Johanna Sofia Margareta Mattsson

An Integrative Analysis of the Tumorigenic Role of TAZ in Human Non-Small Cell Lung Cancer

Purpose: TAZ, also known as WWTR1, has recently been suggested as an oncogene in non–small cell lung cancer (NSCLC). We investigated the clinical relevance of TAZ expression and its functional role in NSCLC tumorigenesis. Experimental Design: We characterized TAZ at the DNA (n = 192), mRNA (n = 196), and protein levels (n = 345) in an NSCLC patient cohort. Gene expression analysis was complemented by a meta-analysis of public datasets (n = 1,382). The effects of TAZ on cell proliferation and cell cycle were analyzed in cell cultures and on tumor growth in mice. TAZ-dependent microarray-based expression profiles in NSCLC cells were combined with molecular profiles in human NSCLC tissues for in silico analysis. Results: Higher TAZ mRNA and protein levels were associated with shorter patient survival. Transduction of TAZ enhanced cell proliferation and tumorigenesis in bronchial epithelial cells, whereas TAZ silencing suppressed cell proliferation and induced cell cycle arrest in NSCLC cells. Microarray and cell culture experiments showed that ErbB ligands (amphiregulin, epiregulin, and neuregulin 1) are downstream targets of TAZ. Our in silico analysis revealed a TAZ signature that substantiated the clinical impact of TAZ and confirmed its relationship to the epidermal growth factor receptor signaling pathway. Conclusion: TAZ expression defines a clinically distinct subgroup of patients with NSCLC. ErbB ligands are suggested to mediate the effects of TAZ on lung cancer progression. Our findings emphasize the tumorigenic role of TAZ and may serve as the basis for new treatment strategies. Clin Cancer Res; 20(17); 4660–72. ©2014 AACR.

Aberrantly activated claudin 6 and 18.2 as potential therapy targets in non-small-cell lung cancer

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.

Profiling cancer testis antigens in non–small-cell lung cancer

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.

Consistent mutation status within histologically heterogeneous lung cancer lesions

Mattsson J S M, Imgenberg‐Kreuz J, Edlund K, Botling J & Micke P 
(2012) Histopathology 61, 744–748

Immunohistochemistry-based prognostic biomarkers in NSCLC: novel findings on the road to clinical use?

Prognostication of non-small cell lung cancer is principally based on stage, age and performance status. This review provides an overview of 342 potential prognostic biomarkers in non-small cell lung cancer described between January 2008 and June 2013, evaluating the association between immunohistochemical protein expression and survival endpoint. Numerous studies proposed prognostic biomarkers, but many were only evaluated in a single patient cohort, and a large number of biomarkers revealed inconclusive findings when analyzed in more than one study. Only 26 proteins first described after 2008 (ALDH1A1, ANXA1, BCAR1, CLDN1, EIF4E, EZH2, FOLR1, FOXM1, IL7R, IL12RB2, KIAA1524, CRMP1, LOX, MCM7, MTA1, MTDH, NCOA3, NDRG2, NEDD9, NES, PBK, PPM1D, SIRT1, SLC7A5, SQSTM1 and WNT1) demonstrated a consistent prognostic association in two or more independent patient cohorts, thus qualifying as promising candidates for diagnostic use. Raised quality standards for study design and antibody validation, and integration of preclinical findings with clinical needs are clearly warranted.

The Impact of the Fourth Edition of the WHO Classification of Lung Tumours on Histological Classification of Resected Pulmonary NSCCs

Introduction: Histopathological classification of lung cancer is of central importance in the diagnostic routine, and it guides therapy in most patients. The fourth edition of the World Health Organization (WHO) Classification of Lung Tumours was recently published and includes changes to the diagnostic procedure for non–small cell carcinomas (NSCCs), with more emphasis on immunohistochemical (IHC) staining. Methods: A total of 656 unselective cases of resected pulmonary NSCC were diagnosed according to the 2004 WHO classification. After IHC staining with cytokeratin 5, p40, p63, thyroid transcription factor 1 (clones 8G7G3/1 and SPT24), and napsin A, the diagnoses were revised in accordance with the new fourth edition of the WHO classification. Results: Reclassification led to a new histological annotation in 36 of the 656 cases (5%). Most notable was the decrease in cases previously classified as large cell carcinomas (56 versus 12 cases). This was partially due to the exclusion of 21 neuroendocrine tumors from this group, with 20 cases ascribed to the adenocarcinoma group on the basis of IHC markers. Only seven cases of adenocarcinoma or squamous cell carcinoma were reclassified after the addition of IHC staining. There was a substantial overlap in staining properties between different markers of squamous and adenocarcinomatous differentiation, respectively, but in 17 to 31 cases (3%–5%), the diagnosis depended on the choice of markers. Conclusions: The fourth edition of the WHO Classification of Lung Tumours leads to changes in histological type in 5% of resected NSCCs. The incorporation of IHC staining into NSCC diagnostics demands awareness that the choice of ancillary stains has an effect on diagnosis.INTRODUCTION: Histopathological classification of lung cancer is of central importance in the diagnostic routine and guides therapy in the majority of patients. The 4(th) edition of the WHO classification was recently published and includes changes to the diagnostic procedure of non-small cell carcinomas (NSCC) with more emphasis on immunohistochemical (IHC) staining.METHODS: 656 unselective cases of resected pulmonary NSCC were diagnosed according to the 2004 WHO classification. After IHC staining with cytokeratin 5, p40, p63, thyroid transcription factor 1 (clones 8G7G3/1 and SPT24) and napsin A the diagnoses were revised in accordance with the new 4(th) edition of the WHO classification.RESULTS: Reclassification led to a new histological annotation in 36 (5%) of the 656 cases. Most notable was the decrease of cases previously classified as large cell carcinomas (56 vs. 12 cases). This was partially due to the exclusion of 21 neuroendocrine tumors from this group, while 20 cases were ascribed to the group of adenocarcinoma based on IHC markers. Only 7 cases of adenocarcinoma or squamous cell carcinoma were reclassified after the addition of IHC staining. There was a substantial overlap in staining properties between different markers of squamous and adenocarcinomatous differentiation, respectively, but in 17-31 cases (3-5%) the diagnosis depended on the choice of markers.CONCLUSIONS: The 4(th) edition of the WHO classification of lung tumours leads to changes of histological type in 5% of resected NSCC cases. The incorporation of IHC staining in NSCC diagnostics demands awareness that the choice of ancillary stains has an effect on diagnosis.

Prognostic impact of COX-2 in non-small cell lung cancer : a comprehensive compartment-specific evaluation of tumor and stromal cell expression

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.

Gene Expression Profiling of Large Cell Lung Cancer Links Transcriptional Phenotypes to the New Histological WHO 2015 Classification

Introduction Large cell lung cancer (LCLC) and large cell neuroendocrine carcinoma (LCNEC) constitute a small proportion of NSCLC. The WHO 2015 classification guidelines changed the definition of the debated histological subtype LCLC to be based on immunomarkers for adenocarcinoma and squamous cancer. We sought to determine whether these new guidelines also translate into the transcriptional landscape of lung cancer, and LCLC specifically. Methods Gene expression profiling was performed by using Illumina V4 HT12 microarrays (Illumina, San Diego, CA) on samples from 159 cases (comprising all histological subtypes, including 10 classified as LCLC WHO 2015 and 14 classified as LCNEC according to the WHO 2015 guidelines), with complimentary mutational and immunohistochemical data. Derived transcriptional phenotypes were validated in 199 independent tumors, including six WHO 2015 LCLCs and five LCNECs. Results Unsupervised analysis of gene expression data identified a phenotype comprising 90% of WHO 2015 LCLC tumors, with characteristics of poorly differentiated proliferative cancer, a 90% tumor protein p53 gene (TP53) mutation rate, and lack of well‐known NSCLC oncogene driver alterations. Validation in independent data confirmed aggregation of WHO 2015 LCLCs in the specific phenotype. For LCNEC tumors, the unsupervised gene expression analysis suggested two different transcriptional patterns corresponding to a proposed genetic division of LCNEC tumors into SCLC‐like and NSCLC‐like cancer on the basis of TP53 and retinoblastoma 1 gene (RB1) alteration patterns. Conclusions Refined classification of LCLC has implications for diagnosis, prognostics, and therapy decisions. Our molecular analyses support the WHO 2015 classification of LCLC and LCNEC tumors, which herein follow different tumorigenic paths and can accordingly be stratified into different transcriptional subgroups, thus linking diagnostic immunohistochemical staining–driven classification with the transcriptional landscape of lung cancer.

HaloPlex Targeted Resequencing for Mutation Detection in Clinical Formalin-Fixed, Paraffin-Embedded Tumor Samples.

In recent years, the advent of massively parallel next-generation sequencing technologies has enabled substantial advances in the study of human diseases. Combined with targeted DNA enrichment methods, high sequence coverage can be obtained for different genes simultaneously at a reduced cost per sample, creating unique opportunities for clinical cancer diagnostics. However, the formalin-fixed, paraffin-embedded (FFPE) process of tissue samples, routinely used in pathology departments, results in DNA fragmentation and nucleotide modifications that introduce a number of technical challenges for downstream biomolecular analyses. We evaluated the HaloPlex target enrichment system for somatic mutation detection in 80 tissue fractions derived from 20 clinical cancer cases with paired tumor and normal tissue available in both FFPE and fresh-frozen format. Several modifications to the standard method were introduced, including a reduced target fragment length and two strand capturing. We found that FFPE material can be used for HaloPlex-based target enrichment and next-generation sequencing, even when starting from small amounts of DNA. By specifically capturing both strands for each target fragment, we were able to reduce the number of false-positive errors caused by FFPE-induced artifacts and lower the detection limit for somatic mutations. We believe that the HaloPlex method presented here will be broadly applicable as a tool for somatic mutation detection in clinical cancer settings.

Various Antibody Clones of Napsin A, Thyroid Transcription Factor 1, and p40 and Comparisons With Cytokeratin 5 and p63 in Histopathologic Diagnostics of Non-Small Cell Lung Carcinoma.

Histopathologic classification of cancer in the lung is important for choice of treatment. Cytokeratin 5 (CK5), p63, and p40 are commonly used immunohistochemical markers for squamous cell carcinoma, and napsin A (NAPA) and thyroid transcription factor 1 (TTF-1) are markers for adenocarcinoma of the lung. The aim of the present study was to evaluate these 5 markers and to compare different commercially available antibody clones in lung cancer. Tissue microarrays including 557 cases of surgically treated primary tumors and 73 matched metastases of non–small cell lung carcinoma were stained with CK5, p63, p40 (monoclonal and polyclonal), NAPA (5 different clones/protocols), and TTF-1 (2 different clones). The sensitivity and specificity to separate squamous cell carcinomas from non–small cell carcinomas of nonsquamous type were 95% and 97%, respectively, for CK5, 95% and 87% for p63, 94% and 96% for p40, 75% to 79% and 96% to 98% for the NAPA clones/protocols and 80% to 85% and 95% to 97% for the TTF-1 clones. A combination of NAPA and TTF-1 resulted in a higher sensitivity (85% to 88%), whereas combining CK5 and p40 did not increase the diagnostic performance. The sensitivity was generally lower in evaluation of lung cancer metastases. The &kgr;-values for comparison of staining results between monoclonal and polyclonal p40 and between the 5 NAPA clones/protocols were 0.97 to 1.0, whereas the corresponding figure for the 2 TTF-1 clones was 0.91 to 0.93. Conclusively, CK5 and p40 are good diagnostic markers for squamous cell carcinoma and superior to p63. In addition, it may be useful to combine NAPA and TTF-1 for increased sensitivity in lung cancer diagnostics. There is no substantial difference between monoclonal and polyclonal p40 and between different NAPA clones, whereas there is a difference between the TTF-1 clones 8G7G3/1 and SPT24.