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Rajagopal Ramesh
Author of
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P2.01 - Advanced NSCLC (ID 159)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Advanced NSCLC
- Presentations: 1
- Now Available
- Moderators:
- Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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P2.01-83 - Role of Exosomal MicroRNAs (miRNAs) as Predictors of Response to Treatment and Prognosis in Non-Small Cell Lung Cancer (NSCLC) (Now Available) (ID 2386)
10:15 - 18:15 | Author(s): Rajagopal Ramesh
- Abstract
Background
Exosomes are 30 - 100 nm cellular entities secreted from cells. Exosome contain messenger RNA, miRNAs, DNA and active peptides . Cancer derived exosomes have distinct miRNA profiles. Signature profile of miRNAs can be utilized for disease characterization including response . We are evaluating exosomal miRNAs from urine of NSCLC patients.
Method
Urine (30-50ml) was collected, before and after treatments in 5 advanced NSCLCs. Exosomes were isolated by differential ultracentrifugation method, miRNA libraries were constructed using 100 ng of total RNA isolated from exosomes and sequenced on the Illumina MiSeq platform. A minimum of 20 million 50base pair sequencing reads were collected and data analyzed using CLC Genomics Workbench Software. miRNAs that was significantly changed ( up- or down-regulated) in the matched samples were identified as candidate biomarkers. Target mi RNAs were analyzed with bioinformatics tools.
Result
miRNA sequencing (RNAseq) showed 111,416 sequence hits for small non-coding RNAs and after aligning with miRBase21, 548 mature miRNAs were identified. The differentially expressed miRNAs clustered separately in pre- and post-treatment samples in all five-patients. Two miRNAs (hsa-miR-6842 and hsa-miR-143) showed marked reduction in post-treatment samples compared to pre-treatment samples. Validation studies by quantitative (q)PCR using miRNA specific primers confirmed the RNAseq results. In silico target analysis identified RAS oncogenic family as a prominent target for these miRNAs.
Table 1; characteristics of patients, miRNAs changes (fold reduction), response and long term outcomes
Patients
1
2
3
4
5
Age/Sex
42/male
74/female
59/male
81/male
68/male
Stage
4
4
4
4
3 (T1N3)
Pathology
Adenocarcinoma
Squamous cell
Adenocarcinoma
Squamous cell
Tissue Molecular Abnormalities
KRAS G12V, STK11 K62fs*99, NFKBIA amplification, NKX2-1 amplification, TP53 R249W
Not tested
STK11 Y156fs*5, CDKN2A p16INK4a E69fs*77 and p14ARF G83fs*51+, MYST3 S1496L, TP53 splice site 919+1G>
Met exon 14 splicing
Not tested
Treatment
Carbo, Permetrexed, Bev X2, f/b carbo/etoposide with radiation
Carboplatin/
Paclitaxel
Carboplatin/
Paclitaxel
Carboplatin/
Paclitaxel
Carboplatin/
Paclitaxel with radiation
Change in
mir-143
-4.371
-1.25
-1.27
-1.139
-15.7
Change in
mir-6842
-21.37
-6.27
-4.21
-3.41
-4.57
Treatment Response
PR
PR
SD
SD
CR
Current Status
Alive, 38 months since diagnosis
Died 18 months after diagnosis
Died 6 months after diagnosis
Alive, 38 months since diagnosis
Alive, 32 months since diagnosis
Out of all mi RNAs, miR-143 and miR 6842 showed maximum reduction in value and changes were more pronounced in patients1 and 5. Both patients received radiation with chemo and had very good response and prolonged control of disease with longer survival. Interestingly both of miRNAs are targeting KRAS mRNA. Patient#1 had K ras mutation while Patient#5 was not tested
Conclusion
Based on our initial experiments we conclude that upon receiving chemo+ radiation combination therapy the miRNA 143 and miRNA 6842 are packaged at lower levels in exosomes by the tumor cells i.e. cells are trying to retain miRNAs within themselves. Since retention of miRNAs will decrease the levels of target miRNA which is KRAS which will result in better treatment outcome. Our study suggests that urine derived exosomal miRNAs 143 and 6842 can be used as prognostic markers to assess treatment response.
This is a preliminary data and study is ongoing.
