Author of

• 10923

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

  • Event: WCLC 2013
  • Type: Poster Session
  • Track: Pathology
  • Presentations: 1
  • Moderators:
  • Coordinates: 10/28/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 10936

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    P1.18-013 - Extracting High quality RNA from FFPE samples for gene expression studies (ID 2092)

    09:30 - 16:30  |  Author(s): H. Do
    • Abstract

    Background
    The use of targeted therapies in the treatment of non-small cell lung cancer is still limited to a relatively small fraction of patients. Chemotherapy remains the mainstay of treatment for most patients today. So far, the best predictors for chemotherapeutic success are based on the expression of certain nucleotide metabolism genes or DNA damage response and repair related genes. However, the samples available for study most commonly comprise FFPE samples, which are characterised by a high degree of RNA fragmentation or degradation.

    Methods
    To address this problem, we have developed a protocol to reliably extract reasonable-quality RNA from FFPE samples. The protocol includes pathology review of the FFPE block, removal of a 2mm core, followed by RNA extraction. Next, the total RNA amount is quantified and a small proportion is accessed for fragmentation e.g. by TapeStation technology and/or a multiplex RT-PCR to determine the amount and size of amplifiable templates. We then assessed the extracted total RNA by various RNA based methodologies.

    Results
    To this end, we prepared core punches from 118 different lung adenocarcinomas and successfully extracted sufficient amounts of total RNA (> 50ng /ul in a 20ul elution) from 111 of the cores (average is 307ng/ul ranging from 53ng to 1.1ug/ul). Fragmentation assessment of 26 of these RNAs showed that all samples contained sufficient amounts of fragments with at least >100 nt. We first tested single gene expression by RT-qPCR. Of 26 samples tested, 24 samples showed robust amplification of a 161 bp fragment of the TBP housekeeping mRNA. We next assessed our RNA using gene expression analysis by NanoString®. We interrogated 150ng total RNA from 10 samples for the expression levels of 45 genes. Data analysis showed robust expression values and no quality control problems in all samples. Finally, we tested whether the RNA was of sufficient quality for next-generation RNA sequencing. We used 100 and 50 bp paired end sequencing on un-size-selected RNA, and 100 bp paired end sequencing after one round of size selection. On average, we obtained 23 million reads per sample, of which 70% mapped to reference sequences after either extensive read clipping or size selection.

    Conclusion
    In conclusion, our extraction protocol enables us to reliably extract total RNA from FFPE samples, which can be used for single-gene expression by RT-qPCR and gene expression of limited gene sets by NanoString® technology. However, the amount of samples and genes tested here were not sufficient to allow identification significant differences between samples, but shows the possibility to use the RNA extracted following our extraction protocol. RNAseq, however, poses a larger problem. The amount of mapped reads is significantly lower compared to high quality RNA from e.g. fresh frozen material or cell lines. The reason for these problems and possible solutions remain elusive. Overall, we present a simple and fast way to accurately extract RNA from FFPE material and show that after QC, single or small gene panels can successfully be assessed. However, large-scale sequencing efforts remain problematic and further optimization is needed.

• 12769

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  P3.18 - Poster Session 3 - Pathology (ID 177)

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

  • 12776

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    P3.18-007 - Metastatic sites with a major component of solid pattern pulmonary adenocarcinoma are associated with shorter survival with systemic therapy and infrequent EGFR mutations (ID 1790)

    09:30 - 16:30  |  Author(s): H. Do
    • Abstract

    Background
    The predominant histologic subtype according to the IASCL/ATS/ERS classification has been associated with prognosis in patients undergoing curative surgical resection. It is recommended in the IASLC/ATS/ERS classification that histologic patterns present in small specimens be recorded in the final histopathology report. We investigated the relationship between histologic patterns in metastatic sites, overall survival and EGFR and KRAS mutations.

    Methods
    We identified patients who underwent surgical resection of non-small cell lung carcinoma in metastatic sites from 2000 to 2011. One biopsy site was selected per patient. A pathologist reviewed all slides to confirm the diagnosis of metastatic lung adenocarcinoma, recording all adenocarcinoma histologic patterns present. EGFR and KRAS mutations were scanned by high resolution melting, and confirmed by Sanger sequencing. Clinical data were extracted from the medical records.

    Results
    The 100 patients comprised 66 males, with a median age of 64 years. 85% had stage IV disease, and 15% had unresected stage III disease with mediastinal lymph node sampling. Most were current or former smokers (79%) of ECOG 0/1 (67%). Just over half the patients received systemic therapy (56%). The overall median survival was 10.2 months (95% CI 8.1 – 12.2 months). Metastatic sites included brain (30%), pleura (25%), bone/skeletal muscle (20%), and lymph nodes (mediastinal 18%; chest wall/neck 7%). It was possible in each biopsy to identify a major histologic pattern (Table 1). For patients receiving systemic therapy, survival was significantly shorter for those with a major component of solid pattern tumour in metastases compared to those with major acinar or micropapillary patterns in metastases (Table 1). No survival difference was noted on the basis of ECOG, stage, EGFR or KRAS mutations. For patients who did not receive systemic therapy, the major histologic pattern showed no association with overall survival (Table 1). Worse ECOG was the only significant factor in determining outcome (ECOG 0/1 vs 2+ – hazard ratio 2.18 (1.14 – 4.16, p=0.018)). EGFR mutations were significantly associated with major non-solid pattern histology in metastases (Fisher’s exact = 0.006). No association was observed by KRAS mutation status (Table 1).

    The major histologic component in sites of metastatic adenocarcinoma – overall survival by the use of systemic therapy; presence of EGFR and KRAS mutations (*Comparison across 4 histological types; OS - overall survival, CI - confidence interval, HR - hazard ratio)

    Solid Micropapillary Acinar Papillary Comments Major Pattern 50% 20% 29% 1% Mutations present n = 50 n = 20 n = 29 n = 1 n=100 EGFR mutation, n (column %) 2 (4%) 5 (25%) 4 (14%) 1 (100%) Fisher's exact = 0.006 * KRAS mutation, n (column %) 18 (36%) 5 (25%) 9 (31%) 0 Fisher's exact = 0.789* Systemic Therapy Given n = 29 n = 13 n = 13 n = 1 n=56 Median OS, months (95% CI) 9.4 (8.3 - 12.2) 18.9 (11.6 - 24.4) 15.9 (10.7 - 24.7) Solid vs MPA HR 0.33 (0.16 - 0.67, p = 0.002) Solid vs Acinar HR 0.32 (0.15 - 0.67, p=0.003) No Systemic Therapy n = 21 n = 7 n = 16 n = 0 n=44 Median OS, months (95% CI) 4.3 (3.3 - 7.4) 4.7 (1.5 - 11.5) 4.4 (1.9 - 16.9) No significant differences

    Conclusion
    Our results suggest that patients with a major component of solid pattern tumour in metastatic sites may be more resistant to systemic therapy as evidenced by shorter overall survival in comparison to those with major micropapillary and acinar pattern tumour in metastases. Furthermore, major solid pattern metastases are unlikely to harbour EGFR mutations. These findings require validation in larger patient cohorts.