Frank Smedts

A- and B-type lamins are differentially expressed in normal human tissues

Abstract A selection of normal human tissues was investigated for the presence of lamins B1, B2, and A-type lamins, using a panel of antibodies specific for the individual lamin subtypes. By use of immunoprecipitation and two-dimensional immunoblotting techniques we demonstrated that these antibodies do not cross-react with other lamin subtypes and that a range of different phosphorylation isoforms is recognized by each antibody. The lamin B2 antibodies appeared to decorate the nuclear lamina in all tissues examined, except hepatocytes, in which very little lamin B2 expression was observed. In contrast to previous studies, which suggested the ubiquitous expression of lamin B1 in mammalian tissues, we show that lamin B1 is not as universally distributed throughout normal human tissues as was to be expected from previous studies. Muscle and connective tissues are negative, while in epithelial cells lamin B1 seemed to be preferentially detected in proliferating cells. These results correspond well with those obtained for lamin B1 in chicken tissues. The expression of A-type lamins is most prominent in well-differentiated epithelial cells. Relatively undifferentiated and proliferating cells in epithelia showed a clearly reduced expression of A-type lamins. Furthermore, most cells of neuroendocrine origin as well as most hematopoietic cells were negative for A-type lamin antibodies.

Keratins 7 and 20 as diagnostic markers of carcinomas metastatic to the ovary

The most common carcinomas metastatic to the ovary that mimic ovarian primaries are colonic adenocarcinomas and endometrial carcinomas. Conventional histochemical staining procedures, even in combination with additional immunohistochemical assays, are of limited value in distinguishing between these metastases and primary ovarian carcinomas. In this study we investigated whether the application of monoclonal antibodies against keratins 7, 8, and 20 could help in differentiating between these categories. The reactivity patterns of 40 carcinomas metastatic to the ovary were compared with those of their primary carcinomas on the one hand and with various primary ovarian carcinomas and mesotheliomas on the other. Colon cancer metastatic to the ovary was keratin 7 negative and keratin 20 positive in 94% of the cases; in contrast, all primary ovarian carcinomas were keratin 7 positive and keratin 20 negative, with the exception of two cases of mucinous cystadenocarcinoma. Ovarian metastases of gastric cancer usually contained keratins 7 and 20. Metastases of endometrial cancer to the ovary and primary ovarian carcinomas usually showed similar keratin expression. We propose that keratin 7 and 20 antibodies may be of help to distinguish between primary ovarian carcinomas and carcinoma metastases in the ovary.

Transition of high-grade cervical intraepithelial neoplasia to micro-invasive carcinoma is characterized by integration of HPV 16/18 and numerical chromosome abnormalities.

Cervical intraepithelial neoplasia (CIN I, II, and III) and cases of CIN III associated with micro‐invasive cervical carcinoma (CIN III & mCA) were analysed for evidence of episomal or integrated human papillomavirus (HPV) 16/18 DNA by fluorescence in situ hybridization (FISH). In parallel, numerical aberrations of chromosomes 1, 17, and X were determined in these lesions as indicators of genomic instability. HPV 16/18 DNA was present in 2 of 12 CIN I, 19 of 23 CIN II/III, and 10 of 12 CIN III & mCA. None of the CIN I and only two of the 19 HPV 16/18‐positive solitary CIN II/III showed an integrated HPV pattern. However, all ten cases of HPV‐positive CIN III & mCA showed this pattern. Transition of CIN II/III to CIN III & mCA therefore correlates strongly with viral integration (p < 0.001). Chromosomal aberrations were detected in 23 of 31 HPV 16/18‐positive lesions (14 solitary CIN I–III and nine CIN III & mCA) and 5 of 16 HPV‐negative lesions. Nine of 21 HPV 16/18‐positive solitary CIN I–III showed tetrasomy for all chromosomes tested, while trisomies for a single chromosome were seen in a further five of these HPV‐positive lesions. In eight of ten HPV‐positive CIN III & mCA, predominantly aneusomies and/or polysomies were detected. A significant correlation (p < 0.02) was found between the chromosome copy number and the physical status of HPV, indicating that in its episomal form HPV induces genomic changes such as tetrasomies and single trisomies, while HPV integration correlates with aneusomies and polysomies, predominantly detected in CIN III & mCA. These data indicate that integration of HPV 16/18 DNA is a pivotal step in the transition of CIN to micro‐invasive carcinoma. Copyright

The Vascular Endothelial Growth Factor Receptor Inhibitor Sunitinib Causes a Preeclampsia-Like Syndrome With Activation of the Endothelin System

Angiogenesis inhibition is an established treatment for several tumor types. Unfortunately, this therapy is associated with adverse effects, including hypertension and renal toxicity, referred to as “preeclampsia.” Recently, we demonstrated in patients and in rats that the multitarget tyrosine kinase inhibitor sunitinib induces a rise in blood pressure (BP), renal dysfunction, and proteinuria associated with activation of the endothelin system. In the current study we investigated the effects of sunitinib on rat renal histology, including the resemblance with preeclampsia, as well as the roles of endothelin 1, decreased nitric oxide (NO) bioavailability, and increased oxidative stress in the development of sunitinib-induced hypertension and renal toxicity. In rats on sunitinib, light and electron microscopic examination revealed marked glomerular endotheliosis, a characteristic histological feature of preeclampsia, which was partly reversible after sunitinib discontinuation. The histological abnormalities were accompanied by an increase in urinary excretion of endothelin 1 and diminished NO metabolite excretion. In rats on sunitinib alone, BP increased (&Dgr;BP: 31.6±0.9 mm Hg). This rise could largely be prevented with the endothelin receptor antagonist macitentan (&Dgr;BP: 12.3±1.5 mm Hg) and only mildly with Tempol, a superoxide dismutase mimetic (&Dgr;BP: 25.9±2.3 mm Hg). Both compounds could not prevent the sunitinib-induced rise in serum creatinine or renal histological abnormalities and had no effect on urine nitrates but decreased proteinuria and urinary endothelin 1 excretion. Our findings indicate that both the endothelin system and oxidative stress play important roles in the development of sunitinib-induced proteinuria and that the endothelin system rather than oxidative stress is important for the development of sunitinib-induced hypertension.

Identification of intermediate cell types by keratin expression in the developing human prostate

The secretory acini of the adult human prostate contain basal, luminal, and intermediate types of exocrine cells. Intermediate cells are thought to play an important role in normal growth and neoplastic transformation. In this study we investigated whether this cell type is present in early stages of prostate development, using keratin antibodies specific for them.

HPV in situ hybridization: impact of different protocols on the detection of integrated HPV.

Although there is consensus that HPV integration is common in invasive cervical carcinomas and uncommon or absent in low‐grade uterine cervical intraepithelial neoplasia (CIN I), estimates for HPV integration in CIN II/III range from 5 to 100% using different PCR‐based and in situ hybridization (ISH) approaches. It has been suggested that HPV integration can be identified using ISH by scoring of punctate signals. The increased sensitivity of fluorescence ISH (FISH) methods, allowing the detection of single copies of HPV, complicates the distinction between integrated and episomal HPV. Recently it has been suggested that, in such assays, the signals originating from integrated virus can be hidden in a background of episomal HPV. We therefore compared 2 different FISH protocols for the detection of integrated HPV in a series of CIN II/III lesions: 1) a mild protocol in which episomal HPV and RNA is retained and 2) a harsh protocol that extensively extracts proteins and RNA, and which promotes the partial loss of episomal HPV but not integrated HPV. A series of 28 HPV 16/18 positive CIN II/III lesions (17 solitary lesions and 11 lesions adjacent to microinvasive carcinoma) were studied. A punctate signal pattern was identified in 7 of these lesions with both protocols. Punctate signal was also present in control samples from lesions that are known to be associated with HPV integration (invasive squamous cell carcinoma (n = 3), adenocarcinoma in situ (n = 3), and invasive adenocarcinoma (n = 1). HPV RNA contributed significantly to the intensity of punctate FISH signal, especially when applying the mild protocol, as shown by omitting DNA denaturation, including RNase pretreatment steps and measuring the fluorescence signal intensity. Also, HPV RNA was frequently detected in addition to episomal/integrated HPV DNA in the majority of the other 21 CIN II/III lesions; this resulted in intense granular/diffuse FISH signals throughout the epithelium. However, in 7 of these lesions, the harsh protocol gave a more consistent punctate pattern in cells throughout the full thickness of the epithelium. This supports the hypothesis that the harsh protocol unmasks integrated HPV more efficiently by extracting RNA and episomal HPV. Overall, with this harsh protocol, a clonally expanded population of cells containing punctate HPV signals was found in 5 of 17 (29%) solitary CIN II/III lesions and in 9 of 11 (88%) CIN II/III lesions associated with microinvasive carcinoma. Combining these data with the results from our previous study, with the harsh protocol in 7 of 40 (18%) solitary CIN II/III lesions and 19/21 (90%) CIN II/III lesions associated with microinvasive carcinoma (p < 0.001), this pattern was found. This indicates that, when robustly defined, a punctate HPV pattern in CIN II/III lesions is associated with the presence of an invasive carcinoma.

BCL‐2 IMMUNOREACTIVITY INCREASES WITH SEVERITY OF CIN: A STUDY OF NORMAL CERVICAL EPITHELIA, CIN, AND CERVICAL CARCINOMA

The presence of the BCL‐2 protein, a marker for inhibition of programmed cell death, was studied in a series of routinely processed cervical tissues, consisting of normal endocervical (n=40) and ectocervical epithelium (n=27), squamous metaplastic epithelium (n=30), CIN (cervical intraepithelial neoplasia) lesions (n=32), and cervical carcinomas (n=13). BCL‐2 was strongly expressed in the basal cell compartment of normal ectocervical squamous epithelium and in nearly all reserve cells, while in endocervical columnar cells it was moderately expressed. In immature squamous metaplastic epithelium, BCL‐2 expression varied. Half of the cases showed only basal cell staining, while the other half showed staining also in suprabasal layers. BCL‐2 could be detected in all premalignant lesions, showing a striking increase in the number of positive cells with increasing severity of CIN, in combination with a mild increase in staining intensity. All adenocarcinomas were positive (n=5), while five of eight squamous cell carcinomas expressed BCL‐2. Based on these results, it is hypothesized that both the larger number of cells staining with BCL‐2 in higher grades of CIN and the increase in staining intensity imply an increasing protection of these neoplastic conditions against programmed cell death. This protection facilitates not only continuing proliferation, but also the induction of genetic instability in dysplastic epithelial cells; it may thus reflect the greater capacity of the more severe CIN lesions to evolve into cervical carcinoma.

Histological assessment of PAXgene tissue fixation and stabilization reagents.

Within SPIDIA, an EC FP7 project aimed to improve pre analytic procedures, the PAXgene Tissue System (PAXgene), was designed to improve tissue quality for parallel molecular and morphological analysis. Within the SPIDIA project promising results were found in both genomic and proteomic experiments with PAXgene-fixed and paraffin embedded tissue derived biomolecules. But, for this technology to be accepted for use in both clinical and basic research, it is essential that its adequacy for preserving morphology and antigenicity is validated relative to formalin fixation. It is our aim to assess the suitability of PAXgene tissue fixation for (immuno)histological methods. Normal human tissue specimens (n = 70) were collected and divided into equal parts for fixation either with formalin or PAXgene. Sections of the obtained paraffin-embedded tissue were cut and stained. Morphological aspects of PAXgene-fixed tissue were described and also scored relative to formalin-fixed tissue. Performance of PAXgene-fixed tissue in immunohistochemical and in situ hybridization assays was also assessed relative to the corresponding formalin-fixed tissues. Morphology of PAXgene-fixed paraffin embedded tissue was well preserved and deemed adequate for diagnostics in most cases. Some antigens in PAXgene-fixed and paraffin embedded sections were detectable without the need for antigen retrieval, while others were detected using standard, formalin fixation based, immunohistochemistry protocols. Comparable results were obtained with in situ hybridization and histochemical stains. Basically all assessed histological techniques were found to be applicable to PAXgene-fixed and paraffin embedded tissue. In general results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue. Compromises made in morphology can be called minor compared to the advantages in the molecular pathology possibilities.

Cell kinetics of prostate exocrine and neuroendocrine epithelium and their differential interrelationship: New perspectives

The prostate gland consists of a complex ductal system lined with exocrine basal and luminal cells, and neuroendocrine epithelial cells. This paper reviews the histologic and molecular cell biologic characteristics of these cells, in normal adult tissue, during prostate morphogenesis, and in the development of benign and malignant neoplastic conditions. Expression of differentiation markers, as well as proliferation and apoptosis markers, growth factors and associated receptors, and abnormalities in genes and chromosomes are reviewed. Accumulating data indicate that (1) pluripotent immortal stem cells are located in the basal cell compartment of the prostate; (2) there is a subpopulation of epithelial cells in the prostate gland (intermediate cells) that have both structural and functional characteristics common to basal and luminal cells, which may be identified in various conditions; and prostate NE cells may have the same common origin as other exocrine cells, and share the same differentiation pathway. A stem cell model is proposed in which both exocrine and endocrine cells are derived from a subpopulation of basal cells (stem cell) that give rise to luminal cells through intermediate cells (pluripotent amplifying cells). These cells are also probably highly implicated in the early development of prostate benign and malignant neoplasia. Prostate Supplement 8:62–73, 1998.

Cytokeratins in smooth muscle cells and smooth muscle tumours

A keratin positive metastatic leiomyosarcoma in the lung, which resulted in diagnostic error, is reported. The results of additional studies of 17 benign and malignant leiomyogenic tumours with various keratin antibodies are presented and discussed in the light of recent bibliographical data.