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An integrative strategy for targeted evaluation of biomarker expression in non-small cell lung cancer
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Despite improvements in therapy, the prognosis for non-small cell lung cancer (NSCLC) patients remains poor, and cure is only possible in localized tumors after surgical resection. A new generation of targeted cancer drugs has led to the expectation that lung cancer therapy can be significantly improved, but these drugs are today only an option in a small subset of NSCLC patients, and their effect is temporary. Therefore, the aim of this thesis was to characterize NSCLC in order to find new treatment targets and to evaluate biomarkers that further optimize therapy selection.

In Paper I, the expression of the potential treatment targets claudin 6 and claudin 18.2 were evaluated based on immunohistochemical- and gene expression analysis. High ectopic protein and gene expression were demonstrated for both claudins in small subgroups of NSCLC. Clinical trials using humanized monoclonal antibodies against both proteins are ongoing in other cancer forms and may be extended to NSCLC.

In Paper II, the prognostic impact of the inflammatory mediator cyclooxygenase 2 (COX-2) was evaluated. No prognostic significance was found in a meta-analysis incorporating gene expression data of 1337 NSCLC patients. Likewise, COX-2 protein expression in tumor cells was not associated with survival in two independent NSCLC cohorts. However, in one of the analyzed cohorts, higher COX-2 expression in the tumor stroma was associated with longer survival and may therefore be a subject for further investigation.

In Paper III, tumor and stromal COX-2 protein expression was examined in patients treated with the COX-2 inhibitor celecoxib in order to evaluate if COX-2 expression is a predictive biomarker for benefit of celecoxib therapy. Celecoxib did not prolong overall survival neither in the whole cohort nor in patients stratified according to COX-2 expression in tumor or stromal cells. Noteworthy, a tendency towards longer survival was again demonstrated in patients with high COX-2 stromal expression.

In Paper IV, the diagnostic methods for identification of ALK rearrangements were assessed in a large representative Swedish NSCLC population. Fluorescence in situ hybridization (FISH), as the diagnostic standard, was compared to two immunohistochemical assays. ALK gene expression levels were incorporated to supplement the molecular data. The frequency of ALK rearrangements was lower than previously reported. The different methods to detect the ALK fusion demonstrated overlapping results. However, the overlap was poor, so the methods cannot be regarded as interchangeable and should thereby be interpreted with caution when used in clinical diagnostics.

In summary, this thesis applied an integrative translational approach to characterize potential new treatment targets and to evaluate the detection of existing predictive biomarkers in NSCLC.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 58 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1229
Keyword [en]
non-small cell lung cancer, prognostic biomarkers, predictive biomarkers, immunohistochemistry, gene expression, COX-2, claudin, ALK
National Category
Other Medical Sciences Cancer and Oncology
Research subject
Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-285844ISBN: 978-91-554-9584-8 (print)OAI: oai:DiVA.org:uu-285844DiVA: diva2:921235
Public defence
2016-06-11, Fåhraeussalen, Rudbecklaboratoriet, Dag Hammarskjöldsväg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2016-05-19 Created: 2016-04-19 Last updated: 2016-06-01
List of papers
1. Aberrantly activated claudin 6 and 18.2 as potential therapy targets in non-small-cell lung cancer
Open this publication in new window or tab >>Aberrantly activated claudin 6 and 18.2 as potential therapy targets in non-small-cell lung cancer
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2014 (English)In: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 135, no 9, 2206-2214 p.Article in journal (Refereed) Published
Abstract [en]

Claudins (CLDNs) are central components of tight junctions that regulate epithelial-cell barrier function and polarity. Altered CLDN expression patterns have been demonstrated in numerous cancer types and lineage-specific CLDNs have been proposed as therapy targets. The objective of this study was to assess which fraction of patients with non-small-cell lung cancer (NSCLC) express CLDN6 and CLDN18 isoform 2 (CLDN18.2). Protein expression of CLDN6 and CLDN18.2 was examined by immunohistochemistry on a tissue microarray (n=355) and transcript levels were supportively determined based on gene expression microarray data from fresh-frozen NSCLC tissues (n=196). Both were analyzed with regard to frequency, distribution and association with clinical parameters. Immunohistochemical analysis of tissue sections revealed distinct membranous positivity of CLDN6 (6.5%) and CLDN18.2 (3.7%) proteins in virtually non-overlapping subgroups of adenocarcinomas and large-cell carcinomas. Pneumocytes and bronchial epithelial cells were consistently negative. Corresponding to the protein expression, in subsets of non-squamous lung carcinoma high mRNA levels of CLDN6 (7-16%) and total CLDN18 (5-12%) were observed. Protein expression correlated well with total mRNA expression of the corresponding gene (rho=0.4-0.8). CLDN18.2 positive tumors were enriched among slowly proliferating, thyroid transcription factor 1 (TTF-1)-negative adenocarcinomas, suggesting that isoform-specific CLDN expression may delineate a specific subtype. Noteworthy, high CLDN6 protein expression was associated with worse prognosis in lung adenocarcinoma in the univariate [hazard ratio (HR): 1.8; p=0.03] and multivariate COX regression model (HR: 1.9; p=0.02). These findings encourage further clinical exploration of targeting ectopically activated CLDN expression as a valuable treatment concept in NSCLC.

Keyword
non-small-cell lung cancer, targeted therapy, gene expression profiling, claudin, tissue microarray
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-235148 (URN)10.1002/ijc.28857 (DOI)000341983700026 ()24710653 (PubMedID)
Note

De 2 första författarna delar förstförfattarskapet. (The 2 first authors contributed equally to the study)

Available from: 2014-11-06 Created: 2014-10-29 Last updated: 2017-12-05
2. Prognostic impact of COX-2 in non-small cell lung cancer: A comprehensive compartment-specific evaluation of tumor and stromal cell expression
Open this publication in new window or tab >>Prognostic impact of COX-2 in non-small cell lung cancer: A comprehensive compartment-specific evaluation of tumor and stromal cell expression
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2015 (English)In: Cancer Letters, ISSN 0304-3835, E-ISSN 1872-7980, Vol. 356, no 2, 837-845 p.Article in journal (Refereed) Published
Abstract [en]

Cyclooxygenase-2 (COX-2) is an enzyme that has been extensively investigated as a prognostic marker in cancer. In non-small cell lung cancer (NSCLC) previous results regarding the prognostic impact of COX-2 expression are inconsistent. Therefore we evaluated the association between transcript levels and overall survival in nine publicly available gene expression data sets (total n=1337) and determined in situ compartment-specific tumor and stromal cell protein expression in two independent cohorts (n=616). Gene expression did not show any correlation with clinical parameters or with overall survival. Protein expression in tumor and stromal cells did not correlate with any clinical parameter or with overall survival in one of the analyzed cohorts, while a significant association of high stromal expression with longer survival was observed in both univariate and multivariate analysis in the other cohort. Stromal expression of COX-2 has not been separately evaluated in NSCLC previously and may be a subject of further investigation, whereas the presented findings from this comprehensive compartment specific evaluation clearly reject the hypothesis of COX-2 tumor cell expression having a prognostic value in NSCLC.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-239073 (URN)10.1016/j.canlet.2014.10.032 (DOI)000348005500057 ()25449785 (PubMedID)
Funder
Swedish Cancer Society
Note

De 2 sista författarna delar sistaförfattarskapet. (The 2 last authors contributed equally to the study)

Available from: 2015-01-06 Created: 2014-12-18 Last updated: 2017-12-05
3. COX-2 expression and effects of celecoxib in addition to standard chemotherapy in advanced non-small cell lung cancer.
Open this publication in new window or tab >>COX-2 expression and effects of celecoxib in addition to standard chemotherapy in advanced non-small cell lung cancer.
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(English)Article in journal (Refereed) Submitted
Abstract [en]

Inhibition of cyclooxygenase-2 (COX-2) is proposed as a treatment option in several cancer types. However, in non-small cell lung cancer (NSCLC), phase III trials have failed to demonstrate a benefit of adding COX-2 inhibitors to standard chemotherapy. The aim of this study was to analyse COX-2 expression in tumor and stromal cells as predictive biomarker for COX-2 inhibition.

Methods: In the multicenter phase III trial, 316 patients with advanced NSCLC were randomized to receive celecoxib (400 mg b.i.d.) or placebo up to one year in addition to a two-drug platinum-based chemotherapy combination. In a subset of 122 patients, archived tumor tissue was available for immunohistochemical analysis of COX-2 expression in tumor and stromal cells.

Results: An updated analysis of all 316 patients included in the original trial, and of the 122 patients with available tumor tissue, showed no survival differences between the celecoxib and placebo arms (HR 1.01; 95%CI 0.81-1.27 and HR 1.12; 95%CI 0.78-1.61, respectively). Similarly, in patients with high COX-2 expression in tumor cells (n=71) or stromal cells (n=55), survival did not differ significantly between patients who received celecoxib or placebo (HR 1.07; 95%CI 0.74-1.54 and HR 0.80; 95%CI 0.56-1.15). No significant interaction effect between COX-2 score in tumor or stromal cells and celecoxib effect on survival was detected (p=0.48 and 0.25, respectively).

Conclusion: In this subgroup analysis of patients with advanced NSCLC, we could not detect any significant interaction between COX-2 expression in tumor or stromal cells and outcome of celecoxib treatment in addition to standard chemotherapy.

Keyword
cyclooxygenase 2, lung cancer, celecoxib, prognosis, inflammation, cancer immunity
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-284590 (URN)
Available from: 2016-04-19 Created: 2016-04-18 Last updated: 2016-06-01
4. Inconsistent results in the analysis of ALK rearrangements in non-small cell lung cancer
Open this publication in new window or tab >>Inconsistent results in the analysis of ALK rearrangements in non-small cell lung cancer
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2016 (English)In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 16, 603Article in journal (Refereed) Published
Abstract [en]

Background: Identification of targetable EML4-ALK fusion proteins has revolutionized the treatment of a minor subgroup of non-small cell lung cancer (NSCLC) patients. Although fluorescence in situ hybridization (FISH) is regarded as the gold standard for detection of ALK rearrangements, ALK immunohistochemistry (IHC) is often used as screening tool in clinical practice. In order to unbiasedly analyze the diagnostic impact of such a screening strategy, we compared ALK IHC with ALK FISH in three large representative Swedish NSCLC cohorts incorporating clinical parameters and gene expression data.

Methods: ALK rearrangements were detected using FISH on tissue microarrays (TMAs), including tissue from 851 NSCLC patients. In parallel, ALK protein expression was detected using IHC, applying the antibody clone D5F3 with two different protocols (the FDA approved Ventana CDx assay and our in house Dako IHC protocol). Gene expression microarray data (Affymetrix) was available for 194 patients.

Results: ALK rearrangements were detected in 1.7% in the complete cohort and 2.0% in the non-squamous cell carcinoma subgroup. ALK protein expression was observed in 1.9% and 1.5% when applying the Ventana assay or the in house Dako protocol, respectively. The specificity and accuracy of IHC was high (>99%), while the sensitivity was between 69% (Ventana) and 62% (in house Dako protocol). Furthermore, only 67% of the ALK IHC positive cases were positive in both IHC assays. Gene expression analysis revealed that 6/194 (3%) tumors showed high ALK gene expression (≥6AU) and of them only three were positive by either FISH or IHC.

Conclusion: The overall frequency of ALK rearrangements based on FISH was lower than previously reported. The sensitivity of both IHC assays was low, and the concordance between the FISH and the IHC assays poor, questioning current strategies to screen with IHC prior to FISH or completely replace FISH by IHC.

Keyword
Anaplastic lymphoma kinase, non-small cell lung cancer, immunohistochemistry, fluorescence in situ hybridization
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-284591 (URN)10.1186/s12885-016-2646-x (DOI)000381219600002 ()27495736 (PubMedID)
Funder
Swedish Cancer Society
Available from: 2016-04-19 Created: 2016-04-18 Last updated: 2017-11-30Bibliographically approved

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