This document discusses molecular testing for lung adenocarcinoma, including common driver mutations, their prevalence, and associated targeted therapies. It describes the WHO classification of lung adenocarcinoma and lists frequently mutated genes found in this cancer. Key points covered include the role of EGFR, ALK, BRAF V600E, ROS1, MET, RET, NTRK, and KRAS mutations and the targeted therapies available to treat cancers driven by these alterations. Testing methods like NGS, PCR, and FISH are used to identify these genomic variants to guide treatment decisions.

1. NGS CASE
PRESENTATION
Mega Lahori
PGY-3

2. Lung
Adenocarcinoma
-
Molecular
Genetics

3. WHO categories of Lung adenocarcinoma
1) AIS, which is a preinvasive, typically solitary lesion that is usually non-mucinous.
2) MIA, which is a solitary and discrete non-mucinous lesion with a maximum area of invasion no greater
than 0.5 cm.
3) Invasive adenocarcinoma variants- lepidic, acinar, papillary, micropapillary, solid, etc

4. Mutation frequency of different genes in Adenocarcinoma of Lung (National Cancer
Institute Lung Cancer Mutation Consortium data)
KRAS (25%)
EGFR (23%)
ALK4 (6%)
BRAF (3%)
PIK3CA (3%)
MET (2%)
HER2neu (1%)
MAPK1 (0.4%)
NRAS (0.2%)
Liang Cheng et al. Molecular pathology of lung cancer: key to personalized medicine.
Modern Pathology volume 25, pages 347–369 (2012)

5. Somatic mutations in lung adenocarcinoma- TCGA 2014

6. Spectrum of oncogenic drivers assigned to 860 patients with lung adenocarcinoma identified
by MSK-IMPACT
Emmet Jordan et al. Prospective comprehensive molecular characterization of lung adenocarcinomas for efficient patient matching to
approved and emerging therapies. Cancer Discov. 2017 Jun; 7(6): 596–609.

7. Comparison of selected gene alteration frequencies in the MSK-IMPACT and
TCGA cohorts

8. Case No. Variants of significance VUS
1 EGFR- E709K, L858R, Amp
PIK3CA- E542K
CDK4- Amp
RUNX1
CCND1
TP53, AMER1, ATM, AXIN1,
CSMD3, EPHA2, EP300,
KMT2C, PALB2, SLX4, TGFBR1,
MYC, HDAC9
2 BRAF- K601E, Amp
RICTOR- Amp
APC, DPYD, HDAC9, HNF1,
PARP4, PMS2, IL7R, CTNND2
3 None TP53, ATR, ATRX, BLM, LARP4B,
MASH3, SMAD4, SPEN, TBX3,
TP62, TSC2, ZFHX3
4 EGFR-T790M, Amp ATM, DNMT3A, EGFR, EGFR-
AS1, MAP3K1
5 CTNNB1- S37F
STK11- L282A
CSMD3, KEAP1, KMT2D, MSH3,
PALB2, POLE, NTRK3, FAM13B,
MYC
Red- Tier 1
Blue- Tier 2

9.  Lung cancer has become a paradigm for the success of targeted therapies in solid tumors. The novel
therapies require an increase in molecular testing.
Molecular testing is used to test for genomic variants associated with oncogenic driver events for which
targeted therapies are available; these genomic variants (also known as molecular biomarkers) include gene
mutations and fusions.
The various testing methods that may be used to assess for the different biomarkers are NGS, Sanger
sequencing, multiplex PCR and FISH.

13. EGFR
• EGFR mutations are responsible for the constitutive activation of the tyrosine kinase receptor.
• Most commonly seen are sensitizing mutations in the EGFR kinase domain (exon 19 deletions or exon 21 L858R
substitutions). These activating mutations result in constitutive activity of the EGFR kinase domain, generating
survival and proliferative signals through the PI3 K-Akt-mTOR and Ras-Raf-MEK pathways.
• EGFR and KRAS mutations are mutually exclusive.
• Drug sensitive EGFR mutations are k/a sensitizing EGFR mutations.
• EGFR mutation is a specific target for therapy by TKIs and is a validated biomarker of treatment response. TK
inhibitors such as erlotinib, gefitinib, and afatinib in the first-line setting yield response rates in excess of 75 %,
and overall survival exceeding 2 years.
• Resistance to TKI therapy is associated with KRAS and BRAF mutations, ALK or ROS1 fusions, specific
acquired EGFR mutations (e.g. T790 M), cMet amplification, ERBB3 overexpression, and epiregulin autocrine
loop activation.

16. ALK4
5% of NSCLC lung cases harbor a transforming fusion gene, EML4–ALK,
which mainly involves adenocarcinoma from non-smokers with wild-
type EGFR and KRAS mutations.
EML4–ALK fusion is formed as the result of a small inversion within the
short arm of chromosome 2 that joins intron 13 of EML4 to intron 19 of
ALK [inv(2)(p21;p23)], generating a constitutively activated protein
tyrosine kinase. Activated ALK is involved in the inhibition of apoptosis
and the promotion of cellular proliferation.
At least seven EML4–ALK variants (V1–V7) have been identified in lung
adenocarcinomas.
EML4–ALK fusion gene is mutually exclusive with the EGFR mutation
Patients with EML4–ALK fusion gene tend to be younger, of Asian
ethnicity, diagnosed at an advanced clinical stage at presentation, male
dominant and more likely to be never-smokers.

18. BRAF V600E
• BRAF is a serine/threonine kinase that is part of the MAP/ERK signaling pathway.
• BRAF V600E is the most common of the BRAF point mutations; it occurs in 1% to 2% of patients with
lung adenocarcinoma.
• Specific targeted therapy is available only for BRAF V600E out of all the BRAF mutations
• Testing for BRAF mutations is recommended in patients with metastatic non-squamous NSCLC and
may be considered in patients with squamous cell NSCLC
• Dabrafenib plus trametinib is recommended for patients with BRAF V600E mutations.

20. ROS1 Rearrangements
• ROS proto-oncogene 1 (ROS1) is a receptor tyrosine kinase- it is very similar to ALK and members of
the insulin receptor family
• ROS1 gene rearrangements (also known as ROS1 fusions) occur in about 1% to 2% of patients with
NSCLC
• Occur more frequently in those who are negative for EGFR mutations, KRAS mutations, and ALK gene
fusions.
• TKIs like crizotinib, ceritinib, and entrectinib are effective for patients with ROS1 fusions.
• Similar to testing for ALK fusions, testing for ROS1 fusions is done using FISH.

22. METex14 Skipping Mutations
C-MET is a tyrosine kinase receptor that is involved in cell survival and proliferation. Oncogenic driver genomic alterations in MET include
METex14 skipping mutations, MET gene copy number gain or amplification, and MET protein overexpression. NCCN NSCLC Panel
recommends testing for METex14 skipping mutations in eligible patients with metastatic NSCLC based on data showing the efficacy of
several agents for patients with METex14 skipping mutations and on the FDA approval for Capmatinib.
RET Rearrangements
RET is a tyrosine kinase receptor that affects cell proliferation and differentiation. RET rearrangements occur in about 1% to 2% of patients
with NSCLC and are more frequent in patients with adenocarcinoma. NCCN NSCLC Panel recommends testing for RET rearrangements in
eligible patients with metastatic NSCLC based on data showing the efficacy of several agents for patients with RET rearrangements and on
the FDA approvals for Selpercatinib (LOXO-292) and Pralsetinib. Patients with RET rearrangements have minimal response (6%) to
immunotherapy.
NTRK Gene Fusions
NTRK gene fusions encode tropomyosin receptor kinase (TRK) fusion proteins (eg, TRKA, TRKB, TRKC) that act as oncogenic drivers for
solid tumors. NTRK fusions occur in 0.2% of patients with NSCLC. NCCN NSCLC Panel recommends NTRK gene fusion testing in
patients with metastatic NSCLC based on clinical trial data showing the efficacy of Larotrectinib and Entrectinib for patients with NTRK
gene fusion–positive disease

23. KRAS
KRAS is a G-protein with GTPase activity that is part of the MAP/ERK pathway; point mutations in KRAS most
commonly occur at codon 12.
Data suggest that approximately 25% of patients with adenocarcinomas in a North American population have
KRAS mutations; KRAS is the most common mutation in this population.
Associated with cigarette smoking.
Patients with KRAS mutations appear to have a shorter survival than patients with wild-type KRAS; therefore,
KRAS mutations are prognostic biomarkers. KRAS mutational status is also predictive of lack of therapeutic
efficacy with EGFR TKIs; it does not appear to affect chemotherapeutic efficacy.
KRAS mutations do not generally overlap with EGFR, ROS1, BRAF, and ALK genetic variants. Therefore, KRAS
testing may identify patients who may not benefit from further molecular testing.
Mutations in KRAS have been proposed as one of the mechanisms of primary resistance anti-EGFR therapies
panitumumab and cetuximab..