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Joanna Chorostowska-Wynimko
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P3.01 - Advanced NSCLC (ID 621)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 10/18/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P3.01-041 - The Robustness of Allele-Specific qPCR Assays for Detection of EGFR Mutations in Plasma Cell-Free DNA from NSCLC Patients (ID 9409)
09:30 - 09:30 | Presenting Author(s): Joanna Chorostowska-Wynimko
- Abstract
Background:
The analysis of EGFR mutations in plasma cell-free DNA (cfDNA) may become an auxiliary tool in the molecular diagnosis of advanced NSCLC patients from whom sufficient tumor tissue/cells specimens cannot be obtained. We evaluated the diagnostic performance of the two most common real-time PCR-based platforms for detection of EGFR mutations in plasma cfDNA that we use in our routine laboratory practice.
Method:
EGFR mutations were analyzed in plasma cfDNA collected from 107 NSCLC patients (non-SQC type, stages I-IV) before treatment (n=80) or at the time of progression on EGFR-TKIs (n=27) using two IVD-marked assays: ‘Cobas EGFR Mutation Test v2’ (Roche) and ‘Therascreen EGFR Plasma RGQ PCR Kit’ (Qiagen).
Result:
The overall concordance between EGFR mutation status in plasma cfDNA and paired tumor tissue samples was 62% (46% for Cobas and 32% for Therascreen test) for all patients regardless the NSCLC stage. The concordance increased to 88% (83% for Cobas and to 56% for Therascreen) when only samples from advanced NSCLC patients were evaluated. Accordingly, the Cobas test showed higher positive percent agreement between cfDNA and tissue results (PPA=83%) than did the Therascreen test (PPA=56%). Both test showed 100% negative percent agreement. The T790M mutation was detected in 26% cfDNA samples from patients upon progression on EGFR-TKIs. In 12 (44%) cases with progressive disease, the plasma cfDNA was the only specimen available for EGFR mutation analysis.
Conclusion:
In our laboratory setting, Cobas test demonstrated superior diagnostic performance over Therascreen assay in detection of EGFR mutations in plasma cell-free DNA from advanced NSCLC patients. Since analysis of mutated EGFR cfDNA in plasma is troublesome laboratory procedure, we recommend to validate the true diagnostic performance of each commercial assay in a specific laboratory conditions despite its IVD certification. The study is on-going.
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P3.02 - Biology/Pathology (ID 620)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 2
- Moderators:
- Coordinates: 10/18/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P3.02-049 - The Evaluation of Circulating miRNA Expression in Plasma as the Epigenetic Marker of EGFR Mutation Status in NSCLC Patients (ID 9764)
09:30 - 09:30 | Author(s): Joanna Chorostowska-Wynimko
- Abstract
Background:
The epidermal growth factor receptor (EGFR) mutation status is so far the only validated predictive biomarker of response to EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy in non-small cell lung cancer (NSCLC) patients. MicroRNA (miRNA) is the class of small non-coding RNA that regulates gene expression. Dysregulation of miRNA function has been implicated in lung carcinogenesis. Stable forms of miRNAs are present in blood (circulating miRNAs) and their expression pattern is disease-specific. This phenomenon implies their potential usefulness as molecular markers in NSCLC diagnosis and therapy. Nevertheless, there is no consensus regarding normalization of the circulating miRNA expression analysis. We aimed to evaluate the potential diagnostic usefulness of several circulating miRNAs, that according to different data normalization approaches might have discriminative value for EGFR mutant-positive and negative NSCLC patients.
Method:
Total RNA was extracted from 100 µL of plasma, material with signs of hemolysis was excluded (as per visual inspection and factor ΔCq value (miR-23a-miR-451a)<5). The exogenous UniSP6 RNA spike-in was used as an internal control of extraction efficacy. Expression of 5 miRNA sequences (miR-504, miR-122, miR-195, miR-10b and miR-21) and UniSP6 in plasma of 60 non-squamous NSCLC patients (43 resectable and 17 advanced stage; 31 patients EGFR+ in paired tumor tissue and plasma specimen) was investigated using reverse transcriptase real-time PCR (RT-qPCR), Next, association between circulating miRNA expression and EGFR mutation status was analyzed according to different data normalization approaches (miR-16 and miR-191 as normalizers).
Result:
Among the 5 circulating miRNAs tested, only plasma miR-504 expression was significantly associated with EGFR mutation status in NSCLC patients regardless the normalizer used (p=0.0158 and p=0.002 for miR-16 and miR-191 normalization, respectively). This association was significantly stronger when only plasma samples from adenocarcinoma patients were analyzed (p=0.0004 and p=0.001 for miR-16 and miR-191 normalization, respectively). Moreover, the highest discriminatory power of circulating miR-504 was showed for patients with exon 19 deletions versus those with wild-type EGFR when the data was normalized according to mir-191 (AUC=0.807 p<0.0001).
Conclusion:
Our study demonstrated the feasibility and potential diagnostic value of mir-504 expression analysis in plasma for discrimination between EGFRm+ and EGFRm- NSCLC patients. However, the normalization strategy is of key importance, strongly impacting circulating miRNA analysis outcome. The study is to be continued.
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P3.02-051 - Low Consistency Between FGFR1 Gene Amplification and Protein Expression in Squamous Cell Lung Cancer (SQCLC) (ID 9789)
09:30 - 09:30 | Presenting Author(s): Joanna Chorostowska-Wynimko
- Abstract
Background:
FGFR type 1 (FGFR1) gene amplification has been identified as one of the key potentially actionable targets in SqCLC, given FGFR1 role in oncogenesis. Equivocal results from the pre-clinical and clinical studies point at the necessity of efficient and reliable predictive identification of potential candidates for therapy with FGFR1 inhibitors. Published data regarding correlation between FGFR1 protein expression and FGRF1 gene amplification are discordant. Our previous analyses demonstrated relatively low intra-tumor heterogeneity of both markers in analyzed SqCLC cases. Therefore, we aimed at verifying their concordance, or lack thereof, in the larger series of SqCLC tumors.
Method:
74 SqCLC tumors were analyzed (2 FFPE sections per tumor). FGFR1 gene copy number was assessed by FISH method using probes specific for the 8p12 locus and the chromosome 8 centromere (CEN8). FGFR1 amplification criteria: FGFR1/CEN8 >2.0 or the average number of FGFR1 signals per cell >6 or >10% of tumor cells containing >15 FGFR1 signals. FGFR1 protein expression was determined by immunohistochemistry (IHC). Expression was defined as staining intensity (graded from 0 to 3+) in >1% of the cancer cells. Correlation between FISH and IHC results was performed using the GraphPad Prism software using Spearman test.
Result:
11/74 (14.86%) SqCLC tumors demonstrated FGFR1 amplification. The average FGFR1 gene copy number per cell ranged from 1.23 to 13.97 (mean+SD 3.61+2.08) while the mean FGFR1/CEN8 ratio was 1,27 (range: 0.53–4.35). The mean content of tumor cells with >15 FGFR1 copies was 10.19%. In IHC(+) tumors (22/74, 29.73%) the percentage of stained cancer cells with intensity >2 was low - only 12/74 (16.22%) samples.The FISH and IHC results were consistent in 79.73% SqCLC tumors (n=59), 55/74 (74.32%) tumors were double-negative, while only 4/74 (5.41%) double-positive. 15/74 pts results were discordant: 7/74 (9.46%) IHC(-) FISH(+), while 8/74 (10.81%) pts IHC(+) FISH(-). There was no correlation between FGFR1 amplification and FGFR1 protein overexpression (P=0.466; r=0.086) in the analyzed series of 74 SqCLC tumors.
Conclusion:
While we demonstrated relative SqCLC tumor homogeneity in terms of FGFR1 amplification and expression for as many as 74% of tumors, most were double negative. In samples demonstrating any positivity, FGFR1 amplification did not relate to protein expression. Therefore, further more detailed comparative evaluation of FGFR1 gene expression or FGFR1 locus might be informative to better understand the determinants of response to FGFR inhibitors. Study funded by NCBiR: 'Development of novel biomarkes and innovative FGFR kinases inhibitor as an anti-cancer therapy' (nr 266776, program: STRATEGMED2).
