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C. Deshpande



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    P2.06 - Poster Session 2 - Prognostic and Predictive Biomarkers (ID 165)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.06-050 - Validation and Utilization of a Clinical Next Generation Sequencing Assay to identify Mutations and Genomic Copy number changes in Lung Cancer (ID 3419)

      09:30 - 09:30  |  Author(s): C. Deshpande

      • Abstract

      Background
      Recent advances in next generation sequencing provide us with the ability to simultaneously analyze both mutation status and copy number changes across a large number of cancer related genes. Here we discuss our experience in developing, validating and applying a 47 gene panel across our lung tumor patient population in a clinical laboratory.

      Methods
      Genomic DNA was extracted from 20 Formalin Fixed Paraffin embedded (FFPE) tissue samples from lung tumors for validation and over 80 patient samples upon clinical implementation. Extracted DNA was analyzed for quality and amplifiable quantity with suitable performance criteria established during validation. Between 10 and 250 ng of total genomic DNA was then subjected to the Truseq Amplicon (Illumina) assay for enrichment of the target regions across 47 different genes. Libraries were then sequenced on the Miseq (Illumina) to an average depth of coverage between 2000 and 4000x with 186 basepair, paired end reads. Data was then analyzed using analysis pipelines composed of various in house and open source tools, to detect insertions, deletions, amplifications and single nucleotide variants to an allele frequency of 5%.

      Results
      Across the clinical sample set, 67% of samples were found to have at least one disease associated mutation, 8% had only an unclear variant, 13% were ‘normal’, while 12% had DNA that was not adequate for testing. The average number of mutations seen in samples with disease associated changes was 1.6, with a range from 1 to 4. Half of the samples analyzed were found to have Tp53 mutations, followed by EGFR changes seen in 11%. While only 16 patients had EGFR changes, nearly half (7) of these had co-mutations, including changes at positions 709, 719, 768 and 790. Many of the EGFR changes were complicated insertion/deletions found in exons 19 and 20, which were missed by standard bio-informatic algorithms and only captured by in house tools. KRAS changes were seen in 21% of patients followed by PTEN, MET, STK11 and APC mutations seen in at least 2 patients. Other known somatic mutations were also identified in ATM, BRAF, CTNNB1, FBXW7, FLT3, GNAS, HRAS, NRAS, PIK3CA and ALK in at least one patient. This included the hotspot mutation F1174L in ALK, which is seen with high frequency in neuroblastoma.

      Conclusion
      The mutation status of many clinically relevant genes can be reliably detected in FFPE samples using a single molecular assay followed by high throughput sequencing. Using this approach known somatic mutations seen frequently in other tumor types can be readily identified in lung tumors, and highlights the future benefits of tumor profiling.

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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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      P2.18-021 - How Accurate are We in Diagnosing Squamous Cell Carcinoma of the Lung in Cytology Specimens and Relevance to Molecular Testing? An Institutional Experience. (ID 3144)

      09:30 - 09:30  |  Author(s): C. Deshpande

      • Abstract

      Background
      Distinction between different subtypes of non–small cell lung carcinoma (NSCLC) particularly separating squamous cell carcinoma (SCC) from non-SCC is important due to the availability of specific targeted therapy based on histologic subtypes. Recent molecular testing guideline for selection of lung cancer patients for epidermal growth factor (EGFR) and anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor therapy from the College of American Pathologists (CAP), International Association for the Study of Lung Cancer (IASLC) and Association for Molecular Pathology (AMP), recommend that in the setting of fully excised lung cancer specimens, EGFR and ALK testing is not recommended in lung cancers that lack any adenocarcinoma component, such as pure SCC (Lindeman et al, J Mol Diagn 2013, 15:1-39). However, more than two-thirds of lung cancer presents as advanced stage disease and cytology specimens may only be available in most of these cases. Accurate distinction between SCC and non-SCC can be challenging in these cytology specimens due to limited cellularity. We report our institutional experience regarding the diagnostic accuracy of classifying SCC in cytology preparations compared with histopathological follow-up.

      Methods
      A retrospective review of the cytology specimens diagnosed as squamous cell carcinomas was carried out from January 2011 – June 2012 from our cytopathology and surgical pathology databases. All cases were correlated with subsequent histopathological diagnoses, immunohistochemical (IHC) and molecular analyses.

      Results
      A total of 200 histopathological specimens diagnosed as squamous cell carcinoma (SCC) were retrieved from our electronic medical records. 30 were excluded due to multiple specimens from the same patient. Corresponding cytology specimens including fine needle aspiration (FNA) specimens, bronchial brushings and bronchial washings were available in 88/170 (52%) cases. Out of these 88 cytology specimens, a diagnosis of malignancy was made in 58 (66%) specimens; these were subcategorized into (1) definite diagnosis of malignancy (32/58=55.2%), (2) favor tumor subtype (14/58=24.1%), (3) unclassified (9/58=15.5%) and (4) suspicious for malignancy (3/58=5.2%). Overall, the diagnoses of malignancy was made in 57/58 (98.3%) while the accurate diagnoses of squamous cell carcinoma was made in 52/55 (95%) cytology specimens. IHC was employed in cases 21/88 (23.9%). Molecular testing was performed on 12/170 (7%) specimens. Molecular testing was successful in these cases with mutations identified in two cases.

      Conclusion
      Our study demonstrates that cytology specimens are an excellent source of making a reliable, quick and accurate diagnosis of lung squamous cell carcinomas in 95% of cases and can serve as an excellent source for IHC and molecular analyses, if necessary.