Integrating Molecular markers into the

Diagnosis of Lung Cancer

2
Lung Adenocarcinoma Classification by
Molecular Tumor Pathway I
Lung Adenocarcinoma Classification by
Molecular Tumor Pathway II
 EGFR mutation positive patient profile:
Responders

 Well to moderately differentiated

lepidic predominant adenocarcinomas
 Non-Mucinous
 Never smoker
 Asian
 Female
 Likelihood of EGFR Mutation Decreases as
Smoking History Increases
 EGFR mutation status (exons 19 and 21) determined in
265 tumor samples
Patients With Exon 19 and

100
21 EGFR Mutations (%)

80
51
60

40
19
20 4

0
Never Former Current
Cigarette-Smoking History

Pham D, et al. J Clin Oncol. 2006;24:1700-1704.

 IPASS: Progression-free survival in
EGFR mutation
positive & negative patients
EGFR mutation positive EGFR mutation negative

Gefitinib (n=132) Gefitinib (n=91)

1.0 Carboplatin / paclitaxel (n=129) 1.0

Probability of progression-free survival

Probability of progression-free survival

Carboplatin / paclitaxel (n=85)

HR (95% CI) = 0.48 (0.36, 0.64) HR (95% CI) = 2.85 (2.05, 3.98)
0.8 p<0.0001 0.8
p<0.0001
No. events gefitinib, 97 (73.5%) No. events gefitinib , 88 (96.7%)
0.6 No. events C / P, 111 (86.0%) 0.6 No. events C / P, 70 (82.4%)

0.4 0.4

0.2 0.2

0.0 0.0
0 4 8 12 16 20 24 0 4 8 12 16 20 24
Months Months
At risk :
Gefitinib 132 108 71 31 11 3 0 91 21 4 2 1 0 0
C/P 129 103 37 7 2 1 0 85 58 14 1 0 0 0

Treatment by subgroup interaction test, p<0.0001

ITT population
Cox analysis with covariates Mok: ESMO, 2008
 Tumor Responses to Crizotinib for
Patients with ALK-positive NSCLC
60
Progressive disease
Stable disease
40
Confirmed partial response
Maximum change in tumor size (%)

20 Confirmed complete response

–20
–30%

–40

–60

–80

–100
*
*Partial response patients with 100% change have non-target disease present
 Trials Evaluating KRAS Mutations and
Resistance to EGFR Inhibitors
Trial EGFR TKI N Response Rate, % Median OS, Mos
KRAS WT KRAS MT KRAS WT KRAS MT
INTEREST[1] Gefitinib 275 9.6 0 7.5 7.8
Jackman[2] Gefitinib or
116* 5 0 11.8 13
erlotinib
Massarelli[3] Gefitinib or
70 10.0 0 9.4 5.0
erlotinib
Shepherd[4] Erlotinib 206 10.2 5.0 7.5 3.7
Miller[5] Erlotinib 101† 32 0 21 13
*Only includes EGFR wild-type patients.
†Includes patients with bronchioloalveolar carcinoma and adenocarcinoma, bronchioloalveolar carcinoma

subtype.

1. Douillard JY, et al. J Clin Oncol. 2010;28:744-752. 2. Jackman DM, et al. ASCO 2008. Abstract 8035.
3. Massarelli E, et al. Clin Cancer Res. 2007;13:2890-2896. 4. Zhu CQ, et al. J Clin Oncol. 2008;26:4268-
4275. 5. Miller VA, et al. J Clin Oncol. 2008;26:1472-1478.
Molecular Markers Treatment and
Relevance in Lung Cancer
Squamous cell
Pemetrexed
carcinoma
(Alimta)

Iressa
Tarceva

ALK-positive ROS-1
driven

Crizotinib
metMab (Xalcori)
(MGCD265)
 Correlation Histologic Subtype,
Molecular and Radiological Findings
Histologic Molecular Radiology (CT) IHC Clinical
Type
AIS or MIA EGFR 10-30% Ground glass TTF-1 + Never smokers
Lepidic EGFR 10-30% Ground glass TTF-1+ Never smokers
(nonmucinous) KRAS 10% with part solid
BRAF 5% nodule
Papillary EGFR 10-30% Solid nodule TTF-1 +
KRAS 3%
P53 30%
Acinar KRAS 20% Solid nodule TTF-1 can be Smokers
EGFR <10% negative
EML4/ALK 5%
Micropapillary KRAS 30% Multifocal TTF-1 + (70%)
EGFR 20% Ground glass
BRAF 20%
Invasive KRAS 80-100% Air bronchogram TTF-1
Mucinous AdCa EGFR no Consolidation negative,
mutation CK20 positive
Recommended Methods for Analyzing
Molecular Markers in Lung Cancer

3
 EGFR mutations associated with sensitivity to
TKI’s
EGFR Tyrosine Kinase Domain Mutually exclusive mutations of
Mutations in Lung Adenocarcinoma EGFR sensitivity
Mutation Percentage of
Response to EXON Mutation
cases
EGFR TKI
KRAS 20%
Sensitivity 18 G719X
19 LREA del BRAF 1%
21 L858R HER 2 3%
L861X
Resistance 19 D761Y
20 D790M

EGFR mutation status by

gene sequencing analyzing
at least 4 exons (18-21)
NCCN Guidelines

 Test only AdCa for

– EGFR (Mutation
analysis)
– Alk (FISH)
 Only EGFR mutated
AdCa and Alk + AdCa
are FDA approved for
TKI
 Cost Crizotinib/month
– US$ 9600.00
FLEX Study using EGFR IHC to predict
outcome to EGFR-TKI (2011)
Problems with EGFR IHC as proposed by
the FLEX Trial
 EGFR by IHC stains positive 80 % of Squamous cell carcinoma
 EGFR by IHC stains Wild type EGFR (not associated with response to
EGFR-TKI)
 Interpretation issues High vs. low Expression (0-3), IHC score
– Standardization of procedure, and interpretation
 Three types of EGFR for IHC
– Total EGFR- correlates poorly with presence of EGFR mutations
– Phosphorylated EGFR –unstable limited use
– Mutated EGFR- scoring cutoff needs to be applied (similar to Her2)
– Currently only two mutation specific antibodies are available

– Negative samples have to be reflexed to molecular analysis

  “Patient age,
percentage of tumor
cells expressing EGFR
and tumor stage were
NOT shown to be of
prognostic value in the
Pirker R et al, Department of Medicine I, Medical
University Vienna, Vienna, Austria
setting of univariate
Lung Cancer. 2012 Aug;77(2):376-82. Epub 2012 analysis”
Apr 11.
 Predictive value of High
EGFR expression in
relation to cetuximab
benefit cannot be
evaluated under current
threshold IHC score
 Guidelines Testing and Reporting of Lung
Cancer Biomarkers (In press)

College of American Pathologists (CAP)

International Association for the Study of Lung Cancer (IASLC)
Association for Molecular Pathology (AMP)
Guidelines Testing and Reporting of Lung
Cancer Biomarkers
 EGFR mutation testing should capture all individual
mutations reported in at least 1% of EGFR-mutant lung
adenocarcinomas
– Exons 18-21
 EGFR immunohistochemistry is NOT recommended for
selection of EGFR TKI therapy
 EGFR copy number analysis (FISH or CISH) is NOT
recommended for selection of TKI therapy
 Whenever possible ALK testing should be done at the
same time as EGFR testing
 ALK testing should be done by split-apart, dual labeled
ALK FISH probes
Practical Approach to Pathologic and
Molecular Diagnosis of Lung Cancer

4
 Pathologic Diagnosis of Lung
Adenocarcinoma

 Determine if primary lung adenocarcinoma

– Metastatic colorectal, breast, uterine
 Determine histologic subtype
– Impacts prognosis : Micropapillary, lepidic predominant
multifocal with AAH
 Exclude squamous cell component
– Immunohistochemistry
 Preserve as much tissue as possible for molecular biology
studies:
– EGFR, ALK
LUNG BIOPSY
Triage of biopsy tissue to optimize diagnosis
and molecular studies at MHS

Interventional radiology is
asked to provide at least two
core biopsies

Block 2 for IHC and

diagnosis
Block 1 for molecular

MOLECULAR
REPORT

PATHOLOGY
REPORT
 Immunohistochemical staining in Lung
Cancer
Adenocarcinoma Squamous Neuroendocrine
TTF-1 P63 Chromogranin
CK 7 CK 5/6 Synaptophysin
Napsin
Mucin

Exclude metastatic AdCa Negative for Ki-67

based on clinical history TTF1
CDX2 Napsin
Villin
Mammaglobin
GCDFP-15

The emphasis is to preserve as much tissue as

possible to allow for molecular studies
TTF-1

CK 7
p63
EGFR Mutation Analysis report
ALK FISH Assay
 ACKNOWLEDGEMENTS
 Luis Raez MD, Thoracic Oncology MHS
– Selected slides on treatment protocols
 Ana María Agrusa, BS
 Rachel Murray, PA
 Departments of Thoracic Oncology and Surgery Memorial
Health Care System, Hollywood,

QUESTIONS