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Transcriptomic and Proteomic Analysis of Tumor Markers in Tissue and Blood from Patients with Lung Cancer
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (Patrick Micke)
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

Despite recent treatment advancements, the survival outcome remains poor for the majority of patients with non-small cell lung cancer (NSCLC). The aim of this thesis was to evaluate protein expression to predict prognosis and identify biomarkers that can be used as targets for immunotherapy or for early detection of NSCLC.

In Paper I an optimized immunohistochemistry (IHC)-based prognostic model was developed for NSCLC. The prognostic performance of the model was compared to the clinicopathological parameters that are used in the clinical setting to predict outcome. The protein model failed to outperform clinicopathological parameters in predicting survival outcome questioning the potential of IHC-based assessment of prognostic markers in NSCLC.

In Paper II the human testis-specific proteome was profiled using RNA-sequencing (RNA-seq) data from testis and 26 other organs. More than 1000 genes demonstrated a testis-enriched expression pattern which makes testis the tissue with the most tissue-specific genes. The majority of the testis-enriched genes were previously poorly described and were further profiled by IHC. This analysis provides a starting point to increase the molecular understanding of testicular biology.

In Paper III the profiling of cancer-testis antigens (CTAs) was performed in NSCLC by using RNA-seq data from 32 normal organs and NSCLC. Ninety genes showed CTA expression profiles. The transcriptomic data were validated by IHC for several CTAs. The comprehensive analysis of CTAs can guide biomarker studies or help to identify targets for immunotherapeutic strategies.

In Paper IV the reactivity of CTAs was evaluated by measuring the abundance of autoantibodies in plasma from patients with NSCLC and benign lung diseases. Twenty-nine CTAs demonstrated exclusive reactivity in NSCLC and six of them were reactive in an independent NSCLC cohort. These findings suggest that some CTAs are immunogenic and could be utilized in immunotherapy.

In Paper V an immunoassay was used on lung adenocarcinoma plasma samples and samples from benign lung diseases. The plasma levels of 92 cancer related proteins were used to build a model that discriminated lung adenocarcinoma from benign controls with a sensitivity of 93% and a specificity of 64%. The results indicate that this assay is promising for the early detection of NSCLC.

In summary, this thesis presents an integrative analysis of lung cancer tissue and blood samples to characterize NSCLC on the transcriptomic and proteomic level and to identify cancer specific proteins.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. , p. 52
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1463
Keywords [en]
non-small cell lung cancer, prognostic biomarkers, cancer-testis antigens, prediction model, tumor markers, autoantibodies, testis, screening
National Category
Medical and Health Sciences
Research subject
Pathology
Identifiers
URN: urn:nbn:se:uu:diva-348349ISBN: 978-91-513-0328-4 (print)OAI: oai:DiVA.org:uu-348349DiVA, id: diva2:1197112
Public defence
2018-06-08, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2018-05-09 Created: 2018-04-11 Last updated: 2018-05-09
List of papers
1. Reaching the limits of prognostication in non-small cell lung cancer: an optimized biomarker panel fails to outperform clinical parameters.
Open this publication in new window or tab >>Reaching the limits of prognostication in non-small cell lung cancer: an optimized biomarker panel fails to outperform clinical parameters.
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2017 (English)In: Modern Pathology, ISSN 0893-3952, E-ISSN 1530-0285, Vol. 30, no 7, p. 964-977Article in journal (Refereed) Published
Abstract [en]

Numerous protein biomarkers have been analyzed to improve prognostication in non-small cell lung cancer, but have not yet demonstrated sufficient value to be introduced into clinical practice. Here, we aimed to develop and validate a prognostic model for surgically resected non-small cell lung cancer. A biomarker panel was selected based on (1) prognostic association in published literature, (2) prognostic association in gene expression data sets, (3) availability of reliable antibodies, and (4) representation of diverse biological processes. The five selected proteins (MKI67, EZH2, SLC2A1, CADM1, and NKX2-1 alias TTF1) were analyzed by immunohistochemistry on tissue microarrays including tissue from 326 non-small cell lung cancer patients. One score was obtained for each tumor and each protein. The scores were combined, with or without the inclusion of clinical parameters, and the best prognostic model was defined according to the corresponding concordance index (C-index). The best-performing model was subsequently validated in an independent cohort consisting of tissue from 345 non-small cell lung cancer patients. The model based only on protein expression did not perform better compared to clinicopathological parameters, whereas combining protein expression with clinicopathological data resulted in a slightly better prognostic performance (C-index: all non-small cell lung cancer 0.63 vs 0.64; adenocarcinoma: 0.66 vs 0.70, squamous cell carcinoma: 0.57 vs 0.56). However, this modest effect did not translate into a significantly improved accuracy of survival prediction. The combination of a prognostic biomarker panel with clinicopathological parameters did not improve survival prediction in non-small cell lung cancer, questioning the potential of immunohistochemistry-based assessment of protein biomarkers for prognostication in clinical practice.Modern Pathology advance online publication, 10 March 2017; doi:10.1038/modpathol.2017.14.

National Category
Cancer and Oncology Medical Genetics
Identifiers
urn:nbn:se:uu:diva-318128 (URN)10.1038/modpathol.2017.14 (DOI)000404718100006 ()28281552 (PubMedID)
Funder
Swedish Cancer Society
Available from: 2017-03-23 Created: 2017-03-23 Last updated: 2018-04-11Bibliographically approved
2. The human testis-specific proteome defined by transcriptomics and antibody-based profiling
Open this publication in new window or tab >>The human testis-specific proteome defined by transcriptomics and antibody-based profiling
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2014 (English)In: Molecular human reproduction, ISSN 1360-9947, E-ISSN 1460-2407, Vol. 20, no 6, p. 476-488Article in journal (Refereed) Published
Abstract [en]

The testis' function is to produce haploid germ cells necessary for reproduction. Here we have combined a genome-wide transcriptomics analysis with immunohistochemistry-based protein profiling to characterize the molecular components of the testis. Deep sequencing (RNA-Seq) of normal human testicular tissue from seven individuals was performed and compared with 26 other normal human tissue types. All 20 050 putative human genes were classified into categories based on expression patterns. The analysis shows that testis is the tissue with the most tissue-specific genes by far. More than 1000 genes show a testis-enriched expression pattern in testis when compared with all other analyzed tissues. Highly testis enriched genes were further characterized with respect to protein localization within the testis, such as spermatogonia, spermatocytes, spermatids, sperm, Sertoli cells and Leydig cells. Here we present an immunohistochemistry-based analysis, showing the localization of corresponding proteins in different cell types and various stages of spermatogenesis, for 62 genes expressed at > 50-fold higher levels in testis when compared with other tissues. A large fraction of these genes were unexpectedly expressed in early stages of spermatogenesis. In conclusion, we have applied a genome-wide analysis to identify the human testis-specific proteome using transcriptomics and antibody-based protein profiling, providing lists of genes expressed in a tissue-enriched manner in the testis. The majority of these genes and proteins were previously poorly characterised in terms of localization and function, and our list provides an important starting point to increase our molecular understanding of human reproductive biology and disease.

Keywords
immunohistochemistry, RNA sequencing, spermatogenesis, testis, tissue specificity
National Category
Evolutionary Biology
Identifiers
urn:nbn:se:uu:diva-227710 (URN)10.1093/molehr/gau018 (DOI)000336495100002 ()
Available from: 2014-07-01 Created: 2014-06-30 Last updated: 2018-04-11Bibliographically approved
3. Profiling cancer testis antigens in non-small-cell lung cancer
Open this publication in new window or tab >>Profiling cancer testis antigens in non-small-cell lung cancer
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2016 (English)In: JCI INSIGHT, ISSN 2379-3708, Vol. 1, no 10, article id e86837Article in journal (Refereed) Published
Abstract [en]

Cancer testis antigens (CTAs) are of clinical interest as biomarkers and present valuable targets for immunotherapy. To comprehensively characterize the CTA landscape of non-small-cell lung cancer (NSCLC), we compared RNAseq data from 199 NSCLC tissues to the normal transcriptome of 142 samples from 32 different normal organs. Of 232 CTAs currently annotated in the Caner Testis Database (CTdatabase), 96 were confirmed in NSCLC. To obtain an unbiased CTA profile of NSCLC, we applied stringent criteria on our RNAseq data set and defined 90 genes as CTAs, of which 55 genes were not annotated in the CTdatabase, thus representing potential new CTAs. Cluster analysis revealed that CTA expression is histology dependent and concurrent expression is common. IHC confirmed tissue-specific protein expression of selected new CTAs (TKTL1, TGIF2LX, VCX, and CXORF67). Furthermore, methylation was identified as a regulatory mechanism of CTA expression based on independent data from The Cancer Genome Atlas. The proposed prognostic impact of CTAs in lung cancer was not confirmed, neither in our RNAseq cohort nor in an independent meta-analysis of 1,117 NSCLC cases. In summary, we defined a set of 90 reliable CTAs, including information on protein expression, methylation, and survival association. The detailed RNAseq catalog can guide biomarker studies and efforts to identify targets for immunotherapeutic strategies.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-310039 (URN)10.1172/jci.insight.86837 (DOI)000387113300012 ()27699219 (PubMedID)
Available from: 2016-12-09 Created: 2016-12-09 Last updated: 2018-04-11Bibliographically approved
4. Detection of autoantibodies against cancer-testis antigens in non-small cell lung cancer
Open this publication in new window or tab >>Detection of autoantibodies against cancer-testis antigens in non-small cell lung cancer
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Cancer testis antigens (CTAs) are defined as proteins that are specifically expressed in testis or placenta and their expression is frequently activated in cancer. Due to their ability to induce an immune response, CTAs may serve as suitable targets for immunotherapy. The aim of this study was to evaluate if there is reactivity against CTAs in the plasma of non-small cell lung cancer (NSCLC) patients through the detection of circulating antibodies. 

To comprehensively analyse auto-antibodies against CTAs the multiplexing capacities of suspension bead array technology was used. Bead arrays were created with 120 protein fragments, representing 112 CTAs. Reactivity profiles were measured in plasma samples from 133 NSCLC patients and 57 cases with benign lung diseases. Altogether reactivity against 69 antigens, representing 81 CTAs, was demonstrated in at least one of the analysed samples. Twenty-nine of the antigens (45 CTAs) demonstrated exclusive reactivity in NSCLC samples. Reactivity against CT47A genes, PAGE3, VCX, MAGEB1, LIN28B and C12orf54 were only found in NSCLC patients at a frequency of 1%-4%. The presence of autoantibodies towards these six antigens was confirmed in an independent group of 34 NSCLC patients.

In conclusion, we identified autoantibodies against CTAs in the plasma of lung cancer patients. The reactivity pattern of autoantibodies was higher in cancer patients compared to the benign group, stable over time, but low in frequency of occurrence. The findings suggest that some CTAs are immunogenic and that these properties can be utilized as immune targets.

Keywords
Lung cancer, adenocarcinoma, squamous cell cancer, cancer immunity, tumor markers, MAGE, PAGE
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-347809 (URN)
Available from: 2018-04-07 Created: 2018-04-07 Last updated: 2018-04-11
5. Multiplex plasma protein profiling identifies novel markers to discriminate patients with adenocarcinoma of the lung
Open this publication in new window or tab >>Multiplex plasma protein profiling identifies novel markers to discriminate patients with adenocarcinoma of the lung
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background:The overall prognosis of non-small cell lung cancer (NSCLC) is poor, and currently only patients with localized disease are potentially curable. Therefore, preferably non-invasively determined biomarkers that detect NSCLC patients at early stages of the disease are of high clinical relevance. The aim of this study was to identify and validate novel protein markers in plasma using the highly sensitive DNA-assisted multiplex proximity extension assay (PEA) to discriminate NSCLC from other lung diseases. 

Methods:Plasma samples were collected from a total of 343 patients who underwent surgical resection for different lung diseases, including 144 patients with lung adenocarcinoma (LAC),68 patients with non-malignant lung disease, 83 with lung metastasis of colorectal cancers and 48 patients with typical carcinoid. One microliter of plasma was analyzed using PEA, allowing detection and quantification of 92 established cancer related proteins. The concentrations of the plasma proteins were compared between disease groups.

Results:The comparison between LAC and benign samples revealed significantly different plasma levels for four proteins; CXL17, CEACAM5, VEGFR2 and ERBB3 (adjusted p-value < 0.05). A multi-parameter classifier was developed to discriminate between samples from LAC patients and from patients with non-malignant lung conditions. With a bootstrap aggregated decision tree algorithm (TreeBagger) a sensitivity of 93% and specificity of 64% was achieved to detect LAC in this risk population. 

Conclusion:By applying the highly sensitive PEA, reliable protein profiles could be determined in microliter amounts of plasma. We further identified proteins that demonstrated different plasma concentration in defined disease groups and developed a signature that holds potential to be included in a screening assay for early lung cancer detection. 

Keywords
lung cancer, tumor markers, blood, serum, screening, biomarker
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-347805 (URN)
Available from: 2018-04-07 Created: 2018-04-07 Last updated: 2018-04-11

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