Eduard Fridman

Novel markers for differentiation of lobular and ductal invasive breast carcinomas by laser microdissection and microarray analysis

BackgroundInvasive ductal and lobular carcinomas (IDC and ILC) are the most common histological types of breast cancer. Clinical follow-up data and metastatic patterns suggest that the development and progression of these tumors are different. The aim of our study was to identify gene expression profiles of IDC and ILC in relation to normal breast epithelial cells.MethodsWe examined 30 samples (normal ductal and lobular cells from 10 patients, IDC cells from 5 patients, ILC cells from 5 patients) microdissected from cryosections of ten mastectomy specimens from postmenopausal patients. Fifty nanograms of total RNA were amplified and labeled by PCR and in vitro transcription. Samples were analysed upon Affymetrix U133 Plus 2.0 Arrays. The expression of seven differentially expressed genes (CDH1, EMP1, DDR1, DVL1, KRT5, KRT6, KRT17) was verified by immunohistochemistry on tissue microarrays. Expression of ASPN mRNA was validated by in situ hybridization on frozen sections, and CTHRC1, ASPN and COL3A1 were tested by PCR.ResultsUsing GCOS pairwise comparison algorithm and rank products we have identified 84 named genes common to ILC versus normal cell types, 74 named genes common to IDC versus normal cell types, 78 named genes differentially expressed between normal ductal and lobular cells, and 28 named genes between IDC and ILC. Genes distinguishing between IDC and ILC are involved in epithelial-mesenchymal transition, TGF-beta and Wnt signaling. These changes were present in both tumor types but appeared to be more prominent in ILC. Immunohistochemistry for several novel markers (EMP1, DVL1, DDR1) distinguished large sets of IDC from ILC.ConclusionIDC and ILC can be differentiated both at the gene and protein levels. In this study we report two candidate genes, asporin (ASPN) and collagen triple helix repeat containing 1 (CTHRC1) which might be significant in breast carcinogenesis. Besides E-cadherin, the proteins validated on tissue microarrays (EMP1, DVL1, DDR1) may represent novel immunohistochemical markers helpful in distinguishing between IDC and ILC. Further studies with larger sets of patients are needed to verify the gene expression profiles of various histological types of breast cancer in order to determine molecular subclassifications, prognosis and the optimum treatment strategies.

Searching for evidence of disease and malignant cell contamination in ovarian tissue stored from hematologic cancer patients

BACKGROUND Storing ovarian tissue for fertility preservation in cancer patients carries the risk of the presence of malignant cells that could lead to recurrence of cancer after reimplantation. Methods to exclude presence of cancer cells were used to improve the safety of cryopreservation-reimplantation procedures. METHODS Fifty-eight patients with hematological malignancies were referred for the storage of ovarian tissue for fertility preservation. Investigation included preoperative imaging and histological evaluation of fresh ovarian tissue. After thawing markers to detect minimal residual disease (MRD) were used and compared with patients disease used as positive control (five patients). RESULTS Preoperative imaging detected disease in the ovaries (two patients). Conventional histology post-tissue harvesting did not disclose malignant cells (56 patients). MRD results post-thawing were negative in Hodgkins disease (CD30 immunohistochemical staining), in T- and B-cell lymphoma (PCR for T-cell receptor and Ig clones, respectively) and in two chronic myelogenous leukemia patients (RT-PCR for BCR-ABL gene expression). However, highly sensitive real-time RT-PCR was positive in one CML patient and, this alarming result avoided tissue transplantation. CONCLUSIONS Preoperative imaging prevented operations and storage of tissue with cancer. Evaluation of stored ovarian tissue for MRD using sensitive markers is essential to increase safety and to prevent reimplantation of tissue with malignant cells.

siRNA Targeted to p53 Attenuates Ischemic and Cisplatin-Induced Acute Kidney Injury

Proximal tubule cells (PTCs), which are the primary site of kidney injury associated with ischemia or nephrotoxicity, are the site of oligonucleotide reabsorption within the kidney. We exploited this property to test the efficacy of siRNA targeted to p53, a pivotal protein in the apoptotic pathway, to prevent kidney injury. Naked synthetic siRNA to p53 injected intravenously 4 h after ischemic injury maximally protected both PTCs and kidney function. PTCs were the primary site for siRNA uptake within the kidney and body. Following glomerular filtration, endocytic uptake of Cy3-siRNA by PTCs was rapid and extensive, and significantly reduced ischemia-induced p53 upregulation. The duration of the siRNA effect in PTCs was 24 to 48 h, determined by levels of p53 mRNA and protein expression. Both Cy3 fluorescence and in situ hybridization of siRNA corroborated a short t(1/2) for siRNA. The extent of renoprotection, decrease in cellular p53 and attenuation of p53-mediated apoptosis by siRNA were dose- and time-dependent. Analysis of renal histology and apoptosis revealed improved injury scores in both cortical and corticomedullary regions. siRNA to p53 was also effective in a model of cisplatin-induced kidney injury. Taken together, these data indicate that rapid delivery of siRNA to proximal tubule cells follows intravenous administration. Targeting siRNA to p53 leads to a dose-dependent attenuation of apoptotic signaling, suggesting potential therapeutic benefit for ischemic and nephrotoxic kidney injury.

Developmental tumourigenesis: NCAM as a putative marker for the malignant renal stem/progenitor cell population

During development, renal stem cells reside in the nephrogenic blastema. Wilms’ tumour (WT), a common childhood malignancy, is suggested to arise from the nephrogenic blastema that undergoes partial differentiation and as such is an attractive model to study renal stem cells leading to cancer initiation and maintenance. Previously we have made use of blastema‐enriched WT stem‐like xenografts propagated in vivo to define a ‘WT‐stem’ signature set, which includes cell surface markers convenient for cell isolation (frizzled homolog 2 [Drosophila] – FZD2, FZD7, G‐protein coupled receptor 39, activin receptor type 2B, neural cell adhesion molecule – NCAM). We show by fluorescence‐activated cell sorting analysis of sphere‐forming heterogeneous primary WT cultures that most of these markers and other stem cell surface antigens (haematopoietic, CD133, CD34, c‐Kit; mesenchymal, CD105, CD90, CD44; cancer, CD133, MDR1; hESC, CD24 and putative renal, cadherin 11), are expressed in WT cell sub‐populations in varying levels. Of all markers, NCAM, CD133 and FZD7 were constantly detected in low‐to‐moderate portions likely to contain the stem cell fraction. Sorting according to FZD7 resulted in extensive cell death, while sorted NCAM and CD133 cell fractions were subjected to clonogenicity assays and quantitative RT‐PCR analysis, exclusively demonstrating the NCAM+ fraction as highly clonogenic, overexpressing the WT ‘stemness’ genes and topoisomerase2A (TOP2A), a bad prognostic marker for WT. Moreover, treatment of WT cells with the topoisomerase inhibitors, Etoposide and Irinotecan resulted in down‐regulation of TOP2A along with NCAM and WT1. Thus, we suggest NCAM as a marker for the WT progenitor cell population. These findings provide novel insights into the cellular hierarchy of WT, having possible implications for future therapeutic options.

The diagnostic role of claudins in serous effusions

We analyzed the diagnostic role of claudins in effusion cytology in 325 effusions, including 218 ovarian, 49 breast, 15 cervical or endometrial, 10 gastrointestinal, and 8 lung adenocarcinomas and 25 malignant mesotheliomas (MMs). Specimens were analyzed for claudin-1 and claudin-3 expression using immunohistochemical analysis. Ovarian and breast adenocarcinoma were further analyzed for claudin-7 expression. Claudin-1 expression was most frequent in ovarian and cervical or endometrial adenocarcinoma compared with other adenocarcinomas and MMs (P < .001). Claudin-3 expression was comparable in adenocarcinomas of different origin but was absent in MMs (P < .001). Reactive mesothelial cells rarely expressed claudins. Claudin-7 expression was higher in ovarian than in breast adenocarcinoma (P < .001). Our data suggest that expression of claudin-3 or claudin-7 is specific for adenocarcinoma and rules out the diagnosis of cells as mesothelial and that absence of claudin-1 expression excludes ovarian carcinoma as the possible origin of metastatic adenocarcinoma. Claudins may, therefore, be of diagnostic value in effusion cytology.

Importance of accurate preoperative diagnosis in the management of aggressive angiomyxoma: report of three cases and review of the literature

Background:Aggressive angiomyxoma is a benign but locally aggressive tumor that occurs mostly in young women. Because excision is often incomplete, the risk of local recurrence is high. This report describes differences in presentation and the importance of accurate preoperative diagnosis of this rare neoplasm.Methods and results:We describe three cases with different presentations. Two were initially misdiagnosed, and local recurrence necessitated reoperation. Accurate diagnosis in the third case was followed by complete excision, with no recurrence.Conclusion:Aggressive angiomyxoma should be considered in the differential diagnosis of young women who present with a well-defined mass in the pelvic tissue. Accurate preoperative diagnosis should alert the surgeon to the need for wide excision, which is essential for prevention of local recurrence.

Aggressive Angiomyxoma of the Scrotum Mimicking Huge Hydrocele: Case Report and Literature Review

Aggressive Angiomyxoma (AAM) is a rare mesenchymal benign myxoid tumor of the pelvis and perineum which occurs almost exclusively in adult females. We are presenting a case of 64 year old male patient with a slowly growing scrotal swelling which has been regarded as hydrocele for 2 years. The patient was referred to scrotal exploration. At surgery a huge mass adjacent to the bulbar urethra was found, not involving the testicles. The morphological picture and the special stains were compatible with aggressive angiomyxoma of the scrotum and peritoneum.

Complex multilocular cystic lesion of rete testis, accompanied by smooth muscle hyperplasia, mimicking intratesticular Leydig cell neoplasm

Non-neoplastic smooth muscle cell proliferation occurs under a variety of circumstances in many body organs. These abnormalities have been described as hypertrophy, hyperplasia or hamartomatous proliferations. In the male genital system, the excessive growth of smooth muscle in spermatic cord or paratesticular tissue is rare. In previously described cases, these lesions presented as masses but lacked the microscopic features of a neoplasm. We describe a complex multilocular cystic lesion composed of cystic transformation of the rete testis associated with smooth muscle proliferation mimicking intratesticular Leydig cell neoplasm. The lesion consists of three separate components: (1) cystic dilatation of the rete testis; (2) diffuse, interstitial smooth muscle proliferation with intraseptal expansion; and (3) extensive stroma with myxoid areas and scattered interstitial Leydig cells. These morphological findings, supported by a wide immunohistochemical panel, are consistent with cystic dilatation of the rete testis associated with smooth muscle hyperplasia, most probably of myoid origin. To the best of our knowledge, no similar complex lesion of the rete testis has yet been reported.

Diffuse Lymphangiomatosis—A Fatal Case With Atypical Skeletal Features

Diffuse lymphangiomatosis is a rare idiopathic condition that occurs mostly in children, is characterized by a non-neoplastic proliferation of lymphatic vessels, leading to organ dysfunction, chylous effusions, and death. A closely related condition—the Gorham-Stout syndrome—is also characterized by lymphangiomatosis and chylous effusions, but also with massive osteolytic changes (“vanishing bone disease”). A 33-year-old woman presented with a 5-year history of worsening chylous effusions and organomegaly. An extensive evaluation has ruled out most diagnoses. A complete radiographic skeletal study did not disclose any osteolytic changes. However, a Tc99 bone scan has demonstrated an absence of osteoblastic activity in some bones. An autopsy confirmed the diagnosis of diffuse lymphangiomatosis, but with histologically normal bone. If this unusual imaging pattern will be reproduced in future cases, a much needed diagnostic aid may help decrease the frequent diagnostic delays in diffuse lymphangiomatosis.

Scrotal epidermal inclusion cyst clinically mimicking polyorchidism in a child: ultrasonic characteristics.

Received: 16 May 2003 Accepted: 16 May 2003 Published online: 17 October 2003 Springer-Verlag 2003 Sir, Epidermal inclusion cysts of the male external genitalia may occur anywhere along the median raphe, which extends from the penile prepuce, through the midline of the scrotum, down to the anus [1]. We present a 5-year-old boy with a median scrotal inclusion cyst clinically mimicking a supernumerary testicle. A 5-year-old healthy boy presented with a slowly growing asymptomatic scrotal lesion. Physical examination revealed a regular well-encapsulated mobile mass located along the median raphe and clearly separated from the urethra (Fig. 1). It was rubbery and nontender on palpation, and measured about 3 cm in diameter. Both testicles were in the normal location and were unremarkable. Scrotal ultrasonography demonstrated normal testicles and an additional mass with a homogeneously hypoechoic consistency (Fig. 2). Doppler study failed to visualize any blood flow within the mass. Trans-scrotal excision was performed and the mass was removed en-bloc. Gross pathological examination of the specimen revealed a smooth well-encapsulated mass. The capsule was very thin and enveloped yellowish smooth solid material. On microscopic examination the mass was cystic, composed of a thin fibrous capsule lined by stratified squamous epithelium and containing keratinous material. Scrotal epidermal inclusion cysts can be solitary or multiple and rarely exceeding 4 cm in diameter [2, 3]. Physical examination typically