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W. Newman



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    P1.18 - Poster Session 1 - Pathology (ID 175)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Pathology
    • Presentations: 1
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      P1.18-015 - Screening for ALK-rearranged NSCLC in selected cases using immunohistochemistry followed by FISH and RT-PCR testing of tumours with increased ALK protein expression in a routine clinical diagnostic setting (ID 2838)

      09:30 - 09:30  |  Author(s): W. Newman

      • Abstract

      Background
      The diagnosis of anaplastic lymphoma kinase (ALK) gene rearrangement in non small cell lung cancer (NSCLC) has acquired therapeutic significance, subsequent to the established response of ALK-rearranged tumours to crizotinib therapy. General recommendations on NSCLC ALK testing will be published later this year by the National Institute for Health and Care Excellence. In advance of this, patients were prospectively screened for ALK-rearranged NSCLC at the Christie Hospital, Manchester, U.K. from May 2012 to May 2013.

      Methods
      Pulmonary adenocarcinomas were selected for testing by ALK immunohistochemistry (IHC) based the presence of any of the following clinicopathologic features associated with ALK rearrangement; never smoker, light ex-smoker, age less than 50 years, signet ring/goblet cell morphology. IHC was performed with the 5A4 clone (Novocastra) according to the European Thoracic Oncology Platform protocol. All IHC-positive cases (intensity score 1+, 2+ or 3+) were tested for ALK rearrangement by both fluorescent in situ hybridisation (FISH) and reverse transcriptase polymerase chain reaction (RT-PCR). FISH analysis using the Abbott Molecular LSI ALK Dual Colour Break Apart Probe required a minimum of 15% of (at least 100) tumour cells with gene rearrangement for a positive diagnosis. RT-PCR testing was employed to detect EML4-ALK fusion transcripts using a series of primers located in EML4 exons 1 to 22, a reverse primer located in ALK exon 20 (Sanders et al., 2011;204:45-52) and sample RNA extracted from a single 40 µM section. Amplified products were Sanger sequenced to establish the fusion variant present.

      Results
      Ninety-one specimens were screened by ALK IHC and of these, 13 demonstrated positive staining. FISH and RT-PCR results were concordant (with the exception of one RT-PCR negative case which failed FISH testing) and 9 cases were diagnosed with ALK-rearrangement (9.9%). The majority of the EML4-ALK fusion transcripts were of variant 1 type (77.8%), with just two subtypes diagnosed as variant 3 (22.2%). The median time from referral for FISH/RT-PCR to the issue of reports was 5 working days.

      Table 1. Summary of clinicopathological features, IHC, FISH and RT-PCR results of cases positive for ALK protein staining on IHC. (ACA =adenocarcinoma)
      Case Age Sex Sample type Histology IHC H-score FISH % + RT-PCR Final ALK diagnosis EGFR mutation
      1 84 F Node excision ACA, signet ring cells 170 55 E13;A20 variant 1 + -
      2 59 M Lung resection ACA, solid with hepatoid cells 190 77 E13;A20 variant 1 + -
      3 56 M Pleural effusion ACA, hepatoid cells 240 64 E13;A20 variant 1 + -
      4 46 M Node biopsy Adenosquamous 300 64 E6;A20 variant 3 + -
      5 64 M Pleural biopsy ACA, solid with hepatoid cells 300 48 E13;A20 variant 1 + -
      6 60 F Node aspirate ACA, signet ring and hepatoid cells 300 66 E13;A20 variant 1 + -
      7 41 F Node biopsy ACA, hepatoid cells 300 71 E13;A20 variant 1 + -
      8 40 M Pleural biopsy ACA, solid with hepatoid cells 300 58 E6;A20 variant 3 + -
      9 65 F Node aspirate ACA, signet ring and hepatoid cells 300 45 E13;A20 variant 1 + -
      10 54 M Pleural effusion ACA 20 5 Negative - -
      11 52 F Pericardial effusion ACA 10 Failed Negative - -
      12 49 F Pleural fluid ACA 35 0 Negative - +
      13 70 F Lung resection ACA, solid with hepatoid cells 54 9 Negative - Unknown

      Conclusion
      In keeping with reported findings ALK-rearranged NSCLC was found in 9.9% of selected adenocarcinomas. Although FISH/RT-PCR was not carried out on IHC-negative cases in this group, the application of IHC as a screening method appears to be a cost-effective means of highlighting ALK-rearranged tumours. RT-PCR testing of formalin-fixed, paraffin-embedded tissue is feasible in the clinical diagnostic setting, and may have an important role in the determination of specific variants detected by IHC.

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    P2.18 - Poster Session 2 - Pathology (ID 176)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Pathology
    • Presentations: 1
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      P2.18-017 - Multiplexed-based mutation profiling of non small cell lung cancer small biopsy samples using the Sequenom LungCarta™ Panel and MassARRAY® System (ID 2856)

      09:30 - 09:30  |  Author(s): W. Newman

      • Abstract

      Background
      The advent of specific therapies for non small cell lung cancer (NSCLC) based on individual tumour genotype has impacted the development of high throughput genomic profiling strategies. A single platform designed for the synchronous screening of multiple mutations across different genes can potentially enable molecular profiling in samples of limited tumour tissue such as small biopsy samples.

      Methods
      Haematoxylin and eosin-stained sections and accompanying reports were reviewed from patients diagnosed with NSCLC (2008 to 2012) in Greater Manchester, U.K. Samples with less than 20% tumour cell content (TCC) were macrodissected to increase the final TCC. In each case DNA was extracted manually from 1 5µM curl/section using the cobas® DNA Sample Preparation Kit. Mutation analysis was performed with the Sequenom LungCarta™ Panel which enables screening of 214 mutations in 26 genes, and utilises multiplexed polymerase chain reactions, single base extension reactions and mass spectrometry (Sequenom MassARRAY® platform).

      Results
      Results Sixty cases comprising 47 lung biopsies, 1 wedge resection, 6 lymph node biopsies, 4 pleural biopsies, 1 brain biopsy and 1 pericardial effusion were classified as 21 adenocarcinomas (ACA), 17 squamous cell carcinomas (SCC), 8 NSCLC favour ACA, 10 NSCLC favour SCC, 1 adenosquamous carcinoma and 3 NSCLC not otherwise specified (NOS). Mutations were successfully detected at a mutant allele frequency of 10% and definite mutations were reported in 28 cases (47%). Possible mutations of low allele frequency or uncertain significance were detected in an additional 15 cases (25%) and also in 10 cases with a definite mutation. In total 32 definite and 39 equivocal mutations have been detected and are currently being validated by a combination of pyrosequencing, next-generation sequencing and immunohistochemistry (IHC).

      Table 1. Unequivocal mutations detected according to histological subtype. ([a]Includes double mutant; TP53 and MAP2K1, [b]includes triple mutant; 2 TP53 and 1 KRAS, [c]includes double mutant; KRAS and MET)
      No. of definite mutations detected No. of mutated samples ACA NSCLC favour ACA SCC NSCLC favour SCC NSCLC NOS % of mutations detected in all ACA or SCC Comment
      13 TP53 12 3[a] 2[b] 5 1 1 17 % ACA 22% SCC 1 confirmed by next generation sequencing. 7 of 8 tested cases were strongly positive for P53 IHC
      12 KRAS 12 8[c] 1[b] 3 31% ACA 11% SCC 7 confirmed by pyrosequencing
      3 MET 3 2[c] 1 10% ACA 0% SCC 1 confirmed by next generation sequencing
      2 EGFR 2 2 7% ACA 0% SCC 2 previously detected by Sanger sequencing
      1 EPHA5 1 1 0% ACA 4% SCC Moderately differentiated SCC
      1 MAP2K1 1 1 3% ACA 0% SCC Poorly differentiated ACA TTF1+

      Conclusion
      The MassARRAY® system of testing for multiple mutations is a sensitive method that facilitates the optimal use of tumour DNA present in small specimens, and can detect concurrent mutations with the potential to influence responses to targeted therapies. Unequivocal mutations were reported in 59% and 37% of cases diagnosed/favoured as ACA and SCC respectively. This may reflect the LungCarta™ panel design, which was based on mutations detected in ACA.