Clinical Integration of NGS for ALK Fusions Identification in NSCLC: �Laboratory Protocols �and Practices

Clinical Integration of NGS for ALK Fusions Identification in NSCLC: �Laboratory Protocols �and Practices
Test Early: ALK status guides first-line TKI selection.

Broad Profiling: NGS preferred to capture ALK fusions and co-mutations.

Liquid Biopsies: ctDNA for monitoring resistance.

Next-Gen TKIs: Overcoming resistance (e.g., lorlatinib).

1. Clinical Integration of
NGS for ALK Fusions
Identification in
NSCLC:
Laboratory Protocols
and Practices at KCCC
Dr.Amir Abdelazim , MD
Biochemical and molecular pathology
[email protected]

2. Content:
•Introduction about ALK gene and laboratory tests
•Situation analysis for NGS lab in KCCC
•Guidelines Recommendations
•Mechanisms for resistance to ALK Inhibitors

3. Introduction

4. ALK fusion
Anaplastic Lymphoma receptor tyrosine Kinase= (ALK)gene
Rearrangements Prevalence: 3–7% of NSCLC (higher in young, non-
smokers, adenocarcinoma).
ALK inhibitors (ALKIs) have shown significant efficacy benefits in ALK-
positive advanced NSCLC.cer (NSCLC)
The availability of accurate and rapid ALK testing methods is
critical to identify patients suitable for ALKIs.
rareALKfusion subtypes

and acquired ALKI-resistance
EML4-ALK fusion (chromosome 2p) → constitutive ALK kinase activation → oncogenesis.

5. ALK Testing Methods
DNA-level fluorescencein situhybridization (FISH) to detect
translocations (break-apart probes).
Quantitative real-time reverse transcription-polymerase chain
reaction (qRT-PCR) to detect fusion mRNA
Immunohistochemistry (IHC) to detect fusion protein expression
Next-generation sequencing (NGS) to detect mRNA-level fusion
sequences
•NGS Advantages:
•Multiplex profiling (ALK + ROS1, RET, etc.), identifies
novel partners, detects co-mutations.
•Pre-Analytical:
•Tissue scarcity (FFPE vs. liquid biopsy validation).
•Technical:
•NGS limitations (RNA degradation, false negatives in low
tumor content).
•Interpretative:
•Variants of unknown significance (VUS

6. (FISH vs. IHC vs. NGS)

7. "High Throughput Sequencing for millions of reads ".
➢"Faster Sequencing Rate" And "Cost Effective" Procedure.
➢Capability to sequence multiple individuals at the same
time.
➢Providing high depth to deliver accurate data and an
insight into unexpected DNA variation

➢Captures a broader spectrum of mutations than Sanger
sequencing
Potential for discovery of novel actionable targets.
Improved turn-around time by avoiding sequential testing
Tissue preservation - many genes simultaneously assessed from single extraction
Why ?NGS…..
•False positives/negatives –
sequencing errors can affect
variant calling.
•Distinguishing germline vs.
somatic mutations requires
additional filtering.
•Tumor heterogeneity – sampling
bias can miss subclonal
mutations.
•Data interpretation complexity
– bioinformatics pipelines are
evolving.
Challenges

8. inv(2)(p21p23) EML4/ALK

9. NGS lab in KCCC

10. What we have in KCCC lab for Lung cancer
• Low limit of detection0.1%
52 genes
•Steps:
•DNA/RNA extraction (tissue/liquid biopsy).
•Library preparation (hybrid capture/amplicon).
•Sequencing (targeted panels).
•Bioinformatics analysis (fusion callers).

11. What ? We have in KCCC lab.

12. What ? We have in KCCC lab.
• Low limit of detection—variant detection down to 0.1%
52 genes
ctDNA are fragments of DNA that are released into the blood
from apoptotic tumor cells
This advance is particularly
important to the estimated 20%
of patients who undergo a
successful biopsy but who are
unable to yield enough tissue to
perform molecular analysis
ctDNA may also have utility in the detection of minimal residual disease and may help
improve diagnostics and prognostication
•Plasma testing acceptable but confirm negatives with tissue.

13. DNA-NGS
RNA-NGS
MSI-qPCR
Report
example

14. Liquid Biopsy and Circulating Tumor DNA
(ctDNA)
•Advantages:
•Non-invasive, real-time monitoring,
captures heterogeneity.
•Limitations: Lower sensitivity
• in low tumor burden.
•Clinical Use:
•Detect resistance mutations
•Monitor treatment response.
Tissue testing seems likely to remain first-line,
although there are great possibilities for liquid
biopsy for surveillance
NGS, next-generation sequencing.
1. Liam CK, et al. Respirology 2020;25:933–943; 2. Tulpule A and Bivona TG, Annu Rev Cancer Biol 2020;4:279–297; 3. Smolle E, et al. Cancers (Basel) 2021;13:699; 4. Mellert H, et al. Diagnostics (Basel) 2021;11:155; 5. Chen Y, et al. Cancer Manag Res 2017;9:801‒811.
Liquid biopsy is important in the assessment of ALK resistance
Tissue biopsies may yield false-negative results as they are limited by tumour
heterogeneity, particularly in the setting of disease resistance

15. Statistic

16. Lung cancer cases received 2022-2024
27435 56
Tissue biopsy Liquid biopsy
TP53
KRAS
EGFR
FAILED
(SEE
COMM
ENT)STK11
SMARC
A4CDK4MDM2PIK3CAEML4(1
3) -
ALK(20)
MYC
ARID1A
negativ
e
NF1
TERT
ERBB2
MET
ROS!-
rearran
gment
CTNNB
1
BRAF
FGFR1
RB1
SETD2
PTEN
CCND1CCNE1
RET
fusions
CDKN2
A
FGF19CREBB
P
FGF3SMAD4U2AF1
ATR
NOTCH
3
PMS2ATMBAP1CCND3FGFR3
MET(13
) -
MET(15
)
MSH6POLERICTORSTAT3AKT1AKT2ATRXAXLBRCA1BRCA2CDK12CDKN2
B
ERBB2(
HER2)
FANCAHRASKITMAP2K
1
MLH1MYCNNTRK1PALB2PDGFR
A
PIK3CBPIK3R1RAD51
D
SMARC
B1
AKT3BRD4(1
1) -
NUTM1
(2)
CBLCCND2CDK2CDK6CDKN1
B
CSKN2
A
EGFR
[Exon
19
deletio
n]
ERBB3ESR1FANCD
2
FANCIFBXW7FGFR2FLT3IDH1JAK2KDRMRE11MTORMYBL1(
14)-
NFIB(9)
MYCLNBNNF2NFE2L2NOTCH
1
NRASNTRK3PPARGRAD51
C
RHOARNF43SEC61G
(2) -
EGFR(9
)
SLX4TSC1

17. Distribution of driver mutation in lung tissue
biopsy done 2022-2024 (309 cases) by NGS
KRAS
EGFR
ALK
ERBB2
MET
ROS!-rearrangment
BRAF
RET fusions
other
KRAS 26.5%
EGFR 21.7%
ALK 4.5%
ERBB2 3.9%
MET 3.9%
ROS1 3.9%
BRAF 3.2%
RET 2.3%
other 30.1%
Actionable Genes in
NSCLC
EGFR, KRAS, HER2, PIK3CA,
ALK, BRAF, ROS1, RET, MET,
and NRAS
ALK fusions :
4.5%

18. Distribution of driver mutation in lung Liquid
biopsy done 2023-2024 (91 cases) by NGS
Actionable Genes in
NSCLC
EGFR, KRAS, HER2, PIK3CA,
ALK, BRAF, ROS1, RET, MET,
and NRAS
KRAS
EGFR
ALK
ERBB2
MET
ROS1
BRAF
RET
other
KRAS 18
EGFR 32
ALK 1
ERBB2 2
MET 10
ROS1 1
BRAF 3
RET 0
other 24

19. Guidelines Recommendations

20. Timeline of the development of ALK
inhibitors in advanced NSCLC
Chemotherapy
driven by NSCLC
subtype
1,2
2L ceritinib
approved
1
2L alectinib
approved
1
2L
brigatinib
approved
1
1L alectinib
approved
1
2
nd
line
Lorlatinib
approved
1
1L
brigatinib
approved
5
1
st
line
Lorlatinib
approved
1
EML4-ALK
translocation
discovered
1
Crizotinib
approved
1
2002 2007 2011 2014 2021201720182020 2024
Ensartinib
2015

21. Clinical Guidelines for ALK Testing
NCCN/ESMO/ASCO
Recommendations:
•Test all advanced NSCLC
(adenocarcinoma/ mixed
histology).
•Prioritize NGS for
comprehensive profiling.
Guideline Update (v3.2025):
Ensartinib upgraded
•1st Gen: Crizotinib
•2nd Gen: Alectinib, Ceritinib, Ensartinib
•3rd Gen: Lorlatinib
Guidelines recommend that molecular
testing should be conducted at the
time of diagnosis and tumor
progression
on targeted therapy

22. Resistance to ALK Inhibitors

23. ALK Signaling Pathway
ALK Fusion Protein(e.g., EML4-ALK):
•Acts like a "stuck accelerator," constantly
sending growth signals.
•Activates Pathways:
•MAPK: Promotes cell division.
•PI3K/AKT: Supports survival.
•STAT3: Drives tumor growth.

24. ALK inhibitor
Resistance:
1.ALK Mutation (new lock shape) → Drug can’t bind.
2. New Pathway (e.g., EGFR) → Bypasses ALK.
ALK Fusion → "Accelerator On" → MAPK/PI3K/STAT3 → Cancer Growth
ALK Inhibitor → Blocks Accelerator → Stops Growth

25. Resistance to ALK Inhibitors
A. On-Target Resistance
•Mutations in ALKchange the "lock" so drugs can’t bind.
•Common Mutations:
•Crizotinib: L1196M (gatekeeper), G1269A.
•Alectinib: G1202R (common), I1171T.
•Lorlatinib: Targets G1202R but may fail againstcompound
mutations(e.g., G1202R + L1196M).
B. Off-Target Resistance
•Alternative Pathwaystake over (e.g., EGFR, MET, KRAS).
•Example: Tumor cells use EGFR instead of ALK to grow.
C. Other Mechanisms
•Tumor heterogeneity: Some cells resist treatment from
the start.(including both ALK dependent and independent
pathways)

26. On-Target Resistance

27. Q1064R
L1122V
P1139S
T1151dup
T1151K
T1151M
L1152P
L1152R
C1156F
I1171S
I1171T
N1178H
V1180L
L1196Q
L1198P
D1203N
S1206C
S1206R
S1206Y
E1210K
G1269S
M1328I
Resistance
InconclusiveG1202R
L1198PM
I1171TN
C1156Y
G1269A

28. Gatekeeper

33. A double L1196M and G1202R mutation
was identified at a frequency of 1.4%

34. A double L1196Q and G1202R mutation
was identified in this report

35. Off-Target Resistance

36. Off-target resistance mechanisms and treatment
options in ALK-positive NSCLC

38. ALK fusions /Variants

39. ALK Fusion Partners : >90 Variants
1.Common: EML4 (~85% of ALK+ NSCLC).
2.Rare: KIF5B, TFG, STRM, HIP1, PTPN3.
3.Very Rare: DCTN1, CLTC, TPR, SQSTM1, BIRC6.
•Most non-EML4 fusions retain ALK kinase domain → respond to ALK TKIs.
SQSTM1-ALK: Unique
breakpoint → resistance
to 1st-gen TKIs but
response to lorlatinib.

40. EML4-ALK isoforms
(potential prognostic differences).
•Breakpoints & Prevalence in KCCC:
3
11
Variant 1Variant 3
14 cases
2022-2024

41. Impact of Variant Structure on ALK Protein
Variant 1 (E13;A20) and Variant 2 (E20;A20) :
•Long truncation of EML4 → stable protein → good response to 1st-gen TKIs
(e.g., crizotinib, alectinib).
Variant 3 (E6;A20):
•Short truncation → unstable protein → higher oncogenic potential and
resistance to crizotinib better with 2
nd
-gen (alectinib, brigatinib) and 3
rd
-gen.

42. Variant 3
Example
(E6;A20)

43. Case study

44. Case study :ALK-positive NSCLC progressing
2022
Tissue
2023
LB

45. Recommended Actions:
1.Confirmatory Testing:
1.Performtissue re-biopsyto assess spatial
heterogeneity
2.Target MET Amplification:
1.MET Inhibitors: Capmatinib, tepotinib, or
crizotinib (dual ALK/MET inhibitor).
2.Combination Therapy: MET inhibitor +
next-gen ALK inhibitor (e.g., lorlatinib) if
residual ALK dependency is suspected.
Interpretations:
1.Resistance Mechanisms:
1.MET Amplification: MET activation sustains tumor
growth independently of ALK signaling.
2.TP53 Mutation: Associated with aggressive and
genomic instability. May contribute to therapeutic
resistance.
2.Why ALK Fusion Was Not Detected:
1.Tumor Heterogeneity: Subclones without the ALK
fusion may dominate due to selective pressure from
prior ALK inhibition.
2.Low ctDNA Shedding: The ALK-positive clone may not
release sufficient ctDNA into circulation for detection.
3.Clinical Implications:
1.MET-driven progression: The tumor is likely relying on
MET signaling, rendering ALK inhibitors ineffective.
2.Loss of ALK Dependency: The ALK fusion may no longer
be the primary driver.

46. Summary & Conclusion

47. Treatment Algorithm for ALK-
Rearranged Advanced NSCLC
1.First-Line Therapy
1.Preferred Options:
1.Alectinib, Brigatinib, or Lorlatinib
2.Alternative Options:
1.Certitinib or Crizotinib (if next-gen TKIs unavailable).
2.Upon Progression:
1.Performre-biopsy (tissue/liquid)to assess resistance
mechanisms.
1.ALK resistance mutations detected:
Switch toLorlatinib(3rd-gen TKI; targets most resistance
mutations).
2.No ALK mutations:
Options: Lorlatinib, chemotherapy
3.CNS Progression:
1.Asymptomatic: Continue current TKI + consider local therapy
(SRS/surgery).
2.Symptomatic: Switch to a TKI with better CNS penetration (e.g.,
Lorlatinib, Alectinib).
4.Post-Lorlatinib Progression:
1.Chemotherapy ± immunotherapy (e.g., platinum/pemetrexed ±
atezolizumab).
2.Enroll inclinical trials(e.g., novel TKI combinations,
immunotherapy).?

48. Conclusion
1.Test Early: ALK status guides first-line TKI selection.
2.Broad Profiling: NGS preferred to capture ALK fusions and co-mutations.
3.Liquid Biopsies:ctDNA for monitoring resistance.
4.Next-Gen TKIs:Overcoming resistance (e.g., lorlatinib).
3rd-Generation ALK Inhibitors – Key points //Lorlatinib
1.TargetsALK resistance mutations(e.g., G1202R, L1196M, F1174L).
2.Not effective againstcompound mutations(e.g., G1202R + L1196M).
3.Active against most EML4-ALK variants (V1, V2, V3) and rare partners (e.g., STRM-ALK).
4.High blood-brain barrier penetration → superior control of brain metastases

49. Thanks