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S. Sakashita
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MINI 14 - Pre-Clinical Therapy (ID 119)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:L. Fernandez-Cuesta, A.F. Gazdar
- Coordinates: 9/08/2015, 10:45 - 12:15, Mile High Ballroom 2c-3c
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MINI14.12 - Genomic Profiling of Patient-Derived Xenografts Identify Passenger Aberrations Associated with Better Prognosis in Non-Small Cell Lung Cancer (ID 1735)
11:50 - 11:55 | Author(s): S. Sakashita
- Abstract
Background:
Patient-derived tumor xenografts (PDXs) increasingly are being used as preclinical models to study human cancers, test novel therapeutics, and identify potential biomarkers, as they more accurately model human cancers than established tumor cell line cultures. However, uncertainty remains as to how well the genomic characteristics of patient non-small cell lung cancer (NSCLC) are recapitulated in these PDX models.
Methods:
PDXs were established by implantation of surgically resected NSCLC patient tumors into the subcutaneous or sub-renal capsule of non-obese diabetic severe combined immune deficient (NOD-SCID mice. Comprehensive genomic profiling including exome, gene copy number, DNA methylation and mRNA expression were conducted on 36 independent PDX models, their matched patient tumors and normal lung tissue. Publicly available cell line and TCGA data were used for comparison. Integrative analysis was performed to identify genomic alterations in PDXs that are associated with significant clinical outcomes in patients.
Results:
From 441 resected NSCLC tumors, 127 serially transplantable and stable PDX models were established. Among 264 NSCLC patients with at least 3-years follow-up, patients whose tumor formed stable PDXs (versus those who did not) showed significantly worse disease free (HR=3.12, 95% CI =2.02-4.83, P<0.0001) and overall survival (HR=4.08, 95% CI =2.16-7.73, P<0.0001), after multivariable adjustment for clinical pathological factors. Genomic and transcriptomic profiling of 36 PDXs showed greater similarity in somatic alterations between PDX and primary tumors than with published cell line data. In addition to known mutations, we found at least 16 non-synonymous somatic mutations in known oncogenes and tumor suppressors that have never been reported. All these mutations had higher observed variant allele frequency in PDXs compared to their matched patient tumors, suggesting that these were tumor sub-clones selected or enriched for growth in the PDXs. Tumor models characterized by a higher number of somatic alterations among 865 frequently altered genes were associated with better overall patient survival (HR=0.15, p=0.00015) compared to patients with corresponding PDXs characterized by higher alteration number; this was validated in the TCGA lung cancer dataset patients (HR=0.28, p=0.000022). These 865 genes were enriched for those encoding for proteins involved in cell adhesion and interactions with the extracellular matrix, and a quarter of the genomic alterations would putatively form neo-antigens implicating a potential role of immune response in the observed improved patient survival.
Conclusion:
PDXs are close preclinical models of patient tumors. Further investigations of passenger mutations may clarify their clinical impact on interactions between tumor cells, stroma, immune microenvironment and patient prognosis.
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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
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-066 - Site-Selected Chromatin-Immunoprecipitation (ChIP) Analysis by Laser Captured Microdissection (ID 1752)
09:30 - 09:30 | Author(s): S. Sakashita
- Abstract
Background:
High throughput sequencing methods such as exome sequencing, RNA sequencing, Chromatin–immunoprecipitation (ChIP) sequencing are essential tools for cancer research. However, these fine and delicate analyses contain several methodological problems. For example, although tumor mass may be suitable for mutation analysis, histological heterogeneity of the tumor tissue causes insufficient results especially for epigenetic or RNA analyses. Besides, the cancer-associated stromal cells and immune cells in the tumor will also affect the results. In this study, we tried ChIP for tiny but pure tumor samples which were selected by laser captured microdissection and verified its availability for ChIP sequence analysis.
Methods:
We used a lung adenocarcinoma frozen tissue harboring EGFR L858R mutation. After formalin fixation (1%, 10min), tumor cells, stroma cells and immune cells were microdissected separately by LMD4000 (Leica) and ChIP was performed to using H3K4me3 anti-body. Then, the quality was confirmed by real-time PCR for CCR7 which is one of the tumor specific markers and CD3 which is representative T lymphocyte marker. Sanger sequence for EGFR L858R mutation was also analyzed for confirmation that each sample was dissected and extracted correctly.
Results:
Only from the sample of tumor cells, we detected EGFR L858R mutation by Sanger sequence but from stromal cells and immune cells, we did not detect EGFR mutation. The result showed that we extracted samples correctly. And H3K4me3 mark at CCR7 gene was detected only from tumor cells and was not detected from the other samples. Moreover, H3K4me3 mark at CD3 gene was detected from stroma cells and immune cells but not tumor cells. These results indicated that microdissection method is useful and necessary method for ChIP analysis.
Conclusion:
Microdissection can be applied for epigenetic analysis like ChIP method. Our results indicated that microdissection method is useful for tumor-cell-specific epigenome profiling by ChIP sequencing.
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-053 - Patient-Derived Xenograft Studies Suggest FGFR1 Amplification Is Insufficient to Predict Response to FGFR Inhibitors in Lung SqCC (ID 3067)
09:30 - 09:30 | Author(s): S. Sakashita
- Abstract
Background:
FGFR1 amplification has been reported in 16%-20% of lung squamous cell carcinoma (SqCC). Early phase clinical trials with anti-FGFR small molecule inhibitors are in progress. It remains unclear whether genomic changes involving FGFR1 is associated with a dependency in FGFR-driven oncogenic activity that could be inhibited with pharmacologic agents. We evaluated a pan-FGFR inhibitor (BGJ398) in four SqCC patient-derived xenograft (PDX) models with amplification of the FGFR1 gene.　
Methods:
FGFR1 gene copy changes were assessed by fluorescence in-situ hybridization. PDX models were established by implanting surgical resected tumor fragments into the subcutaneous tissue of non-obese diabetic severe combined immune deficient (NOD-SCID) mice. Protein and mRNA expression levels were assessed by immunohistochemistry/western blot and RT-qPCR, respectively.
Results:
FGFR1 amplification was observed in 13 of 60 (22%) SqCC patient tumors, with all amplified tumors forming PDX. PDX models with FGFR1 gene amplification displayed higher levels of mRNA and protein compared to non-amplified tumor, excluding polysomy cases. One model demonstrated an average of 50% decrease in tumor volume in the BGJ398 treated group compared to control group, 21 days post-treatment. This model also expressed high FGFR1 and high cMYC protein. BGJ398-resistant PDX models included one model with high FGFR1 but low cMYC protein levels, and two models with low FGFR1 and high cMYC protein levels.
Conclusion:
The lack of growth arrest to a pan-FGFR small molecule inhibitor in the 4 PDX models evaluated suggests that FGFR1 amplification alone was not a sufficient predictive marker for pan-FGFR1 inhibitor activity. FGFR1 protein and MYC protein are putative markers.
