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E.K. Verbeken
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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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MINI22.06 - The Challenge of Molecular Testing for Clinical Trials in Advanced Non-Small Cell Lung Cancer Patients: Analysis of a Prospective Database (ID 1240)
17:15 - 17:20 | Author(s): E.K. Verbeken
- Abstract
- Presentation
Background:
Molecular testing has become important in managing advanced non-small cell lung cancer (NSCLC), both in clinical practice, as well as in clinical trials. For the latter, tissue samples often have to be analysed in a central laboratory. We evaluated the turnaround time and possible delay in start of therapy in this process.
Methods:
We reviewed our prospective database on all molecular testing cases for clinical trial suitability in patients with advanced NSCLC between March 1, 2011 (start) and October 31, 2014. The following time points were considered: T1 (request for tissue sections from the pathology lab); T2 (receipt of sections and shipment); T3 (arrival of sections in central lab (CL)); T4 (receipt of biomarker result from CL).
Results:
251 patients were considered for biomarker-driven trials. Twenty-three cases did not have further analysis, as the request for central molecular testing was cancelled: insufficient tissue (n=11); exclusion criterion (n=10); patient refusal (n=2). Results for the remaining 228 patients were: failure of central biomarker analysis due to insufficient quantity of tissue (n=18), or quality of tissue (n=3, i.e. decalcification or poor fixation). Valuable results were obtained for 207 patients. In 91 of 228 (39.9%) samples sent, a biomarker of interest was documented. This led to 34 clinical trial inclusions. Other patients were no longer eligible due to loss of performance status (n=20), loss of contact (n=14), no trial slot available at the appropriate time (n=18), or exclusion criteria (n=5). The mean waiting time between signing informed consent (T1) and receiving results of the biomarker analysis (T4) was 25.1 calendar (SD 17.3) days (Table). The preparation of the unstained slides by the pathology lab took about 9.1 (SD 6.8) days, the time of the biomarker testing itself accounted for 12.8 (SD 7.3) days. For 18 of 228 (7.9%) patients, repeated sample shipments were needed because of insufficient tumor cells, their mean waiting time between informed consent and receiving the biomarker result was 62.2 (SD 38.4) days. Table: Waiting times (t) in molecular testing for 228 patients.Time interval Mean StDev Median Range Pathology lab (T2-T1) 9.1 6.8 7.0 1 - 70 Shipment (T3-T2) 1.8 1.6 1.0 0 - 17 Analysis (T4-T3) 12.8 7.3 12.0 2 - 58 Request to result (T4-T1) 25.1 17.3 22.0 7 - 184
Conclusion:
While molecular testing is important in many NSCLC trials, our results show that waiting times for central laboratory analysis can cause an important delay in treatment initiation, and even ineligibility for the trial(s) under consideration. Start of therapy based on properly validated local testing, with a posteriori central biomarker testing to guarantee the integrity of the trial, would be more rewarding for quite some patients.
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ORAL 37 - Novel Targets (ID 146)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:S.S. Ramalingam, E. Thunnissen
- Coordinates: 9/09/2015, 16:45 - 18:15, Mile High Ballroom 4a-4f
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ORAL37.05 - Prevalence and Clinical Association of MET Gene Amplification in Patients with NSCLC: Results from the ETOP Lungscape Project (ID 444)
17:28 - 17:39 | Author(s): E.K. Verbeken
- Abstract
Background:
The reported prevalence of MET gene amplification in non-small cell lung cancer (NSCLC) varies from 0-21% and clinical correlations are emerging slowly. In a well-defined NSCLC cohort of the ETOP Lungscape program, we explore the epidemiology, the natural history of MET amplification and its association with MET overexpression, overall survival (OS), relapse-free survival (RFS) and time to relapse (TTR).
Methods:
Resected stage I-III NSCLC, identified based on the quality of clinical data and FFPE tissue availability, were assessed for MET gene copy number (GCN) and expression analysis using silver in-situ hybridization (SISH) and immunohistochemistry (IHC), respectively, on TMAs (MET and centromere-specific probes; anti total c-MET antibody, clone SP44; Ventana immunostainer). MET amplification was defined as MET/centromere ratio ≥2 with average MET GCN ≥4, high MET GCN at two levels as ≥median CGN and ≥5 (irrespective of amplification) and MET IHC+ as 2+ or 3+ intensity in ≥50% of tumor cells. Sensitivity analysis to define the amplification’s thresholds was also performed. All cases were analysed at participating pathology laboratories using the same protocol, after successful completion of an external quality assurance (EQA) program.
Results:
Currently 2709 patients are included in the Lungscape iBiobank (median follow-up 4.8 years, 53.3% still alive). So far, 1547 (57%) have available results for MET GCN with amplification detected in 72 (4.7%; 95%CI: 3.6%, 5.7%) and high MET GCN (≥5) in 65 (4.2%; 95%CI: 3.2%, 5.2%). The median value of average MET GCN per cell is 2.3. IHC MET expression is available for 1515 (98%) of these cases, 350 (23%) of which are MET IHC positive [170 cases (49%) 3+, 180 (51%) 2+]. The median age, for the cohort of 1547 patients, is 66.2 years, with 32.8% women, and 13.5%, 29.7%, 54% never, current, former smokers, respectively. Stage distribution is: IA 23.6%, IB 24.6%, IIA 17%, IIB 12.1%, IIIA 20.9%, IIIB 1.8%, while 52.7%, are of adenocarcinoma and 40.0% of squamous histology. MET amplification and high MET GCN (≥5) are not significantly associated with any histological tumor characteristics or stage (multiplicity adjusted alpha: 0.005). High MET GCN (≥2.3) is less frequent in current smokers (38.3% vs. 55.6% for former or non-smokers, p<0.001). MET amplification and high MET GCN are significantly associated with IHC MET positivity (p<0.001 in all cases). MET amplification is present in 9.7% of IHC MET+ vs 3.1% of IHC MET- patients and high MET GCN (≥5) in 8.6% of IHC MET+ vs 2.8% of IHC MET- patients. MET amplification ranges from 0 to 16% between centers, while high MET GCN (≥5) and (≥2.3) from 0% to 12%, and 11.8% to 98.9%, respectively. MET amplification and both levels of high MET GCN are not associated with OS, RFS or TTR.
Conclusion:
The preliminary results for this large, predominantly European, multicenter cohort demonstrate that MET amplification assessed by SISH prevails in 4.7% of NSCLC, is associated with strong MET expression, and has no influence on prognosis. The large inter-laboratory variability in GCN despite EQA efforts may highlight a critical challenge of MET SISH analysis in routine practice.
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P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.04-009 - Evaluation of RT-PCR Methodology for ALK Assessment in Patients with NSCLC in Europe: Results from the ETOP Lungscape Project (ID 1506)
09:30 - 09:30 | Author(s): E.K. Verbeken
- Abstract
Background:
ALK rearrangement is documented in 2%-7% of NSCLC, depending on the population studied and detection method used. Although the reverse transcriptase-polymerase chain reaction (RT-PCR) was the first used and published method, fluorescence in situ hybridization (FISH) has become the primary standard diagnostic method. Recently, immunohistochemistry (IHC) has also proven to be a reproducible, faster and sensitive technique. This is one of the first studies concurrently comparing all three techniques in resected lung adenocarcinomas from the large ETOP Lungscape cohort.
Methods:
95 cases from the ETOP Lungscape iBiobank, selected based on any degree of IHC staining (clone 5A4 antibody, Novocastra, UK), were examined by ALK FISH (Abbott Molecular, Inc.; Blackhall, JCO 2014) and central RT-PCR. For the latter, formalin-fixed, paraffin-embedded (FFPE) unstained slides were collected from participating centers. Slides were de-paraffinized, Toluidine Blue stained, and tumors macro-dissected. Tissue digestion and RNA extraction were performed (Qiagen RNeasy FFPE Kit). Using primers described in the literature covering most of ALK known translocations, RT-PCR (Superscript One-Step RT-PCR with Platinum Taq – 40 loops) was performed, followed by capillary electrophoresis in two separate mixes. Co-amplification of B-actin was done to validate the procedure and RNA quality. All tests were duplicated.
Results:
76 of 95 RT-PCR had adequate RNA quality (B-actin co-amplification present). Among these, 18 were FISH positive, 16 were RT-PCR positive, including EML4-ALK V3a/b in 7, V1 in 5, V2 in one, and undetermined variants in 3 cases. 53 of 54 FISH negative cases were also RT-PCR negative (98%). 15 of 18 FISH positives harbored a translocation by RT-PCR (83%). Among the 4 discrepant cases, 2 FISH+/RT-PCR- cases had IHC H-scores of 180 and 260, and 98.3% and 95% of rearranged cells by FISH, probably corresponding to variants not covered by the RT-PCR. One had an IHC H-score of 5, and 16% cells rearranged on FISH, most probably corresponding to a FISH false positive case. The last had an IHC H-score of 200, 13% rearranged cells by FISH, and, thus is defined as a false negative FISH result. Provided IHC is defined as positive by an H-score above 120, all but one case (H-Score 20, FISH and RT-PCR positive) gave concordant results by a combination of FISH and RT-PCR. Overall, using as true negative or true positive the concordant result of two of the methods, the third method is characterized by high specificity and sensitivity with corresponding values of 100/98/100% and 94/94/89% for IHC/FISH/RT-PCR, respectively.
Conclusion:
RT-PCR is a very good tool for sorting discordant IHC/FISH cases, however, we do not recommend using this technique as single method due to the lower sensitivity of RT-PCR, as not all variants are covered, and also due to the limitations with RNA preservation.
