Keratin-positive Cells Research Articles

UHRF1 Immunohistochemical Staining Separates Benign Reactive Spindle Cell Mesothelial Proliferations From Sarcomatoid Mesotheliomas.

The separation of benign from malignant mesothelial proliferations is often a difficult pathologic problem. UHRF1 (ubiquitin-like with plant homeodomain and ring finger domains-1) is a regulator of DNA methylation and an epigenetic driver of various human cancers. It has recently been reported that UHRF1 is overexpressed in mesotheliomas. We asked whether UHRF1 immunohistochemistry could be used to separate benign from malignant mesothelial proliferations. Initial studies showed that UHRF1 stained mesothelial cells but also endothelial and other non-neoplastic cells, so that accurate counting of positive mesothelial cells was difficult. Therefore, we ran dual UHRF1-AE1/AE3 stains on 2 tissue microarrays containing 40 reactive mesothelial proliferations and 61 mesotheliomas and only counted UHRF1 staining in keratin-positive cells. On average 10.3±8.6% (mean±SD; range: 0% to 36, median: 6.8%) of epithelioid mesothelioma cells stained compared with 5.3±4.8% (range: 0% to 15%, median: 4.1%) of reactive epithelial mesothelial cells. This difference was statistically significant but there was too much overlap to use diagnostically. In contrast, 37±26% (range: 2.5% to 95%, median: 31%) of cells in sarcomatoid mesotheliomas compared with 1.2±1.2% (range: 0% to 3.0%, median: 1.0%) of cells in reactive spindle cell mesothelial proliferations stained. To confirm this difference we stained whole sections of 21 sarcomatoid mesotheliomas and 19 cases of organizing pleuritis. Staining of mesothelial cells was seen in 2.1±2.4% (range: 0% to 6.8%, median: 1.0%) of organizing pleuritis cases and 44±22% (range: 14% to 90%, median: 41%) of sarcomatoid mesotheliomas. We conclude that dual UHRF1-AE1/AE3 immunohistochemistry is very useful for separating benign spindle cell mesothelial proliferations from sarcomatoid mesotheliomas.

S1725 Case Report: Prostate Cancer Diagnosed on Rectosigmoid Biopsies in a Patient Presenting With Diarrhea

INTRODUCTION: Prostate cancer is the second most commonly diagnosed cancer in men worldwide. Despite the anatomic proximity, rectosigmoid involvement of prostate adenocarcinoma is rare. We present a case of metastatic prostate cancer diagnosed on rectosigmoid biopsies in a patient presenting with diarrhea and fecal urgency. CASE DESCRIPTION/METHODS: A 76-year-old male presented to clinic for evaluation of non-bloody diarrhea with associated fecal urgency. His medical history was significant for prostate adenocarcinoma diagnosed and treated nine years ago with prostatectomy, radiation, and androgen deprivation therapy (ADT). On current presentation, labs showed a normal complete blood cell count, TSH, and tissue transglutaminase IgA. Stool studies were negative for gastrointestinal pathogens. A colonoscopy was performed which demonstrated a normal terminal ileum, sigmoid diverticulosis, and mild radiation proctitis. Random colon biopsies showed changes consistent with collagenous colitis. Treatment for collagenous colitis was initiated, however, three months later the patient continued to have diarrhea as well as new onset abdominal pain, nausea, vomiting, and weight loss. Imaging showed sigmoid colon wall thickening, an ill-defined soft tissue mass in the pelvis, and lymphadenopathy. Flexible sigmoidoscopy revealed congested and erythematous mucosa beginning at 10cm from the anal verge with multiple clean based ulcerations. Targeted biopsies revealed a population of keratin positive cells with limited differentiation and positive PSA staining consistent with poorly differentiated adenocarcinoma of prostate origin, likely from lymphangitic spread. The patient was diagnosed with metastatic castrate-resistant prostate adenocarcinoma and started on ADT. DISCUSSION: Rectosigmoid involvement by prostate adenocarcinoma is rare and review of available literature suggests at least three potential routes of involvement including direct invasion through Denonvilliers’ fascia, lymphatic metastasis, and implantation after transrectal biopsy. Rectosigmoid involvement of prostate adenocarcinoma may present with symptoms including change in bowel habits, rectal urgency, lower gastrointestinal bleeding, and pelvic pain. The case presented here emphasizes the importance of including prostate adenocarcinoma on the differential diagnosis in men presenting with lower gastrointestinal symptoms and abnormal rectosigmoid mucosa on colonoscopy, especially in patients older than 65 years or with a history of prostate adenocarcinoma.Figure 1.: Congested and erythematous mucosa starting approximately 10 cm from the anal verge.Figure 2.: Rectosigmoid ulcerations with surrounding erythematous mucosa.Figure 3.: Ill-defined soft tissue mass in the peri-rectal region with lymphadenopathy.

Histopathology and enhanced detection of tumor invasion of peritoneal membranes.

Tumor invasion of the peritoneal membrane may have an adverse prognostic significance, but its histopathologic features can be diagnostically difficult to recognize. We observed that local peritoneal injury associated with tumor invasion is characterized by activation and proliferation of serosal stromal cells that express cytokeratin, a characteristic property of injured serosal membranes that may have diagnostic utility. To explore this, we examined 120 primary tumors of the gastrointestinal tract and pancreaticobiliary system using cytokeratin and elastic stains to assess for tumor invasion of peritoneal membranes. Peritoneal invasion by tumor was associated with retraction, splaying, and destruction of the elastic lamina and proliferation of keratin-expressing stromal cells of serosal membranes. All 82 peritoneal invasive tumors were characterized by neoplastic cells that invaded the elastic lamina and the serosal connective tissue with neoplastic cells that abutted or were surrounded by keratin-positive stromal cells, whereas all 38 tumors limited to the subserosa showed none of these features. The diagnosis of tumor invasion of peritoneal membranes is enhanced by the combined use of cytokeratin and elastic stains, which in turn would enable better histopathologic correlation with patient treatment and outcome.

Abstract 2421: Circulating tumor cells reveal the genetic evolution of metastatic breast cancer

Abstract BACKGROUND. Anti-cancer treatment failure is caused by the genetic and phenotypic heterogeneous properties of the disease. Early dissemination of circulating tumor cells (CTCs) into the blood circulation of cancer patients allows for parallel genetic evolution of the primary tumor and metastases. This study investigated the genomic make-up of CTCs originating from metastasis as a so called “liquid biopsy” and compared these with the different clones of the archived autologous primary tumor on single cell level. METHODS. From two breast cancer patients, individual CTCs were isolated from blood using Ficoll density gradient followed by micromanipulation of keratin positive cells. Single cells from archived primary breast tumors from the same patients were captured by laser microdissection. DNA was isolated and amplified by whole genome amplification and copy number alterations (CNA) were obtained by shallow, whole genome next generations sequencing (NGS). RESULTS. From the first breast cancer patient, the genomes from 50 single cells from the primary tumor were sequenced. An unsupervised phylogenetic cluster analysis based on CNA revealed five distinct subclones that displayed increasing chromosomal instability from the first to the last cluster. Using support vector machine (SVM) learning, the CNA profiles of 42 CTCs were residing mostly to the first three clones with few chromosomal aberrations, whereas only one CTC resided to the fourth clone and none to the last with many chromosomal aberrations. The tumor from a second breast cancer patient displayed the presence of three genetically distinct clones with increasing chromosomal instability after sequencing 11 single cells. CTCs (n = 12) from this patient at metastatic disease were classified to the last branch using SVM, however with low probability. After repeating unsupervised clustering on the genomes of the primary tumor tissue and CTCs, a fourth branch was formed with CTCs only. CONCLUSION. Our results suggest that therapy resistant metastases in breast cancer patients can originate from tumor clones from early stages of tumor evolution and may genetically still be similar (patient 1). On the other hand, further genetic progression may also take place (patient 2) after which the genetic landscape of the metastasis does not resemble the primary tumor anymore. These results underline the importance of “liquid biopsy” in the diagnosis of metastatic cancer. Citation Format: Simon A. Joosse, Anna Babayan, Katharina Prieske, Daniela Indenbirken, Malik Alawi, Eike C. Burandt, Adam Grundhoff, Volkmar Müller, Klaus Pantel. Circulating tumor cells reveal the genetic evolution of metastatic breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2421.

Decreased expression of thymus-specific proteasome subunit β5t in Down syndrome patients.

The majority of patients with Down syndrome (DS), trisomy 21, have morphologically abnormal thymuses and present with intrinsic immunological abnormalities affecting mainly the cellular immune response. The aim of this study was to examine whether the expression of functionally important molecules is altered in thymic stromal cells in patients with DS. We analysed thymic tissues from patients with trisomy 13 (n = 4), trisomy 18 (n = 14) and trisomy 21 (n = 13) for histological alterations, and for the expression of functionally important molecules such as β5t, a thymoproteasome subunit, and cathepsins L and S. In patients with trisomy 13 and trisomy 18, the thymus was morphologically normal or showed only mild depletion of cortical thymocytes. In contrast, the thymus showed variable histological changes in patients with trisomy 21; six of 13 cases showed severe depletion of thymocytes accompanied by the disappearance of thymic lobular architecture. In such thymuses, spindle-shaped keratin-positive cells were densely distributed, and expression of β5t, but not of cathepsin L, was markedly decreased. The present study suggests that abnormal thymic architecture and decreased expression of functionally important molecules in thymic stromal cells may be involved in immunological abnormalities in DS patients.

Cytokeratins in gastroenterology. Systematic review.

Keratins are proteins that form intermediate filaments of epithelial cell cytoskeleton. The utility of keratin expression determination is based on the fact that epithelial cells acquire a specific pattern of keratin expression during differentiation and maturation, which reflects the specificity of the tissue and the degree of maturation, and generally remains stable during carcinogenesis. Determination of the pattern makes it possible to identify the origin of cells in diagnosing neoplastic lesions as well as in research on pathophysiology or the possibility to apply keratin-positive cell detection in the process of cancer staging and treatment planning. As keratins undergo degradation during apoptosis as caspase substrate the identification of the caspase-derived K18 fragment by the use of specific monoclonal antibody allows us to estimate the apoptosis/necrosis ratio, especially in liver pathology, e.g. nonalcoholic steatohepatitis, chronic hepatitis or graft-versus-host disease or in assessing response to antiviral or antitumour therapy.

The Society of Biomolecular Imaging and Informatics First Annual Conference

The Society of Biomolecular Imaging and Informatics First Annual Conference

著明な貧血と血小板減少と末梢血の異常細胞で発見された転移性乳癌の１例

Worldwide, breast cancer accounts for 22.9% of all cancers excluding non-melanoma skin cancers in women. More than 80% of breast cancer cases are discovered when a woman discovers a lump that feels different from the rest of the breast tissue. We report the case of a 60-year-old woman who had been treated for one year at a psychiatric hospital for suspected schizophrenia, and was referred to our hospital on March 23, 2011 for consultation regarding complete blood count abnormalities: hemoglobin, 4.8g/dl ; Plt, 1.9×104/μl ; WBC, 12,700/μl with 18.5% abnormal cells. Computed tomography revealed an enhanced, irregular mass in her right breast as well as right axillary lymph node swelling, suggestive of breast cancer. BM biopsy confirmed the presence of abnormal, keratin-positive cells, suggesting metastasis to the bone. CA15-3 was 300U/ml (normal range ＜25). The patient died on April 11, 2011, post-admission day 20. We found a rare case of terminal breast cancer that presented with peripheral blood abnormality. Breast cancer in patients who do not notice breast abnormality can present with peripheral blood abnormality.

Subcutaneous preconditioning increases invasion and metastatic dissemination in colorectal cancer models

Mouse colorectal cancer (CRC) models generated by orthotopic microinjection of human CRC cell lines reproduce the pattern of lymphatic, haematological and transcoelomic spread but generate low metastatic efficiency. Our aim was to develop a new strategy that could increase the metastatic efficiency of these models. We used subcutaneous implantation of the human CRC cell lines HCT116 or SW48 prior to their orthotopic microinjection in the cecum of nude mice (SC+ORT). This subcutaneous preconditioning significantly enhanced metastatic dissemination. In the HCT116 model it increased the number and size of metastatic foci in lymph nodes, lung, liver and peritoneum, whereas, in the SW48 model, it induced a shift from non-metastatic to metastatic. In both models the number of apoptotic bodies in the primary tumour in the SC+ORT group was significantly reduced compared with that in the direct orthotopic injection (ORT) group. Moreover, in HCT116 tumours the number of keratin-positive tumour buddings and single epithelial cells increased at the invasion front in SC+ORT mice. In the SW48 tumour model, we observed a trend towards a higher number of tumour buds and single cells in the SC+ORT group but this did not reach statistical significance. At a molecular level, the enhanced metastatic efficiency observed in the HCT116 SC+ORT model was associated with an increase in AKT activation, VEGF-A overexpression and downregulation of β1 integrin in primary tumour tissue, whereas, in SW48 SC+ORT mice, the level of expression of these proteins remained unchanged. In summary, subcutaneous preconditioning increased the metastatic dissemination of both orthotopic CRC models by increasing tumour cell survival and invasion at the tumour invasion front. This approach could be useful to simultaneously study the mechanisms of metastases and to evaluate anti-metastatic drugs against CRC.

Localization of Estrogen Receptor in the Central Lymphoid Organs of Chickens during the Late Stage of Embryogenesis

Immunological function in chicks is greatly affected by estrogen treatment during embryogenesis, but the mechanism of the estrogen effect is not fully understood. To elucidate the effect of estrogen on immune function, we observed estrogen receptor expression in the thymus and bursa of chick embryos by immunohistochemistry. We compared the distribution of estrogen receptor-positive cells with that of keratin-positive epithelial cells. Intense expression of estrogen receptors was detected in thymic and bursal lymphocytes. In peripheral lymphocytes, ER mRNA was detected by RT-PCR analysis. The results of fluorescence-activated cell sorting analysis indicated that the estrogen receptor was expressed in the cytoplasm of the lymphocytes. Furthermore, intense expression of the estrogen receptor was also confirmed in thymic Hassall's corpuscles, bursal follicle-associated epithelial cells, and the bursal interfollicular epithelium. Our results indicate that estrogen affects the differentiation of thymic and bursal lymphocytes, suggesting that the underlying role for estrogen in immune function.

Morphological analysis of biofilm of peritoneal dialysis catheter in refractory peritonitis patient.

A 66-year-old man undergoing peritoneal dialysis (PD) was admitted to our hospital for treatment of PD-related peritonitis. Culture of the PD fluid revealed the presence of Citrobacter freundii, and therapy with ceftazidime was started intraperitoneally. The cell count in PD fluid slowly decreased over time during the first 2weeks of treatment, but increased again on the 14th hospital day. A second culture of the PD fluid revealed the presence of Enterococcus species. A switch in antibiotic therapy to vancomycin did not improve the cell count in the PD fluid. A third culture of the PD fluid revealed the presence of Stenotrophomonas maltophilia. The PD was discontinued and the catheter removed on the 28th hospital day. Examination of the catheter revealed that the inner tip was coated with a fibrous sheet of cells, suggesting biofilm formation. Following catheter removal, the patient was administered intravenous ciprofloxacin, and the inflammatory reaction started to disappear immediately and had completely disappeared after 1 week of treatment. Microscopic analysis of the fibrous structure on the catheter revealed multiple layers of various inflammatory cells. Immunostaining revealed the presence of CD44-positive polynuclear cells, indicating neutrophils, facing the catheter lumen. CD68-positive cells, indicating macrophages, were observed in the following layer, and keratin-positive cells, indicating peritoneal mesothelial cells, were present at the bottom of the structure. Based on the immediate improvement of PD-related peritonitis after catheter removal, we presumed that this biofilm contributed to the intractability of the patient's peritonitis. Morphological analysis of catheter revealed that both the mesothelial cells and the various inflammatory cells may have contributed to biofilm development.

Role of Neuronal and Non-Neuronal Transient Receptor Potential Vanilloid Type 1 and Sensory Neuropeptides in DSS-Induced Peripheral Inflammatory Changes

BACKGROUND & AIMS: The activation of transient receptor potential vanilloid type 1 (TRPV1) leads to release of sensory neuropeptides such as substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) from the nerve endings, and these neuropeptides in turn initiate the biochemical cascade known as neurogenic inflammation. TRPV1 channels and the sensory neuropeptides are expressed not only in sensory nerve fibers but also in non-neuronal cells in the mucosa of large intestine. The aim of this study was to investigate alteration of the sensory neuropeptides and TRPV1 channels in mucosa of DSS-induced colitis mice and to characterize their non-neuronal cells. METHODS: Colitis was induced by 3% dextran sulfate sodium (DSS) solution given as drinking water for 7 days in C57BL/6 mice. To evaluate visceral nociception, colorectal distension was performed in mice treated with vehicle or BCTC on day 7 of DSS treatment. Immunohistochemical analysis was performed using rectal tissues of mice. SP, NKA, CGRP, 5-HT, F4/80, keratin, TNF-α, CD11c, and CD4 were detected by indirect staining with their specific antibodies. TRPV1-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. RESULTS: TRPV1 antagonist BCTC significantly attenuated the visceral hyperalgesia to control level in DSS-induced colitis model. We compared the number of the nerve fibers and non-neuronal cells expressing TRPV1 channels and sensory neuropeptides in normal and DSS-induced colitis model mice. The number of TRPV1and CGRP-expressing nerve fibers is significantly increased in colitis model. The non-neuronal TRPV1 cells were markedly increased but non-neuronal CGRP-expressing cells were not observed in colitis model. No significant alteration in the number of SP and NKA positive nerve fibers was detected. On the other hand, SP and NKA positive cells were drastically increased in colitis model. Next, double labeling studies were carried out to characterize TRPV1-, SP-, and NKA-expressing cells under inflammatory state. We found two types of non-neuronal TRPV1-expressing cells in mucosa of colitis model. One is keratin positive cells located upper part of mucosa. Another is TNF-α and/or F4/80 positive cells located middle part of the mucosa. Both types of TRPV1-expressing cells did not colocalize with CD4 and CD11c. SP and NKA positive cells almost completely colocalized with 5-HT in experimental colitis model mice. CONCLUSION: These results suggest that TRPV1immunopositive epithelial cells and macrophage are associated with visceral hyperalgesia in colitis.

Differential response of primary cultures of breast carcinomas to lapatinib in an in vitro chemoresponse assay

1115 Background: Lapatinib (Tykerb) is a small molecule tyrosine kinase inhibitor that targets the intracellular domain of the epidermal growth factor receptor and HER-2, thereby inhibiting both growth and survival signaling pathways. Lapatinib is currently FDA-approved to treat HER-2-positive breast cancer previously treated with anthracycline and taxane therapies and trastuzumab. Due to the low population response rate of lapatinib, an integrated biomarker that can identify patients with an increased likelihood for response would be of great clinical utility. The current study describes an in vitro chemoresponse assay developed to predict sensitivity and resistance of primary cultures of human breast tumor specimens to lapatinib. Methods: The chemoresponse assay (ChemoFx) for lapatinib was developed using four different immortalized carcinoma cell lines (SK-OV3, BT474, MDA-MB-231, MCF7). In addition to cell lines, the chemoresponse assay was also performed on 55 first passage primary cultures of human breast carcinomas. All cultures were confirmed to contain keratin-positive epithelial cells using fluorescence immunocytochemistry. Cell lines and specimens were treated with a 10 dose concentration range of lapatinib for 72 hours and stained with DAPI; remaining live cells were counted on an inverted fluorescent imaging system. Resulting dose-response curves were analyzed and categorized as responsive, intermediate responsive, or non-responsive using a proprietary scoring algorithm. Results: All four cell lines (BT474, MDA-MB-231, MCF7, SK-OV3) were responsive to lapatinib treatment, with EC50 values of approximately 10 uM. Dose-response curves of the 55 primary breast cultures revealed that 9% of the specimens tested were responsive to lapatinib, 15% had an intermediate response, and 76% were non-responsive. These results are consistent with a reported clinical response rate of 10% for lapatinib. Conclusions: Initial results with the described integrated cell-based assay demonstrate that in vitro chemoresponse testing may be useful in predicting patient response to lapatinib. This type of in vitro biomarker is likely to increase the efficacy of the current chemotherapy decision-making process for oncologists and their patients. No significant financial relationships to disclose.

What is a small round blue cell tumour anyway?

The spectrum of ‘small round blue cell' neoplasms is broad, but within the sinonasal tract (SNT), tumours of the dispersed neuroendocrine cell system narrow to olfactory neuroblastoma (ONB), pituitary adenoma, melanoma, and neuroendocrine carcinoma. A single totipotential cells shows multidirectional differentiation along epithelial and neuroendocrine lines. ONB arise from the neuroepithelial olfactory cells involving the cribriform plate of the ethmoid. With a bimodal age distribution (2nd and 6th decades), symptoms are non-specific, frequently delaying diagnosis. Histologically, a lobular architecture is maintained irrespective of the tumour grade. Circumscribed lobules are outlined by S100 protein positive supporting cells, while the neoplastic cells are arranged in a syncytial architecture with a background of neuronal processes. Pseudorosettes (Homer Wright) are more common than true rosettes (Flexner-Wintersteiner). The cells are small to medium sized, set in a richly vascularised fibrovascular stroma. The nuclei are round with delicate, ‘salt-and-pepper' chromatin distribution with small nucleoli. Pleomorphism, mitoses, and necrosis increase with tumour grade. The cells show neuroendocrine immunoreactivity (chromogranin, synaptophysin, NSE, CD56), with rare keratin positive cells. Grade and stage affect prognosis. There is no EWS/FLI-1 gene fusion. Many tumours in the SNT mimic ONB and must be excluded due to differences in treatment and prognosis.

Osteofibrous Dysplasia and Adamantinoma in Children and Adolescents: A Clinicopathologic Reappraisal

Osteofibrous dysplasia (OFD) and adamantinoma are rare and most commonly arise in the tibia of young individuals. Although OFD has typical histopathologic features, areas resembling OFD have often been noted at the periphery of otherwise classic adamantinomas, and some have suggested that OFD may be either a precursor to or a regressive phase of adamantinoma. The so-called OFD-like adamantinoma encompasses some features of both OFD and adamantinoma. We studied the clinical, imaging, histopathologic, immunohistochemical, ultrastructural, and molecular features of 16 OFD and 8 adamantinomas (5 OFD-like and 3 classic) in an attempt to further define their morphology, clinical course, and relationship. Patients with OFD were generally younger than those with adamantinoma. Osteoblastic and osteoclastic activity was more prominent in OFD than in OFD-like adamantinoma. In addition to the inconspicuous small clusters of epithelial cells in OFD-like adamantinoma, isolated keratin-positive cells with a unique ultrastructural hybrid fibroblastic-epithelial phenotype were found in the stroma of all OFD and OFD-like adamantinomas. Fluorescence in situ hybridization analysis revealed trisomies 7, 8, and/or 12 in the spindle cell stroma of OFD, OFD-like, and classic adamantinoma, supporting a neoplastic origin of OFD and a common histogenesis for all 3 lesions. Trisomies were not observed in osteoblasts or osteoclasts suggesting that the osseous component is reactive and non-neoplastic. Of the 11 OFD patients with follow-up (median, 4.5 y), all 3 who underwent incisional biopsy had persistent, nonprogressive disease and 2 of 8 who underwent curettage or wide excision had recurrence; none developed adamantinoma. All 6 adamantinoma patients with follow-up (3 classic and 3 OFD-like) were treated with wide excision. One with classic adamantinoma died of pulmonary metastases 9 years after presentation; the other 5 were free of disease with a median follow-up of 12 years. None of the classic adamantinomas evolved into OFD-like adamantinoma or OFD. Although the histopathology, immunohistochemistry, ultrastructure, and cytogenetics indicate that these lesions are closely related, our data and the literature suggest that only classic adamantinoma has malignant potential. OFD, OFD-like adamantinoma, and classic adamantinoma appear to show a progressive complexity of cytogenetic aberrations, perhaps indicative of a multistep neoplastic transformation.

Podocytes Contribute to the Formation of Glomerular Crescents

The cellular composition of crescents in glomerular disease is controversial. The role of podocytes in crescent formation has been especially difficult to study because podocytes typically lose their characteristic terminally differentiated phenotype under disease conditions, making them difficult to identify. We reasoned that the intermediate filament protein nestin, a marker of progenitor cells that has recently been identified in podocytes, may allow the investigation of podocyte involvement in glomerular crescents. In a series of 35 biopsies with crescentic glomerular disease, all showed nestin-positive cells in the crescents, ranging in number from occasional to approximately 50% of crescent cells. Other podocyte markers, such as podocin and WT1, failed to identify cells in crescents, and no contribution by endothelial or myogenic cells was noted. CD68-positive cells were observed in 80% of cases but were never as numerous as the nestin-positive cells. Nestin and CD68 were not coexpressed by the same cells, providing no evidence of trans-differentiation of podocytes into a macrophage phenotype. Keratin-positive cells were found in crescents in 51% of cases, but only as occasional cells. Up to one third of crescent cells were cycling in 48% of biopsies, and double immunostaining identified these cells as a mixture of nestin-positive cells and "null" cells (negative for nestin, CD68, and keratin). In addition to our observations in human disease, we also identified nestin-positive proliferating podocytes in the crescents of 2 mouse models of crescentic glomerulonephritis. We conclude that podocytes play a role in the formation of glomerular crescents.

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.

Developmental changes of keratin expression in chick embryo olfactory epithelium in relation to cellular differentiation and neurogenesis in vivo and in vitro

Olfactory embryogenesis was studied using an anti-chick keratin antibody on chick embryo sections as well as in vitro. Olfactory placodes form at embryonic day 3 (ED3) in the anterior facial ectoderm and invaginate to form the nasal pits. At ED5, the epidermal ectoderm and respiratory epithelium show the same dense cytokeratin immunoreaction. In contrast, absence of keratin expression in the basal part of olfactory epithelial primordium, in the deeper nasal pit area, coincides with one of the critical first steps of olfactory neurogenesis. However, beginning with periphero-central olfactory synaptogenesis at ED8, a new basal cell population starts to express keratin in the olfactory epithelium. Keratin positive cells appear to correspond, by their epithelial localisation and morphology, to sustentacular and basal cells. This interpretation was confirmed in vitro with ED14 chick primary olfactory cultures where TrKA immunoreactivity was used as a marker of horizontal basal cells (HBCs). After ED15, late keratin expression was detected in forming Bowman's glands. The density of keratin expressing basal cells was measured between ED10 and ED20, and appeared highest in the median part of the olfactory epithelium, the area of most active olfactory neurogenesis and neuronal maturation. Thus, keratin expression corresponds to a specialisation of horizontal basal cells as active neuronal stem cells.

Axillary dissection is not required for all patients with breast cancer and positive sentinel nodes.

Sentinel node (SN) biopsy for breast cancer enhances staging sensitivity, often demonstrating only micrometastases (<2 mm) or isolated, keratin-positive cells. When SN metastasis is present, the value of additional axillary dissection is unclear and not all patients benefit from axillary lymph node dissection (ALND). Prospective cohort study, median 32-month follow-up. Multidisciplinary breast cancer centers. Forty-six women having SN metastases diagnosed between May 1, 1996, and September 1, 2001, who refused ALND or were recommended to omit ALND owing to serious comorbid conditions. Isosulfan blue dye-directed SN biopsy. Axillary lymph node dissection was not performed. Standard breast irradiation was given. Adjuvant systemic therapy was provided as determined by an oncologist. Interval clinical evaluation was performed. Axillary and systemic failure rates. Mean patient age was 61.6 years (age range, 36-92 years). Mean tumor size was 1.65 cm (range, 0.4-5.5 cm). Thirty-five (76%) of 46 tumors were ductal carcinomas and 39 (87%) of 45 were estrogen receptor-positive. A mean of 2.6 SNs were identified (median, 2; range, 1-7). Thirty-nine patients (85%) had a single positive SN; the remaining 7 patients (15%) had 2 positive SNs. Seven patients (15%) had macrometastases (>2 mm); 16 (35%) had micrometastases (<2 mm); and 23 (50%) had cellular metastases. Only 16 positive SNs (35%) were seen on hematoxylin-eosin staining, while 30 SNs (65%) had positive immunohistochemical staining. There have been no axillary recurrences. One patient (2%) developed distant metastases during follow-up (range, 4-61 months). Patients with SN metastases who did not have ALND had a low incidence of regional failure. To confirm this observation, we suggest that patients with SN metastases are ideal candidates for trials evaluating the necessity of ALND.

Low-grade mucoepidermoid carcinoma of salivary glands: characteristic immunohistochemical profile and evidence of striated duct differentiation.

The purpose of the present study is to determine the presence and distribution of epithelial and myoepithelial cells in mucoepidermoid carcinoma (MEC) of salivary glands and to compare them with normal salivary gland tissue and other primary carcinomas. This is in order to establish novel diagnostic criteria and to better understand MEC histogenesis. Formalin-fixed paraffin-embedded tissues from ten well-differentiated MECs, three adenoid cystic carcinomas (ACC), four acinic cell carcinomas (AC), and three epithelial-myoepithelial carcinomas (EMCC) of salivary glands were studied with immunohistochemistry using antibodies that recognise antigens indicative of epithelial and myoepithelial cell differentiation. An anti-mitochondrial antibody was also employed. Normal salivary tissue was present for comparative study in non-tumorous areas of the same section from 12 cases. MEC contained numerous keratin-positive cells. Anti mitochondrial antibody was diffusely positive in all ten of these tumours. Smooth muscle actin, h-caldesmon, and smooth muscle heavy chain myosin, which are indicative of myoepithelial cell differentiation, were negative. Rare cells in only one case were stained by calponin. Cytokeratin 14 (CK14) and anti mitochondrial antibody stained cells located mainly at the periphery of neoplastic nests and cystic spaces, while CK7 was mainly present in cells bordering gland lumina (zoning pattern). The immunohistochemical cell profile was similar to that seen in striated normal ducts. All others tumours studied showed a different immunohistochemical pattern, mostly consisting of a lack of mitochondrion-rich cells and the presence of myoepithelial cells in ACC and EMCC. Immunoreactivity in MEC for CK7, CK14 and mitochondrial antibodies appears as a peculiar pattern of staining, different from that of other salivary gland tumors; this seems helpful for diagnostic purposes. In addition, a differentiation of the "striated duct phenotype" is suggested.