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

Lung Adenocarcinoma Pemetrexed EGFR-TKI’s Met inhibitors (Crizotinib) Squamous cell carcinoma Bevacizumab

Therapeutic Implications

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

The need for standardized criteria

Lung Cancer Diagnosis

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

ADENOCARCINOMA

Mixed subtype

Acinar

Papillary

Solid

Bronchioloalveolar carcinoma

(nonmucinous)

Bronchioloalveolar carcinoma

(mucinous)

Fetal

Mucinous (colloid)

Signet ring

SMALL CELL CARCINOMA

LARGE CELL CARCINOMA

Large cell neuroendocrine carcinoma

(LCNEC)

Large cell carcinoma with NE

morphology (LCNEM)

ADENOSQUAMOUS CARCINOMA

Sarcomatoid carcinoma

SQUAMOUS CELL CARCINOMA

Papillary

Clear cells

Small cell

Basaloid

WHO 2004 Classification

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

Review the cytology and biopsy together

Effusion Aspirate Washing Brushing FOB

TBBs Core SLBx

Cell Block

H & E Stain: The Gold Standard

Useful Diagnostic material

Classical morphology

Lepidic, papillary, acinar

Adenocarcinoma

Keratinization, pearls, Intercellular bridges Squamous Cell

Carcinoma

NE morpholog

Large cell

Small cell

NSCLC ?LCNEC

SCLC

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

Mukhopadhyay, USCAP, March 2011

Mucin

Morphological Approach to Classify Lung Cancer

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

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

Morphologic features with criteria for mitotic rate and necrosis.

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

Fixation time: 6- 48 hours

Required Tissue Conditions

Fixatives: formalin and alcohol

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.

EML4-ALK : Echinoderm microtubule associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) gene fusions.

EGFR : Epidermal Growth Factor Receptor

KRAS: V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog

Nature medicine volume 18 | number 3 | March 2012

ROS1: V-Ros Avian UR2 Sarcoma Virus Oncogene Homolog.

Driver Mutations in Lung Adenocarcinoma

EGFR

KRAS

EML4-ALK

Mutually Exclusive

– Confers response to TKI

– Confers resistance to TKI – Confers sensitivity to Crizotinib

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

Young Female Asian, never/light smokers

EGFR KRAS EML4-ALK

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

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

Lung Adenocarcinoma Morphology with EGFR Mutations

Method to Test for EGFR mutations

Mutations Amplification Protein Expression

Preferred method

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

Arch Pathol Lab Med; Vol 137, June 2013

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

Multiplex PCR

Method to Test for EGFR mutations

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

Mutations Amplification Protein Expression

Not Preferred

Detection by FISH

-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

Method to Test for EGFR mutations

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

Mutations Amplification Protein Expression

Detection by IHC

* In frame deletion

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

Thershold= 200 Validated by the Round Robin Test

Arch Pathol Lab Med—Vol 137, September 2013

Dako (Glostrup, Denmark) pharmDx kit.

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 KRAS EML4-ALK

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

Nature 2013; 497: 577–578

• 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

Lung Adenocarcinoma Morphology with KRAS Mutations

Chimeric protein with constitutive ALK kinase activity

EGFR KRAS EML4-ALK

Soda. Nature. 448, 2 August 2007

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

• 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

Lung Adenocarcinoma Morphology with EMLA4-ALK Chromosomal Aberration

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

Mutations Amplification Protein Expression

Screening Tool

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

Yousem, USCAP. March, 2011

Makes sense to assess the drug target directly

EML4-ALK mutations- Protein expression

Thunnissen, Virchow Arch 2012; 461:145-257

ALK gene translocation or inversion

Over-expression of the ALK protein

Over-activity of the ALK tyrosine kinase

Crizotinib

Small Biopsies

Cytology and biopsy

Panel of immunohistochemical stains

Report

Molecular studies

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

Classification

TTF1 & NaspinA adenocarcinoma

p63 & CK5/6 squamous cell

carcinoma

• Avoid NSCLC therapeutic implications • Neuroendocrine markers morphology is suspected

Tissue prioritized for biomarkers 1- EGFR 2-ALK

Lindeman, et al, Arch Pathol Lab Med; April 3,2013

“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

Gender, ethnicity, and smoking habits are not recommended for selection

Basic criteria

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

Inadequate for molecular testing discuss need for further sampling.

Tissue specimens should be managed to maximize the amount of tissue available for molecular studies.

J Thorac Oncol, 2011; 6: 244–285

Erlotinib/Gefitinib: EGFR Crizotinib: ALK

Adenocarcinoma Stage III and IV

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 EML4-ALK 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

0

20

40

60

80

100

120

140

160

180

AC SCC Small cell NE NSCLC Others

Distribution of 851 Lung Cancer Cases by Diagnosis

2001-2010

Males

Females

AC

(n= 242)

NSCLC-NOS

(n= 150*)

SCC

(n= 181)

Lung Cancer 2001-2010

(n= 851)

Small cell

(n= 113)

SCC

(n= 28)

NSCLC-NOS

(n= 25)

AC

(n= 37)

Total AC

(n= 279)

* 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

NE

(n= 34) Others**

(n= 131)

Excluded

Excluded

Included

NSCLC-NOS

(n= 25)

AC

(n= 37) SCC

(n= 28)

NSCLC-NOS

Submitted for IHC

(n= 91*)

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

Results of IHC staining for NSCLC-NOS

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

Napsin A Sensitivity = 75.2%

Lung Adenocarcinoma

(n= 106)

No KRAS mutation (67) Mutated KRAS (39)

Mutated EGFR (9) No EGFR mutation (58)

Mutated Alk No Alk mutation

Reverse hybridization

Multiplex PCR

IHC followed by FISH

Mutational Analysis for Lung AC

Mutational Analysis Methodology

Reverse Hybridiztaion Multiplex PCR

Summary of mutations in KRAS exon 2, and EGFR

KRAS Mutations Number of Cases

c.34G>T, p.G12C 19

c.34G>A, p.G12C 1

c.35G>C, p.G12A 11

c.35G>A, p.G12A 2

c.35G>A, p.G12D 5

c.35G>T, p.G12V 2

c.37G>T, p.G13C 2

c.38G>A, p.G13A 2

c.38G>A, p.G13D 2

EGFR Mutations

Exon 18 0

Exon 19 deletions 8

Exon 20 0

L858R-Exon 21 1

KRAS: 37% EGFR: 8.5%

Variable (N=106)

KRAS mutation N (%)

No KRAS mutation N (%)

p-value

Age (in years) Mean(sd)

64.0 (8.7)

61.0 (11.2)

0.172

Gender Female Male

13 (32.5%) 27 (67.5%)

21 (31.8%) 45 (68.2%)

0.942

Tumor differentiation Poor

Moderate Well

25 (62.5%) 15 (37.5%)

0 (0.0%)

41 (62.1%) 20 (30.3%)

5 (7.6%)

0.207

Smoking Yes No

Not Available

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

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

0.286

Size (T) <=3 >3

Not Available

6 (15.0%) 9 (22.5%)

25 (62.5%)

6 (9.1%)

22 (33.3%) 38 (57.6%)

0.389

LN (N) Yes No

Not Available

7 (17.5%) 7 (17.5%)

26 (65.0%)

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

0.879

Metastais (M) Yes No

Not Available

6 (15.0%)

10 (25.0%) 24 (60.0%)

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

0.658

Variable (N=106)

EGFR mutation N (%)

No EGFR mutation N (%)

p-value

Age (in years) Mean(sd)

59.0 (8.7)

61.8 (10.6)

0.552

Gender Female Male

6 (85.7%) 1 (14.3%)

20 (28.2%) 51 (71.8%)

0.005*

Tumor Differentiation Poor

Moderate Well

3 (42.9%) 0 (0.0 %) 4 (57.1%)

46 (64.8%) 22 (31.0%)

3 (4.2%)

<0.001*

Smoking Yes No

Unknown

2 (28.6%) 1 (14.3%) 4 (57.1%)

24 (33.8%)

7 (9.9%) 40 (56.3%)

0.999

Size ≤ 3 cm > 3 cm

NA

1 (11.1%) 2 (22.2%) 6 (66.7%)

11 (11.3%) 29 (29.9%) 57 (58.8%)

0.881

Lymph Node Status Yes No

Not Available

0 (0.0%)

2 (22.2%) 7 (77.8%)

19 (19.6%) 19 (19.6%) 59 (60.8%)

0.424

Metastasis Yes No NA

2 (22.2%) 1 (11.1%) 6 (66.7%)

18 (18.6%) 22 (22.7%) 57 (58.8%)

0.792