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N. Baixeras
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MA 20 - Recent Advances in Pulmonology/Endoscopy (ID 685)
- Event: WCLC 2017
- Type: Mini Oral
- Track: Pulmonology/Endoscopy
- Presentations: 1
- Moderators:C. Lee, S. Sasada
- Coordinates: 10/18/2017, 14:30 - 16:15, F205 + F206 (Annex Hall)
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MA 20.06 - Discerning Lung Cancer Cell Patterns with Confocal Endomicroscopy (ID 8640)
15:05 - 15:10 | Author(s): N. Baixeras
- Abstract
- Presentation
Background:
Probe-based confocal endomicroscopy (pCLE) allows confocal microscopy of lung tissue in vivo but limited evidence is available. The objective was to discriminate pCLE patterns of lung cancer in vivo.
Method:
Fluorescence properties of methylene blue (MB) were examined ex vivo in confocal microscope. Next, 15 regions of the central airways were studied in vivo with pCLE and a representative image chosen for analysis with ImageJ software. Biopsy was performed for final diagnosis.
Result:
Ex vivo study showed no differences between 1% and 2% MB concentrations and rapid extinction of fluorescence after 10 minutes of MB application (figure). In vivo study included samples of bronchial mucosa (n = 6), inflammation (n = 3) and tumor (n = 6). pCLE image evaluation (table) showed inflammation and tumor nuclei were bigger (except SCLC) and occupied a greater area. Fluorescence of tumor nuclei was more intense. Non fluorescent area was inferior for both inflammation and tumor samples. Number of nuclei could not discriminate between normal and tumor.Figure 1 Table. Imaging features evaluated in pCLE framesArea occupied by nuclei (µm[2]) Intensity of nuclei (UA) Mean size of nuclei (µm[2]) Non-fluorescent area (µm[2]) Number of nuclei (µm[2]) Bronchial epithelium (mean(SD)) 97,769(9,451) 126(9) 107(10) 67,100(12,567) 937(84) Inflammation (mean(SD)) 117,381(22,166) 122(27) 127(15) 35,124(32,630) 933(225) B cell lymphoma 138,354 145 185 49,269 746 Adenocarcinoma 155,033 198 177 5,225 875 Squamous cell carcinoma 102,805 145 155 54,301 663 Small cell lung cancer 107,201 157 63 11,257 1,687 Non-small cell lung cancer 113,173 187 122 32,359 926 Hamartoma 120,188 145 114 25,438 1,058
Conclusion:
1. MB fluorescence is unaffected by stain concentration 2. There is exponential extinction of MB over time 3. Lung cancer cell pattern distinction in vivo is feasible Funded by Fundació MaratóTV3, SEPAR and FUCAP
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P1.01 - Advanced NSCLC (ID 757)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 10/16/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P1.01-075 - Simultaneous Multiplex Profiling of Gene Fusions, METe14 Mutations and Immune Genes in Advanced NSCLC by NCounter Technology (ID 9481)
09:30 - 09:30 | Author(s): N. Baixeras
- Abstract
Background:
Assessment of several immune-genes and tumor drivers is critical for individualized treatment of non-small cell lung cancer (NSCLC). We have previously demonstrated the ability of the transcript-based nCounter Technology for the detection of ALK, ROS1 and RET gene fusions, using a customized codeset (Reguart et al. Clinical Chemistry 2017). Here, we present the first results of the validation in advanced NSCLC samples of a new CodeSet designed to simultaneously characterize clinically relevant gene fusions, MET alterations and the expression of immune genes.
Method:
We have designed an in-house custom set to detect driver fusions involving 4 genes (ALK, ROS, RET, NTRK), MET exon 14 skipping mutation, MET overexpression and immune genes (PD1, PDL-1, CD4, CD8, FOXP3, GZMM, IFNG). A panel of ALK-ROS-RET-NTRK positive cell lines (H2228, H3122, SU-DHL-1, HCC78, BaF3 pBABE, LC2/ad and a NTRK-positive cell line), Hs746T (METex14), EBC-1 (overexpressing MET) and a negative cell line (PC9) were used for the initial validation of the panel. Subsequently, 58 FFPE samples from advanced NSCLC patients, previously characterized by FISH, RT-PCR and IHC, have been analyzed. Total amount of 100-150 ng RNA was used for detection. Workflow includes RNA isolation, hybridization and digital counting with for a total turnaround of 3 days. Raw counts were normalized using positive controls, negative controls and house-keeping genes.
Result:
.Results obtained with the cell lines positive for ALK, ROS1, RET and NTRK1 fusion genes were exactly coincident with their genotypes, with fusion transcripts successfully detected in all cases by 3’/5’ imbalance and direct fusion probes. In addition, METex14 splicing transcripts were detected in the Hs746T cells at significant levels, higher than those of wt MET mRNA. In contrast, METex14 mRNA counts were almost undetectable in the rest of cell lines. Regarding FFPE samples from advanced patients, 46 could be successfully analyzed by nCounter. Among 13 patients positive for ALK and ROS1 fusions, 12 were confirmed by nCounter. Regarding the METex14 splicing variant, 5 out of 6 patients previously detected by RT-PCR were also positive by nCounter.
Conclusion:
Preliminary data suggest that multiplex detection of clinically relevant drivers can be successfully achieved using nCounter Technology. The assay is simple, requires short hands-on-time, needs low input RNA and is highly efficient in detecting gene rearrangements and METex14 splicing variants. Results will be prospectively validated in a larger cohort of advanced NSCLC patients and we will determine if clusters of different inmune-phenotypes exist among oncogenic-driven NSCLC tumors.
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P2.08 - Locally Advanced Nsclc (ID 709)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Locally Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 10/17/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P2.08-006 - Immunological Biomarkers Characterization in Locally Advanced Non-Small Cell Lung Cancer Treated with Concurrent Chemo-Radiotherapy (ID 9584)
09:30 - 09:30 | Author(s): N. Baixeras
- Abstract
Background:
The immune microenvironment of locally advanced non-small cell lung cancer (NSCLC) has not been systematically studied. Our aim was to determine the prognostic value of immunological biomarkers expression in a cohort of patients (pts) in this clinical setting.
Method:
We retrospectively reviewed 46 bronchial biopsies from locally advanced NSCLC. Pts were treated between 2010 and 2014 with concurrent chemo-radiotherapy (cCRT) at the Catalan Institute of Oncology. The following immunological markers were assessed by immunohistochemistry: PD-L1, ≥5% membrane expression on tumor cells was considered positive (+); HLA-Class I expression was classified into 0,1+,2+ according to membrane intensity; CD8+ tumor infiltrating lymphocytes (CD8 TILs) classified into low ≤5% or high >5% intratumoral infiltration. Chi-square test for assessing correlation and survival analysis by Kaplan-Meier method were used.
Result:
From 46 pts: Median age was 65 (43-81); gender: male 94%, female 6%; ECOG≤1 96%; smoking status: current 67%, former 30%, never 3%; histology: squamous cell carcinoma (SCC) 63%, adenocarcinoma (ADC) 24%, NSCLC (NOS+large cell) 13%; cN0-1 30%, cN2 57%, cN3 13%. Platinum doublet CT: Cisplatin 57%, Carboplatin 43%. PD-L1 was positive in 38% of cases and was positively correlated with HLA-I expression (p= 0.015) and CD8-TILs (p= 0.008). No correlations between PD-L1/CD8 TILs status and G3-4 radio-induced toxicities (pneumonitis, esophagitis) were found. At a median follow-up of 48 months (m), 53% of pts had relapsed. According to immune phenotype, median overall survival (mOS) was 20m (PD-L1 +, CD8 high; n=10) vs 17 m (PDL1 negative, CD8 low; n=19) vs not reached (PD-L1 negative, CD8 high; n=5) (p=0.23). Considering CD8 TILs, mOS in high CD8 (n=15) was 35m vs 18 m in low CD8 (n=26) (p=0.22).
Conclusion:
PD-L1, HLA-I and TILs CD8 expression was positively correlated. The potential role of TILs CD8+ as a prognostic biomarker in this cohort of pts that comprised mostly SCC histology, is promising. These results should be investigated in a larger cohort.
