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S. Viteri
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MINI 22 - New Technology (ID 134)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
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MINI22.01 - Detecting ALK, ROS1 and RET Fusion Genes in Advanced Non-Small Cell Lung Cancer (NSCLC) Using a Novel Multiplexed NCounter-Based Assay (ID 2254)
16:45 - 16:50 | Author(s): S. Viteri
- Abstract
- Presentation
Background:
Gene fusions of anaplastic lymphoma kinase (ALK), ROS1, and RET are targetable oncogenes present in approximately 9% of advanced NSCLC. Current assays for detecting gene fusions are based on FISH (FDA-approved companion diagnostic test for ALK), immunohistochemistry (IHQ) and qRT-PCR. These tests, however, are complex and have disadvantages in terms of turnaround, sensitivity, cost and throughput. The nCounter platform allows joint detection, in a single tube, without any enzymatic reaction and in 72 hours, of multiple fusion genes by transcript-based method from formalin-fixed paraffin-embedded (FFPE) samples.
Methods:
A custom set consisting of 5´and 3´ probes and/or fusion-specific probes to detect ALK, ROS1 and RET fusion transcripts was evaluated. A panel of ALK-ROS-RET positive cell lines (H2228, H3122 [EML4-ALK], SU-DHL-1 [NPM-ALK], HCC78 [SLC34A2-ROS], BaF3 pBABE [CD74-ROS], LC2/ad [RET]) and control fusion negative cell lines (PC9, H1975 [EGFR mut], H460, H23 [KRAS mut]) were used for nCounter validation. To determine the minimum of tumor surface area for detection, ALK translocated cell line H2228 was tested in FFPE at increasing cell numbers (2500, 5000, 10.000, 25000, 50000) corresponding to a surface area of 0.27, 0.55, 1.1, 2.75 and 5.5 mm2, respectively, in the FFPE block. A total of 38 FFPE samples positive by FISH, IHC and/or qRT-PCR for ALK (n=30), ROS (n=7) and RET (n=1) were also analyzed. Total RNA was isolated from cell lines and FFPE and < 225 ng were used for hybridization. Raw counts were normalized using positive controls, negative controls and 4 house-keeping genes (GAPDH, GUSB, OAZ1 and POLR2A) as described in Lira et al. J Mol Diagn 2013. Positive and negative ALK fusion translocation was defined by a 3’/5’ ratio score of > 2.0 and ≤ 2.0 respectively. Response to crizotinib by RECIST criteria was retrospectively collected in patients with ALK-positive NSCLC.
Results:
nCounter sensitivity to predict fusion transcripts ALK, ROS and RET in cell lines by using both methods (3’/5’ and direct reporter probes) was 100%. Results indicate that samples containing as few as 10% positive tumor cells and a 2.75 mm2 tumor surface area were sufficient for adequate gene fusion detection. The accuracy of prediction (AUC) of ALK 3’-5’ ratio score in 45 independent samples was 82.6% (95% CI 69.3-95.6) with a kappa coefficient score of 0.637. Among 28 samples ALK-FISH-positive, ALK 3’-5’ scoring was positive in 27 samples (96%). One sample was non-evaluable by ALK 3’-5’ scoring. Among the 17 samples ALK-FISH-negative, ALK 3’-5’ score was negative and positive in 10 (59%) and 7 (41%) samples, respectively. All patients with ALK-FISH-negative samples but ALK 3’-5’ score positive (n=7) were positive for ALK IHC and 5 of them were treated with crizotinib. Response assessment was available in 3 of these patients and response rate was 100%. One patient non-evaluable by FISH but positive 3’-5’ scoring also responded to crizotinib.
Conclusion:
The ALK/ROS1/RET nCounter-based assay is a highly sensitive screening modality that might identify FISH-negative/non-evaluable NSCLC patients who could benefit from ALK inhibitors.
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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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P2.04-048 - Analysis of Gene Expression in the Re-Replication Pathway and Selective Blockade with Checkpoint Inhibitors as a Therapeutic Option in NSCLC (ID 2594)
09:30 - 09:30 | Author(s): S. Viteri
- Abstract
Background:
Targeted lung cancer therapy has undoubtedly made a difference to the treatment of EGFR mutation and ALK translocation carriers. However, targeted therapies for other subgroups like squamous cell carcinoma are still scarce. Re-replication of the genome could initiate gene amplification and cause chromosomal translocation and loss, contributing to tumor progression. It has been shown that cell cycle checkpoints and DNA damage response are activated when re-replication is induced. Cell cycle checkpoints, mediated by CHK1 and 2, are essential to prevent re-replication and maintain genomic integrity. Specific CHK1 inhibitors such as LY2603618 have been shown to delay tumor growth when given in combination with pemetrexed in NSCLC xenograft models.
Methods:
We selected a panel of NSCLC adenocarcinoma and squamous cell carcinoma cell lines representing different genetic backgrounds with TP53, KRAS and EGFR mutations. In addition, six PC9-derived, TKI resistant cell lines were included (PC9-ER, PC9-GR1 to GR5). Expression of genes involved in the re-replication pathway (MDC1, ATR, ATM, CHEK2, Rap80, Cdc1, Cdc6, MYC, SLX4, CHEK1, BRCA1, BRCA2, p53, ORC4, ORC5, ORC6 and GMNN) was analyzed by RT-PCR. All cell lines were treated with CHK1 and a CHK1/2 inhibitors, and the IC50 was determined by the MTT assay
Results:
We observed different expression levels of key genes involved in the re-replication pathway. Interestingly, a p53 mutated squamous cell line (SK MES1), which has high expression levels of CHK1 and CHK2 (22.31 and 18.66, respectively), showed the lowest IC50 in our study (IC50= 0.024 mM) with a CHK1 selective inhibitor (LY2603618). Also, two EGFR-resistant cell lines, one harbouring the T790M mutation, were highly sensitive to CHK1 inhibition (IC50 of 0.19µM for PC-GR5; 0.40 µM for PC9-GR4). Interestingly, when using a dual CHK1-CHK2, the IC50 is significantly higher in the SK MES1 cell line (84.62 µM vs 0.024 µM) when compared to single CHK1 inhibitonHalf maximal inhibitory concentrations (IC50s) of CHK1 and CHK1-2 inhibitors
Cell line CHK1 (IC50 µM, mean) CHK1-2 (IC50 µM, mean) SK-MES1 0.027 84.62 A549 0.8 15 HCC78 1.2 33.4 H2228 2 0.5 H3255 8.1 12.6 H1975 22.6 9.6
Conclusion:
A great advance has been made in targeted therapy for NSCLC during the last 10 years. Nevertheless, few specific therapeutic options exist for squamous cell carcinoma of the lung nowadays. Different expression of genes involved in the re-replication pathway, and the sensitivity of some NSCLC cell lines (such as SK-MES1, a squamous carcinoma cell line) to selective CHK-1 and dual CHK1-CHK2 inhibitors identify this pathway as a possible therapeutic target worthy of further investigation.
