Clinical Presentation and Therapy of Primary Immune Thrombocytopenia Resistant to Splenectomy

Background: A satisfactory therapeutic response is achieved with splenectomy in 60–80% of the patients diagnosed with immune thrombocytopenic purpura (ITP). There is an ongoing consensus on the short-term efficacy of splenectomy, however, its long-term efficacy remains controversial. Our aim was to establish the frequency of resistance and relapse after splenectomy, the occurrence of complications, the therapeutic strategies and the drug efficacy in splenectomy-resistant ITP. Methods: We retrospectively analyzed 138 adult ITP patients who had been previously diagnosed and treated at the Clinic of Hematology, Clinical Center of Serbia, and who underwent splenectomy between 1987 and 2018. Results: Of the 138 ITP patients, 20.3% (n=28/138) were refractory to splenectomy, 11.6% (n=16/138) relapsed and 8.7% (n=12/138) were primarily resistant. The average post-splenectomy follow-up period was 117 months (range 3-474). The average follow-up period of the patients resistant to splenectomy was 147 months (range 23-474). Of the patients refractory to splenectomy, 67.8% (n=19/28) showed a good therapeutic response: 49% (n=14/28) complete remission and 18.8% (n=5/28) partial remission. The response was usually achieved using the following drugs: romiplostim (100%), eltrombopag (75%), cyclosporine (66.67%), mycophenolate mofetil (50%), danazol (50%) and corticosteroids (40.9%). Hemorrhagic and non-hemorrhagic complications occurred in 78.6% (n=22/28) and 28.6% (n=8/28) of the patients, respectively. Conclusion: Splenectomy remains a very efficient therapeutic modality for the treatment of ITP patients with a high percentage of splenectomy-resistant patients achieving remission. Thrombopoietin receptor agonists have shown exceptional results so far in the treatment of refractory ITP patients

Expression Profiles of Long Non-Coding RNA GAS5 and MicroRNA-222 in Younger AML Patients

Acute myeloid leukemia (AML) is a heterogeneous malignant disease both on clinical and genetic levels. AML has poor prognosis and, therefore, there is a constant need to find new prognostic markers, as well as markers that can be used as targets for innovative therapeutics. Recently, the search for new biomarkers has turned researchers' attention towards non-coding RNAs, especially long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs). We investigated the expression level of growth arrest-specific transcript 5 (GAS5) lncRNA in 94 younger AML patients, and also the expression level of miR-222 in a cohort of 39 AML patients with normal karyotype (AML-NK), in order to examine their prognostic potential. Our results showed that GAS5 expression level in AML patients was lower compared to healthy controls. Lower GAS5 expression on diagnosis was related to an adverse prognosis. In the AML-NK group patients had higher expression of miR-222 compared to healthy controls. A synergistic effect of GAS5(low)/miR-222(high) status on disease prognosis was not established. This is the first study focused on examining the GAS5 and miR-222 expression pattern in AML patients. Its initial findings indicate the need for further investigation of these two non-coding RNAs, their potential roles in leukemogenesis, and the prognosis of AML patients

A gene expression profile associated with relapse of cytogenetically normal acute myeloid leukemia is enriched for leukemia stem cell genes

Some 50 – 80% of patients with acute myeloid leukemia (AML) achieve a complete remission with contemporary chemotherapy protocols, yet the majority of them eventually relapse with resistant disease: some patients no longer respond to chemotherapy at disease recurrence; others accomplish second and even third remissions whose decreasing duration nevertheless indicates that the pool of residual leukemic cells, i.e. of cells that persisted during treatment with cytotoxic drugs, increases with every round of therapy [1]. Either of these clinical courses therefore refl ects an enhanced chemotherapy resistance of leukemic cells at relapse as compared to the cell population at diagnosis. Molecular changes enabling malignant cells to survive exposure to cytotoxic drugs may already have been present in a subset of the leukemic cell population at presentation, or may emerge during treatment [2,3], but in any case are thought to be selected as a consequence of drug therapy, and to play a major role in therapy resistance at relapse. Remarkably, however, even though various types of molecular alterations may be acquired at relapse, neither specifi c cytogenetic alterations nor functionally relevant point mutations as identifi ed by whole genome sequencing were associated with relapse in a recurrent manner [2,3]. Certain copy number variations and known AML associated point mutations were newly present at relapse in small proportions of patients (usually 10%), but the latter were lost in other patients, indicating that they are unlikely to represent drivers of therapy resistance at disease recurrence [4]. Th ese fi ndings could either indicate that chemotherapy resistance at relapse is acquired through a large variety of different mechanisms, or that molecular changes of other types than those mentioned above are of more general relevance in this context. Indeed, an earlier study has suggested that the expression of specifi c genes may change in a consistent manner between diagnosis and relapse of AML [5]. However, only a limited number of genes and mostly unpaired samples were probed in this investigation. Th erefore, in the present study, genes whose expression changed in a relapse-specifi c manner were sought in a set of paired AML samples and on a genome-wide scale. To limit the genetic heterogeneity of the study population, only samples from patients with cytogenetically normal (CN) AML were used.Letter to the Edito

Immunoglobulin heavy chain gene rearrangements in patients with Gaucher disease

Uvod: Nekoliko studija u literaturi navode dokaze o povećanoj incidenci hematoloških komplikacija u bolesnika sa Gošeovom bolešću, uključujući monoklonsku i poliklonsku gamapatiju i hematološke malignitete, a posebno multipli mijelom. Metode: Određivana je serumska koncentracija imunoglobulina kao i rearanžman gena za teški lanac imunoglobulina - IGH, PCR analizom. Klonalni PCR produkti su direktno sekvencirani i analizirani koristeći adekvatne alate i baze podataka. Monoklonski proteini seruma su detektovani i identifikovani metodom elektroforeze. Rezultati: Među 27 bolesnika, klonalni IGH rearanžman je otkriven kod osmoro, od kojih je petoro imalo i monot klonski protein u serumu. Hipergamaglobulinemija je otkrivena u 9/27 bolesnika. Podaci o praćenju za 17 bolesnika su pokazali da je klonalni rearanžman ostao isti u četiri bolesnika, dok je u jednog bolesnika iščezao tokom perioda praćenja. Preostalih 12/17 bolesnika nisu imali klonalni IGH rearanžman niti su ga stekli nakon perioda praćenja. Zaključak: Iako klonska ekspanzija može da nastane relativno rano u toku Gošeove bolesti, barem sudeći prema rearanžmanu IGH gena, detektovani klonovi mogu biti tranzitorni. Pažljivo kliničko praćenje ovih bolesnika je obavezno, uključujući i nadzor nad limfoidnim neoplazmama, posebno multiplim mijelomom.Background: Several studies support the evidence of increased incidence of hematological complications in Gaucher disease including monoclonal and polyclonal gammopathies and blood malignancies, especially multiple myeloma. Methods: Serum concentrations of immunoglobulins and PCR analysis of the IGH gene rearrangements were performed. The clonal PCR products were directly sequenced and analyzed with the appropriate database and tools. Serum monoclonal proteins were detected and identified by electrophoresis. Results: Among 27 Gaucher patients, clonal IGH rearrangement was discovered in eight, with 5/8 having also serum monoclonal protein. Elevated immunoglobulins were detected in 9/27 patients. Follow-up data for 17 patients showed that the clonal rearrangement remained the same in four of them, however, in one patient it disappeared after the follow-up period. The remaining 12/17 patients were without previous IGH clonal rearrangement and remained so after the follow-up. Conclusions: Although clonal expansion may occur relatively early in the disease course, at least judging by the IGH gene rearrangements in Gaucher patients, the detected clones may be transient. A careful clinical follow-up in these patients is mandatory, including monitoring for lymphoid neoplasms, especially multiple myeloma

Acquired von Willebrand syndrome in multiple myeloma

Acquired von Willebrand syndrome (AvWS) is an uncommon complication of multiple myeloma (MM), and it is believed to be connected with paraprotein. The aim of this study was to determine the incidence of AvWS in patients with MM, and estimate the role of paraprotein in its occurrence. The study included 40 patients with MM. The plasma level of paraprotein, platelet adhesion on glass pearls, plasma von Willebrand factor antigen concentration, and ristocetin-induced platelet aggregation (RIPA) were measured initially. Absence of RIPA was found in six patients with MM (15%); however, all six of them had normal levels of von Willebrand factor antigen. Paraprotein was isolated from the serum of these patients. Platelet aggregation was measured in six healthy donors before and after addition of the isolated paraprotein. RIPA was significantly decreased in healthy donors in the presence of paraprotein (P lt 0.001). The same test was repeated with added human immunoglobulins for intravenous use without any change in RIPA. A significant negative correlation between plasma paraprotein level and RIPA was found (P lt 0.001). These investigations have shown that paraprotein is associated with AvWS in patients with MM

Expression profiles of long non-coding RNA gas5 and microrna-222 in younger AML patients

Introduction: Acute myeloid leukemia (AML) is a heterogeneous malignant disease, that accounts for 80% of all acute leukemias in adults. Imprecise risk stratification and lack of personalized treatment creates a constant need to find new prognostic markers and targets for innovative therapeutics. Recently, thissearch has pointed towards non-coding RNAs(ncRNA). Numerousstudies have shown dysregulation of lncRNA GAS5 in cancers, but it was poorly investigated in AML. Since GAS5 actslike a molecularsponge for miR-222, co-expression profiles of these non-coding RNAs could be novel prognostic markersin AML. Methods: GAS5 expression levels were analysed in 94 AML patients and 14 healthy controls using RealTime PCR and miR-222 expression levels were analysed in a subgroup of 39 patients with normal karyotype (AML-NK). ROC curve analyses were used to find a cut-off value between GAS5high and GAS5low, while the median value was used for distinguishing between miR-222high and miR-222low. Results: We showed that GAS5 expression in AML patients was lower compared to healthy controls. Lower GAS5 expression on diagnosis was related to an adverse prognosis. The disease-free survival and the overallsurvival were lower in the GAS5low group butsurvival analysisfailed to confirm thisfinding. In the AML-NK group patients had higher expression of miR-222 compared to healthy controls. A synergistic effect of GAS5low/miR-222high status on disease prognosis was not established. Conclusion:Our findingsindicate the potential prognostic significance of GAS5 expression and the need for further investigation of these two non-coding RNAs and their potential roles in leukemogenesis

Expression levels of BCL2, BAX and MDR1 as pharmacotranscriptomic and prognostic markers of prognosis in acute myeloid leukemia

Acute myeloid leukemia (AML) is a malignancy of hematopoetic tissue which occurs due to a halt in differentiation, loss of proliferation control and dysregulated apoptosis of myeloid progenitor cells. In many cancers, as well as AML, dysregulation of apoptosis constitutes the basis of pathogenesis and this phenomenon is important for chemotherapy success. Pharmacotranscriptomic markers of AML prognosis could be targets of specific therapy. The anti-apoptotic gene BCL2 (B-cell lymphoma protein 2), the pro-apoptotic BAX (BCL2-associated X) and genes involved in drug resistance, like MDR1 could have a significant impact on AML prognosis and therapy response. Bone-marrow samples at diagnosis were collected from 51 adult patients with AML-NK. Expressions of BCL2, BAX and MDR1 were analysed using the real-time polymerase chain reaction method. Statistical evaluation was performed. The presence of chemoresistance was found to be associated with overexpression of BCL2 (BCL2+) (p=0.018), while underexpression of BAX in patients has shown a greater affinity towards relapse (p=0.034). Evaluating the expressions of BCL2 and BAX in a combined effect has shown that 87% of patients with BAX/BCL2low status were resistant to therapy (p=0.024). BCL2+ status was associated with high expression of MDR1 (p<0.001). Likewise, high expression of MDR1 was associated with the absence of NPM1 and FLT3-ITD mutations (p=0.048 and p=0.010, respectively). This is the first study that focused only on AML-NK patients, when it comes to analysis of BCL2, BAX and MDR1 gene expression profiles. The results of this preliminary study have shown that high BCL2 expression would likely lead to resistance from chemotherapy, making anti-BCL2 treatment a viable option in patients with this expression profile. A study on a larger group of patients could clarify the prognostic importance of the studied genes in adult AML-NK patients and improve the precision medicine approach in the field of hematology.Book of abstracts: International Conference of Biochemists and Molecular Biologists in Bosnia and Herzegovina - ABMBBIH May, 202

Експресиони образац дуге некодирајуће РНК GAS5 и микроРНК- 222 код млађих пацијената оболелих од АМЛ

Akutna mijeloidna leukemija (AML) je heterogeno maligno oboljenje kako na kliničkom tako i na genetičkom nivou. AML ima lošu prognozu, i zato postoji konstantna potreba za novim prognostičkim markerima, kao i markerima koji mogu biti mete za inovativne terapeutike. U skorije vreme potraga za novim biomarkerima je naučnicima skrenula pažnju ka nekodirajućim RNK, naročito dugim nekodirajućim RNK (dnkRNK) i mikroRNK (miRNK). U ovom radu ispitali smo nivo ekspresije „growth arrest-specific transcript 5“ (GAS5) dnkRNK kod 94 mlađih osoba obolelih od AML, kao i nivo ekspresije miR-222 u grupi od 39 osoba obolelih od AML sa normalnim kariotipom (AML-NK), u cilju da ispitamo njihov prognostički potencijal. Naši rezultati su pokazali da je nivo ekspresije GAS5 kod obolelih od AML niži u poređenju sa zdravim kontrolama, i češći je u grupi sa nepovoljnom prognozom. U AML-NK grupi pacijenti su imali povišenu ekspresiju miR-222 u poređenju sa zdravim kontrolama. Sinergistički efekat „GAS5low/miR-222 high“ nije utvrđen. Ovo je prva studija koja je ispitala interakciju ekspresije GAS5 i miR-222 kod obolelih od AML. Naši rezultati indikuju potrebu za daljim ispitivanjem ove dve nekodirajuće RNK i njihove potencijalne uloge u leukemogenezi i prognostici AML pacijenata.Акутна мијелоидна леукемија (АМЛ) је хетерогено малигно обољење како на клиничком тако и на генетичком нивоу. АМЛ има лошу прогнозу, и зато постоји константна потреба за новим прогностичким маркерима, као и маркерима који могу бити мете за иновативне терапеутике. У скорије време потрага за новим биомаркерима је научницима скренула пажњу ка некодирајућим РНК, нарочито дугим некодирајућим РНК (днкРНК) и микроРНК (миРНК). У овом раду испитали смо ниво експресије „growth arrest-specific transcript 5“ (GAS5) днкРНК код 94 млађих особа оболелих од АМЛ, као и ниво експресије miR-222 у групи од 39 особа оболелих од АМЛ са нормалним кариотипом (АМЛ-НК), у циљу да испитамо њихов прогностички потенцијал. Наши резултати су показали да је ниво експресије GAS5 код оболелих од АМЛ нижи у поређењу са здравим контролама, и чешћи је у групи са неповољном прогнозом. У АМЛ-НК групи пацијенти су имали повишену експресију miR-222 у поређењу са здравим контролама. Синергистички ефекат „GAS5low/miR-222 high“ није утврђен. Ово је прва студија која је испитала интеракцију експресије GAS5 и miR-222 код оболелих од АМЛ. Наши резултати индикују потребу за даљим испитивањем ове две некодирајуће РНК и њихове потенцијалне улоге у леукемогенези и прогностици АМЛ пацијената.Knjiga sažetaka: Treći Kongres biologa Srbije, Zlatibor, Srbija 21 - 25. 9. 2022