Chapter 4 138 Results PD-1T signature development To develop a predictive mRNA signature that reflects tumor infiltration by PD-1T TILs, we first selected 94 pretreatment samples from advanced stage NSCLC patients treated with nivolumab (training cohort). 66/94 (70%) of these samples had sufficient RNA available for gene expression analysis using the Nanostring nCounter platform. Only archival samples were used, and therefore the majority with insufficient RNA were blocks that were already (partially) used for previous molecular analysis. 58/94 (62%) samples were successful in obtaining high quality mRNA expression data to be used for signature development, as 8 samples did not meet RNA quality criteria (Fig. 1). Of these 58 patients, 12 showed disease control at 12 months (DC 12m) (n=12) and 46 developed progressive disease (PD) within 12 months of treatment (Fig. 1). DC12 was chosen as primary clinical outcome measure based on previous observations that patients with DC 12m were more accurately identified by the biomarker as compared to DC 6m (i.e. higher sensitivity). This was also found for a group with no long-term benefit (i.e. higher negative predictive value (NPV))7. Clinicopathological characteristics and treatment outcomes are summarized in Supplementary Table S3. In line with our previous work, the number of PD-1T TILs per mm2 was significantly higher in patients with DC 12m compared to patients with PD (P<0.01) (Fig. 2A). PD1T IHC high versus low status was called using a previously established cut-off of 90 PD-1T TILs per mm2 (Fig. S1A,B)7. 12/12 (100%) patients with DC 12m were classified as PD-1T IHC high. 29/46 (63%) patients with PD were classified as PD-1T IHC low, and 17/46 (37%) as PD-1T IHC high (Fig. 1, 2A). To obtain a distinctive mRNA expression gene set with maximum contrast, we performed differential gene expression analysis between PD-1T IHC high patients with DC 12m (n=12) and PD-1T IHC low patients with PD (n=29) (Fig. 1, 2A). After correction for multiple testing, 54 genes were significantly higher expressed and 2 genes were significantly lower expressed in the PD-1T IHC high DC 12m group. Some of the top ranked genes included LAG3, CTLA4, CXCR6 and CXCL13, which have previously shown to be highly expressed in PD-1T TILs8 (Table S4). In addition, various pathways related to active immune responses in the tumor microenvironment (TME), including type I interferon signaling, regulation of lymphocyte chemotaxis and natural killer cell mediated cytotoxicity were significantly upregulated in the PD-1T IHC high DC 12m group (Fig. 2B).
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