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
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