008) WJTOG0403 40%(97) - - 5%(20)Douillard et al (2010) INTEREST 20% (169) - - 8% (128)Asahina et al (2006) Phase 2 (NEJ) 26% (72) - - 10% (10)Yang et al (2008) NCT173875 61%(82) - - 63%(8)Bell et al (2005) IDEAL/INTACT 17% (213) - 5% (178) -Rosell et al (2009) Spanish LCG 17%(1634) - 11%(287) -Herbst et al (2005) TRIBUTE 17%(120) 6%(31) 9%(77) -Richardson et al (2009) RADIANT 25% (337) 2%(191) 4%(53) -

TOTAL 20%(2641) 4%(222) 9%(595) 11%(263)

ADC: Adenocarcinoma; SQC: Squamous cell carcinoma; LCC: Large cell carcinoma.

Which Patients to Test for EGFR Mutation

• Advanced (stage IIIB-IV) or recurrent non-squamous histology:

– Adenocarcinoma

– Large cell carcinoma

– Poorly differentiated NSCLC (NOS)

• Squamous, if clinical factors favour higher chance for finding a mutation, e.g. never smoker

• Patients who are being considered for EGFR TKI therapy instead of chemotherapy

Summary of Current Practice for EGFR mutation testing in Canada

• Advanced NSCLC with non-squamous histology

• Testing is performed in a certified clinical diagnostic laboratory (follows recognized laboratory standards)

• Minimum test includes exon 19 deletion and exon 21 L858R mutation

• A pathologist examines one HE section to determine tumour cellularity and identify areas for microdissection or coring

• In some centers EGFR mutation status is incorporated into the final pathology report

On the HorizonMutation-specific antibodies for the detection of EGFR mutations in non-

small-cell lung cancer.

Yu J, Kane S, Wu J, Benedettini E, Li D, Reeves C, Innocenti G, Wetzel R, Crosby K, Becker A, Ferrante M, Cheung WC, Hong X, Chirieac LR, Sholl LM, Haack H, Smith BL,

Polakiewicz RD, Tan Y, Gu TL, Loda M, Zhou X, Comb MJ. Clin Cancer Res. 2009 May 1;15(9):3023.

Brevet, M et al (2010) J Mol Diagn 12(2): 169.

CST Antibodies: Highly Specific but More Sensitive for L858R

than 19 deletions Mutation + Mutation - Total

E19 deletionIHC+ 23 (74%) 2 (1%) 25

IHC- 8 (26%) 161 (99%) 169

Total 31 (100%) 163 (100%) 194

E21 L858RIHC+ 20 (95%) 2 (1%) 22

IHC- 1 (5%) 171 (99%) 172

Total 21 (100%) 173 (100%) 194

Other Investigated Candidate Predictive Markers

for Erlotinib/Gefitinib Therapy

• EGFR immunohistochemistry• EGFR high gene copy number• KRAS mutation• Serum proteomic signature

EGFR IHC, FISH and KRAS

• EGFR IHC is not a good predictive marker for response or survival benefit

• FISH for EGFR gene copy number increase is not currently used due to technical challenges and unconfirmed value as a predictive biomarker

• KRAS role in EGFR TKI therapy is currently uncertain

New Therapies in Advanced NSCLC

Agent Patient Selection Marker

Reason for Selection

GefitinibEGFR mutation (1st line) Efficacy

Erlotinib

Bevacizumab + cisplatin doublet Non-squamous Toxicity

Pemetrexed + cisplatin Non-squamous Efficacy

Cetuximab Possibly EGFR IHC Efficacy

Crizotinib ALK rearrangement Efficacy

Important Points about Histology Diagnoses

• WHO diagnostic classification system (Edition 4)– Is intended for diagnosis based on examination

of the whole tumour– Does not include immunohistochemical data

(IHC)– Does not include NSCLC NOS as an

independent category– Does not include guidelines of diagnosing

NSCLC in small biopsies • Up to 75% of lung cancers present at

advanced stage1 and diagnosis is based on biopsy or cytology specimens alone

• Data from clinical trials showing the importance of histology are based solely upon light microscopy

1. http://csqi.cancercare.on.ca/cms/one.aspx?portalId=63405&pageId=67837

William D. Travis, MD, Elisabeth Brambilla, MD, Masayuki Noguchi, MD, Andrew G. Nicholson, MD, Kim R. Geisinger, MD, Yasushi Yatabe, MD, David G. Beer, PhD, Charles A. Powell, MD, Gregory J. Riely, MD, Paul E. Van Schil, MD, Kavita Garg, MD, John H. M. Austin, MD, Hisao

Asamura, MD, Valerie W. Rusch, MD, Fred R. Hirsch, MD, Giorgio Scagliotti, MD,Tetsuya Mitsudomi, MD, Rudolf M. Huber, MD, Yuichi Ishikawa, MD, James Jett, MD, Montserrat

Sanchez-Cespedes, PhD, Jean-Paul Sculier, MD, Takashi Takahashi, MD, Masahiro Tsuboi, MD, Johan Vansteenkiste, MD, Ignacio Wistuba, MD, Pan-Chyr Yang, MD, Denise Aberle, MD,

Christian Brambilla, MD, Douglas Flieder, MD, Wilbur Franklin, MD, Adi Gazdar, MD, Michael Gould, MD, MS, Philip Hasleton, MD, Douglas Henderson, MD, Bruce Johnson, MD, David Johnson, MD, Keith Kerr, MD, Keiko Kuriyama, MD, Jin Soo Lee, MD, Vincent A. Miller, MD,

Iver Petersen, MD, PhD, Victor Roggli, MD, Rafael Rosell, MD, Nagahiro Saijo, MD, Erik Thunnissen, MD, Ming Tsao, MD, and David Yankelewitz, MD

International Association for the Study of LungCancer/American Thoracic Society/European

Respiratory Society International MultidisciplinaryClassification of Lung Adenocarcinoma.

J Thorac Oncol. 2011 Feb;6(2):244.

IASLC/ERS Classification for Resected NSCLC (2011)

Strong Pathologic Recommendations

1. The use of term “BAC” be discontinued

2. Adenocarcinoma in situ (AIS) defines small (≤3 cm) solitary ADC with pure lepidic growth and potentially 100% disease-specific survival (DFS) if completely resected

3. Minimally invasive adenocarcinoma (MIA) defines small (≤3 cm) solitary ADC with pure lepidic growth and small foci of invasion measuring ≤0.5 cm, and near 100% DFS if completely resected

Strong Pathologic Recommendations – cont’d

4. Lepidic Predominant ADC replaces “mixed subtype” to define invasive ADC with predominantly non-mucinous lepidic pattern (formerly non-mucinous BAC)

5. Addition of “micropapillary predominant ADC” as a major histologic subtype due to its association with poor prognosis

Strong Pathologic Recommendations – cont’d

6. For small biopsies/cytology, NSCLC be further classified into more specific histologic types (e.g. adeno, squamous), whenever possible

7. The term NSCLC-NOS be used as little as possible and only applied when a more specific diagnosis is NOT possible by morphology/special stains

Minimally Invasive Adenocarcinoma (MIA)

• Small, solitary (≤ 3 cm) adenocarcinoma with predominantly lepidic pattern and ≤ 5 mm invasion in greatest dimension in any one focus

• Great majority are non-mucinous• Can be applied to multiple tumors only if others

are regarded as synchronous primaries and not intrapulmonary metastasis

MIA (Criteria of Invasion)• Invasive components:

– Histological patterns other than lepidic (e.g. acinar, papillary, micropapillary, solid)

– Tumor cells infiltrating myofibroblastic stroma

• MIA excluded with presence of: – Lymphatic, blood vessels or pleural invasion

– Tumor necrosis

• Microinvasive areas found in one tumor:– Multiple foci of MIA invasive areas possible

– Individual invasive areas measured separately

– Size of largest invasive area measured in largest dimension ≤ 0.5 cm

Recommendations on Tumor Size and Specimen Processing

• AIS/MIA diagnosis requires entire histologic sampling of entire tumor

• Evidence for AIS and MIA with 100% disease-free survival mainly from tumors ≤ 2.0 or 3.0 cm

• The terms AIS and MIA should not be used for small biopsies or cytology specimens

– If a noninvasive pattern is present in a small biopsy, it should be referred to as a lepidic growth pattern

Invasive Adenocarcinoma – Lepidic and Micropapillary prodominant

• Lepidic predominant ADC replaces “mixed subtype” to define invasive ADC with predominantly non-mucinous lepidic pattern (formerly non-mucinous BAC)

– If tumor has lepidic growth yet alveolar spaces are filled with papillary or micropapillary structure, tumor is classified as papillary or micropapillary ADC, respectively

• “Micropapillary predominant ADC” is added as a major histologic subtype and is associated with poor prognosis

Predominant Patterns Recognized

lepidic acinar lepidic

micropapillary solid Solid + mucin

Histological Variants• Invasive Mucinous Adenocarcinoma:

– Majority have invasive component and high association with KRAS mutation– Most tumors formerly classified as “mucinous BAC” belong to this category

• Colloid Carcinoma: – Abundant extracellular mucin– Includes rare mucinous cystic adenocarcinoma

• Fetal Adenocarcinoma:– Distinct histological, clinical (younger age) and genetic (b-catenin mutation)

features– Mostly low grade with good prognosis

• Enteric Adenocarcinoma:– Rare as primary lung ADC– Need to exclude gastrointestinal primary

Variants of Invasive Adenocarcinoma

Invasive mucinous

ADC

Fetal Enteric

Colloid