Proteomic analysis of infiltrating ductal carcinoma tissues by coupled 2-D DIGE/MS/MS analysis.

There is a growing interest in protein expression profiling aiming to identify novel diagnostic markers in breast cancer. Proteomic approaches such as two-dimensional differential gel electrophoresis coupled with tandem mass spectrometry analysis (2-D DIGE/MS/MS) have been used successfully for the identification of candidate biomarkers for screening, diagnosis, prognosis and monitoring of treatment response in various types of cancer. Identifying previously unknown proteins of potential clinical relevance will ultimately help in reaching effective ways to manage the disease. We analyzed breast cancer tissues from five tumor and five normal tissue samples from ten breast cancer subjects with infiltrating ductal carcinoma (IDC) by 2-D DIGE using two types of immobilized pH gradient (IPG) strips: pH 3-10 and pH 4-7. From all the spots detected, differentially expressed (p < 0.05 and ratio > 2) were 50 spots. Of these, 39 proteins were successfully identified by MS, representing 29 different proteins. Ten proteins were overexpressed in the tumor samples. The 2-D DIGE/MS/MS analysis revealed an increase in the expression levels in tumor samples of several proteins not previously associated with breast cancer, such as: macrophage-capping protein (CAPG), phosphomannomutase 2 (PMM2), ATPase ASN1, methylthioribose-1-phosphate isomerase (MRI1), peptidyl-prolyl cis-trans isomerase FKBP4, cellular retinoic acid-binding protein 2 (CRABP2), lamin B1 and keratin, type II cytoskeletal 8 (KRT8). Ingenuity Pathway Analysis (IPA) revealed highly significant (p = 10(-26)) interactions between the identified proteins and their association with cancer. These proteins are involved in many diverse pathways and have established roles in cellular metabolism. It remains the goal of future work to test the suitability of the identified proteins in samples of larger and independent patient groups.

MicroRNAs in Breast Cancer -Our Initial Results.

MicroRNAs (miRNAs) are small [∼21 nucleotide (nt)] non coding RNAs (ncRNAs) that regulate gene expression posttranscriptionally. About 3.0% of human genes encode for miRNAs, and up to 30.0% of human protein coding genes may be regulated by miRNAs. Currently, more than 2000 unique human mature microRNAs are known. MicroRNAs play a key role in diverse biological processes including development, cell proliferation, differentiation and apoptosis. These processes are commonly dysregulated in cancer, implicating miRNAs in carcinogenesis, where they act as tumor supressors or oncogenes. Several miRNAs are associated with breast cancer. Here we present our initial results of miRNA analyses of breast cancer tissues using quantitative real time-polymerase chain reaction (ReTi-PCR) (qPCR) involving stem-loop reverse transcriptase (RT) primers combined with TaqMan® PCR and miRNA microarray analysis.

Do we have to treat subclinical rejections in early protocol renal allograft biopsies?

The aim of the present study was to evaluate whether treatment of subclinical, borderline rejections (SR/BR) or histological findings of chronic allograft nephropathy (CAN) in protocol biopsies in the first month posttransplantation after living related kidney transplantation has a beneficial effect on graft histology and renal function at 6 months. Among the 40 paired biopsies, only 6/80 showed no histological lesions. BR was found in 13/40 and 12/40, and SR in 15/40 and 21/40 of patients on the 1- and 6-month biopsies, respectively. The mean histological index/total sum of scores for acute and chronic changes (HI) increased at 6-month biopsy: 5.3 +/- 2.9 vs 7.8 +/- 3.6 (P < .001). Similarly, the mean sum of histological markers for chronicity (CAN score) of 2.1 +/- 1.5 increased to 4.6 +/- 2.3 (P < .001) on the 6-month biopsy. When divided according to whether there was treatment of BR and SR, the treated BR/SR group on 1-month biopsy had a mean HI score of 7.11 +/- 1.9, which remained almost the same (7.11 +/- 2.32) at 6 months. Among the untreated BR/SR group it increased from 4.95 +/- 1.99 to 8.16 +/- 4.30. However, there was no difference in graft function between the groups from 1 to 6 months. In conclusion, a protocol 1-month biopsy may be valuable to establish the prevalence of BR/SR in stable allografts. The presence of an untreated BR/SR upon a 1-month biopsy showed greater susceptibility for histological deterioration on the 6-month biopsy due to an accelerated CAN process.

Protocol biopsies in kidney transplant recipients: histologic findings as prognostic markers for graft function and outcome.

The aim of the present study was to identify subclinical and borderline rejections as well as histological markers of chronic allograft nephropathy (CAN) among protocol biopsies performed at 1 and 6 months after living related kidney transplantation to assess their possible implications for graft function. Twenty paired allograft biopsies performed at 1 and 6 months were reviewed according to the Banff scoring scheme. The mean ages of donors and recipients were 59.6 +/- 13.8 and 34.4 +/- 8.7 years, respectively. Among all biopsies only 10% (4/40) showed no histopathological lesions. At the first month borderline rejection was shown in 35% and subclinical rejection in 10% of patients. At 6 months the proportion of findings was even higher, namely, 40% and 30%, respectively. When divided according to donor age, donors above 55 years showed a mean CAN score of 2.33 +/- 1.56 which increased to 5.0 +/- 2.26 on the 6 month biopsy (214.3%). Unexpectedly, the proportion of these changes in the younger donor group also increased by 173.3%, which might have been explained by the greater number of borderline and subclinical rejections in the younger donor group at the 1 month biopsy. In conclusion, 1 month biopsy may be valuable to determine borderline and subclinical rejection and to prognosticate the outcome of renal allograft function. Our findings suggest a greater susceptibility of histological deterioration among the older donor population. However, the presence of an untreated rejection in the younger donor pool leads to a rapid impairment of the graft function accelerating the process of chronic allograft nephropathy.

Sentinel lymph node detection in breast cancer - first experience.

INTRODUCTION: Breast cancer accouns for 22.9% of all cancers in women and 13.7% of cancer deaths. Positive axillary lymphnodes (ALN) predict the development of distant metastases. The status of the sentinel lymphnode (SLN) is crutial for the treatment selection. AIM: To determine the benefits of SLN detection in patients with breast cancer. MATERIAL AND METHODOLOGY: 38 female patients (pts), age 44 ± 12 years, with T1-2 N0 M0 breast cancer, without enlarged ALN on ultrasound (US), were included. SLN detection was performed using gamma camera and gamma detection probe after periareolar subcutaneous and/or peritumoral injection of (99m-Technetium-SENTISCINT). Blue dye was administered 20 min before the operation. SLN was extirpated and ex tempore histopathology was performed. RESULTS: Ex tempore SLN evaluation was negative and the lymphatic pathways preserved in 28/38 (74%) pts. In 10/38 (26%) pts SLN was positive, followed by radical surgery. In 3/28 ex tempore negative patients, histopathological analysis showed metastatic involvement (false negative). In 3/10 ex tempore positive patients micro metastases 0,2-2 mm were detected. 12 pts had 2 SLN, 8/12 (66%) had negative and 4/12 (34%) had positive SLN. 3 pts had a rare double drainage to axilla and a. mammaria int. CONCLUSION: Our results confirm that SLN detection technique is non-invasive, safe and reliable and should be incorporated into the guidelines for breast cancer pts (T1-2 N0 M0). The most reliable option for colloid application is the combined technique of periareolar and peritumoral injection. Patients with drainage to a. mammaria interna should be selected for adjuvant protocols.

Novel RET mutations in macedonian patients with medullary thyroid carcinoma: genotype-phenotype correlations.

Medullary thyroid carcinomas (MTCs) are rare neoplasms comprising 2-10% of all thyroid malignnancies. More than 75% are sporadic tumors and the remainder is familial and MEN2 related. Both sporadic and syndromic MTCs frequently show mutations in the RET proto-oncogene. It has been noted that some MTC cases present an indolent, and some an aggressive clinical course. Ki-67 expression is generally low, with documented exceptions, whereas high expression of Bcl-2 has been reported in majority of the cases. Some studies have shown that Ki-67 and Bcl-2 expressions have prognostic value, as well as RET mutational status. We analyzed 20 unrelated MTC cases for Ki-67, Bcl-2 expression and RET mutations and tested their intercorrelations, correlations to the morphologic features and stage of the tumors, as well as their influence on survival. In 13 of the 20 analyzed cases we found 23 sequence changes distributed in exons 8, 10-13 and 16. There were 11 different missense mutations, single nucleotide deletion with frameshift, and 8 different synonymous mutations. Only 4 of the sequence changes have been previously published. Twelve patients (60%) had tumors expressing one or more missense mutations or single nucleotide deletion and 7 of them (35%) had at least one damaging or possibly damaging RET mutation. Most of the tumors had low Ki-67 expression (mean 6.48% of cells) and high Bcl-2 expression (mean 68.3%). Significantly better survival was observed in cases with low Ki-67 (< 6.5%; p < 0.05), high Bcl-2 expression (> 68.3%; p < 0.01) and younger age at diagnosis (< 51 years; p < 0.05).

Ischemic dilatative cardiomyopathy and aneurysms of the left ventricular cavity: transplantation vs alternative surgery.

Patients with terminal end-stage heart failure due to severe coronary disease associated with dilatative cardiomyopathy have an annual mortality of 30-50%. Between July 1997 and December 1999, 21 patients at the University Hospital in Frankfurt, and 25 patients from Skopje underwent total circular repair with simultaneous coronary artery bypass.

Immunohistochemical and ultrastructural features of Gaucher's cells--five case reports.

Gaucher's disease is an autosomal recessive lysosomal storage disease resulting from glucocerebrosidase deficiency. In this report, five patients with adult Gaucher's disease are described. The clinical course of these patients was characterized by progressive diffuse aseptic necrosis in the large bones, so-called Erlenmeyer's flask deformity, and hepatosplenomegaly. Splenomegaly was accompanied by hypersplenism with anemia and thrombocytopenia, therefore splenectomy was performed. The diagnosis of Gaucher's disease was based on the finding of Gaucher's cells on bone marrow biopsy. Tissue blocks were cut and routinely processed. Slides staining for iron (Peris' blue) and PAS (periodic acid--Schiff) including immunohistochemical staining for CD68 and HLA-DR was performed in all five cases. Gaucher's cells were seen as large cells with granular or fibrillar distended cytoplasm, with the characteristic 'wrinkled tissue paper' appearance, and eccentric nuclei. PAS staining showed strongly positive granular or fibrillar material in the cytoplasm. Immunohistochemical stain for CD68 and HLA-DR helped identify isolated Gaucher's cells, which are hystiocytic in nature. This stain accentuates their fine linear striations. Small pieces were ultrastructurally analyzed.

Gastrointestinal malignant lymphomas in Macedonia.

The gastrointestinal tract including oral cavity is the most common location of extranodal lymphomas. In this retrospective study, the histomorphological, immunohistochemical and clinical features of 21 Macedonian cases with diagnosed malignant lymphomas were investigated. The series included 15 males and 6 females, mean age 44 (4-78) years. The most common locations were hard palate (n = 7), gastric site (n = 5), small intestinal wall (n = 2), large intestinal wall (n = 5), and lingual root (n = 2). All cases were B cell lymphomas, 23.8% of them low grade B cell lymphoma of mucosa associated lymphoid tissue, 4.7% mantle cell lymphoma, 47.6% diffuse large cell lymphoma, and 23.8% Burkitt type lymphoma. There was no T cell lymphoma. Most of the cases were positive for CD20 and CD79a. Monoclonality was confirmed by light chain restriction, except for nine cases where it failed due to poor tissue preservation. The fact that eight cases were in the clinically advanced third and fourth stage implied a conclusion that not only primary non-Hodgkin's lymphomas but also secondary lesions could invade the gastrointestinal tract. Immunohistochemical staining was helpful in differentiation between benign and malignant infiltration in low grade lymphomas, and in distinguishing diffuse large cell lymphomas from undifferentiated epithelial neoplasms.

Perindopril treatment in streptozotocin induced diabetic nephropaty.

Diabetic nephropathy (DN) is one of the most common causes of terminal stadium damage to the kidneys. The angiotensin-converting enzyme (ACE) represents a significant risk factor for the progression of DN. ACE inhibitors are medications of particular interest knowing the role of angiotensin II in the development of DN. This study aimed to examine the effects of ACE inhibitor treatment perindopril (PER), administered to rats with streptozotocin (STZ) induced DN, that developed albuminuria, renal hypertrophy and mild glomerulussclerosis. DN was induced by a STZ (60 mg/kg ip) single injection to normotensive Wistar rats. The administration of STZ caused diabetes mellitus (DM) with symptoms and signs of DN including poor general condition, body-weight loss, kidney weight increase as well as increased values of BUN and serum creatinine, accompanied by increased diuresis as well as distinct albuminuria. The majority of these symptoms were manifested 4 weeks after, and even more distinctly 8 and 12 weeks after administering STZ. The perindopril treatment (6 mg/kg BW), starting 4 weeks after administering STZ, resulted in a significant improvement of all symptoms and signs of DN, significantly lowering the values of BUN and serum creatinine, albuminuria and diuresis. The histopathological examination of the renal samples at 8 and 12 weeks after the beginning of the study have shown that perindopril significantly lowers the progression of glomerulopathy, and significantly improves the glomerulosclerotic index, as well as the progression of renal histological abnormalities induced with STZ. Thus perindopril treatment ameliorates STZ-induced nephropathic changes in DM rats.