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2509 Background: Anti-PD1 and PD-L1 antibodies (mAb) are immune checkpoint inhibitors (ICIs) to treat patients with metastatic non–small cell lung cancer (NSCLC). Unfortunately, only a handful of patients respond to ICIs. Methods: A cohort of patients with metastatic NSCLC (n=133) treated with anti-PD1 or anti-PD-L1 mAb in two independent care centers was evaluated. An independent cohort of 132 patients from another hospital was used as a validation. Immunoscore IC, an in vitro diagnostic test (CE-IVD), was used on a routine single FFPE slide, and duplex immunohistochemistry CD8 and PD-L1 staining was quantified using digital pathology. Quantitative and spatial parameters related to cell location, number, proximity, and clustering were analyzed. An Immunoscore IC–based model discriminated patients into 2 categories or 3 categories. Results: Anti–PD-L1 clone (HDX3) had similar characteristics as other anti–PD-L1 clones (22C3, SP263) with a mean overall agreement above 95%. Intra- and inter-laboratory concordances for classifying patients at 1% cut-off according to digital anti–PD-L1 (HDX3) were 100% and 94%, respectively. Routine laboratory evaluation of PD-L1 expression showed an agreement with digital anti–PD-L1 quantification of 92% and 97% at 1% and 50% cut-offs, respectively. Using univariate Cox model after FDR correction, 5 pathological dichotomized variables were significantly associated with PFS (all p < 0.0001). These variables included: CD8 free of PD-L1, CD8 clusters, CD8 cells in proximity of PD-L1 cells, CD8 number, PD-L1 cells in proximity of CD8 cells. Similar results were found using univariate Cox analysis on continuous variables (all p < 0.003) in two independent cohorts of patients. Using multivariate Cox model Immunoscore IC classification improved the discriminating power of prognostic model, which included clinical variables and pathologist PD-L1 assessment. In two categories, the Immunoscore IC risk score was significantly associated with both patients’ PFS (p < 0.0001) and OS (p < 0.0001) in the training cohort and in the validation cohort (PFS: p = 0.0047, OS: p < 0.0001). Further increased hazard ratios were found when stratifying patients into 3 categories of Immunoscore IC. At 6 months, PFS rates were 10% versus 60% in the training cohort and 20% versus 62% in the validation cohort for high-risk and low-risk Immunoscore IC score, respectively. All patients (100%) with high-risk Immunoscore IC score relapsed in less than 18 months, in contrast to 34% and 33% of low-risk Immunoscore IC patients who did not relapse for more than 36 months in the training and validation cohorts, respectively. Conclusions: These data underline that Immunoscore IC is a potent tool to predict the efficacy of ICIs in patients with NSCLC. Immunoscore IC characterized patients who are resistant to ICIs.

Introduction The detection of a ROS1 rearrangement in advanced and metastatic lung adenocarcinoma (LUAD) led to a targeted treatment with tyrosine kinase inhibitors with favorable progression-free survival and overall survival of the patients. Thus, it is mandatory to screen for the ROS1 rearrangement in all these patients. ROS1 rearrangements can be detected using break-apart fluorescence in situ hybridization (FISH), which is the gold standard; however, ROS1 immunohistochemistry (IHC) can be used as a screening test because it is widely available, easy and rapid to perform, and cost-effective. Methods We evaluated the diagnostic accuracy and interpathologist agreement of two anti-ROS1 IHC clones, SP384 (Ventana, Tucson, Arizona) and D4D6 (Cell Signaling, Danvers, Massachusetts), in a training cohort of 51 positive ROS1 FISH LUAD cases, and then in a large validation cohort of 714 consecutive cases of LUAD from six routine molecular pathology platforms. Results In the two cohorts, the SP384 and D4D6 clones show variable sensitivity and specificity rates on the basis of two cutoff points greater than or equal to 1+ (all % tumor cells) and greater than or equal to 2+ (>30% stained tumor cells). In the validation cohort, the D4D6 yielded the best accuracy for the presence of a ROS1 rearrangement by FISH. Interpathologist agreement was moderate to good (interclass correlation 0.722–0.874) for the D4D6 clone and good to excellent (interclass correlation: 0.830–0.956) for the SP384 clone. Conclusions ROS1 IHC is an effective screening tool for the presence of ROS1 rearrangements. However, users must be acutely aware of the variable diagnostic performance of different anti-ROS1 antibodies before implementation into routine clinical practice.

Background: The detection of a ROS1 rearrangement in advanced or metastatic lung adenocarcinoma (LUAD) lead to a targeted treatment with tyrosine kinase inhibitors with improved progression free survival (PFS) and overall survival (OS) of the patients. Thus, it is mandatory to screen systematically for the ROS1rearrangement in this patient population. ROS1 rearrangements can be detected using fluorescence in situ hybridization (FISH), however ROS1 immunohistochemistry (IHC) can be used as a screening test since is largely available, easy, rapid to perform, and cost-effective. However, some false positive and negative IHC results are observed when using the D4D6 clone leading to confirmatory ROS1 FISH in IHC positive samples. Patients and methods: We evaluated the sensitivity and specificity of anti-ROS1 SP384 (Ventana, Tucson, AZ) and D4D6 (Cell Signaling, Danvers, MA) antibodies in a multicenter population of 336 LUAD cases enriched for ROS1 FISH positive cases (n=51) provided from 6 French molecular pathology platforms. Three senior lung pathologists independently scored the SP384 slides as positive or negative around a cutoff of staining in >30% tumor cells at a ≥2+ intensity level, and for the D4D6 clone around a cutoff of ≥2+ intensity level in any tumor cells. Inter-reader precision between pathologists was assessed. Results were correlated to the PFS and the OS of patients treated with crizotinib. Results: Sensitivity and specificity rates were 100% (95%CI 93.2-100%) and 99.31% (95%CI 97.51-99.92%) for the SP384 clone, and 90.6% (95%CI 79.34-96.87%) and 99.65% (95%CI 98.05-99.99%) for the D4D6 clone, respectively. Inter-reader agreement was 97.5% (95%CI 94.8-99.7) for the SP384 clone and 86.3% (95%CI 91.3-95.6) for the D4D6 clone. Overall, when compared to ROS1 FISH analysis, the SP384 clone had an accuracy of 99.4%, while D4D6 clone of 98.2%. Using log-rank test, we observed that LUAD with positive ROS1 SP384 status had a longer PFS than those characterized by the D4D6 clone (P=0.021). Conclusions: Interpretation above a cutoff of >30% tumor cells with staining at a ≥2+ intensity level, the ROS1 SP384 clone demonstrates superior sensitivity to D4D6 clone, while preserving similar specificity rate for the detection of ROS1 rearrangements. The presented data provide evidence that the SP384 clone may be used for effective stratification prior to confirmation with orthogonal methods. Citation Format: Véronique Hofman, Isabelle Rouquette, Sandra Lassalle, Simon Heeke, Jean-Christophe Sabourin, Nicolas Piton, Julien Mazières, Jean-Michel Vignaud, Clémence Yguel, Anne Laure Lepage, Frédéric Bibeau, Elodie Long-Mira, Katia Zahaf, Hugues Begueret, Jonathan Benzaquen, Michel Poudenx, Charles-Hugo Marquette, Marius Ilie, Paul Hofman. Multicenter study evaluating the ROS1 status in lung adenocarcinoma using the novel SP384 immunohistochemistry clone. Towards a new algorithm for ROS1 status assessment in routine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3127.