Inge Platteel

TRAIL-receptor expression is an independent prognostic factor for survival in patients with a primary glioblastoma multiforme.

SummaryPurposeIn order to improve the survival of patients with a glioblastoma multiforme tumor (GBM), new therapeutic strategies must be developed. The use of a death inducing ligand such as TRAIL (TNF Related Apoptosis Inducing Ligand) seems a promising innovative therapy. The aim of this study was to quantify the expression of the death regulating receptors TRAIL-R1, TRAIL-R2 and TRAIL on primary GBM specimens and to correlate this expression with survival.Experimental designExpression of TRAIL and TRAIL-receptors was assessed by immunohistochemistry, both quantitatively (% of positive tumor cells) and semi-quantitatively (staining intensity) within both the perinecrotic and intermediate tumor zones of primary GBM specimens. RT-PCR of GBM tissue was performed to show expression of TRAIL receptor mRNA.ResultsImmunohistochemistry showed a slight diffuse intracytoplasmic and a stronger membranous staining for TRAIL and TRAIL receptors in tumor cells. Semi-quantitative expression of TRAIL showed a significantly higher expression of TRAIL in the perinecrotic zone than in the intermediate zone of the tumor (P=0.0001). TRAIL-R2 expression was significantly higher expressed than TRAIL-R1 (P=0.005). The antigenic load of TRAIL-R2 was positively correlated with survival (P=0.02). Multivariate analysis of TRAIL-R1 within the study group (n=62) showed that age, gender, staining intensity, antigenic load, % of TRAIL-R1 expression, were not statistically correlated with survival however radiotherapy was significantly correlated (multivariate analysis: age: P=0.15; gender: P=0.64; staining intensity: P=0.17; antigenic load: P=0.056; % of TRAIL-R1 expression: P=0.058; radiotherapy: P=0.0001). Subgroup analysis of patients who had received radiotherapy (n=47) showed a significant association of % of TRAIL-R1 expression and the antigenic load of TRAIL-R1 with survival (multivariate analysis: P=0.036, respectively, P=0.023).Multivariate analysis of TRAIL-R2 staining intensity and antigenic load, within the study group (P=0.004, respectively, P=0.03) and the subgroup (P=0.002, respectively, P=0.004), showed a significant association with survival. RT-PCR analysis detected a negative relation between the amount of TRAIL-R1 mRNA and the WHO grade of astrocytic tumors (P=0.03).ConclusionsTRAIL-R1 and TRAIL-R2 expression on tumor cells are independent prognostic factors for survival in patients with a glioblastoma multiforme. Both receptors could be targets for TRAIL therapy. As TRAIL-R2 is more expressed, in comparison with TRAIL-R1, on GBM tumor cells, TRAIL-R2 seems to be of more importance as a target for future TRAIL therapy than TRAIL-R1.

The angiogenic makeup of human hepatocellular carcinoma does not favor vascular endothelial growth factor/angiopoietin-driven sprouting neovascularization.

Quantitative data on the expression of multiple factors that control angiogenesis in hepatocellular carcinoma (HCC) are limited. A better understanding of the mechanisms underlying angiogenesis in HCC will improve the rational choice of anti‐angiogenic treatment. We quantified gene and protein expression of members of the vascular endothelial growth factor (VEGF) and angiopoietin systems and studied localization of VEGF, its receptors VEGFR‐1 and VEGFR‐2, Angiopoietin (Ang)‐1 and Ang‐2, and their receptor, in HCC in noncirrhotic and cirrhotic livers. We employed real‐time reverse transcription polymerase chain reaction (RT‐PCR), western blot, and immunohistology, and compared the outcome with highly angiogenic human renal cell carcinoma (RCC). HCC in noncirrhotic and cirrhotic livers expressed VEGF and its receptors to a similar extent as normal liver, although in cirrhotic background, VEGFR‐2 levels in both tumor and adjacent tissue were decreased. Ang‐1 expression was slightly increased compared with normal liver, whereas Tie‐2 was strongly down‐regulated in the tumor vasculature. Ang‐2 messenger RNA (mRNA) levels were also low in HCCs of both noncirrhotic and cirrhotic livers, implying that VEGF‐driven angiogenic sprouting accompanied by angiopoietin‐driven vascular destabilization is not pronounced. In RCC, VEGF‐A levels were one order of magnitude higher. At the same time, endothelially expressed Ang‐2 was over 30‐fold increased compared with expression in normal kidney, whereas Ang‐1 expression was decreased. Conclusion: In hepatocellular carcinoma, tumor vascularization is not per se VEGF/angiopoietin driven. However, increased CD31 expression and morphological changes representative of sinusoidal capillarization in tumor vasculature indicate that vascular remodeling is taking place. This portends that therapeutic intervention of HCC at the level of the vasculature is optional, and that further studies into the molecular control thereof are warranted. (HEPATOLOGY 2008.)

P53-specific T cell responses in patients with malignant and benign ovarian tumors : Implications for p53 based immunotherapy

Despite intensive treatment, 70% of the ovarian cancer patients will develop recurrent disease, emphasizing the need for new approaches such as immunotherapy. A promising antigenic target for immunotherapy in ovarian cancer is the frequently overexpressed p53 protein. The aim of the study was to evaluate the nature and magnitude of the baseline anti‐p53 immune response in ovarian cancer patients. P53‐specific T cell responses were detected in both half of the ovarian cancer patients as in the group of control subjects, consisting of women with benign ovarian tumors and healthy controls. Importantly, while in the control group p53‐specific immunity was detected among the CD45RA+ naïve subset of T cells only, the p53‐specific T‐cell responses in ovarian cancer patients were also present in the CD45RO+ memory T‐cell subset, suggesting that in the cancer patients sufficient amounts of cancer‐derived p53 was presented to induce the formation of a p53‐specific memory T‐cell response. Further characterization of the p53‐specific memory T‐cell responses revealed that in addition to the type 1 cytokine IFN‐γ also the type 2 cytokines IL‐4 and IL‐5, as well as the immunosuppressive cytokine IL‐10 were produced. Notably, p53‐specific T cells were not only detected in the peripheral blood, but also among tumor infiltrating lymphocytes and in tumor‐draining lymph nodes. In conclusion, the existence of a weak mixed T‐helper type 1 and 2 p53‐specific T‐cell repertoire supports the rationale of using p53 long peptides in vaccination strategies aiming at the induction of p53‐specific Th1/CTL immunity.

Common and rare EGFR and KRAS mutations in a Dutch non-small-cell lung cancer population and their clinical outcome

Introduction In randomly assigned studies with EGFR TKI only a minor proportion of patients with NSCLC have genetically profiled biopsies. Guidelines provide evidence to perform EGFR and KRAS mutation analysis in non-squamous NSCLC. We explored tumor biopsy quality offered for mutation testing, different mutations distribution, and outcome with EGFR TKI. Patient and Methods Clinical data from 8 regional hospitals were studied for patient and tumor characteristics, treatment and overall survival. Biopsies sent to the central laboratory were evaluated for DNA quality and subsequently analyzed for mutations in exons 18–21 of EGFR and exon 2 of KRAS by bidirectional sequence analysis. Results Tumors from 442 subsequent patients were analyzed. For 74 patients (17%) tumors were unsuitable for mutation analysis. Thirty-eight patients (10.9%) had EGFR mutations with 79% known activating mutations. One hundred eight patients (30%) had functional KRAS mutations. The mutation spectrum was comparable to the Cosmic database. Following treatment in the first or second line with EGFR TKI median overall survival for patients with EGFR (n = 14), KRAS (n = 14) mutations and wild type EGFR/KRAS (n = 31) was not reached, 20 and 9 months, respectively. Conclusion One out of every 6 tumor samples was inadequate for mutation analysis. Patients with EGFR activating mutations treated with EGFR-TKI have the longest survival.

Genomic Profile of Endometrial Tumors Depends on Morphological Subtype, Not on Tamoxifen Exposure

Tamoxifen has been a very effective treatment for breast cancer for several decades, however, at the same time increases the risk of endometrial cancer, especially after prolonged exposure. In addition, tamoxifen has been associated with a higher proportion of unfavorable uterine tumor subtypes (carcinosarcomas and serous adenocarcinomas) with worse survival. We investigated whether endometrial tumors, which developed after prolonged tamoxifen treatment for breast cancer, are genetically different from endometrial tumors without preceding tamoxifen exposure. Array CGH was used on archival formalin‐fixed paraffin embedded endometrial tumors to determine genomic aberrations. We compared the genomic profiles of 52 endometrial tumors from breast cancer patients after long‐term (≥2 years) tamoxifen use (endometrioid adenocarcinomas, n = 26; carcinosarcomas, n = 14; and serous adenocarcinomas, n = 12) with endometrial tumors from unexposed breast cancer patients (n = 45). Genomic profiles were correlated with tamoxifen exposure, tumor subtypes, and histopathological characteristics of the endometrial tumors. The common uterine corpus cancers of the endometrioid subtype show few genomic aberrations. Tumors with many genomic aberrations were in general ER‐negative. In contrast, carcinosarcomas and serous adenocarcinomas showed many aberrations; however, they were indistinguishable from each other. Tumors that developed after prolonged tamoxifen use did not show more or different aberrations than unexposed tumors. This was true for all tumor subtypes. Thus, endometrial carcinomas that develop after prolonged tamoxifen use cannot be distinguished from nonusers on basis of their tumor genomic profile.

Molecular Characterization of the Vascular Features of Focal Nodular Hyperplasia and Hepatocellular Adenoma: A Role for Angiopoietin-1

Focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA) are two hepatic nodular lesions of different etiologies. FNH, a polyclonal lesion, is assumed to be a regenerative reaction following a vascular injury, whereas HCA is a monoclonal, benign neoplastic lesion. In addition to features that are predominantly found in either FNH or HCA (e.g., dystrophic vessels in FNH and single arteries in HCA), FNH and HCA share morphological vascular abnormalities such as dilated sinusoids. We hypothesized that these anomalous vascular features are associated with altered expression of growth factors involved in vascular remodeling. This was based on reports of morphologically abnormal hepatic vasculature and nodular lesions in transgenic models of hepatocytic overexpression of angiopoietin‐1 (Ang‐1), a member of the angiopoietin family, which is crucially involved in vascular morphogenesis and homeostasis. We investigated gene and protein expression of members of the angiopoietin system and vascular endothelial growth factor A (VEGF‐A) and its receptors in 9 FNH samples, 13 HCA samples, and 9 histologically normal livers. In comparison with normal samples, a significant increase in Ang‐1 was found in FNH (P < 0.01) and HCA (P < 0.05), whereas no significant changes in Ang‐2, receptor tyrosine kinase with immunoglobulin‐like and EGF‐like domains 2, VEGF‐A, or vascular endothelial growth factor receptor 2 (VEGFR‐2) were observed. Conclusion: Because of the different etiological contexts of a preceding vascular injury in FNH and a neoplastic growth in HCA, Ang‐1 might exert different effects on the vasculature in these lesions. In FNH, it could predominantly stimulate recruitment of myofibroblasts and result in dystrophic vessels, whereas in HCA, it may drive vascular remodeling that produces enlarged vessels and arterial sprouting that generates single arteries. Hepatology 2010

Rapid BRAF mutation tests in patients with advanced melanoma: Comparison of immunohistochemistry, Droplet Digital PCR, and the Idylla Mutation Platform

BRAF mutational testing has become a common practice in the diagnostic process of patients with advanced melanoma. Although time-consuming, DNA sequencing techniques are the current gold standard for mutational testing. However, in certain clinical situations, a rapid test result is required. In this study, the performance of three rapid BRAF mutation tests was compared. Thirty-nine formalin-fixed paraffin-embedded melanoma tissue samples collected between 2007 and 2014 at a single center were included. These samples were analyzed by immunohistochemistry using the anti-BRAF-V600E (VE1) mouse monocolonal antibody (BRAF-VE1 IHC), a V600E-specific Droplet Digital PCR Test, and the Idylla BRAF- Mutation Test (Idylla). Results were compared with the results of conventional BRAF mutation testing, performed using high-resolution melting analysis followed by Sanger sequencing. Next-generation sequencing was performed on samples with discordant results. The Idylla test and Droplet Digital PCR Test correctly identified all mutated and wild-type samples. BRAF-VE1 IHC showed one discordant result. The Idylla test could identify BRAF-V600 mutations other than BRAF-V600E and was the fastest and least laborious test. The Idylla Mutation Test is the most suitable test for rapid BRAF testing in clinical situations on the basis of the broad coverage of treatment-responsive mutations and the fast procedure without the need to perform a DNA isolation step.