1. Molecular Diagnosis of NSCLC- WHAT’S NEW
showcase
Dr. Anurag Mehta
Director Lab & Molecular Services
RGCI & RC

2. Established
Genome directed therapy is the standard treatment for “Metastatic Lung Adenocarcinoma”.
Several driver alterations are druggable.
Three targets are actionable by FDA approved drugs. ROS 1 is a new addition
Currently, no targeted therapy is available for SCC & SCLC

3. Acquired Resistance through secondary mutations
Actionable Targets
FDA approved
EGFR mutation
ALK fusion
ROS1 fusion
Acquired Resistance through secondary mutations
Off label use, small studies
MET CNG
RET fusion
BRAF V600E & others
MET Exon 14 skipping mutation
HER 2 exon 20 mutation
Inadequate assertions
NTRK1
NRG1
FGFR
ERBB4

4. What’s new?
ROS 1 fusion is a new addition as a target by a FDA approved drug. (Crizotinib-Mar 2016)
No designated companion diagnostic.
FISH is the established method. Unlike in ALK; FISH is easier to interpret.
RNA methods ( RT - PCR, anchored multiplexed PCR / RACE PCR)
Next generation DNA sequencing
IHC has good sensitivity but lower specificity. PPV is not satisfactory. Can be used as a screening tool but confirmation with FISH is necessary. IHC is not binary. Grading is necessary but subjective. Process is not harmonized.

5. What’s new? New draft guidelines from CAP, IASLC & AMP
Optimize all phases of laboratory testing
Testing for secondary mutations leading to acquired resistance
Recommendations for selection of appropriate testing methodology/ platform(s) & in special context.
Also have recommendations for expanded testing- other than main 3.
Tissue proficient testing – multiplexed sequence based testing.

7. What’s new? 2016 statement Explanation Recommendation:
Physician’s should use molecular testing for the appropriate genetic targets on either primary or metastatic lung lesions to guide initial therapy selection.
To determine EGFR and ALK status for initial treatment selection, primary tumors or metastatic lesions
are equally suitable for testing.
emanate

8. What’s new? 2016 statement Explanation Recommendation:
In some clinical settings in which tissue is limited and/or insufficient for molecular testing, physicians may utilize a mutation - specific IHC assay for EGFR testing
New Recommendation statement
When performing ALK testing, physicians can utilize IHC as an equivalent alternative to FISH.
No scientific need to perform both methods
New Recommendation Statement

9. 2016 statement Explanation
What’s new?
2016 statement
Explanation
Physicians may use ROS1 IHC as a screening test in lung adenocarcinoma patients; however, positive ROS1 IHC
results should be confirmed by a molecular or cytogenetic method.
New Recommendation Statement
Recommendation:
In some clinical settings in which tissue is limited and/or insufficient for molecular testing, physicians may use a cell -free plasma DNA (cfDNA) assay for EGFR.
( Liquid biopsy has been permitted as a valid diagnostic tool for Primary EGFR mutation testing)

10. Explanation What’s new? Strong Recommendation: 2016 statement
In lung adenocarcinoma patients who harbor sensitizing EGFR mutations and have progressed after treatment with an EGFR –targeted TKI, physicians must use EGFR T790M mutational testing when selecting patients for third generation EGFR – targeted therapy
New Recommendations
Recommendation:
Laboratories testing for EGFR T790M mutation in patients with acquired resistance to EGFR –targeted kinase inhibitors should deploy assays capable of detecting EGFR T790M mutations in as little as 4% of viable cells (2% of EGFR alleles).

11. What’s new? 2016 statement Expansion Expert Consensus Opinion:
Physicians may use cell free plasma DNA (cfDNA) methods to identify EGFR T790M mutations in lung adenocarcinoma patients with progression or acquired resistance to EGFR -targeted tyrosine kinase inhibitors; testing of the tumor sample is recommended if the plasma result is negative.
New statements
Multiplexed genetic sequencing panels are preferred over multiple single gene tests to identify other treatment options beyond EGFR, ALK and ROS1.

12. What’s new? 2016 statement Expansion No Recommendation:
There is currently insufficient evidence to support a recommendation for or against routine testing for ALK mutational status for lung adenocarcinoma patients with sensitizing ALK mutations who have progressed after treatment with an ALK targeted tyrosine kinase inhibitor.
ALK acquired resistance testing is not mature for clinical usage
Expert Consensus Opinion: BRAF, RET, MET, HER2 & KRAS molecular testing is currently not indicated as a routine stand-alone assay outside the context of a clinical trial. As part of larger testing panels performed either initially or when routine EGFR, ALK, and ROS1 testing are negative, it is appropriate to include BRAF, RET, MET, HER2 & KRAS in the panel done as an initial test or to identify other treatment options.
New statement

13. Advances in molecular diagnostics
Mutation specific IHC for EGFR has been accepted in special setting.
Liquid biopsy as a primary assay for EGFR mutation is acceptable
IHC for ALK testing is adequate
Molecular testing beyond three!!
Multiple marker testing in a single experiment
Testing for resistance mutation in EGFR (T790M)- role of liquid biopsy

14. Single gene assays. Not tissue proficient.
Actionable mutation
Currently the best Methods to test those
Biomarker
Genetic aberration for testing
Most suited method
Alternate tests
EGFR
Mutation
ARMS
Sequencing/NGS/IHC
EML4-ALK
Rearrangement
IHC
FISH
Ros-1
IHC- D4D6. Lack specificity
C-Met
Amplification(CNG) --
Mutation- exon 14 skipping
NGS/c-DNA – amplicon- sequencing
HER2
Exon 20 mutation
RT PCR
NGS
BRAF
Pyrosequencing
NGS/ IHC- lacks both sensitivity and specificity
RET
Multiplex PCR/NGS
Single gene assays. Not tissue proficient.
BRAF V 600E and G469A, and D594G. V600E account for 50% of BRAF mutations.

15. NGS panels preferred over single gene tests
Results are quicker
Overcomes issue of sample availability
Enables expanded testing beyond EGFR, ALK, ROS1
Can help patients find appropriate treatment/ clinical trials.

17. Targeted NGS
“Oncomine Dx Target Test Panel”
All driver mutations are analyzed in 1 experiment.

22. Detect 104 actionable SNV and INDELS, 63 lung gene fusions, and up to 28 total and phospho-proteins
Low sample inputs; as little as 5 ng DNA, 50 ng RNA, and 250 ng protein or just 2 FFPE slides
Integrated SNV, Fusion, and protein measurements allow for simplified data analysis
Compatible with many sample types, including cell or tissue lysate and FFPE

23. Feature Specifications Number of Targets
63 probes: 35 for specific fusion detection, 24 for positional gene expression imbalance detection, and 4 internal reference genes.
Input Material
50–100 ng Purified Total RNA (~5000–10,000 cells) 150–300 ng FFPE extracted RNA
Sample Type(s)
FFPE, fresh frozen tissue, cell extracts, cell lysates
Species
Human
Time to Results
<24 hours
Data Analysis
nSolver™ Analysis software
Customize
Up to 24 additional genes or additional fusions

24. What else is new? Testing on progression- acquired resistance
Biomarker testing for immunotherapy

25. Testing on progression

26. Awareness is high.
Liquid Biopsy can be used.
If the plasma based test is negative a tissue biopsy need to be done
The only plasma based assay approved by FDA is Roche RT PCR test with LOD of 4%.
Most laboratories doing cf-NA based testing employ ddPCR wit LOD of .01% what to do with cases having a mutant allele fraction of < 2%
Treatment with TKI
Acquired resistance Secondary Mutation
Testing for secondary mutation - T790M
EGFR sensitizing mutation

27. TIGER-X summary: The liquid biopsies were in agreement with tissue assays for the T790M mutation more than 80% of the time. The tissue, plasma, and urine tests complemented each other, with each test identifying cases of T790M positivity missed by the other tests. The discordant tissue results may be attributed to tumor heterogeneity and/or inadequate biopsy sampling. Overall, these findings support the use of plasma and urine tests to detect EGFR mutations in patients with NSCLC, particularly when tumor tissue is not available or inadequate for testing.
Tiger x: Considering the tissue sample as the reference, sensitivity for detecting T790M mutation in plasma and urine was 80.9% and 81.1%, respectively. Response rates were similar in the T790M-mutant population irrespective of whether the status was identified in plasma, tissue, or urine

28. 180
120 New
60 PD
Dana Farber study
EGFR & KRAS analysis
Tissue/ CfNA by DDPCR
For T 790M
Sensitivity 77%
Specificity 63%
PPV – 79%
L858R, Del 19, KRAS
Sensitivity – 80% for EGFR
66% for KRAS
PPV-100%
Shorter TAT 3 Vs. 12
Plasma genotyping is capable of circumventing many limitations of standard tissue genotyping including
Slow turn around time (TAT)
Limited tissue for testing
Failed biopsies.
Prospective Validation of Rapid Plasma Genotyping for the Detection of EGFR and KRAS Mutations in Advanced Lung Cancer. Adrian G. Sacher, MD; Cloud Paweletz, PhD; Suzanne E. Dahlberg, PhD; Ryan S. Alden, BSc; Allison O’Connell, BSc; Nora Feeney, BSc; Stacy L. Mach, BA; Pasi A. Jänne,MD, PhD; Geoffrey R. Oxnard, MD. JAMA Oncol. doi: /jamaoncol

29. Tx Biopsy RT PCR Therascreen
Extended Validation comparing paired Tissue and Plasma samples against FDA approved Qiagen Therascreen
Sl No
Age/Sex
Tx Biopsy RT PCR Therascreen
dd PCR-Tx DNA
ddPCR- cf DNA
Primary Mutation
T790M
Primary 1
46/F
Del 19
Del 19 (45.3%, 23.9 copies/ul)
Negative
Del 19 (23.3%) 2
56/F
DNA not amplified, no wild and mutant droplets
Del 19 (0.8%) 3
48/M
Del 19 (44.4%, 0.62 copies/ul)
Del 19 (9.5%) 4
62/M
Del 19 (14.13%, 21.5 copie/ul)
Del 19 (0.28%) 5 6
65/M
Del 19 (51.02%, 631 copies/ul)
0.008%, 0.1 copies/ul
Del 19 (0.88%)
Positive (0.029%) 7
52/F
L858R
L858R (46%, 1.6 copies/ul) 8
65/F
Del 19 (8.34%, 7.1 copies/ul)
0.1%, 0.08 copies/ul 9
54/M
Del 19, T790M
Del 19 (124.7%, 176 copies/ul)
12%, 25.1 copies/ul
Del 19 (3.56%)
Positive (0.67%) 10
55/M
L858R (49.9%, 158 copies/ul)
0.02%, 0.09 copies/ul
L858R (7.5%)

30. Statistical evaluation of our data for L858R & del 19
Value
Sensitivity
90.48%
Specificity %
PPV
100.00%
N PV
75.00 %
For T 790 M
Concordance : 50%
Tested 70 samples on progression
43 of these are + for T790M- 60.2%

31. 2. To detect secondary mutation as cause of acquired resistance

32. ALK rearrangement
Treat with a cognate TKI
Acquired Resistance
The response to next generation of TKI is variable dependent on nature of genetic alteration
Is there a role for secondary mutation testing

33. ALK resistance On target mechanisms Off target mechanisms
L1196M……………7%
G1269A…………...4%
C1156Y……………2%
S1206Y……………2%
G1202R…………...2%
1151Tins…………..2%
E1210K …………...2%
ALK amplification…8%
EGFR
C-KIT
IGR1R
SRC
MEK/ERK
C1156Y (2%), G1202R (2%), I1171T (2%), S1206Y (2%), and
E1210K (2%).

35. Experimental. Evidence for Mutation testing not considered enough

36. Biomarkers for Immunotherapy
Checkpoint inhibitors
PD1- PDL1 axis is disrupted using anti PD1 or anti PDL1 Antibodies.

37. The existing biomarker test used is identifying expression of PDL1 on tumor cells & also in microenvironment.
Problems
Several assays on several platforms for different drugs
Clinical validity: PPV & NPV is low

38. NCCN guideline for PDL1 testing
Ist line - Pembrolizumab
Ist line - Pembrolizumab
Companion is Dako 22C3. 50% + cells
Following the Astra Zeneca and Blueprint study SP263 performed on Ventana platform has been given CE mark
2nd line Nivolumab or Atezolumumab
No Test
NCCN guideline for PDL1 testing
Companion is Dako 22C3. 50% + cells
Following the Astra Zeneca and Blueprint study SP263 performed on Ventana platform has been given CE mark
2nd line Nivolumab or Atezolumumab
No Test

39. Media Release
Roche’s VENTANA PD-L1 (SP263) Assay gains CE label expansion to inform treatment decisions in lung cancer patients being considered for KEYTRUDA (pembrolizumab) immunotherapy
Lung cancer remains the leading cause of cancer deaths, with nearly 1.6 million deaths worldwide.
PD-L1 is a protein involved in the suppression of the immune system, which can impact the body’s ability to fight cancer.
The VENTANA PD-L1 (SP263) Assay1 is now available for countries accepting the CE mark to identify untreated and previously treated metastatic non-small cell lung cancer (mNSCLC) patients eligible for KEYTRUDA immunotherapy.

40. An effort is on to improve biomarker testing for immunotherapy- Planned through
Total Mutation Burden
Immune gene profiling from tumor tissue
Looking for BRCA1- 2, POLE, POLD mutations and MSI in a tumor.
Multiplexing of IHC

41. Conclusions
Major advances
Rationalization of molecular testing
Testing beyond 3 approved target is encouraged and many physicians practice it.
Multiplexed, multi gene sequencing is being advocated.
Liquid biopsy is a new tool
For secondary mutation detection in patients progressing on 1st and 2nd line EGFR TKIs
For primary predictive biomarker testing..
Immunotherapy biomarker testing is being reorganized.

42. Thank you for your patience

43. Commercially available assays for ROS1 testing
FISH fluorescence in situ hybridisation, IHC immunohistochemistry, IVD in vitro diagnostic, NGS next-generation sequencing, RT-PCR reverse transcription polymerase chain reaction, RUO research use only
Method
Manufacturer
Reagent
Regulatory status
FISH
Cytocell
ROS1 Dual Color Break Apart Probe
CE-IVD
ZytoVision/Zytomed
ZytoLight SPEC ROS1 Dual Color Break Apart Probe
Abbott
ROS 1 Break-Apart FISH
RUO
IHC
Cell Signaling Technologies
ROS1 D4D6 rabbit monoclonal antibody
RT-PCR
AmoyDx
ALK and ROS1 gene fusion detection kit
NGS
Thermo Fisher
Oncomine Fusion panel (ALK, ROS1, RET and NTRK1)
ArcherDx
FusionPlex™ ALK, RET, ROS1 v2 Panel