Patrick Micke's research on integrative lung cancer pathology

New therapies are needed for non-small cell lung cancer

Non-small cell lung cancer (NSCLC) represents 85 % of all diagnosed cases. Thorough molecular characterization of clinical NSCLC tissue has led to the discovery of druggable molecular aberrations, such as mutated epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) fusion proteins, which has improved the treatment for a subgroup of NSCLC patients.

However, because of the development of resistance, the treatment response is only temporary and cure is not possible. Moreover, in most NSCLC patients the tumours do not harbour druggable molecular targets, thus, effective treatment options are missing for these patients.
 

The Uppsala NSCLC cohort

The basis of our research is the tissue biobank within the Department of Pathology. Since the early 1990s fresh frozen tissue of operated lung cancer specimens has systematically been collected to provide unfixed high quality tissue for advanced research techniques. Since 2015 the tissue sampling has been incorporated in the infrastructure of Uppsala-Umeå Comprehensive Cancer Consortium (U-CAN; www.u-can.uu.se), complementing the tissue samples with corresponding blood samples before and after therapy.

Together with the research group of Johan Botling we have composed two large cohorts including tissue samples from 712 operated NSCLC patients (Uppsala I and Uppsala II). Formalin-fixed paraffin embedded (FFPE) tissues from both cohorts were compiled on tissue microarrays (TMAs) for immunohistochemical and fluorescence in situ hybridization analysis. Whole tissue sections from each patient are also available.

Furthermore, we have performed genome wide analysis of gene expression and gene copy number levels using Affymetrix chips for 194 cases from the Uppsala I cohort and performed RNA sequencing (RNAseq) on 199 cases from the Uppsala II cohort.
 

Aberrations associated with clinical outcome have been identified

Based on these clinically and molecularly annotated data sets we were able to identify specific aberrations on genomic and transcriptomic levels that indicate molecular subclasses that were associated with clinical outcome (Micke et al., 2011; Botling et al., 2013, Micke et al., 2014). In addition to the epithelial tumor cell characteristics, we have described stromal components that were correlated to relevant patient characteristics and survival (Edlund et al., 2012; Paulsson et al., 2014).

We have also identified B-cell signatures and the infiltration of plasma cells as the most reproducible prognostic stromal features in several types of cancer (Schmidt et al., 2012), including lung cancer (Lohr et al., 2013).

These results highlight the impact of the host’s immune response in the tumorigenic process and inspired us to further evaluate the natural immune landscape of NSCLC.

The immune landscape of NSCLC

The introduction of T-cell activating checkpoint inhibitors has revolutionized the treatment of NSCLC. However, only a subset of patients benefit from this therapy. An improved understanding of cancer immunity may reveal molecular markers that better predict treatment response or that can be targeted to enhance immunotherapy. In addition, immune markers can be explored for early detection or prognostication of NSCLC as well as for clinical follow-up.

The Uppsala NSCLC cohort (including tissue microarrays, mRNA/SNP microarray data, RNAseq and detailed clinical data) is the optimal basis to comprehensively characterize the immune landscape of NSCLC with regard to (i) immune cell infiltrates, (ii) expression of immune regulators and markers of T-cell activation, and (iii) stroma environment determined by fibroblast, vascular and extracellular matrix features.

These ongoing studies include staining and careful annotation of tissue specimens from 712 NSCLC patients from both retrospective cohorts. In parallel, we are constructing a prospective cohort including all patients treated with immunotherapy at the Uppsala University Hospital and will analyze tissue and blood samples in association with clinical response.

The combination of an extensively annotated NSCLC cohort with prospective clinical evaluation, is unified by the overall aim to better understand the immune regulatory mechanisms in NSCLC and by this identify immune markers to improve cancer detection and treatment selection.

The landscape of cancer-testis antigens in NSCLC

As a prerequisite of an effective immune response, immune cells have to recognize cancer specific structures on the tumor cells. Cancer testis antigens (CTAs) present such immunogenic proteins that are aberrantly expressed in cancer but not in normal tissue, with the exceptions of testis and placenta.

Based on a previous successful collaboration with the Human Protein Atlas (Lindskog et al., 2014), RNAseq data from 199 NSCLC specimens were compared to the non-malignant transcriptome of 142 samples from 32 different organs. We identified 90 CTAs, of them 55 previously not described, subsequently enabling us to provide a map of the CTA expression in NSCLC (Djureinovic et al., 2016).

These findings stimulated us to further explore these CTAs as biomarkers for lung cancer screening or ultimately exploit them as targets for therapy by analyzing NSCLC plasma samples.