Mats G. Karlsson

Biomarker Discovery in Non–Small Cell Lung Cancer: Integrating Gene Expression Profiling, Meta-analysis, and Tissue Microarray Validation

Purpose: Global gene expression profiling has been widely used in lung cancer research to identify clinically relevant molecular subtypes as well as to predict prognosis and therapy response. So far, the value of these multigene signatures in clinical practice is unclear, and the biologic importance of individual genes is difficult to assess, as the published signatures virtually do not overlap. Experimental Design: Here, we describe a novel single institute cohort, including 196 non–small lung cancers (NSCLC) with clinical information and long-term follow-up. Gene expression array data were used as a training set to screen for single genes with prognostic impact. The top 450 probe sets identified using a univariate Cox regression model (significance level P < 0.01) were tested in a meta-analysis including five publicly available independent lung cancer cohorts (n = 860). Results: The meta-analysis revealed 14 genes that were significantly associated with survival (P < 0.001) with a false discovery rate <1%. The prognostic impact of one of these genes, the cell adhesion molecule 1 (CADM1), was confirmed by use of immunohistochemistry on tissue microarrays from 2 independent NSCLC cohorts, altogether including 617 NSCLC samples. Low CADM1 protein expression was significantly associated with shorter survival, with particular influence in the adenocarcinoma patient subgroup. Conclusions: Using a novel NSCLC cohort together with a meta-analysis validation approach, we have identified a set of single genes with independent prognostic impact. One of these genes, CADM1, was further established as an immunohistochemical marker with a potential application in clinical diagnostics. Clin Cancer Res; 19(1); 194–204. ©2012 AACR.

Increased expression of inducible nitric oxide synthase in psoriatic skin and cytokine-stimulated cultured keratinocytes

Summary Since nitric oxide (NO) has been implicated in the pathogenesis of various hyperproliferative and inflammatory diseases, the mRNA expression of constitutive nitric oxide synthase (cNOS) and inducibie nitric oxide synthase (iNOS) were investigated in psoriatic skin by reverse transcriptase coupled to the polymerase chain reaction (PCR). The study showed that the mRNA expression of brain nitric oxide synthase (bNOS), one of two isoforms of cNOS, was weak in both psoriatic plaques lesions and uninvolved skin, while mRNA transcripts for the second isoform, endothelial nitric oxide synthase (eNOS). were not detectable using the present method. In contrast, the mRNA expression of iNOS was markedly increased in lesional skin as compared to uninvolved skin. Cultured human keratinocytes exposed to a combination of interleukin‐lβ (IL‐1β) and tumour necrosis factor‐α (TNF‐α) for 4 h. showed strong gene expression of iNOS, while in 24h. the expression had returned to baseline expression. In summary, the study demonstrates that mRNA for the inducible form of NOS is over‐expressed in psoriatic lesions. The cause of this may be the local presence of inflammatory cytokines. These findings imply that iNOS may play an important part in local regulation of NO synthesis in psoriasis and other inflammatory dermatoses.

Expression of Ephb2 and Ephb4 in breast carcinoma

Eph receptor tyrosine kinases and their cell-surfacebound ligands, the ephrins, play key roles in diverse biological processes. Eph receptors comprise the largest family of receptor tyrosine kinases consisting of eight EphA receptors (with five corresponding ephrinA ligands) and six EphB receptors (with three corresponding transmembrane ephrinB ligands). Originally identified as neuronal pathfinding molecules, EphB receptors and ephrinB ligands are later proved to be crucial regulators of vasculogenesis and embryogenesis. More studies indicate that Eph receptors are involved in angiogenesis and tumorigenesis. This study aimed to investigate the expression of EphB2 and EphB4 in breast carcinomas. Semiquantitative RT-PCR and immunohistochemistry were used to examine the expression patterns of EphB2 and EphB4. Clinicopathological and survival correlations were statistically analyzed in a series of 94 breast carcinomas, 9 normal specimens and 4 breast carcinoma cell lines. 1(1%), 16(17%), 29(31%), 48(51%) of the 94 tumors were negative, weak, moderate and strong EphB2 protein expression, respectively. 6(6%), 27(29%), 28(30%), 33(35%) of the tumors were negative, weak, moderate and strong EphB4 expression, respectively. Both EphB2 and EphB4 RT-PCR products could be detected in all specimens. Increased EphB2 protein expression was negatively associated with overall survival, and there was a trend that increased EphB2 protein expression was correlated with shorter disease free survival, while EphB4 protein expression was associated with histological grade and stage. EphB4 membrane staining was increased with S phase fraction and associated with DNA aneuploidy. These findings indicate that both EphB2 and EphB4 are involved in the development of breast cancer and that both molecules could be potential predictive markers.

Clinicopathological associations of CD44 mRNA and protein expression in primary breast carcinomas

Aims:  The purpose of this study was to examine the occurrence of CD44 isoforms in breast carcinomas and their role in predicting clinical outcome.

p53 and Rb immunostaining in locally advanced bladder cancer: relation to prognostic variables and predictive value for the local response to radical radiotherapy.

The association between known prognostic variables and altered immunostaining for the nuclear proteins retinoblastoma (Rb) and p53 was studied in a homogeneous series of locally advanced bladder cancer. The predictive value of this immunostaining for the local response to intended radical radiotherapy was investigated. Among 262 patients treated with intended radical radiotherapy between 1967 and 1986, a total of 154 patients were evaluable with respect to local response to treatment. The paraffin-embedded specimen from the tumour prior to irradiation was immunostained with the monoclonal antibodies PMG3-245 for Rb and 1801 for p53 nuclear proteins after heating in a microwave oven for 40 min at 650 W. An altered expression of Rb and p53 was observed in 18 and 42% of the tumours, respectively. p53 overexpression was associated with higher tumour grade. However, the results of the p53 and Rb immunostaining procedures had no predictive value for tumor response to radiation treatment, local control or cancer-specific mortality.

Epidermal growth factor receptor expression: predictive value for the outcome after cystectomy for bladder cancer?

To determine whether epidermal growth factor receptor (EGFR) immunostaining of tumour cells is associated with cancer‐specific death after cystectomy for locally advanced bladder cancer.

The expression of EGFR family ligands in breast carcinomas.

Expression of EGF, HB-EGF, TGF-oc, HRG-oc, HRG-f1, and HRG-P3 in 100 frozen breast carcinoma materials was immunohistochemically studied. Among these tumors, 67% were positive for EGF, 53% for HB-EGF, 57% for TGF-a, 60% for HRGax, 53% for HRG-31, and 63% for HRG-f3 in the neoplastic epithelial cells. No significant associations between expression of the growth factors and clinicopathological features like tumor size, histologic grade, node status, ploidy, ER status, and c-erbB-4 expression were observed, with the exceptions that significant relations were present between EGF expression and tumor size (p = 0.01) and between HRG-P3 expression and node status (p =0.02). The expressions of these growth factors showed no associationwith cancer-specific survival by the Kaplan Meier analysis.

A miRNA expression signature that separates between normal and malignant prostate tissues.

BackgroundMicroRNAs (miRNAs) constitute a class of small non-coding RNAs that post-transcriptionally regulate genes involved in several key biological processes and thus are involved in various diseases, including cancer. In this study we aimed to identify a miRNA expression signature that could be used to separate between normal and malignant prostate tissues.ResultsNine miRNAs were found to be differentially expressed (p <0.00001). With the exception of two samples, this expression signature could be used to separate between the normal and malignant tissues. A cross-validation procedure confirmed the generality of this expression signature. We also identified 16 miRNAs that possibly could be used as a complement to current methods for grading of prostate tumor tissues.ConclusionsWe found an expression signature based on nine differentially expressed miRNAs that with high accuracy (85%) could classify the normal and malignant prostate tissues in patients from the Swedish Watchful Waiting cohort. The results show that there are significant differences in miRNA expression between normal and malignant prostate tissue, indicating that these small RNA molecules might be important in the biogenesis of prostate cancer and potentially useful for clinical diagnosis of the disease.

PIK3CA, HRAS and KRAS Gene Mutations in Human Penile Cancer

PURPOSE The knowledge of somatic mutations that arise in penile cancer is limited. We examined the dysregulation of components in the phosphatidylinositol 3-kinase and Ras pathways. MATERIALS AND METHODS Using single stranded conformational analysis and direct sequencing we performed mutational analysis of the PIK3CA, PTEN, HRAS, KRAS, NRAS and BRAF genes in 28 penile tumors. RESULTS We identified somatic missense mutations in 11 of the 28 penile cancer samples (39%). In the PIK3CA gene 8 mutations (29%) were identified that were E542K or E545K. In the HRAS gene a G12S and a Q61L mutation were found (7%). The KRAS gene contained 1 mutation (3%), that is a G12S change. PIK3CA mutations were found in all grades and stages, whereas HRAS and KRAS mutations were found in larger and more advanced tumors. The mutations were mutually exclusive, suggesting that dysregulation of either pathway is sufficient for the development and progression of penile carcinoma. CONCLUSIONS The high frequency of mutations in the PIK3CA, HRAS and KRAS genes leads us to believe that dysregulation of the phosphatidylinositol 3-kinase or Ras pathway is significant for the development and progression of penile carcinoma.

PREDICTIVE VALUE OF p53 AND pRb IMMUNOSTAINING IN LOCALLY ADVANCED BLADDER CANCER TREATED WITH CYSTECTOMY

PURPOSE We elucidate the association between altered immunostaining for retinoblastoma gene protein (pRb) and p53 nuclear proteins, and cancer specific death in patients treated with cystectomy for locally advanced bladder cancer. MATERIALS AND METHODS The hospital records of 173 patients treated with cystectomy for advanced urothelial bladder cancer between 1967 and 1992 were retrospectively reviewed. Representative biopsies obtained before treatment were sectioned and stained using the standard immunohistochemical technique with antibody DO-7 (p53) and antibody PMG3-245 (pRb). A tumor was considered to have an altered p53 expression if 20% or more of tumor cells exhibited nuclear staining. Similarly, if no tumor cell had nuclear immunostaining the tumor was considered to have an altered pRb expression. RESULTS An altered expression was observed for p53 in 98 tumors (57%) and for pRb in 60 (35%). In a proportional hazards analysis no association was found between an altered expression of pRb or p53 and cancer specific death. This finding was also true in another analysis when the results of immunostaining for pRb and p53 were combined. CONCLUSIONS An altered expression for pRb and/or p53 was not correlated to cancer specific death. Thus, these parameters could not be used as predictors of treatment outcome after cystectomy for locally advanced bladder cancer.