The immune cell infiltrate in the microenvironment of vulvar Paget disease.

BACKGROUND: Non-invasive vulvar Paget disease (VPD) is a rare skin disorder mainly affecting elderly women. Recently, the immune modulator imiquimod was reported as an effective treatment option. Knowledge about the immune microenvironment of VPD is lacking. METHODS: This study investigates the basic characteristics of the immune infiltrate in VPD (n = 10); moreover the influence of imiquimod was studied (n = 6). Immunohistochemistry for CD4, CD8, CD14, CD20, CD56 and FoxP3 was performed. The infiltrates of VPD were compared to vulvar high-grade squamous cell intraepithelial lesions (HSIL) (n = 43), a HPV induced vulvar premalignancy with known response to imiquimod cream, and healthy controls (n = 30). Immune cell counts in samples taken before and after treatment were compared. RESULTS: The microenvironment in VPD differs from the healthy vulvar skin and vulvar HSIL. VPD is characterized by a decrease in immune cells in the epithelium and an abundant number of immune cells in the stroma, consisting predominantly of T cells. The intraepithelial CD8+/Foxp3+ ratio and number of CD56+ increased after imiquimod therapy, whereas the numbers of CD14+ cells decreased which may point to a treatment-induced type 1 immune response. CONCLUSIONS: The epithelium in VPD contains less immune cells, but a dense stromal immune infiltrate. Changes in immune cell counts after immune modulation in relation to clinical responses should be further investigated.

L1 cell adhesion molecule (L1CAM) is a strong predictor for locoregional recurrences in cervical cancer.

BACKGROUND: L1 cell adhesion molecule (L1CAM) has been shown to be a prognostic marker in various cancer types, and has been suggested to play a role in epithelial mesenchymal transition (EMT). Here, we determined the prognostic significance of L1CAM in cervical cancer and its association with vimentin expression on tumor cells, indicative of EMT. METHODS: Formalin-fixed, paraffin-embedded primary tumor samples from 372 cervical cancer patients were collected for immunohistochemical analysis of L1CAM expression. In 109 FFPE specimens, the percentage of vimentin expressing tumor cells was determined by flow cytometry. RESULTS: Positive L1CAM expression (≥10% of tumor cells) was associated with disease-free survival, validated using RNAseq TCGA data. L1CAM expression was independently associated with locoregional recurrence-free survival (hazard ratio 2.62, 95% CI 1.33 - 5.17, P = 0.006), and strongly associated with percentage of vimentin expressing tumor cells (P = 0.003). Expression of both L1CAM and vimentin indicated a subgroup with the highest risk of recurrence (hazard ratio 3.15, 95% CI 1.25 - 7.92, P = 0.015). CONCLUSION: L1CAM might be a promising new prognostic marker for locoregional recurrences in cervical cancer, and its association with vimentin expression suggests that L1CAM might affect tumor aggressiveness, possibly through EMT.

Stathmin is a highly sensitive and specific biomarker for vulvar high-grade squamous intraepithelial lesions.

AIMS: Differentiating between human papilloma virus-dependent vulvar low-grade and high-grade squamous intraepithelial lesions (LSILs and HSILs) remains difficult in selected cases. Stathmin, a protein involved in cell cycle progression, might be a useful additional marker for this differentiation. The aim of this study was to investigate the additional diagnostic value of stathmin expression in vulvar intraepithelial neoplastic (VIN) lesions. METHODS: Immunohistochemical analysis was used to evaluate stathmin, P16 and Ki67 expression in 91 samples, including LSILs (n=16), HSILs (n=50), differentiated VIN (dVIN; n=10), lichen sclerosis (LS; n=10) and normal vulvar tissue (n=5). RESULTS: Stathmin was expressed in more than one-third of the epithelium in all HSILs and in 20% of LSILs. P16 and Ki67 were expressed in more than one-third of the epithelium in 94% of HSILs and in 13% and 40% of LSILs, respectively. Stathmin was expressed in more than one-third of the epithelium in 10% of the dVIN and in none of the LS or normal lesions. P16 and Ki67 expression was not present in more than one-third of the epithelium in any of these lesions. The sensitivity of stathmin for differentiating between LSILs and HSILs was 100% compared with a sensitivity of 94% for both p16 and Ki67. The specificity of stathmin, p16 and Ki67 was 80%, 87% and 60%, respectively. CONCLUSIONS: Stathmin is a highly sensitive and specific biomarker for the diagnosis of vulvar HSIL. In addition to the more commonly used immunohistochemical markers p16 and Ki67, stathmin can be a useful diagnostic tool for identifying HSILs, especially in cases in which differentiating between LSIL and HSIL is difficult.

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.

Patterns of parametrial involvement in radical hysterectomy specimens of cervical cancer patients.

A tumor in the parametria, either continuous with or separate from the primary malignancy, is an unfavorable prognostic factor in cervical cancer. The incidence of a parametrial tumor localized in blood or lymph vessels, or in tissue, and the relationship of these involvement patterns with pathologic characteristics and prognosis were investigated. Seventy-nine of 763 surgically treated cervical cancer patients (10%) had a tumor in the parametria in hysterectomy specimens. The available patient material was reviewed to discriminate between continuous and discontinuous parametrial tumor growth. The involvement pattern for discontinuous growth was specified on the basis of immunohistochemical staining with different specific markers. Fifty percent of the parametrial tumor involvement found postoperatively was caused by continuous extension of the primary process into the parametria. In the other 50%, the parametrial tumor was separate from the primary process. In this discontinuous group, we found a frequent presence of tumor in the lymph nodes and/or lymph vessels (together 79%) and even a rare appearance of tumor in the blood vessels (14%). A tumor was further found in unspecified vessels in 2 patients (5%), and as isolated foci in 6 patients (14%). Fourteen patients (33%) had more than 1 involvement pattern. Positive pelvic lymph nodes were more frequent in the discontinuous group. The involvement pattern was no independent predictor of overall survival. Parametrial blood vessel involvement was related to the development of distant metastases. The majority (79%) of parametrial involvement in the discontinuous group is caused by lymphatic metastases. Parametrial blood vessel involvement might be an independent predictor for the development of distant metastasis.

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.

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.

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.

High numbers of tumour-infiltrating activated cytotoxic T lymphocytes, and frequent loss of HLA class I and II expression, are features of aggressive B cell lymphomas of the brain and testis.

Loss of both HLA class I and class II expression in B cell lymphomas is a mechanism of escape from a cytotoxic T lymphocyte (CTL) immune response and will therefore give a strong selective survival advantage in tumours expressing strong immunogenic antigens. We investigated loss of HLA expression using specific antibodies on tissue sections from 254 B cell lymphomas originating from nodal and different extranodal sites in relation to numbers of tumour-infiltrating T cells. Complete loss of HLA class I and II was observed in a minority of the nodal, stomach, and skin lymphomas but in the majority of the lymphomas originating from the testis and the CNS. Interestingly, relatively high percentages of activated CTLs were detected in both primary testicular and CNS lymphomas compared to lymphomas at other sites, with highest percentages in the testis (p < 0.0001). We conclude that loss of both HLA class I and II expression occurs very frequently in lymphomas originating from the testis and the CNS as compared to nodal and some other extranodal sites. The presence of high percentages of activated CTLs in the testicular and CNS lymphomas suggests that loss of HLA expression provides a strong growth advantage for lymphoma cells in these immune-privileged sites.

High-resolution multi-parameter DNA flow cytometry enables detection of tumour and stromal cell subpopulations in paraffin-embedded tissues.

The accuracy of DNA ploidy measurements of paraffin-embedded tissues is limited by the lack of resolution and the inability to identify the DNA diploid population unequivocally in bimodal DNA histograms. A multi-parameter DNA flow cytometric method has been developed that enables the simultaneous detection of neoplastic and stromal cells in samples from dewaxed 50 microm sections or 2 mm diameter punches of archival tissue blocks. The method combines heat pretreatment in sodium citrate buffer and subsequent enzymatic dissociation with a collagenase/dispase mixture. Cells were simultaneously stained for keratin (FITC), vimentin (R-PE), and DNA (PI) before flow cytometric analysis. The method was applied to 12 paraffin-embedded cervical carcinomas and four colorectal carcinomas. In all cervical cancers, distinct keratin-positive and vimentin-positive cell populations were observed. While the exclusive vimentin-positive cell fractions always yielded unimodal DNA content distributions, bimodal distributions were observed for the keratin-positive cell fractions in nine cervical carcinomas, whereas one cervical carcinoma showed three distinct G0G1 populations. Coefficients of variation of the G0G1 peaks ranged from 1.70% to 4.79%. Average background, aggregate, and debris values were 14.7% (vimentin-positive fraction) and 33.8% (keratin-positive fraction). Flow sorting confirmed that the exclusively vimentin-positive cell fractions represent different normal stromal and infiltrate cells that can serve as an internal ploidy reference enabling discrimination between DNA hypo-diploid and DNA hyper-diploid tumour cell subpopulations. The neoplastic origin of the keratin-vimentin co-expressing cells from two cervical carcinomas was confirmed by genotyping of flow-sorted samples revealing loss of heterozygosity (LOH) of 6p. This improved method obviates the need for fresh/frozen tumour tissue for high-resolution DNA ploidy measurements and enables the isolation of highly purified tumour subpopulations for subsequent genotyping.

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.