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

NonSmall Cell Carcinoma (80%)

Small Cell Carcinoma (20%)

Availability of New Molecular Biomarkers

Therapeutic Implications
Lung Adenocarcinoma
Pemetrexed
EGFR-TKIs
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: 244285

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 MedVol 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

The current WHO 2004 classification system

recognizes 4 major types of lung NETsTC,
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

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 TKIs (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 MedVol 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: 577578

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: 16011612.

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: 16011612

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: 244285

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 dont 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
>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%)
<0.001*

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

46 (64.8%)
22 (31.0%)
3 (4.2%)
0.999

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

24 (33.8%)
7 (9.9%)
40 (56.3%)
0.881

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

11 (11.3%)
29 (29.9%)
57 (58.8%)
0.424

0 (0.0%)
2 (22.2%)
7 (77.8%)

19 (19.6%)
19 (19.6%)
59 (60.8%)
0.792

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

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