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D. Buitrago



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    MO01 - Lung Cancer Biology - Techniques and Platforms (ID 90)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO01.04 - Comparison of Microarray and RNA Sequencing Platforms for Profiling MicroRNAs in Formalin-Fixed, Paraffin-Embedded Non-Small Cell Lung Cancer Specimens (ID 3145)

      10:40 - 10:45  |  Author(s): D. Buitrago

      • Abstract
      • Slides

      Background
      MicroRNAs are useful biomarkers for various disease states, and their preservation in formalin-fixed, paraffin-embedded (FFPE) tissue makes them particularly useful for clinicogenetic studies. Although global microRNA expression in FFPE samples is routinely measured with microarrays, the utility of RNA sequencing for such profiling has yet to be established. In this study, to appraise the suitability of RNA sequencing, microRNAs in RNA from lung cancer FFPE samples were quantified by both a microarray and a sequencing platform.

      Methods
      The affinity spin column–based Roche High Pure FFPE RNA kit was used to extract total RNA from 8 resected stage I lung adenocarcinoma FFPE tumor specimens (~3 mm[3]) with ≥50% tumor content. RNA was quantified by RiboGreen fluorometric and absorbance spectrometric analysis at 260 nm, and its quality was examined by electrophoresis on an RNA Pico chip in an Agilent Bioanalyzer 2100. MicroRNAs in 120 ng of RNA were profiled using the 8x60K Agilent Human miRNA Microarray (release 16.0) platform. MicroRNAs were also quantified by use of the Illumina HiSeq 2000 sequencing system (1x 50 bp reads), with multiplexed sequencing libraries prepared using 1 ug of RNA with the Illumina Truseq Small RNA Preparation Kit (version 2.0). Microarray data were processed using the AgiMicroRna Bioconductor package in R. Sequencing data were demultiplexed using CASAVA software and were mapped against mature human microRNAs in the miRBase database (version 16) using STAR aligner software. Absolute microRNA count values were then normalized among samples by use of the edgeR Bioconductor package.

      Results
      Results of RiboGreen fluorometric analysis suggested that an average of 16 ug (range, 6-35 ug; SD, 8 ug) of RNA was obtained from the FFPE specimens. Significant degradation of RNA was observed, as expected, with Bioanalyzer RNA integrity number values between 1.9 and 2.5. An average of 1.3 million sequencing reads (range, 9.1-16.9 million; SD, 3.5 million) were obtained, but only 1.4% (range, 0.4%-2.1%; SD, 1.4%) of them mapped to known microRNAs. Of the 1205 human microRNAs detectable with the microarray platform, 302 were identified as expressed in the 8-sample set, and 593 were identified as expressed in the sequencing platform. For the 177 microRNAs detected by both microarray and sequencing methods, the interplatform Spearman correlation coefficient was >0.5 for only 51 of them. Reverse-transcription PCR assays are being performed to identify the platform that yields the most accurate microRNA profile.

      Conclusion
      MicroRNA profiling by RNA sequencing and microarray techniques produced different results. The RNA sequencing method described here does not appear to be suitable for profiling microRNAs in RNA from FFPE samples. It is possible that depletion of ribosomal RNA fragments from FFPE RNA samples may improve the quality of data obtained from RNA sequencing.

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    O09 - General Thoracic Surgery (ID 100)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Surgery
    • Presentations: 1
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      O09.01 - Sites, Symptoms, CT Scan Findings and Survival in Patients with Recurrence After Curative-Intent Surgical Resection for Stage I Lung Adenocarcinoma (ID 2907)

      16:15 - 16:25  |  Author(s): D. Buitrago

      • Abstract
      • Presentation
      • Slides

      Background
      The purpose of this study is to examine the patterns of recurrence for stage I lung adenocarcinoma and to identify clinicopathologic factors associated with post-recurrence survival (PRS).

      Methods
      We performed a retrospective review of 1027 patients with stage I lung adenocarcinoma who underwent a surgical resection between 1999 and 2009 (median follow-up 35 months). The manner of recurrence detection, either by scheduled CT scan, presentation with new symptoms, or by other means, was noted. Tumors were classified using the new IASLC/ATS/ERS nomenclature and grading as low (adenocarcinoma in situ, minimally invasive adenocarcinoma, or lepidic-predominant), intermediate (papillary-predominant or acinar-predominant), and high (micropapillary-predominant, solid-predominant, colloid-predominant, or invasive mucinous) grade. The Kaplan-Meier method was used to analyze recurrence-free survival (RFS). Log-rank tests and Cox proportional hazard models were used to analyze the association between predictive factors and PRS.

      Results
      Of the 1027 patients with follow-up data available, 151(15%) had recurrent disease (table), five-year RFS was 80%. Of the 151 patients with recurrence, 80 (52%) were detected by a scheduled CT scan (51 locoregional and 29 distant). Symptomatic recurrences were seen in 70 (46%) patients (9 locoregional and 61 distant). Overall, 5-year PRS was 27.8%. On multivariate analysis, recurrences identified by new symptoms (HR, 2.15; 95% CI, 1.36- 3.40; p=0.001), a recurrence free interval ≤ 24 months (HR, 2.52; 95% CI, 1.31- 4.84; p=0.006), and tumors with high architectural grade (HR, 1.69; 95% CI, 1.07- 2.67; p=0.024) and vascular invasion (HR, 1.79; 95% CI, 1.14- 2.81; p=0.012) were significantly associated with a worse PRS (Figure).Figure 1Figure 2

      Conclusion
      Our study demonstrates the recurrence patterns in patients who underwent surgical resection for stage I lung adenocarcinoma. We identify a symptomatic recurrence, a recurrence-free interval ≤ 24 months, high architectural grade, and vascular invasion, as independent factors associated with worse post recurrence survival.

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    O22 - Mesothelioma III (ID 122)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Mesothelioma
    • Presentations: 1
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      O22.02 - CD8 T-cell Infiltration and Tumor IL-7R Expression are Independent Prognostic Factors in Epithelioid Malignant Pleural Mesothelioma (ID 2935)

      16:25 - 16:35  |  Author(s): D. Buitrago

      • Abstract
      • Presentation
      • Slides

      Background
      Following our publication (Cancer Immunol Immunother 2011) demonstrating the prognostic importance of chronic inflammatory cell infiltration in epithelioid malignant pleural mesothelioma (MPM), we investigated the prognostic significance of the immune microenvironment in the tumor nest and the tumor-associated stroma in epithelioid MPM.

      Methods
      A tissue microarray (TMA) was constructed from 170 epithelioid MPM cases, with 6 representative tumor cores and 3 representative stromal areas. Immunohistochemical analyses for immune cell infiltration (CD3, CD4, CD8, CD20, FoxP3) and interleukin receptors (IL-7R and IL-12Rβ2) were performed. TMA slides were analyzed for immune cell infiltration of tumor and stroma (low vs high, divided by use of the median), as well as for immune marker expression (sum of intensity and distribution). Overall survival (OS) was estimated using Kaplan-Meier analysis, and log-rank tests and Cox proportional hazards models were used to analyze the association between each marker and OS.

      Results
      Analysis of single immune cell infiltration for all patients revealed that high tumor CD8+ T-cell infiltration, high CD20+ B-cell infiltration, and low tumor IL-7R expression correlated with higher OS (Figure). Combined tumor CD8+ and CD4+ cell infiltration significantly correlated with better OS (5-year OS, 36% [n=61] vs. 20% [n=96]; p=0.008). In a multivariate analysis including age, stage, lymph node metastases, lymphatic invasion, and vascular invasion, high CD8+ T-cell infiltration and low tumor IL-7R expression were independent predictors of OS (Table). Figure 1Figure 2

      Conclusion
      Tumor CD8+ T-cell infiltration and tumor IL-7R expression are independently associated with survival, which highlights the biologic and prognostic significance of the immune microenvironment for patients with epithelioid MPM.

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