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ORAL 37 - Novel Targets (ID 146)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:S.S. Ramalingam, E. Thunnissen
- Coordinates: 9/09/2015, 16:45 - 18:15, Mile High Ballroom 4a-4f
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ORAL37.01 - FISHing TRK Activation by Gene Rearrangements in Non Small Cell Lung Cancer (ID 834)
16:45 - 16:56 | Author(s): S. De
- Abstract
- Presentation
Background:
The tropomyosin-receptor kinase (TRK) family includes genes important in nervous system development, NTRK1 (N1), NTRK2 (N2) and NTRK3 (N3). Oncogenic activation was identified long ago as N1 fusions in colon cancer and numerous fusions have been recently identified affecting all family members in multiple tumor types. This study developed FISH reagents for molecular diagnosis of NTRK rearrangements and investigated their prevalence in NSCLC. The ultimate goal is to validate a clinical assay for selection of patients who may benefit from novel tyrosine kinase inhibitors (TKIs) targeting these fusion proteins.
Methods:
Three FISH break-apart (BA) probe sets (LDTs) were tailored for diagnosis of rearrangements in N1, N2 and N3 and tested in specimens with known genomic status for these genes: cell lines KM12 (N1), CUTO3 (N1), MO-91 (N3), xenograft CULC001 (N1), and clinical specimens, and used to screen resected NSCLC. The LSI NTRK1 Cen and Tel probes (Abbott Molecular) were also tested. A specimen was positive for individual rearrangement when ≥15% tumor cells had split or single 3’,5’ signals. Moreover, a 6-target, 2-color FISH probe including the 3’N1, 3’N2 and 3’N3 sequences labeled in red and the 5’N1, 5’N2 and 5’N3 sequences labeled in green (TRKombo) was designed for rapid screening of TRK rearrangements in clinical specimens.
Results:
Results were obtained in 443, 410, and 434 examined NSCLC and positive patterns were detected in 5, 5 and 1 specimens, respectively for N1, N2, and N3. These 11 positive patients had age ranging from 38y to 76y, gender 6 male:5 female, and were current (4), former (5) or never (2) smokers. Histology was predominantly adenocarcinoma (7) but also included squamous cell (3) and neuroendocrine morphology (1). Unique to the N1 assay was the observance of FISH signal fusions where the 5’N signals appeared as doublet in >20% of the NSCLC specimens, which was determined to be copy number variation due to segmental duplication. Other atypical patterns were observed for all three targets and included doublets of the FISH fusion signals (18%, 14% and 9% respectively) and gene clusters (~5% for each). Twenty specimens (pre-clinical models and clinical cases) characterized as positive by the LDT N1 and by next generation sequencing (NGS) or atypical by the LDT NTRK1 BA were blindly analyzed with the LSI NTRK1 probe set and the results were reproducible, with brighter intensity of the fluorescent signals for the LSI probe. These specimens (positive by FISH and several atypicals) are currently under investigation to characterize the sequence specific genomic rearranged region by using a custom targeted, capture-based NGS panel (NimbleGen, Roche). The TRKombo screening probe performed well in blinded experiment using validation set including pre-selected positive and negative specimens and is under testing in clinical tissue sections.
Conclusion:
N1, N2 and N3 fusions were detected by FISH in a subset of lung carcinomas including adeno, squamous and neuroendocrine tumors. Optimization of molecular panels for diagnosis of these rearrangements is relevant since they represent a sizeable number of cases across multiple tumor types and there are numerous targeted inhibitor agents under development.
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ORAL 41 - Immune Biology, Microenvironment and Novel Targets (ID 159)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:S.K. Padda, R. Nemenoff
- Coordinates: 9/09/2015, 18:30 - 20:00, Four Seasons Ballroom F1+F2
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ORAL41.06 - Transcriptional Profiling of Distinct Macrophage Subsets in Lung Tumor Microenvironment Reveals Their Functional Heterogeneity (ID 3181)
19:24 - 19:35 | Author(s): S. De
- Abstract
Background:
Lung cancer is the leading cause of cancer-related deaths in both men and women. While extensive research has focused on genetic mutations in neoplastic epithelial cells, it has now become apparent that cancer progression and metastasis involve complex interactions between cancer cells and the cells of the tumor microenvironment. Myeloid cells of mononuclear phagocyte lineage are a significant component of the tumor microenvironment in lung cancer. Depending on the activation state, myeloid cells have been implicated in tumor – promoting processes such angiogenesis, tissue remodeling and immunosuppression, but also in anti-tumor immunity such as supporting immune surveillance and direct cytotoxicity. The goal of this study was to identify distinct populations of monocyte/macrophage cells and to gain insight into their functions through transcriptional profiling.
Methods:
We used an orthotopic immunocompetent mouse model, in which Lewis Lung carcinoma cells, a cell line derived from mouse adenocarcinoma, were injected directly into the left lung lobe of syngeneic C57BL/6 mice. Whole left lung lobes bearing primary tumors were harvested at 2 and at 3 weeks after cancer cell injection, together with lungs from uninjected mice. Tissues were processed into single-cell suspensions and analyzed by multi-color flow cytometry. The flow cytometry strategy employed a combination of myeloid specific surface markers such as CD11b, CD11c, CD64, and SiglecF to identify distinct monocyte/macrophage subpopulations. We recovered these cell populations by flow cytometry-based cell sorting, isolated RNA, and performed transcriptional profiling by RNA-seq. Sequencing data were analyzed by TopHat/Cufflinks/CuffDiff software package and EdgeR. To define the lineage of the isolated cells we correlated their transcriptional profiles to published profiles of immune cells from blood and lung of naïve mice. Further, we used hierarchical clustering and web-based bioinformatic pathway analysis tool to discover functions and pathways enriched in specific myeloid populations.
Results:
Based on the combination of myeloid markers and transcriptional profiling, we identified 4 distinct populations of monocyte/macrophage cells: MacA, which represent alveolar macrophages, MacB1, which represent a mixture of dendritic cells and Ly6C- monocytes, MacB2, which represent Ly6C+ monocytes, and MacB3, which represent interstitial/infiltrating macrophages. While the numbers of MacA and MacB1 remain unchanged with cancer progression, MacB2 and MacB3 expand rapidly. Pathway analysis indicated that each population of cells regulates distinct functions in the tumor microenvironment, such as lipid metabolism, cytokine or chemokine secretion, production and remodeling of extracellular matrix, antigen presentation.
Conclusion:
These data provide critical insights into the heterogeneous nature and diverse functions of myeloid cells in tumor microenvironment of lung cancer. This study has the potential for development of therapeutics that target specific subsets of myeloid cells that could complement conventional cancer-cell-targeted therapies.
