Enno J. Dreef

Conventional and tissue microarray immunohistochemical expression analysis of mismatch repair in hereditary colorectal tumors.

Immunohistochemistry (IHC) of mismatch repair (MMR) proteins in colorectal tumors together with microsatellite analysis (MSI) can be helpful in identifying families eligible for mutation analysis. The aims were to determine sensitivity of IHC for MLH1, MSH2, and MSH6 and MSI analysis in tumors from known MMR gene mutation carriers; and to evaluate the use of tissue microarrays for IHC (IHC-TMA) of colon tumors in its ability to identify potential carriers of MMR gene mutations, and compare it with IHC on whole slides. IHC on whole slides was performed in colorectal tumors from 45 carriers of a germline mutation in one of the MMR genes. The TMA cohort consisted of 129 colon tumors from (suspected) hereditary nonpolyposis colorectal cancer (HNPCC) patients. Whole slide IHC analysis had a sensitivity of 89% in detecting MMR deficiency in carriers of a pathogenic MMR mutation. Sensitivity by MSI analysis was 93%. IHC can also be used to predict which gene is expected to harbor the mutation: for MLH1, MSH2, and MSH6, IHC on whole slides would have correctly predicted the mutation in 48%, 92%, and 75% of the cases, respectively. We propose a scheme for the diagnostic approach of families with (suspected) HNPCC. Comparison of the IHC results based on whole slides versus TMA, showed a concordance of 85%, 95%, and 75% for MLH, MSH2, and MSH6, respectively. This study therefore shows that IHC-TMA can be reliably used to simultaneously screen a large number of tumors from (suspected) HNPCC patients, at first in a research setting.

A Meta-Analysis of Gene Expression Data Identifies a Molecular Signature Characteristic for Tumor-Stage Mycosis Fungoides

Mycosis fungoides (MF) is the most common type of primary cutaneous T-cell lymphoma (CTCL). To identify a molecular signature characteristic of MF tumor stage, we used a bioinformatic approach involving meta-analysis of publicly available gene expression data sets combined with previously generated gene expression data. Results for a selection of genes were further refined and validated by quantitative PCR and inclusion of additional controls. With this approach, we identified a profile specific for MF tumor stage, consisting of 989 aberrantly expressed genes, the majority of which (718 genes) are statistically significantly more expressed in MF compared with normal skin, inflamed skin, and normal T cells. As expected, the signature contains genes reflecting the highly proliferative characteristic of this T-cell malignancy, including altered expression of cell cycle and kinetochore regulators. We uncovered details of the immunophenotype, suggesting that MF originates from IL-32-producing cells and identified previously unreported therapeutic targets and/or diagnostic markers, for example, GTSF1 and TRIP13. Loss of expression of the NF-κB inhibitor, NFKBIZ, may partly explain the enhanced activity of NF-κB, which is a hallmark of MF and other CTCLs.

L1 cell adhesion molecule is a strong predictor for distant recurrence and overall survival in early stage endometrial cancer: pooled PORTEC trial results.

BACKGROUNDnL1 cell adhesion molecule (L1CAM) expression has been implicated as risk factor for disease recurrence in endometrial cancer (EC), most likely due to its role in promoting tumour cell motility. We tested the performance of L1CAM expression in predicting the risk of recurrence in the randomised post operative radiation therapy in endometrial carcinoma (PORTEC)-1 and -2 trials.nnnMETHODSnIn the PORTEC trials, stage I EC patients were randomised to external beam radiotherapy (EBRT) versus no additional treatment (PORTEC-1, n=714), or to EBRT versus vaginal brachytherapy (PORTEC-2, n=427). Tumour samples of 865 (75.8%) patients were available for L1CAM expression analysis by immunohistochemistry. An established scoring system for EC was used, with >10% L1CAM staining defined as positive.nnnRESULTSnPositive L1CAM expression was significantly correlated with risk of distant recurrence, with a hazard ratio (HR) of 5.1 (95% confidence interval (CI) 3.1-8.7) but not with vaginal relapse, while a trend for pelvic nodal relapse was found. Tumours with the highest expression levels (>50% positive) had the strongest risk of distant recurrence (HR 5.3, CI 2.7-10.4). In multivariate Cox analysis with the risk factors age, depth of invasion, grade, lympho-vascular space invasion (LVSI) and treatment, L1CAM expression remained an independent prognostic factor for distant recurrence (HR 3.5, CI 1.92-6.30) and overall survival (HR 2.1, CI 1.41-2.98).nnnCONCLUSIONnL1CAM expression is a strong independent predictor for distant recurrence and overall survival in stage I endometrial cancer. These results warrant prospective validation of L1CAM as marker for selecting patients who could benefit from more extensive diagnostic and/or therapeutic procedures.

EMMPRIN-induced MMP-2 activation cascade in human cervical squamous cell carcinoma.

Tumor progression and recurrence of cervical cancer is associated with upregulation of matrix metalloproteinase 2 (MMP‐2). We evaluated the location, origin and activity of MMP‐2 in cervical squamous cell carcinomas in comparison with MT1‐MMP (MMP‐14), TIMP‐2 and extracellular matrix metalloproteinase inducer (EMMPRIN). Positive immunostaining for MMP‐2 in malignant cells was detected in 83% of the patients. Two patterns of tumor cell MMP‐2 staining were observed: either homogenous in all tumor cells or confined to the cells neighboring the stroma (tumor‐border staining pattern, TBS). Fluorescence in situ zymography showed active MMP‐2 mainly around tumor nodules displaying TBS. The MMP‐2 staining of TBS tumors correlated significantly with the presence of TIMP‐2 and MT1‐MMP, proteins involved in docking MMP‐2 to the cell surface and essential for MMP‐2 activation. In situ mRNA hybridization in TBS tumors demonstrated more abundant presence of MMP‐2 mRNA in neighboring myofibroblasts than in the adjacent tumor cells. Moreover, the TBS MMP‐2 pattern correlated with the presence of EMMPRIN (p = 0.023), suggesting that tumor cells induce MMP‐2 production in nearby stromal cells. This pro‐MMP‐2 could subsequently be activated on tumor cells via the presence of MT1‐MMP and TIMP‐2. The biological relevance of this locally activated MMP‐2 was underscored by the observation that only the TBS pattern of MMP‐2 significantly correlated with decreased survival. In conclusion, the colocalization of EMMPRIN, MT1‐MMP and TIMP‐2 in human cervical carcinomas seems to be involved in a specific distribution pattern of tumor cell bound MMP‐2, which is related with local proteolytic activity and therefore might be associated with worse prognosis of the patients.

Expression of endoglin (CD105) in cervical cancer

In this study, we have investigated the role of endoglin (CD105), a regulator of transforming growth factor (TGF)-β1 signalling on endothelial cells, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-A (VEGF-A) in cervical cancer. We have measured the number and determined the location of both newly formed (CD105-positive) and the overall number of (CD31-positive) blood vessels, and bFGF and VEGF-A expression using immunohistochemistry in 30 cervical carcinoma specimens. Vascular endothelial growth factor-A mRNA expression was determined using RNA-in situ hybridisation. CD105- and CD31-positive vessels and bFGF- and VEGF-A-positive cells were predominantly present in the stroma. The presence of CD105- and CD31-positive vessels in the stroma did neither correlate with the number of VEGF-A-positive cells nor the number of bFGF-positive cells. However, the number of CD105- and CD31-positive vessels was associated with the expression of VEGF-A mRNA in the epithelial cell clusters (P=0.013 and P=0.005, respectively). The presence of CD105-positive and CD31-positive vessels was associated with the expression of αvβ6 (a TGF-β1 activator; P=0.013 and P=0.006, respectively). Clinically, the number of CD105-positive vessels associated with the number of lymph node metastasis (P<0.001). Furthermore, the presence of CD105-positive vessels within the epithelial cell clusters associated with poor disease-free survival (P=0.007).

Elevated expression of SerpinA1 and SerpinA3 in HLA-positive cervical carcinoma.

In cervical cancer, an important mechanism by which tumour cells escape immune surveillance is loss of HLA class I, enabling tumours to evade recognition and lysis by cytotoxic T lymphocytes. Some tumours, however, escape from immune surveillance without accumulating defects in antigen presentation. We hypothesized that tumours with no or partial loss of HLA class I develop alternative mechanisms to prevent immune elimination. To investigate this hypothesis, genome‐wide expression profiling using Illumina arrays was performed on cervical squamous cell carcinomas showing overall loss of HLA class I, partial, and normal HLA class I protein expression. Statistical analyses revealed no significant differences in gene expression between tumours with partial (n = 11) and normal HLA class I expression (n = 10). Comparison of tumours with normal/partial HLA class I expression (n = 21) with those with overall loss of HLA class I expression (n = 11) identified 150 differentially expressed genes. Most of these genes were involved in the defence response (n = 27) and, in particular, inflammatory and acute phase responses. Especially SerpinA1 and SerpinA3 were found to be up‐regulated in HLA‐positive tumours (3.6‐ and 8.2‐fold, respectively), and this was confirmed by real‐time PCR and immunohistochemistry. In a group of 117 tumours, high SerpinA1 and SerpinA3 expression in association with normal/partial HLA expression correlated significantly with poor overall survival (p = 0.035 and p = 0.05, respectively). Thus, HLA‐positive tumours are characterized by higher expression of genes associated with an inflammatory profile. In addition, expression of the acute phase proteins SerpinA1 and SerpinA3 in HLA‐positive tumours is associated with worse prognosis. Copyright

Expression of B-cell transcription factors in primary cutaneous B-cell lymphoma

Expression patterns of eight transcription factors involved in different stages of B-cell development were investigated in a large group of primary cutaneous B-cell lymphomas and compared with expression patterns during normal B-cell development. The following transcription factors were investigated: Pax-5, PU.1, Oct2, BOB.1, Bcl-6, Mum1/IRF4, Blimp-1 and FOXP1. Primary cutaneous large B-cell lymphomas, leg type showed aberrant coexpression of Bcl-6 and Mum1/IRF4 and in addition strong expression of FOXP1. Expression of FOXP1 and Mum1/IRF4 strongly suggests an activated B-cell type of origin. In contrast, primary cutaneous follicle center lymphomas showed expression of Bcl-6, Pax-5, PU.1, Oct2 and BOB.1, but not of Mum1/IRF4, Blimp-1 and FOXP1. Primary cutaneous marginal zone B-cell lymphoma showed expression of Pax-5, PU.1, Oct2 and BOB.1, but not Bcl-6 by the neoplastic B-cells, and Mum1/IRF4 and Blimp-1 by the neoplastic plasma cells. In conclusion, in primary cutaneous follicle center lymphoma and primary cutaneous marginal zone B-cell lymphoma expression patterns were observed similar to their supposed benign counterparts, germinal center B-cells and postgerminal center B-cells, respectively, which might reflect their indolent clinical behaviour and excellent prognosis. In contrast, the activated B-cell expression pattern in the group of primary cutaneous large B-cell lymphoma, leg type may contribute to its poor prognosis and Mum1/IRF4 and FOXP1 may serve as additional diagnostic markers for this type of primary cutaneous B-cell lymphoma.

Chronic right ventricular pressure overload results in a hyperplastic rather than a hypertrophic myocardial response

Myocardial hyperplasia is generally considered to occur only during fetal development. However, recent evidence suggests that this type of response may also be triggered by cardiac overload after birth. In congenital heart disease, loading conditions are frequently abnormal, thereby affecting ventricular function. We hypothesized that chronic right ventricular pressure overload imposed on neonatal hearts initiates a hyperplastic response in the right ventricular myocardium. To test this, young lambs (aged 2–3 weeks) underwent adjustable pulmonary artery banding to obtain peak right ventricular pressures equal to left ventricular pressures for 8 weeks. Transmural cardiac tissue samples from the right and left ventricles of five banded and five age‐matched control animals were studied. We found that chronic right ventricular pressure overload resulted in a twofold increase in right‐to‐left ventricle wall thickness ratio. Morphometric right ventricular myocardial tissue analysis revealed no changes in tissue composition between the two groups; nor were right ventricular myocyte dimensions, relative number of binucleated myocytes, or myocardial DNA concentration significantly different from control values. In chronic pressure overloaded right ventricular myocardium, significantly (P < 0.01) more myocyte nuclei were positive for the proliferation marker proliferating cellular nuclear antigen than in control right ventricular myocardium. Chronic right ventricular pressure overload applied in neonatal sheep hearts results in a significant increase in right ventricular free wall thickness which is primarily the result of a hyperplastic myocardial response.

Primary lymphoma of bone: extranodal lymphoma with favourable survival independent of germinal centre, post-germinal centre or indeterminate phenotype

Aims: To determine prognostic significance of immunohistochemical markers and investigate possible germinal centre (GC) derivation in primary lymphoma of bone (PLB). Methods: Immunohistochemical expression of BCL-6, CD10, BCL-2, p53, CD30, CD44 and MUM-1 was studied in 36 patients with PLB. All cases were clinically staged and cases of secondary bone involvement of primary nodal lymphomas were excluded, prior to immunostaining. Clinical charts were reviewed for clinical symptoms and therapy given; survival post-biopsy was calculated. Results: All patients presented with pain and a palpable mass. The majority showed centroblastic-multilobated morphology; half of the cases (19/36) had a GC phenotype (CD10+BCL-6+ or CD10−BCL-6+MUM-1−), whereas 8/36 cases had a non-GC phenotype (CD10−BCL-6− or CD10−BCL-6+MUM-1+). Nine cases were of indeterminate phenotype (CD10−BCL-6+; MUM-1 not available). Eight of 22 evaluated patient samples showed immunoreactivity for MUM-1. Most patients (31/36) received combination therapy in the form of polychemotherapy and radiotherapy. The five-year overall survival was 75%. No significant difference in survival was found between the three different tumour phenotypes, or for the tested antigens individually. Age at presentation and stage of disease had a significant influence on five-year overall survival. Survival rates were 90% for the patients <60 years of age and 40% for those ⩾60 years. Survival rates were 90% for stage I and 41% for stage IV. Conclusion: This study illustrates the homogeneity of PLB. The majority of cases are of the GC phenotype which has a favourable prognosis.

Detection of Parvovirus B19 Infection in First and Second Trimester Fetal Loss

Fetal and placental tissues and maternal sera from a series of 273 cases of first and second trimester fetal loss were collected to detect the frequency of parvovirus B19 infection. In addition, fetal tissues were studied for the presence of congenital anomalies. Serology of maternal sera, histology of fetal tissues and placenta, polymerase chain reaction (PCR), in situ hybridization (ISH), and immunohistochemistry (IHC) were used for the detection of parvovirus B19 infection. Sera were tested for B19-specific immunoglobulin M (IgM) and/or IgG using an enzyme-linked immunosorbent assay technique. Based on serology, 149 cases not related to B19 infection were excluded from further analysis. Two of the remaining 124 cases (0.7% of all 273 cases) had parvovirus B19-specific IgM and IgG at the time of abortion, indicating a recent maternal parvovirus B19 infection. In our histological examination, 10 cases contained nuclear vacuolization in fetal erythroid progenitor cells, either in fetal tissues (n = 2) or in placental tissue (n = 8). However, this vacuolization was considered a fixation artifact and not identical to parvovirus B19-specific nuclear inclusions described in previous reports. Only 1 of these 10 cases had parvovirus B19 DNA detectable in placental tissue by PCR analysis. Neither in this case nor in any of the other cases tested was parvovirus B19 DNA or protein detectable by ISH or IHC, respectively. In none of 41 cases in which fetal tissues were available were congenital anomalies found. In conclusion, the frequency of maternal parvovirus B19 infection in this series of fetal losses is low (0.8%). This low frequency does not allow any conclusions with regard to the occurrence of congenital anomalies resulting from parvovirus B19 infection and the usage of nuclear histology for the detection of fetal parvovirus B19 infection is considered a nonspecific parameter that requires confirmation by PCR.