Transitional Cell Type Research Articles

Abstract PR014: Macrophage induced epithelial to mesenchymal transition in sarcomatoid renal cell carcinoma

Abstract Intro: Sarcomatoid de-differentiation in renal cell carcinoma (sRCC) leads to aggressive tumors that may be uniquely responsive to adjuvant immunotherapy. sRCC is thought to arise by epithelial to mesenchymal transition (EMT) of the parental tumor, most commonly clear cell RCC (ccRCC). However, factors that drive sRCC are unknown and no biomarkers exist to predict the transition. Furthermore, the immune microenvironment in sRCC is not well understood and may provide additional therapeutic targets. Here, we use spatial biology and in vitro studies to explore EMT/immune cell crosstalk in sRCC. Methods: Single cell spatial transcriptomics was performed on a human sRCC specimen with Nanostring CosMx platform. Semi-supervised clustering referenced to scRNAseq data was used to define cell types and map them spatially. Differential gene expression and cell distance analysis were performed. Multiplex immunofluorescent staining (mIF) was performed by Vectra Polaris. In vitro work used ccRCC cell line, 786-O, and sRCC cell line, UOK-127. M0, M1, or M2 macrophages were derived from THP-1 cells. Analysis of macrophage markers and EMT markers were performed by western blot and RT-qPCR. Results: Single cell spatial transcriptomic data revealed a ccRCC population, 2 sRCC cell types, and a novel transitional cell type along the EMT continuum and spatially between ccRCC and sRCC. Importantly, matched H&E shows the transitional cell type present in areas histologically defined as ccRCC, demonstrating molecular evidence of transition towards sRCC prior to histologic changes. Cell to cell distance revealed strong correlation of M2 macrophages with transitional and sRCC cells. The ccRCC population expressed CCL20, a factor known to assist in macrophage recruitment, and CCL20 was upregulated in the transitional cell type. FZD4 was the highest fold changed gene lost from the ccRCC to transitional cells. In vitro culture of RCC cells with M2 macrophages led to CCL20 upregulation and loss of FZD4. Overexpression of CCL20 and knockdown of FZD4 in RCC cells both led to EMT. Exposure of macrophages to CCL20 led to M2 polarization. To validate the role of these genes, analysis of the human protein atlas (877 RCC specimens) revealed high expression of CCL20 and low expression of FZD4 are both associated with poor survival. mIF of 20 human sRCC specimens demonstrated CD163 (M2 macrophage marker) was associated with sRCC or ccRCC areas with high expression of mesenchymal marker N-cadherin. Conclusion: We report the first detection of a transitional cell type along the de-differentiation pathway from ccRCC to sRCC. This cell type is detectable in areas histologically defined as ccRCC, demonstrating strong potential as a transcriptional biomarker to inform adjuvant immunotherapy. We show that CCL20 on tumor cells leads to macrophage recruitment and polarization, resulting in FZD4 loss and CCL20 upregulation, which induce EMT and propagates a vicious cycle of EMT/macrophage crosstalk in sRCC. This suggests potential for therapeutic targeting of macrophages or CCL20 in RCC. Citation Format: Allison May, Claire Williams, Stephanie The, Jake McGue, Sean Kim, Nathan Schurman, Greg Shelley, Tyler Robinson, Simpa Salami, Rohit Mehra, Timothy Frankel, Aaron Udager, Evan Keller. Macrophage induced epithelial to mesenchymal transition in sarcomatoid renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR014.

Modeling fibrotic alveolar transitional cells with pluripotent stem cell-derived alveolar organoids.

Repeated injury of the lung epithelium is proposed to be the main driver of idiopathic pulmonary fibrosis (IPF). However, available therapies do not specifically target the epithelium and human models of fibrotic epithelial damage with suitability for drug discovery are lacking. We developed a model of the aberrant epithelial reprogramming observed in IPF using alveolar organoids derived from human-induced pluripotent stem cells stimulated with a cocktail of pro-fibrotic and inflammatory cytokines. Deconvolution of RNA-seq data of alveolar organoids indicated that the fibrosis cocktail rapidly increased the proportion of transitional cell types including the KRT5 - /KRT17 + aberrant basaloid phenotype recently identified in the lungs of IPF patients. We found that epithelial reprogramming and extracellular matrix (ECM) production persisted after removal of the fibrosis cocktail. We evaluated the effect of the two clinically approved compounds for IPF, nintedanib and pirfenidone, and found that they reduced the expression of ECM and pro-fibrotic mediators but did not completely reverse epithelial reprogramming. Thus, our system recapitulates key aspects of IPF and is a promising system for drug discovery.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

Cell lineage specification and signalling pathway use during development of the lateral plate mesoderm and forelimb mesenchyme.

The lateral plate mesoderm (LPM) is a transient tissue that produces a diverse range of differentiated structures, including the limbs. However, the molecular mechanisms that drive early LPM specification and development are poorly understood. In this study, we use single-cell transcriptomics to define the cell-fate decisions directing LPM specification, subdivision and early initiation of the forelimb mesenchyme in chicken embryos. We establish a transcriptional atlas and global cell-cell signalling interactions in progenitor, transitional and mature cell types throughout the developing forelimb field. During LPM subdivision, somatic and splanchnic LPM fate is achieved through activation of lineage-specific gene modules. During the earliest stages of limb initiation, we identify activation of TWIST1 in the somatic LPM as a putative driver of limb bud epithelial-to-mesenchymal transition. Furthermore, we define a new role for BMP signalling during early limb development, revealing that it is necessary for inducing a somatic LPM fate and initiation of limb outgrowth, potentially through activation of TBX5. Together, these findings provide new insights into the mechanisms underlying LPM development, somatic LPM fate choice and early initiation of the vertebrate limb.

To Study the Association of Cyclin D1 Expression with Fuhrman’s Nuclear Grading in Renal Cell Carcinoma

Background: In adults, renal cell cancer (RCC) consists of tumors arising from the renal parenchyma and renal pelvis. Renal parenchyma cancer is mainly renal cancer i.e. adenocarcinoma. Renal pelvis malignancy is mainly transitional cell type. Presently, prognostic markers for renal cell cancer are tumor stage and nuclear grade. Aim: To observe the expression of cyclinD1 in different variants of RCC and to associate cyclinD1 expression with Fuhrman nuclear grades of RCC. Results: Out of 93 patients, 54(58.1%) were of more than 40 years of age, with M: F ratio of 1: 1. Clear cell RCC was the most common histological type i.e. 72out of 93(77.4%) biopsies. Low grades RCC were 50 out of 93(53.8%) and High grade RCC were in 43 out of 93(46.6%) biopsies and out of these 43 high grade RCC 23(24.7%) were found to invade surrounding tissue. As far as CyclinD1 expression score was concerned, 60 out of 93(64.4%) biopsies were found to have score 1 and 2 with no and upto30% +ve cells and 33(35.5%) biopsies were of score 3 and 4 with 30-60% and >60% positive cells respectively. In this study, 28 out of 93(30.2%) biopsies of low Fuhrman’s nuclear grade showed cyclin D1 score 3 and 4 respectively. Conclusion: In our study cyclin D1 protein was over expressed in low grade RCC showing that high protein levels may be related with the disease progression in patients of RCC. Cyclin D1 protein expression in future can be used as adjuvant to Fuhrman’s nuclear grading and will increase the predictive accuracy of the current disease progression models for RCC. Keywords: CyclinD1, RCC, Low and High Fuhrman nuclear grade

Investigating the Transitional Cell Type in the Kidney Collecting Duct

The kidney cortical collecting duct (CCD) has long been thought to contain two distinct cell types, principal cells (PC) and intercalated cells (IC), which facilitate 4-5% of sodium and 10% of water reabsorption in the kidney. Recent single cell RNA-Seq data has identified a distinct transitional cell expressing both PC and IC markers and a number of distinct markers. In this study, the presence of CCD cell types was investigated in mice and rats under the disease model of the syndrome of apparent mineralocorticoid excess (SAME, conferred by a mutation of the Hsd11b2gene, expressed by PCs) and also in two CCD derived cell lines. Inactivation of HSD11B2in humans results in a severe, and often fatal, phenotype in early neonatal life, whilst milder mutations, with residual enzyme activity cause hypertension, sodium retention and hypokalemia in adulthood. The Hsd11b2 gene knockout (KO) model of SAME in mice and rats was studied in the context of CCD cell plasticity and it was hypothesised to reduce the proportion of PCs within the CCD. Cells in the CCD were counted in WT and Hsd11b2 KO animals following immunofluorescent staining for PC- and IC-specific markers (Aqp2 and V-ATPase B1, respectively) in paraffin-embedded kidney sections. ImageJ and GraphPadPrism were used to analyse data, which are expressed as mean ± SEM. Significance was assessed using Tukey's multiple comparisons test, p<0.0001. PC proportion was significantly reduced in KO mice CCDs compared to controls at 4 weeks (52.1 ± 1.9 % vs. 73.0 ± 1.9 %) and 17 weeks (54.1 ± 1.8 % vs. 72.2 ± 1.3 %). A significant increase in the number of cells expressing both PC and IC markers was observed between WT and KO at 4 weeks (8.4 ± 1.2 % vs.25.9 ± 2.0 %) and at 17 weeks (6.5 ± 0.8 % vs. 22.1 ± 2.0 %). Similarly, in the rat KO a significant reduction in the proportion of PCs was noted compared to controls at 5 weeks (60.69 ± 2.51 % vs. 73.30 ± 1.84 %) and 24 weeks (38.40 ± 2.07 % vs. 66.80 ± 1.94 %). This study shows CCD cell plasticity in models of SAME, but also provides further evidence for a PC+/IC+cell in the kidney. The mCCDcl1­ and mpkCCDcl4­ cell lines are typically used as models of PC function. Immunofluorescent staining using antibodies against PC, IC and transitional cell markers (Aqp2, V-ATPase B1 and Parm1, respectively) was carried out on fixed, polarised cells and images analysed using ImageJ software. It was found that both cell line lines contained cells that co-expressed PC and IC markers, similar to the cell type seen in mouse and rat kidneys. Furthermore, a large proportion of cells in both cell lines expressed the transitional cell marker, Parm1. These results provide further support for the presence of a transitional cell type in the CCD, especially in pathophysiological states. The observation of similar cells in CCD cell lines also indicates their use as in vitro models for investigating the role and plasticity of CCD cell types.

Single-Cell Transcriptomics Reveal Immune Mechanisms of the Onset and Progression of IgA Nephropathy

IgA nephropathy (IgAN) is the leading cause of kidney failure due to an incomplete understanding of its pathogenesis. We perform single-cell RNA sequencing (RNA-seq) on kidneys and CD14+ peripheral blood mononuclear cells (PBMCs) collected from IgAN and normal samples. In IgAN, upregulation of JCHAIN in mesangial cells provides insight into the trigger mechanism for the dimerization and deposition of IgA1 in situ. The pathological mesangium also demonstrates a prominent inflammatory signature and increased cell-cell communication with other renal parenchymal cells and immune cells, suggesting disease progress from the mesangium to the entire kidney. Specific gene expression of kidney-resident macrophages and CD8+ Tcells further indicates abnormal regulation associated with proliferation and inflammation. A transitional cell type among intercalated cells with fibrosis signatures is identified, suggesting an adverse outcome of interstitial fibrosis. Altogether, we systematically analyze the molecular events in the onset and progression of IgAN, providing a promising landscape for disease treatment.

Neuronal metaplasia: A pituitary adenoma with gangliocytic component and growth hormone (GH) secretion

BackgroundPituitary adenomas (PA) possessing neuronal components are rare. These tumors are usually benign and arise within the sellar region. Most are associated with acromegaly and few with Cushing disease or hyperprolactinemia. Few studies have focused on the origin and presence of neuronal components in the sellar region.Clinical caseA 53‐year‐old woman developed headaches and numbness on one side of her face. Initial work‐up found significant enlargement and/or elongation over the past five years to her hands, feet and face, along with blurred/double vision. She had a history of hypertension, polyuria, and polydypsia. MRI revealed a sellar/suprasellar mass extending toward the cavernous regions and into the chiasm. The lesion was interpreted as a pituitary macroadenoma with mass effect on the optic chiasm and optic nerves.Based on the clinical and radiologic findings, she was diagnosed with acromegalic features secondary to macroadenoma and was started on Octreotide. One month after starting drug treatment the patient underwent transsphenoidal surgery. Morphologic examination of the tissue revealed a homogenous population of predominantly acidophilic tumor cells with focal chromophobic cells. The tumor was admixed with ganglion cells with acidophilic cytoplasm, embedded in fibrillar, neuropil‐like matrix. Immunohistochemistry (IHC) of the tumor, showed intense positivity for GH. Cytokeratin immunostaining showed a distinct pattern of spherical paranuclear fibrous bodies in most cells. Multiple focal areas of neuropil populated with multi‐nucleated Nissl stained ganglion cells were noted. IHC showed that the ganglion cells expressed Hu, as well as variable immunopositivity for multiple pituitary hormones. The neuropil was also positive for neurofilament protein. Microscopic evaluation of the histologic and IHC findings, along with ultrastructural analysis, were characteristic of a sparsely granulated GH‐producing PA with a neuronal component corresponding to neuronal metaplasia.ConclusionAt present, this entity is classified as “ganglicytoma” by the WHO. The name PA with neuronal metaplasia is a more appropriate designation than the other terminologies currently used. Analysis of published data is indicative that such a lesion is likely to have developed from uncommitted progenitor cells within the PA. Metaplasia best explains the presence of transitional cell types as indicated by the histological presence of NFP, Hu, and Nissl with production of multiple pituitary hormones in our sparsely granulated GH‐producing PA. The neuropil herein must have formed from a defective progenitor cell existing within the pituitary tissue. The exact relationship between GH‐producing PAs and the presence of neuronal cells within it is perplexing. The pathogenesis and nosology of PA with neuronal components is not yet fully resolved and further study is necessary to explore the cause, progression and influencing factors of this disease.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Granular Cell Tumor of the Neurohypophysis: 3 Cases and a Systematic Literature Review of 98 Cases

Granular cell tumors (GCTs) of the neurohypophysis are rare neoplastic diseases of the pituitary. Because of the rare nature of the disease, previous descriptions have been limited to single-case studies, small series, or simple reviews. We reported 3 cases in our medical center and systematically reviewed 98 cases who met inclusion criteria. Data were gathered on different aspects of GCTs' characteristics, including demographics, clinical features, laboratory features, histological features, immunocytochemical features, radiologic features, and treatment to elucidate any associations useful in determining pathogenesis and appropriate treatment. Female sex was significantly associated with GCTs (P < 0.001). The most common presenting symptom and tentative diagnosis were ophthalmologic defects in 61.2% and pituitary adenoma preoperatively in 18.4%, respectively. Most neurohypophysial GCTs possess immunopositivity for S-100 protein (59.1%, 26/44) and negative staining for glial fibrillary acidic protein (69.8%, 30/43). These findings lend strong support to the suggestion that tumors probably remain in the transitional cell type. To our knowledge, our systematic review presents the largest number of symptomatic cases ever enumerated, with 98 cases meeting the inclusion criteria.

Updates on FIGO Staging Classification and the New WHO Classification for Cancer of the Ovary

Background: The knowledge of different subtypes of ovarian cancer is improving with the progress in molecular pathological research. The WHO classification was revised,in parallel with the implementation of the new FIGO staging classification. The former is mainly based on the histopathological findings and defines the actual type of tumor. It has an important impact on prognosis and therapy of the patient.&#x0D; Materials and methods: FIGO staging classification for cancer of the ovary, fallopian tube, and peritoneum published by Jaime Prat and the new WHO Classification of Ovarian Cancer published by Robert Kurman and coauthors in 2014 are summarized.&#x0D; Results: The International Federation of Gynecology and Obstetrics recently significantly revised staging criteria for cancer of the ovary. The latest revision was based on the concept that high-grade serous tubal intraepithelial carcinoma (STIC) may be the origin of some high-grade serous carcinomas of the ovary and peritoneum. Therefore, staging criteria for the ovary, fallopian tube, and peritoneum have been unified. Understanding this background and other important revised points are essential. The previous focus of mesothelial origin of ovarian cancer has been eliminated in new classification. Instead, a discussion of tubal carcinogenesis of hereditary and some other high-grade serous carcinomas is featured. Regarding serous cancers, the previously assumed pathogenesis pathway may be correct for some, but not for all. The earlier transitional cell type of ovarian cancer has been removed while seromucinous tumors have been added as a new entity. The role of some borderline tumors as one possible step in the progression from benign to invasive lesions is incorporated. The article summarizes the essential updates concerning serous, mucinous, seromucinous, endometrioid, clearcell, and Brenner tumors.&#x0D; Conclusion: The new WHO classification takes into account the recent findings on the origin, pathogenesis, and prognosis of different ovarian cancer subtypes. In both FIGO and WHO classification, the tubal origin of hereditary and some non-hereditary high-grade serous cancers is mentioned in contrast to the hitherto theory of mesothelial origin of tumors. Seromucinous tumors represent a new entity.&#x0D; Bangladesh J Obstet Gynaecol, 2017; Vol. 32(1): 33-41

The diversity and plasticity of adult hepatic progenitor cells and their niche.

The liver is a unique organ for homoeostasis with regenerative capacities. Hepatocytes possess a remarkable capacity to proliferate upon injury; however, in more severe scenarios liver regeneration is believed to arise from at least one, if not several facultative hepatic progenitor cell compartments. Newly identified pericentral stem/progenitor cells residing around the central vein is responsible for maintaining hepatocyte homoeostasis in the uninjured liver. In addition, hepatic progenitor cells have been reported to contribute to liver fibrosis and cancers. What drives liver homoeostasis, regeneration and diseases is determined by the physiological and pathological conditions, and especially the hepatic progenitor cell niches which influence the fate of hepatic progenitor cells. The hepatic progenitor cell niches are special microenvironments consisting of different cell types, releasing growth factors and cytokines and receiving signals, as well as the extracellular matrix (ECM) scaffold. The hepatic progenitor cell niches maintain and regulate stem cells to ensure organ homoeostasis and regeneration. In recent studies, more evidence has been shown that hepatic cells such as hepatocytes, cholangiocytes or myofibroblasts can be induced to be oval cell-like state through transitions under some circumstance, those transitional cell types as potential liver-resident progenitor cells play important roles in liver pathophysiology. In this review, we describe and update recent advances in the diversity and plasticity of hepatic progenitor cell and their niches and discuss evidence supporting their roles in liver homoeostasis, regeneration, fibrosis and cancers.

The new WHO classification of ovarian, fallopian tube, and primary peritoneal cancer and its clinical implications.

Molecular pathological research has contributed to improving the knowledge of different subtypes of ovarian cancer. In parallel with the implementation of the new FIGO staging classification, the WHO classification was revised. The latter is mainly based on the histopathological findings and defines the actual type of tumor. It has, therefore, also an important impact on prognosis and therapy of the patient. The new WHO Classification of Ovarian Cancer published 2014 by Robert Kurman and co-authors is summarized. The major changes compared to the hitherto existing classification are presented. The new classification eliminates the previous focus of mesothelial origin of ovarian cancer. Instead, it features a discussion of tubal carcinogenesis of hereditary and some other high-grade serous carcinomas. The previously assumed pathogenesis pathway may be correct for some, but not for all, serous cancers. The new classification was established to classify ovarian cancer in a more consistent way. The earlier transitional cell type of ovarian cancer has been removed while seromucinous tumors have been added as a new entity. The role of some borderline tumors as one possible step in the progression from benign to invasive lesions is incorporated. The article summarizes the essential updates concerning serous, mucinous, seromucinous, endometrioid, clear-cell, and Brenner tumors. The new WHO classification takes into account the recent findings on the origin, pathogenesis, and prognosis of different ovarian cancer subtypes. The tubal origin of hereditary and some non-hereditary high-grade serous cancers is mentioned in contrast to the hitherto theory of mesothelial origin of tumors. Seromucinous tumors represent a new entity.

Evolutionary implication of B-1 lineage cells from innate to adaptive immunity.

The paradigm that B cells mainly play a central role in adaptive immunity may have to be reevaluated because B-1 lineage cells have been found to exhibit innate-like functions, such as phagocytic and bactericidal activities. Therefore, the evolutionary connection of B-1 lineage cells between innate and adaptive immunities have received much attention. In this review, we summarized various innate-like characteristics of B-1 lineage cells, such as natural antibody production, antigen-presenting function in primary adaptive immunity, and T cell-independent immune responses. These characteristics seem highly conserved between fish B cells and mammalian B-1 cells during vertebrate evolution. We proposed an evolutionary outline of B cells by comparing biological features, including morphology, phenotype, ontogeny, and functional activity between B-1 lineage cells and macrophages or B-2 cells. The B-1 lineage may be a transitional cell type between phagocytic cells (e.g., macrophages) and B-2 cells that functionally connects innate and adaptive immunities. Our discussion would contribute to the understanding on the origination of B cells specialized in adaptive immunity from innate immunity. The results might provide further insight into the evolution of the immune system as a whole.

Neutrophil-lymphocyte ratio as a predictor of recurrence and progression in patients with high-grade pT1 bladder cancer.

We investigated the value of the preoperative neutrophil-lymphocyte ratio (NLR) in predicting recurrence and progression of high-grade pT1 non-muscle-invasive tumour in patients with bladder cancer during a 5-year follow-up period. We retrospectively reviewed data of 1100 patients with bladder cancer; these patients underwent transurethral resection and were monitored at multiple centres from 2008 to 2013. In total, 166 consecutive and newly diagnosed patients with high-grade pT1 tumours were included in this study. The NLR was calculated by dividing the absolute neutrophil count by the absolute lymphocyte count. Of the 166 patients, 152 were male. The patients were evaluated as two separate groups in terms of recurrence and progression. The mean follow-up period was 24.2 months (interquartile range 13.8-36.6 months). A statistically significant difference was found between recurrence and tumour size (p = 0.001), number of tumours (p < 0.001), NLR (p < 0.001), and smoking (p = 0.007). No statistically significant correlation was found between NLR and progression. According to receiver operating characteristic (ROC) analysis, the optimum cut-off value for the NLR was ≥2.43 (74% sensitivity, 60% specificity, p < 0.001; area under the curve [AUC] 0.687, 95% confidence interval [CI] 0.607-0.767). Multivariate logistic regression analysis determined that the following factors were independent predictors of recurrence in patients with high-grade pT1 non-muscle-invasive bladder cancer: tumour number (OR 5.32, 95% CI 2.10-12.90), NLR of ≥2.43 (OR 2.587; 95% CI 1.156-5.789), and smoking (OR 4.17, 95% CI 1.31-13.21). A high preoperative NLR may play an important role in predicting recurrence of superficial transitional cell type high-grade pT1 bladder tumours. Prospective studies are required to validate the role of NLR as a prognostic marker in high-grade pT1 bladder tumours.

Impact of density of schistosomal antigen expression in urinary bladder tissue on the stratification, cell type, and staging, and prognosis of carcinoma of the bladder in Egyptian patients.

BackgroundInfection with urinary schistosomiasis and its severity are oncogenic factors for developing carcinoma of the bladder, whether it is urothelial carcinoma (UC) of a transitional cell type (TCC) or non-urothelial of squamous cell carcinoma (SCC). In UC it is not defined whether it is schistosomal or not. This led to controversial results in expression of tumour markers, tumour prognosis, and response to therapy.ObjectivesWe assessed the application by immunohistochemistry method (IHC) for detection of schistosomal antigen in bladder cancer tissue samples to differentiate UC associated with or without schistosomiasis. Urothelial carcinoma stage, grade, and progression were correlated with the density, intensity, and index of schistosomal antigen expression. Follow up was done for 2-5 years.Design and participantsArchival tissue samples of 575 patients were studied: 515 urothelial carcinoma, 30 patients with SCC associated with schistosomiasis, and a control group of 30 patients without schistosomiasis.MeasurementsExpression of schistosomal antigen in tissue was done by IHC using monoclonal antibodies (MAbs) against schistosomal antigens (SA). Correlation of intensity of antigen expression to clinical and pathological data was analysed.Results and limitationsWe identified 3 parameters of antigen expression: density, intensity and index with 4 grades for each. SCC-group was 100% positive. UC was positive in 61.4% distributed as follows: Ta: 37.5%, T1: 62%, and muscle invasive T2-4 were 64%. Upstaging, metastases and recurrence were correlated with high index in T1 and T2-4 tumours.ConclusionUrothelial carcinoma associated with schistosomiasis was defined by the positive expression of schistosomal antigens in tissues detected by lHC using MAbs against schistosomal haematobium. Upstaging and progression of T1 and T2-4 were correlated with high density, intensity, and index of antigen expression. Non-schistosomal UC had negative expression for schistosomal antigen, which was detected in 36.5% of cases. These results would be of significance in differentiating schistosomal from non-schistosomal bladder cancer of UC and would predict the prognosis in T1, T2-3 tumours. Implementation of IHC using MAbs against SA in UC would help in planning the proper therapy. Schistosomiasis should be considered as an oncogene for UC in endemic areas.

Genetic alterations of chromosomes, p53 and p16 genes in low- and high-grade bladder cancer.

A majority of patients with bladder cancer present with superficial disease and subsequently, some patients show progression to muscle invasive or metastatic disease. Bladder cancer has a complex genetic process and identification of the genetic alterations which occur during progression may lead to the understanding of the nature of the disease and provide the possibility of early treatment. The aim of the present study was to compare the structural and numerical chromosomal differences and changes in the p16 and p53 genes between low-grade (LG) and high-grade (HG) bladder cancer (BC) using cytogenetic and molecular cytogenetic methods. Between March 2009 and March 2010, cytogenetic analyses were carried out on tumor and blood samples in 34 patients with transitional cell type BC, and on blood samples of 34 healthy patients as a control group. Fluorescence in situ hybridization probes for the p16 and p53 genes were also used to screen the alterations in these genes in 32 patients with BC. The patients were divided into two groups (LG and HG) and the findings were compared. A total of 11 (32.3%) patients exhibited LGBC, 22 (64.7%) exhibited HGBC and one (3%) patient exhibited carcinoma in situ. There were no differences between the LGBC and HGBC groups according to the number of chromosomal aberrations (P=0.714); however, differences between alterations of the p16 and p53 genes were significant (P=0.002 and P=0.039). Almost all structural abnormalities were found to be located to the 1q21, 1q32, 3p21 and 5q31 regions in patients with HG tumors. In conclusion, the p16 and p53 genes were altered more prominently in patients with HG tumors compared with LG tumors. The structural abnormalities in the 1q21, 1q32, 3p21 and 5q31 regions were observed more frequently in patients with HG tumors. These regions may play significant roles in the progression of BC, but further studies are required to find candidate genes for a panel of BC.

English

A 30-year old female presented with a 8-month history of left nasal obstruction associated with recurrent episodes of epistaxis. Left nasal cavity was filled with polypoidal mass. Computed tomography revealed a mass present in the left nasal cavity, appears to be arising from ethmoidal air cells and lateral nasal wall. Diagnostic Nasal Endoscopy was done and biopsy was taken. Report came as sinonasal malignancy of transitional cell type. Endoscopy assisted, surgical excision of the mass was done and later she was referred higher centre for further management. Non-keratinizing carcinoma (Transitional type) of the sinonasal tract is a rare entity and there are only few reports concerning this type of malignancy. This may be partly because of its diagnostic difficulty due to many synonyms such as Schneiderian carcinoma, cylindrical cell carcinoma and transitional cell carcinoma.

Signaling and Immunophenotypic Diversity in Pediatric Acute Myeloid Leukemia As Defined by 31-Parameter Single-Cell Mass Cytometry

Abstract 2565Acute myeloid leukemia (AML) encompasses a broad spectrum of cellular morphologies. This spectrum exists both within each patient in the form of a malignant developmental hierarchy, and across patients in the form of distinct disease classes with distinguishing mutational or pathological hallmarks. Flow cytometry has long been an essential tool for classifying AML surface markers, and has enabled improved diagnosis, minimal residual disease monitoring, and cell sorting for functional studies. Previous work by our laboratory and others (Irish et al., Cell 2004; Kornblau et al., Clin. Cancer Res. 2010) has shown that clinically relevant heterogeneity also exists at the level of single-cell intracellular signaling capabilities, particularly in response to ex vivo perturbations. Due to the constraints of fluorescent flow cytometry, the limited number of simultaneous parameters measured per cell in these studies was limited (e.g., 3–6). We hypothesized that there may exist additional layers of heterogeneity in both surface marker phenotypes and signaling responses which were invisible to the low-parameter systems used in these studies.To address this challenge, we used a next-generation 31-parameter ‘mass cytometry' platform to survey the diversity of signaling responses and their relationship to subsets defined by surface marker patterns. We measured the simultaneous co-expression of 16 surface markers and 15 dynamic intracellular signaling epitopes (e.g. phosphorylated kinase substrates) in a cohort of pediatric AML diagnosis bone marrow samples (n=18) and healthy adult bone marrow controls (n=3). Signaling dynamics were measured under 18 stimulation conditions, including a battery of cytokines, chemical stimuli, and small molecule kinase inhibitors. The resulting single-cell data was overlaid and compared using a new, unsupervised flow cytometry analysis and visualization program–SPADE (Spanning-tree Progression Analysis of Density-Normalized Events)–which links rare, transitional, and abundant cell types alike along a branching tree organized by phenotypic progression. Thus, we distilled the diversity of cellular phenotypes, their relative frequencies, and the corresponding signaling dynamics of each cell population onto a single graph structure representing the distinct and overlapping expression patterns in the 16-dimensional “immunophenotypic space” explored by the 21 individuals.Striking signaling and immunophenotypic diversity was observed between the AML samples, particularly in response to pervanadate stimulation. Pervanadate liberates intracellular kinase cascades by broadly inhibiting tyrosine phosphatases, and thus reveals the cell’s potential for kinase activity and the interplay of signaling circuits in different cell populations. Interestingly, two transcription factors implicated in AML pathogenesis – CREB and c-Cbl–often exhibited mutually exclusive phosphorylation patterns in different subsets of primitive (CD34+) cells. Specifically, CREB was readily phosphorylated at Ser133 upon pervanadate exposure in the CD34+CD123- cells, while c-Cbl phosphorylation at Tyr700 was restricted to CD34+CD123+ cells. As CD123 (IL-3Ra) has been implicated as a marker of AML stem cells, the finding that these cells are poised for c-Cbl signaling may suggest a potential therapeutic avenue.This work demonstrates the utility of 31-parameter mass cytometry for the simultaneous characterization of multiple cancer stem cell markers and comparison of their expression patterns to the normal tissue hierarchy. These results will inform future studies of AML signaling biology, prognostic biomarkers, and indicators of minimum residual disease.EFS and SCB contributed equally to this work. Disclosures:No relevant conflicts of interest to declare.

Expression of RFC/SLC19A1 is Associated with Tumor Type in Bladder Cancer Patients

Urinary bladder cancer (UBC) ranks ninth in worldwide cancer. In Egypt, the pattern of bladder cancer is unique in that both the transitional and squamous cell types prevail. Despite much research on the topic, it is still difficult to predict tumor progression, optimal therapy and clinical outcome. The reduced folate carrier (RFC/SLC19A1) is the major transport system for folates in mammalian cells and tissues. RFC is also the primary means of cellular uptake for antifolate cancer chemotherapeutic drugs, however, membrane transport of antifolates by RFC is considered as limiting to antitumor activity. The purpose of this study was to compare the mRNA expression level of RFC/SLC19A1 in urothelial and non-urothelial variants of bladder carcinomas. Quantification of RFC mRNA in the mucosa of 41 untreated bladder cancer patients was performed using RT-qPCR. RFC mRNA steady-state levels were ∼9-fold higher (N = 39; P<0.0001) in bladder tumor specimens relative to normal bladder mRNA. RFC upregulation was strongly correlated with tumor type (urothelial vs. non-urothelial; p<0.05) where median RFC mRNA expression was significantly (p<0.05) higher in the urothelial (∼14-fold) compared to the non-urothelial (∼4-fold) variant. This may account for the variation in response to antifolate-containing regimens used in the treatment of either type. RFC mRNA levels were not associated with tumor grade (I, II and III) or stage (muscle-invasive vs. non-muscle invasive) implying that RFC cannot be used for prognostic purposes in bladder carcinomas and its increased expression is an early event in human bladder tumors pathogenesis. Further, RFC can be considered as a potential marker for predicting response to antifolate chemotherapy in urothelial carcinomas.

15- CARCINOMA OF THE URINARY BLADDER: AGE DIFFERENCES.

15- CARCINOMA OF THE URINARY BLADDER: AGE DIFFERENCES.