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Janusz Milanowski



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    P1.11 - Screening and Early Detection (Not CME Accredited Session) (ID 943)

    • Event: WCLC 2018
    • Type: Poster Viewing in the Exhibit Hall
    • Track:
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/24/2018, 16:45 - 18:00, Exhibit Hall
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      P1.11-03 - MicroRNA with Ability to Reciprocal Regulation of Dicer and Drosha – Plasma Expression Status and Diagnostic Value in Non-Small Cell Lung Cancer (ID 12133)

      16:45 - 18:00  |  Author(s): Janusz Milanowski

      • Abstract
      • Slides

      Background

      Examination of microRNAs expression in plasma could be useful in screening and in early detection of non-small cell lung cancer (NSCLC). One of the most important enzymes in miRNA biogenesis are Dicer and Drosha. Disrupted expression of miRNA with ability to reciprocal regulation of Dicer and Drosha could participate in cancer development.

      a9ded1e5ce5d75814730bb4caaf49419 Method

      Plasma expression of miR-27-3p, miR-31, miR-182 and miR-195 were analysed in 138 Polish NSCLC patients (median age 65 years, 83 male and 55 female) and in 45 healthy people (median age 62 years, 28 male and 17 female). 57 (41.3%) NSCLC patient were in I-IIIA stage and 81 (58.7%) patients were in IIIB-IV stage. Relative expression of microRNAs between studied groups was compared using U Mann-Whitney test. For assessment of diagnostic accuracy (test sensitivity and specificity), the receiver operating curves (ROC) with area under curve (AUC) analysis were generated.

      4c3880bb027f159e801041b1021e88e8 Result

      We demonstrated that plasma levels of miR-27-3p, miR-31 and miR-182 were significantly higher (p<0.000001, p=0.00008, p=0.006 respectively) and miR-195 significantly lower (p=0.000002) in NSCLC patients in comparison with healthy donors. Moreover, patients with early stages (I-IIIA) of NSCLC showed significantly higher expression of miR-27a-3p (p<0.000001), miR-31 (p=0.0003) and miR-182 (p=0.000003) than healthy persons. Expression of miR-195 was significantly lower in patients with early stages (I-IIIA) of NSCLC than in healthy donors (p<0.000001). AUC for miR-27a was 0.95 (94% sensitivity and 81% specificity, p<0.00001), for miR-31 was 0.71 (73% sensitivity and 61% specificity, p=0.001), for miR-182 was 0.77 (70% sensitivity and 79% specificity, p<0.00001) and for miR-195 was 0.82 (74% sensitivity and 80% specificity, p=0.00001).

      8eea62084ca7e541d918e823422bd82e Conclusion

      Expression of miR-27a, miR-31, miR-182 and miR-195 could distinguish patients with NSCLC from healthy people. The examination of these microRNAs in plasma could be used in non-invasive lung cancer diagnosis. Deregulation of Dicer and Drosha expression by microRNAs could have oncogenic character.

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    P1.13 - Targeted Therapy (Not CME Accredited Session) (ID 945)

    • Event: WCLC 2018
    • Type: Poster Viewing in the Exhibit Hall
    • Track:
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/24/2018, 16:45 - 18:00, Exhibit Hall
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      P1.13-14 - The Molecular Landscape of Lung Adenocarcinomas with Activating EGFR Gene Mutations Determined by Targeted-Sequencing (ID 12476)

      16:45 - 18:00  |  Author(s): Janusz Milanowski

      • Abstract
      • Slides

      Background

      The analysis of EGFR activating mutations and ALK rearrangement is a routine procedure in qualification of NSCLC patients for molecularly targeted therapy. Targeted sequencing, as a type of NGS, allows more comprehensive analysis of low-frequency variations in suppressors and oncogenes that are commonly mutated in solid tumors. In the following study we analyzed a molecular landscape of NSCLC patients harboring EGFR activating mutations who response for EGFR TKIs.

      a9ded1e5ce5d75814730bb4caaf49419 Method

      The studied group included 19 Caucasian patients (7 male and 12 female, median age 65±12 years, 4 current smokers, 12 non-smokers and 3 former-smokers) with lung adenocarcinoma. 12 (63%) deletions in exon 19, 4 (21%) substitutions Leu858Arg in exon 21, 2 (11%) insertions A763_Y764 in exon 20 and 1 (5%) substitution Thr790Met in exon 20 of EGFR gene were detected using real-time PCR technique. Four non-smoking patients (median age 65±12 years) with lung adenocarcinoma and wild-type (wt) of EGFR gene were a control group. The DNA for the analysis was isolated from FFPE tissue or cellblocks and the mutational landscape was evaluated using Illumina’s TruSight Tumor 26 panel on miSeq platform (Illumina, USA).

      4c3880bb027f159e801041b1021e88e8 Result

      The targeted sequencing confirmed lack of EGFR gene mutations in control group and the presence of EGFR mutations in 84% (16/19) of patients with these mutations confirmed in routine diagnostics - all deletions in exon 19 and substitutions in exon 21 were confirmed. Targeted sequencing did not identify rare mutations in exon 20. NGS compared to real-time PCR technique achieved 86,95% accuracy with 84,21% of sensitivity and 100% of specificity or PPV and NPV values reached 100% and 57,10%, respectively. The targeted sequencing allowed to identify 166 variants in 25 out of 26 genes covered by the analysis. The most frequently mutated suppresors were TP53, MSH6 and APC (38%; 19% and 17% respectively) and oncogenes were MET, PDGFR and EGFR-AS1 (31%, 14% and 13% respectively). The frequency of particular variants was significantly higher in smokers than in non-smokers (p=0,0002; χ²=16,94). U-Man Whitney test showed that the median number of genetic alternations in EGFR mutated group was significantly higher than in wt EGFR group (p=0.0091; median: 17 vs 11 respectively).

      8eea62084ca7e541d918e823422bd82e Conclusion

      The targeted sequencing allowed to detect the most common activating mutations in EGFR gene and numerous variants both in suppressors and oncogenes. NGS showed too low sensitivity for detection of rare EGFR mutations. The molecular landscape was more unpredictable and varied in smokers compare to non-smokers.

      6f8b794f3246b0c1e1780bb4d4d5dc53

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    P2.04 - Immunooncology (Not CME Accredited Session) (ID 953)

    • Event: WCLC 2018
    • Type: Poster Viewing in the Exhibit Hall
    • Track:
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/25/2018, 16:45 - 18:00, Exhibit Hall
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      P2.04-05 - Correlation Between PD-L1 Gene Promoter Polymorphisms and Expression of PD-L1 mRNA and Protein in NSCLC Patients. (ID 12554)

      16:45 - 18:00  |  Author(s): Janusz Milanowski

      • Abstract
      • Slides

      Background

      Immunotherapy, the most novel approach to cancer treatment, gives a promising option for non-small cell lung cancer (NSCLC) patients. Most drugs targeting PD-1 or PD-L1 have been proven to be effective when cancer tissue expresses PD-1 or PD-L1 protein on its surface. The promoter region of PD-L1 gene is responsible for regulating the gene expression, thus in effect the expression of the protein. We decided to check whether single nucleotide polymorphisms (SNPs) in PD-L1 gene promoter region may affect this expression.

      a9ded1e5ce5d75814730bb4caaf49419 Method

      The study included 47 NSCLC patients (20 male, 27 female; median age 65±7.6 ). PD-L1 protein expression was analysed using two IHC assays with SP142 and 22C3 antibodies, on corresponding analysis platforms. mRNA was analysed using qRT-PCR method. PD-L1 gene promoter region polymorphisms [rs822335(C/T) and rs822335(C/G)] were analysed using real-time PCR method, on Illumina Eco platform. The analysis was performed in histological (resected tissue) and cytological (cellblocks from bronchoscopy biopsies) material (FFPE blocks).

      4c3880bb027f159e801041b1021e88e8 Result

      There were significant positive correlations between expression of mRNA of PD-L1 gene and the percentage of PD-L1 protein positive cells: tumor cells in the reaction with 22C3 antibody (R=+0.39,p=0.025), tumor cells in the reaction with SP142 antibody (R=+0.45, p=0.009) as well as infiltrating immune cells in the reaction with SP142 antibody (R=+0.49, p=0.006).

      The studied group consisted of 21 CC homozygotes, 23 CT heterozygotes and 3 TT homozygotes in rs822335, and 12 CC homozygotes, 24 CG heterozygotes and 11 GG homozygotes in rs822336 polymorphic sites.

      Slightly higher (p=0.08) percentage of tumor cells with PD-L1 expression was observed in patients with CC genotype compared to patients with CT and TT genotypes in rs822335 polymorphism. We observed significantly higher expression of mRNA for PD-L1 gene in patients with GG genotype compared to patients with CG genotype (p=0.05) in rs822336 polymorphism. Additionally, odds ratio analysis showed that a higher chance of high mRNA expression of PD-L1 gene occurred in patients with GG genotype as compared to patients with CC genotype (OR=7.0, ch2=4.46, p=0.035).

      8eea62084ca7e541d918e823422bd82e Conclusion

      Analysis of PD-L1 expression on tumor cells presents numerous problems – availability of material, tumor heterogeneity, different methods of detection. We have presented that analysis of PD-L1 gene promoter polymorphisms may be useful in determination of PD-L1 expression. It might be carried out in blood samples, when surgical material is not available. The usefulness of PD-L1 gene polymorphism, as a predictor of immunotherapy, should be investigated in clinical trials.

      6f8b794f3246b0c1e1780bb4d4d5dc53

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    P3.13 - Targeted Therapy (Not CME Accredited Session) (ID 979)

    • Event: WCLC 2018
    • Type: Poster Viewing in the Exhibit Hall
    • Track:
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/26/2018, 12:00 - 13:30, Exhibit Hall
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      P3.13-08 - Assessment of EGFR Gene Mutations In cf-DNA in Monitoring of Response to EGFR TKIs in Patients with Lung Adenocarcinoma (ID 12474)

      12:00 - 13:30  |  Author(s): Janusz Milanowski

      • Abstract
      • Slides

      Background

      Molecular analysis of cf-DNA in NSCLC patients enables detection and monitoring of EGFR mutations. It allows for detection of the acquired resistance for 1st and 2nd generation of EGFR-TKIs caused Thr790Met substitution. The third generation of EGFR-TKIs (osimertinib) could overcome the resistance in patients with Thr790Met mutation.

      a9ded1e5ce5d75814730bb4caaf49419 Method

      The studied group included 23 Caucasian patients (8 male and 15 female, median age 71±9) with diagnosed lung adenocarcinoma and with EGFR mutations detected in tumor samples. Blood samples were collected before administration of EGFR-TKIs in all patients, and re-collected repeatedly from 10 patients during therapy. EGFR mutations and content of mutated cf-DNA were analyzed using ctEGFR Mutation Analysis Kit (Entrogen, USA) in Rotor-Gene Real-Time PCR device (Qiagen, Germany).

      4c3880bb027f159e801041b1021e88e8 Result

      Analysis of EGFR activating mutations in cf-DNA showed 82.61% concordance/sensitivity (19/23) with tumor samples. The mean content of mutated cf-DNA was 7.44% and it was significantly lower (p<0.000035) than in tumor samples (34.54%). Concentration of mutated cf-DNA positively correlated with advanced stage of the disease. Monitoring of EGFR status in cf-DNA showed reduction and stabilization of mutant DNA content with the emergence of response to EGFR-TKIs treatment. Primary Thr790Met substitution was undetectable in cf-DNA and in tumor samples. Acquired resistance in Thr790Met mechanism during EGFR-TKIs treatment was detected only in one patient (1/10). This patient responded to osimertinib therapy.

      8eea62084ca7e541d918e823422bd82e Conclusion

      Analysis of EGFR mutations in cf-DNA shows lower sensitivity than in DNA isolated from tumor samples. Multiple evaluations of EGFR status may be useful in monitoring of therapy and allows for early detection of acquired resistance.

      6f8b794f3246b0c1e1780bb4d4d5dc53

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