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Fernando Lopez-Rios



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    MA26 - New Therapies and Emerging Data in ALK, EGFR and ROS1 (ID 930)

    • Event: WCLC 2018
    • Type: Mini Oral Abstract Session
    • Track: Targeted Therapy
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/26/2018, 13:30 - 15:00, Room 201 BD
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      MA26.07 - ROS1 (SP384) Immunohistochemistry Inter-Reader Precision Between 12 Pathologists (ID 12387)

      14:10 - 14:15  |  Author(s): Fernando Lopez-Rios

      • Abstract
      • Presentation
      • Slides

      Background

      ROS1 positive non-small cell lung cancer (NSCLC) patients can be treated with specific tyrosine kinase inhibitors including crizotinib. ROS1 positivity is often clinically detected by fluorescence in situ hybridization (FISH), however ROS1 IHC can be used to screen samples prior to FISH confirmation of ROS1 status. The ROS1 (SP384) antibody detects ROS1 with high sensitivity, specificity, and consistency. Consistent interpretation of a ROS1 IHC assay between pathologists is important patient evaluation. Here we present inter-reader precision of 12 pathologists across 60 FFPE cases stained with ROS1 (SP384).

      a9ded1e5ce5d75814730bb4caaf49419 Method

      A retrospective cohort of 60 FFPE NSCLC cases stained with H&E, Rabbit Monoclonal Negative Control Ig, and ROS1 (SP384) were selected to represent positive, negative, and borderline ROS1 IHC status. Twelve practicing lung pathologists independently scored the cases as positive or negative around a cutoff of cytoplasm staining in > 30% tumor cells at a ≥2+ intensity level using Pathotrainer software (Pathomation bvba). Scoring was blinded to other readers and ROS1 status of the cases. Overall percent agreement (OPA), negative percent agreement (NPA), and positive percent agreement (PPA) were calculated in comparison to the group mode. Average overall percent agreement (AOPA), average positive agreement (APA), and average negative agreement (ANA) were calculated pairwise for each reader pair. Following independent assessment, participating pathologists conducted a discordant case review establishing consensus reads for all 60 cases and compared 44 cases to available FISH results.

      4c3880bb027f159e801041b1021e88e8 Result

      OPA of each of the 12 readers to the mode was 96.4% (95% CI 93.9-98.6) with PPA of 96.3% (95% CI 92.7-99.4) and NPA of 96.5% (95% CI 92.8-99.5). Pairwise AOPA between each of the 12 readers was 94.5% (95%CI 91.2-97.7) with APA 94.0% (95% CI 89.5-97.6) and ANA 95.0% (95%CI 91.2-97.9).

      Consensus IHC scores were concordant with FISH 90.0% (40/44 cases).

      8eea62084ca7e541d918e823422bd82e Conclusion

      Inter-reader precision around a cutoff of >30% tumor cells with cytoplasmic staining at a ≥2+ intensity level was high in interpreting ROS1 (SP384) in NSCLC samples. Case review highlighted confirmation with FISH in questionable cases and staining patterns to be considered when interpreting ROS1 (SP384) IHC.

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    P1.09 - Pathology (Not CME Accredited Session) (ID 941)

    • 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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      P1.09-09 - Evaluation of a Novel ROS1 Immunohistochemistry Clone (SP384) for the Identification of ROS1 Rearrangements in NSCLC Patients (ID 12744)

      16:45 - 18:00  |  Author(s): Fernando Lopez-Rios

      • Abstract

      Background

      After the approval of crizotinib in ROS1 rearranged NSCLCs, the importance of accurately identifying those patients has never been greater. Although the recently updated guideline for molecular testing supports the use of ROS1 IHC as a screening test, to the best of our knowledge, only one ROS1 clone is commercially available and most published comparison studies involve a relatively small numer of positive cases. This situation prompted us to investigate a novel ROS1 IHC antibody in a large series of ROS1 positive NSCLCs samples.

      a9ded1e5ce5d75814730bb4caaf49419 Method

      Thirty-nine ROS1 FISH-positive (i.e., gold standard) samples from patients with NSCLCs procured at 22 hospitals were used for this study. In addition, 20 consecutive ROS1 FISH-negative samples from NSCLCs diagnosed at the referral institution were included as negative controls. The material available for all tumors had been formalin-fixed and paraffin-embedded. The specifics of formalin fixation were unknown. All specimens were independently screened for ROS1 expression by two IHC antibodies (clone D4D6 from Cell Signaling Technology and clone SP384 provided by Ventana Medical Systems, Inc.) according to previously published methodology or the manufacturer´s instructions. FISH-validated ROS1-positive external controls were included in all the slides. The slides were reviewed by two pathologists blinded to FISH results. The results of both ROS1 IHC assays were evaluated using a modified H-score: strong cytoplasmic staining (3+), clearly visible using a ×2 or ×4 objective; moderate staining (2+), requiring a ×10 or ×20 objective to be clearly seen; and weak staining (1+), cannot be seen until a ×40 objective is used. Both anti-ROS1 IHC staining results were finally interpreted using a binary scoring system: positive (3+ or 2+) or negative (1+ or 0).

      4c3880bb027f159e801041b1021e88e8 Result

      In ROS1 FISH-negative cases, positive immunoreactivity (3+ or 2+) was observed in 25% and 5% of samples by SP384 and D4D6, respectively. In ROS1 FISH-positive cases, positive expression above the threshold was always present with both antibodies except for one sample that was only stained with SP384. In 4 positive cases (10.3%) by SP384 and 22 positive tumors (56.4%) by D4D6, we noted significant intratumoral heterogeneity, ranging from weak to strong protein expression.

      8eea62084ca7e541d918e823422bd82e Conclusion

      We have studied a very large series of ROS1 FISH-positive NSCLCs with a novel IHC clone, which showed excellent sensitivity. The predominantly homogeneous and intense staining may support the use of a dichotomous scoring approach, before confirmation with FISH or a molecular method.

      Funding: I+D+I 2013-2016/Feder. ISCIII: PI14/01176

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    P2.09 - Pathology (Not CME Accredited Session) (ID 958)

    • 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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      P2.09-11 - TMB Estimated with Targeted NGS in Early Stage Squamous Cell Carcinoma: Correlation with PD-L1 Expression and Lymphocyte Density (ID 12774)

      16:45 - 18:00  |  Author(s): Fernando Lopez-Rios

      • Abstract

      Background

      It has been proposed that a combination of assays may refine the prediction of response to checkpoint inhibitors, with tumour mutation burden (TMB) being lately the strongest biomarker associated with efficacy. Although some targeted next generation sequencing (NGS) assays provide a good estimate of whole exome sequencing TMB, they are only available throughout referral laboratories. We sought to investigate the possibility of profiling TMB in-house with a commercially available targeted NGS assay, and to correlate the results with survival, the status of TP53, PD-L1 overexpression and tumor-infiltrating lymphocytes (TILs).

      a9ded1e5ce5d75814730bb4caaf49419 Method

      The study included samples from 40 patients diagnosed with early-stage lung squamous cell carcinoma. PD-L1 immunohistochemistry (IHC) was performed with two clones (SP263 and SP142, Ventana Medical Systems). CD8+ TILs were scored with a digital algorithm. The status of TP53 (exons 4–10) was investigated with direct sequencing. Ion TorrentTM OncomineTM Tumor Mutation Load Assay (ThermoFisher Scientific), a targeted NGS assay which covers 1.7Mb across 409 cancer driver genes, was used to assess TMB. NGS was performed on genomic DNA from FFPE tumor samples using the Ion S5TM system. The results were analyzed with the Ion ReporterTM software. According to the manufacturer’s instructions, TMB was calculated based on the number of non-synonymous somatic mutations, after removing polymorphisms and known or predicted driver mutations from all the variants. We investigated the correlation between TMB and PD-L1 expression, lymphocyte density, TP53 status and survival.

      4c3880bb027f159e801041b1021e88e8 Result

      TMB data was available for all 40 patients. The mean and median number of mutations was 13 and 11, respectively. CD8+/mm2 TILs within the peritumoral stromal compartment ranged from 312 to 4793 and within the intraepithelial compartment ranged from 17 to 2002. Sixty percent and 32% of cases were positive in tumor cells with SP263 or SP142, respectively (1% cut-off). Immune cells PD-L1 expression (1% cut-off) was observed in 92% of samples with both clones. No correlation was found between PD-L1 expression or lymphocyte density and TMB. Tumors with TP53 mutations showed non-significant higher numbers of mutations than those wild-type (p=0.075). Accordingly, there was a similar trend for overall survival using the median number of mutations as a cut-off (p=0.137, HR, 5.29).

      8eea62084ca7e541d918e823422bd82e Conclusion

      It is feasible to use targeted NGS to estimate TMB. In our pilot study, there was no obvious correlation with other immunooncology predictive biomarkers.

      Funding: AES 2017/Feder. ISCIII: PI17/01001

      6f8b794f3246b0c1e1780bb4d4d5dc53

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    PC04 - Targeted Therapy for NSCLC (ID 843)

    • Event: WCLC 2018
    • Type: Pro-Con Session
    • Track: Targeted Therapy
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/24/2018, 15:15 - 16:45, Room 106
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      PC04.06 - Focused Panels for Molecular Profiling for Routine Use (ID 11619)

      16:15 - 16:27  |  Presenting Author(s): Fernando Lopez-Rios

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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