Assessment of programmed cell death ligand 1 (PD-L1) immunohistochemical staining is used for decision on treatment with programmed cell death 1 and PD-L1 checkpoint inhibitors in lung adenocarcinomas and squamous cell carcinomas. This study aimed to compare the staining properties of tumor cells between the antibody clones 28-8, 22C3, SP142, and SP263 and investigate interrater variation between pathologists to see if these stainings can be safely evaluated in the clinical setting. Using consecutive sections from a tissue microarray with tumor tissue from 55 resected lung cancer cases, staining with five PD-L1 assays (28-8 from two different vendors, 22C3, SP142, and SP263) was performed. Seven pathologists individually evaluated the percentage of positive tumor cells, scoring each sample applying cutoff levels used in clinical studies: <1% positive tumor cells (score 0), 1-4% (score 1), 5-9% (score 2), 10-24% (score 3), 25-49% (score 4), and >50% positive tumor cells (score 5). Pairwise analysis of antibody clones showed weighted kappa values in the range of 0.45-0.91 with the highest values for comparisons with 22C3 and 28-8 and the lowest involving SP142. Excluding SP142 resulted in kappa 0.75-0.91. Weighted kappa for interobserver variation between pathologists was 0.71-0.96. Up to 20% of the cases were differently classified as positive or negative by any pathologist compared with consensus score using ≥1% positive tumor cells as cutoff. A significantly better agreement between pathologists was seen using ≥50% as cutoff (0-5% of cases). In conclusion, the concordance between the PD-L1 antibodies 22C3, 28-8 and SP263 is relatively good when evaluating lung cancers and suggests that any one of these assays may be sufficient as basis for decision on treatment with nivolumab, pembrolizumab, and durvalumab. The scoring of the pathologist presents an intrinsic source of error that should be considered especially at low PD-L1 scores.

Disease recurrence in surgically treated lung adenocarcinoma (AC) remains high. New approaches for risk stratification beyond tumor stage are needed. Gene expressionbased AC subtypes such as the Cancer Genome Atlas Network (TCGA) terminalrespiratory unit (TRU), proximal-inflammatory (PI) and proximal-proliferative (PP) subtypes have been associated with prognosis, but show methodological limitations for robust clinical use. We aimed to derive a platform independent single sample

Accurate histological classification and identification of fusion genes represent two cornerstones of clinical diagnostics in non-small cell lung cancer (NSCLC). Here, we present a NanoString gene expression platform and a novel platform-independent, single sample predictor (SSP) of NSCLC histology for combined, simultaneous, histological classification and fusion gene detection in minimal formalin fixed paraffin embedded (FFPE) tissue. The SSP was developed in 68 NSCLC tumors of adenocarcinoma (AC), squamous cell carcinoma (SqCC) and large-cell neuroendocrine carcinoma (LCNEC) histology, based on NanoString expression of 11 (CHGA, SYP, CD56, SFTPG, NAPSA, TTF-1, TP73L, KRT6A, KRT5, KRT40, KRT16) relevant genes for IHC-based NSCLC histology classification. The SSP was combined with a gene fusion detection module (analyzing ALK, RET, ROS1, MET, NRG1, and NTRK1) into a multicomponent NanoString assay. The histological SSP was validated in six cohorts varying in size (n = 11–199), tissue origin (early or advanced disease), histological composition (including undifferentiated cancer), and gene expression platform. Fusion gene detection revealed five EML4-ALK fusions, four KIF5B-RET fusions, two CD74-NRG1 fusion and three MET exon 14 skipping events among 131 tested cases. The histological SSP was successfully trained and tested in the development cohort (mean AUC = 0.96 in iterated test sets). The SSP proved successful in predicting histology of NSCLC tumors of well-defined subgroups and difficult undifferentiated morphology irrespective of gene expression data platform. Discrepancies between gene expression prediction and histologic diagnosis included cases with mixed histologies, true large cell carcinomas, or poorly differentiated adenocarcinomas with mucin expression. In summary, we present a proof-of-concept multicomponent assay for parallel histological classification and multiplexed fusion gene detection in archival tissue, including a novel platform-independent histological SSP classifier. The assay and SSP could serve as a promising complement in the routine evaluation of diagnostic lung cancer biopsies.

Introduction: Endometrioid endometrial carcinoma is a cancer type with generally excellent prognosis when diagnosed at an early stage, but there is a subset of patients with relapsing disease in spite of early diagnosis and surgical treatment. There is a need to find prognostic markers to identify these patients with increased risk of relapse. Depth of myometrial invasion, histological grade, and presence of lymphovascular invasion are known risk factors. DNA content (ploidy) and proliferation measured as S-phase fraction (SPF) have been discussed as prognostic markers but need additional evaluation. Material and methods: We evaluated relapse-free survival (RFS) with respect to ploidy and SPF, which was analyzed by flow cytometry on fresh tumor tissue, in a cohort of 1001 women treated for stage I endometrioid endometrial carcinoma in northern Sweden during the period of 1993-2010, with a median follow up time of 12.0 years. Data were obtained from historical records. Results: In simple analysis, both aneuploidy and high SPF were associated to increased risk of relapse with hazard ratios (HR) 2.37 (95% CI 1.52-3.70) and 1.94 (95% CI 1.24-3.02), respectively. Our data also confirmed stage, tumor grade, and ploidy as independent prognostic markers in an age adjusted cox regression multivariable analysis but we did not find SPF to contribute to prognosis. However, the combination of aneuploidy and high SPF identified a group of patients with increased risk of relapse, HR 2.02 (95% CI 1. 19-3.44). Conclusion:In this study, which is the largest study of ploidy and SPF in stage I endometrioid endometrial carcinoma using fresh frozen tissue, aneuploidy was shown to be an independent prognostic marker. Furthermore, the combination of aneuploidy and high SPF could be used to identify patients with increased risk of relapse.

e20637 Background: Several checkpoint inhibitors targeting the PD1 axis are now approved for the treatment of non-small cell lung cancer (NSCLC). The best predictor for therapy response is PD-L1 expression on tumor cells assessed by immunohistochemistry (IHC). However, the PD-L1 assays have been developed independently for each available drugs. In this study, we evaluated the different assays to determine the comparability between the assays and the concordance between pathologists under conditions reflecting the clinical situation. Methods: We tested four IHC assays (28-8, 22C3, SP263 and SP142) and one in-house protocol (28-8 on the Ventana system) on a tissue microarray (TMA) including 55 resected tissue specimens of NSCLC patients. The TMAs were independently annotated by seven pathologists with regard to the percentage of tumor cell membrane staining using a six-grade scale ( < 1%; 1-≤5%; 5%-≤10%; 10-≤25%; 25-≤50%; ≥50%). Results: The staining scores were comparable for the three assays with clones 28-8, 22C3 and SP263 as well as the in-house 28-8 protocol (κ-value: 0.75-0.91). The use of the SP142 clone resulted in a lower percentage of positive tumor cells and positive cases (κ: 0.44-0.63). When a reference score was defined based on the majority of scores, the proportion of positive cases (≥1%) were 38% for 28-8, 29% for 22C3, 42% for SP263, 37% for 28-8 in-house and 16% for SP142%. With ≥1% cut-off 3-8 of the 55 cases (5-15%) were differently classified as positive or negative when comparing any two assays excluding SP142. Analysing the scoring of the seven pathologists a clear inter-rater variability (κ: 0.71-0.96) was observed independent from the assay. With a ≥1% cut-off, 20% of cases were annotated differently by at least one pathologist. Conclusions: The concordance between the three antibody clones 28-8, 22C3 and SP263 was acceptable and was in the similar range as inter-rater variability of different pathologists. The results suggest that assessment of tumor cell staining for Nivolumab, Pembrolizumab and Durvalumab might be performed with only one of these assays. The assessment of the pathologist presents an intrinsic source of error that should be considered especially at low PD-L1 scores.