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L. Bubendorf
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MA17 - Genetic Drivers (ID 409)
- Event: WCLC 2016
- Type: Mini Oral Session
- Track: Biology/Pathology
- Presentations: 1
- Moderators:M. Satouchi, G.R. Simon
- Coordinates: 12/07/2016, 14:20 - 15:50, Lehar 1-2
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MA17.02 - Genome-Wide Copy Number and Mutational Analysis in Longitudinal Biopsies of Matched Primary and Metastatic Pulmonary Adenocarcinomas (ID 4702)
14:26 - 14:32 | Author(s): L. Bubendorf
- Abstract
Background:
There are still limited data on the extent of intratumoral heterogeneity of cancer gene mutations and genome-wide copy number aberrations between primary tumors and metastases in non-small cell lung cancers (NSCLC). Deconvolution of the intermixture of tumor and stromal components remains a major challenge for such analysis. To overcome these limitations, we applied a refined nuclei flow sorting approach on matched longitudinal biopsies (primary/metastasis) from pulmonary adenocarcinomas.
Methods:
Multiparameter Ploidy Profiling (MPP) comprises the isolation of nuclei from frozen or formalin-fixed and paraffin embedded (FFPE) tissues, followed by multiparameter flow sorting by DAPI for DNA content (ploidy) and TTF1 as a lineage marker to enrich for tumor cell nuclei. Homogenous TTF1 expression was ascertained by immunohistochemistry. Sorted populations were subjected to genomic profiling by high resolution aCGH and NGS with the Ion Torrent™ Comprehensive Cancer Panel. This approach allows for the detection of genome-wide copy number aberrations and provides all exon-coverage of 409 well-known cancer genes. Sequencing was performed with a mean depth of 965x.
Results:
MPP was successfully applied on 44 frozen or FFPE tissue specimens from 19 patients. Clonally unrelated secondary primaries were found in three patients, defined by the absence of both shared copy number (CN) transition and somatic mutations. The concordance rate between primary tumor and corresponding metastases was 65.2% and reached 85.5% for mutations and copy number amplifications/deletions in the top 12 affected genes (including CDKN2A, KRAS, ATM, KEAP1, EGFR and STK11). The correlation of the allele frequencies between primary tumors and metastases was linear (r=0.87, p<0.001), irrespective of the time interval between the tissue resections. Overall, ploidy was not different between primary tumors and metastases. Additionally, the metastases did not bear a higher burden of private events (CN transitions and somatic mutations) than the primary tumors.
Conclusion:
MPP is a powerful method to increase the precision of downstream analysis due to unprecedented purity of tumor DNA. Our data argue for a high concordance rate of mutations and CN transitions between primary tumors and their corresponding metastases. Intriguingly, the ploidy remains remarkably stable during progression even after long time-periods, which suggests chromosomal stability with a limited degree of macroevolutionary shifts over time and space. Taken together, our data suggest the presence of at least two evolutionary patterns: 1) early/branched and 2) late/linear progression, with a continuum from high to low genetic divergence of the primary tumor and metastases to their most recent common ancestor.
Information from this presentation has been removed upon request of the author.
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MTE02 - Advances in Pathology (Ticketed Session) (ID 296)
- Event: WCLC 2016
- Type: Meet the Expert Session (Ticketed Session)
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 07:30 - 08:30, Schubert 2
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MTE02.01 - Advances in Pathology (ID 6540)
07:30 - 08:30 | Author(s): L. Bubendorf
- Abstract
- Presentation
Abstract not provided
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P1.02 - Poster Session with Presenters Present (ID 454)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.02-025 - Evaluation of NGS and RT-PCR Methods for ALK Assessment in European NSCLC Patients: Results from the ETOP Lungscape Project (ID 5001)
14:30 - 14:30 | Author(s): L. Bubendorf
- Abstract
Background:
The reported prevalence of ALK rearrangement in NSCLC ranges from 2%-7%, depending on population and detection method. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proven to be a reproducible and sensitive technique. Reverse transcriptase-polymerase chain reaction (RT-PCR) has been advocated and most recently the advent of targeted Next-Generation Sequencing (NGS) for ALK and other fusions has become possible. This is one of the first studies comparing all 4 techniques in resected NSCLC from the large ETOP Lungscape cohort.
Methods:
96 cases from the ETOP Lungscape iBiobank (N=2709) selected based on any degree of IHC staining (clone 5A4 antibody, Novocastra, UK) were examined by FISH (Abbott Molecular, Inc.; Blackhall, JCO 2014), central RT-PCR and NGS. H-score 120 is used as cutoff for IHC+. For both RT-PCR and NGS, RNA was extracted from the same formalin-fixed, paraffin-embedded tissues. For RT-PCR, primers were used covering the most frequent ALK translocations. For NGS, the Oncomine™ Solid Tumour Fusion Transcript Kit was used, allowing simultaneous sequencing of 70 ALK, RET and ROS1 specific fusion transcripts associated with NSCLC, as well as novel ALK translocations using 5’-3’ ALK gene expression ‘Imbalance Assay’.
Results:
NGS provided results for 90 cases, while RT-PCR for 77. Overall, 70 cases have results for all 4 methods, with fully concordant 60 (85.7%) cases (49 ALK-, 11 ALK+). Before employing the ‘Imbalance Assay', in 5 of the remaining 10 cases, NGS differs from the other methods (3 NGS-, 2 NGS+), while in the other 5, NGS agrees with RT-PCR in all, IHC in 2, and FISH in 1. Using the concordant result of at least two of the three methods as true negative/positive, the specificity and sensitivity of the fourth is 96/94/100/96% and 94/94/89/72% for IHC/FISH/RT-PCR/NGS, respectively (incorporating imbalance: NGS sensitivity=83%). Imbalance scores are presented here for 18 NGS- cases: 9 ‘NGS-/FISH+/IHC+’, 9 ‘NGS-/FISH-/IHC-‘. Among the ‘NGS-/FISH+/IHC+’, there is strong evidence of imbalance in 4 cases (score’s range: 0.0144-0.0555), uncertain in 5 (range: 0.0030-0.0087), and no evidence (scores≤0.0004) in the 9 negative cases.
Conclusion:
NGS is a useful screening tool for ALK rearrangement status, superior to RT-PCR when RNA yield is limited. When using NGS, it is critically important to integrate the 5’-3’ imbalance assay and to confirm with one or more additional methods in the ‘imbalance’ cases. Data further highlight the possibility of missing actionable rearrangements when only one screening methodology is available.
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P1.08 - Poster Session with Presenters Present (ID 460)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Surgery
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.08-065 - Resection of Isolated Brain Metastasis Improves Outcome of Non Small-Cell Lung Cancer (NSCLC) Patients: A Retrospective Multicenter Study (ID 6132)
14:30 - 14:30 | Author(s): L. Bubendorf
- Abstract
Background:
Metastatic non-small cell lung cancer (NSCLC) is an incurable disease. Selected patients with solitary brain metastasis from NSCLC can achieve long-term survival following metastasectomy. We analyzed the outcome of all consecutive and unselected patients undergoing resection of brain metastases in two cancer centers in Switzerland to assess safety and efficacy of brain metastasis resection in NSCLC.
Methods:
119 consecutive NSCLC patients undergoing surgical resection of brain metastases from two centers in Switzerland (University Hospital Basel, Cantonal Hospital St. Gallen) between 2000 and 2014 were analyzed. Measured outcomes were extent of resection, resection status, postoperative complications and overall survival (OS). We used the log-rank test to compare unadjusted survival probabilities and multivariable Cox regression to investigate potential prognostic factors with respect to OS.
Results:
Median age was 60.5 years, 56% were male, 74% were smokers, 55% had adenocarcinoma. Median OS of the whole cohort was 18.0 months. 1-year survival rate was 63%, 12% of patients were alive after 5 years. In total, 146 brain metastases were resected; the maximum number of resected metastases was 4 (median: 1). Median diameter of resected metastases was 25 millimeters (range, 6-70 mm). About half of metastases were localized in the frontal cortex or the cerebellum. 86% of patients received postoperative radiotherapy. 63% of patients were treated with whole brain radiation, 12.6% received stereotactic radiotherapy. Median dose of postoperative radiotherapy was 30 Gy. Patients not receiving adjuvant radiotherapy (n=11) had a significantly worse outcome (median OS 9.0 vs. 20.2 months, p=0.002). Patients with more than one brain metastasis (n=21) had a significantly worse outcome compared to those with a solitary metastasis (median OS 13.5 vs. 19.5 months, p=0.006). Also patients with extracerebral metastases (n=33) had a significantly poorer outcome (median OS 14.0 vs. 23.1 months, p=0.005). Patients with non-squamous histology (n=98) had a better outcome than patients with squamous cell carcinoma (median OS 22.6 vs. 12.0 months, p=0.019). 21% of patients experienced postoperative complications, including need for surgical reintervention (5.8%), neurological deficits (4.2%), infection (4.2%), stroke (3.4%) and others (11.8%). The occurrence of postoperative complications was not associated with outcome. In the multivariate analysis existence of extracerebral metastases and resection of more than one brain metastasis were independent negative prognostic factors.
Conclusion:
Patients with isolated brain metastasis from NSCLC in the absence of extracranial metastasis should be evaluated for metastasectomy. Prospective trials are needed to characterize the patient population experiencing the greatest benefit from a surgical procedure.
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P3.01 - Poster Session with Presenters Present (ID 469)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.01-021 - Reproducibility of Comprehensive Histologic Assessment and Refining Histologic Criteria in P Staging of Multiple Tumour Nodules (ID 5365)
14:30 - 14:30 | Author(s): L. Bubendorf
- Abstract
Background:
Multiple tumor nodules (MTNs) are being encountered, with increasing frequency with the 8[th] TNM staging system recommending classification as separate primary lung cancers (SPLC) or intrapulmonary metastases (IM). Pathological staging requires assessment of morphological features, with criteria of Martini and Melamed supplanted by comprehensive histologic assessment of tumour type, predominant pattern, other histologic patterns and cytologic features. With publication of the 2015 WHO classification of lung tumours, we assessed the reproducibility of comprehensive histologic assessment and also sought to identify the most useful histological features.
Methods:
We conducted an online survey in which pathologists reviewed a sequential cohort of resected multifocal tumours to determine whether they were SPLC, IM, or a combination. Specific histological features for each nodule were entered into the database by the observing pathologist (tumour type, predominant adenocarcinoma pattern, and histological features including presence of lepidic growth, intra-alveolar cell clusters, cell size, mitotic rate, nuclear pleomorphism, nucleolar size and pleomorphism, nuclear inclusions, necrosis pattern, vascular invasion, mucin content, keratinization, clear cell change, cytoplasmic granules¸ lymphocytosis, macrophage response, acute inflammation and emperipolesis). Results were statistically analyzed for concordance with submitting diagnosis (gold standard) and among pathologists. Consistency of each feature was correlated with final determination of SPLC vs. IM status (p staging) by chi square analysis and Fisher exact test.
Results:
Seventeen pathologists evaluated 126 tumors from 48 patients. Kappa score on overall assessment of primary v. metastatic status was 0.60. There was good agreement as measured by Cohen’s Kappa (0.64, p<0.0001) between WHO histological patterns in individual cases with SPLC or IM status but proportions for histology and SPT or IM status were not identical (McNemar's test, p<0.0001) and additional histological features were assessed. There was marked variation in p values among the specific histological features. The strongest correlations (<0.05) between p staging status and histological features were with nuclear pleomorphism, cell size, acinus formation, nucleolar size, mitotic rate, nuclear inclusions, intra-alveolar clusters and necrosis pattern. Correlation between lymphocytosis, mucin content, lepidic growth, vascular invasion, macrophage response, clear cell change, acute inflammation keratinization and emperipolesis did not reach a p value of 0.05.
Conclusion:
Comprehensive histologic assessment shows good reproducibility between practicing lung pathologists. In addition to main tumour type and predominant patterns, nuclear pleomorphism, cell size, acinus formation, nucleolar size, and mitotic rate appear to be useful in distinguishing between SPLC and IM.
