Immunophenotypic characterization of acute myeloid leukemia in adults and its role in the diagnosis,monitoring, and prognosis of the disease

U tezi su ispitivani savremeni aspekti primene imunofenotipizacije multiparametarskom protočnom citofluorimetrijom (IMPC) u dijagnostici i praćenju AML. Ispitivanjem je obuhvaćeno 320 odraslih bolesnika sa de novo AML, od kojih je 294 uključeno u retrospektivno ispitivanje (dijagnoza, klasifikacija i prognoza), dok je ostalih 26 bolesnika bilo uključeno u prospektivno ispitivanje (praćenje minimalne rezidualne bolesti - MRB). Bolesnici su klasifikovani kao AML-neklasifikovana (48%), AML sa rekurentnim genetskim poremećajima (37,4%) i AML sa znacima mijelodisplazije (14,6%). IPCM omogućava postavljanje dg AML analizom ks kod 98% bolesnika. Ispitivanjem 44 različita hLDM utvrđeno je da je njihova ekspresija na blastima deregulisana, o čemu govore aberacije u njihovoj ekspresiji kod svakog bolesnika. Heterogen ćelijski sastav populacije leukemijskih ćelija (leukemijski blasti i prekursori) utvrđen je kod 55% bolesnika sa AML. Imunološka i citomorfološka klasfikacija AML su saglasne kod 73% bolesnika, odnosno imunološka i SZO klasifikacija kod 68% bolesnika. Ispitivanje MRB sprovedeno je primenom jedne (42%) ili dve (58%) kombinacije IFSL po bolesniku. Primenom IMPC, pokazana je visoka učestalost MRB kod naših bolesnika sa AML posle lečenja indukcionom (69%) odnosno konsolidacionom terapijom (50%). Nivo MRB u ks bolesnika ≥0,1% NĆ posle indukcione terapije, svrstava bolesnike u grupu visokog rizika za razvoj relapsa bolesti. Ispitivanje prognostičkog značaja hLDM kod bolesnika sa AML, pokazalo je značajnu vezu između rane monocitne diferencijacije leukemijskih ćelija, ekspresije CD22 molekula i pojave rane smrti i (p<0,05), odnosno niže incidence kompletne remisije (p<0,05)...In the thesis are examined modern aspects of application immunophenotyping and multiparameter flow cytometry (IMPC) in the diagnosis and monitoring of AML. The study included 320 adult patients with de novo AML, of which 294 included in the retrospective study (diagnosis, classification and prognosis), while the other 26 patients were included in a prospective study (minimal residual disease - MRD). Patients were classified as AML-unclassified (48%), AML with recurrent genetic abnormalities (37.4%) and AML with signs of myelodysplasia (14.6%). IPCM allows setting dg AML by analysis of bone marrow (bm) in 98% of patients. By examining of 44 different HLDM, it was found that its expression is deregulated on the blasts, whereas at least one type of immunophenotypic aberrations was found per patient. Heterogeneous cellular composition of the population of leukemic cells (leukemic blasts and precursors) was found in 55% of patients with AML. Immunological and cytomorphological classification of AML agree with 73% of patients, respectively immunological and WHO classification in 68% of patients. MRD trial was conducted by one (42%) or two (58%) combination of IFSL per patient. By applying the IMPC, a high incidence of MRD was detected in our patients with AML, after induction (69%) and/or consolidation therapy (50%). The level of MRD in bm of patients ≥0,1% NC after induction therapy, classified patients in the high risk group for the development of relapse. The prognostic significance of HLDM in patients with AML, showed a significant association between early monocytic differentiation of leukemia cells, the expression of CD22 molecule and the appearance of early death (p<0.05), and lower incidence of complete remission (p<0.05)..

The role of immunophenotyping in differential diagnosis of chronic lymphocytic leukemia

Introduction. Accurate diagnosis of chronic lymphocytic leukemia (CLL) acquires immunophenotyping by flow cytometry in order to facilitate differential diagnosis between CLL and other mature B-cell neoplasms (MBCN). Objective. The aim of this study was to define immunological profile of CLL cells. Methods. Immunophenotyping by flow cytometry was performed on peripheral blood specimens at diagnosis in the group of 211 patients with de novo MBCN. Results. Absolute count of B-cells was significantly increased in all MBCN patients comparing to healthy control group (p<0.05). B-cell monoclonality was detected in 96% of all MBCN patients, by using surface immunoglobulin (sIg) light chain restriction. B-cell antigens, CD19, CD20, CD22, were expressed with very high frequency in CLL and other MBCN. In comparison with other MBCN, in CLL group, the frequency of expression was higher for CD5 and CD23 (p<0.0001), though lower for FMC7 antigen (p<0.0001). CLL patients were characterized by lower expression patterns of CD20, CD22, CD79b, and sIg (p<0.0001) as well as higher expression pattern of CD5 antigen (p<0.05). Correlation between the final diagnosis of MBCN and values of CLL scoring system showed that the majority of CLL patients (97%) had higher values (5 or 4) whereas the majority of other MBCN patients (96%) had lower score values (0-3). Conclusion. Our results have shown that characteristic immunophenotype which differentiates CLL from other MBCN is defined by following marker combination - CD19+ CD20+low CD22+low CD5+high CD23+ FMC7- CD79b+low sIg+low. CLL score values of 5 or 4 points are highly suggestive for diagnosis of CLL

Prognostic significance of cebpa mutations in patients with normal-karyotype - acute myeloid leukemia

Introduction: CEBPA gene encodes a transciptional factor with an essential role in granulocyte differentiation process. In acute myeloid leukemia (AML), mutationsin CEBPA gene have been associated with favorable outcome and up to now only the presence of double mutated CEBPA gene (CEBPAdm) was included in WHO classification. Prognostic influence of CEBPA mutationsin C-terminal (bZIP) region recently have been proposed as a marker for better overalsurvival (OS), higher probability of achieving complete remission (CR) and a lower risk of relapse. Since AML with normal karyotype (AML-NK) is a group with intermediate prognosis with the need for new prognostic markers, we analyzed the influence of bZIP CEBPA mutations as an additional molecular marker in Serbian AML-NK patients. Methods: CEBPA mutational screening was performed using a multiplex polymerase chain reaction– based fragment length analysis. A total of 61 bone marrow samples were collected from de novo AMLNK patients. Results: In our analysis, frequency of CEBPA mutations in Serbian AML-NK patients was 15% (12/61 patients). Six out of 12 patients had mutation in bZIP region (CEBPAbZIP+). All six CEBPAbZIP+ patients (100%) achieved CR after induction chemotherapy versus 62% of CEBPAbZIP- patients. CEBPAbZIP+ patients showed a significantly longer OS (CEBPAbZIP+ 31.5 months vs CEBPAbZIP- 10 months) and disease free survival (DFS) (CEBPAbZIP+ 30 months vs CEBPAbZIP- 10.5 months). Conclusion: Our analysis of Serbian AML-NK patients showed CEBPAdm was not associated with better prognosis but our results indicate that CEBPAbZIP+ status is a good candidate for a prognostic molecular marker

Coexistence of trisomy 12 and del(13)(q14.3) in two patients with chronic lymphocytic leukemia

We describe two patients with diagnosis of chronic lymphocytic leukemia (CLL) in whom interphase fluo­rescence in situ hybridization (FISH) analysis revealed trisomy 12 and del(13)(q14.3) occurring in the same clone. These abnormalities are rarely seen together and the prognostic relevance of their coexistence is still unclear. According to some data, a probable adverse prognosis for this group of patients is suggested. Our patients have been in a stable phase of the disease for more than one year since the given abnormalities were documented in their karyotypes. Further study is necessary to determine the prognostic significance of coexistence of these abnormalities in CLL patients

Influence of applied CD34+ cell dose on the survival of Hodgkin's lymphoma and multiple myeloma patients following autologous stem cell transplants

Background/Aim. Autologous stem cell transplants (ASCTs) improve the rate of overall survival (OS) in patients with hematological malignancies such as multiple myeloma (MM) after induction chemotherapy, aggressive non-Hodgkin's lymphomas (NHL), and relapsed, chemotherapy-sensitive Hodgkin's lymphoma (HL). The study aim was to evaluate influence of applied CD34+ cell quantity on clinical outcome, as well as early post-transplant and overall survival (OS) of HL and MM patients following ASCT. Methods. This study included a total of 210 patients (90 HL/120 MM) who underwent ASCT. Stem cell (SC) mobilization was accomplished by granulocyte-colony stimulating factor (G-CSF) 10–16 μg/kg body mass (bm) following chemotherapy. For proven poor mobilizers, mobilization with G-CSF (16 μg/kgbm) and Plerixafor (24 or 48 mg) was performed. To our best knowledge, it was the first usage of the Plerixafor in our country in the ASCT-setting. Harvesting was initiated merely at "cut-off-value" of CD34+ cells ≥ 20 × 106/L in peripheral blood with "target-dose" of CD34+ cells ≥ 5 × 106/kgbm in harvest. The CD34+ cell count and viability was determined using flow cytometry. Results. The majority of HL patients (76.7%) were infused with > 5.0 × 106/kgbm CD34+ cells, while 68.3% of MM patients were treated by approximately 4.0–5.4 × 106/kgbm CD34+ dose, respectively. Beneficial response (complete/partial remission) was achieved in 83.3% (HL) and 94.2% (MM) patients. Among parameters that influenced survival of HL patients with positive response to the therapy, multivariate analysis (pre-ASCT performance status, CD34+ cell quantity applied, rapid hematopoietic, i.e. lymphocyte and platelet recovery) indicated that higher CD34+ cell dose used, along with pre-ASCT performance status correlated with superior event-free survival (EFS) and OS following ASCT. In MM patients, multivariate analysis (renal impairment, infused CD34+ cell quantity, early platelet recovery) indicated that the number of CD34+ cells infused was the most important parameter that influenced both EFS and OS after ASCT. Conclusion. Data obtained in this study undoubtedly confirmed that CD34+ cell dose applied is an independent factor that may contribute to superior clinical outcome and OS of HL and MM patients following ASCT

Acute promyelocytic leukemia lacking t(15;17): Molecular evidence of atypical PML/RAR-α transcriptional variant by gene sequencing

Uvod. Precizno dijagnostikovanje akutne promijelocitne leukemije (APL), ne samo na osnovu morfoloških i kliničkih parametara, već i na molekularnom nivou, veoma je važno radi primene adekvatne ciljane terapije. Prikaz bolesnika. Prikazali smo bolesnicu, staru 62 godine, sa dijagnozom APL. Primenom standardne citogenetičke analize, kao i primenom fluorescentne in situ hibridizacije (FISH), nije bilo potvrđeno prisustvo t(15;17) kod opisane bolesnice. Primenom metode reverzna transkriptazalančana reakcija polimeraze (RT-PCR), identifikovana su dva atipična promyelotic leukemia/retinoic acid receptor alpha (PML/RAR-α) fuziona transkripta. Oba transkripta su predstavljala izoforme. Duži transkript je zadržao "okvir čitanja" i kodirao je funkcionalan PML/RAR-α aberantni protein, dok je kraći transkript bio van "okvira čitanja". Zaključak. Naša studija ukazuje na potrebu za primenom molekularne metodologije u svakodnevnoj kliničkoj praksi. Precizna karakterizacija PML/RAR-α fuzionih transkipta čini osnovu za identifikovanje retkih bolesnika čije lečenje zahteva dodatni oprez. Prema našim saznanjima, ovo je tek peti slučaj opisanog atipičnog PML/RAR-α transkripta koji u sebi sadrži celokupan PML egzon 7a, a među njima jedini koji se nije mogao detektovati primenom citogenetičke i FISH analize. Svi ovde predstavljeni slučajevi su imali smrtni ishod. Zbog toga, naši rezulatati, zajedno sa sličnim slučajevima opisanim u literaturi, naglašavaju značaj detaljne identifikacije atipičnih PML/RAR-α fuzija, ne samo u svrhu prepoznavanja njihove uloge u procesu leukemogeneze, veći i u smislu procene njihovog uticaja na ishod lečenja.Introduction. The accurate diagnosis of acute promyelocytic leukemia (APL), not only on the morphological and clinical, but also on the molecular level, is very important for application of targeted therapies. Case report. A 62year-old woman presented with APL. By using conventional cytogenetic analysis as well as applying the fluorescence in situ hybridization (FISH) analysis it has not been possible to confirm the presence of t(15;17) in the presented patient. Using reverse transcriptase polymerase chain reaction (RT-PCR) two atypical promyelotic leukemia/retinoic acid receptor alpha (PML/RAR-α) fusion transcripts were identified. Both detected transcripts were isoforms. The larger transcript was in-frame, coding for functional aberrant PML/RAR-α protein, while the shorter transcript was an out-of-frame. Conclusion. Our study highlights the need for the application of molecular methodology in daily clinical practice. Precise characterization of PML/RAR-α fusion transcript creates a basis for identifying rare individual cases that require special caution when treating such patients. To our knowledge this is only the fifth case of atypical PML/RAR-α transcript containing full PML exon 7a, and among them the only one that was cytogenetically cryptic and FISH negative. All of the herein presented cases had lethal outcome. Therefore, our findings with the additional review of the literature, emphasizes the importance of detailed identification of atypical PML/RAR-α fusions, not only for the purpose of knowing their role in leukemogenesis, but also for the assessment of the impact that they can have on the outcome of the treatment

Proinflammatory Cytokine IL-6 and JAK-STAT Signaling Pathway in Myeloproliferative Neoplasms

The recent JAK1/2 inhibitor trial in myeloproliferative neoplasms (MPNs) showed that reducing inflammation can be more beneficial than targeting gene mutants. We evaluated the proinflammatory IL-6 cytokine and JAK-STAT signaling pathway related genes in circulating CD34(+) cells of MPNs. Regarding laboratory data, leukocytosis has been observed in polycythemia vera (PV) and JAK2V617F mutation positive versus negative primary myelofibrosis (PMF) patients. Moreover, thrombocytosis was reduced by JAK2V617F allele burden in essential thrombocythemia (ET) and PMF. 261 significantly changed genes have been detected in PV, 82 in ET, and 94 genes in PMF. The following JAK-STAT signaling pathway related genes had augmented expression in CD34(+) cells of MPNs: CCND3 and IL23A regardless of JAK2V617F allele burden; CSF3R, IL6ST, and STAT1/2 in ET and PV with JAK2V617F mutation; and AKT2, IFNGR2, PIM1, PTPN11, and STAT3 only in PV. STAT5A gene expression was generally reduced in MPNs. IL-6 cytokine levels were increased in plasma, as well as IL-6 protein levels in bone marrow stroma of MPNs, dependent on JAK2V617F mutation presence in ET and PMF patients. Therefore, the JAK2V617F mutant allele burden participated in inflammation biomarkers induction and related signaling pathways activation in MPNs

Gene expression profile of circulating CD34(+) cells and granulocytes in chronic myeloid leukemia

Purpose: We compared the gene expression profile of peripheral blood CD34(+) cells and granulocytes in subjects with chronic myeloid leukemia (CML), with the accent on signaling pathways affected by BCR-ABL oncogene. Methods: The microarray analyses have been performed in circulating CD34(+) cells and granulocytes from peripheral blood of 7 subjects with CML and 7 healthy donors. All studied BCR-ABL positive CML patients were in chronic phase, with a mean value of 2012 +/- SD of CD34(+) cells/mu l in peripheral blood. Results: The gene expression profile was more prominent in CML CD34(+) cells (3553 genes) compared to granulocytes (2701 genes). The 41 and 39 genes were significantly upregulated in CML CD34(+) cells (HINT1, TXN, SERBP1) and granulocytes, respectively. BCR-ABL oncogene activated PI3K/AKT and MAPK signaling through significant upregulation of PTPN11, CDK4/6, and MYC and reduction of E2F1, KRAS, and NFKBIA gene expression in CD34(+) cells. Among genes linked to the inhibition of cellular proliferation by BCR-ABL inhibitor Imatinib, the FOS and STAT1 demonstrated significantly decreased expression in CML. Conclusion: The presence of BCR-ABL fusion gene doubled the expression quantity of genes involved in the regulation of cell cycle, proliferation and apoptosis of CD34(+) cells. These results determined the modified genes in PI3K/AKT and MAPK signaling of CML subjects

The emergence of non-secretory multiple myeloma during the non-cytotoxic treatment of essential thrombocythemia: A case report

Introduction. The emergence of multiple myeloma as a second malignancy in patients with essential thrombocythemia is extremely rare. Several cases have been published so far, pointing out the impact of a cytotoxic effect during treatment of essential thrombocythemia on the development of multiple myeloma. Case presentation. We report the case of a 52-year-old Caucasian man who presented to our hospital because of leukocytosis, a slightly decreased hemoglobin level and thrombocytosis. After a complete hematological work-up, essential thrombocythemia was diagnosed. The patient was included in a multicenter clinical study, treated with anagrelide and his platelet counts were maintained in the normal range for more than 3 years. A sudden drop in his hemoglobin level with normal leukocyte and platelet count occurred at the same time as a back pain. Magnetic resonance imaging of his spine revealed the existence of a pathological fracture of Th4, the collapse of the upper edge of Th7 and osteolytic lesions of multiple thoracic vertebrae. Repeated hematological examinations, including bone biopsy with immunohistochemistry, disclosed diagnosis of multiple myeloma of the non-secretory type. Conclusions: To the best of our knowledge this is the first published case in which multiple myeloma developed during the treatment of essential thrombocythemia with the non-cytotoxic drug anagrelide. Our attempts to find a common origin for the coexistence of multiple myeloma and essential thrombocythemia have not confirmed the genetic basis of their appearance. Further studies are needed to determine the biological impact of this coexistence

Immunophenotypic characterization of acute myeloid leukemia in adults and its role in the diagnosis,monitoring, and prognosis of the disease

U tezi su ispitivani savremeni aspekti primene imunofenotipizacije multiparametarskom protočnom citofluorimetrijom (IMPC) u dijagnostici i praćenju AML. Ispitivanjem je obuhvaćeno 320 odraslih bolesnika sa de novo AML, od kojih je 294 uključeno u retrospektivno ispitivanje (dijagnoza, klasifikacija i prognoza), dok je ostalih 26 bolesnika bilo uključeno u prospektivno ispitivanje (praćenje minimalne rezidualne bolesti - MRB). Bolesnici su klasifikovani kao AML-neklasifikovana (48%), AML sa rekurentnim genetskim poremećajima (37,4%) i AML sa znacima mijelodisplazije (14,6%). IPCM omogućava postavljanje dg AML analizom ks kod 98% bolesnika. Ispitivanjem 44 različita hLDM utvrđeno je da je njihova ekspresija na blastima deregulisana, o čemu govore aberacije u njihovoj ekspresiji kod svakog bolesnika. Heterogen ćelijski sastav populacije leukemijskih ćelija (leukemijski blasti i prekursori) utvrđen je kod 55% bolesnika sa AML. Imunološka i citomorfološka klasfikacija AML su saglasne kod 73% bolesnika, odnosno imunološka i SZO klasifikacija kod 68% bolesnika. Ispitivanje MRB sprovedeno je primenom jedne (42%) ili dve (58%) kombinacije IFSL po bolesniku. Primenom IMPC, pokazana je visoka učestalost MRB kod naših bolesnika sa AML posle lečenja indukcionom (69%) odnosno konsolidacionom terapijom (50%). Nivo MRB u ks bolesnika ≥0,1% NĆ posle indukcione terapije, svrstava bolesnike u grupu visokog rizika za razvoj relapsa bolesti. Ispitivanje prognostičkog značaja hLDM kod bolesnika sa AML, pokazalo je značajnu vezu između rane monocitne diferencijacije leukemijskih ćelija, ekspresije CD22 molekula i pojave rane smrti i (p<0,05), odnosno niže incidence kompletne remisije (p<0,05)...In the thesis are examined modern aspects of application immunophenotyping and multiparameter flow cytometry (IMPC) in the diagnosis and monitoring of AML. The study included 320 adult patients with de novo AML, of which 294 included in the retrospective study (diagnosis, classification and prognosis), while the other 26 patients were included in a prospective study (minimal residual disease - MRD). Patients were classified as AML-unclassified (48%), AML with recurrent genetic abnormalities (37.4%) and AML with signs of myelodysplasia (14.6%). IPCM allows setting dg AML by analysis of bone marrow (bm) in 98% of patients. By examining of 44 different HLDM, it was found that its expression is deregulated on the blasts, whereas at least one type of immunophenotypic aberrations was found per patient. Heterogeneous cellular composition of the population of leukemic cells (leukemic blasts and precursors) was found in 55% of patients with AML. Immunological and cytomorphological classification of AML agree with 73% of patients, respectively immunological and WHO classification in 68% of patients. MRD trial was conducted by one (42%) or two (58%) combination of IFSL per patient. By applying the IMPC, a high incidence of MRD was detected in our patients with AML, after induction (69%) and/or consolidation therapy (50%). The level of MRD in bm of patients ≥0,1% NC after induction therapy, classified patients in the high risk group for the development of relapse. The prognostic significance of HLDM in patients with AML, showed a significant association between early monocytic differentiation of leukemia cells, the expression of CD22 molecule and the appearance of early death (p<0.05), and lower incidence of complete remission (p<0.05)..