Monitoring M-BCR-ABL expression level in CML patients by RQ-PCR: experience of a single Center.

UNLABELLED: Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome and the BCR-ABL fusion gene that encodes an abnormal tyrosine kinase. Development of specific tyrosine kinase inhibitors completely changed the management of these patients. MATERIALS AND METHODS: Between April 2008 and July 2012, at the Molecular Biology Laboratory, University of Medicine and Pharmacy of Targu Mures, Romania, we monitored the M-BCR-ABL transcript level by real time quantitative PCR in case of 15 CML patients diagnosed at the Hematology and Transplant Center of Targu Mures. RESULTS: Modification of M-BCR-ABL expression level shows statistically significant correlation (p=0.013) with the clinical course of these patients. CONCLUSIONS: Molecular biology techniques have an important role in monitoring CML patients and regular analysis is recommended.

Morphometric and ultrasonographic study of the human fetal hip joint during intrauterine development.

UNLABELLED: The main method for the early screening of the developmental dysplasia of the hip (DDH) is the ultrasound imaging. There are several studies about the ultrasound imaging of newborns' hips, but only a few studies include the prenatal period of life. Our aim was to examine the prenatal development of the hip joint through the evolution of the α angle seen on the ultrasound, described in the Graf R method, combined with anatomical dissection. MATERIALS AND METHODS: Thirty-one post-mortem fetal hips were analyzed trough anatomical dissection, in 25 cases trough ultrasound imaging, in which the α angle was measured. Based on the morphometric examination, we applied the sine rule and we calculated the α1 angle, which also represents the coverage of the femoral head. RESULTS: Based on the morphometric examination, not only the diameters of the femoral head and of the acetabulum, but also the joint cavity (X) showed an increase during development. Both of the α angles (measured α, calculated α1) showed a decrease as the fetus developed. CONCLUSIONS: The decrease of the angles (α, α1) and the increase of the joint cavity during development correspond to the findings of the main research papers: the hip joint is less stable in the perinatal life. The α angle can be accurately determined only after the ossification of the acetabulum had started, in our case after the fetus is older than 18 weeks.

Insulinoma diagnosed as drug-refractory epilepsy in an adolescent boy: a case report.

Solitary insulinoma is a rare pancreatic tumor in all age groups with an estimated incidence of 1 in 250 000 persons a year. It is even rarely in childhood and mostly shows benign behavior. Cases with uncertain or malignant biology are extremely rare with less than 30 cases described in the literature. Here we report a case of pediatric insulinoma, the first in our department files in the past 20 years, with rapid clinical course following a clinical misdiagnosis as juvenile myoclonic epilepsy, which was complicated with low glucose level (20 mg/dL) and neuroglycopenia. Our case underlines some unusual features of a pediatric insulinomas presented without past medical and family history, after surgery complicated with mental retardation and recurrent epileptiform episodes. Despite the small tumor size, low Ki67 index/mitotic rate and benign immunophenotype marked by positivity for pro-insulin but negativity for ß-HCG, the diagnosis was concluded as insulinoma of uncertain biological behavior due to vascular tumor invasion in agreement with the 2003 WHO Classification for Pancreatic Endocrine Neoplasms. Besides these features, perineural invasion can differentiate insulinomas of uncertain outcome from benign insulin producing tumors. Pediatric insulinomas may present misleading symptoms of epilepsy in neglected cases coming from poor socioeconomic background. Chronic insufficient blood glucose level might contribute to mental retardation and epilepsiform myoconvulsions to be prevented. Differentiation between insulinoma with benign and uncertain behavior is difficult where histological pattern and tumor immunophenotype are less important than the critical morphological parameters. Life long follow-up including regular control of blood glucose and abdominal status of patients are essential for proper assessment of clinical outcome of pediatric insulinoma.

Hyperplastic polyps and serrated adenomas: precancerous lesions with mixed immunophenotype.

Our immunohistochemical study wants to be a contribution to clarifying the adenoma-carcinoma sequence and serrated pathway of colorectal carcinogenesis. Thus, we performed immunohistochemical analysis of hyperplastic polyps (HP), serrated adenomas (SA), and classical adenomas (tubular adenomas - TA and tubulovillous adenomas - TVA) and carcinomas developed from adenomas (CA) using expression of p53, Ki-67, c-myc, APC, MSH2 and Ets-1 proteins. Because of correlation of the expression of these proteins, we propose several immunophenotypes, which show modifications along the known carcinogenetic mechanisms. Along the adenoma-carcinoma sequence we noted an increase in the expression of p53, Ki-67, c-myc and Ets-1, and a decrease in APC expression. The majority of TAs and TVAs are characterized by p53+÷Ki-67+, p53+÷c-myc+, p53+÷APC+, and Ets-÷p53+, Ets-÷Ki-67+ immunophenotypes. The majority of HPs and SAs are Ets-÷p53-, Ets-÷Ki-67+, Ets-÷c-myc+, APC+÷MSH2-. In approximately 1÷3 of the hyperplastic polyps and serrated adenomas, we noted that the decrease in expression of MSH2 is associated with an increase in the expression of p53, c-myc, Ki-67, and Ets-1. Thus, we can conclude that a group of hyperplastic polyps and serrated adenomas display similar immunohistochemical characteristics to tubular and tubulovillous adenomas, which delineates a group of precancerous lesions that can develop via mixed carcinogenic pathways.

Follow-up of childhood chronic myelogenous leukemia with monitoring the BCR-ABL fusion gene expression in peripheral blood.

UNLABELLED: Chronic myelogenous leukemia (CML) accounts for 15-20% of adult leukemias but is very rare in children (2%). Fewer than 10% of CML patients are younger than 20 years. CML is a myeloproliferative disorder characterized by the presence of the Philadelphia chromosome or the BCR-ABL fusion oncogene. The objective of this paper is to present the monitoring of imatinib therapy in two children with CML by the BCR-ABL fusion gene expression assessment from peripheral blood with quantitative real-time polymerase chain reaction (PCR) method. PATIENTS AND METHODS: The 18 and six months follow-up of the patients included clinical examination, routine laboratory tests, bone marrow aspirate investigation including cytogenetic tests and the major BCR-ABL fusion gene expression measurement with qRT-PCR method from the peripheral blood. RESULTS: Patient No. 1 diagnosed with chronic phase CML showed excellent adherence to daily 400 mg imatinib treatment and achieved complete hematologic (CHR) and cytogenetic response (CCR) by three months and major molecular response (MMR) by 12 months, with lack of side effects due to imatinib. Patient No. 2 experienced severe hematologic toxicity, which necessitated temporary withdrawal of the drug. Transient non-compliance together with imatinib dose reduction has driven to treatment failure. In this case, mutational analysis is warranted. CONCLUSIONS: BCR-ABL fusion gene expression level measurement from peripheral blood with qRT-PCR method is an excellent tool in the follow-up of CML patients.

Histopathological and immunohistochemical features of gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most frequent mesenchymal tumors of the gastrointestinal tract. Major advances in their definition and classification and the understanding of their molecular mechanisms have recently been made. These advances have become a model of targeted therapy in oncology. The diagnosis of GISTs relies on histological arguments - proliferation of spindle cells, seldom of epithelioid cells or both spindle and epithelioid cells - and on immunohistochemical arguments - expression of CD117 usually associated with CD34 expression. The evaluation of the prognosis is essential and based on a simple algorithm using two prognostic parameters, tumor size and mitotic index. The aim of this paper is a complex histopathological assessment, using both classic and modern (immunohistochemistry) techniques, of the GISTs comprised in the study. GISTs occur mainly in older adults (median age 60-69 years), anywhere along the gastrointestinal tract but also retroperitoneal. Most of them were nodular (75%), tumor necrosis and mucosal ulceration being the most frequent encountered secondary alterations; these modifications proved to be significantly correlated with large tumor size and high malignancy. Immunohistochemical evaluation revealed that 77 (97%) cases of GISTs presented a positive reaction for CD117, 50 (63%) cases were positive for CD34, 19 (24%) were positive for SMA and only 10 (13%) were positive for S100. Immunohistochemical evaluation remains an important tool of pathology in the diagnosis of GISTs, in the differential diagnosis from other gastrointestinal mesenchymal tumors and represents the gold standard for diagnosis of these tumors and an eligibility criterion for imatinib therapy.

[Bleomycin therapy for lymphangioma]. / Tratament cu bleomicina pentru limfangioame.

Lymphangiomas are uncommun congenital malformations of the lymphatic system, that involve the skin and subcutaneous tissues. Of the several types of treatment, surgical excision has been the preferred. There is a high recurrence rate because lymphangiomas tend to infiltrate the surrounding tissues. The bleomycin is a cytotoxic antitumoral antibiotic, that causes modifications of DNA. It has been also successfully used in intralesional injection treatment of cystic hygromas and haemangiomas, based specifically on a high sclerosing effect on vascular endothelium. We report the cases of five patients, with congenital lymphangioma, localized on the leg, in cervical and latero-thoracal region, treated with repeated intralesional bleomycin injections. The treatment indication was given by the location of this lesions and the infiltration of the surrounding vital tissues, that made the complete surgical excision impossible. Intralesional injection of bleomycin into the lymphangiomas was given at a dose, not exceeding 0,5 mg/kg of body weight, at intervals of 4 weeks. Complete resolution (n = 4) or significant improvement (n = 1) occurred in all patients treated. No other treatment was needed. We didn't notice local or general adverse effects. With this method we set the purpose to treat effectively this congenital malformations, obviating the need for invasive primary surgery or systemic treatment regimens. Toward other methods, intralesional bleomycin injections have a minimal risk of side effects (ulceration, pulmonary fibrosis).

Morphological identification of neuron types in the rat hippocampus.

The cerebral cortex ensures an optimal interaction of mammals, including humans, with their environment, by encoding, storing and combining information about the surrounding world and the internal milieu. Probably the simplest and the most popular region for studying the cortical network is the hippocampal CA1 area, because it has the least heterogeneous neuronal population, the somata and dendrites of principal neurons (pyramidal cells) are arranged into well defined layers and the extrinsic and intrinsic inputs are segregated. The relatively homogeneous pyramidal cell population is supported by a very heterogeneous GABAergic interneuron population, which provides not only general inhibition, but also regulates the precise timing of pyramidal cell activity. Interneurons usually innervate distinct domains of the surface of their target cell. The strategic placement of inhibitory synapses, indicate that GABAergic interneurons belonging to different classes serve distinct functions in the hippocampal network. Neuron types are usually defined according to various morphological, molecular and physiological features. Under typical experimental conditions only some of these parameters are available, therefore an important scientific question is: which partial measures are sufficient for correct recognition of a class of cell. By immunohistochemistry it is possible to stain all neurochemically identical neurons in a given brain region, therefore it is the most widely used method for identifying neuron classes. This review presents the neuron types identified so far in the area CA1 of the rat hippocampus with special emphasis on the immunocytochemical characterization of these cells.

Quantitative characterization of regional differences in the GABAA-receptor alpha1-subunit mRNA expression in the rat brain.

Inhibition in the central nervous system is largely mediated by local-circuit neurons that release GABA (gamma-amino-butyric acid). GABAA-receptors play a major role in virtually all brain physiological functions and serve as targets for numerous classes of drugs, used both in clinical practice and as research tools. These receptors are heteropentamers, alpha1 being the most widely occurring subunit; therefore it is the best candidate to be studied in pathological conditions where the inhibitory system might be altered (e.g. epilepsy). We compared quantitatively the regional distribution of GABAA-receptor alpha1-subunit (GABAAR-alpha1) expression in three brain areas: neocortex, hippocampus and cerebellum by RT-qPCR. TaqMan probe was used in order to avoid detection of non-specific amplification products and synaptophysin as internal control. This substance was chosen because it has a stable expression restricted to neurons, and contrary to GAPDH, the most commonly used reference gene for expression analysis, synaptophysin expression is not modified in animal models of epilepsy. Expression of synaptophysin was higher than expression of GABAAR-alpha1 in all samples from the central nervous system. The latter was significantly different among the studied brain areas. It was the smallest in the hippocampus, intermediate in the neocortex and the highest in the cerebellum. Interanimal differences were small for any brain region under study. These results indicate that combination of TaqMan real-time PCR method with synaptophysin as internal control can reliably measure the relative expression of GABAAR-alpha1 mRNA, and are suitable for investigating the modifications that appear under pathological conditions and/or diverse experimental paradigms.

Diagnostic and differential diagnostic criteria of lymphoid neoplasms in bone marrow trephine biopsies: a study of 87 cases.

The aim of this study is to present the diagnostic and differential diagnostic criteria of the bone marrow specimen involved by lymphomas based on the histomorphological immunophenotype features and clonality of the tumor cells, patterns of lymphoproliferation and diagnostic pitfalls. BMB material obtained from the right posterior iliac crest was represented from 87 untreated and treated patients with BM involving malignant lymphoma, stained with Hematoxylin-Eosin, Giemsa, Periodic Acid Schiff and Gömöri's Silver. In order to perform immunohistochemistry examination we used a large antibody panel. B-cell clonality was determined in six cases. We found eight reactive lymphoproliferative responses and 79 lymphoid neoplasms of which 45 were diagnosed as de novo lymphoma, the rest of 34 samples being examined for staging. The predominant lymphoma was CLL (30 cases), over followed by DLBCL (18 cases). The most frequent patterns of involvement were the interstitial (29%) and mixed (15%) ones. In eight cases, we found reactive lymphoid aggregates. The B-cell clonality test showed four monoclonal, one oligoclonal and one polyclonal diseases form. Diagnosis of lymphoma versus reactive aggregate has been based on the combination of a lot of antibodies and involvement pattern. Although investigation of gene rearrangement was necessary for the establishment of the correct diagnosis in only 6.9% of cases, it should be emphasized that it is of great importance in disease monitoring.

Plasmoblastic lymphoma associated with human immunodeficiency virus.

Plasmoblastic lymphoma (PBL) is a subtype of the diffuse large B-cell lymphoma, typically present as extranodal disease associated with human immune deficiency virus (HIV) infection. PBLs are often the initial manifestation of AIDS. Here we present a case of PBL concerning the oral cavity. A 34-year-old woman presented a tumor in the oral cavity that involved the maxilla and gingiva (confirmed by CT-scan). The gingival biopsy showed a massive infiltration by large lymphoid cells with round, vesicular nuclei, prominent nucleoli, fine chromatin and an significant amount of basophilic cytoplasm which express CD79a, CD138, cytoplasmic lambda light chain and LCA, without staining for CD20, CD38, CD3 and CTK. Serological analysis confirmed HIV positivity. PBLs lack most B-lineage markers, but many express CD79a in at least some of the cells, therefore generate difficulties in differential diagnosis. Overall assessment and correlation of the histopathological and immunohistochemical features with the clinical findings and serology investigation are the most helpful diagnostic tools and can lead to the final diagnosis.

The expression of cytoskeleton regulatory protein Mena in colorectal lesions.

The actin regulatory proteins Ena/VASP (Enabled/Vasodilator stimulated phosphoprotein) family is involved in the control of cell motility and adhesion. They are important in the actin-dependent processes where dynamic actin reorganization it is necessary. The deregulation of actin cycle could have an important role in the cells' malignant transformation, tumor invasion or metastasis. Recently studies revealed that the human orthologue of murine Mena is modulated during the breast carcinogenesis. In our study, we tried to observe the immunohistochemical expression of mammalian Ena (Mena) in the colorectal polyps and carcinomas. We analyzed 10 adenomatous polyps (five with dysplasia) and 36 adenocarcinomas. We used the indirect immunoperoxidase staining. BD Biosciences have provided the Mena antibody. We observed that Mena was not expressed in the normal colorectal mucosa neither in polyps without dysplasia, but its expression was very high in polyps with high dysplasia. In colorectal carcinomas, Mena marked the tumoral cells in 80% of cases. In 25% of positive cases, the intensity was 3+, in 60% 2+ and in the other 15% 1+. The Mena intensity was higher in the microsatellite stable tumors (MSS) and was correlated with vascular invasion, with intensity of angiogenesis marked with CD31 and CD105 and with c-erbB-2 and p53 expression. This is the first study in the literature about Mena expression in colorectal lesions.

The correlation between the immunostains for p53 and Ki67 with bcl-2 expression and classical prognostic factors in colorectal carcinomas.

UNLABELLED: The prognostic role of p53 and Ki67 in colorectal carcinomas (CRC) is very controversial in the literature. In our study, we tried to find if their immunostains are correlated with bcl-2 expression or other classical prognostic factors (sex, age, localization and size of tumor, the grade and staging of tumor). We studied 507 cases with CRC and chose 38 cases in which we realized these correlations. Fourteen cases were mucinous CRC, the other 24 cases being non-mucinous CRC (six well differentiated, 13 moderate and five poorly differentiated). For statistical analysis, we used the Statistical Program Graph Pad In Stat 3-Trial Version. We considered the significant association when p<0.05, with 95% confidence interval. RESULTS: The median value was 75% for p53 expression, respectively 35% for Ki67 expression. Bcl-2 was positive in 47% of cases but not correlated with p53 or Ki67. We found a significantly statistical decrease p53 immunostain with grade of tumor (70% in well differentiated, respectively 40% in poorly differentiated CRC) and increase of Ki67 median expression (25% in well differentiated, respectively 60% in poorly differentiated CRC). Ki67 was correlated with age of patients, lymph node involvement, being more expressed in N2 (80%) than in N0 (22.5%) and with Dukes MAC staging (25% in B1, 60% in C2). P53 was correlated with age of patients and pT component, after pTNM staging (75% in pT2, 40% in pT4). P53 was not correlated with Ki67. CONCLUSION: The CCR prognostic is not determined only by proliferative capacity of tumoral cells.

Comparative detection of high-risk HPV (16, 18, 33) in cervical bioptic material of county hospital of Tg. Mures.

The purpose of this study was to collect data about the incidence of high-risk HPV (16, 18, 33) types in paraffin embedded cervical bioptic material, including LSIL, HSIL and cervical cancers using immunohistochemistry and nested PCR methods. In our study were included randomly selected 10 LSIL, 18 HSIL and 30 cervical cancer cases. We analyzed the expression of HPV in this specimens with immunohistochemistry used DAKO K1H8 antibody and CHEMICON Mab HPV 16, 16 antibody using LSAB method and Tiramin amplification method, and nested PCR for HPV 16, 18 and 33. In LSIL cases three, in HSIL cases eight and in carcinoma 20 cases were positive for HPV 16 or 18 for immunohistochemistry or PCR. Although this proportion in lower than those reported in the literature, our work signals the existence of the infection in our country and presents a relatively cheap diagnostic method.

Prognostic significance and detection of the internal tandem duplication of the FLT3 gene in acute myeloid leukemia.

The FMS-like tyrosine kinase-3 (FLT3), which belongs to the class III receptor tyrosine kinase family, expressed by immature hematopoietic cells, plays an important role in the proliferation, differentiation and survival of stem cells. The activating mutations of FLT3 gene have been reported to be of prognostic significance. The most common somatic alteration of the FLT3 gene is the Internal Tandem Duplication (FLT3/ITD), which is caused by the elongation of the juxtamembrane (JM) domain of FLT3. The duplicated fragment size varies from 3 to more than 400 base pair, always occurs in multiples of three while the reading frame is preserved. The elongated segment of DNA can be amplified by polymerase chain reaction (PCR), and the products are separated by gel electrophoresis. The FLT3/ITD is found in 20-40% of adult AML patients and is the most frequent mutation in leukemia. Using native peripheral blood and bone marrow from AML and non-AML patients (total of 19 samples), and samples from the RNA bank (total of eight samples), the authors purpose was to work out a method for FLT3/ITD detection, which can be used in routine diagnostics. All samples produced detectable PCR products, which proofs that this procedure can be used for the detection of FLT3/ITD mutations in daily clinical practice.

Morphometric investigation of carotid body in sudden infant death syndrome.

BACKGROUND: The pathomechanism of sudden infant death syndrome (SIDS) has not been clarified yet. The high rate of early progenitor cells in carotid body has been reported as a pathognomic feature for SIDS. AIM AND STUDY DESIGN: The morphometric analysis was done by NIKON Eclipse microscope with a morphometric program Lucia G. SUBJECTS: This study was designed to investigate the structure and developmental state of carotid body in SIDS and non-SIDS cases. A comparison was made between the rates of dark and early progenitor cells. OUTCOME MEASURES: The Kruskal-Wallis test showed a significantly higher number of progenitor cells in the SIDS group than in controls (p=0.0003). RESULTS AND CONCLUSION: In this study on Hungarian SIDS cases we confirmed the observation that infants who died suddenly have an underdeveloped carotid body.

Immunohistochemical features of paragangliomas.

Immunohistochemistry is part of the routine diagnosis of the neuroendocrine tumors. In our study, we included 52 paragangliomas with various localizations by routine histology and immunohistochemistry. In order to increase the diagnostic specificity, a complex immunohistochemistry panel has been performed consisting of Bcl-2, Ki-67, Bax and Pituitary Adenylate Cyclase-Activating Peptide (PACAP), somatostatin, VIP and Calcitonin Gene Related Peptide (CGRP). After heat induced antigen retrieval, the immunostaining was performed by StreptABC using DAB as a chromogen. We were the first to demonstrate the presence of Bax and PACAP in paragangliomas. Some of the used markers are of prognostic value. The relationship between Bcl-2 and Bax is decisive in generating the final response to the input apoptotic signals. The Ki-67 antigen staining has gained wide acceptance in prognostic evaluation of other tumor types. We noted a small number of Ki-67 positive cases, which signifies a low mitotic activity of these tumors and a relatively high number of Bax positivities (32.9%) and the much lower number of Bcl-2 positivities (11.39%), and could explain the benign behaviour of paragangliomas.