Heat-induced antigen retrieval for immunohistochemical reactions in routinely processed paraffin sections.

The development of heat-induced antigen (epitope) retrieval (HIER) technologies has led to dramatic improvements in our ability to detect antigens in formalin fixed, archival tissues. Paradoxically, wet heat treatment at temperatures greater than 95 degrees C in appropriate buffer solutions can reconstitute the antigenicity of many proteins that have been rendered nonreactive during the fixation and paraffin embedding process, which heretofore could only be identified in fresh or frozen tissues. The reason for this effect is unclear, but it has been suggested that the vigorous heat treatment partially reverses or disrupts the aldehyde cross-links occurring in proteins during formalin fixation and restores the original conformation of antigenic epitopes. The great success of antigen/epitope retrieval technologies further emphasizes the importance of preanalytical steps in immunohistochemistry. Over the past several years, since this technology was first reported, there have been numerous modifications to the original formulation. It is the purpose of this chapter to discuss the critical issues required for optimal HIER and to provide guidelines for the use of popular HIER buffers and heating devices used for routine immunohistochemical detection.

Collagen XVII/BP180 protein expression in squamous cell carcinoma of the skin detected with novel monoclonal antibodies in archived tissues using tissue microarrays and digital microscopy.

Collagen XVII/BP180, a hemidesmosomal adhesion protein, is lost during normal keratinocyte maturation; however, it may be reexpressed upon malignant transformation. In this work, highly sensitive monoclonal antibodies 6D1 and 9G2 were produced, characterized, and used for the detection of collagen XVII in a tissue microarray series of archived samples of nonmelanocytic epithelial neoplasias, including 5 verruca vulgaris, 14 seborrheic keratosis, 38 actinic keratosis, 38 basal cell carcinoma (BCC), 15 basosquamous carcinoma, 58 squamous cell carcinoma (SCC), and 9 normal skin. Digital microscopy and a new tissue microarray software linking image and patient data allowed easy and validated evaluation and quality archiving of stained samples. In normal skin and benign epidermal lesions, collagen XVII protein was restricted to basal keratinocytes. However, possibly as a sign of undifferentiated/transformed state, it was widely expressed in SCC showing elevated levels around invasive tumor fronts with some staining in tumor adjacent stroma, endothelium, and histiocytes. Collagen XVII immunostaining of atypical keratinocytes in most actinic/solar keratosis supports the view of their malignancy and common origin with SCC. Squamous component of basosquamous carcinoma showed moderate reaction, whereas islets of BCC were mainly negative reflecting the diverse genotype and phenotype, and pathogenesis of SCC and BCC. These results suggest that collagen XVII neoexpression may be associated with early atypia/malignant transformation of keratinocytes. Further investigations are under way to analyze the potential of these antibodies for tracing progression and metastatic potential of skin tumors.

Epigenetic changes and suppression of the nuclear factor of activated T cell 1 (NFATC1) promoter in human lymphomas with defects in immunoreceptor signaling.

The nuclear factor of activated T cell 1 (Nfatc1) locus is a common insertion site for murine tumorigenic retroviruses, suggesting a role of transcription factor NFATc1 in lymphomagenesis. Although NFATc1 is expressed in most human primary lymphocytes and mature human T- and B-cell neoplasms, we show by histochemical stainings that NFATc1 expression is suppressed in anaplastic large cell lymphomas and classical Hodgkin's lymphomas (HLs). In HL cell lines, NFATc1 silencing correlated with a decrease in histone H3 acetylation, H3-K4 trimethylation, and Sp1 factor binding but with an increase in HP1 binding to the NFATC1 P1 promoter. Together with DNA hypermethylation of the NFATC1 P1 promoter, which we detected in all anaplastic large cell lymphoma and many HL lines, these observations reflect typical signs of transcriptional silencing. In several lymphoma lines, methylation of NFATC1 promoter DNA resulted in a "window of hypomethylation," which is flanked by Sp1-binding sites. Together with the under-representation of Sp1 at the NFATC1 P1 promoter in HL cells, this suggests that Sp1 factors can protect P1 DNA methylation in a directional manner. Blocking immunoreceptor signaling led to NFATC1 P1 promoter silencing and to a decrease in H3 acetylation and H3-K4 methylation but not DNA methylation. This shows that histone modifications precede the DNA methylation in NFATC1 promoter silencing.

Immunophenotypic profiling of nonsmall cell lung cancer progression using the tissue microarray approach.

This study, using tissue microarrays, aimed at the immunomorphologic profiling of nonsmall cell lung cancer (NSCLC) cases to reveal clinically relevant disease groups and biomarkers associated with patients' survival and tumor progression including brain metastatic potential. Donor tissue blocks were form 59 patients, including 33 primary tumors without distant metastasis and 26 brain metastatic primary tumors as well as the brain metastases. Sections were immunostained for 29 markers targeting molecules of cell adhesion, cell growth, cell cycle, and apoptosis regulation. beta-Catenin expression was the only independent prognostic marker associated with better outcome. Elevated expression of collagen XVII, CD44v6, and caspase-9, and the reduced production of beta-catenin and cellular apoptosis susceptibility protein were significantly associated with the metastatic potential of primary NSCLC. Expression of positive cell cycle regulators cyclin D1 and cyclin D3 was also increased in metastatic primary tumors. Metastatic tumor progression into the brain was accompanied by prominent p16, syndecan-1, p53 (DO7), and caspase-3 protein levels. Hierarchical clustering of complex immunoprofiles based on the differentially expressed markers grouped NSCLCs of the poorest outcome with high correlation including 2/3 of brain metastases of mixed histology. The brain metastatic potential of NSCLCs may be linked to the elevated levels of cyclinD1, cyclinD3, p16, p53, caspase-3, caspase-9, CD44v6, and collagen XVII and the down-regulation of beta-catenin and cellular apoptosis susceptibility protein. Unsupervised immunoprofiles based on differentially expressed biomarkers may help selecting lung cancers with aggressive behavior.

Mitosis-specific promoter of the alfalfa cyclin-dependent kinase gene (Medsa;CDKB2;1) is activated by wounding and ethylene in a non-cell division-dependent manner.

Cyclin-dependent serine/threonine kinases (CDKs) have pivotal roles in regulating the eukaryotic cell cycle. Plants possess a unique class of CDKs (B-type CDKs) with preferential protein accumulation at G2/M-phases; however, their exact functions are still enigmatic. Here we describe the functional characterization of a 360-bp promoter region of the alfalfa (Medicago sativa) CDKB2;1 gene in transgenic plants and cell lines. It is shown that the activity of the analyzed promoter was characteristic for proliferating meristematic regions in planta and specific for cells in the G2/M-phases in synchronized cell cultures. Immunohistochemical analysis of transgenic root sections further confirmed the correlation of the expression of the CDKB2;1 promoter-linked reporter genes with the accumulation of the correspondent kinase. It was found that, in addition to auxin (2,4-dichlorophenoxyacetic acid) treatment, wounding could also induce both the reporter and endogenous genes in transgenic leaf explants. Furthermore, ethylene, known as a wound-response mediator, had a similar effect. The gene activation in response to wounding or ethephon was faster and occurred without the induction of cell cycle progression in contrast to the control auxin treatment. In silico analysis of this promoter indeed revealed the presence of a set of cis-elements, indicating not only cell cycle- but wound- and ethylene-dependent regulation of this CDK gene. Based on the presented data, we discuss the functional significance of the complex regulation of mitosis-specific CDK genes in plants.

Familial clustering of nasopharyngeal carcinoma in a non-endemic geographical region. Report of two Hungarian cases and a review of the literature.

The aim of this study was to investigate the familial clustering of nasopharyngeal carcinoma (NPC) in a non-endemic geographical region on the basis of two case reports and a review of the literature. Following an upper respiratory infection, NPC (WHO type III) was detected in a 57-year-old female (Case 1) who presented with nasal symptoms and a year later in her 36-year-old son (Case 2) who presented with enlarged lymph nodes. After a full diagnostic work-up, cT2a cN0 cM0 (stage IIA; Case 1) and cT2a cN2 cM0 (stage III; Case 2) disease were identified, and telecobalt irradiation was administered to both patients. The mother achieved complete remission and has been disease-free during a 14-year follow-up period. After initial complete remission, the son experienced regional (cervical) and base of the skull relapses within 2 years, which were treated unsuccessfully by means of radical neck dissection, a second course of radiotherapy and chemotherapy. Epstein-Barr virus (EBV) was detected in pathology sections from both patients. The authors review 20 additional well-documented cases of familial clustering of NPC in non-endemic geographical regions from the English language literature. This clinical entity typically has WHO type III histology; it may occur following an upper respiratory tract infection, and EBV-related serological titers were elevated in all 20 investigated cases. No consequent promoting factors were identified. The present two cases and the review of the literature strongly suggest that familial clustering of NPC in non-endemic geographical areas may be related to EBV infections. The difference in outcome of our two cases may be explained by the fact that the disease in Case 2 was diagnosed 1 year later than that in Case 1 and hence at a more advanced stage.

A tumor-suppressor function for NFATc3 in T-cell lymphomagenesis by murine leukemia virus.

Nuclear factor of activated T cell (NFAT) transcription factors play a central role in differentiation, activation, and elimination of lymphocytes. We here report on the finding of provirus integration into the Nfatc3 locus in T-cell lymphomas induced by the murine lymphomagenic retrovirus SL3-3 and show that NFATc3 expression is repressed in these lymphomas. The provirus insertions are positioned close to the Nfatc3 promoter or a putative polyadenylated RNA (polyA) region. Furthermore, we demonstrate that NFATc3-deficient mice infected with SL3-3 develop T-cell lymphomas faster and with higher frequencies than wild-type mice or NFATc2-deficient mice. These results identify NFATc3 as a tumor suppressor for the development of murine T-cell lymphomas induced by the retrovirus SL3-3.

Transient upregulation of connexin43 gap junctions and synchronized cell cycle control precede myoblast fusion in regenerating skeletal muscle in vivo.

The spatio-temporal expression of gap junction connexins (Cx) was investigated and correlated with the progression of cell cycle control in regenerating soleus muscle of Wistar rats. Notexin caused a selective myonecrosis followed by the complete recapitulation of muscle differentiation in vivo, including the activation, commitment, proliferation, differentiation and fusion of myogenic cells. In regenerating skeletal muscle, only Cx43 protein, out of Cx-s 26, -32, -37, -40, -43 and -45, was detected in desmin positive cells. Early expression of Cx43 in the proliferating single myogenic progenitors was followed by a progressive upregulation in interacting myoblasts until syncytial fusion, and then by a rapid decline in multinucleate myotubes. The significant upregulation of Cx43 gap junctions in aligned myoblasts preceding fusion was accompanied by the widespread nuclear expression of cyclin-dependent kinase inhibitors p21(waf1/Cip1) and p27(kip1) and the complete loss of Ki67 protein. The synchronized exit of myoblasts from the cell cycle following extensive gap junction formation suggests a role for Cx43 channels in the regulation of cell cycle control. The potential of Cx43 channels to stimulate p21(waf1/Cip1) and p27(kip1) is known. In the muscle, proving the involvement of Cx43 in either a direct or a bystander cell cycle regulation requires functional investigations.

Low level of cyclin D1 protein expression in thyroid microcarcinomas from an autopsy series.

In a recent epidemiological screening study in an autopsy series, we found a high prevalence of microcarcinomas (MCs) (21/443 = 4.74%). We found no iodine intake-, gender-, or age-dependent differences in the prevalence of MCs. The results suggest a different and benign behavior of MCs compared to clinical cancer. The role of cyclin D1 overexpression in the pathogenesis of thyroid tumors is not known clearly; however, overexpression of this protein was reported in well-differentiated papillary cancers and in incidentally found metastasizing MCs. To date, cyclin D1 expression has not been investigated in autopsy-derived thyroid MCs. Eight MCs were available for immunostaining and comparison with 15 clinically detected papillary thyroid cancers. Fourteen out of 15 clinical carcinomas expressed cyclin D1 (93.3%), while in the MCs this ratio was 1 out of 8 (12.5%) (p = 0.0001). The only cyclin D1-positive MC was multifocal (both lobes of the gland were affected). We concluded that the benign behavior of most autopsy-derived MCs may be associated with the lack of cyclin D1 overexpression.