Olga Grzybowska-Izydorczyk

Expression and prognostic significance of the inhibitor of apoptosis protein (IAP) family and its antagonists in chronic lymphocytic leukaemia.

Impaired apoptosis is still considered to be an important event in the development and progression of chronic lymphocytic leukaemia (CLL). However, mechanisms of this defect have not been fully elucidated. In this study, expression of inhibitor of apoptosis proteins, IAPs (cIAP1, cIAP2, XIAP and survivin), and their antagonists (Smac/DIABLO and HtrA2/Omi) was comprehensively analysed in 100 untreated CLL patients, using flow cytometry and Western blot techniques. Expression of anti-apoptotic cIAP1 and cIAP2 in leukaemic cells was significantly higher than in non-tumour lymphocytes (p=0.000001 and p=0.014, respectively), whereas the IAP-antagonist, Smac/DIABLO, was decreased in CLL (p=0.010). Higher expression of all analysed IAPs (cIAP1, p=0.002; cIAP2, p=0.026; XIAP, p=0.002; survivin, p=0.00006) and lower levels of Smac/DIABLO (p=0.006) were found in patients with progressive disease, compared to those with stable CLL. High baseline expression of cIAP1 and survivin correlated with worse response to treatment. Co-expression of these proteins was associated with shorter overall survival of CLL patients (p=0.005). In conclusion, CLL cells show the apoptosis-resistant profile of IAPs/IAP-antagonist expression. Upregulation of IAPs is associated with a progressive course of the disease. Co-expression of cIAP1 and survivin seems to be an unfavourable prognostic factor in CLL patients. Further studies with longer follow up are warranted to confirm and expand these findings.

Rituximab plus cladribine with or without cyclophosphamide in patients with relapsed or refractory chronic lymphocytic leukemia.

Objectives: The aim of our study was to determine the feasibility, effectiveness and toxicity of combined regimens consisting of rituximab and cladribine (2‐CdA) (RC) and RC plus cyclophosphamide (RCC) in the treatment of patients with recurrent or refractory chronic lymphocytic leukemia (CLL).

CD38 Gene Polymorphisms Contribute to Genetic Susceptibility to B-Cell Chronic Lymphocytic Leukemia: Evidence from Two Case-Control Studies in Polish Caucasians

Given the recent findings on the importance of CD38 signaling in the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL), we hypothesized that single nucleotide polymorphisms (SNP) in the CD38 gene may be related to B-CLL risk. We evaluated two potentially functional CD38 SNPs, intronic rs6449182 (184C>G) and missense rs1800561 (418C>T, Arg140Trp) in two hospital-based case-control studies (study A and validation study B). Genotyping was done using PCR-based assays in a total of 460 Polish Caucasian patients with B-CLL and 503 age-matched and gender-matched controls. We found that frequencies of both variant alleles (rs6449182 G and rs1800561 T) were significantly higher in B-CLL. In study A, logistic regression analysis revealed an association between B-CLL and genotypes: rs6449182 CG [odds ratio (OR), 3.57; 95% confidence interval (95% CI), 2.4-5.3], rs6449182 GG (OR, 5.2; 95% CI, 2.36-11.5), and rs1800561 CT (OR, 6.72; 95% CI, 1.5-30.1), although no homozygous rs1800561 TT genotype was detected in either study. These results were confirmed in study B, which showed an association between B-CLL and genotypes rs6449182 CG (OR, 4.00; 95% CI, 2.7-6.0), rs6449182 GG (OR, 12.84; 95% CI, 4.3-38.7), and rs1800561 CT (OR, 10.12; 95% CI, 1.3-81.6), and in the combined analysis of both studies. We also observed that rs6449182 G carriers had more advanced clinical stage (P = 0.002) and tended to be younger at diagnosis (P = 0.056). Furthermore, we found higher CD38 transcript levels and higher proportions of CD38-positive cells in carriers of rs6449182 G and rs1800561 T alleles (P < 0.05 for all comparisons). In conclusion, our data show that CD38 SNPs may affect CD38 expression and contribute to the increased risk of B-CLL carcinogenesis. (Cancer Epidemiol Biomarkers Prev 2009;18(3):945–53)

Polymorphisms of TNF and IL‐10 genes and clinical outcome of patients with chronic lymphocytic leukemia

Genetic variations in tumor necrosis factor (TNF) and interleukin‐10 (IL‐10) were reported to influence susceptibility to and outcome of patients with non‐Hodgkin lymphoma. Therefore, we investigated whether single nucleotide polymorphisms in TNF and IL‐10 may play a role in the clinical course of patients with chronic lymphocytic leukemia (CLL). TNF‐308G>A, IL‐10‐3575T>A, and IL‐10‐1082A>G seem to be functionally relevant, were genotyped in 292 previously untreated patients with CLL. The control group consisted of 192 randomly selected blood donors. The patients carrying TNF‐308GG and IL‐10‐1082AA genotypes presented a higher 3‐year treatment‐free survival (56.6 vs. 40.6%, P = 0.05) as well as a 10‐year overall survival (OS) rates (92.3 vs. 57.6%, P = 0.005) than those with other TNF‐308 and IL‐10‐1082 genotype combinations. Multivariate analysis demonstrated the Rai stage (P = 0.0002), IGHV mutation status (P = 0.01), TNF‐308G>A (P = 0.03), and TNF/IL‐10 polymorphism‐based risk groups (P = 0.05) to be independent factors predicting OS. When the mutated IGHV patients were analyzed, the homozygotes TNF‐308GG and IL‐10‐1082AA presented a higher 10‐year OS rate than those carrying other TNF‐308 and IL‐10‐1082 genotypes (100 vs. 67.7%, P = 0.01). In the unmutated IGHV patients, only the TNF‐308G>A polymorphism influenced OS. The genetic variations in TNF and IL‐10 genes work as independent predictors of survival and may play a role in the clinical course of CLL. It suggests inherited ability of the host to shift the balance between the Th1 and Th2 response, which in turn might contribute to the pathogenesis and prognosis of B‐cell malignancies.

mTOR kinase inhibitors as a treatment strategy in hematological malignancies

The mammalian target of rapamycin (mTOR) kinase is a key element of intracellular signal transduction, responsible for the regulation of cell growth and proliferation. Since abnormal activation of the mTOR pathway was found in several tumors, including human malignancies, it may be an attractive target for antineoplastic treatment. The first identified mTOR inhibitor was rapamycin (sirolimus). Subsequently, the most potent rapamycin analogues (rapalogues), such as everolimus, temsirolimus and deforolimus, have been developed. After encouraging preclinical experiments, several clinical trials testing the rapalogues in monotherapy or in combinations with other cytotoxic agents have been conducted in patients with hematological malignancies. Results of these studies, described in this review, indicate that inhibition of the mTOR pathway may be a very promising strategy of anti-tumor treatment in several types of lymphomas and leukemias. Recently, a second generation of more effective mTOR inhibitors has been developed. These are currently being assessed in preclinical, Phase I or I/II clinical studies.

Poor prognosis of Hodgkin variant of Richter transformation in chronic lymphocytic leukemia treated with cladribine

It is with great interest that we read the comprehensive analysis of all 86 cases of Hodgkin variant of Richter transformation (Hodgkin-RT) in chronic lymphocytic leukemia (CLL) reported in the world literature during the last 35 years (Bockorny et al, 2012). A very interesting finding from this study was that patients in whom CLL had been treated with fludarabine had significantly shorter survival after transformation compared to the ones not treated with this agent (0·7 vs. 2·1 years, P = 0·019) (Bockorny et al, 2012). Very poor prognosis in the fludarabine-treated patients is in contrast with the general conviction that Hodgkin-RT outcome is superior to the outcome of more common Richter transformation to diffuse large B-cell lymphoma (DLBCL-RT) (Brecher & Banks, 1990). The authors hypothesized that shorter survival may reflect a more aggressive course of Hodgkin-RT in patients who had received fludarabine, and possibly also other purine nucleoside analogs (PNAs) (Bockorny et al, 2012). Besides fludarabine, PNAs used in CLL include cladribine and pentostatin (Karlsson et al, 2002; Kay et al, 2010). Unlike the practice of most European countries, in Poland cladribine is often used in CLL and has been included in large clinical trials of the Polish Adult Leukemia Study Group (PALG) coordinated by our centre (Robak et al, 2006). In order to evaluate the clinical course of HodgkinRT in patients treated previously with cladribine we performed a single-centre retrospective analysis to compare Hodgkin-RT incidence, characteristics and outcome with those of DLBCL-RT. We searched electronic databases of the Departments of Haematology and Pathology of the Medical University of Lodz to identify patients who developed biopsyor fine-needle aspiration–proven RT between January 2000 and November 2011. Hospital records and ambulatory charts were reviewed to determine clinical, laboratory and pathological features, treatment and outcome of CLL and RT. Hodgkin-RT and DLBCL-RT characteristics were compared by Fisher exact test or Mann–Whitney test. Survival after transformation was plotted using the Kaplan-Meier method and comparisons made by the log-rank test. Statistical analysis was performed using Predictive Analytics SoftWare (PASW) Statistics 18 (SPSS Inc., Chicago, IL, USA); P < 0·05 was considered significant. Among 786 patients with CLL admitted to our centre since January 2000, 40 (5·1%) patients developed RT including 33 (4·2%) DLBCL-RT, six (0·8%) Hodgkin-RT and one plasmablastic lymphoma. There were 23 male and 17 female patients with a median age of 67·2 years (range 35·8–84·9). RT was diagnosed after a median of 2·6 years (range 0–12·3) from CLL diagnosis. Median survival after transformation was 1·1 years, 95% confidence interval (95%CI) 0·4– 1·8 years. The patients with Hodgkin-RT comprised five men and one woman with a median age of 70·6 years (range 55·8– 76·3). One patient was reported earlier (Robak et al, 2003). All patients received cladribine-based regimens before transformation, namely five patients received CC (cladribine and cyclophosphamide) and one patient received RCC (CC plus rituximab). Histological subtypes of Hodgkin lymphoma (HL) were mixed cellularity (four patients), nodular sclerosis (one patient), and not determined in one patient. All patients presented with extensive disease involvement (Ann Arbor stage IIIB in three patients and IVB in three patients). Moreover, concomitant progression of CLL was documented in two patients. Treatment was mostly HL-type therapy including ABVD (adriamycin, bleomycin, vinblastine, and dacarbazine) in three patients (in one combined with radiation) and COPP (cyclophosphamide, oncovin, procarbazine and prednisone) in one patient, while one patient received COP (cyclophosphamide, oncovin and prednisone) and one patient with poor performance status received steroids and vincristine. Two partial remissions were achieved, while four patients did not respond to chemotherapy. During the follow-up period, five patients died of disease progression at 0·5, 2, 4, 11 and 13 months from transformation, whilst one patient remained alive after 7 months of observation (Fig 1B). The comparison of patients with DLBCL-RT and Hodgkin RT is shown in Table I. Presenting characteristics and time-to-transformation were comparable in both types of RT. However, a trend suggesting more common preceding treatment with PNA in Hodgkin-RT was observed (P = 0·063). Most interestingly, we found significantly shorter survival after transformation in Hodgkin-RT compared to DLBCL-RT (P = 0·019) (Table I, Fig 1A). In contrast, no survival difference was seen between DLBCL-RT patients who had or had not received PNAs (P = 0·77), (Fig 1B). The results of this study indicate that Hodgkin-RT in cladribine-treated CLL patients is characterized by very short survival, even compared to DLBCL-RT. This is in line with the very poor prognosis of Hodgkin-RT patients treated previously with fludarabine (Bockorny et al, 2012), and suggests Correspondence

Elevated plasma levels of the angiogenic tetrapeptide acetyl-ser-asp-lys-pro are found in some patients with hematologic malignancies.

The evidence that angiogenesis plays a critical role in the pathogenesis of different non-solid hematologic malignancies is growing. Indeed, increased bone marrow microvessel density and higher levels of proangiogenic factors have been shown to be associated with the physiopathology of a number of different myeloproliferative disorders [1]. The tetrapeptide acetyl-ser-asp-lys-pro (AcSDKP), which was originally identified as an inhibitor of hematopoietic stem cell proliferation [2], has also recently been shown to be a potent angiogenic factor [3,4]. Interestingly, elevated levels of this tetrapeptide were detected in the plasma and bone marrow of mice bearing a transplantable acute myeloid leukemia (AML). Indeed, the levels of AcSDKP in overtly leukemic animals were 5–10 times greater than those observed in non-leukemic controls [5]. Further, a correlation was apparent between increasing levels of AcSDKP and disease progression. Further to the observations made in our animal model of hematologic malignancy, we measured AcSDKP levels in the plasma of leukemic patients using an enzyme-linked immunoabsorbent assay (EIA) [6]. The levels of AcSDKP were measured in plasma samples from 29 patients (mean age 58 years: range 37–78) with AML and 53 patients (mean age 67 years: range 45–92) with chronic lymphocytic leukemia (CLL) and compared with AcSDKP levels in the plasma of 20 healthy individuals (mean age 55 years: range 37–71). AcSDKP concentrations were found to be significantly elevated in the plasma of both groups of patients (AML: median 1.96+ 0.18 pmol/mL and CLL: median 3.71+ 0.41 pmol/mL) when compared with those from healthy individuals (median 1.28+ 0.07 pmol/mL) (Figure 1). It should be noted that in this initial analysis, the patients were chosen at random with no attempt made to match samples with respect to sex, age, or grade of disease. While the authors acknowledge that this fact introduces a considerable degree of inherent variability into the dataset, we feel that the findings warrant further clinical investigation. Consistent with our animal study, we expect that the analysis of plasma samples from more closely sex-, age-, and disease-matched patients will reveal a strong correlation between the levels of AcSDKP and the extent of hematologic malignancy. However, as shown in Table I, no variation in AcSDKP concentration was noted in both healthy donors and leukemic patients between the two age ranges studied (age 460 years compared with age 560 years). Therefore, as there is no difference between the two age groups observed in controls as well as in leukemic patients, the reported high levels of the tetrapeptide are thus most likely to be due to the presence of the hematologic malignancies. While there is still no direct evidence of a role for AcSDKP in hematologic disease, our findings suggest that the plasma levels of this tetrapeptide

Prognostic value of inhibitor of apoptosis protein family expression in patients with acute myeloid leukemia

The inhibitor of apoptosis protein (IAP) family acts as an inhibitor of apoptosis pathways. The potential prognostic value of the expression of selected IAP family members, XIAP, cIAP-1, cIAP-2 and survivin protein, was evaluated with regard to treatment response and survival of 56 newly diagnosed adult patients with acute myeloid leukemia (AML). The presence of these IAP members influenced the achievement of a complete response (CR). In addition, overall survival (OS) was influenced by low survivin expression in univariate and multivariate analysis (p = 0.014 and p = 0.013, respectively). A strong correlation was observed between members of the IAP family (XIAP and cIAP-1, XIAP and cIAP-2, cIAP-1 and cIAP-2, p < 0.001 for all comparisons), while Smac/DIABLO demonstrated an inverse correlation with XIAP, cIAP-1 and cIAP-2 (p < 0.001 for all comparisons). Further studies should be undertaken to better demonstrate the mode of action of IAP members, as well as their prognostic and therapeutic potentials.

Safety and feasibility of therapeutic platelet depletion with Spectra Optia in a pregnant woman

INTRODUCTION Thrombocytapheresis is an alternative treatment beneficial in rare circumstances, when cytoreductive agents are contraindicated, drug therapy gave no response or the expected response would be too slow. Here we present a case of a pregnant woman who underwent 5 thrombocytaphereses using Spectra Optia device to reduce circulating platelets (PLT) count and prepare for Cesarian section. PATIENT CHARACTERISTICS AND PERFORMED TREATMENT A 39-year-old woman with diagnosed chronic myeloid leukemia (CML) was treated with interferon because of too high PLT count. The treatment was well tolerated but the effect was not satisfactory (PLT count remained high). Because of high risk of bleeding during childbirth, the healthcare providers decided to perform thrombocytapheresis to reduce circulating PLT count below 1000×10E3/μl, and to prepare the patient for a planned Cesarean section. The results are presented as mean±SD. RESULTS Five therapeutic aphereses procedures were performed, with a Spectra Optia device (TerumoBCT). A mean of 1.3±0.3 total blood volume was processed and we observed a mean PLT drop of 42.3±17.7%. Each apheresis procedure resulted in a PLT level ≤1000×10E3/μl. PLT CE1 was high 50.6±2.6% and reproducible. The white blood cell (WBC) loss was low (18.5%±11.0%). No adverse effects were observed. CONCLUSION Therapeutic platelet depletion using the Spectra Optia™ Apheresis System can be effective and safe during pregnancy. Thrombocytapheresis procedures were reproducible and Spectra Optia system successfully adjusted settings to each procedure conditions. Thrombocytapheresis seems to be a viable and safe option even in pregnant women.

VEGF, ANGPT1, ANGPT2, and MMP-9 expression in the autologous hematopoietic stem cell transplantation and its impact on the time to engraftment

As a site of complicated interactions among cytokines, bone marrow niche has been the subject of many scientific studies, mainly in the context of the proteins influencing damage or recovery of endothelium after allogeneic hematopoietic stem cell transplantation (HSCT). In this study, we aimed at exploring mutual correlations of bone marrow niche cytokines involved in the homing and mobilization of hematopoietic stem cells, as well as in angiogenesis. The aim of our study was to evaluate levels of cytokines: VEGF, angiopoietin-1 (ANGPT1), angiopoietin-2 (ANGPT2), and matrix metalloproteinase 9 (MMP-9) during autologous HSCT and to examine their influence on hematological recovery. Forty-three patients with hematological malignancies (33 multiple myeloma, 10 lymphoma) were enrolled in the study. Plasma samples were taken at five time points: before conditioning treatment (BC), on transplantation day (0) and 7 (+7), 14 (+14), and 21 (+21) days after HSCT. The cytokine levels were evaluated by ELISA method. Our study revealed decreased levels of VEGF, ANGPT1, and MMP-9 in the early post-transplant period as compared to the baseline (BC). ANGPT2 was decreased after conditioning treatment, but tended to increase from day +7. On day +7, positive correlations between ANGPT1 level as well as MMP-9 and the time to engraftment were observed. As opposite to ANGPT1, negative correlation between ANGPT2 level on day +7 after HSCT and the time to hematological recovery was noticed. Our study suggests that investigated cytokines are an important part of bone marrow environment and significantly influence the time to engraftment after HSCT.