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E. Thunnissen
Moderator of
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ORAL 37 - Novel Targets (ID 146)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 8
- Moderators:S.S. Ramalingam, E. Thunnissen
- Coordinates: 9/09/2015, 16:45 - 18:15, Mile High Ballroom 4a-4f
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ORAL37.01 - FISHing TRK Activation by Gene Rearrangements in Non Small Cell Lung Cancer (ID 834)
16:45 - 16:56 | Author(s): M. Varella-Garcia, S.L. Kako, C.M. Nguyen, S. Saichaemchan, W. Ariyawutyakorn, S. De, S.B. Keysar, A. Jimeno, M. Roncalli, A. Santoro, L. Toschi, A.T. Le, D.L. Aisner, R.C. Doebele
- Abstract
- Presentation
Background:
The tropomyosin-receptor kinase (TRK) family includes genes important in nervous system development, NTRK1 (N1), NTRK2 (N2) and NTRK3 (N3). Oncogenic activation was identified long ago as N1 fusions in colon cancer and numerous fusions have been recently identified affecting all family members in multiple tumor types. This study developed FISH reagents for molecular diagnosis of NTRK rearrangements and investigated their prevalence in NSCLC. The ultimate goal is to validate a clinical assay for selection of patients who may benefit from novel tyrosine kinase inhibitors (TKIs) targeting these fusion proteins.
Methods:
Three FISH break-apart (BA) probe sets (LDTs) were tailored for diagnosis of rearrangements in N1, N2 and N3 and tested in specimens with known genomic status for these genes: cell lines KM12 (N1), CUTO3 (N1), MO-91 (N3), xenograft CULC001 (N1), and clinical specimens, and used to screen resected NSCLC. The LSI NTRK1 Cen and Tel probes (Abbott Molecular) were also tested. A specimen was positive for individual rearrangement when ≥15% tumor cells had split or single 3’,5’ signals. Moreover, a 6-target, 2-color FISH probe including the 3’N1, 3’N2 and 3’N3 sequences labeled in red and the 5’N1, 5’N2 and 5’N3 sequences labeled in green (TRKombo) was designed for rapid screening of TRK rearrangements in clinical specimens.
Results:
Results were obtained in 443, 410, and 434 examined NSCLC and positive patterns were detected in 5, 5 and 1 specimens, respectively for N1, N2, and N3. These 11 positive patients had age ranging from 38y to 76y, gender 6 male:5 female, and were current (4), former (5) or never (2) smokers. Histology was predominantly adenocarcinoma (7) but also included squamous cell (3) and neuroendocrine morphology (1). Unique to the N1 assay was the observance of FISH signal fusions where the 5’N signals appeared as doublet in >20% of the NSCLC specimens, which was determined to be copy number variation due to segmental duplication. Other atypical patterns were observed for all three targets and included doublets of the FISH fusion signals (18%, 14% and 9% respectively) and gene clusters (~5% for each). Twenty specimens (pre-clinical models and clinical cases) characterized as positive by the LDT N1 and by next generation sequencing (NGS) or atypical by the LDT NTRK1 BA were blindly analyzed with the LSI NTRK1 probe set and the results were reproducible, with brighter intensity of the fluorescent signals for the LSI probe. These specimens (positive by FISH and several atypicals) are currently under investigation to characterize the sequence specific genomic rearranged region by using a custom targeted, capture-based NGS panel (NimbleGen, Roche). The TRKombo screening probe performed well in blinded experiment using validation set including pre-selected positive and negative specimens and is under testing in clinical tissue sections.
Conclusion:
N1, N2 and N3 fusions were detected by FISH in a subset of lung carcinomas including adeno, squamous and neuroendocrine tumors. Optimization of molecular panels for diagnosis of these rearrangements is relevant since they represent a sizeable number of cases across multiple tumor types and there are numerous targeted inhibitor agents under development.
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ORAL37.02 - Protein Tyrosine Phosphatase Non Receptor 11 PTPN11/Shp2 as a Driver Oncogene and a Novel Therapeutic Target in Non-Small Cell Lung Cancer NSCLC (ID 1590)
16:56 - 17:07 | Author(s): Y. Elamin, S. Toomey, A. Carr, K. Gately, S. Rafee, P. Morris, J. Crown, O. Breathnach, K.J. O'Byrne, B. Hennessy
- Abstract
Background:
PTPN11/Shp2 somatic mutations occur in 25% of Juvenile myelomonocytic leukemias (JMML) and less commonly in adult solid tumors. PTPN11/Shp2 activates the mitogen-activated protein kinase (MAPK) and the phosphatidylinositide 3-kinase (PI3K) pathways. Accordingly, PTPN11/Shp2 mutations were shown to sensitize leukemia cells to MEK and PI3K inhibitors.
Methods:
We applied mass-spectrometry based genotyping (Sequenom Inc., Germany) to DNA extracted from tumor and matched normal tissue of 356 NSCLC patients (98 adenocarcinomas and 258 squamous cell (SCC)). PTPN11/Shp2 constructs with mutations (E76A, A72D) were generated and stably expressed in IL-3 dependent BaF3 cells and NSCLC cell lines (H1703, H157). The acquisition of MAPK and PI3K pathways activation was evaluated using western blotting and reverse phase protein array (RPPA). PTPN11/Shp2 phosphatase activity was measured in whole cell protein lysates using Shp2 assay kit (R&D Systems).
Results:
Somatic PTPN11/Shp2 hotspot mutations occurred in 3 (3.1%) and 9 (3.4%) of adenocarcinomas and SCCs, respectively. Mutant PTPN11/Shp2, compared to PTPN11/Shp2 wild type, promoted ten-fold IL-3 independent BaF3 cell survival. BaF3, H1703, and H157 cells expressing mutant PTPN11/Shp2 exhibited increased PTPN11/Shp2 phosphatase activity, phospho-ERK1/2, and phospho-AKT levels. Sequencing of NSCLC cell lines revealed that NSCLC H661 cell line has a PTPN11/Shp2 activating mutation (N58S). H661 had significantly higher PTPN11/Shp2 phosphatase activity when compared to PTPN11 wild-type H1703 and Calu3 NSCLC cells. Since the biological functions of PTPN11/Shp2 are mediated through its phosphatase domain, we stably expressed the inactivating PTPN11/Shp2 phosphatase domain mutation (C459S) in H661, H1703 and H157 cells resulting in catalytically inactive PTPN11/Shp2. This led to decreased phospho-ERK1/2 levels in all three cell lines. Importantly, the inactivation of PTPN11/Shp2 resulted in decreased phospho-AKT levels in H661 cells (PTPN11-mutated) and had no effect on phospho-AKT levels in the PTPN11/Shp2-wild type H1703 and H157 cells. Taken together, this data suggests that PTPN11/Shp2 activating mutations are oncogenic in NSCLC cells. Moreover, these findings reveal that PTPN11/Shp2 mutations may selectively activate the PI3K pathway in NSCLC cells. Parental H661 (PTPN11-mutated, KRAS and PIK3CA-wild type), parental H1703 (PTPN11, KRAS and PIK3CA-wild type) and parental H157 (KRAS-mutated, PTPN11 and PIK3CA-wild type) cells were treated with the novel MEK (BAY86-9766) and PI3K (BAY80-6946) inhibitors. IC50 values (table 1) suggest that PTPN11-mutated NSCLC cells have modest sensitivity to MEK inhibitors and profound sensitivity to PI3K inhibitors.Table 1 IC 50 valuse
Cell Line BAY86-9766 (nM) BAY80-6946 (nM) H661 2880 ± 600 13 ± 4.7 H157 1450 ± 520 < 50% inhibition @ 200 H1704 < 50% inhibition @ 10000
Conclusion:
PTPN11/Shp2 demonstrates the in vitro features of a driver oncogene, and potentially represents a new target in NSCLC.
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ORAL37.03 - Discussant for ORAL37.01, ORAL37.02 (ID 3464)
17:07 - 17:17 | Author(s): L.E. Raez
- Abstract
- Presentation
Abstract not provided
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ORAL37.04 - Comprehensive Genomic Profiling (CGP) of Advanced Cancers Identifies MET Exon 14 Alterations That Are Sensitive to MET Inhibitors (ID 3156)
17:17 - 17:28 | Author(s): G.M. Frampton, S.M. Ali, J.W. Goldman, C. Lee, J. Weiss, J.A. Bufill, R. Salgia, M. Jahanzeb, K. Konduri, P. Forde, D. Morosini, J.S. Ross, P.J. Stephens, V. Miller, I. Ou
- Abstract
- Presentation
Background:
Amplifications and activating mutations in the c-MET proto-oncogene are known oncogenic drivers that have proven responsive to targeted therapy. Mutations causing skipping of MET exon 14 are also oncogenic, but less well characterized. We undertook comprehensive genomic profiling (CGP) of a large series of advanced cancers to further characterize MET exon 14 alterations.
Methods:
DNA was extracted from 40 microns of FFPE sections from 38,028 advanced cancer cases. CGP was performed on hybridization-captured, adaptor ligation based libraries to a mean coverage depth of >500x using three versions of the FoundationOne test. Hybridization capture baits for the MET gene were identical for all three versions of the test. Base substitution, indel, copy number alteration, and rearrangement variant calls were examined to identify those nearby to the splice junctions of MET exon 14. These genomic alterations were then manually inspected to identify those likely to affect splicing of exon 14, or delete the exon entirely.
Results:
221 cases harboring MET ex14 alterations were identified. These patients had a median age of 70.5 years (range 15-88), with 97 males and 124 females. The cases were lung carcinoma (193), carcinomas of unknown primary (15), brain glioma (6), and one each of adrenal cortical carcinoma, hepatocellular carcinoma, histiocytic sarcoma, renal cell carcinoma, rhabdomyosarcoma, skin merkel cell carcinoma, and synovial sarcoma. The majority were stage IV. Identification of this alteration has lead to treatment with MET inhibitors such as crizotinib, and to durable partial responses or better exceeding 3 months in histiocytic sarcoma (1), sarcomatoid lung carcinoma (1), and nsclc (1+). Multiple patients (5+) have initiated treatment on either crizotinib or MET inhibitors in clinical development, and additional outcome data will be reported. One patient with locally advanced unresectable disease harbored a MET exon 14 skipping alteration. On initiation with treatment with an MET inhibitor, symptomatic relief was observed in 3 days, radiographic response was observed at two weeks, and resection was performed 8 weeks after initiation of the MET inhibitor.
Conclusion:
MET exon 14 alterations define a hereto unrecognized population of advanced cancer cases, particularly in NSCLC. Multiple case reports demonstrate that these alterations confer sensitivity to multiple small molecule MET inhibitors. This finding expands the population of advanced NSCLC patients who can derive benefit from MET-targeted therapies.
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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): L. Bubendorf, U. Dafni, S.P. Finn, A. Warth, S. Savic, E. Thunnissen, A. Sejda, E.K. Verbeken, V. Tischler, A. Marchetti, I. Sansano, E.M. Speel, R. Cheney, D. Nonaka, K. Monkhorst, A. Di Lorito, M. Consuelo Calabuig, M. Reidy, H. Hager, W. Mathieson, M. Martorell, P. Baas, H. Dienemann, E. Smit, F. Blackhall, A. Dingemans, E. Felip, A. Adjei, P. Meldgaard, W. Weder, J. Vansteenkiste, R. Dziadziuszko, Z. Tsourti, K. Kerr, K. Schulze, A. Das-Gupta, S. Peters, R. Stahel
- 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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ORAL37.06 - Defining MET Copy Number Driven Lung Adenocarcinoma Molecularly and Clinically (ID 2379)
17:39 - 17:50 | Author(s): S.A. Noonan, L. Berry, D. Gao, X. Lu, A.E. Barón, P. Chesnut, N. Hart, J. Sheren, D.L. Aisner, D.T. Merrick, R.C. Doebele, M. Varella-Garcia, R. Camidge
- Abstract
- Presentation
Background:
Increases in MET copy number define an oncogenic driver state sensitive to MET inhibition (Camidge et al, ASCO 2014). However, the level at which the genomic gain is relevant remains uncertain. When testing is performed by fluorescence in situ hybridization (FISH), variable cut-points in both mean MET/cell and MET/CEP7 ratio have been used. Partially overlapping datasets from the Lung Cancer Mutation Consortium (LCMC1) and Colorado Molecular Correlates (CMOCO) Laboratory were explored for a distinct MET-copy number driven lung adenocarcinoma subtype.
Methods:
MET was assessed by FISH. Data from non-adenocarcinomas and EGFR mutant patients with acquired resistance to an EGFR inhibitor were excluded. Positivity criteria were mean MET/cell ≥5 (low ≥5-<6, intermediate ≥6-<7, high ≥7) or MET/CEP7 ≥1.8 (low ≥1.8-≤2.2, intermediate >2.2-< 5, high ≥5). MET metrics were compared by race, sex, smoking status, stage at diagnosis, number of metastatic disease sites, site of metastases, presence of other known drivers (EGFR, KRAS, ALK, ERBB2, BRAF, NRAS, ROS1 and RET), response to first line chemotherapy and overall survival using Fisher’s exact tests, chi-square tests, Spearman correlations and log-rank tests, as appropriate. Statistical significance was set at the 0.05 level without adjustment for multiple comparisons.
Results:
1164 unique adenocarcinomas were identified (60% female, 85% Caucasian, 66% ex/current smokers). MET/CEP 7 data was available on 1164 and mean MET/cell on 700. 52/1164 (4.5%) had MET/CEP7 ≥1.8 (48% female, 83% Caucasian, 69% smokers). 50/52 (98%) had ≥1 other oncogenic driver tested (25/50 (50%) positive). 113/700 (16%) had mean MET/cell ≥ 5 (57% female, 82% Caucasian, 58% smokers). 109/113 (96%) had ≥ 1 other oncogenic driver tested (73/109 (67%) positive). Among patients with ≥1 additional driver oncogene tested, alternate drivers in low, indeterminate and high categories of mean MET/cell were 44/60 (67%), 17/24 (70%) and 12/28 (43%) respectively and for MET/CEP7: 16/29 (55%), 9/18 (50%) and 0/4 (0%) respectively. MET positive with additional drivers were excluded from further analyses. Men exceeded women in MET/CEP7 (men 4% vs women 1.6%, p = 0.019) and mean MET/cell positive cases (men 9.6% vs women 5.4%, p = 0.058). 6.4% of adrenal metastasis cases were MET/CEP7 positive vs 2% all other sites, p=0.031. Mean MET/cell: 12% adrenal vs 5% other sites, p=0.082. MET/CEP7 or mean MET/cell positive and negative groups did not differ by other variables (p > 0.05).
Conclusion:
The proportion of ‘MET positive’ adenocarcinomas varies by definition and positivity cut-point. Mean MET/cell ≥5 defines nearly 4x more positives than MET/CEP7 ≥1.8 and no mean MET/cell positive category was free from overlap with other drivers. As only high MET/CEP7 had no overlap with other drivers, MET/CEP7 ≥ 5 is the clearest candidate for a pure MET-copy number driven state, however cases free from other drivers do exist at lower MET positivity levels. MET/CEP7 positive cases free from other known drivers are more likely to be male, but unlike other known oncogenic states, race and smoking status are not significant in determining positivity. MET positivity may have a specific biological phenotype, being more likely to present with adrenal metastases.
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ORAL37.07 - Lung Cancer Mutation Consortium Pathologist Panel Evaluation of MET Protein (ID 2129)
17:50 - 18:01 | Author(s): T.A. Boyle, F. Khalil, M. Mino-Kenudson, A. Moreira, L. Sholl, G. Sica, M.Z. Knight, A.A. Kowalewski, K. Ellison, C.J. Rivard, L. Berry, H. Chen, K. Kugler, B.E. Johnson, D.J. Kwiatkowski, P.A. Bunn, Jr, F.R. Hirsch
- Abstract
- Presentation
Background:
MET is a receptor tyrosine kinase with frequently activated signaling in lung cancers. Multiple studies indicate that MET overexpression correlates with poor clinical prognosis. Tumors with MET amplification and overexpression may respond better to MET inhibitors than tumors with low expression. The prevalence of MET overexpression in lung cancer cohorts has varied from 20%-80%, as has the proportion of patient’s testing positive for prospective clinical trials with entry based on MET overexpression. The Lung Cancer Mutation Consortium (LCMC) Pathologist Panel endeavored to standardize evaluation of MET protein expression with “Round Robin” conferences.
Methods:
508 FFPE non-small cell lung cancer specimens were stained by immunohistochemistry for MET protein expression (SP44 antibody, Ventana). Seven pathologists from LCMC sites with specialized training in MET scoring evaluated 78 Aperio-scanned images of MET-stained slides in two successive rounds of 39 different cases per round. The percentage of tumor cells with membranous and/or cytoplasmic staining at different intensities were evaluated with H-scores ranging from 0 to 300. Overall group and individual pathologist’s scores were compared with intraclass correlation coefficients (ICCs). Between rounds, a “Round Robin” teleconference was conducted to review discordant cases and improve consistency of scoring. Steps to improve scoring included: review of a Roche MET training document, sharing pictures of cases with concordant scores (Figure 1), and provision of H&E images for the second round to facilitate identification of tumor areas. Figure 1
Results:
The overall average MET H-score for the 78 cases was 165.3 (H-score range: 42.5-279.7). The average H-score was <125 for 14 specimens, 125-175 for 35 specimens, and >175 for 29 specimens. The overall group ICC comparing the consistency of H-scores from all 7 pathologists improved from 0.50 (95% confidence interval: 0.37-0.64, “fair” correlation) for the first scoring round to 0.74 (95% confidence interval: 0.64-0.83, “good” correlation) for the second round. A comparison of the individual pathologist’s ICCs demonstrated improved individual scoring consistency for all seven pathologists between rounds with an average of 0.64 (“moderate” correlation, range 0.43-0.76) for the first round and 0.82 (“almost perfect” correlation, range 0.75-0.93) for the second round.
Conclusion:
Development of standardized, reproducible strategies for evaluation of complex biomarkers, such as MET, are critical to clinical trial design. The consistency of scoring for MET protein expression and other biomarkers may be improved by continuous training and communication between pathologists with easy access to H&E images and other visual aids.
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ORAL37.08 - Discussant for ORAL37.04, ORAL37.05, ORAL37.06, ORAL37.07 (ID 3465)
18:01 - 18:11 | Author(s): G.J. Weiss
- Abstract
- Presentation
Abstract not provided
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Author of
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MINI 01 - Pathology (ID 93)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:W.A. Franklin, A.G. Nicholson
- Coordinates: 9/07/2015, 10:45 - 12:15, Mile High Ballroom 2c-3c
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MINI01.09 - Discussant for MINI01.05, MINI01.06, MINI01.07, MINI01.08 (ID 3297)
11:30 - 11:40 | Author(s): E. Thunnissen
- Abstract
- Presentation
Abstract not provided
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MS 01 - How to Treat Multiple GGO's (ID 19)
- Event: WCLC 2015
- Type: Mini Symposium
- Track: Treatment of Localized Disease - NSCLC
- Presentations: 1
- Moderators:J. Donington, M. Tsuboi
- Coordinates: 9/07/2015, 14:15 - 15:45, 102+104+106
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MS01.04 - The Pathologic Classification of GGO's - Clinical Pathologic Correlation (ID 1851)
15:20 - 15:40 | Author(s): E. Thunnissen
- Abstract
Abstract:
Definition. Ground glass opacity (GGO) is a finding on thin-section CT that is defined as “hazy increased attenuation of the lung with preservation of bronchial and vascular margins”.[1]This is in contrast to consolidation that is defined as a “homogeneous increase in pulmonary parenchymal attenuation that obscures the margins of vessels and airway walls” (also called ‘solid´ component). Introduction. The resolution of the CT differs few orders of magnitude from the resolution of the microscope. Therefore, GGO is not one disease: pathological examination reveals several different diseases. The radiological GGO change is actually due to a reduction of air, while a certain amount of air remains present. At the microscopic level this may be caused by either i) partial filling of the alveolar airspaces, ii) thickening of the parenchymal interstitium and alveolar walls, iii) relative increase in perfusion, or iv) any combination of these factors.[1,2]Alveolar spaces may become partially filled by several ways, such as transudative fluid, blood, inflammatory cells or debris, or amorphous material as seen in cardiogenic pulmonary edema, diffuse alveolar hemorrhage, pneumonia, and pulmonary alveolar proteinosis. Alveolar walls and septal interstitium may become thickened secondary to edema, neoplastic proliferation, fibrosis, and noncaseating granulomatous deposition as seen in cardiogenic pulmonary edema, nonspecific interstitial pneumonia, and sarcoidosis. Partial alveolar filling and interstitial thickening coexist in many disease entities. Thus GGO is a non-specific finding that may be caused by various disorders, including inflammatory disease, pulmonary fibrosis, alveolar haemorrhage or neoplasms. GGO may be either (multi)focal (=localised GGO) or diffuse (present bilaterally in most of the lobes, nicely reviewed by El-Sherief et al.[1]), usually associated with inflammatory diseases. Localized GGO may be pure associated with a solid component (mixed GGO) or without (pure GGO). Localized GGO may contain benign[3,4](organising pneumonia, non-specific fibrosis, atypical adenomatous hyperplasia: AAH; aspergillosis) premalignant (adenocarcinoma in situ: AIS, formerly also called BAC)[5]and malignant diseases[3]((minimal) invasive adenocarcinoma with prominent lepidic component[4,5]). The clinical significance of localized GGO is its high incidence of malignancy compared with solid nodules. The reported range varies from 10-90%.[6–8] CT-guided thoracic needle biopsy is a useful tool for tissue diagnosis and may support the patient management with GGO lesions.[9]Fluoroscopic or image guidance has been studied as well.[10–12] The core biopsy procedure is preferred over aspiration.[13]As usual with biopsies underestimation/ underdiagnosis due to sampling variation is not excluded.[11,14]As GGO contains many diseases the term “Natural history of GGO” is a misnomer.[15,16] Radiology To distinguish GGOs with growth from those without growth, a 3-year follow-up observation period is a reasonable benchmark based on the data that the volume-doubling time (VDT) of pure GGOs ranges from approximately 600 to 900 days and that of part-solid GGOs ranges from 300 to 450 days.[7,8,17] AAH is often associated with malignancy and is shown on CT as persistent well-defined oval or round nodular GGOs without solid components, and it does not change on the follow-up CT.[18] Clinicoradiological characteristics of a benign course are smaller size (< 10 mm.), round/oval shape, lack of consolidation[18,19]or scattered consolidation.[20] Clinicoradiological characteristics of a progressive course are smoking history[21]and initial lesion diameter (> 1 cm) [5,19,22,23], lobulated or speculated margin,[4]growth[16], increase in attenuation,[24]greater irregularity of pixel texture (entropy).[5]Growth of localised GGO may be slow: for one case progression was reported within 10 years.[25] Biomarkers EGFR mutations may be present in pure and mixed GGO lesions, representing preinvasive and invasive cancer.[26–28]Interestingly, for p53 immunohistochemistry positive staining was seen in a small group (n=6) associated with growth or a solid component.[26]MIB1 (ki67) is higher in mixed GGO than in pure GGO.[29]CEA not relevant for the distinction of the progressive GGO.[30] Remarkably, with laborious cytogenetic analysis spontaneous metaphases appeared after 24-48 h. in 9 cases of pure GGO. Abnormal FISH was associated with poor outcome.[15] In case of multiple GGO synchronous BAC and/or ADC can have different EGFR or K-ras mutational profiles suggesting these lesions arise as independent events rather than intrapulmonary spread or systemic metastasis.[31] Management Surgical handling is hampered since AAH not or much less well easy palpable than AIS.[30] Since GGO may contain only AIS or minimal invasive adenocarcinoma partial resection may be the method of choice.[30]and systematic lymph node dissection may be avoided.[32] For multiple localized GGO wait and see is an option[33] Prognosis of mixed GGO invasive adenocarcinomas better for solid size than size including the GGO.[34,35]A larger solid component is worse than less solid component.[36] References 1. El-Sherief, A. H. et al. Clear Vision Through the Haze: A Practical Approach to Ground-Glass Opacity. Curr. Probl. Diagn. Radiol. 43, 140–158 (2014). 2. Hewitt, M. G., Miller, W. T., Reilly, T. J. & Simpson, S. The relative frequencies of causes of widespread ground-glass opacity: A retrospective cohort. Eur. J. Radiol. 83, 1970–1976 (2014). 3. Lee, H. J. et al. Nodular ground-glass opacities on thin-section CT: size change during follow-up and pathological results. Korean J. Radiol. 8, 22–31 4. Kim, H. Y. et al. Persistent Pulmonary Nodular Ground-Glass Opacity at Thin-Section CT: Histopathologic Comparisons 1. Radiology 245, 267–275 (2007). 5. Son, J. Y. et al. Quantitative CT Analysis of Pulmonary Ground-Glass Opacity Nodules for the Distinction of Invasive Adenocarcinoma from Pre-Invasive or Minimally Invasive Adenocarcinoma. PLoS One 9, e104066 (2014). 6. Ichinose, J. et al. Invasiveness and malignant potential of pulmonary lesions presenting as pure ground-glass opacities. Ann. Thorac. Cardiovasc. Surg. 20, 347–52 (2014). 7. Lee, H. Y. & Lee, K. S. Ground-glass Opacity Nodules. J. Thorac. Imaging 26, 106–118 (2011). 8. Kobayashi, Y. & Mitsudomi, T. Management of ground-glass opacities : should all pulmonary lesions with ground-glass opacity be surgically resected ? 2, 354–363 (2013). 9. Yang, J.-S. et al. Meta-analysis of CT-guided transthoracic needle biopsy for the evaluation of the ground-glass opacity pulmonary lesions. Br. J. Radiol. 87, 20140276 (2014). 10. Hur, J. et al. Diagnostic Accuracy of CT Fluoroscopy–Guided Needle Aspiration Biopsy of Ground-Glass Opacity Pulmonary Lesions. Am. J. Roentgenol. 192, 629–634 (2009). 11. Yamagami, T. et al. Diagnostic performance of percutaneous lung biopsy using automated biopsy needles under CT-fluoroscopic guidance for ground-glass opacity lesions. Br. J. Radiol. 86, 20120447 (2013). 12. Chavez, C. et al. Image-guided bronchoscopy for histopathologic diagnosis of pure ground glass opacity: a case report. J. Thorac. Dis. 6, E81–4 (2014). 13. Choi, S. H. et al. Percutaneous CT-guided aspiration and core biopsy of pulmonary nodules smaller than 1 cm: analysis of outcomes of 305 procedures from a tertiary referral center. AJR. Am. J. Roentgenol. 201, 964–70 (2013). 14. Lu, C.-H. et al. Percutaneous Computed Tomography-Guided Coaxial Core Biopsy for Small Pulmonary Lesions with Ground-Glass Attenuation. J. Thorac. Oncol. 7, 143–150 (2012). 15. Bettio, D., Venci, A., Cariboni, U., Di Rocco, M. & Infante, M. Fluorescent in situ hybridization (FISH) in the differential diagnosis of ground-glass opacities in the lung. Lung Cancer 71, 319–322 (2011). 16. Chang, B. et al. Natural History of Pure Ground-Glass Opacity Lung Nodules Detected by Low-Dose CT Scan. CHEST J. 143, 172 (2013). 17. Oda, S. et al. Volume-Doubling Time of Pulmonary Nodules with Ground Glass Opacity at Multidetector CT. Acad. Radiol. 18, 63–69 (2011). 18. Park, C. M. et al. CT findings of atypical adenomatous hyperplasia in the lung. Korean J. Radiol. 7, 80–6 19. Lee, S. M. et al. Invasive Pulmonary Adenocarcinomas versus Preinvasive Lesions Appearing as Ground-Glass Nodules: Differentiation by Using CT Features. Radiology 268, 265–273 (2013). 20. Matsunaga, T. et al. Lung cancer with scattered consolidation: detection of new independent radiological category of peripheral lung cancer on thin-section computed tomography. Interact. Cardiovasc. Thorac. Surg. 16, 445–449 (2012). 21. Kobayashi, Y. et al. The association between baseline clinical-radiological characteristics and growth of pulmonary nodules with ground-glass opacity. Lung Cancer 83, 61–66 (2014). 22. Kitami, A. et al. One-dimensional mean computed tomography value evaluation of ground-glass opacity on high-resolution images. Gen. Thorac. Cardiovasc. Surg. 60, 425–430 (2012). 23. Fan, L., Liu, S. Y., Li, Q. C., Yu, H. & Xiao, X. S. Multidetector CT features of pulmonary focal ground-glass opacity: Differences between benign and malignant. Br. J. Radiol. 85, 897–904 (2012). 24. Eguchi, T. et al. Tumor Size and Computed Tomography Attenuation of Pulmonary Pure Ground-Glass Nodules Are Useful for Predicting Pathological Invasiveness. PLoS One 9, e97867 (2014). 25. Min, J. H. et al. Stepwise evolution from a focal pure pulmonary ground-glass opacity nodule into an invasive lung adenocarcinoma: An observation for more than 10 years. Lung Cancer 69, 123–126 (2010). 26. Aoki, T. et al. Adenocarcinomas with Predominant Ground-Glass Opacity: Correlation of Morphology and Molecular Biomarkers. Radiology 264, 590–596 (2012). 27. Usuda, K. et al. Relationships between EGFR mutation status of lung cancer and preoperative factors - are they predictive? Asian Pac. J. Cancer Prev. 15, 657–62 (2014). 28. Yoshida, Y. et al. Molecular Markers and Changes of Computed Tomography Appearance in Lung Adenocarcinoma with Ground-glass Opacity. Jpn. J. Clin. Oncol. 37, 907–912 (2007). 29. Ohta, Y. et al. Pathologic and Biological Assessment of Lung Tumors Showing Ground-Glass Opacity. Ann. Thorac. Surg. 81, 1194–1197 (2006). 30. OHTSUKA, T., WATANABE, K., KAJI, M., NARUKE, T. & SUEMASU, K. A clinicopathological study of resected pulmonary nodules with focal pure ground-glass opacity. Eur. J. Cardio-Thoracic Surg. 30, 160–163 (2006). 31. Chung, J.-H. et al. Epidermal Growth Factor Receptor Mutation and Pathologic-Radiologic Correlation Between Multiple Lung Nodules with Ground-Glass Opacity Differentiates Multicentric Origin from Intrapulmonary Spread. J. Thorac. Oncol. 4, 1490–1495 (2009). 32. Ye, B. et al. Factors that predict lymph node status in clinical stage T1aN0M0 lung adenocarcinomas. World J. Surg. Oncol. 12, 42 (2014). 33. Kim, H. K. et al. Management of Multiple Pure Ground-Glass Opacity Lesions in Patients with Bronchioloalveolar Carcinoma. J. Thorac. Oncol. 5, 206–210 (2010). 34. Nakamura, S. et al. Prognostic impact of tumor size eliminating the ground glass opacity component: modified clinical T descriptors of the tumor, node, metastasis classification of lung cancer. J. Thorac. Oncol. 8, 1551–7 (2013). 35. Tsutani, Y. et al. Prognostic significance of using solid versus whole tumor size on high-resolution computed tomography for predicting pathologic malignant grade of tumors in clinical stage IA lung adenocarcinoma: A multicenter study. J. Thorac. Cardiovasc. Surg. 143, 607–612 (2012). 36. Shimada, Y. et al. Survival of a surgical series of lung cancer patients with synchronous multiple ground-glass opacities, and the management of their residual lesions. Lung Cancer 88, 174–180 (2015).
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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. Thunnissen
- 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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P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-039 - Ex-Vivo Artifacts and Histopathological Pitfalls in the Lung (ID 2602)
09:30 - 09:30 | Author(s): E. Thunnissen
- Abstract
Background:
Surgical and pathological handling of lung physically affects lung tissue. This leads to artifacts that alter the morphological appearance of pulmonary parenchyma.
Methods:
In this study four mechanisms of ex-vivo artifacts and corresponding diagnostic pitfalls are described and illustrated.
Results:
The four patterns of artifacts are: 1) Surgical collapse, due to the removal of air and blood from pulmonary resections; 2) Ex-vivo contraction of bronchial and bronchiolar smooth muscle; 3) Clamping edema of open lung biopsies, and 4) Spreading of tissue fragments and individual cells through a knife surface. Morphologic pitfalls include diagnostic patterns of adenocarcinoma, asthma, constrictive bronchiolitis, and lymphedema.
Conclusion:
Four patterns of pulmonary ex-vivo artifacts are important to recognize, in order to avoid morphologic misinterpretations, possibly improving reproducibility in histopathological diagnosis of lung cancer
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P1.05 - Poster Session/ Prevention and Tobacco Control (ID 215)
- Event: WCLC 2015
- Type: Poster
- Track: Prevention and Tobacco Control
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.05-006 - One Cigarette Takes 12.6 Minutes of Your Life (ID 2560)
09:30 - 09:30 | Author(s): E. Thunnissen
- Abstract
Background:
Smoking is the largest cause of premature mortality. Smoking cessation is important, but is difficult to reach. A general underestimation of personal risk in smokers or a degree of misunderstanding around key risk factors for disease may be substantial. [1]The aim of this abstract is to calculate the reduction in average life expectancy per cigarette.
Methods:
Men born in 1900-1930 who smoked only cigarettes and continued smoking died on average about 10 years younger than lifelong non-smokers. Cessation at age 60, 50, 40, or 30 years gained, respectively, about 3, 6, 9, or 10 years of life expectancy.[2]Assuming that these men started at age 15 years and died at age of 72 this results on average in 57 years of smoking. Also assumed is that each day one pack of 20 cigarettes is smoked.
Results:
Smoking for 57 years 20 cigarettes per day results in a total of 416,100 cigarettes. The total number of minutes in 10 years is 5,256,000. The average decrease in life expectancy is 12.6 minutes/ cigarette or 4.2 hours /pack, equals more than a day/week. Discussion: If a smoker is aware of the reduced life expectancy then smoking of one cigarette may be looked-upon as a mini-suicide attempt. Taken also into account the passive smoking effect, the smoker may be seen as a mini-suicide-nano-terrorist.
Conclusion:
Conclusion The reduction in average life expectancy is 12.6 minutes per cigarette or 4 hours per pack. This knowledge may be of help to raise more awareness for the dangers of smoking. 1. Bethea J, Murtagh B, Wallace SE. “ I don ’ t mind damaging my own body ” A qualitative study of the factors that motivate smokers to quit. 2015;1–9. 2. Doll R, Peto R, Hall E, Wheatley K, Gray R. Mortality in relation to consumption of alcohol: 13 years’ observations on male British doctors. BMJ. 1994;309(6959):911–8.
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P2.08 - Poster Session/ Thymoma, Mesothelioma and Other Thoracic Malignancies (ID 225)
- Event: WCLC 2015
- Type: Poster
- Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.08-023 - Aberrant Neuroendocrine Lung Tumor Nomenclature in Daily Practice, How Common Is It? (ID 2120)
09:30 - 09:30 | Author(s): E. Thunnissen
- Abstract
Background:
The WHO 2015 classification for pulmonary carcinoids (PC) and high-grade neuroendocrine carcinomas (NEC) has in essence, not been changed compared to the previous one, despite known limitations in the diagnostic process such as 1) the need for resection material or large biopsies 2) reported inter-observer variability and 3) sporadic exposure in daily practice. Furthermore, nomenclature used in previous or different classification systems for neuroendocrine tumors may result in an aberrantly applied description of the WHO 2004 diagnoses. Here we evaluate if nomenclature established in daily pathology practice in the Netherlands is according to that advised by the WHO 2004 for PC, NEC and non-small cell lung cancer (NSCLC) with neuroendocrine differentiation established by immunohistochemistry (IHC).
Methods:
Written conclusions (diagnoses) of pathology reports (2003-2012) were retrieved from the Dutch Pathology Registry. Conclusions describing PC, NEC and carcinomas with neuroendocrine features/differentiation were selected by multiple queries on anatomic location, diagnosis and keywords (e.g. carcinoma + endocrine). All conclusions were screened in concordance with an experienced pathologist (JLD & RJS) and data on sampling method, diagnoses and origin of primary tumor were collected. Conclusions were excluded if established on autopsy cases or if it reported differential diagnoses, diagnoses of non-pulmonary/unknown primary, non-neuroendocrine or small cell lung cancer. We compared the retrieved diagnoses with the advised WHO 2004 nomenclature after which all diagnoses were clustered (e.g. typical/well differentiated/grade I carcinoid into “PC”). For statistical analysis the X[2] test was used.
Results:
4612 conclusions were eligible for analysis of which N=698 (15%) described a diagnoses that did not match the WHO nomenclature. Foremost non-WHO diagnoses were: (poorly differentiated) neuroendocrine carcinoma; high-grade neuroendocrine carcinoma/tumor; NSCLC neuroendocrine carcinoma; neuroendocrine tumor and low grade (well differentiated) neuroendocrine carcinoma/tumor (carcinoids). After discussion, we clustered N=2005 (43%) diagnoses into PC, N=1788 (39%) in high-grade NEC, N=763 (17%) in carcinoma with neuroendocrine features/differentiation and N=56 (1%) in neuroendocrine tumor n.o.s., respectively. Deviations from the WHO nomenclature occurred in 8% (N=157) of PC and 21% (N=377) of high-grade NEC and this occurred mainly on biopsy/cytology specimens (75% (N=399)). In (NSCLC) carcinomas with neuroendocrine features/differentiation diagnoses deviated from the WHO in 14% (N=108). Additionally, both the terms neuroendocrine “features” and “differentiation” were used to address positive neuroendocrine IHC staining (16% vs 25%) though differentiation was used slightly more often (p=0.001). Finally, 52% (N=1045) of PC diagnoses were established on biopsy/cytology specimens and a strong increase in diagnoses of large cell neuroendocrine carcinoma (LCNEC) on biopsy/cytology specimens was observed (<2008 N=174 vs. ≥2008 N=464, p<0.001).
Conclusion:
In daily practice 8% of PC, 21% of high-grade NEC and 14% of (NSCLC) carcinomas with neuroendocrine features/differentiation diagnoses deviated from the WHO 2004 nomenclature. This occurred mainly on biopsy/cytology specimens. Also, the diagnosis (NSCLC) carcinoma with neuroendocrine ‘differentiation/features’ was unclear and should be specified (i.e. IHC or morphologically based (or both)). Finally, often the diagnosis LCNEC was established on biopsy/cytology specimens whereas this is not advised by the WHO. Whether these findings are due to personal preferences or difficulties applying current classification to limited samples, require further investigation.
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P3.01 - Poster Session/ Treatment of Advanced Diseases – NSCLC (ID 208)
- Event: WCLC 2015
- Type: Poster
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.01-022 - A Prospective Multicenter Study for ALK IHC+ Metastasized NSCLC (ID 2566)
09:30 - 09:30 | Author(s): E. Thunnissen
- Abstract
Background:
Pulmonary adenocarcinomas may harbor driver mutations, that sensitize tumors to drugs that specifically target the genetic alteration. Metastasized NSCLC with an EML4-ALK translocation are sensitive to a range of tyrosine kinase inhibitors, of which crizotinib is most extensively studied. ALK-positive NSCLC was determined in a phase III trial with fluorescence in situ hybridisation (ALK FISH+). ALK immunohistochemistry (IHC) seems to run parallel with ALK FISH positivity. However discrepant cases occur, which include ALK IHC+ FISH-. The aim of this study is to collect cases with ALK IHC+ and compare within this group response to crizotinib treatment of ALK FISH+ cases with ALK FISH- cases.
Methods:
A prospective multicenter investigator initiated research study was started in Europe. This study is supported by Pfizer. Cases diagnosed with ALK IHC+ lung cancer (5A4 or D5F3) treated with crizotinib are collected centrally. Slides are submitted centrally for validation of ALK IHC (with ETOP and Ventana protocol), ALK FISH (with Vysis probes) and DNA analysis.
Results:
The study started on April 1 2014 and is still open. Currently 10 centers are actively participating. 1443 cases have been examined with ALK IHC of which 39 (2.7%) recorded positive. 24 cases have been submitted to the database. The validation process is still ongoing. The fraction of ALK IHC+ FISH- cases is low. Two cases with ALK IHC+ FISH- metastastatic NSCLC responded to crizotinib treatment. In two cases ALK positivity could not be confirmed (ALK IHC- and ALK FISH-). These patients had progressive disease following crizotinib treatment.
Conclusion:
A clinically relevant question what the effect of ALK inhibitor treatment is on metastatic NSCLC ALK IHC+ FISH- compared to ALK IHC+ FISH+ is examined. Other centers with interested collaborating physicians are invited to participate.
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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: 2
- 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. Thunnissen
- 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.
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P3.04-040 - Comparison of Histology with Genome-Wide Copy Number Profiling in Patients with Metachronous or Synchronous Tumors (ID 3035)
09:30 - 09:30 | Author(s): E. Thunnissen
- Abstract
Background:
Multiple synchronous and metachronous lung tumors are frequently encountered in patients with lung cancer. In addition, tumors of head and neck (usually squamous cell carcinoma) have a chance for a second primary malignancy in the lung. For treatment purposes it is important to know whether tumors are related (clonal = metastases) or not (multiple primaries). Histopathological comparison of the synchronous or metachronous tumors has been associated with molecular analysis. The purpose of this study is to examine the value of histopathological scoring with genome-wide copy number profiling for determination of clonality.
Methods:
From cases in which array CGH for clonality analysis performed between 2006 and 2012 were selected if at least one intrathoracic tumor was present. In the first years genome-wide copy number profiling was performed with arrayCGH and later with shallow sequencing. Results of the genome-wide copy number profiling were compared to histological (sub)typing.
Results:
100 tumor pairs from 59 patients were examined. 32 pairs were discovered simultaneously (synchronous), the other 68 were metachronous. The histopathological diagnosis was similar in 74 cases (74%). genome-wide copy number profiling revealed evidence for clonality in 55% of the pairs, no-clonality in 28% and was undetermined in 17%. Comparing of histology with genome-wide copy number profiling revealed concordancy in 54 pairs ( 74%; 44 clonal en 10 non-clonal). In 18 of the 62 pairs where histology was similar the genome-wide copy number profiling revealed a non-clonal pattern. In 11 out of 21 pairs where histology differed between the pairs, genome-wide copy number profiling revealed a clonal pattern. Thus histology was not prognostic in 29/83 pairs (35%).
Conclusion:
For the determination of clonality in lung cancer histological examination is discordant with genome-wide copy number profiling in 35% of the comparisons. As histology is a poor predictor of clonality, genome-wide copy number profiling is preferred for clonality analysis between tumors.
