Mutation and immune profiling of non-small cell lung cancer

Several novel therapies that target molecular alterations and immune checkpoints in lung cancer have been introduced in the last decade. Still, only a minority of patients obtain long term disease control and overall survival remains poor. The aim of this thesis was to characterize the landscape of genetic alterations and immune cell infiltrates in tumor tissues from a large representative patient cohort of non-small cell lung cancer (NSCLC). The mutational status of 82 genes related to lung cancer development were evaluated, in paper I, by a targeted re-sequencing approach adapted to work on “real-life” samples of mixed quality. We observed a remarkably high prevalence of activating KRAS mutations. Otherwise, the mutation spectrum resembled other western lung cancer populations. Poor survival was linked to subgroups of lung adenocarcinoma with mutations in TP53, STK11 and SMARCA4, independent of concomitant KRAS mutations. In lung squamous cell carcinoma, patients with mutations in CSMD3 had better survival. The infiltration of tumor-associated immune cells was assessed by immunohistochemical analysis in paper II. Previously described immune response patterns termed “inflamed” and “desert” were confirmed in our dataset. In addition, we discovered a new immune phenotype characterized by overall sparse presence of most immune cell types except for a distinct infiltration of NK and plasma cells. This novel immune class displayed a favorable prognosis and was therefore designated “oasis”. In paper III, infiltration of macrophage subtypes was evaluated by immunohistochemical analysis of CD68, CD163, MSR1 and MARCO. The majority of macrophages exhibited a tumor promoting phenotype and expression of MARCO, a targetable scavenger receptor, was detected in a distinct subset of NSCLC patients. Further investigation of the functional roles of MARCO in a human NSCLC setting was carried out in paper IV. Here, MARCO expression on cultured myeloid cells could be induced by NSCLC cell lines. The MARCO+ cells displayed an immunosuppressive phenotype and could effectively suppress the cytolytic effect of NK cells and CD8+ T cells. A monoclonal antibody targeting MARCO removed these inhibitory effects of the MARCO+ cells. In summary, this thesis contributes knowledge on the genetic and immunologic underpinning of lung cancer that forms the basis for current and future treatment strategies in the evolving era of personalized oncology and pathology

Mutation and immune profiling of non-small cell lung cancer

Several novel therapies that target molecular alterations and immune checkpoints in lung cancer have been introduced in the last decade. Still, only a minority of patients obtain long term disease control and overall survival remains poor. The aim of this thesis was to characterize the landscape of genetic alterations and immune cell infiltrates in tumor tissues from a large representative patient cohort of non-small cell lung cancer (NSCLC). The mutational status of 82 genes related to lung cancer development were evaluated, in paper I, by a targeted re-sequencing approach adapted to work on “real-life” samples of mixed quality. We observed a remarkably high prevalence of activating KRAS mutations. Otherwise, the mutation spectrum resembled other western lung cancer populations. Poor survival was linked to subgroups of lung adenocarcinoma with mutations in TP53, STK11 and SMARCA4, independent of concomitant KRAS mutations. In lung squamous cell carcinoma, patients with mutations in CSMD3 had better survival. The infiltration of tumor-associated immune cells was assessed by immunohistochemical analysis in paper II. Previously described immune response patterns termed “inflamed” and “desert” were confirmed in our dataset. In addition, we discovered a new immune phenotype characterized by overall sparse presence of most immune cell types except for a distinct infiltration of NK and plasma cells. This novel immune class displayed a favorable prognosis and was therefore designated “oasis”. In paper III, infiltration of macrophage subtypes was evaluated by immunohistochemical analysis of CD68, CD163, MSR1 and MARCO. The majority of macrophages exhibited a tumor promoting phenotype and expression of MARCO, a targetable scavenger receptor, was detected in a distinct subset of NSCLC patients. Further investigation of the functional roles of MARCO in a human NSCLC setting was carried out in paper IV. Here, MARCO expression on cultured myeloid cells could be induced by NSCLC cell lines. The MARCO+ cells displayed an immunosuppressive phenotype and could effectively suppress the cytolytic effect of NK cells and CD8+ T cells. A monoclonal antibody targeting MARCO removed these inhibitory effects of the MARCO+ cells. In summary, this thesis contributes knowledge on the genetic and immunologic underpinning of lung cancer that forms the basis for current and future treatment strategies in the evolving era of personalized oncology and pathology

Comprehensive analysis of RNA binding motif protein 3 (RBM3) in non-small cell lung cancer

Aims High expression of the RNA-binding motif protein 3 (RBM3) correlates with improved prognosis in several major types of cancer. The aim of the present study was to examine the prognostic value of RBM3 protein and mRNA expression in non-small cell lung cancer (NSCLC). Methods and results Immunohistochemical expression of RBM3 was evaluated in surgically treated NSCLC from two independent patient populations (n = 213 and n = 306). Staining patterns were correlated with clinicopathological parameters, overall survival (OS), and recurrence-free interval (RFI). Cases with high nuclear RBM3 protein expression had a prolonged 5-year OS in both cohorts when analyzing adenocarcinomas separately (P = .02 and P = .01). RBM3 remained an independent prognostic factor for OS in multivariable analysis of cohort I (HR 0.44, 95% CI 0.21-0.90) and for RFI in cohort II (HR 0.38, 95% CI 0.22-0.74). In squamous cell carcinoma, there was instead an insignificant association to poor prognosis. Also, the expression levels of RBM3 mRNA were investigated in 2087 lung adenocarcinomas and 899 squamous cell carcinomas assembled from 13 and 8 public gene expression microarray datasets, respectively. The RBM3 mRNA levels were not clearly associated with patient outcome in either adenocarcinomas or squamous cell carcinomas. Conclusions The results from this study support that high protein expression of RBM3 is linked to improved outcome in lung adenocarcinoma

Stromal FAP is an independent poor prognosis marker in non-small cell lung adenocarcinoma and associated with p53 mutation

Objectives Fibroblasts regulate tumor growth and immune surveillance. Here, we study FAP, PDGFβR and α-SMA fibroblast markers in a well-annotated clinical cohort of non–small-cell lung cancer (NSCLC) for analyses of associations with immune cell infiltration, mutation status and survival. Materials and Methods A well-annotated NSCLC cohort was subjected to IHC analyses of stromal expression of FAP, PDGFβR and α-SMA and of stromal CD8 density. Fibroblast markers-related measurements were analyzed with regard to potential associations with CD8 density, cancer genetic driver mutations, survival and PD-L1 expression in the whole NSCLC cohort and in subsets of patients. Results High stromal FAP expression was identified as an independent poor prognostic marker in the whole study population (HR 1.481; 95 % CI, 1.012–2.167, p = 0.023) and in the histological subset of adenocarcinoma (HR 1.720; 95 % CI, 1.126–2.627, p = 0.012). Among patients with adenocarcinoma, a particularly strong association of FAP with poor survival was detected in patients with low stromal CD8 infiltration, and in other subpopulations identified by specific clinical characteristics; elderly patients, females, non-smokers and patients with normal ECOG performance status. α-SMA expression was negatively associated with CD8 infiltration in non-smokers, but none of the fibroblast markers expression was associated with CD8 density in the whole study population. Significant associations were detected between presence of p53 mutations and high α-SMA (p = 0.003) and FAP expression (p &lt; 0.001). Conclusion The study identifies FAP intensity as a candidate independent NSCLC prognostic biomarker. The study also suggests continued analyses of the relationships between genetic driver mutations and the composition of tumor stroma, as well as continued probing of marker-defined fibroblasts as NSCLC subset-specific modifiers of immune surveillance and outcome.De två första författarna delar förstaförfattarskapet</p

Evaluation of NTRK immunohistochemistry as a screening method for NTRK gene fusion detection in non-small cell lung cancer

Purpose: The small molecule inhibitors larotrectinib and entrectinib have recently been approved as cancer agnostic drugs in patients with tumours harbouring a rearrangement of the neurotrophic tropomyosin receptor kinase (NTRK). These oncogenic fusions are estimated to occur in 0.1–3 % of non-small cell lung cancers (NSCLC). Although molecular techniques are most reliable for fusion detection, immunohistochemical analysis is considered valuable for screening. Therefore, we evaluated the newly introduced diagnostic immunohistochemical assay (clone EPR17341) on a representative NSCLC cohort. Methods: Cancer tissue from 688 clinically and molecularly extensively annotated NSCLC patients were comprised on tissue microarrays and stained with the pan-TRK antibody clone EPR17341. Positive cases were further analysed with the TruSight Tumor 170 RNA assay (Illumina). Selected cases were also tested with a NanoString NTRK fusion assay. For 199 cases, NTRK RNA expression data were available from previous RNA sequencing analysis. Results: Altogether, staining patterns for 617 NSCLC cases were evaluable. Of these, four cases (0.6 %) demonstrated a strong diffuse cytoplasmic and membranous staining, and seven cases a moderate staining (1.1 %). NanoString or TST170-analysis could not confirm an NTRK fusion in any of the IHC positive cases, or any of the cases with high mRNA levels. In the four cases with strong staining intensity in the tissue microarray, whole section staining revealed marked heterogeneity of NTRK protein expression. Conclusion: The presence of NTRK fusion genes in non-small cell lung cancer is exceedingly rare. The use of the immunohistochemical NTRK assay will result in a small number of false positive cases. This should be considered when the assay is applied as a screening tool in clinical diagnostics

Expression of scavenger receptor MARCO defines a targetable tumor-associated macrophage subset in non-small cell lung cancer

Tumor-associated macrophages (TAMs) are attractive targets for immunotherapy. Recently, studies in animal models showed that treatment with an anti-TAM antibody directed against the scavenger receptor MARCO resulted in suppression of tumor growth and metastatic dissemination. Here we investigated the expression of MARCO in relation to other macrophage markers and immune pathways in a non-small cell lung cancer (NSCLC) cohort (n = 352). MARCO, CD68, CD163, MSR1 and programmed death ligand-1 (PD-L1) were analyzed by immunohistochemistry and immunofluorescence, and associations to other immune cells and regulatory pathways were studied in a subset of cases (n = 199) with available RNA-seq data. We observed a large variation in macrophage density between cases and a strong correlation between CD68 and CD163, suggesting that the majority of TAMs present in NSCLC exhibit a protumor phenotype. Correlation to clinical data only showed a weak trend toward worse survival for patients with high macrophage infiltration. Interestingly, MARCO was expressed on a distinct subpopulation of TAMs, which tended to aggregate in close proximity to tumor cell nests. On the transcriptomic level, we found a positive association between MARCO gene expression and general immune response pathways including strong links to immunosuppressive TAMs, T-cell infiltration and immune checkpoint molecules. Indeed, a higher macrophage infiltration was seen in tumors expressing PD-L1, and macrophages residing within tumor cell nests co-expressed MARCO and PD-L1. Thus, MARCO is a potential new immune target for anti-TAM treatment in a subset of NSCLC patients, possibly in combination with available immune checkpoint inhibitors