Molecular Testing NSCLC by Anurag mehta

1. Molecular testing in Lung Cancer: Identification of
newer biomarkers
Dr Anurag Mehta
Director Laboratory Services
Rajiv Gandhi Cancer Institute
Delhi
S P O T L I G H T I N N S C L C

2. Now
KRAS
25%
ALK
7%
EGFR Sensitizing
17%
No Known Oncogenic Driver
Detected
31%
EGFR Other 4%
MET exon 14 skipping 3%
> 1 Mutation 3%
HER2 exon 20 insertion2%
ROS1 2%
BRAFV600E2%
RET 2%
NTRK < 1%
PIK3CA 1%
MEK1 < 1%
5 years back
KRAS G12C
1. 5 additional actionable biomarkers for 1st line use.
2. Sotorasib – accelerated approval KRAS G12C (28/05/2021) after 1 line
3. 2 biomarkers directed therapies being used more often with level 2 evidence
4. New drugs for established drivers
Adenocarcinoma
2 druggable biomarkers
NRG fusions
Boolell V, Alamgeer M, Watkins DN et.al. The evolution of
therapies in NSCLC. Cancer(Basel)2015;7:1815-1846

3. LCMC demonstrates that the best outcomes are seen in
patients with identified drivers placed on targeted therapy:
3.5-year median survival but without targeted therapy, 2.4
years
Kris MG, Johnson BE, Berry LD, Kwiatkowski DJ et.al. JAMA.
2014;311(19):1998.
Genome directed therapy provides improved survival

4. Overall
survival
(months)
Combination
CT(1973)
SCLC and NSCLC
50
40
30
20
10
0
Platinum
doublets
(2002)
NSCLC
Platinum
doublets
and
bevacizumab
(2006)
Non-squamous
NSCLC
Gefitinib
(2009)
East Asian,
never or light
smoker
NSCLC
Erlotinib
(2009)
Spanish
EGFR- mutant
NSCLC
Gefitinib
(2009)
Japanese
EGFR- mutant
NSCLC
Crizotinib
(2017)
ALK-mutant
NSCLC
>45.8
months
Extra 10
months
81
months
Any ATKI
2019
Colorado
38.6
months
Osimertinib
FLAURA
2020
80
1. Adapted from Pao W & Chmielecki J. Nat Rev Cancer 2010;10:760–774
2. Mok T et al. Presented at ESMO 2017:LBA50
3. Clin Oncol 36:2251-2258. © 2018
4. Ramalingam SS et. al. N Eng J Med. 2020 Jan 2;382(1):41-50
5. Jose M. Pacheco et. al. J Thorac Oncol. 2019 April ; 14(4): 691–700
Treat as many - by
Genome directed therapy

5. Know actionable Drivers
• EGFR
• ALK fusions
• ROS1 fusions
• BRAFV600E
• RET fusions
• Δ MET exon 14
• NTRK fusions
• KRAS G12C
• PDL1
Tissue is a bigger issue
Other challenges

6. RET rearrangement
1. Seen in 1-2% of NSCLC.
2. Mechanism of action is similar to ALK and ROS1
fusions.
• The chimeric protein formed by RET fusion is
ligand free and drives cancer due to
uninterrupted activated state
3. The RET gene sits in the 3’ position- retains the
kinase domain and fuses with a partner at 5’
(KIF5B mostly)

7. Method Pros Cons Consider Using?
IHC
Most Labs, less
expensive
Performance Variable NO
FISH Some Labs
False Negative
False Positive
Rare
circumstances
RT-PCR
Some Labs
Not Tissue proficient
Extensive multiplexing
Long intronic regions – preclude DNA
based rtPCR .
mRNA based rtPCR preferred
YES
No commercially
available Kit
? AmoyDX
DNA NGS
A few Labs
Tissue proficient
Panel design crucial
Must Cover all break regions
Mate pair reading
YES
RNA NGS
A few Labs
Tissue proficient
Dependent upon RNA quality YES
Expression
imbalance
Some Labs
Fast, relatively
inexpensive
Need well established cut off
YES, especially if
part of panel
evaluating
multiple kinase
fusions
40%
Testing for RET fusion rearrangement
Promising but yet to be fully validated.
C. Belli, F. Penault-Llorca, M. Ladanyi, et.al. ESMO recommendations on the standard methods to detect RET fusions and
mutations in daily practice and clinical research, Annals of Oncology, Volume 32, Issue 3, 2021,

8. 8
Advance
NSCLC
FFPE
not available
Liquid
Biopsy
NGS
rtPCR
FFPE available
NGS Available
DNA sequencing
RNA sequencing
preferred
NGS
not available
FISH/RTPCR
C. Belli, F. Penault-Llorca, M. Ladanyi, et.al. ESMO recommendations on the standard methods to detect RET fusions and
mutations in daily practice and clinical research, Annals of Oncology, Volume 32, Issue 3, 2021,

9. 1. RET fusion testing now should be part of any expanded panel of molecular
tests
2. Majority of RET fusions detectable with DNA NGS but increased sensitivity
with RNA NGS
3. Single gene alternative
• FISH albeit limited sensitivity and specificity
• IHC is available – not fully validated
• rtPCR is a possible method

10. MET exon 14 skipping
1. A new actionable biomarker
2. n- 3% for all NSCLC. LUAD, SCC, NOS
3. Mutually exclusive with other driver mutations
4. Recently approved highly effective treatments

11. Testing options for Δ MET exon 14
Testing Substrate
DNA
• Look for mutations /indels- 120
• Many may still be unknown
mRNA
 An altered sequence missing bases assigned to exon 14
• A sequence of shorter length- 140 bp
• Near perfect sensitivity
• Shorter template makes it easy to amplify despite fragmentation of NA in FFPE tissue
Optimization of Routine Testing for MET Exon 14 Splice Site Mutations in NSCLC Patients. Journal of Thoracic Oncology Vol.
13 No. 12: 1873-1883. 2018

12. NGS- Most favored platform
Effectiveness of DNA sequencing: design dependent.
• Oncomine and Illumina trusight fail to identify 5’ alterations ( No amplicon).
• DNA sensitivity- 63%
RNA: most sensitive
More MET-ex14 mutations identified by RNA NGS than DNA NGS
• DNA NGS: 16/644 (2.5%)
All mutations identified by DNA assay were at or around splice site for intron 14
since assay did not include intron 13 splice acceptor site
• RNA NGS: 25/644 (3.9%) Jurkiewicz. ASCO 2020. Abstr 9036.

13. DELETIONS
•c.2888 (2-35 bp del)
•c.2903_3028+67del
•c.2905_2940del
•c.3010_3028+8del
•c.3018_3028+8del
•c.3020_3028+24del
•c.3025_3027GAA>Gfs
•c.3028+1_3028+9del
MISSENSE
•c.3008A>G (Y1003)
•c.3028+2T>C
•c.3028+1G>T
•c.3028G>A (D1010)

14. Good call
False Call
Acceptable call
1. Confirm by single gene assay
2. The other value may be testing for ∆MET
exon 14 in SCC

15. PCR
1. DNA: PCR and sequence by Sanger Sequencing - identify SNVs and Dels
• Must know all the alterations - skipping
• Design the primers/probes accordingly
2. RNA: Amplify – sequence or fragment length analysis
• technical considerations- integrity of mRNA

17. RISH
EXON 13 EXON 14 EXON 15
EXON 12
EXON 13 EXON 15
EXON 12

18. Summary
1. All NSCLC should be tested.
2. RNA sequencing preferred as a part of composite NGS for broad molecular
testing.
3. Labs not having access to NGS can use rt PCR or fragment sizing assay - a simple
and robust method for MET exon14 skipping

19. NTRK fusion rearrangement
 Very rare (0.3% of NSCLC) but actionable if identified!
 Larotrectinib and Entrectinib both show significant clinical activity in
patients with NTRK fusion–positive disease
 Multiple testing methods available to identify rearrangement

20. Testing for NTRK Fusions
Options:
1. IHC- false positivity
2. FISH – 3 probes
3. RT-PCR- ???
4. DNA or RNA NGS
FISH
RT-PCR
RNA NGS
Marchiò. Ann Oncol. 2019;30:1417.
ESMO Working Group Recommendations:
Approach for NTRK Fusion Testing in NSCLC
NTRK Testing
YES NO
NO YES
Use IHC as a screening tool
No TRK
expression
Detection of TRK
expression
Use frontline NGS,
preferably with RNA
testing when possible
Is there a sequencing
platform available?
Histologic type
consistent with recurrent
NTRK fusions?

21. Testing for KRASG12C
1. QIAGEN therascreen® KRAS RGQ PCR Kit- Tissue
2. Guardant 360 – Plasma ( sensitivity -70%)
3. KRAS mutational testing – readily available – rtPCR, ddPCR.
4. NGS can be used both for tissue and plasma

22. FLOURESCENCE IN SITU HYBRIDIZATION (FISH)1,6
Fluorescent probes label specific gene regions causing them to
fluoresce on microscopy
IMMUNOHISTOCHEMISTRY (IHC)2,6
Antibodies detect specific proteins expressed by cells; a chemical
reaction generates a colored deposit for cells expressing the
antibody, identified using microscopy
REAL TIME PCR
RT-PCR (Reverse transcriptase-polymerase chain reaction)
Target RNA is reverse transcribed to DNA amplified by PCR
NEXT-GENERATION SEQUENCING (NGS)4,
Using micro- and nano-technology to run parallel sequencing
A number of molecular technologies are available
Vincent MD et al. Curr Oncol 2012;19:S33–S44; 2. Ramos-Vara JA. Vet Pathol 2005;42:405–426; 3. Peake I. J Clin Pathol 1989;42:673–676; 4. Grada A &
Weinbrecht K. J Invest Dermatol 2013;133:e11; 5. Kerr KM. J Thorac Oncol 2014;9:593–595 6. Tsao MS et al (eds). IASLC Atlas of ALK Testing in Lung Cancer
2013. https://www.iaslc.org/publications/iaslc-atlas-alk-testing-lung-cancer. Accessed May 28, 2014

23. Lap 1 a ( save Tissue)
Acinar, papillary, Lepidic
NSCC- ADC
Solid P40+
TTF1+
Solid P40-
TTF1-
Solid P40 -
TTF1
+
NSCC- favour ADC
NSCC- favour ADC
NSCC- NOS
Extract DNA x10
ALK x 2 Ros x 2
Ros x 1
FISH
PDL1 x 1
EGFR & BRAF V600E X 10
Extract RNA X 10
cDNA
NGS
SNV, CNV, Fusions
Lap 1 b ( save Tissue)
Lap 2
Additional 8-10% cases to treat /
recruit in a trial
ARMS PCR
Cobas/ Therascreen
Cytology- cell Blocks
Plasma for EGFR & BRAF
Liquid Biopsy for the other markers
Used saved DNA

24. Biomarker Genetic aberration for testing
Frequency
(RGCIRC)
Single gene assay FDA approved method
EGFR Sensitizing Mutation 29.4% ARMS
1. Roche: Cobas EGFR Mutation Test v2
2. Qiagen: Therascreen EGFR RGQ PCR Kit
3. NGS: Oncomine Dx Target Test
4. Foundation One CDx
EML4-ALK Rearrangement 8% IHC/ FISH/RTPCR
1. VENTANA ALK (D5F3) CDx Assay
2. Vysis ALK Break Apart FISH Probe Kit
3. Foundation One CDx
Ros-1 Rearrangement 1.17%
FISH
IHC-D4D6(CONFIRM POSITIVE BY
FISH
1. NGS: Oncomine Dx Target Test
BRAFV600E Mutation 2% rtPCR
1. NGS: Oncomine Dx Target Test
2. Foundation One CDx
Mutation- exon
14 skipping
Mutations leading to truncated
mRNA
3%
Reverse transcriptase -PCR with
sequencing or fragment sizing
FoundationOne CDx NGS for Capmatinib
Archer DX based RNA seq and ctDNA - NGS
NTRK Fusion rearrangement 2% IHC/FISH/rtPCR FoundationOne CDx - larotrectinib
RET Rearrangement FISH
Oncomine Dx Target Test-Selpercatinib
Oncomine Dx Target Tes- Pralsetinib
NGS is an acceptable platform for testing several biomarkers together.
Biomarker testing using Tissue/cytology- Single gene assays versus NGS

25. In conclusion, single gene assays and NGS methods offer a good complementary workflow, with rapid results based on
a restricted analysis using probes, followed by NGS analysis. We demonstrated that targeted NGS is a cost and
time effective platform to detect multiple mutations simultaneously in various genes with
high reproducibility and sensitivity. Thus, targeted NGS at diagnosis may provide useful information. This
work is a proof of concept that targeted NGS is accessible in routine including large screening and reasonable cost.
NGS should not be restricted to specific patients because many of the non-validated molecular alterations susceptible
to targeted therapies have low prevalence.
Is NGS a valid testing methodology?

26. NGS HAS BEEN USED TO FACILITATE PARALLEL TESTING FOR MULTIPLE GENE MUTATIONS IN
A NATIONWIDE PROGRAM
Barlesi F et al. Lancet 2016;387:1415–1426
• French National Cancer Institute-funded nationwide program
• Systematic routine analysis of EGFR mutations, ALK rearrangements, KRAS, BRAF, HER2
and PIK3CA mutations in 17,664 patients with advanced NSCLC over 1 year
Frequency of molecular aberrations from analyzed samples
 Genetic aberration recorded in ~50%
 Screen failure rates varied from 1 to 4%
 Presence of genetic aberration affected
1st-line treatment in ~51% of patients

27. Successes
• Reliable in detecting known standard-of-care mutations, with good sensitivity and
specificity1
• NGS testing panels may provide data on all known mutations allowing access to targeted
treatment in 1st or later lines of therapy1
• Accurate identification of more clinically actionable alterations compared to current
diagnostic tests2
• Acceptable turnaround time in obtaining analysis results3
NGS testing panels have been validated, though challenges exist
1. McCourt CM et al. PLoS One 2013;8:e69604;
2. Frampton GM et al. Nat Biotech 2013;31:1023–1031;
3. Barlesi F et al. Lancet 2016;387:1415–1426

28. 1. Because of highly effective targeted therapies (and little
likelihood of benefit from ICIs), testing for
EGFR/ALK/ROS1/BRAF/NTRK/METex14/RET / KRASG12C
shall be offered to all patients with advance
nonsquamous NSCLC
• Exception – MET exon 14 skipping – test all
histologies
2. Additional drugs likely to be approved in the not-too-
distant future for HER2 and EGFR exon 20 insertions
3. Broad testing with NGS for both required and emerging
biomarkers is now well recognized
Conclusions
For Pathologist
1. Take care of the tissue.
2. Use minimum IHC to type biopsies -LUAD/SCC/NOS
3. For squamous NSCLC, consider testing in young,
never or light smokers, or if biopsy specimen is small
or of mixed histology and for MET exon 14 skipping
4. Primary tumors and metastatic lesions are equally
suitable
5. Bone biopsy potentially suboptimal due to
decalcification and degradation of DNA
6. Bring NGS in to practice at greater pace
7. Liquid biopsy is an option worth considering

29. • cDNA based assay
• Forward: TGGGTTTTTCCTGTGGCTGA
• Reverse: AGGATACTGCACTTGTCGGC
Primers for fragment sizing assay for Met exon 14 skipping
Total size of fragment= 235
Skipped 14- 140
TGGGTTTTTCCTGTGGCTGAAAAAGAGAAAGCAAATTAAAGATCTGG
GCAGTGAATTAGTTCGCTACGATGCAAGAGTACACACTCCTCATTTGG
ATAGGCTTGTAAGTGCCCGAAGTGTAAGCCCAACTACAGAAATGGTT
TCAAATGAATCTGTAGACTACCGAGCTACTTTTCCAGAAGATCAGTTT
CCTAATTCATCTCAGAACGGTTCATGCCGACAAGTGCAGTATCCT