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Paolo Graziano
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P1.01 - Advanced NSCLC (ID 158)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2019, 09:45 - 18:00, Exhibit Hall
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P1.01-59 - Expanding Access to Large-Scale Genomic Mutational Analyses for Patients with Advanced NSCLC in Italy (ID 2410)
09:45 - 18:00 | Author(s): Paolo Graziano
- Abstract
Background
In NSCLC, large-scale mutational analysis facilitates access to targeted treatments but is still not routinely employed due to significant technological barriers. The Alleanza Contro il Cancro (ACC) network of Italian Cancer Centers developed an affordable targeted sequencing panel for the identification of multiple genetic alterations with potential clinical utility, and designed a prospective multicentric trial to recruit 1000 newly diagnosed advanced NSCLC patients, aiming to i) compare panel performance against a set of externally validated biomarkers, including alterations in standard-of-care (EGFR, ROS1 and ALK) and non-standard-of-care (KRAS, BRAF, MET) biomarkers; ii) identify alterations in a large dataset of driver and potentially actionable genes; iii) correlate genotypes to survival outcomes and toxicity; iv) carry out ancillary studies on additional biomarkers and/or on specific patient groups (e.g. mutational burden, cfDNA, extensive characterization of immunotherapy-treated patients); v) build a centralized data repository for mutation interpretation and clinical recommendation.
Method
Through systematic literature mining and ad-hoc developed bioinformatic pipelines we identified: i) a set of 164 potentially actionable genes in solid tumors; ii) additional 18 genes with predicted driver function in NSCLC; iii) 70 actionable fusion transcripts; iii) 141 SNPs associated with pharmacogenomics markers. We designed a custom enrichment panel (~800 kb target) and compared PCR- and hybridization-based enrichment on semiconductor or by-synthesis sequencing to be subsequently deployed in a large observational trial. Sequencing is decentralized, to allow rapid turnaround time, but raw and processed data are collected in a single informatic infrastructure for centralized quality control and continuous bioinformatic pipeline improvement.
Result
PCR/semiconductor sequencing was selected for deployment based on cost and feasibility (2-day, highly automated workflow). 182 patients have been enrolled to date (90% stage IV, 10% IIIB). Of 65 patients with treatment information available, 28 (43%) subsequently received immunotherapy and 13 (20%) targeted therapy. For 56 patients with complete sequencing data, EGFR and KRAS status was concordant in 9/10 and 38/41 cases; discordant cases are being validated with orthogonal methods. Clinically significant MET amplifications were called in 2/2 cases. Remaining target regions did not show pathogenic alterations. Multiple alterations in potentially actionable genes were identified.
Conclusion
Large-scale sequencing is reliable, feasible and sustainable across multiple hospitals and provides clinically relevant results. The increased availability of genomic information may result in enhanced access to tailored therapies. Data and sample integration in centralized, shared repositories will allow multiple ancillary studies.
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P1.14 - Targeted Therapy (ID 182)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Targeted Therapy
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2019, 09:45 - 18:00, Exhibit Hall
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P1.14-26 - ALK Fusion Variant Detection by Targeted RNA-Seq in TKIs Treated ALK-Positive Lung Adenocarcinoma (ID 1860)
09:45 - 18:00 | Author(s): Paolo Graziano
- Abstract
Background
Clinical outcomes of ALK positive (ALK+) Non-Small-Cell Lung Cancer (NSCLC) and the identification of the most effective anaplastic lymphoma kinase inhibitor (ALKi) according to the specific ALK fusion variants are not well assessed. We retrospectively characterized fusion variant distribution in a cohort of ALK+ lung adenocarcinomas (ADC) with paired clinical data about treatments and outcomes.
Method
Diagnostic tumor tissue from advanced ALK+ (by FISH and/or IHC) ADC diagnosed from 2010 to 2018 and treated with single or multiple ALKis were collected (expanded cohort from Gobbini et al. Lung Cancer, 2017). The OncomineTM Solid Tumor Fusion Transcript Kit on an Ion PGM™ system and the Ion Reporter™ software were used to identify targeted ALK fusion gene products (ThermoFisher).
Result
Specific fusion variant transcripts were found in 34/55 (62%) of collected samples. As expected, EML4-ALK fusion transcripts were the most common (31/34 samples, 91%), but HIP-ALK transcripts were also detected (3/34 - 9%). Among EML4-ALK fusions the following variants were detected: V1 (n=11); V2 (n=2); V3a/b (n=12 ) V5a/b (n=5 ) and E6A19 (n=1). Patient median age was 60 year [range 36-85], 22 were male and 12 female. Three patients were current, 11 former and 20 never smokers. Crizotinib, alectinib, ceritinib, brigatinib and lorlatinib were the ALKis used. Independently of the therapy line, 12 patients received crizotinib only, while 22 patients received crizotinib followed by one or two other ALKis. Regardless of the type of transcript, those patients who received more than one ALKi had a better median overall survival compared to those receiving crizotinib only, as expected (74 vs 21 months, HR: 5.31; 95%CI: 1.464-19.26, log rank p=0.0006). Furthermore, a significant difference in the mean duration of the different ALKi treatment was found according to the ALK variants (Chi-square p<0.0001), suggesting a private ALKi efficacy profile for specific fusion variants. Finally, the 3 HIP-ALK cases showed a better outcome with respect the EML4-ALK variants (not reached vs 51 months).
Conclusion
Our analysis suggests that different ALK fusion variant might affect ALKi treatment duration in ALK+ lung ADC.
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P2.14 - Targeted Therapy (ID 183)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Targeted Therapy
- Presentations: 2
- Moderators:
- Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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P2.14-14 - Comparison of Molecular Testing Modalities for Detection of NRG1 Rearrangements in Invasive Mucinous Adenocarcinoma (ID 2179)
10:15 - 18:15 | Author(s): Paolo Graziano
- Abstract
Background
The NRG1 (Neureguline-1) fusion gene has been recently described as a new molecular feature of non small cell lung cancer (NSCLC), strictly related to invasive mucinous adenocarcinoma (IMA) subtype. Despite of NRG1 fusion partners, all Nrg1 chimeric ligands were predicted to retain the EGF-like domain of the wild-type NRG1 III-β3 isoform that produces oncogenic signals through ErbB2-ErbB3 heterodimers and leads to phosphorylation of ErbB3. To date, the NRG1 fusions were quite exclusively identified by RNA sequencing and only in few cases confirmed by fluorescent in situ hybridization (FISH) analysis, mainly due to the cellular features of IMA subtypes that produce interference in fluorescent signals detection. An accurate detection of NRG1 rearrangement/fusions in clinical tumor samples is actually demanded. In this study we compared the performance of two molecular testing approaches to detect NRG1 breaks in paraffin embedded formalin fixed (FFPE) IMA lung tissues.
Method
A total of 19 lung FFPE IMAs were screened by immunohistochemistry (IHC) to evaluate the expression of phosphorylated-ErbB3 (pErbB3) receptor. Samples positive for pErbB3 staining were tested by using by break-apart FISH to detect putative NRG1 rearrangements and RNA-targeted next generation sequencing (NGS) to identify fusion variants.
Result
Eleven cases showed an increased expression of pErbB3 in cancer cells compared to the adjacent non-involving bronchial epithelium which demonstrated a basal level staining of the protein. pErbB3 positive cases were investigated by FISH and showed <15% of rearranged nuclei (range 17-47%, mean 29%). In addition to the canonical signal split pattern, the FISH NRG1 assay reveals that cells also showed rearrangement patterns in form of isolated 3’ signals, thus indicating a strength analogy with ALK and ROS1 fusions, where a 5’ gene deletion was frequently observed. CD74-NRG1 fusion variant was identified by NGS analysis in four cases, whereas in three cases a 3’/5’ NRG1 imbalance was detected. For both approaches, we identified assay characteristics that likely contributed to false-negative results.
Conclusion
Our investigations confirm the usefulness of IHC/FISH combined approach for NRG1 broken tumors identification, but also highlight the crucial role of NGS to identify NRG1 functional chimeric transcripts. Such combined molecular testing should enhance the selection of NRG1-positive patients to include in clinical trials with specific compounds designed to inhibit the RTK downstream signal.
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P2.14-32 - Epigenetic Silencing of SPARC in NSCLCs (ID 2345)
10:15 - 18:15 | Author(s): Paolo Graziano
- Abstract
Background
The silencing of SPARC gene through methylation of its promoter region has been commonly observed in many solid tumors and is frequently associated with tumor progression and an aggressive clinical outcome. At present, the data concerning the mechanisms of SPARC deregulation in lung cancer are almost incomplete and correlation analysis with disease clinical course and specific therapeutic strategies is ongoing. Here we present the epigenetic profile of SPARC gene promoter in a collection of NSCLC cell lines and tissue samples and assess its prognostic value in surgical NSCLC resected patients.
Method
Four cell lines (3 adenocarcinoma, ADC and 1 large cell carcinoma, LCC) and 66 primary NSCLC tissues from surgically resected patients (30 squamous cell carcinoma, SqCC and 36 ADC) and 11 lung non-neoplastic tissues were epigenetically scanned. Promoter methylation analysis was performed using a quantitative methylation specific PCR assay in real-time (QMSP). The downstream effect of epigenetic silencing was also investigated in A549 and H1573 NSCLC cell lines by 5-Aza-2’-deoxycytidine treatment to demonstrate if the demethylating agent was able to restore SPARC mRNA expression levels. SPARC methylation levels were correlated with clinicopathological features.
Result
A tumor-specific DNA methylation of the SPARC gene promoter region was found as a specific feature of NSCLC (p=00643 Mann-Whitney test) and was also observed in all cell lines analyzed. In particular, it was detected in 56% of SqCCs (20/36) and 64% of ADCs (19/30), with SqCC showing the highest levels of methylation. Overall, we found promoter hypermethylation in 59% of NSCLCs. Moreover, a direct correlation with mRNA levels was confirmed by in vitro 5-azacytidine treatment. In SqCCs, SPARC methylation levels correlated with a negative prognosis (p<0,012 Supremum Test for Functional Form, HR=1,93; 95%CI).
Conclusion
Our results further suggest that epigenetic deregulation of the SPARC gene could be involved in the cancerogenesis of NSCLC. Additional studies on a larger cohort of NSCLCs and correlation with clinic-pathological features may contribute to disease progression prediction and new molecular basis to codify the response to therapy in lung cancer patients.
