Author of

• 11866

  +

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

  • Event: WCLC 2013
  • Type: Poster Session
  • Track: Pathology
  • Presentations: 1
  • Moderators:
  • Coordinates: 10/29/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 11883

    +

    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): M. Adaway
    • 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.