Janus Kinase V617F Mutation Detection in Patients with Myelofibrosis.

Myelofibrosis (MF) is characterized by a presence of an extra fibrous tissue in the bone marrow and additional hematopoiesis. The somatic mutation in the Janus kinase 2 (JAK2) gene (V617F) occurs gradually and is detected in about 50.0% of myelofibrosis or essential thrombo-cytopenia (ET) patients. Our aim was to determine the genotype status according to the carriers of the V617F mutation in patients with MF at the Hematology Ward of the University Hospital "Ivan Rilski" in Sofia, Bulgaria. DNA samples were isolated from venous blood of patients with various hematological disorders. DNA was amplified by polymerase chain reaction (PCR) and subsequent restriction analysis was performed using a BsaXI restriction enzyme. The genotype status was determined on 2.0% agarose gel. We analyzed 38 patients initially suspected of carrying MF or osteomyelofibrosis (OMF). After trepanobiopsy, 20 out of 38 patients were confirmed as myelofibrotic (52.6%), 5/38 (13.2%) were diagnosed as ET, 1/38 (2.6%) was diagnosed as myeloproliferative neoplasm (MPN), 6/38 (15.8%) had polycythemia vera (PV). In six patients, the presence of disease was rejected. Patients with MF were divided into three groups according to the JAK2 V617F genotype status: homozygous for the mutation (3/20 or 15.0%), heterozygous (9/20 or 45.0%) and homozygous for the wild type allele (8/20 or 40.0%). The triggering factor of MF is still unknown. It was considered that this factor could have a genetic nature. Mutations in three genes were mainly accepted as an actual predisposing events to this disease: point mutations leading to amino acid substitutions in JAK2 (V617F) and in MPL (W515L, W515K), as well as insertion or deletion in CALK We have proven that carriers of the V617F mutation prevailed in the group of patients with MF (altogether 12 patients or 60.0%). Previous studies also showed that JAK2 V617F is present in more than half of MF patients within their blood-forming cells. Therefore, the risk of evolution to MF could be associated with V617F-mutant allele burden in patients with MPN.

[Sentinel lymph node dissection: preliminary results in vulvar cancer].

AIM: The aim of the study was to investigate and assess sentinel lymph node dissection methods in squamous cell vulvar cancer in cases of individualized therapeutic approach. MATERIALS AND METHODS: In the period January 2000-2010, 113 patients with squamous cell vulvar carcinoma were diagnosed, treated and followed up at the Clinic of Gynecologic Oncology of University Hospital - Pleven. All patients underwent primary surgical treatment and were surgically staged. Groin dissection was performed on 77 (72.64%) patients with invasive carcinoma (deep stromal invasion > 1 mm). Sentinel lymph node dissection was performed on seven patients after preoperative application of subcutaneous peritumor infiltration of Blue V in four places. The sentinel lymph nodes were identified following skin incision parallel to the inguinal ligaments. The nodes were dissected and sent for prompt frozen section histological evaluation. All patients underwent inguinal lymph node dissection as indicated by findings. RESULTS AND DISCUSSION: In the seven patients, one to three lymph nodes were found and frozen sections were histologically analyzed. The analysis did not reveal metastases. Primary tumor diameters varied from 0.5 to 4 cm. During follow-up, no cancer recurrences were found in any of the seven patients. CONCLUSION: Sentinel lymph node dissection is a reliable method in early vulvar cancer when certain criteria are applied in patient selection. The method allows avoiding inguinofemoral lymphadenectomy in a group of carefully selected patients with vulvar cancer.

[Laparoscopic complications of gynecologic surgery].

Despite the growth of laparoscopic surgery, its complications must not be underestimated and patients must be informed of the risks of so-called 'minimally' invasive surgery.

[Evaluation of the expression of S100A1 protein in serous, mucinous and endometroid ovarian carcinoma].

According to literature approximately 60% from all ovarian malignances express epithelial phenotype. According to their histomorphological characteristics, epithelial ovarian tumors are divided into eight groups. In some particular cases, separate histological types are hard to distinguish one from another. Recent studies show the presence of beneficial effect of adjuvant radiotherapy on most of the early ovarian carcinomas (all, except serous carcinomas). In renal cell carcinomas SI00A 1 is used to distinguish between different subtypes of the malignancy. Forty cases of ovarian carcinomas were analyzed in a retrospective study. Immunohistochemical evaluation of the S100A1 protein expression was carried out on representative archival formalin -fixed -paraffin-embedded tissue materials. Positivity for S100A1 was observed in 31 (77.50%) of the studied cases. Twenty-seven out of thirty-two (84.38%) cases of serous ovarian carcinoma were found to express S100A1. S100A1 expression was observed in one out of the two mucinous and three out of the six endometroid ovarian carcinomas. Immunopositivity was nuclear, cytoplasmic or nuclear and cytoplasmic in serous carcinomas, nuclear in the one positive mucinous carcinoma and sytoplasmic in the three imunopositive endometroid ovarian carcinomas. The S100A1 immunohistochemical marker is not likely to be useful in clinical practice to distinguish between different histological subtypes of ovarian cancer. The large percentage of S100A1 positivity in serous ovarian carcinomas needs a better understanding.

PRIMARY OVARIAN SYNOVIAL SARCOMA - A CASE REPORT.

Synovial sarcoma is an aggressive soft tissue sarcoma, which most often occurs in the limbs near the joints. It accounts for 5-10% of all soft tissue sarcomas. It extremely rarely affects the pelvis. So far, only 4 cases of primary involvement of the adnexa have been described. We present a case of a 77-year-old female patient diagnosed with a rapidly growing pelvic formation, subsequently diagnosed as monophasic synovial sarcoma of the ovary. Synovial sarcoma derived from the adnexa is a rare disease that is virtually unknown. The diagnosis is complex, and there is a poor prognosis.

Corona mortis, aberrant obturator vessels, accessory obturator vessels: clinical applications in gynaecology.

Corona mortis (CMOR) is a heterogeneous and often dubious term that causes much confusion in medical literature, especially in regard to its modern day significance in pelvic surgery. Some authors define CMOR as any abnormal anastomotic vessel between the external iliac and obturator vessels, whereas others define it as any vessel coursing over the superior pubic branch, regardless whether it is a vascular anastomosis, an accessory obturator vessels, an obturator vessel related to the external iliac system or a terminal small vessel. There is no standard classification of CMOR and obturator vessels variations, although there are multitudes of classifications describing the diverse variations in the obturator foramen region. We define accessory obturator, aberrant obturator vessels and CMOR as different structures, as CMOR is an anatomical term that reflects a clinical situation rather than an anatomical structure. A new clinical classification for aberrant, accessory obturator vessels and CMOR is proposed regarding the anatomical variations, and the location of vessels to the deep femoral ring. The clinical significance of accessory obturator, aberrant vessels and CMOR is delineated in oncogynaecological and urogynaecological surgery.

Multifunctional antioxidant activity of HBED iron chelator.

The use of N,N'-bis (2-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid (HBED) for iron chelation therapy is currently being tested. Besides its affinity for iron, bioavailability, and efficacy in relieving iron overload, it is important to assess its anti- and/or pro-oxidant activity. To address these questions, the antioxidant/pro-oxidant effects of HBED in a cell-free solution and on cultured Chinese hamster V79 cells were studied using UV-VIS spectrophotometry, oximetry, spin trapping, and electron paramagnetic resonance (EPR) spectrometry. The results indicate that HBED facilitates Fe(II) oxidation but blocks O2(.-)-induced reduction of Fe(III) and consequently pre-empts production of .OH or hypervalent iron through the Haber-Weiss reaction cycle. The efficacy of HBED as a 1-electron donor (H-donation) was demonstrated by reduction of the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)-derived nitrogen-centered radical cation (ABTS(.+)), accompanied by formation of a short-lived phenoxyl radical. HBED also provided cytoprotection against toxicity of H2O2 and t-BuOOH. Our results show that HBED can act both as a H-donating antioxidant and as an effective chelator lacking pro-oxidant capacity, thus substantiating its promising use in iron chelation therapy.

Comparative cellular catabolism and retention of astatine-, bismuth-, and lead-radiolabeled internalizing monoclonal antibody.

UNLABELLED: Monoclonal antibodies (mAbs) labeled with alpha-emitting radionuclides such as (211)At, (212)Bi, (213)Bi, and (212)Pb (which decays by beta-emission to its alpha-emitting daughter, (212)Bi) are being evaluated for their potential applications for cancer therapy. The fate of these radionuclides after cells are targeted with mAbs is important in terms of dosimetry and tumor detection. METHODS: In this study, we attached various radionuclides that result in alpha-emissions to T101, a rapidly internalizing anti-CD5 mAb. We then evaluated the catabolism and cellular retention and compared them with those of (125)I- and (111)In-labeled T101. T101 was labeled with (211)At, (125)I, (205,6)Bi, (111)In, and (203)Pb. CD5 antigen-positive cells, peripheral blood mononuclear cells (PBMNC), and MOLT-4 leukemia cells were used. The labeled T101 was incubated with the cells for 1 h at 4 degrees C for surface labeling. Unbound activity was removed and 1 mL medium added. The cells were then incubated at 37 degrees C for 0, 1, 2, 4, 8, and 24 h. The activity on the cell surface that internalized and the activity on the cell surface remaining in the supernatant were determined. The protein in the supernatant was further precipitated by methanol for determining protein-bound and non-protein-bound radioactivity. Sites of internal cellular localization of radioactivity were determined by Percoll gradient centrifugation. RESULTS: All radiolabeled antibodies bound to the cells were internalized rapidly. After internalization, (205,6)Bi, (203)Pb, and (111)In radiolabels were retained in the cell, with little decrease of cell-associated radioactivity. However, (211)At and (125)I were released from cells rapidly ((211)At < (125)I) and most of the radioactivity in the supernatant was in a non-protein-bound form. Intracellular distribution of radioactivity revealed a transit of the radiolabel from the cell surface to the lysosome. The catabolism patterns of MOLT-4 cells and PBMNC were similar. CONCLUSION: (211)At catabolism and release from cells were somewhat similar to that of (125)I, whereas (205,6)Bi and (203)Pb showed prolonged cell retention similar to that of (111)In. These catabolism differences may be important in the selection of alpha-radionuclides for radioimmunotherapy.

Vascular-targeted radioimmunotherapy with the alpha-particle emitter 211At.

Astatine-211, an alpha-particle emitter, was employed in a model system for vascular-targeted radioimmunotherapy of small tumors in mouse lung to compare its performance relative to other radioisotopes in the same system. Astatine-211 was coupled to the lung blood vessel-targeting monoclonal antibody 201B with N-succinimidyl N-(4-[211At]astatophenethyl) succinamate linker. Biodistribution data showed that the conjugate delivered 211At to the lung (260-418% ID/g), where it remained with a biological half-time of about 30 h. BALB/c mice bearing about 100 lung tumor colonies of EMT-6 cells, each about 2000 cells in size, were treated with 211At-labeled monoclonal antibody 201B. The administered activity of 185 kBq per animal extended the life span of treated mice over untreated controls. Injections of 370 kBq, corresponding to an absorbed dose of 25-40 Gy, were necessary to eradicate all of the lung tumors. Mice receiving 740 kBq of 211At-labeled monoclonal antibody 201B developed pulmonary fibrosis 3-4 months after treatment, as did mice treated with 3700 kBq of the alpha-particle emitter 213Bi-labeled monoclonal antibody 201B in previous work. Animals that were injected with 211At bound to untargeted IgG or to glycine, as control agents, also demonstrated therapeutic effects relative to untreated controls. Control groups that received untargeted 211At required about twice as much administered activity for effective therapy as did groups with lung-targeted radioisotope. These results were not consistent with radioisotope biodistribution and dosimetry calculations that indicated that lung-targeted 211At should be at least 10-fold more efficient for lung colony therapy than 211At bound to nontargeting controls. The data showed that 211At is useful for vascular-targeted radioimmunotherapy because lung tumor colonies were eradicated in the mice. Work in this model system demonstrates that vascular targeting of alpha-particle emitters is an efficient therapy for small perivascular tumors and may be applicable to human disease when specific targeting agents are identified.

Preparation and in vivo evaluation of linkers for 211At labeling of humanized anti-Tac.

The syntheses, radiolabeling, antibody conjugation, and in vivo evaluation of new linkers for 211At labeling of humanized anti-Tac (Hu-anti-Tac), an antibody to the alpha-chain of the IL-2 receptor (IL-2Ralpha) shown to be a useful target for radioimmunotherapy are described. Synthesis of the organometallic linker precursors is accomplished by reaction of the corresponding bromo- or iodoaryl esters with bis(tributyltin) in the presence of a palladium catalyst. Subsequent conversion to the corresponding N-succinimidyl ester and labeling with 211At of two new linkers, N-succinimidyl 4-[211At]astato-3-methylbenzoate and N-succinimidyl N-(4-[211At]astatophenethyl)succinamate (SAPS), together with the previously reported N-succinimidyl 4-[211At]astatobenzoate and N-succinimidyl 3-[211At]astato-4-methylbenzoate, are each conjugated to Hu-anti-Tac. The plasma survival times of these conjugates are compared to those of directly iodinated (125I) Hu-anti-Tac. The N-succinimidyl N-(4-[211At]astatophenethyl)succinamate compound (SAPS) emerged from this assay as the most viable candidate for 211At-labeling of Hu-anti-Tac. SAPS, along with the directly analogous radio-iodinated reagent, N-succinimidyl N-(4-[125I]astatophenethyl)succinamate (SIPS), are evaluated in a biodistribution study along with directly iodinated (125I) Hu-anti-Tac. Blood clearance and biological accretion results indicate that SAPS is a viable candidate for further evaluation for radioimmunotherapy of cancer.