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).

Morphology of renal interstitial fibroblasts.

INTRODUCTION: Renal fibrogenesis is a process common to all progressive kidney diseases. The main executive cell in this process is the fibroblast, by secreting and remodelling the extracellular matrix. The number of fibroblasts is minor in a healthy kidney interstitium, but it increases during the process of fibrosis. Their morphology and immunophenotype vary due to different intrinsic and extrinsic factors which makes their identification and visualization, as well as determination of their origin, very difficult. MATERIAL AND METHODS: We performed morphological and immunohistochemical analyses on kidney biopsies with interstitial fibrosis, using the following antibodies: Vimentin, α-SMA, S100A4, Cadherin 9 and CD34. We also did light-microscopy analyses of semithin sections of tissue embedded in epoxy resin and stained with Toluidine blue. RESULTS: Our observations show that different cells in the fibroblastic population show positivity for different markers, thus contributing to the theory that there are different subpopulations of fibroblasts, with different origins, that take part in renal fibrogenesis.

Histological features, Ki-67 and Bcl-2 immunohistochemical expression and their correlation with the aggressiveness of pheochromocytomas.

Pheochromocytomas and paragangliomas are rare neoplasms. Approximately 10% may present malignant behaviour. There are no reliable morphological signs of malignancy, except for the presence of metastasis. We performed morphological and immunohistochemical analysis on 15 pheochromocytomas and 5 paragangliomas aiming to find correlations between the morphological features of the tumours, immunohistochemical expressions of Ki-67 and Bcl-2, and the biological behaviour of the tumours. According to the biological behaviour of the tumors, the patients were divided into an indolent disease group (ID), and an aggressive disease group (AD). The morphological analysis included the PASS core parameters, greatest tumour diameter and weight, as well as age and gender of the patients, survival and disease-free periods after operation. According to histomorphological parameters, tumours were divided into tumours with "benign-like" morphology and tumours with "malignant-like" appearance. The disease course was neither correlated to the PASS score, nor to the individual parameters comprising it. The rest of the morphological parameters and the immunohistochemical expressions of Ki-67 and Bcl-2 were not able to predict the disease course, although we found significantly higher Ki-67 expression in paragangliomas in comparison to pheochromocytomas (p<0.01). Some of the PASS parameters (vascular invasion and presence of atypical mitoses) were positively correlated to the tumour weight (R=0.75; p<0.01, and R=0.56; p<0.05, respectively). The disease course was in positive correlation to the tumour weight, presence of vascular invasion and atypical mitoses; however there were no statistically significant differences regarding those parameters between the ID and AD groups (p>0.05).

Acute renal failure in a patient with diffuse large B-cell lymphoma: case report.

INTRODUCTION: Renal parenchymal involvement is common in systemic lymphomas. In almost all cases, renal involvement appears to be a secondary process, either by direct extension from a retroperitoneal mass or via haematogenous spread in the setting of disseminated disease. Secondary renal involvement in systemic lymphomas is generally presented as multiple masses, but also as a solitary nodule. Acute renal failure by a lymphoma infiltration of the kidney is extremely rare. Primary renal non-Hodgkin's lymphoma is even more uncommon and it is a debated issue because of the absence of lymphoid tissue in normal kidneys. CASE PRESENTATION: We report on the case of a 62-year old woman, who had melena, abdominal pain, malaise and fever. She was hospitalized at the Nephrology Clinic due to severe anemia and signs of acute renal failure. The peripheral blood smear showed the presence of dysplastic erythroblasts and hypo-granular neutrophils. Ultra-sound was performed, which showed enlarged kidneys with signs of urinary obstruction of the first degree, with swollen, hypoechogenic parenchyma. After not responding to the conducted treatment, the patient died from heart failure. An autopsy was performed and Non-Hodgkin's, diffuse large B-cell lymphoma infiltrating multiple parenchymal organs was determined as the main cause of death. CONCLUSION: Diffuse large B-cell lymphoma with multiple organ affection and secondary renal involvement, presented as an acute renal failure is a rare case. We report on this case to update the literature concerning this topic and highlight the importance of renal biopsy in the diagnostics.

Renal histopathology and clinical course in patients with Wegener's granulomatosis--single centre experience from the Republic of Macedonia.

The aim of this study was to evaluate the clinical course of patients with Wegener's granulomatosis (WG) with renal involvement, to examine histopatological form seen in renal biopsies and present follow-up of the patients. A retrospective analysis was carried out of 18 patients presenting with WG and active renal disease at the University Nephrology Department, Ss. Cyril and Methodius University, Skopje, R. Macedonia. All patients were ANCA positive and had a percutaneous renal biopsy taken on their admission. 12 patients were male, 6 female, aged 48.61±13.77 (M±SD). All had extrarenal symptoms prior to admission. Oligoanuria was present in 7/18 (38.9%) of the patients, serum urea levels of the whole group were 40.67±18.13 mmol/l (M±SD) and for serum creatinine 691.06±384.93 µmol/l (M±SD). Necrotizing glomerulonephritis with crescents was present in 11/18 (61.11%) of the patients, the others presented diffuse proliferative extracapillary glomerulonephritis. All patients were treated with steroids and cyclophosphamide, and plasmapheresis was performed in 7/18 (38.9%) of the patients. Probability rate for surviving after one month was 0.6111 and after three months 0.3889 (Kaplan-Meier). The current treatment of WG in our study did not prevent serious complications and development of ESRD in a large number of our patients. This systemic disorder is still a serious problem and early diagnosis and alternative strategies for the management of the disease will be an important objective for further studies.

Limb-sparing in patients with non-metastatic high-grade osteosarcoma.

PURPOSE: To improve survival rates and functional outcome in patients with non-metastatic, high-grade osteosarcoma of the extremities, using the Scandinavian Sarcoma Group XIV neoadjuvant chemotherapy protocol. PATIENTS AND METHODS: The analysis included 37 patients treated during the period 2000-2005. Age range was 8 to 65 year (median 23). Seven (7/37) patients were excluded from the study. The remaining 30 patients received 2 cycles of preoperative chemotherapy (high dose methotrexate, cisplatin and doxorubicin). Surgery was carried out in the 9th week. Twenty-seven (90%) patients had limb-salvage operation and in the remaining 3 amputation was performed. Based on the histopathological assessment of the removed tumor patients were classified in two groups (regarding good or poor response to chemotherapy). All 30 patients received 3 courses of postoperative chemotherapy with the same regimen. Patients with poor response received 3 more cycles of chemotherapy with high dose ifosfamide. Follow-up was 2-8 years (mean 52 months). RESULTS: Histopathological assessment showed poor response to neoadjuvant chemotherapy in 57% of the patients but no significant difference in 3-year survival between the 2 groups was noted. Three-year survival of the patients with local recurrence was 40 vs. 88% of those without local recurrence (p=0.013). Three-year survival of the patients with distant metastases was 20 vs. 92% of those without distant metastases (p=0.0002). Three-year overall survival (OS) was 80% and disease-free survival (DFS) 60% for all 30 patients. CONCLUSION: Neoadjuvant chemotherapy in patients with high-grade osteosarcoma of the extremities gives the opportunity for limb-sparing operation and at the same time improves survival rates.

Treatment of non-metastatic high-grade osteosarcoma (study of 30 cases treated with Scandinavian osteosarcoma protocol XIV and surgery).

UNLABELLED: The aim of this study is to improve 3-years survival rates and functional outcome in high-grade osteosarcoma patients treated with amputations and limb-sparing surgery, introducing Scandinavian Sarcoma Group chemotherapy protocol (SSG XVI). PATIENTS AND METHODS: During the period 2000-2005, thirty seven patients with high-grade, non-metastatic osteosarcoma on the extremities were treated at the Clinic for Orthopaedic Surgery in Skopje. Mail patients were 21 (57%) and female were 16 (43%). Patients age varied from 8 to 63 years (mean 18 +/- 13). Seven patients (7/37) did not comply with including criteria and were excluded from the study. The rest 30 patients were introduced to two courses of pre-operative chemotherapy (high doses of Methotrexate, Cisplatin and Adriamycin). Surgical treatment was in 9-th week of the protocol. In 27/30 (90%) of the patients limb-sparing surgery was done, and in 3/30 (10%) amputations were performed. Histopathological assessment of the tumour after the neo-adjuvant chemotherapy divided the patients into group with bad and group with good response. All the patients had 3 more courses of chemotherapy after surgery (same as the preoperative). Patients with bad response were introduced to 3 more cycles of 5 days with high-dose of Ifosfamide. Follow-up was from 2 to 8 years, mean 51 months. RESULTS: Histopathological assessment showed that 57% of the patients had bad response to neo-adjuvant chemotherapy, but there was no statistical significance in the survival time of the groups (p = 0.06). Three-years survival time was 40% of the patients with local recurrence in comparison with 80% of the patients with no local recurrence. Three-years survival time was 20% of the patients with distant metastases in comparison with 92% of the patients with no metastases. Overall survival time (OS) was 80%. After 3 years 60% of the patients were disease-free (DFS). CONCLUSION: High-grade osteosarcoma of the extremities treated with modern chemotherapy protocols enables limb-sparing in the same time with improved survival time of the patients. Introducing high-dose Ifosfamide in treatment of patients with bad response after neo-adjuvant chemotherapy improves their functional results as well as the survival time. Key words: osteosarcoma, neo-adjuvant chemotherapy, limb-sparing.

Morphological changes in the tubulointerstitial compartment in primary glomerulopathies.

The renal interstitium structurally supports the functional renal units and is involved in almost all renal functions. The degree of renal disfunction strongly correlates to the changes in the tubulointerstitial compartment present in almost all types of glomerular diseases. A phenomenon arising in such an environment is epithelial-mesenchymal transition, i.e. a change of the cell;s epithelial phenotype into a mesenchymal one. Histochemical, immunohistochemical and morphometric analyses were made of 50 renal biopsies with primary glomerulopathies, as well as light-microscopy analyses of semi-thin sections embedded in epoxy resin. Double immunohistochemical stainings with pairs of epithelial and mesenchymal antibodies were also done. The results were analyzed and correlated with the clinical data of the renal function of the patients. The immunohistochemical analyses of the atrophic tubular epithelial cells showed a loss of expression of Cytokeratin and E-cadherin, an enhanced expression of HLA-DRalpha, and a de novo expression of Vimentin and alphaSMA as markers for epithelial-mesenchymal transition. The double immunohistochemical stainings with Cytokeratin/Vimentin and Cytokeratin/alphaSMA showed a simultaneous expression of these antigens in atrophic tubular cells. Their proliferative index was mildly enhanced. Interstitial fibrosis was present in 98% of the analysed biopsies. The analyses show correlations among all the changes in the tubulointerstitial compartment as well as the concentration of creatinine in the serum as a parameter of renal function. The study emphasizes the usefulness of the implementation of histomorphometrical and immunohistochemical techniques as well as ultrastructural and molecular analyses in the process of nephropathological diagnosis.

Evaluation of telomerase activity in patients with chronic B lymphocytic leukemia versus age matched controls. Correlation between telomerase activity and bone marrow infiltration.

Telomerase is a ribonucleoproteic enzyme associated with cellular immortality and malignancy. This enzyme, besides the catalytic subunit bearing reverse transctiptase activity, contains an RNA template complementary to TTAGGG telomeric repeats, thus permitting de novo synthesis of telomeric DNA onto chromosomal telomeric ends. Increased telomerase activity has been reported in Chronic Lymphocytic Leukemia (CLL) by many authors. In order to investigate the telomerase activity in patients with CLL and its correlation to commonly used morphologic prognostic markers, 38 frozen blood lymphocyte samples from patients with CLL and 47 age-matched controls were investigated for telomerase activity using the Telomerase PCR ELISA-plus kit from Roche. Trepanobiopsies from the same patients were analysed for the type of bone marrow infiltration as well. Analysis showed highly variable Relative Telomerase Activity (RTA) in B-CLL patients, ranging from comparable or even lower than the mean RTA of controls (in Binet A stage patients) to manifold increase in the majority of patients with advanced stage disease. The sex and age of the patients showed no influence on RTA in CLL patients, in contrast to the control group, where the age influenced telomerase activity. We found a positive correlation between the RTA and disease stages (Binet), as well as between RTA and the type of BM infiltration.

Imaging diagnosis of musculoskeletal hydatid disease.

Hydatid disease has a worldwide distribution and causes health problems in endemic countries. Musculoskeletal hydatid disease may be a difficult differential diagnosis. The aim of this retrospective study is to present some different imaging features of musculoskeletal hydatid disease. We evaluated imaging findings of 15 patients with musculoskeletal hydatid disease over a 15-year period. Ten (67%) were men and 5 (33%) women, ages ranging between 12 and 69 years. All underwent radiographic evaluation. CT was done in 9 patients and MRI in 3 patients with soft tissue involvement. All underwent surgery, followed by histological examination. Bone involvement occurred in 11 patients and soft-tissue involvement in 4 patients respectively. Bone lesions in the spine were found in 4 (36%), in the pelvis 2 (18%), in the proximal femur 2 (18%), in the tibia 2 (18%) and 1 (9%) in the first metatarsal. On plain film we identified three different patterns of presentation of bone HD, defined as "typical" osteolytic lesion (33%), "tumour-like" features (41.6%) and lesions resembling infection (25%). Soft-tissue hydatidosis was localized on the distal part of the thigh, paravertebral and deltoid muscle. MR imaging showed a cystic mass, containing multiple vesicles, and in one of them the pathognomonic for hydatid disease, the "water lily" sign, was present. We present three cases of primary musculoskeletal hydatid disease localized on metatarsal bone, proximal femur and deltoid muscle. Musculoskeletal hydatid disease, presenting with a variety of patterns, may resemble a tumour or an inflammatory process. Because of the rare presentation of the disease it should be kept in mind in the diagnostic work-up of musculoskeletal tumours.

High-grade surface osteosarcoma - chondroblastic type.

We present the case of an 8-year-old boy with pain in the distal part of the right thigh that occurred after a ball injury, without haematoma or oedema. The plain film showed exostosis of the distal part of the right femur. In another institution the "exostosis-like" formation was surgically removed and the histological finding was an aggressive type of chondroblastoma. A few months later, on the site of the surgical intervention, a significantly enlarged painful solid mass appeared. Radiography showed a large dense mass at the level of distal femoral metadiaphysis with tumour matrix mineralization, and Codman's sign. Computed tomography showed a large soft tissue mass, which arose from the surface of the underlying cortical bone and signs of high-grade malignancy. CT is a valuable method in predicting high-grade malignancy. Arteriography was also done. After operative treatment - seq. Campanacci, histology showed high-grade surface osteosarcoma of chondroblastic type. Pre- and post-operative chemotherapy was carried out. The boy died 2 years after diagnosis because of recurrent disease. High-grade surface osteosarcoma has the worst prognosis, compared with other surface osteosarcomas. Therefore, timely diagnosis is indispensable.