Vlatko Pejša

Bone marrow renin-angiotensin system expression in polycythemia vera and essential thrombocythemia depends on JAK2 mutational status

Recent observations raise possibility for constitutively active, mutated Jak2 to modulate expression of RAS genes in CMPD. We analyzed the expression of AGT, renin, AT2R1 and ACE genes in normal and bone marrows of PV and ET patients with the respect to the presence of V617F JAK2 mutation. PV and ET had different expression patterns of major RAS components compared to normal BM which was primarily associated with the JAK2V617F mutation and less with PV or ET disease phenotype. However, AT2R1 was exclusively markedly upregulated only in PV, while ET showed moderate expression irrespective of the JAK2 mutational status.

The Degree of Anisocytosis Predicts Survival in Patients with Primary Myelofibrosis

Background: Red cell distribution width (RDW) provides a quantitative measure of anisocytosis and it is associated with the presence of subclinical systemic inflammation and a poor outcome in a variety of diseases when elevated. Anisocytosis is a feature of primary myelofibrosis (PMF) but it’s prognostic role in PMF has not yet been evaluated. Aims: To determine whether anisocytosis bears prognostic significance in patients with PMF and its relation to disease features. Methods: 33 newly-diagnosed patients with PMF were analyzed in this study. Baseline RDW values were obtained in addition to other routine blood analyses (CRP, LDH, complete blood count and iron metabolism parameters) and JAK2 V617F mutational status. Patients were staged according to IPSS prognostic scoring system, liver and spleen size were assessed by palpation. The Mann Whitney U test, the Pearson correlation and the Χ2 test/ the Fisher test were used where appropriate. Survival analyses were performed using methods of Kaplan and Meier, the log-rank test and the Cox regression analysis. All statistical tests were two-sided and P values <0.05 were considered significant. Results: Median RDW was 19.0% (15.2% - 22.5%). RDW correlated significantly with hemoglobin (p=0.005), CRP (p=0.031), spleen size (p=0.036) and IPSS score (p=0.003). Patients with more pronounced anisocytosis had an inferior overall survival (OS) – very-high RDW (≥19.0%) vs. high RDW (15.1% - 18.9%) subgroup, HR 5.37, p=0.002. RDW remained significantly associated with OS (p=0.002) in a multivariate model including IPSS score, hemoglobin level and CRP. Summary/Conclusion: PMF pathogenesis surpasses inflammation as only cause of anisocytosis. A higher degree of anisocytosis is associated with more advanced disease features and a decreased overall survival. RDW encompasses standard prognostic score and may help in the rapid detection of patients with an unfavorable prognosis.

No adverse effect of ABVD chemotherapy in a patient with chronic hepatitis C and Hodgkin’s disease

SummaryThere is insufficient information on the effects of chemotherapy protocols for Hodgkin’s disease (HD) and the course of coexisting hepatitis C virus (HCV) infection. A single literature case reported a patient with HD who developed fulminant hepatitis and hepatic coma after receiving chemotherapy. The case described here is of a female patient previously exposed to prolonged war stress, complicated by intravenous drug abuse and chronic hepatitis C. One year after diagnosis of HCV infection she was diagnosed with HD (nodular sclerosis type II, clinical stage IIIB). The patient received six cycles of ABVD chemotherapy (doxorubicin, bleomycin, vinblastine and dacarbazine) resulting in complete remission of HD. There was no hepatitis flare either during or after chemotherapy. In conclusion, there were no adverse effects of the ABVD regimen on the course of HCV infection in this patient who was successfully treated for HD. Because concurrent HCV infection and HD is extremely rare, we discuss here the possibility of the synergistic contribution of chronic war stress and hepatitis C infection in the pathogenesis of HD.

Heat shock protein 27 (HSP27/HSPB1) expression is increased in patients with primary and secondary myelofibrosis and may be affecting their survival

Marko Lucijanic , Ana Livun , Katarina Marija Tupek, Tajana Stoos-Veic, Gorana Aralica, Iva Gecek, Vlatko Pejsa and Rajko Kusec Department of Hematology, University Hospital Dubrava, Zagreb, Croatia; Clinical Institute of Laboratory Diagnosis, Division of Molecular Diagnosis and Genetics, University Hospital Dubrava, Zagreb, Croatia; Department of Clinical Cytology and Cytometry, University Hospital Dubrava, Zagreb, Croatia; School of Medicine, University of Osijek, Osijek, Croatia; Department of Pathology, University Hospital Dubrava, Zagreb, Croatia; School of Medicine, University of Zagreb, Zagreb, Croatia

High absolute basophil count is a powerful independent predictor of inferior overall survival in patients with primary myelofibrosis

ABSTRACT Objectives: To investigate the clinical and prognostic significance of absolute basophil count (ABC) in patients with primary myelofibrosis (PMF). Methods: We retrospectively investigated 58 patients with PMF treated in our institution in the period from 2006 to 2017. ABC was obtained in addition to other hematological and clinical parameters. Patients were separated into high and low ABC groups using the Receiver operating characteristic curve analysis. Results: ABC was higher in PMF patients than in healthy controls (P < 0.001). Patients with high ABC had higher white blood cells (P < 0.001), higher red cell distribution width (P = 0.035), higher lactate dehydrogenase (P < 0.001), more frequently had circulatory blasts (P < 0.001), constitutional symptoms (P = 0.030) and massive splenomegaly (P = 0.014). ABC was also positively correlated with absolute monocyte count (AMC) (P < 0.001) and other components of differential blood count. There was no difference in ABC regarding driver mutations or degree of bone marrow fibrosis. Univariately, high ABC was significantly associated with inferior overall survival (hazard ratio (HR) 4.79, P < 0.001). This effect remained statistically significant (HR 4.27, P = 0.009) in a multivariate Cox regression model adjusted for age, gender, Dynamic International Prognostic Scoring System (HR 2.6, P = 0.001) and AMC (HR 8.45, P = 0.002). Discussion: High ABC reflects higher disease activity and stronger proliferative potential of disease. ABC and AMC independently predict survival and therefore seem to reflect different underlying pathophysiologic processes. Hence, both have a potential for improvement of current prognostic scores. Conclusion: Basophils represent a part of malignant clone in PMF and are associated with unfavorable disease features and poor prognosis which is independent of currently established prognostic scoring system and monocytosis.

Rituximab with dose-adjusted EPOCH as first-line treatment in patients with highly aggressive diffuse large B-cell lymphoma and autologous stem cell transplantation in selected patients.

Aim To assess the benefit of rituximab with dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (R-DA-EPOCH) regimen as a first-line treatment for patients with diffuse large B-cell lymphoma (DLBCL) presenting with unfavorable or aggressive features, and autologous stem cell transplantation (ASCT) as a part of the first-line treatment for selected DLBCL patients with additional aggressive features. Methods We retrospectively analyzed 75 newly diagnosed DLBCL patients with Ki-67+≥80% or International Prognostic Index ≥2 who were treated with R-DA-EPOCH between 2005 and 2015. Of 24 DLBCL patients with additional aggressive features (Ki-67+≥90% or age-adjusted IPI≥2) who were planned to receive consolidation with ASCT, 17 patients underwent the procedure. We determined the overall response rate (ORR), complete remission (CR), partial remission (PR), 5-year overall survival (OS), and progression free survival (PFS) in all DLBCL patients and specifically those planned to receive ASCT. Results All 75 patients included in the analysis started one or more cycles of therapy. The ORR, CR, and PR rates were 80%, 55%, and 25%, respectively. The response was non-evaluable in 10 of 75 patients due to treatment discontinuation. The OS and PFS rates for all 75 patients were 70% and 61%, respectively, and 80% and 79%, respectively, for 24 planned-to-receive-ASCT patients. Age (≤65 vs >65 years) had no prognostic impact on OS and PFS (P = 0.994 and P = 0.827, respectively). Conclusion Our retrospective analysis of one of the largest DLBCL patient cohorts outside the US National Cancer Institute showed that R-DA-EPOCH is a very effective therapeutic option as a first-line treatment of DLBCL patients with unfavorable prognostic features irrespective of their age. ASCT provided additional benefit for DLBCL patients with additional aggressive features.

Prognostic implications of low transferrin saturation in patients with primary myelofibrosis

OBJECTIVES Transferrin saturation (TSAT) 20% or less is considered to represent functional iron deficiency in the context of malignant disease, phenomenon mediated through inflammatory changes of iron homeostasis. We aimed to investigate clinical and prognostic significance of low TSAT in patients with primary (PMF) and secondary myelofibrosis (SMF), malignant diseases characterized by strong inflammatory milieu. METHODS We retrospectively analyzed 87 patients with myelofibrosis and compared TSAT with disease specific parameters. RESULTS One-third of patients had TSAT ≤20%. Lower TSAT was significantly associated with Janus-kinase-2 (JAK2) mutation (P = 0.007), transfusion independency (P = 0.003), higher platelets (P = 0.004), lower mean-corpuscular-volume (P < 0.001), lower ferritin (P < 0.001), higher absolute-neutrophil-count (P = 0.027), lower absolute-lymphocyte-count (P = 0.041) and lower albumin (P = 0.018). PMF patients presenting with low TSAT (≤20%) experienced significantly shorter overall-survival (OS) (HR = 2.43; P = 0.017), whereas TSAT did not affect OS of SMF patients (HR = 1.48; P = 0.623). Low TSAT remained significantly associated with inferior OS in PMF in a series of multivariate Cox regression models comparing its properties to anemia, transfusion dependency, ferritin and Dynamic-International-Prognostic-System (DIPSS). CONCLUSIONS Low TSAT has detrimental effect on survival of PMF patients. This effect is independent of anemia and of ferritin levels that seem to be better at representing iron overload in PMF patients.

Higher Sclerostin/SOST expression is associated with lower percentage of circulatory blasts and better prognosis in patients with myelofibrosis

Dear Editor, Sclerostin (a product of SOST gene) acts as a main negative regulator of bone metabolism, exerting its properties through inhibition of canonical-WNT signaling-pathway (cWNT) in osteoblasts [1]. It is produced by osteocytes and bone marrow (BM) cells. cWNT activation is implicated in pathogenesis of Philadelphia chromosome-negative myeloproliferative neoplasms (PhMPNs) [2, 3], diseases characterized by remodeling of BM stroma and development of BM fibrosis/ osteosclerosis during course of the disease. We aimed to investigate Sclerostin/SOSTexpression in BM tissues of patients with primary (PMF) and secondary post PhMPN myelofibrosis (SMF) and to assess its clinical correlations. We retrospectively investigated Sclerostin/SOST expression in BM of 66 diseased patients (51 PMF, 15 post-PV/ post-ET-SMF, diagnoses were established according to the WHO [4] and the IWG-MRT [5] criteria) and 18 ageand sex-matched controls (limited-stage aggressive non-Hodgkin lymphoma patients without BM involvement) using immunohistochemistry (IHC; Sclerostin 21933-1-AP rabbit polyclonal Proteintech primary antibody) and real-time polymerase chain reaction (RT-PCR; SOST Hs00228830_m1 Thermo Fischer Scientific TaqMan assay; evaluated from BM aspirates). Sclerostin expression was expressed as a percentage of positive cells. SOST mRNA expression was normalized to Abl and expressed as a ΔCT value. Correlations with clinical parameters were made. Optimal cut-off values for survival were determined using the ROC curve analysis. The MannWhitney U test, the Spearman rank correlation, the CoxMantel log rank test [6], and the Cox regression analysis were used. Analyses were done using MedCalc Statistical Software ver.18 (MedCalc Software bvba, Ostend, Belgium). P values < 0.05 were considered statistically significant. All procedures were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. All patients provided written informed consent. The median age of patients was 67 years; 59% were males, 67% had grades II-III BM fibrosis, and 70, 11, and 2% were JAK2, CALR, and MPL mutated, respectively. Median followup of our cohort was 74 months with median overall survival of 69 months. Sclerostin/SOST expression did not significantly differ between PMF and SMF, nor between patients and controls. However, non-significantly higher expression was observed in myelofibrosis patients (both PMF and SMF) than that in controls; result was near statistical significance for SOST (P = 0.056). In diseased patients, higher Sclerostin expression measured by IHC was significantly correlated with lower percentage of circulatory blasts (rho − 0.28, P = 0.042) and transfusion dependency (P = 0.049). Higher SOST expression measured by RT-PCR was similarly significantly correlated with lower percentage of circulatory blasts (rho − 0.44, P = 0.042), but also higher platelets (rho 0.4, P = 0.031) and smaller spleen size (rho − 0.6, P = 0.001). We found no significant association of Sclerostin/SOST expression with JAK2, CALR, and MPL mutation status or degree of bonemarrow fibrosis. Patients with higher Sclerostin expression (HR = 0.35, P = 0.006) and higher SOST expression (HR = 0.44, P = 0.044) had superior overall survival than patients presenting with lower Sclerostin/SOST expression as shown in a Fig. 1. This association remained significant for SOST (HR = 0.21, P = 0.025) after adjusting for age, gender, and circulatory blasts (HR = 1.06, P = 0.002). * Marko Lucijanic markolucijanic@yahoo.com

Guidelines for Diagnosis and Treatment of Chronic Lymphocytic Leukemia. Krohem B-Cll 2017.

SUMMARY – Recent developments in the diagnosis and treatment of chronic lymphocytic leukemia (B-CLL) have led to change of approach in clinical practice. New treatments have been approved based on the results of randomized multicenter trials for first line and for salvage therapy, and the results of numerous ongoing clinical trials are permanently providing new answers and further refining of therapeutic strategies. This is paralleled by substantial increase in understanding the disease genetics due to major advances in the next generation sequencing (NGS) technology. We define current position of the Croatian Cooperative Group for Hematologic Disease on diagnosis and treatment of CLL in the transition from chemo-immunotherapy paradigm into a new one that is based on new diagnostic stratification and unprecedented therapeutic results of B-cell receptor inhibitors (BRI) and Bcl-2 antagonists. This is a rapidly evolving field as a great number of ongoing clinical trials constantly accumulate and provide new knowledge. We believe that novel therapy research including genomic diagnosis is likely to offer new options that will eventually lead to time limited therapies without chemotherapy and more effective clinical care for B-CLL based on individualized precision medicine.

Loss of response to azacitidine is associated with deletion 12p13 in a patient with myelodysplastic syndrome with unique translocation t(13;17)(q12;q25) after prior breast cancer and acute promyelocytic leukemia

Dear Editor, With increasingly successful treatment of malignancies, therapy-related leukemia (TRL) and myelodysplastic syndrome (MDS) are increasingly observed, especially in association with prior radiotherapy and the use of alkylating agents or topoisomerase-II inhibitors [1, 2]. They are characterized by lesions of chromosomes 5 and 7 and poor survival (median 7–10 months) [3, 4]. Azacitidine may be a reasonable treatment choice [5] although safety concerns in patients with complex cytogenetics [6] or previous acute promyelocytic leukemia (APL) exist [7]. Here, we present a case of two consecutive, individually rare TRLs following initial solid tumor and subsequent treatments with the development of unique translocation (13;17)(q12;q25). Azacitidine was successfully used achieving 1-year-long remission when patient relapsed, revealing a distinctive karyotype (Fig. 1). A female patient aged 61 was diagnosed with breast cancer in 2006. She had mastectomy, received four cycles of doxorubicin/cyclophosphamide, four cycles of paclitaxel, and radiotherapy accomplishing remission. In 2009, APL was diagnosed, PML/RARα positive (47, XX,+8,t(15;17)(q24;q21)[10]). Antracycline/ATRA-based induction and three consolidation therapies were followed by 2year ATRA/6-MP/methotrexate maintenance. Remission was soon achieved, and patient remained without detectable PML/ RARα transcript thereafter. In 2013, the patient developed anemia requiring transfusion support. MDS (18 % blasts) with complex cytogenetics (45,XX,del(5)(q22q33),-7,t(13;17)(q12;q25), del(12)(p13),add(20)(q11)[cp15]) was diagnosed. Azacitidine was instituted as salvage therapy, and the patient became transfusion independent, achieving hematological remission. After 11 cycles, azacitidine had to be stopped due to the development of anemia and thrombocytopenia. Repeated revisions showed increased number of blasts (15 % progressing to 72 %) and alt e r ed cy togene t i c s (45 ,XX,de l (5 ) (q22q33 ) , 7 , t(13;17)(q12;q25),del(12)(p13)[20]). Fluorescence in situ hybridization (FISH) was performed on the actual sample and retrospectively (at the time of MDS diagnosis) revealing 12p13 deletion was initially present in a subclone comprising 35 % of cells and now prevailing in 90 % of cells. 17p13 deletion was absent in both samples. The patient receives supportive treatment and remains alive more than 18 months since diagnosis. * Ozren Jaksic ojaksic@kbd.hr