Elimelech Okon

Morphological study of fully and partially isolated early human follicles

OBJECTIVE To compare the development of fully and partially isolated human follicles by using various culture systems. DESIGN Human ovarian material was incubated with collagenase and deoxyribonuclease. Fully and partially isolated follicles (30-50 microm) were dissected and studied under light and electron microscopy. The follicles were then cultured on and within various matrices. Fully isolated follicles were also cocultured with stromal cells. SETTING Rabin Medical Center, a major care and referral center. PATIENT(S) Women undergoing laparoscopy. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Microscopy studies, follicular measurements. RESULT(S) Electron microscopy studies revealed an excess of lipid droplets in the granulosa cells of freshly isolated follicles. An increase in follicular size and granulosa cell number was observed only in the fully isolated follicles cultured within collagen gels for 24 hours. Most of the partially isolated follicles detached from the collagen gels. When cultured on collagen, extracellular matrix, and poly-L-lysine, both the fully and the partially isolated follicles deteriorated within the first 24 hours; coculture with stromal cells had no beneficial effect. CONCLUSION(S) The excess in lipid droplets in granulosa cells of isolated follicles might suggest that the isolation process does not yield completely healthy follicles. However, despite this finding, our studies show that fully isolated follicles, but not partially isolated follicles, can grow within, but not on, a culture matrix.

Role and prognostic significance of the Ki‐67 index in non‐Hodgkin's lymphoma

Expression of Ki‐67, a nuclear antigen protein present in all cycling cells, is used to determine the growth fraction of tumors. The aim of this study was to evaluate the role and prognostic significance of the Ki‐67 proliferation index (PI) in non‐Hodgkins lymphoma. Ki‐67 was assayed immunohistochemically in tissue samples of 319 patients with newly‐diagnosed non‐Hodgkins lymphoma. In 268 patients, the Ki‐67 PI was correlated with clinical course and outcome. The mean Ki‐67 PI ranged from 26.6% in indolent lymphomas to 97.6% in very aggressive lymphomas (P < 0.001). The index was <45% in 82.8% of indolent lymphomas and >45% in 85% of aggressive lymphomas (AUC = 0.877, P < 0.001). In patients with diffuse large B‐cell lymphoma (n = 141), a Ki‐67 PI of 70% was found to significantly discriminate patients with good or bad prognosis (AUC = 0.65, P = 0.004). Three‐year survival was 75% ± 5.6% in patients with a low Ki‐67 index compared with 55.9% ± 6% in patients with a high index (P = 0.015). In patients with a low IPI (≤2), 3‐year survival was 94% ± 4% in those with a Ki‐67 index ≤70% and 64% ± 8.1% in those with a higher index (P = 0.002); in patients with bulky disease (>10 cm), the corresponding 3‐year survival by Ki‐67 index was 100% and 25% ± 12% (P = 0.012). Our results suggest that the mean Ki‐67 PI differs by type of lymphoma. A cut‐off value of 45% can help differentiate indolent from aggressive disease. In diffuse large B‐cell lymphoma, a cut‐off value of 70% can distinguish patients with a good and bad prognosis when combined with other prognostic factors of low IPI score and bulky disease. Am. J. Hematol. 2009.

c-kit expression in primary and metastatic merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine tumor of the skin that is associated with a high incidence of recurrence and metastasis. The therapeutic arsenal for this malignancy is limited and once it spreads, there is no effective treatment. c-kit expression has been demonstrated previously in primary MCCs thus raising the possibility of treating MCCs with imatinib mesylate, the tyrosine kinase inhibitor that has shown promise in the management of c-kit expressing tumors. In this study we examine 25 additional primary MCCs and also 6 of their lymph node metastases. Formalin-fixed, paraffin-embedded tissues were stained immunohistochemically with an antibody directed against the KIT receptor. Percentage and intensity of staining were analyzed semiquantitatively using a three-tiered system. Twenty-one of the 25 (84%) primary tumors stained positively for KIT, of which 14 (67%) showed widespread positivity. Five of the 6 lymph nodes (83%) were similarly positive. High mitotic rate and vascular invasion in the primary tumors tended to be associated with prominent staining in the lymph node metastases. No association was found between c-kit expression and outcome. We confirm that the majority of primary MCCs express c-kit and further find that metastases are positive for the KIT receptor as well. Thus, c-kit expression may be an early event in the transformation of MCC, but not a marker for tumor progression.

Possible Direct Cytoxicity Effects of Cyclophosphamide on Cultured Human Follicles: An Electron Microscopy Study

AbstractPurpose: To evaluate the direct effect of cyclophosphamide on cultured human ovarian follicles. Methods: Human ovarian cortical slices from premenopausal women were incubated with medium containing cyclophosphamide (0.0005–0.5 mg/mL) for 2–48 h and assessed by transmission electron microscopy. Noncultured specimens and samples cultured without cyclophosphamide were used as controls. Results: There were significantly more damaged granulosa cell nuclei after incubation with 0.5 mg/mL cyclophosphamide for at least 4 h. There were also more changes in the basement membrane after incubation with cyclophosphamide at concentrations of 0.05 and 0.5 mg/mL. Conclusions: Although the cyclophosphamide dose that caused damage to the granulosa cell nuclei was above the pharmacological level, our results suggest that cyclophosphamide, and not only its active metabolite phospharamide mustard, might have a destructive effect on human follicles, as it remains in the circulation longer. This effect could be mediated by damage to the granulosa cells and perhaps the basement membrane.

HLA-DR and β2 microglobulin expression in medullary and atypical medullary carcinoma of the breast: histopathologically similar but biologically distinct entities

Aims—To examine the expression of HLA-DR and β2 microglobulin in medullary carcinoma and atypical medullary carcinoma of the breast to determine if the effective presentation of tumour antigens to the immune system can differentiate between these two histopathologically similar entities. Methods—Expression of HLA-DR and β2 microglobulin was examined by immunohistochemical methods in five samples of medullary carcinoma of the breast, which has a relatively favourable prognosis, six samples of atypical medullary carcinoma of the breast, which has a prognosis closer to that of regular invasive duct carcinoma, and 20 samples of invasive duct carcinomas, 10 with an accompanying lymphocytic infiltrate. Results—A positive and significant correlation was found between tumour type and both HLA-DR and β2 microglobulin expression. Expression was most prominent in medullary carcinoma, followed by atypical medullary carcinoma and invasive duct carcinoma with and without lymphocytic infiltrates. The mean intensity and percentage of HLA-DR tumour immunostaining were significantly higher in medullary carcinoma than in the other three tumour groups, as was the mean intensity of β2 microglobulin immunostaining. Mean percentage of β2 microglobulin immunostaining was significantly higher in medullary carcinoma than in invasive duct carcinoma without lymphocytic infiltrates, and showed a trend to increase from invasive duct carcinoma with lymphocytic infiltrates to atypical medullary carcinoma and medullary carcinoma. Conclusions—Medullary carcinoma and atypical medullary carcinoma of the breast differ in their expression of HLA-DR and β2 microglobulin. The relatively favourable prognosis of medullary carcinoma of the breast may be related to effective tumour antigen presentation to the immune system through MHC-I and MHC-II expression. Immunotherapy aimed at MHC-I and MHC-II induction might have a beneficial effect in breast cancer.

Microvessel density in chemosensitive and chemoresistant diffuse large B-cell lymphomas

Preliminary reports involving a number of different kinds of tumors have indicated that microvessel quantification may be useful in predicting disease outcome. The aim of this study was to examine the relationship between microvessel density (MVD) as a parameter of tumor angiogenesis and the response to chemotherapy in diffuse large B-cell (DLBC) lymphomas.A total of 36 DLBC lymphoma patients were evaluated, 23 of them with a chemosensitive; responsive disease (median survival 8 y) and 13 with a chemoresistant, refractory disease (median survival 8 months). Microvessel quantification was performed by immunohistochemical staining, using monoclonal antibodies against factor VIII related antigen (F8RA) and against platelet/endothelial cell adhesion molecule-CD31.We found that F8RA stained a significantly higher number of blood vessels (about 2.5 times more) than CD-31; 7 samples were not stained with CD-31 but were positive for F8RA. There was no significant difference between the density of microvessel staining of the two groups. In the chemosensitive DLBC lymphomas positive for F8RA, the mean number of microvessels stained was 54.5±36.1 per microscopic field (200×) examined (range 6–149) whereas in the chemoresistant group the corresponding mean number was 43.1±25.5 (range 11–94).F8RA appears to be more sensitive for staining DLBC lymphomas microvessels than CD-31. Our data demonstrate that there is no correlation between tumor MVD and response to chemotherapy in patients with DLBC lymphomas.

Syncytial variant of nodular sclerosing Hodgkin's disease. A new clinicopathologic entity

The clinicopathologic and histopathologic features of an unusual morphologic variant of nodular sclerosing Hodgkins Disease (HD) termed the syncytial variant has been reported recently.1 We report eight new cases with this unique morphology, identified by reviewing the histopathology of 58 patients with nodular sclerosing HD treated in our institute between 1982 and 1988. The clinicopathologic and histopathologic data of these cases are reported and we emphasize this new entity and the difficulties encountered in the morphologic diagnosis. All of the eight patients had a difficult initial clinical course, and were in a clinically advanced stage of the disease at the time of diagnosis. We cite a statistical comparison of the clinical features of the different forms of nodular sclerosing HD.

Expression of VEGF-C, VEGF-D and their receptor VEGFR-3 in diffuse large B-cell lymphomas.

Lymphangiogenesis—the new growth of lymphatic vessels is an important route for the metastatic spread of human cancer. The receptor tyrosine kinase VEGFR-3 is expressed predominantly on lymphatic endothelium, and activation by its ligands VEGF-C and VEGF-D induces lymhpangiogenesis. VEGF-C, VEGF-D and VEGFR-3 have been found to play an important role in the lymphangiogenesis of several cancers. The present study investigated the expression of these factors by immunohistochemical staining of diagnosis specimens from 38 patients with diffuse large B-cell lymphoma (DLBCL). VEGF-C, VEGF-D and VEGFR-3 were expressed in both lymphoma cells and endothelial cells of blood and lymphatic tissue in all but one patient (who was negative for VEGF-D in lymphoma). There was a significant correlation in the intensity of staining between VEGF-C and -D in lymphoma and blood vessels (P < 0.001), and between the intensity of staining of VEGF-D and the patient International Prognostic Index score (P = 0.049) and borderline significance with overall survival (P = 0.051). Mean microvessel count was 58 (range 23 – 120), and it increased in association with high-intensity VEGF-C staining in lymphoma cells. Our findings indicate the importance of lymphangiogenic factors in the pathogenesis of DLBCL and suggest a potential therapeutic role for antilymphangiogenesis agents.

High Incidence of Cholelithiasis in Older Patients with Homozygous Beta-Thalassemia

The records of 48 patients with homozygous beta-thalassemia were reviewed for evidence of cholelithiasis by abdominal ultrasonography or plain abdominal X-ray. The presence of cholelithiasis was reported in 25 patients (52%). The incidence increased with age to 83% in patients over 31 years of age. Gallstones were more frequent among patients with beta-thalassemia intermedia and with less blood transfusion requirements. Eleven patients with cholelithiasis (44%) became symptomatic. Eight received operations on an elective or semielective basis. Another patient had incidental cholecystectomy during splenectomy. The preoperative evaluation included abdominal ultrasonography, nucleotide biliary scan and evaluation of the cardiopulmonary status. Due to the longer survival of patients with homozygous beta-thalassemia, the problem of cholelithiasis is becoming more frequent.

Farage, a Novel Early B Cell Lymphoma Cell Line with Trisomy 11

Farage, a new cell line established from a lymph node biopsy of a patient with non-Hodgkins lymphoma (NHL), constitutes a clonal expansion of cells at a distinct stage of B-cell differentiation. The cells lack both T and myeloid surface markers, express B cell surface antigens including CD19, CD20, CD22, HLA-DR, were positive for C3 receptors and EBNA and expressed BCL-2. No immunoglobulin determinants could be demonstrated on the cell surface. Intracellular IgM and kappa chains were detected, in an unusual but distinct localization and appeared to be localized to the nucleus or to the perinuclear area without any spread to the cytoplasm, as seen in the early B cells. Southern blot DNA analysis showed rearrangement of one of the IgJH alleles. The Farage cells were negative for B cell activation antigens including CD25, CD11b, HC2 and Bly-7. The cells were negative for two anti CD-10 (CALLA) reagents but weakly positive with one. Interestingly they were strongly positive for both nuclear and cytoplasmic T...