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D. Casadevall
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P1.02 - Poster Session with Presenters Present (ID 454)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 2
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.02-039 - Assessment of KRASmutations (by Digital PCR) in Circulating Tumoral DNA from Lung Adenocarcinoma Patients (ID 5593)
14:30 - 14:30 | Author(s): D. Casadevall
- Abstract
Background:
KRAS mutations are detected in approximately 25% of lung adenocarcinomas (LA). Targeted therapies against KRAS are under investigation. The use of tumor biopsy for molecular testing may be challenging due to the invasiveness of the procedure, the limited material for multiple biomarker analyses and tumor heterogeneity. Mutation detection in circulating cell-free tumor DNA (ctDNA) can overcome these caveats and also be used for tracking tumor dynamics. The aim of this study was to evaluate KRAS mutation detection in plasma samples from LA.
Methods:
Plasma samples from 35 patients with histologically confirmed KRAS mutant LA were collected at initiation of chemotherapy. KRAS mutations were assessed using digital PCR technology (QuantStudio3D Digital PCR System, Thermofisher Scientific). Correlation between ctDNA and tumor biopsies in terms of mutation detection was analysed. In 5 cases plasma samples were obtained during the course of the disease to monitor clonal dynamics.
Results:
Most cases were male (71%), with stage IV disease (83%), and showed KRAS mutation on codon12 (94%). KRAS mutation was found in plasma samples in 28/35 cases, showing a concordance with the tumor of 80%. In patients whose disease was limited to thorax (stages II, III, and IVa) KRAS mutation was detected in 7/10 (70%) plasma samples. Plasma/tumor biopsy concordance in cases with extra-thoracic metastases was 84% (21/25). The 4 false negative cases had low burden of extra-thoracic disease, with bone (2 cases), brain (1 case), and abdominal lymph node (1 case) as the only metastatic location outside the thorax. KRAS clonal dynamics in plasma showed a good correlation with treatment responses in some cases (figure 1).Figure 1
Conclusion:
High concordance in the detection of KRAS mutations was found between plasma and tumor tissue using digital PCR technology, particularly in cases with extra-thoracic disease. Digital PCR allows for tracking clonal dynamics in KRAS mutant LA.
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P1.02-048 - MET Exon 14 Skipping Mutations and Gene Amplifications Are Not Simultaneous Events in NSCLC (ID 5881)
14:30 - 14:30 | Author(s): D. Casadevall
- Abstract
Background:
Mutations in the MET exon 14 RNA splice acceptor and donor sites, which lead to exon skipping, have been described with responsiveness to crizotinib. Until now, patient selection has been made in view of MET amplification/high polysomy and protein overexpression, with discrepancies in positivity criteria. We investigated the prevalence of abnormal MET mutational status and the subsequent gene copy number alterations (CNAs) in NSCLC.
Methods:
We routinely tested 190 lung adenocarcinomas and adenosquamous carcinomas for MET exon 14 and flanking introns mutations by PCR-direct sequencing, and for MET gene CNAs by FISH (Abbott Molecular). Amplifications were defined as mean gene by mean centromere ratio ≥2.2, and high gains as mean gene ≥5.0. RT-PCR was performed to validate mutations leading to exon 14 skipping. We collected clinical-pathological data together with EGFR and KRAS mutational status and ALK, ROS1 and RET rearrangements.
Results:
MET alterations were found in 34 patients (17.9%): 11 mutated cases (5.8%), eight gene amplifications (4.2%), and 15 high gains (9.1%). Six out of 11 mutations were confirmed to lead to exon 14 skipping (3.2%). Remarkably, none of these exon 14 skipped cases had concurrent MET amplification nor high gains. Although, KRAS p.G12C and EGFR 19 exon mutations were found concomitant with MET mutation in two of these cases. Among the 14 confirmed MET altered cases (6 exon 14 skipped and 8 amplified): 13 patients were male (93%), with a median age of 65.7 years (range: 40-91), nine current smokers (64%) (40 pack-years, range: 20-60), and eight diagnosed in an advanced stage disease (III and IV) (57%). Correlation with c-MET protein expression by IHC is ongoing, and data will be presented at the meeting.
Conclusion:
As no concurrent MET mutated and amplified cases were found, our data support prospective identification of both, MET exon 14 skipping mutations and gene amplifications. These mutations define a new subset of NSCLC patients that should be analyzed independently of the status of MET gene copy number.
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P2.01 - Poster Session with Presenters Present (ID 461)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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P2.01-063 - PDL1, JAK2 and PTEN Copy Number Alterations Synergistically Upregulate PD-L1 Expression in NSCLC (ID 5871)
14:30 - 14:30 | Author(s): D. Casadevall
- Abstract
Background:
Predictive biomarkers research in anti-PD-1/PD-L1 immunotherapy treatment is still at an early stage of development. Recently, amplification of PDL1 gene (9p24.1) has been described in NSCLC in correlation with PD-L1 protein expression. In addition, other tumor-constitutive alterations such as JAK2 amplifications (322kb upstream of PDL1) or PTEN deletions are also known to modulate PD-L1 expression. We aimed to determine PDL1, JAK2 and PTEN copy number alterations (CNAs) and subsequent PD-L1 protein expression in NSCLC.
Methods:
A total of 171 NSCLC patients (121 ADC and 50 SCC) were included. Clinical, histopathological and molecular data were collected. In resected early-stage diseases, two distinct histologic areas from FFPE tumoral tissue were included for each patient in 8 tissue microarrays (TMAs). PD-L1 expression analysis was assessed by IHC using PD-L1 #SP142 clone (Ventana) and positive cut off was defined at >1%. Moreover, H-score semi-quantitative approach was used to generate a score from 0 to 300. PDL1, JAK2 and PTEN CNAs were studied by FISH using commercial and non-commercial probes hybridized with respective centromere enumeration probes. Amplifications were defined as mean gene by mean centromere ratio ≥2.0, deletions as mean gene by mean centromere ratio ≤0.8, gains as mean gene ≥2.5, and high gains as mean gene ≥4.0.
Results:
PD-L1 expression was positive in 40 out of 171 cases (23.3%), with an average H-score of 177 and significantly associated with ADC solid histological pattern (p=0.012), KRAS mutations (p=0.001), the presence of TILs (p=0.001), and active smoking status (p=0.031). PDL1 gene CNAs were seen in 68/159 assessable cases (42.8%). We found 14 tumors with PDL1 amplification (8.8%), 21 PDL1 high gains (13%) and 33 PDL1 gains (20.8%). Twelve out of 14 FISH amplified cases had PD-L1 positive expression. Thus, FISH predicted positive PD-L1 IHC result with a low sensitivity (31.6%) but a high specificity (98.6%). Among PD-L1 expressing tumors (n=40), seven cases had JAK2 amplifications (6 of them with PDL1 gene coamplification) and eight showed PTEN deletions (3 of them were PDL1 amplified). Differences in H-score intensity between these groups were observed: JAK2-PDL1 coamplified cases had near 2-fold increase in PD-L1 expression than PDL1 alone (average H-score: 282 vs. 148).
Conclusion:
PDL1 gene amplification is synergistically regulating PD-L1 protein expression. In addition, JAK2 amplification upregulates PD-L1 expression, following the concept of cooperative oncogenic effects of genes within the PDJ amplicon. PDL1, JAK2 and PTEN CNAs analysis may be relevant for anti-PD-1/PD-L1 patient selection.
