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									                                       Comparison of IHC, FISH and RT-PCR for the Detection of EML4-ALK
                                             Translocation Variants in Non-Small Cell Lung Cancer
                                                                         Michelle L.     Wallander 1,                    Katherine B.                       Geiersbach 2,                       Sheryl           Tripp 1           and Lester J.            Layfield 2

                                                                    1ARUP   Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT
                                                    2Department          of Pathology, University of Utah Health Sciences Center and ARUP Laboratories, Salt Lake City, UT



Introduction                                                                  Results                                                                                                                                                                                Results
                                                                                                                                                                                   #28: FISH Positive
Lung cancer is the most common and deadly form of cancer in the                                                                       #28: IHC Positive (2+)                     (Avg. 70% Rearranged)
                                                                                                                                                                                                                                                                      ALK protein expression was detectable by IHC in 9 of 52 (17%)
United States with a 5-year survival rate of only 15.7%. Recently, it                                                                                                                                                                                                samples.
has become apparent that non-small cell lung cancer (NSCLC) can                                                                                                                                                                                                       ALK break apart FISH was positive in 4 of 52 (8%) samples.
be further divided into clinically relevant subsets based on specific
molecular alterations within the tumor. In 2007, Soda et al identified                                                                                                                                                                                                EML4-ALK variant 3a/b RT-PCR was positive in 2 of 52 (4%) of
a small chromosome 2p inversion (~12 Mb) in a subset of Japanese                                                                                                                                                                                                     samples.
NSCLC patients, which resulted in the fusion of the echinoderm
                                                                                                                                                                                                                                                                      EML4-ALK variant 1 RT-PCR was positive in 9 of 52 (17%) of
microtubule-associated protein-like 4 (EML4) gene and the                                                                                                                                                                                                            samples.
anaplastic lymphoma kinase (ALK) gene. Multiple different EML4-                                                            EML4-ALK Variant 3a/b RT-PCR                                  MRPL19 RT-PCR
ALK translocation variants have since been identified that contain                                                                                                                                                                                                    The total incidence of EML4-ALK translocations in our NSCLC
various truncations of EML4 but always contain the tyrosine kinase                                                       V3a/b V3a/b                                             V3a/b V3a/b                                                                         series, which was enriched for adenocarcinomas with wild-type
                                                                                                                          Pos   Neg      #28        #28     NTC                   Pos   Neg     #28     #28     NTC
domain of ALK, starting at exon 20. The most common (~70%)                                                                                                                                                                                                           EGFR status, was 21.1% (11 of 52).
translocations in NSCLC involve EML4 exon 13 (variant 1) or EML4                                                                                                     -500 bp-
                                                                                                                                                                                                                                                                      Concordance between IHC, FISH and RT-PCR methodologies
exon 6 (variant 3a/b; 3b contains an additional 33-bp due to                                                                                                                                                                                                         was 100% for EML4-ALK variant 3a/b (2 of 2 samples).
alternative splicing).
                                                                                                      V3b (138 bp)-
                                                                                                                                                                                                                       -MRPL19 (94 bp)
                                                                                                                                                                                                                                                                      No concordance was found between IHC, FISH and RT-PCR
The incidence of EML4-ALK translocations in NSCLC is low (2-7%)                                       V3a (105 bp)-                                                  - 50 bp -                                                                                       methodologies for EML4-ALK variant 1 (0 of 9 samples).
but can be higher (13%) if the population is enriched for
adenocarcinoma, never or light smokers, younger age and lack of                                                                                                                                                                                                           FISH and V1 RT-PCR concordance: 11% (sample # 51)
EGFR or KRAS mutations. Early-phase clinical trials of ALK
                                                                                                                Sample     EGFR         ALK IHC
                                                                                                                                                          ALK FISH    ALK FISH ALK FISH
                                                                                                                                                                                                 Avg FISH
                                                                                                                                                                                                                 EML4-ALK                                                 IHC and V1 RT-PCR concordance: 11% (sample # 39)
inhibitors in patients with known EML4-ALK translocations have                                                                                            Reader 1    Reader 2 Reader 3                         V3a/b RT-PCR
yielded promising results. Therefore, a need exists for molecular                                                # 16      wildtype      positive           86%          50%         74%       70% (positive)     positive
                                                                                                                                                                                                                                                                      One EML4-ALK variant 1 positive case (sample # 18) had a
testing to identify the correct target patient population. We                                                    # 28      wildtype      positive           70%          73%         67%       70% (positive)     positive                                           concurrent EGFR mutation (L858R).
compared ALK immunohistochemistry (IHC), ALK break apart FISH
                                                                              Detection of EML4-ALK variant 3a/b by IHC, break apart FISH and RT-PCR. All 3 methodologies are shown for one representative                                                            EML4-ALK was not detected by RT-PCR in normal tissue (lymph
and RT-PCR in a subset of lung adenocarcinomas for the detection
                                                                              positive sample (#28). Two of two (100%) variant 3a/b positive samples displayed loss of the green probe by FISH.                                                                      node or lung) from 4 RT-PCR positive samples that had sufficient
of EML4-ALK variants 1 and 3a/b.                                                                                                                                                                                                                                     tissue for analysis (data not shown).

Materials and Methods                                                         Results                                                                                                                                                                                Conclusions
                                                                                                                                                                                                      EML4-ALK Variant 1 RT-PCR
 52 FFPE lung adenocarcinomas were selected from the University                                                                #51: FISH Positive                                                                                                                   Concordance among the three methodologies (IHC, FISH and RT-
                                                                                        #51: IHC Negative (0)                 (Avg. 74% Rearranged)                                      V1       V1
of Utah surgical pathology files                                                                                                                                                                                                                                     PCR) differed for the two tested EML4-ALK translocation variants.
                                                                                                                                                                                         Pos     Neg      #51    #51         #30    #30    NTC
                                                                                                                                                                                                                                                                     Variant 3a/b was detectable by all three methods. Conversely,
 The study population was enriched for wildtype EGFR status as                                                                                                                                                                                   -500 bp            limited ALK protein expression and subjectivity in FISH scoring
determined by direct sequencing of exons 18-21 (WT: n = 46;                                                                                                                                                                                                          resulted in no concordance between all three methods for the
Mutant: n = 6).                                                                                                                                                                                                                                                      detection of EML4-ALK variant 1. Agreement among FISH readers
 ALK IHC: monoclonal mouse CD246, clone ALK1 (Dako)                                                                                                                      V1 (109 bp)-
                                                                                                                                                                                                                                                  - 50 bp
                                                                                                                                                                                                                                                                     was poor for variant 1. This was likely due to the expected small
                                                                                                                                                                                                                                                                     split in probe signals. These results suggest that the detection of
 ALK FISH: Vysis LSI ALK Dual Color, Break Apart Rearrangement                                                                                                                                                                                                      other EML4-ALK variants that involve the 3’ region of EML4 (exons
Probe (Abbott Molecular)                                                                                                        #30: FISH Negative                                                        MRPL19 RT-PCR                                              15-20) may also be challenging by FISH.
                                                                                        #30: IHC Negative (0)                 (Avg. 18% Rearranged)
    Positive cutoff: ≥ 40% cells rearranged                                                                                                                                             V1      V1                                                                  RT-PCR was the most sensitive and least subjective methodology
                                                                                                                                                                                         Pos    Neg      #51     #51    #30        #30    NTC                        for EML4-ALK variant 1 detection. While multiple specific primer
 RT-PCR:
                                                                                                                                                                                                                                                 -500 bp             sets would need to be designed for the detection of all known
    Variant 1 primers (109 bp)                                                                                                                                                                                                                                      EML4-ALK translocation variants from FFPE tissue by RT-PCR, this
      EML4 Ex13 Fwd: 5’-TAGAGCCCACACCTGGGAAA-3’                                                                                                                                                                                                                     approach would likely yield the greatest assay sensitivity.
       ALK Ex20 Rev: 5’-CGGAGCTTGCTCAGCTTGTA-3’                                                                                                                      MRPL19 (94 bp)-
                                                                                                                                                                                                                                                 -50 bp              Additionally, our results confirm that the incidence of EML4-ALK
                                                                                                                                                                                                                                                                     translocations in NSCLC is greater (21%) in enriched patient
   Variant 1 positive control: FFPE HEK293 cells transiently                                                                                                                                                                                                        populations.
  expressing pcDNA3-EML4-ALK variant 1
                                                                                                                                                          ALK FISH    ALK FISH ALK FISH                         EML4-ALK V1
    Variant 3a/b primers (105 bp & 138 bp)
                                                                                                                Sample     EGFR         ALK IHC
                                                                                                                                                          Reader 1    Reader 2 Reader 3
                                                                                                                                                                                                 Avg. FISH
                                                                                                                                                                                                                  RT-PCR                                             References
                                                                                                                 # 14      wildtype     negative             7%          10%         25%       14% (negative)     positive
       EML4 Ex6 Fwd: 5’-GCATAAAGATGTCATCATCAACCAAG-3’                                                           # 18       L858R       negative            16%          44%         20%       26% (negative)     positive                                           Soda M, Choi YL, Enomoto M et al. Identification of the transforming EML4-ALK fusion gene in
       ALK Ex20 Rev: 5’-CGGAGCTTGCTCAGCTTGTA-3’                                                                  # 30      wildtype     negative             4%          32%         19%       18% (negative)     positive                                           non-small cell lung cancer. Nature, 2007; 448:561-566.
                                                                                                                                       weak patchy                                                                                                                   Shaw AT, Yeap BY, Mino-Kenudson M et al. Clinical features and outcome of patients with non-
                                                                                                                 # 39      wildtype                         6%            6%         23%       11% (negative)     positive
                                                                                                                                         positive                                                                                                                    small-cell lung cancer who harbor EML4-ALK. J Clin Oncol, 2009; 27:4247-4253.
    Variant 3a/b positive control: H2228 cells
                                                                                                                 # 42      wildtype     negative            12%          50%         7%        23% (negative)     positive                                           Kwak EL, Bang YJ, Camidge DR et al. Anaplastic lymphoma kinase inhibition in non-small-cell
    MRPL19 primers (94 bp)                                                                                      # 47      wildtype     negative             9%          35%         11%       18% (negative)     positive                                           lung cancer. N Engl J Med, 2010; 363:1693-1703.
                                                                                                                 # 50      wildtype     negative            11%          39%         19%       23% (negative)     positive
       MRPL19 Ex4 Fwd:5’-GGAAGAGGACTTGGAGCTACT-3’                                                               # 51      wildtype     negative            84%          52%         87%       74% (positive)     positive
       MRPL19 Ex5 Rev: 5’-TCCTGGACCCGAGGATTAT-3’                                                                 # 57      wildtype     negative            17%          22%         30%       23% (negative)     positive                                           Acknowledgements
                                                                                                                                                                                                                                                                     .

 The use of human tissue for this analysis was approved by the               Detection of EML4-ALK variant 1 by IHC, break apart FISH and RT-PCR. All 3 methodologies are shown for two variant 1 positive                                                          We graciously thank Maria Martelli and Brunangelo Falini for providing controls.
University of Utah Institutional Review Board (#22487).                       samples, as determined by FISH/RT-PCR (#51) and RT-PCR (#30). The remaining variant 1 data is summarized in the table.

								
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