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D. Jain



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    P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)

    • Event: WCLC 2015
    • Type: Poster
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      P1.04-080 - miR-326 Is Down-Regulated in Non-Small Cell Lung Cancer and Targets NFIB, a Lung Developmental Gene: A Pilot Study (ID 1326)

      09:30 - 17:00  |  Author(s): D. Jain

      • Abstract
      • Slides

      Background:
      Lung cancer is the leading cause of cancer mortality worldwide. Non-small cell lung cancer (NSCLC) is the most common subtype, accounting for about 80% of all lung cancers. miRNAs are small RNAs of 21-24 nucleotides in length, which play major role in cell proliferation and differentiation and their differential expression is known to be associated with various cancers including lung cancer. Role of miR-326 has been previously studied as a marker of bone metastasis in lung cancer. Moreover, we have previously shown that miR-326 plays a critical role in the epithelial to mesenchymal transition (EMT) by targeting transforming growth factor (TGF)-β1 and other members of TGF-β signaling pathway. The aim of present study is to check the expression and correlation of miR-326 and lung epithelial developmental gene nuclear factor IB (NFIB) in non-small cell lung cancer tissue samples as cancer metastasis is accompanied by EMT.

      Methods:
      We have examined eight pathologically confirmed non-small cell lung cancer cases. All patients were men and smokers with age ranged from 29 to 74 years (mean 54.6 years). Surgical resection was performed in all the cases which were either stage II or III. Histopathologically, 4 cases were squamous cell carcinomas, 3 were adenocarcinomas including one case of invasive mucinous carcinoma and one case was low grade mucoepidermoid carcinoma. RNA was isolated from fresh frozen tissue to check for miR-326 and NFIB levels by real time PCR. Protein expression was checked by immunohistochemistry (NFIB; 1:200; Abcam)) and in-situ hybridization (miR-326; Exiqon). Adjoining lung tissue served as normal control in each case.

      Results:
      Expression of both miR-326 and NFIB was found to be down regulated in non-small cell lung cancer tissue at both RNA and protein level (Fig 1A-C). Our in silico experiments identified a target site of miR-326 at the 3’UTR of NFIB gene; presumably it stabilizes the transcripts of NFIB (Fig 1D). Figure 1



      Conclusion:
      Our preliminary data suggests that miR-326 stabilizes the transcripts of NFIB in normal epithelial cells and maintain epithelial cell integrity. Dysregulation of miR-326 and NFIB in non-small cell lung cancer indicate that miR-326 and NFIB work synergistically and may contribute to the development of non-small cell lung cancer.

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    P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)

    • Event: WCLC 2015
    • Type: Poster
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      P3.04-006 - ALK Immunohistochemistry in NSCLC: Evaluation of Performance of D5F3-IHC without Using Automated Ventana System (ID 2377)

      09:30 - 17:00  |  Author(s): D. Jain

      • Abstract
      • Slides

      Background:
      Since the advent of targeted therapy, molecular testing for common mutations has become vital in the diagnostic algorithm of non- small cell lung cancer (NSCLCs). ALK-EML4 fusion is a rare abnormality detected in 3–13% patients of adenocarcinomas (ADC). Although Fluorescent In-Situ Hybridization (FISH) is a gold standard technique for detection of ALK rearrangement, it is expensive, time-consuming and requires specialized equipment and expertise for interpretation. Immunohistochemistry (IHC) with ALK rearrangement-specific antibodies is considered as a more economical method for routine diagnostic practice. Ultrasensitive automated Ventana D5F3-IHC revealed a very high correlation with FISH and approved by China FDA for targeted therapy; however, the automated IHC apparatus are not widely used in most general laboratories. In this study, we evaluated performance of ALK IHC using manual semiquantitative method in a cohort of 133 adenocarcinomas, to achieve the frequency of ALK positivity in Indian patients and correlation with automated Ventana D5F3-IHC.

      Methods:
      We tested 133 cases of primary lung ADCs, which were negative for EGFR mutation, for ALK rearrangement by D5F3-IHC.Thirty three of them were tested by both automated Ventana (D5F3) and manual methods (Cell Signaling Technology, Danvers, MA, USA). The intensity of cytoplasmic staining was classified as 0 (negative) or 1+/2+/3+ (weak/medium/strong). Binary score of positive (strong granular cytoplasmic staining in any percentage of tumor cells) and negative (absence of strong granular cytoplasmic staining) was used for Ventana IHC which was taken as gold standard. A comparison analysis and clinicopathological features were recorded.

      Results:
      Male to female ratio of the patient population was 2.3:1. ALK rearrangement was positive in 10 (7.5%) cases, out of which 7 were men and 50% were non- smokers. Median age for all ADCs was 55 years and for ALK rearrangement positive cases was 47 years. Three of 10 ALK IHC positive cases showed signet ring cell morphology. On comparison, all cases positive by Ventana (10 cases) (Figure 1A) showed positive results by manual method. Six cases showed 3+ (Figure1B) whereas 2+ (Three cases) and 1+ (one case) staining intensity was observed. The latter 4 cases were positive by FISH. All negative cases by Ventana system were negative by manual method. Figure 1



      Conclusion:
      Mutation specific IHC serves as a rapid tool for detection of ALK rearrangement in low resource settings. Manual IHC is equally effective in detection of ALK rearranged cases as automated methods. IHC positive cases may subsequently be analyzed by FISH thus reducing the cost of automated systems.

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