Lung Cancer Pathology: Updates

Leading Cancer Sites, Worldwide GLOBOCAN 2008

A 10-Year Prediction of Lung Cancer Incidence and Mortality Rates in 22 Arab Countries After Ten Years (2020)

Elsayed I. Salim et alAsian Pacific Journal of Cancer Prevention, Vol 12, 2011

Histopathological Classification of Lung Cancer

Lung Cancer Subtypes

Non–Small Cell Carcinoma (80%)

Small Cell Carcinoma (20%)

Availability of New Molecular Biomarkers Therapeutic Implications Lung Adenocarcinoma Pemetrexed EGFR-TKI’s Met inhibitors (Crizotinib) Squamous cell carcinoma Bevacizumab

Lung Cancer Diagnosis A multidisciplinary process requiring pathological diagnosis correlated with: Clinical Radiologic Molecular Surgical

The need for standardized criteria

International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society International Multidisciplinary Classification of Lung Adenocarcinoma Travis et al , J Thorac Oncol , 2011; 6: 244–285

WHO 2004 Classification ADENOCARCINOMA Mixed subtype Acinar Papillary Solid Bronchioloalveolar carcinoma (nonmucinous) Bronchioloalveolar carcinoma (mucinous) Fetal Mucinous (colloid) Signet ring

SQUAMOUS CELL CARCINOMA

Papillary Clear cells Small cell Basaloid SMALL CELL CARCINOMA

LARGE CELL CARCINOMA Large cell neuroendocrine carcinoma (LCNEC) Large cell carcinoma with NE morphology (LCNEM)

ADENOSQUAMOUS CARCINOMA Sarcomatoid carcinoma

Bronchioloalveolar Carcinoma

Revisiting Histomorphological Features and Integration of Immunohistochemistry and Molecular Biology

Journal of Clinical Oncology, Vol 30, No 13, 2012: pp 1401-3

Grading Architecture is the basis of the grading system: Poor Favorable Intermediate

(solid and micropapillary) (nonmucinous lepidic [formerly BAC]) (papillary and acinar)

Useful Diagnostic material H & E Stain: The Gold Standard

Effusion Aspirate Washing Brushing

Cell Block

Review the cytology and biopsy together

FOB TBBs Core SLBx

Classical morphology Lepidic, papillary, acinar

Adenocarcinoma

Keratinization, pearls, Intercellular bridges

NE morpholog

Squamous Cell Carcinoma Large cell Small cell

NSCLC ?LCNEC

SCLC

10% - 40% of NSCLC cannot be subtyped by morphology alone

Morphological Approach to Classify Lung Cancer

Mucin Brown, et al Arch Pathol Lab Med—Vol 137, September 2013

ACA, adenocarcinoma ; DG3 , desmoglein 3 and CK5 cytokeratin 5; NPV, negative predictive value; PPV, positive predictive value; SCC, squamous cell carcinoma; TTF-1, thyroid transcription factor 1

Mukhopadhyay, USCAP, March 2011

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The current WHO 2004 classification system recognizes 4 major types of lung NETs—TC, AC, LCNEC, and SCLC

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Morphologic features with criteria for mitotic rate and necrosis.

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Ki-67 can serve as a useful ancillary tool in the diagnosis of lung NETs, small biopsy and cytology specimens

Neuroendocrine (NE) immunohistochemical markers should only be performed in cases where there is suspected NE morphology:

• NE markers: • CD56 • Chromogranin • Synaptophysin • CK AE1/3 • TTF1

What are the pitfalls in biopsy diagnosis of small cell carcinoma? • • •

•

Artifacts Not correlating biopsy and cytology Difficult cases in differential diagnosis of SCLC versus NSCLC Combined SCLC

Required Tissue Conditions  Fixatives: formalin and alcohol  Fixation time: 6- 48 hours  Cell blocks cut at 2- 4 micra  Prepare extra slides to avoid loss during trimming

NSCLC is a multifaceted disease complex requiring personalized approach for its treatment.

Driver Mutations in Lung Adenocarcinoma EGFR : Epidermal Growth Factor Receptor KRAS: V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog EML4-ALK : Echinoderm microtubule associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) gene fusions. ROS1: V-Ros Avian UR2 Sarcoma Virus Oncogene Homolog. Nature medicine volume 18 | number 3 | March 2012

Mutually Exclusive EGFR

– Confers response to TKI

KRAS

– Confers resistance to TKI

EML4-ALK – Confers sensitivity to Crizotinib

EGFR

KRAS

EML4-ALK

Young Female Asian, never/light smokers

Sun S. et.al, nature reviews cancer 2007; 7 oct.: 778-790

Sun S., Lung cancer in never smokers- a different disease Nature Reviews Cancer 2007, 7: 778-790

Lung Adenocarcinoma Morphology with EGFR Mutations

Well differentiated invasive adenocarcinomas with lepidic growth, showing low grade features (acini, papillae) without necrosis and with minimal host immune response.

Method to Test for EGFR mutations Mutations

Preferred method PCR-based EGFR mutation testing for exons 19 and 21 (90% of cases)

Arch Pathol Lab Med; Vol 137, June 2013

Amplification

Protein Expression

Method to Test for EGFR mutations Multiplex PCR

19 deletions in exon 19 without distinguishing between them T790M in exon 20 L858R in exon 21 L861Q in exon 21 G719X ( detects G719S, G719A, G719C, but does not distinguish between them) in exon 18 S7681 in exon 20 3 insertions in exon 20 but does not differentiate between them

Method to Test for EGFR mutations

Mutations

Amplification

Protein Expression

Not Preferred Detection by FISH

Mutations have high response to TKI’s (75%) regardless of amplification.

Method to Test for EGFR mutations Mutations

Amplification

Protein Expression Detection by IHC

-Pan-EGFR AB is not recommended for detection of mutations -Abs to ID* exons 19 (15 bp deletions) and 21 L858R -has high sensitivity and specificity -for screening, -biopsies insufficient for molecular analysis

-Non-15 bp deletion of Exon 19, IHC is limited * In frame deletion

Hasanovic et al, Lung Cancer 2012; 77: 299-305

Randomized phase III First-Line Erbitux in Lung Cancer (FLEX)

Dako (Glostrup, Denmark) pharmDx kit.

Thershold= 200 Validated by the Round Robin Test Arch Pathol Lab Med—Vol 137, September 2013

Overall survival for patients according to treatment group and EGFR expression group

www.thelancet.com/oncology Vol 13 January 2012

www.thelancet.com/oncology Vol 13 January 2012

www.thelancet.com/oncology Vol 13 January 2012

EGFR Currently, there are no direct inhibitors of KRAS, although there are inhibitors of targets downstream to KRAS.

Nature 2013; 497: 577–578

KRAS

EML4-ALK

Lung Adenocarcinoma Morphology with KRAS Mutations • Most frequent mutated oncogene ( around 30% ) • Old male smokers with high stage disease. • Moderate /poorly differentiated with solid growth, mucinous differentiation; necrosis; and mucinous BAC.

Yousem, USCAP, March, 2011

EGFR

KRAS

EML4-ALK

Soda. Nature. 448, 2 August 2007

Chimeric protein with constitutive ALK kinase activity

Janku et al, J Thorac Oncol 2011; 6: 1601–1612.

Lung Adenocarcinoma Morphology with EMLA4-ALK Chromosomal Aberration

•

•

Young men with never/light smoking history. High grade adenocarcinoma with acinar, or solid growth with mucinous and signet ring differentiation. Yousem, USCAP, March, 2011

Janku et al, J Thorac Oncol 2011;6: 1601–1612

Method to Test for EML4-ALK mutations

Mutations

Amplification

Protein Expression

Preferred method

Approach: Ab screen, if negative/weak and unique clinical profile, proceed to FISH

Clin Cancer Res 2010;16:1561-1571

Detection Method for EML4-ALK mutations Amplification

Protein Expression

Mutations

•New antibody variation of ALK1-D5F3 AB provides high sensitivity and specificity

Screening Tool

Yousem, USCAP. March, 2011

EML4-ALK mutations- Protein expression ALK gene translocation or inversion Over-expression of the ALK protein

Crizotinib

Over-activity of the ALK tyrosine kinase

Makes sense to assess the drug target directly Thunnissen, Virchow Arch 2012; 461:145-257

Small Biopsies TTF1 & NaspinA adenocarcinoma

Cytology and biopsy

Panel of immunohistochemical stains p63 & CK5/6 squamous cell carcinoma

Report

2004 WHO classification and/or Proposed IASLC/ATS/ERS Classification

Molecular studies • •

Avoid NSCLC Neuroendocrine markers

therapeutic implications morphology is suspected

“Molecular Testing Guidline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors by the CAP/IASLC/AMP”*

Erlotinib/Gefitinib: EGFR Crizotinib: ALK

Basic criteria Tissue prioritized for biomarkers 1- EGFR 2-ALK Gender, ethnicity, and smoking habits are not recommended for selection Lindeman, et al, Arch Pathol Lab Med; April 3,2013

Specimen that can be used for molecular evaluations

• • •

Cytology specimen • Fine needle aspiration [FNA] Core or transbronchial biopsy Surgical resection • Fresh Tissues • FFPE Tissues • Frozen fixed • Alcohol fixed Tissue specimens should be managed to maximize the amount of tissue available for molecular studies. Inadequate for molecular testing for further sampling.

discuss need J Thorac Oncol, 2011; 6: 244–285

Adenocarcinoma Stage III and IV

Erlotinib/Gefitinib: EGFR Crizotinib: ALK

Resistance Seconadry mutation in EGFR & ALK

Crizotinib

Inhibits ROS 1 occurs in 1%-2%

ROS1 is a receptor tyrosine kinase of the insulin receptor family

Evolution Of Lung Cancer Histology Over Time

www.thelancet.com Vol 382 August 24, 2013

Screening for the Prevalence of KRAS, EGFR and EML4ALK Mutations in a Lung Adenocarcinoma Patient Cohort at Two Lebanese Medical Centers

American University of Beirut Medical Center Hammoud Hospital University Medical Center Supported by: Lebanese National Council for Scientific Research

180

Distribution of 851 Lung Cancer Cases by Diagnosis 2001-2010

160 140 120

100 Males Females

80 60 40

20 0

AC

SCC

Small cell

NE

NSCLC

Others

Lung Cancer 2001-2010 (n= 851)

AC (n= 242)

NSCLC-NOS (n= 150*)

SCC (n= 181)

Small cell (n= 113)

NE (n= 34)

Others** (n= 131)

Excluded AC (n= 37) Total AC (n= 279) Included

SCC NSCLC-NOS (n= 28) (n= 25)

Excluded

* Details are present in slides 2 & 3 ** Others include metastatic neoplasms & rare carcinoma or neoplasm that don’t belong to the most common categories mentioned above

Results of IHC staining for NSCLC-NOS NSCLC-NOS Submitted for IHC (n= 91*)

NSCLC-NOS (n= 25)

AC (n= 37)

* One case diagnosed as small cell carcinoma; not shown in the figure

SCC (n= 28)

Lung cancer NSCLC poorly differentiated, adenocarcinoma (H&E, Napsin A) (400x)

Napsin A Sensitivity = 75.2%

Mutational Analysis for Lung AC Lung Adenocarcinoma (n= 106) Reverse hybridization

Mutated KRAS (39)

No KRAS mutation (67) Multiplex PCR

Mutated EGFR (9)

No EGFR mutation (58)

IHC followed by FISH Mutated Alk

No Alk mutation

Mutational Analysis Methodology

Reverse Hybridiztaion

Multiplex PCR

Summary of mutations in KRAS exon 2, and EGFR

KRAS: 37% EGFR: 8.5%

KRAS Mutations c.34G>T, p.G12C c.34G>A, p.G12C c.35G>C, p.G12A c.35G>A, p.G12A c.35G>A, p.G12D c.35G>T, p.G12V c.37G>T, p.G13C c.38G>A, p.G13A c.38G>A, p.G13D EGFR Mutations

Number of Cases 19 1 11 2 5 2 2 2 2

Exon 18 Exon 19 deletions Exon 20 L858R-Exon 21

0 8 0 1

Variable (N=106) Age (in years) Mean(sd) Gender Female Male Tumor differentiation Poor Moderate Well Smoking Yes No Not Available Size (T) 3 Not Available LN (N) Yes No Not Available Metastais (M) Yes No Not Available

KRAS mutation N (%)

No KRAS mutation N (%)

p-value 0.172

64.0 (8.7)

61.0 (11.2) 0.942

13 (32.5%) 27 (67.5%)

21 (31.8%) 45 (68.2%) 0.207

25 (62.5%) 15 (37.5%) 0 (0.0%)

41 (62.1%) 20 (30.3%) 5 (7.6%) 0.286

23 (57.5%) 4 (10.0%) 13 (32.5%)

36 (54.6%) 14 (21.2%) 16 (24.2%) 0.389

6 (15.0%) 9 (22.5%) 25 (62.5%)

6 (9.1%) 22 (33.3%) 38 (57.6%) 0.879

7 (17.5%) 7 (17.5%) 26 (65.0%)

12 (18.2%) 14 (21.2%) 40 (60.6%) 0.658

6 (15.0%) 10 (25.0%) 24 (60.0%)

14 (21.2%) 13 (19.7%) 39 (59.1%)

Variable (N=106) Age (in years) Mean(sd) Gender Female Male Tumor Differentiation Poor Moderate Well Smoking Yes No Unknown Size ≤ 3 cm > 3 cm NA Lymph Node Status Yes No Not Available Metastasis Yes No NA

EGFR mutation N (%)

No EGFR mutation N (%)

p-value 0.552

59.0 (8.7)

61.8 (10.6) 0.005*

6 (85.7%) 1 (14.3%)

20 (28.2%) 51 (71.8%)