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N. Leonard
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P1.03 - Chemotherapy/Targeted Therapy (ID 689)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Chemotherapy/Targeted Therapy
- Presentations: 1
- Moderators:
- Coordinates: 10/16/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P1.03-039 - Therapeutic Inhibition of the Cancer Stem Cell Marker, ALDH1, a Promising Mechanism by Which Cisplatin Sensitivity Can Be Restored in NSCLC (ID 9909)
09:30 - 09:30 | Author(s): N. Leonard
- Abstract
Background:
Cisplatin remains the cornerstone of current chemotherapeutic combination startegies in the treatment of NSCLC. Despite initial cisplatin sensitivity tumours develop resistance, which in turn undermines the efficacy of cisplatin as a therapuetic agent. Numerous mechanisms, signalling pathways and theories have been suggested and elucidated in terms of cisplatin resistance and in the development of it, however, to date the clinical issue of resistance has not been overcome. A current avenue of interest is the cancer stem cell (CSC) hypothesis, in which the survival and expansion of highly resistant CSCs during chemotherapeutic treatment are thought to be a contributing factor of resistance and recurrence. Specific inhibition of key CSC markers in combination with chemotherapy may undermine the inherent resistance of the CSC population and sensitise these cells to the cytotoxic effects of therapy. One such CSC marker observed across numerous tumours is aldehyde dehydrogenase 1 (ALDH1), our hypothesis suggests that inhibition of the ALDH1-positive CSC population within cisplatin resistant NSCLC will resensitise the cellls to the cytotoxic effects of cisplatin.
Method:
Using an isogenic panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines ALDH1 was identified as a CSC marker present within the CisR sublines of each NSCLC histology and characterised as CSCs. ALDH1 was inhibited using two pharmacological ALDH1 inhibitors, diethlylaminobenzaldehyde (DEAB) and disulfiram (commercially known as Antabuse used in the treatment of alcoholism). ALDH1 inhibition was confirmed by flow cytometry. PT and CisR cell lines were treated with inhibitor alone and in combination with cisplatin and assessed in terms of proliferation, clonogenic survival and apoptosis relative to cisplatin-only treatment.
Result:
Both DEAB and the FDA-approved disulfiram significantly decreased the presence of the ALDH1-positive CSC subpopulation across all CisR cell lines. DEAB and disulfiram in combination with cisplatin induced a significant decrease in proliferation and clonogenic survival as well as significant increases in cisplatin-induced apoptosis across CisR sublines when compared to cisplatin alone.
Conclusion:
DEAB and disulfiram significantly reduced the presence of the highly resistant ALDH1-positive CSC subpopulation. This pharmacological CSC depletion in conjunction with cisplatin was associated with the resensitisation of cisplatin resistant cells to the cytotoxic effects of cisplatin, thus restoring cytotoxic efficacy. The resensitisation effect of the disulfiram-based combination strategy, as well as its FDA-approval and extentsive safety profile highlights this strategy as one of great promise. In summary, these data suggest a role for ALDH1 inhibition in the resensitisation and possible circumvention of cisplatin resistance.
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P2.02 - Biology/Pathology (ID 616)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/17/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P2.02-064 - A Novel 5-miR Signature Shows Potential as a Diagnostic Tool and as a Predictive Biomarker of Cisplatin Response in NSCLC (ID 9957)
09:30 - 09:30 | Author(s): N. Leonard
- Abstract
Background:
MicroRNAs are a class of small non-coding RNAs that range in size from 19-25 nucleotides. They have been shown to regulate a number of processes within tumour biology, including metastasis, invasion and angiogenesis. More recently, miRNAs have been linked to chemoresistance in solid tumours, including lung cancer. Their role in cisplatin resistance has yet to be determined.
Method:
MicroRNA expression within a panel of age-matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines was profiled using the 7[th] generation miRCURY LNA arrays (Exiqon) and subsequently validated by qPCR. Significantly altered miRNAs within the CisR sublines were manipulated using antagomirs (Exiqon) and Pre-miRs (Ambion) and functional studies were carried out in the presence and absence of cisplatin. To examine the translational relevance of these miRNAs, their expression was examined in a cohort of chemo-naïve patient-matched normal and lung tumour tissue and serum from NSCLC patients of different histologies. A xenograft model of cisplatin resistance was carried out in which 1x10[3] H460 PT or CisR cells were injected into 5-7week old NOD/SCID mice. Tumour volume was measured over time and harvested once the tumour mass measured 500mm[3] and formalin-fixed and paraffin embedded (FFPE). Expression of the 5-miR signature was analysed within FFPE murine tumours and compared between PT and CisR tumours.
Result:
Profiling and subsequent validation revealed a 5-miR signature associated with our model of cisplatin resistance (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p, miR-4286). Inhibition of the miR-30 family and miR-34a-5p reduced clonogenic survival of CisR cells when treated cisplatin. Expression of the miRNA signature was significantly altered in both adenocarcinoma (AD) and squamous cell carcinoma (SCC) relative to matched normal lung tissue and between SCC and AD tissue. miR-4286 was significantly up-regulated in SCC sera compared to normal control and AD sera. Similarly to the cell line expression of the miRNAs, the miR-30 family members and miR-34a-5p were up-regulated in the CisR xenograft FFPE tissue relative to PT.
Conclusion:
A novel miRNA signature associated with cisplatin resistance was identified in vitro, genetic manipulation of which altered clonogenic response to cisplatin. The 5-miR signature showed both diagnostic and prognostic biomarker potential across a number of diagnostically relevant biological media.
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P3.02 - Biology/Pathology (ID 620)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/18/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P3.02-053 - Optimization and Characterization of Assays to Identify Met Exon 14 Skipping in FFPE Embedded NSCLC Samples (ID 9881)
09:30 - 09:30 | Author(s): N. Leonard
- Abstract
Background:
The hepatocyte growth factor (HGF)receptor (MET), is frequently altered in NSCLC. Despite having a significant number of diverse mutations/alterations, randomized trials with MET inhibitors have proved disappointing, with no clinical benefit (1). More recently MET exon 14 skipping alterations have emerged as potential therapeutic targets as MET exon as they inhibit the degradation of Met, prolonging its oncogenic activity (2). Patients with Met exon 14 skipping have been found to sensitive to MET inhibitors such as crizotinib, and clinical trials of MET TKIs in METex14 mutated NSCLC are ongoing (1).
Method:
A one-step RT-PCR end-point PCR assay to examine for the detection of Met exon14 skipped mRNAs in FFPE was designed, optimized and tested on a cohort of NSCLC patients. Positive samples were confirmed by targeted next-generation sequencing of these samples. Finally RNA in situ hybridization (RISH) was optimised on a cMET exon 14 skipped cell line (NCI-H596) and subsequently performed on full-face sections using a specific BaseScope™ Assay (Techne).
Result:
Initial studies found that standard end-point PCR resulted in significant false-positives. However, a one-step RT-PCR methodology resolved this issue. Met exon 14 skipped samples were then examined in a cohort of pulmonary sarcomatoid carcinomas (PSCs). In agreement with another study of Caucasian patients (3), we identified Met Exon 14 skipped mutations in 2/20 (10%) of patients. Expression of Met exon 14 skipped was confirmed using targeted resequencing by NGS. RISH was also examined in the same samples.
Conclusion:
These results demonstrate the optimization of a methodology to robustly detect Met exon 14 mutated patients in FFPE material by a PCR based assay, with results comparable to those obtained in similar studies. This methodology can be utilised by any standard hospital diagnostic laboratory without the need for any specialized technology such NGS, RISH or FISH. A qPCR based version of this assay is currently being optimized and the results will be presented at the meeting. References Reungwetwattana, T. et al., (2017). Lung Cancer 103: 27–37 Pilotto, S. et al. (2017). Ann Transl Med 5(1):2 Saffroy, R. et al (2017). Oncotarget. 2017 Mar 21. doi: 10.18632/oncotarget.16403. [Epub ahead of print]
