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Abstract: Mastocytosis is a condition characterized by accumulation of clonal mast cells (MCs) that often involves the skin. Pathologists are often challenged with skin biopsies with a question of cutaneous lesions of mastocytosis (CLM) including cutaneous mastocytosis, mastocytosis in the skin, or systemic mastocytosis. The histopathological criteria for CLM remain poorly defined due to heterogeneity of the published literature and the lack of comparative prospective studies. MC count is greatly influenced by detection and counting techniques, criteria for viable MCs used, anatomical location biopsied, and the dermal level that is analyzed. Although MC numbers in CLM can be significantly higher compared with healthy controls and a patient with other inflammatory skin diseases, in some instances, considerable overlap exists. Based on the largest studies published, it is suggested that a number of MCs between 75 and 250 MCs/mm 2 are a range in which CLM should be considered and, above 250 MC/mm 2 , a diagnosis of CLM can be made. A recent study showed a high specificity of >95% of a MC count >139 MC/mm 2 compared with patients with other inflammatory skin diseases. Noteworthy, the total number and percentage of MCs is significantly higher in children compared with adults, particularly in polymorphic maculopapular cutaneous mastocytosis. In difficult cases, ancillary techniques such as D816V mutation analysis on formalin-fixed paraffin-embedded tissue have a high sensitivity and specificity. There is no enough evidence that immunohistochemistry of CD25, CD2, or CD30 has any additional value in the diagnosis, subtyping, or clinical course of mastocytosis., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Abstract: Cutaneous mastocytosis is characterized by the abnormal accumulation of mast cells in the skin. However, mast cell counting is not always easy and reproducible with classical methods. This work aims to demonstrate the reliability, usability, and virtues of a new software used on digital tablets for counting mast cells in cutaneous specific lesions of mastocytosis, to assess differences in mast cell counts between clinical subtypes of mastocytosis in the skin, and to consider the feasibility of applying a diagnostic mast cell count cutoff to urticaria pigmentosa, which is the most frequent form of cutaneous mastocytosis. Using a new digital tablet software that was accessible by multiple observers through its own wireless network and allowed high resolution of the image without data compression, we counted the number of mast cells on slides of patients and control skins immunostained for CD117. We found that our counting method was highly reproducible and that the new software allowed very quick counting. We evidenced strong differences in the mast cell count between most of the clinical subtypes of mastocytosis in the skin. However, when applied to a subset of patients with urticaria pigmentosa, a diagnostic cutoff in the mast cell count lacked sensitivity. Thus, our digital method for counting CD117-immunostained mast cells was highly accurate and was of a significant value for the diagnosis of mastocytosis in the skin. However, some subtypes with low mast cell counts will still require the application of additional diagnostic criteria., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Merkel cell carcinoma (MCC) is a rare and aggressive malignancy from neuroendocrine cells in the skin. Despite being one of the most life-threatening of skin cancers, little is known about the potential signaling mechanism that drives carcinogenesis in MCC. The purpose of this study is to assess the impact of Merkel cell polyomavirus (MCPyV), p53, and c-kit on the histological features and clinical prognosis of MCC treated in our regional hospitals., Method: The design was a retrospective study. The specimens were taken between 1993 and 2013 in 2 referral hospitals of Southern Spain. Data were collected retrospectively and analyzed using SPSS software., Results: Thirteen lesions from 13 subjects were included in the study. Positivity for c-kit was associated with the absence of MCPyV viral DNA (P = 0.048) and positivity for p53 (P = 0.002). More rate of mitoses per high-power field was presented significantly in those specimens with: positivity for c-kit (P = 0.046), positivity for p53 (P = 0.05), lesions with infiltrative growth pattern (P = 0.008), and lymphovascular invasion (P = 0.034). We observed an inverse relationship between p53 expression and MCPyV infection (Pearson's coefficient: -0.524; P = 0.046) and between c-kit expression and MCPyV infection (Pearson's coefficient: -0.548; P = 0.05), whereas the relationship was positive between p53 expression and c-kit expression (Pearson's coefficient: 0.884; P < 0.001)., Conclusion: We conclude that presence of MCPyV DNA has no effect on overall survival. MCCs with p53 and c-kit expressions are associated with the absence of or low MCPyV DNA showing an inverse relationship. A multifactorial molecular pathogenesis where positivity for p53 and c-kit are associated with other mechanisms different than MCPyV (such as pro-mitotic factors) may lead to aggressive clinical behavior.

Objectives: To identify whether mast cell densities in vulvar biopsies from the vestibule are associated with vulvodynia., Methods: We enrolled 100 women aged 19 to 59 years with confirmed vulvodynia cases, 100 racially matched controls, and 100 black control women. All had vulvar biopsies performed at the 7 o'clock position of the vestibule, which were then immunostained to detect c-KIT protein. The numbers of c-KIT positive mast cells per ×400 magnification field were manually counted, and t tests and logistic regression were used to assess the association with case-control status., Results: Of the biopsies, 235 were adequate samples for c-KIT testing for mast cells. The mast cell density was substantially lower in black control women (13.9 ± 10.9) in comparison to white control women (22.5 ± 13.2 p < 0.001): hence the analysis was confined to white cases and racially matched control women. Compared with racially matched controls, cases were younger, more likely to be married, and reported a higher household income. The average number of mast cells per ×400 magnification field overall was 19.1 ± 13.2 (range, 0-62). There was no difference in the mast cell count between racially matched cases (22.4 ± 13.9 per ×400 field) and controls (22.5 ± 13.2) in either the univariate or multivariable analyses. Within the group of cases, there was no difference in mast cell density based on the presence or absence of a variety of urogenital symptoms., Conclusions: No difference in mast cell density in biopsies of the vestibule was found between white cases and racially matched controls. Black control women have a lower mast cell density compared with white control women.

Rationale: Cardiac progenitor cells (CPCs) improve left ventricular remodeling and function after acute or chronic myocardial infarction. However, the long-term (>5 weeks) effects, potential tumorigenicity, and fate of transplanted CPCs are unknown., Objective: To assess the outcome of CPC therapy at 1 year., Methods and Results: Female rats underwent a 90-minute coronary occlusion; 4 hours after reperfusion, they received intracoronarily vehicle or 1 million male, syngeneic CPCs. One year later, CPC-treated rats exhibited smaller scars and more viable myocardium in the risk region, along with improved left ventricular remodeling and regional and global left ventricular function. No tumors were observed. Some transplanted (Y-chromosome(POS)) CPCs (or their progeny) persisted and continued to proliferate, but they failed to acquire a mature cardiomyocyte phenotype and were too few (4-8% of nuclei) to account for the benefits of CPC therapy. Surprisingly, CPC transplantation triggered a prolonged proliferative response of endogenous cells, resulting in increased formation of endothelial cells and Y-chromosome(NEG) CPCs for 12 months and increased formation, for at least 7 months, of small cells that expressed cardiomyocytic proteins (α-sarcomeric actin) but did not have a mature cardiomyocyte phenotype., Conclusions: The beneficial effects of CPCs on left ventricular remodeling and dysfunction are sustained for at least 1 year and thus are likely to be permanent. Because transplanted CPCs do not differentiate into mature myocytes, their major mechanism of action must involve paracrine actions. These paracrine mechanisms could be very prolonged because some CPCs engraft, proliferate, and persist at 1 year. This is the first report that transplantation of any cell type in the heart induces a proliferative response that lasts at least 1 year. The results strongly support the safety and clinical utility of CPC therapy., (© 2016 American Heart Association, Inc.)

Rationale: The mechanism of functional restoration by stem cell therapy remains poorly understood. Novel manganese-enhanced MRI and bioluminescence reporter gene imaging were applied to follow myocardial viability and cell engraftment, respectively. Human-placenta-derived amniotic mesenchymal stem cells (AMCs) demonstrate unique immunoregulatory and precardiac properties. In this study, the restorative effects of 3 AMC-derived subpopulations were examined in a murine myocardial injury model: (1) unselected AMCs, (2) ckit(+)AMCs, and (3) AMC-derived induced pluripotent stem cells (MiPSCs)., Objective: To determine the differential restorative effects of the AMC-derived subpopulations in the murine myocardial injury model using multimodality imaging., Methods and Results: SCID (severe combined immunodeficiency) mice underwent left anterior descending artery ligation and were divided into 4 treatment arms: (1) normal saline control (n=14), (2) unselected AMCs (n=10), (3) ckit(+)AMCs (n=13), and (4) MiPSCs (n=11). Cardiac MRI assessed myocardial viability and left ventricular function, whereas bioluminescence imaging assessed stem cell engraftment during a 4-week period. Immunohistological labeling and reverse transcriptase polymerase chain reaction of the explanted myocardium were performed. The unselected AMC and ckit(+)AMC-treated mice demonstrated transient left ventricular functional improvement. However, the MiPSCs exhibited a significantly greater increase in left ventricular function compared with all the other groups during the entire 4-week period. Left ventricular functional improvement correlated with increased myocardial viability and sustained stem cell engraftment. The MiPSC-treated animals lacked any evidence of de novo cardiac differentiation., Conclusion: The functional restoration seen in MiPSCs was characterized by increased myocardial viability and sustained engraftment without de novo cardiac differentiation, indicating salvage of the injured myocardium., (© 2015 American Heart Association, Inc.)

Distinct genetic aberrations between melanomas in different anatomical locations have been confirmed in recent years. However, the associations between immunohistochemical expression, tumor sites, and clinical parameters are not clear. We examined the correlation of protein expression and gene mutation of c-kit with clinicopathological parameters and lesion locations in patients with malignant melanoma (MM). We collected 170 melanocytic lesions, including 106 cutaneous MM from acral melanoma (AM) and nonacral melanoma (NAM) sites, 24 dysplastic nevi, and 40 common melanocytic nevi. Tissue microarray was constructed, and immunohistochemical expression for c-kit was assessed with correlation with clinical parameters. Mutation in exons 11, 13, 17, and 18 of KIT gene in genomic DNA by polymerase chain reaction sequencing was also analyzed. Immunostaining scores for c-kit were found to be statistically higher in Dysplastic Nevi than in common melanocytic nevi and MM. In addition, cytoplasmic c-kit staining was significantly correlated with poor survival in patients with AM but not in those with NAM. Twenty-nine cases of MM (including 9 NAM and 20 AM) are analyzed for mutation in exons 11, 13, 17, and 18 of KIT gene in genomic DNA by polymerase chain reaction sequencing, and no genetic mutation is found. Our findings confirm that KIT mutations, in contrast to previous white cohorts, are not common in both AM and NAM of the Chinese and do not necessarily correlate with c-kit expression. The significantly different association between the expression of c-kit immunoreactivities and the mortality risks of melanomas on acral versus nonacral sites might change site-specific targeted therapeutic concepts in melanoma in the future.

Endometrial stromal sarcomas (ESS) are rare uterine malignant mesenchymal neoplasms, which are currently treated by surgery, as effective adjuvant therapies have not yet been established. Tyrosine kinase inhibitors have rarely been applied in ESS therapy, with few reports describing imatinib responsivity. The aim of this study was to analyze the status of different tyrosine kinase receptors in an ESS series, in order to evaluate their potential role as molecular targets. Immunohistochemistry was performed for EGFR, c-KIT, PDGFR-α, PDGFR-β, and ABL on 28 ESS. EGFR, PDGFR-α, and PDGFR-β gene expression was investigated by real-time polymerase chain reaction (qRT-PCR) on selected cases. "Hot-spot" mutations were screened for on EGFR, c-KIT, PDGFR-α, and PDGFR-β genes, by sequencing. All analysis was executed from formalin-fixed, paraffin-embedded specimens. Immunohistochemical overexpression of 2 or more tyrosine kinase receptors was observed in 18 of 28 tumors (64%), whereas only 5 tumors were consistently negative. Gene expression profiles were concordant with immunohistochemical overexpression in only 1 tumor, which displayed both high mRNA levels and specific immunoreactivity for PDGFR-α, and PDGFR-β. No activating mutations were found on the tumors included in the study. This study confirms that TKRs expression is frequently observed in ESS. Considering that the responsiveness to tyrosine kinase inhibitors is known to be related to the presence of specific activating mutations or gene over-expression, which are not detectable in ESS, TKRs immunohistochemical over-expression alone should not be considered as a reliable marker for targeted therapies in ESS. Specific post-translational abnormalities, responsible for activation of TKRs, should be further investigated.

Background: SCIPIO is a first-in-human, phase 1, randomized, open-label trial of autologous c-kit(+) cardiac stem cells (CSCs) in patients with heart failure of ischemic etiology undergoing coronary artery bypass grafting (CABG). In the present study, we report the surgical aspects and interim cardiac magnetic resonance (CMR) results., Methods and Results: A total of 33 patients (20 CSC-treated and 13 control subjects) met final eligibility criteria and were enrolled in SCIPIO. CSCs were isolated from the right atrial appendage harvested and processed during surgery. Harvesting did not affect cardiopulmonary bypass, cross-clamp, or surgical times. In CSC-treated patients, CMR showed a marked increase in both LVEF (from 27.5 ± 1.6% to 35.1 ± 2.4% [P=0.004, n=8] and 41.2 ± 4.5% [P=0.013, n=5] at 4 and 12 months after CSC infusion, respectively) and regional EF in the CSC-infused territory. Infarct size (late gadolinium enhancement) decreased after CSC infusion (by manual delineation: -6.9 ± 1.5 g [-22.7%] at 4 months [P=0.002, n=9] and -9.8 ± 3.5 g [-30.2%] at 12 months [P=0.039, n=6]). LV nonviable mass decreased even more (-11.9 ± 2.5 g [-49.7%] at 4 months [P=0.001] and -14.7 ± 3.9 g [-58.6%] at 12 months [P=0.013]), whereas LV viable mass increased (+11.6 ± 5.1 g at 4 months after CSC infusion [P=0.055] and +31.5 ± 11.0 g at 12 months [P=0.035])., Conclusions: Isolation of CSCs from cardiac tissue obtained in the operating room is feasible and does not alter practices during CABG surgery. CMR shows that CSC infusion produces a striking improvement in both global and regional LV function, a reduction in infarct size, and an increase in viable tissue that persist at least 1 year and are consistent with cardiac regeneration., Clinical Trial Registration: This study is registered with clinicaltrials.gov, trial number NCT00474461.

Melanocytic tumors occur as much in humans as in dogs and are frequently associated with receptor tyrosine kinase dysregulation. The transmembrane c-kit protein is a receptor tyrosine kinase that is crucial in melanocytic homeostasis and, when mutated, is associated with tumor development in those cells. In human studies, its expression is generally detected in melanocytomas and primary malignant melanomas, being lost with tumor progression and metastasis. In this study, we aimed to analyze c-kit expression in canine cutaneous melanocytic tumors and its association with tumor behavior, in order to investigate the dog's potential in comparative pathology and c-kit's potential in the diagnosis of these tumors. The expression of c-kit was evaluated immunohistochemically in 39 canine cutaneous melanocytic tumors and scored in terms of the labeling location, extension, and intensity. The labeling location was essentially cytoplasmic, and the labeling extension and intensity were generally higher in melanocytomas (83.3% diffuse-labeled cells) than those in malignant melanomas (22.2% negative-labeled cells). The differences found in the labeling extension were statistically significant (P < 0.001). There was no association between c-kit immunoexpression in malignant melanomas and the clinicopathological criteria, except between the labeling intensity and the degree of intralesional pigmentation (P = 0.048). Our results for labeling extension are in agreement with similar human studies, reinforcing the dog's potential as a model organism for investigation in this type of cancer. In addition, the loss of c-kit expression in malignant melanomas might be a criterion of tumor aggressiveness, indicating that this receptor may be useful in the diagnosis of these tumors.

Introduction: We evaluated the immunohistochemical expression of p53, Ki67, CD30, and CD117 and correlated it with histological features and presence of clinical metastasis at diagnosis of testicular seminomas., Materials and Methods: A retrospective study of 62 patients was performed in patients with pure seminoma. The retroperitoneum was staged with computed tomography scan and the thorax with simple x-rays and/or computed tomography scan. Pathologists were unaware of the clinical stage of the patients. Manual microarrays were created from a tissue representative of tumor. The expression of p53, Ki67, CD30, and CD117 was evaluated as negative, any degree of expression, and expression in more than 50% of neoplastic cells. Univariate and multivariate analysis were performed., Results: Sixty-two cases were analyzed: 43 cases were in clinical stage I (69.4%), 17 were in clinical stage II (27.4%), and 2 were in clinical stage III (3.2%). Fifty-six cases expressed CD117 (90%), 42 p53 (68%), 8 CD30 (13%), and all cases Ki67. There were no differences in p53, Ki67, CD30, and CD117 expression between testicular seminoma with and without clinical metastasis at diagnosis, regardless of the magnitude of expression. Neither of them found positive association between these marker expressions and morphologic risk factors such as tumor size greater than 6 cm and rete testis invasion., Conclusions: This study shows that expression of p53, Ki67, and CD30 and loss of CD117 expression fail to predict the presence of clinical metastasis at diagnosis of testicular seminoma and do not correlate with other histopathological risk factors in clinical stage I patients.

Objectives: Ramon y Cajal discovered interstitial cells in the pancreas associated with intrinsic nerves. It was our aim to provide evidence for or against the hypothesis that the pancreatic duct harbors interstitial cells of Cajal (ICCs) that may function as pacemakers for duct motility., Methods: We used immunohistochemistry using c-Kit as the ICC marker and protein gene product 9.5 for nerves. Electron microscopy further characterized the cells and their interrelationships., Results: c-Kit-positive cells were associated with smooth muscle cells and nerve fibers of the duct wall and were rich in mitochondria, rough endoplasmic reticulum, and intermediate filaments; they possessed occasional caveolae and had a discontinuous basal lamina. They were connected by small gap junctions to each other and to smooth muscle cells. c-Kit-positive cells around large blood vessels were similar. c-Kit-positive cells within acini were similar in structure but were not associated with smooth muscle cells., Conclusions: The c-Kit-positive cells around the main duct were identified as ICCs and have the morphological criteria to likely function as pacemaker cells for the previously observed spontaneous rhythmic pancreatic duct contractions. Interstitial cells of Cajal around the large blood vessels likely affect vessel wall rhythmicity.

Background: Despite numerous studies demonstrating the efficacy of cellular adoptive transfer for therapeutic myocardial regeneration, problems remain for donated cells with regard to survival, persistence, engraftment, and long-term benefits. This study redresses these concerns by enhancing the regenerative potential of adoptively transferred cardiac progenitor cells (CPCs) via genetic engineering to overexpress Pim-1, a cardioprotective kinase that enhances cell survival and proliferation., Methods and Results: Intramyocardial injections of CPCs overexpressing Pim-1 were given to infarcted female mice. Animals were monitored over 4, 12, and 32 weeks to assess cardiac function and engraftment of Pim-1 CPCs with echocardiography, in vivo hemodynamics, and confocal imagery. CPCs overexpressing Pim-1 showed increased proliferation and expression of markers consistent with cardiogenic lineage commitment after dexamethasone exposure in vitro. Animals that received CPCs overexpressing Pim-1 also produced greater levels of cellular engraftment, persistence, and functional improvement relative to control CPCs up to 32 weeks after delivery. Salutary effects include reduction of infarct size, greater number of c-kit(+) cells, and increased vasculature in the damaged region., Conclusions: Myocardial repair is significantly enhanced by genetic engineering of CPCs with Pim-1 kinase. Ex vivo gene delivery to enhance cellular survival, proliferation, and regeneration may overcome current limitations of stem cell-based therapeutic approaches.

Study Design: Case report and clinical discussion., Objective: To describe a rare case of vertebral involvement of systemic mastocytosis and its multimodal therapy., Summary of Background Data: Vertebral fractures in young men are rare events. A thorough diagnostic work-up that unravels the underlying cause of osteoporosis and appropriate therapy are crucial to prevent further fractures., Methods: A 36-year-old man was evaluated for severe back pain and was found to suffer from progressive osteoporosis and multiple vertebral fractures. Bone biopsy analysis revealed tryptase-positive mast cells that were positive for c-KIT, thus confirming the diagnosis of systemic mastocytosis., Results: In addition to zoledronic acid (4 mg per month) and prednisolone (50 mg per day) treatment, the patient underwent kyphoplasty. The procedure was associated with arterial hypotension which was most likely because of pressure-induced mast cell degranulation. Follow-up visits demonstrated stable bone mineral density and tolerable back pain while on zoledronic acid., Conclusion: Systemic mastocytosis is a rare cause of vertebral fractures in young men. Because of the potential risk of pressure-induced release of the allergy mediator histamine, kyphoplasty for vertebral involvement of systemic mastocytosis should be conducted with appropriate precautions.

Gastrointestinal stromal tumors (GISTs) rarely metastasize to the skin. We describe 5 patients with GIST with subcutaneous and cutaneous metastases. The mean age at metastasis was 54 years (range 30-68 years) with a male predominance (4:1). Primary tumors occurred in the stomach (n = 3), small bowel (n = 1), and abdomen, not otherwise specified (n = 1). The average time from primary tumor resection to the resection of skin metastases was 59 months (range 11-155 months). The metastases occurred in the scalp (n = 2), cheek (n = 1), and abdomen (n = 2) with 3 patients presenting with solitary nodules and 2 patients with multiple nodules. The average size was 2 cm (range 0.6-4 cm). Histologically, 2 cases were spindled and 3 cases demonstrated mixed epithelioid and spindle cell morphology. All were confirmed to have CD117 reactivity. KIT genotyping was performed in 4 of 5 cases. Two cases harbored a mutation in exon 11, and the remaining 2 cases were wild type in exons 9, 11, 13, and 17. All 5 patients had multiple concurrent or subsequent abdominal and/or hepatic metastases. In 4 patients with an average follow-up of 32 months (range 6-75 months), after the resection of the metastases, 2 were alive with disease and 2 died of disease. Cutaneous metastases seem to be a late complication of GIST, but their presence does not necessarily herald a rapid demise of the patient.

Gastrointestinal stromal tumors (GIST) are the most common malignant mesenchymal tumors of the gastrointestinal tract. The principal treatment modality for primary GIST is surgery whereas for metastatic GIST, imatinib has an established role. In patients with locally advanced and metastatic GIST, the role of surgery in the imatinib era is still unclear. Fifteen patients with locally advanced (n=9) and/or metastatic GIST (n=6) were treated with imatinib followed by resection. Detailed histopathological examination was performed before and after treatment with imatinib, which was given for a median of 11 months before surgery. Ten patients showed a radiographic partial response, four patients had stable disease, and one patient progressed. At the time of surgery, the median tumor diameter was 6.5 cm. In all the nine patients with locally advanced GIST, a R0 resection could be performed. Histopathological examination showed imatinib effects in all tumors, including the case with progressive disease. All patients with locally advanced disease (n=9) were alive after a median follow-up of 40 months (range: 18-59), of which seven patients were free of disease. Four of the six patients treated for metastatic GIST died of disease after 30, 45, 50, and 74 months of follow-up. Remarkably, in five of six patients in whom CD117 expression was diminished or lost in the resection specimen, disease recurrence was observed. In patients with retained CD117 expression, one of the nine patients had recurrent disease. In conclusion, preoperative imatinib treatment in patients with locally advanced GIST resulted in a decrease of tumor load in most patients, enabling complete surgical resection. For patients with metastatic GIST, the role of surgery remains less clear. Loss or decrease of CD117 expression in the resected specimen after imatinib treatment may be associated with disease recurrence.

Objective: CD117/KIT overexpression is common in neoplasms such as gastrointestinal stromal tumors and predicts clinical response to tyrosine kinase inhibitors. Pancreatic adenocarcinoma has a poor prognosis, and therefore, targeted molecular therapy may be beneficial. Marked differences in the incidence of CD117/KIT expression have been reported in pancreatic adenocarcinoma. The aim of this study was to test the hypothesis that CD117/KIT expression is unusual in pancreatic adenocarcinoma., Methods: CD117/KIT immunohistochemistry was performed on 23 archival pancreatic adenocarcinoma samples using 2 primary antibodies., Results: Satisfactory internal and external positive control labeling was achieved for both primary antibodies. No tumor cell labeling was identified using one primary antibody, whereas all cases showed cytoplasmic CD117/KIT staining with the second. However, CD117/KIT expression was also identified using the latter within nuclei and benign pancreatic epithelium, suggesting that artifactual staining was occurring., Conclusions: Pancreatic adenocarcinoma does not express CD117/KIT as assessed using the primary immunohistochemical antibody usually used in our laboratory for CD117/KIT detection. The variation in reported incidence of CD117/KIT expression in pancreatic adenocarcinoma is because of methodological differences in immunohistochemical technique. Ideally, immunohistochemical studies of this molecule should be combined with mutational status testing of the c-kit gene.

It has been shown that tyrosine kinase oncoprotein c-kit and antiapoptotic molecule bcl-2 are overexpressed in several types of malignancy, including small cell carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC) of the lung. Whether these 2 molecules are coexpressed in lung neuroendocrine tumors has not been investigated. Here, we analyzed immunohistochemical results to determine expression and coexpression patterns of c-kit and bcl-2 in the spectrum of lung neuroendocrine tumors. Using a polyclonal antibody against c-kit and a monoclonal antibody against bcl-2, our data demonstrated that all 7 cases (100%) of SCLC included in this study were positive for both c-kit and bcl-2. Among 14 LCNECs, 7 (50%) stained positive for c-kit and 9 (64%) for bcl-2. All cases of high grade neuroendocrine carcinomas (SCLCs and LCNECs) that showed positive staining for c-kit coexpressed bcl-2. In contrast, all typical and atypical carcinoids (TC and AC) were negative for c-kit, and only 1 of 16 (6.3%) TCs and 1 of 6 (16.7%) ACs stained positive for bcl-2. These results indicate a progressive increase in the frequency of c-kit and bcl-2 expression and coexpression, from carcinoid tumors (TC and AC) to LCNEC and to SCLC. High grade neuroendocrine carcinomas are more likely to coexpress c-kit and bcl-2 when compared with carcinoid tumors. The high frequency of coexpression of these 2 molecules in high grade neuroendocrine carcinomas of the lung suggests that they may be involved in the carcinogenic pathway, given their important roles in carcinogenesis. Therapeutic targeting on both c-kit and bcl-2 molecules might be beneficial in the management of patients with high grade neuroendocrine carcinomas of the lung in the future.

Background: Accurate bone marrow (BM) blast counts (BCs) are essential for diagnosis (dx) of myelodysplasia (MDS), MDS/myeloproliferative (MDS/MPD) disease, or acute myeloid leukemia (AML), and may be difficult in hemodiluted bone marrow aspirates (BMAs). Erythroid precursors (EPs) may be indistinguishable from myeloblasts in BM sections (aspirate clots/cores). We compare the usefulness of immunohistochemistry (IHC) [ie, CD34, CD117, myeloperoxidase (MPO), Hemoglobin A1 (HbA1), and terminal deoxynucleotidyl transferase (TdT)] of BM sections (IHC-BM) with BMA, bone marrow touch preparation (BMTP), and flow cytometry (FC) BCs., Design: The initial BC (48), percentage (%) of Eps (38) (both based on initial 100 to 600-cell counts), and FC expressions of CD34, CD117, Glycophorin A(GLY A), and TdT (44) were tabulated from 50 BMs (MDS, MDS/MPD, or AML). BMAs (48) and BMTPs (25) subsequently received 500-cell counts. IHC-BM was performed (45:formalin, 5:B5-fixed) [CD34 (46), CD117 (45), HbA1 (45), TdT (42), and MPO (45)]., Results: Retrospective BMA BCs revealed a 31% (15/48) discrepant rate between the original/retrospective BMA BCs; 80% revealed an underestimated initial BC. There was a 28% discordance rate between the retrospective BMA and BMTP reviews; 77% showed a higher BMTP BC. IHC showed significantly higher BCs in 19% (9/47), resulting in a different dx (5). However, CD34 and CD117 IHCS revealed lower BCs in 38% and 48%, respectively. The CD34 IHC results were primarily due to CD34-negative blasts by FC. The CD117 IHC results were largely unexplained. EPs were CD34 and CD117-negative., Conclusions: (1) Evaluation for MDS/AML requires 500-cell counts of BMAs and/or BMTPs. (2) CD34 and/or CD117 blasts by FC indicate IHC-BM may increase BC accuracy. (3) CD34 is more reliable than CD117 by IHC; however, in combination, they are most reliable and should be performed on BM clots/cores due to variable reactivity.

Objective: This review describes the pathologic and epidemiologic features of gastrointestinal stromal tumor (GIST) as well as the contemporary management of this tumor. The integration of surgery and treatment with targeted molecular agents in the treatment of GIST is highlighted., Summary Background Data: GIST is the most common mesenchymal tumor of the gastrointestinal tract. Its cellular origin from the interstitial cell of Cajal and distinctness from smooth muscles tumors were only recently appreciated. The discovery of the centrality of KIT proto-oncogene mutations in the pathogenesis of this tumor, and the development of imatinib mesylate, a specific inhibitor of KIT tyrosine kinase function have revolutionized the treatment of GIST., Methods: We conducted a review of the English literature on GIST. The pathology, epidemiology, diagnosis, and treatment of this tumor are summarized with particular emphasis on recent developments in the field., Results: GIST is a rare tumor that usually arises from the stomach or small intestine. It is characterized by immunohistochemical staining for KIT. Treatment of primary localized tumors is surgical. The benefit of adjuvant treatment with the KIT tyrosine kinase inhibitor imatinib is the subject of investigation. The treatment of unresectable, recurrent, or metastatic GIST is primarily imatinib treatment. The integration of surgery or ablative modalities is often employed, particularly when all disease is amenable to gross resection or destruction, or when GIST becomes resistant to imatinib. Newer tyrosine kinase inhibitors, such as sunitinib are the subject of ongoing investigation., Conclusions: The treatment paradigm for GIST has required the integration of surgery and molecular therapy and this will likely serve as a paradigm for the treatment of other solid tumors as targeted agents are developed.

Cotyledonoid dissecting leiomyoma (Sternberg tumor) is a very rare variant of uterine smooth muscle tumor with an unusual and alarming gross appearance. Including our case, approximately 20 cases were reported in the literature. A 27-year-old woman presented with pelvic mass. A 41-cm fungating rubbery mass with placenta-like appearance adhering to the posterior uterine surface and extending to the left broad ligament and pelvic cavity was observed. An intraoperative frozen section was requested, and a diagnosis of a benign smooth muscle tumor was given. Total abdominal hysterectomy and bilateral salpingo-oophorectomy were performed with removal of the pelvic tumor extension. Thorough tumor sampling was performed and showed nodules of interlacing bundles of bland-looking smooth muscle cells, separated by expanded, edematous, and highly vascularized stroma. No atypia, mitotic activity, or coagulative necrosis was seen. Immunohistochemical studies confirmed the smooth muscle nature of the tumor. Cotyledonoid leiomyoma has a distinctive gross appearance which usually raises the suspicion of sarcoma. Intraoperative frozen section is a mandatory and helpful procedure to avoid overtreatment of such cases.

Gastrointestinal stromal tumor (GIST) are the most common mesenchymal tumors of the gastrointestinal (GI) tract. Most GISTs arise in the stomach and small bowel, whereas a small number occur elsewhere in the GI tract. Rare cases are identified outside the GI tract and are collectively known as extragastrointestinal stromal tumors (EGISTs). Because of their malignant potential and recent advances in the management of GISTs with imatinib mesylate (Gleevec, Glivec), it is imperative that these tumors are correctly diagnosed. In this study, we reviewed the clinical and pathologic characteristics of 3 cases of EGIST presenting as vulvovaginal/rectovaginal septal masses that were originally misdiagnosed, presumably due to their unusual anatomic locations. The original diagnoses were leiomyoma in one case and leiomyosarcoma in 2 cases. The lesions were localized to the rectovaginal septum () or vagina () and ranged from 4 to 8 cm in diameter. All 3 lesions had a spindle cell morphology that mimicked a smooth muscle tumor. Mitotic figures numbered from 12/50 to 16/50 high power fields (HPFs; median 15). Immunohistochemistry revealed that all 3 cases were strongly positive for KIT (CD117) and CD34 and negative for smooth muscle actin, desmin, pan-cytokeratin, and estrogen receptor. KIT sequence analysis revealed oncogenic mutations in all 3 cases. The first tumor recurred at 2 years and the second tumor recurred at 10 years; the third case is too recent for meaningful follow-up. EGISTs that present as gynecologic masses are rare but may be more common than is currently recognized. Misdiagnosis may lead to inappropriate therapy because conventional chemotherapy and radiotherapy are not effective in the treatment of GISTs, whereas imatinib mesylate (Gleevec, Glivec) has a proven role in managing these tumors. Thus, it is imperative to consider EGISTs in the differential diagnosis of mesenchymal neoplasms in the vulvovaginal/rectovaginal septum.

To harvest bone for autologous grafting is a daily problem encountered by craniofacial and oral surgeons. Stem cells derived from human dental pulp are able to differentiate in osteoblasts and are a potential source of autologous bone produced in vitro. The authors describe their preliminary results in this new field with its potential application in craniomaxillofacial surgery. Dental pulp was gently extracted from 34 human permanent teeth (all third molars) of patients 19 to 37 years of age. After they were digested, the cells were selected using a cytometer for c-kit, STRO-1, CD34, CD45, and then for CD44 and RUNX-2. This study, made on a considerable number of cases, provided evidence that dental pulp is extremely rich in stem cells, which were c-kit+/CD34+/STRO-1+/CD45-, capable of differentiation toward several stromal-derived differentiated cells and mainly osteoblasts. These findings, supported by the large number of cases, are of great interest for tissue regeneration, tissue-based clinical therapies, and transplantation.

We assessed the usefulness of several immunohistochemical stains in distinguishing these two neoplasms, including cytokeratin 7, cytokeratin 20 (CK20), neuron-specific enolase, chromogranin, synaptophysin, neurofilaments (NF), thyroid-transcription factor-1 (TTF-1), CD56 antigen, S-100 protein, vimentin, c-erbB-2 oncoprotein, and CD117 antigen. All 13 cases of Merkel cell carcinoma evaluated were positive for CK20, and negative for TTF-1. Twelve of 13 Merkel cell carcinoma cases were positive for NF. Eleven of 13 cases of small cell lung carcinoma were positive for TTF-1. All small cell lung carcinoma cases were negative for NF, and all but one were negative for CK20. In terms of the remaining antigens, there were no differences of significance between the two neoplasms. These findings suggest that a set of three immunohistochemical stains, including CK20, NF, and TTF-1, is useful in affording a distinction between Merkel cell carcinoma and small cell lung carcinoma.

Gastrointestinal stromal tumors (GISTs) are well-recognized mesenchymal neoplasms of the intestinal tract. A diagnosis of GIST is not always possible using IHC techniques for detection of c-kit. The authors describe a 64-year-old man who presented with an upper abdominal quadrant mass. Histology showed a predominantly epithelioid neoplasm with focal "spindle cell" areas. IHC studies were positive for muscle markers and negative for c-kit. The morphologic and immunophenotypic appearance could be compatible with either a smooth muscle tumor or a GIST. Because of the differences in treatment protocols and prognosis between these two entities, molecular studies to detect c-kit or platelet-derived growth factor receptor (PDGFR) activating mutations were performed. No mutations were found in the c-kit gene, but a mutation was detected in the PDGFR gene. This additional molecular study allowed the authors to formulate the precise diagnosis of a c-kit-negative GIST with strong smooth muscle marker expression.

High-mobility group box 1 protein (HMGB1) is a chromatin protein that is released by inflammatory and necrotic cells. Extracellular HMGB1 signals tissue damage, stimulates the secretion of proinflammatory cytokines and chemokines, and modulates stem cell function. The present study examined exogenous HMGB1 effect on mouse left-ventricular function and myocyte regeneration after infarction. Myocardial infarction was induced in C57BL/6 mice by permanent coronary artery ligation. After 4 hours animals were reoperated and 200 ng of purified HMGB1 was administered in the peri-infarcted left ventricle. This intervention resulted in the formation of new myocytes within the infarcted portion of the wall. The regenerative process involved the proliferation and differentiation of endogenous cardiac c-kit+ progenitor cells. Circulating c-kit+ cells did not significantly contribute to HMGB1-mediated cardiac regeneration. Echocardiographic and hemodynamic parameters at 1, 2, and 4 weeks demonstrated a significant recovery of cardiac performance in HMGB1-treated mice. These effects were not observed in infarcted hearts treated either with the unrelated protein glutathione S-transferase or a truncated form of HMGB1. Thus, HMGB1 appears to be a potent inducer of myocardial regeneration following myocardial infarction.

In order to determine whether imatinib mesylate (Gleevec), a tyrosine kinase inhibitor that binds the CD-117 (c-kit) receptor, may be of value in the treatment of malignant melanoma, an immunohistochemical analysis of 40 cases of primary and metastatic melanoma was undertaken. Thirty-five of the 40 cases showed 1+ or stronger labelling for CD-117 (up to a maximum of 4+). Three patients with neoplasms showing 4+ staining were selected for imatinib therapy. None responded. c-kit (CD-117) expression in melanoma appears to be common; however, the value of imatinib therapy remains to be proven.

Recent evidence infers a contribution of smooth muscle cell (SMC) progenitors and stromal cell-derived factor (SDF)-1alpha to neointima formation after arterial injury. Inhibition of plaque area and SMC content in apolipoprotein E-deficient mice repopulated with LacZ+ or CXCR4-/- BM or lentiviral transfer of an antagonist reveals a crucial involvement of local SDF-1alpha and its receptor CXCR4 in neointimal hyperplasia via recruitment of BM-derived SMC progenitors. After arterial injury, SDF-1alpha expression in medial SMCs is preceded by apoptosis and inhibited by blocking caspase-dependent apoptosis. SDF-1alpha binds to platelets at the site of injury, triggers CXCR4- and P-selectin-dependent arrest of progenitor cells on injured arteries or matrix-adherent platelets, preferentially mobilizes and recruits c-kit-/platelet-derived growth factor receptor (PDGFR)-beta+/lineage-/sca-1+ progenitors for neointimal SMCs without being required for their differentiation. Hence, the SDF-1alpha/CXCR4 axis is pivotal for vascular remodeling by recruiting a subset of SMC progenitors in response to apoptosis and in concert with platelets, epitomizing its importance for tissue repair and identifying a prime target to limit lesion development.

Recent studies in mice have challenged the ability of bone marrow cells (BMCs) to differentiate into myocytes and coronary vessels. The claim has also been made that BMCs acquire a cell phenotype different from the blood lineages only by fusing with resident cells. Technical problems exist in the induction of myocardial infarction and the successful injection of BMCs in the mouse heart. Similarly, the accurate analysis of the cell populations implicated in the regeneration of the dead tissue is complex and these factors together may account for the negative findings. In this study, we have implemented a simple protocol that can easily be reproduced and have reevaluated whether injection of BMCs restores the infarcted myocardium in mice and whether cell fusion is involved in tissue reconstitution. For this purpose, c-kit-positive BMCs were obtained from male transgenic mice expressing enhanced green fluorescence protein (EGFP). EGFP and the Y-chromosome were used as markers of the progeny of the transplanted cells in the recipient heart. By this approach, we have demonstrated that BMCs, when properly administrated in the infarcted heart, efficiently differentiate into myocytes and coronary vessels with no detectable differentiation into hemopoietic lineages. However, BMCs have no apparent paracrine effect on the growth behavior of the surviving myocardium. Within the infarct, in 10 days, nearly 4.5 million biochemically and morphologically differentiated myocytes together with coronary arterioles and capillary structures were generated independently of cell fusion. In conclusion, BMCs adopt the cardiac cell lineages and have an important therapeutic impact on ischemic heart failure.

Background: Adult stem cells can contribute to myocardial regeneration after ischemic injury. Bone marrow and skeletal muscles contain a population of CXCR4+ cells expressing genes specific for muscle progenitor cells that can be mobilized into the peripheral blood. The aims of the study were (1) to confirm the presence of early tissue-committed cells expressing cardiac, muscle, and endothelial markers in populations of mononuclear cells in peripheral blood and (2) to assess the dynamics and magnitude of the mobilization of CD34+, CD117+, CXCR4+, c-met+, CD34/CD117+, and CD34/CXCR4+ stem cells into peripheral blood in relation to inflammatory and hematopoietic cytokines in patients with ST-segment-elevation acute myocardial infarction (STEMI)., Methods and Results: Fifty-six patients with STEMI (<12 hours), 39 with stable angina, and 20 healthy control subjects were enrolled. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used for detection of tissue-specific markers. The number of the cells was assessed by use of a flow cytometer on admission, after 24 hours, and after 7 days. RT-PCR revealed increased expression of mRNA (up to 3.5-fold increase) for specific cardiac (GATA4, MEF2C, Nkx2.5/Csx), muscle (Myf5, Myogenin, MyoD), and endothelial (VE-cadherin, von Willebrand factor) markers in peripheral blood mononuclear cells. The number of CD34/CXCR4+ and CD34/CD117+ and c-met+ stem cells in peripheral blood was significantly higher in STEMI patients than in stable angina and healthy subjects, peaking on admission, without further significant increase after 24 hours and 7 days., Conclusions: The study demonstrates in the setting of STEMI a marked mobilization of mononuclear cells expressing specific cardiac, muscle, and endothelial markers as well as CD34/CXCR4+ and CD34/CD117+ and c-met+ stem cells and shows that stromal cell-derived factor-1 is an important factor influencing the mobilization.

Interstitial cells of Cajal (ICCs) were identified in the intact fixed media of the rabbit portal vein (RPV) using c-kit staining. The following experiments were performed using single cell preparations of the enzyme-dispersed vessel. Surviving contacts between the processes of single ICCs and the bodies of smooth muscle cells (SMCs) were observed in electron micrographs and by confocal microscopy. Spontaneous rhythmical [Ca2+]i oscillations were observed in ICCs after loading with the calcium indicator fluo-3 and were associated with depolarizations of the ICCs recorded by tight-seal patch pipette. To investigate signal transmission from ICCs to SMCs in dispersed cell pairs, or within small surviving fragments of the ICC network, an ICC was stimulated under voltage-clamp, while changes in [Ca2+]i in the stimulated cell as well as in a closely adjacent SMC or ICCs were monitored using fast x-y confocal imaging of fluo-3 fluorescence. After stimulation of single voltage-clamped ICC by a depolarizing step similar in duration to depolarizations associated with spontaneous [Ca2+]i oscillations, a depolarization and transient elevation of [Ca2+]i was observed in a closely adjacent SMCs after a delay of up to 4 seconds. In contrast, signal transmission from ICC to ICC was much faster, the delay being less than 200 ms. These results suggest that the an ICC may, in addition to generating an electrical signal (such as a slow wave) and thereby acting as a pacemaker for vascular SMCs of RPV, also release some unknown diffusible substance, which depolarizes the SMCs.

Collateral growth is characterized by macrophage accumulation, suggesting an important role of circulating cells. To study origin and function of macrophages during arteriogenesis, we related the extent of macrophage accumulation to vascular proliferation and investigated the fate of fluorescently (CMFDA) labeled blood cells that were injected at the time of femoral artery occlusion. The effect of bone marrow depletion via cyclophosphamide before femoral artery occlusion on collateral proliferation and macrophage accumulation was studied, and we looked for the presence of bone marrow-derived stem cells in the vicinity of growing collateral vessels. Finally, we investigated the arteriogenic effect of macrophage activation via MCP-1 in bone marrow-depleted animals. Maximal macrophage accumulation occurred during the first 3 days after femoral artery occlusion and paralleled the extent of vascular proliferation. Fluorescently labeled leukocytes homed to spleen and wound but they were absent in proliferating collateral arteries during maximal macrophage accumulation. Depletion of circulating cells did neither affect macrophage accumulation nor collateral growth. Staining of monocyte-depleted animals for BrdUrd and ED2, alphaSMA, or VE-Cadherin demonstrated local proliferation of macrophages and vascular cells, whereas C-Kit, SSEA1, or Thy1-positive bone marrow-derived stem cells were not detectable. Enhancement of macrophage accumulation via MCP-1 was independent of circulating monocytes and promoted arteriogenesis in the absence of direct effects on vascular cells. We propose that the initial phase of vascular growth is characterized by local proliferation of tissue resident precursors rather than by migration of blood born cells. The full text of this article is available online at http://circres.ahajournals.org.

Objective: We sought to determine the influence of thermal (burn) injury with sepsis and norepinephrine on the clonogenic potential and functional cytokine response to lipopolysaccharide (LPS) stimulation in nonmyeloid committed (CD117) and myeloid committed (ER-MP12) bone marrow progenitor cells., Summary and Background Data: We have previously demonstrated that norepinephrine stimulated myelopoiesis after burn injury and sepsis, but the site of this stimulation in monocyte development is unknown. In the present study the influence of norepinephrine on the developmental hierarchy of bone marrow cells after thermal injury and sepsis was determined by assessing the clonogenic potential and LPS-stimulated cytokine responses of mature macrophages derived from CD117 and ER-MP12 bone marrow progenitor cells., Methods: Tissue and bone marrow norepinephrine content was ablated by chemical sympathectomy with 6-hydroxydopamine treatment. CD117 and ER-MP12 bone marrow cells were isolated using antibody-linked magnetic microbeads. Clonogenic potential in response to colony-stimulating factors was determined. Both progenitor cell types were differentiated to mature macrophages in vitro and tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 cytokine responses to LPS provocation were determined., Results: The macrophage- and granulocyte-macrophage colony-stimulating factor responsive clonogenic potential was increased with burn sepsis, suggesting an expansion of both progenitor populations. Such increases were greatly reduced with prior depletion of norepinephrine. TNF-alpha and IL-6 cytokine responses to LPS were markedly influenced by the specific progenitor cells involved as well as the injury conditions and the status of norepinephrine prior to injury. In burn sepsis the depletion of norepinephrine resulted in a dramatic decrease in both IL-6 and TNF-alpha production by both progenitor-derived macrophages., Conclusions: Depletion of norepinephrine attenuated burn and burn sepsis-induced bone marrow progenitor clonal growth in response to macrophage- and granulocyte-macrophage colony-stimulating factor. Functional phenotypes of bone marrow progenitor-derived macrophages are greatly influenced by norepinephrine and the milieu created by thermal injury and sepsis.

Background: Increasing evidence points to a role for circulating endothelial progenitor cells, including populations of CD34- and CD133-positive cells present in peripheral blood, in maintenance of the vasculature and neovascularization. Immature populations, including CD34-positive cells, have been shown to contribute to vascular homeostasis, not only as a pool of endothelial progenitor cells but also as a source of growth/angiogenesis factors at ischemic loci. We hypothesized that diminished numbers of circulating immature cells might impair such physiological and reparative processes, potentially contributing to cerebrovascular dysfunction., Methods and Results: The level of circulating immature cells, CD34-, CD133-, CD117-, and CD135-positive cells, in patients with a history of atherothrombotic cerebral ischemic events was analyzed to assess possible correlations with the degree of carotid atherosclerosis and number of cerebral infarctions. There was a strong inverse correlation between numbers of circulating CD34- and CD133-positive cells and cerebral infarction. In contrast, there was no correlation between the degree of atherosclerosis and populations of circulating immature cells. Analysis of patients with cerebral artery occlusion revealed a significant positive correlation between circulating CD34- and CD133-positive cells and regional blood flow in areas of chronic hypoperfusion., Conclusions: These results suggest a possible contribution of circulating CD34- and CD133-positive cells in maintenance of the cerebral circulation in settings of ischemic stress. Our data demonstrate the utility of a simple and precise method to quantify circulating CD34-positive cells, the latter providing a marker of cerebrovascular function.

Nuclear transfer techniques have been proposed as a strategy for generating an unlimited supply of rejuvenated and histocompatible stem cells for the treatment of cardiac diseases. For this purpose, c-kit-positive fetal liver stem cells obtained from cloned embryos were injected in the border zone of infarcted mice to induce tissue reconstitution. Cloned embryos were derived from somatic cell fusion between nuclei of cultured LacZ-positive fibroblasts and enucleated oocytes of a different mouse strain. We report that regenerating myocardium replaced 38% of the scar at 1 month. The rebuilt tissue expressed LacZ and was composed of myocytes and vessels connected with the coronary circulation. Myocytes were functionally competent and expressed contractile proteins, desmin, connexin43, and N-cadherin. These structural characteristics indicated that the new myocytes were electrically and mechanically coupled. Similarly, the formed coronary arterioles and capillary structures contained blood and contributed, therefore, to tissue oxygenation. Cardiac replacement resulted in an improvement of ventricular hemodynamics and in a reduction of diastolic wall stress. These beneficial effects were obtained by stem cell transdifferentiation and commitment to the cardiac cell lineages. Myocardial growth was independent from fusion of the injected stem cells with preexisting partner cells. In conclusion, c-kit-positive stem cells derived by nuclear transfer cloning restore infarcted myocardium. Although problems currently plague nuclear transplantation, including the potential for epigenetic and imprinting abnormalities, stem cells derived from cloned embryos are sufficiently normal to repair damaged tissue in vivo. Importantly, the magnitude of myocardial regeneration obtained in this study is significantly superior to that achieved with adult bone marrow cells.

Introduction: The development of a bioreactor providing a three-dimensional network of interwoven capillary membranes with integrated oxygenation and decentralized mass exchange enables the culture of primary human liver cells from discarded donor organs for extracorporeal liver support., Methods: Primary liver cells were isolated from 54 discarded organs (donor age 56.7+/-13.2 years). Between 2.8x10(10) and 6.4x10(10) parenchymal cells (PC) were cocultured with nonparenchymal cells (NPC) of the same organ in bioreactors (n=36). The metabolic activity of the cells was regularly determined during culture. The cell morphology and ultrastructure were investigated after culture periods of 1 to 5 weeks., Results: Cell metabolism was maintained over at least 3 weeks after a phase of adaptation lasting 2 to 3 days. Through the use of transmission electron microscopy and immunohistochemistry, it was demonstrated that PC and NPC spontaneously formed tissue-like structures. Vascular cavities (CD 31 immunoreactivity [IR]) and bile duct-like channels (CK 19 IR), both exhibiting proliferation activity (Ki-67 IR), were regularly distributed. Some of the bile duct-like channels showed similarities to the Canals of Hering found in the natural liver. Cells expressing morphologic and antigenic characteristics of adult liver stem cells (CD 34 IR and c-kit IR) and areas with cells that showed both hepatocyte and biliary characteristics were detected., Conclusion: The results show that primary human liver cells obtained from discarded donor organs recover and can be maintained in bioreactors for clinical liver support therapy. In addition, initial observations on adult liver stem-cell culture in bioreactors are presented.

Background: Murine umbilical cord blood cells (UCBCs) were studied for their ability to reconstitute the hematopoietic system., Methods: On average, 150 microL of cord blood per fetus containing 1.2 to 2 x 10(4) nucleated cells were collected from day 18.5 fetal umbilical cord, and 3 to 6 x 10(3) cells per fetus were obtained after separation by gradient centrifugation., Results: Although lineage marker-, c-Kit+, and Sca-1+ cells were detectable among UCBCs, cells designated to be in the side population (SP) by Hoechst 33342 staining were hardly detectable within this population; the frequency of cells of this phenotype was less than 1 of 105. Instead, the lineage marker-, c-Kit+, and Sca-1- population contained a considerable number of SP cells. Nevertheless, UCBCs obtained from fetuses of green fluorescent protein-transgenic mice successfully reconstituted the blood cells of lethally irradiated recipients. Fluorescent cells could be readily detected in every blood cell lineage and among immature cell populations. Furthermore, fluorescent SP cells sorted from the recipient bone marrow cells could also reconstitute the blood cells in the secondary recipients, indicating that UCBCs also replenished bone marrow stem cells., Conclusion: Murine UCBC could fully reconstitute the hematopoietic system of lethally irradiated recipients including hematopoietic stem cells in bone marrow.

CD 117 (KIT) is a transmembrane, tyrosine kinase growth factor receptor which is expressed on numerous diverse fetal and adult cells including hematopoietic cells, mast cells, melanocytes, germ cells, and the interstitial cells of Cajal. Its expression in tumors is also diverse, but with selective use and attention to specific staining patterns, this marker is useful in the identification of gastrointestinal stromal tumors, mast cell tumors, and seminomatous germ cell tumors.

Neurofibromatosis type 1 could be associated with multiple gastrointestinal stromal tumours, although their presence is not considered among the major diagnostic criteria. We present here a case of a 50-year-old female complaining of abdominal pain, with about 100 small intestinal stromal tumours. This finding prompted us to suspect a neurofibromatosis which was clinically confirmed afterwards. Light microscopy examination revealed a low-grade stromal tumour with skeinoid fibres. Mixed neural-interstitial cells of Cajal origin or, alternatively, neural differentiation of interstitial cells of Cajal are discussed on the basis of immunophenotype (CD117+, CD34+) and ultrastructure. A 2-year follow-up did not indicate an aggressive course in the case of this neoplasm.

Background: To date, over 40 in utero transplants have been performed in humans; the only successes were documented in the treatment of severe combined immunodeficiency syndromes. Hemoglobinopathies and metabolic disorders are candidate diseases for this approach; however, when applied clinically, the results have been discouraging. To address the role of the fetal immune system in the outcome of in utero transplantation, we have developed a murine model of in utero transplantation in immunologically intact murine recipients and have studied chimerism and tolerance/immunity to allogeneic donor cells through the lives of the animals., Methods: We have performed experiments in which purified murine sca-1+/lin- cells and c-kit+/lin- cells of C57BL/6 (H2b) mice were injected into Balb/c (H2d) fetal recipients at early gestational ages. Chimerism was tested by highly sensitive semiquantitative polymerase chain reaction assay and tolerance/immunity to donor cells was studied by in vivo (skin grafts, responses to postnatal boosts) and in vitro (mixed lymphocyte culture, cytotoxicity, and cytokine release) assays., Results: One hundred percent (10/10) of mice transplanted with c-kit+ cells and 44% (4/9) of mice transplanted with sca+ cells showed circulating donor cells within the first 6 months of life (P=0.031). Mice in the sca+ group rejected donor skin grafts at a mean time of 9.1+/-0.2 days, whereas mice in the c-kit+ group rejected donor skin grafts at a mean time of 15.1+/-0.7 days (P=0.001). The difference between the transplanted groups and non-transplanted controls was also significant (P<0.05). All mice transplanted with sca+/lin- cells showed greater response to donor cells than to third-party cells at all effector to target ratios (P=0.002). Differences in response to donor alloantigen between sca+ and c-kit+ groups were significant (P=0.003). Cytokine quantification demonstrated higher TH1 than TH2 cytokine release in all groups, and the response to donor cells was higher in the sca+ compared with c-kit+ mice (P=0.031)., Conclusion: These results demonstrate a low level of chimerism and tolerance in mice transplanted in utero with sca+/lin- and c-kit+/lin- cells. The possibility of active in utero immunization to donor cells is supported by accelerated skin graft rejection in mice transplanted with sca+ cells and enhanced in vitro immune responses in mice with persistent microchimerism.

c-kit protooncogene encodes a type III transmembrane receptor kinase, the stem cell factor receptor, or KIT. The ligand of the KIT. stem cell factor, is a cytokine that stimulates mast cell growth and differentiation. We have studied immunohistochemically KIT expression in 23 canine mast cell tumors (MCTs), 10 histiocytomas, 5 malignant melanomas, and in 2 cell lines derived from mast cells (HMC-1, human and C2, canine). As expected, KIT was detected both in the human mast cell leukemia cell line (HMC- ) and in the canine mastocytoma cell line C2. In normal canine skin, KIT expression was confined to mast cells. All canine MCTs expressed KIT, although the intensity of the staining reaction varied considerably among the 23 neoplasms. Grade III tumors showed the highest expression of KIT, whereas grade I tumors showed the lowest expression of KIT. Two patterns of KIT expression were detected in mast cells. In normal canine mast cells and in some neoplastic mast cells, KIT appeared mainly on the cell membrane. However, in many canine MCTs, KIT is accumulated in the cytoplasm, usually near the cell nucleus. The meaning of these two patterns is not clear. Expression of KIT could not be detected immunohistochemically in any of the other neoplasias investigated. According to our results, it can be concluded that most, if not all, canine MCT express KIT. Furthermore, there is an inverse correlation between the degree of differentiation and the expression of KIT. Moreover, according to our results, KIT can be used as a reliable immunohistochemical marker for canine mast cells and undifferentiated mast cell tumors.

To investigate the role of c-KIT receptor in melanocytic tumour development and progression, we analysed the expression and localization of c-KIT by immunohistochemistry and Western blotting. In contrast to the positive staining shown by melanocytes and naevus cells in the epidermis of common naevi (n=20), all dysplastic naevi (n=13) were negative, as were dermal melanocytic cells of blue naevi (n = 4) and common naevi (n = 26). Three out of four superficial spreading melanomas lost c-KIT expression both in the epidermal and dermal parts, while nodular melanomas showed no expression of c-KIT except in partially positive cells, and six out of seven metastatic melanomas were negative. In acral lentiginous melanomas (n = 8), in contrast to other types of melanoma, all cases with melanoma cells growing basally in the epidermis showed strong c-KIT positivity, but melanoma cells growing at the upper layers of the epidermis and vertically into the dermis lost c-KIT expression. Using the Western blot method on cultured pigment cells, human epidermal melanocytes, junctional naevus cells and one out of three metastatic melanoma cell lines showed 125 and 145 kDa bands corresponding to c-KIT, whereas dermal naevus cells did not. These results suggest that dysplastic naevi are distinct from ordinary naevi in terms of c-KIT expression and that basally growing cells in acral lentigenous melanomas could be at an initial stage of tumour progression, before c-KIT loss occurs.