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J. Chacartegui
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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: 2
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-027 - Erlotinib Induce miRNA Alterations in T790M EGFR Mutated NSCLC: Preclinical Study (ID 2483)
09:30 - 09:30 | Author(s): J. Chacartegui
- Abstract
Background:
Lung cancer is one of the leading causes of cancer-related deaths worldwide. In the most common type, non-small cell lung cancer (NSCLC), an array of oncogenic driver mutations affecting growth factor receptors and signaling pathways, such as EGFR, KRAS, c-Met or ALK translocation has driven the development of directed-drug therapies with tyrosine kinase inhibitors (TKIs) and monoclonal antibodies. Nevertheless, the appearance of resistance mechanisms, such as c-Met amplification or de novo mutations in EGFR, decreases the effectiveness of these therapies. Therefore, we analyzed the levels of miRNA which have proved to be involved in disease progression (miR-30b-5p,195-5p,221-3p) in NSCLC cells that are resistant (H1975) or sensitive (HCC827) against first generation TKI (erlotinib) treatment, to assess whether they are markers of response to the treatment.
Methods:
The H1975 cell line (EGFR mutations T790M/L858R) was cultured in RPMI 1640 medium, supplied with 10% FBS, 2 mM L-glutamine and 1% penicillin/streptomycin (Gibco). The sulforhodamine B assay was performed to assess cell proliferation. The cells were treated during 72h with 10µM erlotinib (SelleckChem) or DMSO. After collection, cells were lysed and RNA was extracted through commercial kit (RNA Mini Spin, GE Healthcare). The miRNAs profile analysis was performed through TaqMan Real-Time PCR and miRNA RNU-48 was used as endogenous control. Data was processed according to the formula 2[-ΔΔct]. Control values (H1975 + vehicle) are used as baseline and results are shown in logarithmic scale (Image).
Results:
Cells treated with 10µM erlotinib show an increased expression of miR-30b-5p, miR-195-5p compared to control values. On the other hand, we detected that the expression of the miR-221-3p was strongly down-regulated in respect the control values.
Conclusion:
H1975 carries the T790M mutation in EGFR, associated in NSCLC with appearance of resistance to TKIs treatment. However, we observed after treatment an increase of onco-suppressor miR-30b-5p and decrease of the oncogenic mir-221-3p, which are reported to correlate with good prognosis (Garofalo 2013, Zhong 2014). Moreover, mir-195-5p, another miRNA related with onco-suppression, is also upregulated, which has been reported to correlate with good response (Liu, 2015). Overall, our data suggests that erlotinib treatment alters miRNA in an EGFR mutated cell line. Further analysis of miRNA in exosomes produced by H1975, and comparison with HCC827 exosomal and cellular miRNA levels is currently undergoing in our group to confirm their value as response to treatment biomarkers.Figure 1
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P2.04-094 - Exosomal MiRNA Analysis of Non-Small Cell Lung Cancer (NSCLC) Liquid Biopsies. Mirror of the Disease Status?: Proof of Concept Study (ID 1395)
09:30 - 09:30 | Author(s): J. Chacartegui
- Abstract
Background:
The discovery of the alterations in EGFR, c-Met or ALK in NSCLC has driven the development of targeted-drug therapy using tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is needed. The exosomes extracted from blood samples could be non-invasive and regularly updated biomarkers. Here we analyze selected exosomal miRNAs to evaluate its biomarker potential in NSCLC.
Methods:
1 ml of serum/plasma sample from 11 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. Exosome were isolated through commercial-kit or D~2~O/sucrose density-gradient ultracentrifugation. After exosome characterization (Western-Blot, Transmission Electron Microscopy) a panel of miRNAs (30b-5p, 30c-5p, 103,195,221-3p,222-3p), correlated with NSCLC disease (Garofalo et al, 2013), was analyzed. The miRNAs profile analysis was performed through TaqMan Real-Time PCR and mir-1228-3p was used as endogenous control. The data was processed according to the formula 2[-ΔΔct]. Control values are used as baseline and results are shown in logarithmic scale (figure).
Results:
Patients without molecular alterations (WMA): The levels of miR-30b-5p/30c-5p/103/221-3p/222-3p were down-regulated relative to the healthy controls. The patient with docetaxel treatment (P2) has an increased down-regulation of these miRNAs compared to the non-treated patient (P1). Patient with BRAF G464V: We observed an increased up-regulation of miR-30b-5p/30c-5p/103/221-3p/222-3p just after stopping Erlotinib treatment (P4a) compared with one month after the treatment (P4b). Patients with C-Met 3+ over-expression: We detected an increase of expression of miR-30b-5p/30c-5p and a decrease of expression of mir221-3p/222-3p in a patient treated with Crizotinib (P6) compared to a non-treated patient (P5). Patients with EGFR (exon 19 del): We observed a decrease of expression of mir221-3p/222-3p in a patient treated with Afatinib beyond progression (P8) compared to a non-treated patient (P7). Patients with ALK t(2p23): We detected a decrease of expression of miR-30b-5p/30c-5p/103 compared to healthy controls. No differences between treated (P9-P11) and non-treated (P3) patients were observed. Nevertheless, mir221-3p/222-3p differs significantly between patients treated with Ceritinib one week (P10) and one month (P11) after the treatment was started.
Conclusion:
This panel of exosomal miRNAs derived from patients with varying mutations is responsive to different treatments. The down-regulation of miR-30b-5p/30c-5p in exosomes of patients with WMA and ALK t(2p23) mutations, mirrored the reported low levels of these miRNAs in NSCLC tissue (Zhong et al.,2014). Follow-up analysis to correlate clinical progression and exosome miRNAs profile is currently ongoing. Figure 1
