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M. Pintilie



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    P1.05 - Poster Session 1 - Preclinical Models of Therapeutics/Imaging (ID 156)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.05-007 - Large scale establishment of genetically diverse patient-derived primary tumor xenografts from resected early stage non-small cell lung cancer (NSCLC) patients (ID 1539)

      09:30 - 16:30  |  Author(s): M. Pintilie

      • Abstract

      Background
      The fidelity of established NSCLC cell line models to reflect patient tumors has been challenged. Patient-derived primary tumor xenografts (PTXGs) established directly from patient tumors in immunodeficient mice reproduce closely the histology of the primary tumors, thus are potentially better preclinical models to investigate novel therapies. We previously reported that early stage NSCLC patients whose tumors form PTXGs have significantly greater risk of relapse after surgery (Clin Cancer Res 2011; 17: 134-141). We report here a more extended analysis of clinical-molecular-pathological features of early stage NSCLC that are associated with engraftment and its impact on patient outcome.

      Methods
      Resected NSCLC tumors were harvested within 30 minutes after surgery and were implanted into severely immunodeficient mice to establish PTXGs. Tumors that grew were propagated for up to 3 passages. The mutational profiles of the primary tumors were assessed by the MassARRAY platform that included 133 mutations with ‘putative’ driver function, which have been reported in COSMIC database as recurrent in NSCLC. All identified mutations were verified by direct sequencing in both the primary and PTXG tumors. Engraftment rate among clinical factors were tested using the Fisher’s exact or Mann-Whitney tests. The Kaplan-Meier method was used to estimate 3-year overall (OS) and disease-free survival (DFS) probabilities. The effect of engraftment on OS and DFS adjusting for clinical variables was assessed using a Cox proportional hazards model.

      Results
      From April 2005 to December 2010, 261 rigorously verified resected primary non-carcinoid NSCLCs were engrafted; 38 xenografts that were lymphoma were excluded from further analysis. For the remaining 223 primaries, 101 (45.3%) successfully engrafted and formed PTXG lines. Engraftment rates were 33.8% (48/142) for adenocarcinoma (AdC), 67.7% (42/62) for squamous cell carcinoma (SqCC), 66.7% (4/6) for large cell neuroendocrine carcinoma, and 53.8% (7/13) for others. The tumors forming PTXGs were more likely to be poorly differentiated (p=0.00012) and of larger tumor size and higher pT stage (p<0.0001), but were not correlated with the pN stage. Among 95/101 (94.1%) PTXG cases profiled for mutations, 6 had mutations in the EGFR tyrosine kinase domain, 18 in KRAS/HRAS, 5 in PIK3CA, 2 in paxillin and 1 in STK11/LKB gene; 56 (62.2%) were negative for mutations. The median follow-up time was 2.7 years (range 0.04 – 7.5 years). Patients whose tumors engrafted had decreased DFS (HR 2.68, 95% CI 1.16-4.60, Wald p<0.0001) and OS (HR 3.14, 95% CI 1.56-6.33, Wald p=0.0014). Significantly poorer survival was maintained in AdC. Among 33 patients with EGFR mutation, only 6 (18.2%) engrafted. Engraftment was associated with significantly poorer DFS (HR 4.76; 1.43-15.86, log-rank p=0.005) and OS (HR 8.55, 95% CI 0.77-94.3, log-rank p=0.035) in this population.

      Conclusion
      The ability to form PTXGs of early stage NSCLC is confirmed as a very strong poor prognostic marker. Although EGFR mutant tumors usually do not engraft, engraftment of EGFR mutant tumors is associated with poor patient survival. PTXGs appear to represent biologically aggressive NSCLC.

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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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      P2.06-023 - Gene expression signature and immunohistochemical assessment of NRF2 pathway activation for adjuvant chemotherapy benefit in lung squamous cell carcinoma (SqCC) (ID 1990)

      09:30 - 16:30  |  Author(s): M. Pintilie

      • Abstract

      Background
      Genomic profiling of SqCC has identified somatic alterations in NRF2 or its negative regulators (NFE2L2 mutations/amplifications, KEAP1 or CUL3 mutations/deletions) in ~1/3 of tumors. These alterations result in activation of the NRF2 transcriptional program, but the clinical significance of this pathway in lung SqCC patients is unknown. We hypothesize that a gene expression signature that reflects somatic NRF2-activating alterations may be identified and correlated with NRF2 protein over-expression. Furthermore, such gene expression or its immunohistochemical correlates may have prognostic significance and/or may be predictive of adjuvant chemotherapy benefit in early stage resectable lung SqCC patients.

      Methods
      Logistic regression (LR) and SAM analysis were applied independently to 104 SqCC cases from The Cancer Genome Atlas (TCGA) that had both microarray gene expression and mutation data to identify genes associated with NRF2 pathway mutational status. Overlapping genes were used to define the signature, which was then tested in 3 independent SqCC microarray datasets to evaluate its prognostic value. Correlation of the signature with NRF2 and KEAP1 mutations and with NRF2 and KEAP1 immunoreactive protein expression by immunohistochemistry (IHC) was evaluated. We also tested the gene expression signature as a potential predictor of adjuvant chemotherapy benefit in a subset of NCIC JBR.10 adjuvant chemotherapy trial patients with microarray data.

      Results
      A 28-gene signature that distinguished SqCC with or without aberration of the NRF2 pathway genes (NFE2L2/KEAP1/CUL3) in the TCGA dataset was identified. This gene signature that putatively represents NRF2 pathway activation status separates consistently SqCC into 2 groups in independent datasets. Both NRF2/KEAP1 mutation and NRF2 protein expression by IHC were significantly correlated with the NRF2 pathway activation signature (p<0.001 for each comparison). KEAP1 protein expression was not associated with the gene expression signature. No prognostic effect of the activated signature was observed in three independent datasets. In the JBR.10 patient cohort, a trend toward improved survival with adjuvant chemotherapy was observed in patients with the NRF2 “wild type” signature (HR 0.32, 95%CI 0.065-1.6 p=0.16), but not in patients with the “activated” signature (HR 2.28, 95%CI 0.24–22, p=0.48; interaction p=0.15).

      Conclusion
      A gene expression signature based on mutational activation of the NRF2 pathway may be predictive of benefit from adjuvant cisplatin/vinorelbine in SqCC. Patients with NRF2 pathway activating somatic alterations may have reduced benefit from this therapy. NRF2 immunohistochemistry could potentially be useful to identify NRF2-activated lung SqCC patients who may not benefit from adjuvant chemotherapy but this requires further validation.