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Mutation and immune profiling of non-small cell lung cancer
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 69
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1592
Keywords [en]
Non-small cell lung cancer, Tumor microenvironment, Tumor-associated macrophages, Immune infiltrates, PD-L1, Mutation patterns, Immune therapy, MARCO, TP53, STK11, KRAS
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-390321ISBN: 978-91-513-0733-6 (print)OAI: oai:DiVA.org:uu-390321DiVA, id: diva2:1344250
Public defence
2019-10-11, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2019-09-19 Created: 2019-08-20 Last updated: 2019-10-15
List of papers
1. Mutation patterns in a population-based non-small cell lung cancer cohort and prognostic impact of concomitant mutations in KRAS and TP53 or STK11
Open this publication in new window or tab >>Mutation patterns in a population-based non-small cell lung cancer cohort and prognostic impact of concomitant mutations in KRAS and TP53 or STK11
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2019 (English)In: Lung Cancer, ISSN 0169-5002, E-ISSN 1872-8332, Vol. 130, p. 50-58Article in journal (Refereed) Published
Abstract [en]

OBJECTIVES: Non-small cell lung cancer (NSCLC) is a heterogeneous disease with unique combinations of somatic molecular alterations in individual patients, as well as significant differences in populations across the world with regard to mutation spectra and mutation frequencies. Here we aim to describe mutational patterns and linked clinical parameters in a population-based NSCLC cohort.

MATERIALS AND METHODS: Using targeted resequencing the mutational status of 82 genes was evaluated in a consecutive Swedish surgical NSCLC cohort, consisting of 352 patient samples from either fresh frozen or formalin fixed paraffin embedded (FFPE) tissues. The panel covers all exons of the 82 genes and utilizes reduced target fragment length and two-strand capture making it compatible with degraded FFPE samples.

RESULTS: We obtained a uniform sequencing coverage and mutation load across the fresh frozen and FFPE samples by adaption of sequencing depth and bioinformatic pipeline, thereby avoiding a technical bias between these two sample types. At large, the mutation frequencies resembled the frequencies seen in other western populations, except for a high frequency of KRAS hotspot mutations (43%) in adenocarcinoma patients. Worse overall survival was observed for adenocarcinoma patients with a mutation in either TP53, STK11 or SMARCA4. In the adenocarcinoma KRAS-mutated group poor survival appeared to be linked to concomitant TP53 or STK11 mutations, and not to KRAS mutation as a single aberration. Similar results were seen in the analysis of publicly available data from the cBioPortal. In squamous cell carcinoma a worse prognosis could be observed for patients with MLL2 mutations, while CSMD3 mutations were linked to a better prognosis.

CONCLUSION: Here we have evaluated the mutational status of a NSCLC cohort. We could not confirm any survival impact of isolated driver mutations. Instead, concurrent mutations in TP53 and STK11 were shown to confer poor survival in the KRAS-positive adenocarcinoma subgroup.

Keywords
KRAS, Mutation patterns, Non-small cell lung cancer, STK11, TP53, Targeted resequencing
National Category
Clinical Laboratory Medicine
Research subject
Pathology
Identifiers
urn:nbn:se:uu:diva-380587 (URN)10.1016/j.lungcan.2019.01.003 (DOI)000463276900008 ()30885352 (PubMedID)
Funder
Swedish Cancer Society, 2013/711Swedish Cancer Society, 2016/827
Available from: 2019-03-29 Created: 2019-03-29 Last updated: 2019-08-20Bibliographically approved
2. Extending the immune phenotypes of lung cancer: Oasis in the desert
Open this publication in new window or tab >>Extending the immune phenotypes of lung cancer: Oasis in the desert
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Introduction: Tumor infiltrating immune cells are key elements of the tumor microenvironment and mediate the anti-tumor effects of immunotherapy. The aim of the study was to characterize patterns of immune cell infiltration in non-small cell lung cancer (NSCLC) in relation to tumor mutations and clinicopathological parameters. 

Methods: Lymphocytes (CD4+, CD8+, CD20+, FOXP3+, CD45RO+), macrophages (CD163+), plasma cells (CD138+), NK cells (NKp46+) and PD-L1+ were annotated on a tissue microarray including 357 operated NSCLC cases. Somatic mutations and tumor mutational burden were analyzed by targeted sequencing for 82 genes, and transcriptomic immune patterns were established in 197 patients based on RNAseq data. 

Results: We identified somatic mutations (TP53, NF1, KEAP1, CSMD3, LRP1B) that correlated with specific immune cell infiltrates. Hierarchical clustering revealed four immune classes: with (1) high immune cell infiltration (“inflamed”), (2) low immune cell infiltration (“desert”), (3) a mixed phenotype, and (4) a new phenotype with an overall muted inflammatory cell pattern but with an imprint of NK and plasma cells. This latter class exhibited low expression of immune response-related genes (e.g. CXCL9, GZMB, INFG, TGFB1), but was linked to better survival and therefore designated “oasis”. Otherwise, the four immune classes were not related to the presence of specific mutations (EGFR, KRAS, TP53) or histologic subtypes. 

Conclusion: We present a compartment-specific immune cell analysis in the context of the molecular and clinical background of NSCLC and identified the novel immune class “oasis”. The immune classification helps to better define the immunogenic potency of NSCLC in the era of immunotherapy. 

Keywords
Immune infiltration, PD-L1, checkpoint therapy, tumor microenvironment
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-390316 (URN)
Available from: 2019-08-08 Created: 2019-08-08 Last updated: 2019-08-20
3. Expression of scavenger receptor MARCO defines a targetable tumor-associated macrophage subset in non-small cell lung cancer
Open this publication in new window or tab >>Expression of scavenger receptor MARCO defines a targetable tumor-associated macrophage subset in non-small cell lung cancer
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2018 (English)In: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 143, no 7, p. 1741-1752Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
WILEY, 2018
Keywords
lung cancer, MARCO, tumor-associated macrophages, immune therapy, PD-L1
National Category
Cancer and Oncology Clinical Laboratory Medicine
Research subject
Pathology
Identifiers
urn:nbn:se:uu:diva-364230 (URN)10.1002/ijc.31545 (DOI)000443392100020 ()29667169 (PubMedID)
Funder
Swedish Cancer Society
Available from: 2018-10-24 Created: 2018-10-24 Last updated: 2019-08-20Bibliographically approved
4. Antibody targeting of tumor associated macrophages in lung cancer remodel the tumor microenvironment and revives immune targeting of tumor cells
Open this publication in new window or tab >>Antibody targeting of tumor associated macrophages in lung cancer remodel the tumor microenvironment and revives immune targeting of tumor cells
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Immunotherapy for cancer has revolutionized clinical practice and enabled cures for previously lethal cancers. However, the clinical responses are variable and highly influenced by immune regulatory compartments in the tumor microenvironment. This is especially true for immune-excluded tumors, where clinical trials aiming to recover T cell anti-tumor activity have been disappointing. Thus, in NSCLC and other cancers there is a clinical need for additional and combinatory treatments. We have previously shown that antibodies targeting scavenger receptors expressed on tumor-associated macrophages (TAMs), reduces tumor growth and impair metastasis in murine cancer models. Here we investigated targeting of the scavenger receptor MARCO on human TAMs in NSCLC. We found that expression of this receptor in the tumor correlated with immune-exclusion phenotype. Also, we found that lung cancer cell lines converted healthy myeloid cells towards TAM like cells with high expression of MARCO. These human MARCO+ myeloid cells stopped cytotoxic T cells and natural killer (NK) cells from killing tumors and inhibited their overall activity. We then generated anti-human MARCO antibodies and found that these could repolarize TAMs leading to augmented cytolytic ability of NK cells and T cells to kill tumor cells and recovered their proliferation and IFNγ production capacity. Overall, our data demonstrate that it is feasible to use antibodies to alter human TAM immune suppression of NK and T cell anti-tumor activities.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-390320 (URN)
Available from: 2019-08-08 Created: 2019-08-08 Last updated: 2019-08-20

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