New prognostic factors in chronic lymphocytic leukemia diagnosed using an immunophenotypic method

Chronic lymphocytic leukemia (CLL) is a clonal lymphoproliferative disease which is characterized by proliferation and accumulation in blood, bone marrow and other tissues, small B-derived cells accompanied by coexpression of CD5 antigen. Diagnosis of CLL shall be based on standard criteria, but at the same time in determining the treatment strategies some prognostic factors will be helpful. Some of them are widely known and used, but with the progress of research, looking for new prognostic factors is useful for improving the strategy.Material and MethodsIn 43 patients, previously untreated, studies were performed by flow-cytometry to determine the prevalence of antigen CD49d, CD11c, CD54 on CLL cells. To determine their significance as prognostic factors correlation of their expression with the recognized standard prognostic factors, such as Rai stage, expression of CD38 and ZAP-70 as well as the presence of cytogenetic changes were done.ResultsIn our studies, a positive correlation between the expression of CD38 and CD49d, and ZAP-70 and CD49d on CLL cells were found. It was shown that among patients with CD11c + up 85.7% there were ZAP-70 positive. In addition, a significantly higher incidence of leukemic cells CD49d+ antigen was present in patients in Rai stage III / IV vs. 0/ II. There were no correlations between the number of cells expressing the antigens CD 11c and CD54 and the degree of advancement of Rai and expression of CD38. We also showed no correlation between the expression of CD54 and the expression of ZAP-70 either. In the group of patients CD38 (−), alternatively CD49d (+) and / or CD11c (+) up to 90.9% there were ZAP-70 (+).Conclusions1.Frequency of the incidence of the expression of CD49d correlates with clinical (Rai stage system) and/or risk factors determined by immunophenotype methods (CD38, ZAP-70).2.There is a rationale to continue CD11c antigen estimation as a potential prognostic factor based on our results that 86% of CD11c+ cases presented ZAP-70 positivity.Przewlekła białaczka limfocytowa (PBL) jest klonalną chorobą limfoproliferacyjną charakteryzującą się namnażaniem i nagromadzeniem w krwi, szpiku oraz innych tkankach małych limfocytów B z koekspresją antygenu CD5. O rozpoznaniu PBL decydują standardowe kryteria, natomiast dla ustalenia strategii leczenia pomocne są tzw. czynniki prognostyczne. Niektóre z nich są powszechnie stosowane, ale wraz z postępem badań poszukuje się nowych czynników rokowniczych.Materiał i metodaU 43 pacjentów, uprzednio nieleczonych, metodą cytometrii przepływowej zbadano częstość występowania na komórkach PBL molekuł adhezyjnych CD49d, CD11c, CD54. Dla określenia ich znaczenia, jako czynników prognostycznych, skorelowano ich ekspresję z: stopniem zaawansowania wg Rai, ekspresją CD38 i ZAP-70 oraz aberracjami cytogenetycznymi.Wynikistwierdzono dodatnią korelację pomiędzy ekspresją CD38 i CD49d oraz pomiędzy ZAP-70 i CD49d na komórkach PBL. Wykazano, że 85,7% pacjentów CD11c(+) było ZAP-70(+). Ponadto stwierdzono znamiennie większą częstość występowania na komórkach białaczkowych CD49d w grupie chorych w stadium Rai III/IV vs 0/II. Nie wykazano korelacji pomiędzy liczbą komórek z ekspresją antygenów CD11c i CD54 a stopniem zaawansowania Rai i CD38. Nie wykazano także korelacji pomiędzy ekspresją CD54 a ekspresją ZAP-70. W grupie pacjentów CD38(−) i równocześnie CD49d(+) i/lub CD11c(+) aż 90,9% było ZAP-70(+).Wnioski1.Częstość występowania ekspresji CD49d koreluje z klinicznymi (Rai) i fenotypowymi (CD38, ZAP-70) czynnikami ryzyka.2.Uzasadnionym jest kontynuowanie badań antygenu CD11c jako potencjalnego czynnika prognostycznego, gdyż stwierdzono, że 86% przypadków CD11c+ wykazuje dodatnią ekspresję ZAP-70

Establishment and Characterization of the Novel High-Grade Serous Ovarian Cancer Cell Line OVPA8

High-grade serous ovarian carcinoma (HGSOC) is the most frequent histological type of ovarian cancer and the one with worst prognosis. Unfortunately, the majority of established ovarian cancer cell lines which are used in the research have unclear histological origin and probably do not represent HGSOC. Thus, new and reliable models of HGSOC are needed. Ascitic fluid from a patient with recurrent HGSOC was used to establish a stable cancer cell line. Cells were characterized by cytogenetic karyotyping and short tandem repeat (STR) profiling. New generation sequencing was applied to test for hot-spot mutations in 50 cancer-associated genes and fluorescence in situ hybridization (FISH) analysis was used to check for TP53 status. Cells were analyzed for expression of several marker genes/proteins by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS), and immunocytochemistry (ICC). Functional tests were performed to compare OVPA8 cells with five commercially available and frequently used ovarian cancer cell lines: SKOV3, A2780, OVCAR3, ES2, and OAW42. Our newly-established OVPA8 cell line shows morphologic and genetic features consistent with HGSOC, such as epithelial morphology, multiple chromosomal aberrations, TP53 mutation, BRCA1 mutation, and loss of one copy of BRCA2. The OVPA8 line has a stable STR profile. Cells are positive for EpCAM, CK19, and CD44; they have relatively low plating efficiency/ability to form spheroids, a low migration rate, and intermediate invasiveness in matrigel, as compared to other ovarian cancer lines. OVPA8 is sensitive to paclitaxel and resistant to cisplatin. We also tested two FGFR inhibitors; OVPA8 cells were resistant to AZD4547 (AstraZeneca, London, UK), but sensitive to the new inhibitor CPL304-110-01 (Celon Pharma, Łomianki/Kiełpin, Poland). We have established and characterized a novel cell line, OVPA8, which can be a valuable preclinical model for studies on high-grade serous ovarian cancer