Prognostic significance of ligands belonging to tumour necrosis factor superfamily in acute lymphoblastic leukaemia

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Prognostic significance of ligands belonging to tumour necrosis factor superfamily in acute lymphoblastic leukaemia L. Bolkun a,∗ , D. Lemancewicz a,b , E. Jablonska c , A. Szumowska a , U. Bolkun-Skornicka d , M. Moniuszko e,f , J. Dzieciol b , J. Kloczko a a

Department of Haematology, Medical University of Bialystok, Poland Department of Human Anatomy, Medical University of Bialystok, Poland c Department of Immunology, Medical University of Bialystok, Poland d Department of Pharmaceutical Technology, Medical University of Bialystok, Poland e Department of Regenerative Medicine and Immune Regulation, Medical University of Bialystok, Poland f Department of Allergology and Internal Medicine, Medical University of Bialystok, Poland b

a r t i c l e

i n f o

Article history: Received 14 September 2014 Received in revised form 16 December 2014 Accepted 18 December 2014 Available online xxx Keywords: Acute lymphoblastic leukaemia APRIL BAFF TRAIL

a b s t r a c t Altered activities of ligands belonging to tumour necrosis factor (TNF) superfamily, namely B-cell activating factor (BAFF), a proliferation-inducing ligand (APRIL) and apoptosis inducing ligand (TRAIL) were demonstrated in several haematological diseases including acute lymphoblastic leukaemia (ALL). BAFF, APRIL and TRAIL provide crucial survival signals to immature, naive and activated B cells. These ligands are capable of activating a broad spectrum of intracellular signalling cascades that can either induce apoptosis or protect from programmed cell death. BAFF and APRIL, which can directly activate the NF-␬B pathway, have been identified as crucial survival factors for ALL cells. Here, we have analyzed serum BAFF, APRIL and TRAIL concentrations in 48 patients with newly diagnosed ALL and 44 healthy volunteers. The levels of APRIL and BAFF were significantly higher in ALL patients as compared to healthy volunteers. In contrast, concentrations of TRAIL were significantly lower in ALL patients. Moreover, following induction, the levels of APRIL, but not BAFF or TRAIL, were significantly lower in a group of patients with complete remission (CR) as compared to non-respondent (NR) ALL patients. Furthermore, we demonstrated statistically significant differences in concentrations of APRIL between CR MRD-negative and CR, MRD-positive ALL patients. Notably detection of higher concentrations of APRIL was associated with shorter leukaemiafree survival and overall survival. Altogether, our data indicate that APRIL can play an important role in the pathogenesis of ALL and the measurement of APRIL levels can improve prognostication in ALL patients. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Acute lymphoblastic leukaemia (ALL) is currently ranked as the most common malignant disease in children and a major cause of mortality due to hematopoietic malignancies in adults [1]. Notwithstanding high rates of remission induction and survival achieved through current treatment regimens, the response to therapy is still poor in a subset of patients. This fact necessitates a thorough comprehension of the survival signals and microenvironment that contribute to the establishment of the leukaemic clone and its resistance to therapy. Implications of the dysregulation,

∗ Corresponding author at: Department of Haematology, Medical University of Bialystok, 24a Sklodowskiej-Curie, Bialystok 15-276, Poland. Tel.: +48 606925377; fax: +48 857447004. E-mail address: [email protected] (L. Bolkun).

which is capable of providing survival and co-stimulatory signals, are still broad. The ability to manipulate such a powerful axis could increase or even grant total independence of environmentally regulated homeostatic control. Tumour necrosis factor-alpha (TNF-␣) is a pleiotropic cytokine that is capable of exerting numerous biological effects. In particular, TNF-␣ can have proliferative and/or survival effects on ‘normal’ untransformed cells and some human tumour cells in contrast to anti-proliferative, apoptotic and cytotoxic effects exerted against other tumours (both in vitro and in vivo) [2]. Both B-cell activating factor (BAFF), and a proliferation-inducing ligand (APRIL) are members of the TNF superfamily that provide crucial survival factors for immature, naive and activated B cells [3,4]. BAFF and APRIL are produced as type II transmembrane proteins, (likewise many ligands belonging to the TNF family), and are then proteolytically cleaved at a furin protease site and released in a soluble form [5]. Previous studies demonstrated that APRIL and BAFF share two TNF receptors

http://dx.doi.org/10.1016/j.leukres.2014.12.012 0145-2126/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Bolkun L, et al. Prognostic significance of ligands belonging to tumour necrosis factor superfamily in acute lymphoblastic leukaemia. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2014.12.012

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superfamily members: B-cell maturation antigen (BCMA), transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) [6–8]. In addition, APRIL binds to heparin sulfate proteoglycan, which serves as a platform to mediate ligand multimerization and cross-linking [9]. Our relatively limited understanding of the role of BAFF, APRIL and their receptors in normal B-cell homeostasis and in several tumour models does not preclude the possibility of their involvement in the pathogenesis of haematological malignancies. Certain studies reported aberrant expressions of BAFF and APRIL by tumour B cells isolated from a subset of patients with chronic lymphocytic leukaemia [10,11]. BAFF and APRIL were found to protect B-CLL cells from spontaneous and drug-induced apoptosis and to enhance cell survival [10]. Previous performed studies have already shown promise for BAFF as a potential prognostic marker in CLL, especially when used in combination with CD38, ZAP70 expression and the mutational status [12]. Similarly, in follicular lymphoma, expressions of both BAFF and BAFF-R are elevated and coincide with inferior progression-free survival (PFS). The overexpression of BAFF, suggested being secondary to the elevated expression of BAFF-R, apparently increased sensitivity to BAFF [13]. Furthermore, the most recent studies found that myeloma cell lines and primary myeloma cells express BAFF, APRIL and their receptors, and that both BAFF and APRIL act as growth factors for myeloma cells [14]. BAFF was also demonstrated to protect myeloma cells from dexamethasone-induced apoptosis [15]. In addition, the possible use of BAFF and APRIL as markers of disease activity and progression [16] as well as their role in angiogenesis [17] has recently been investigated. TNF-related apoptosis inducing ligand (TRAIL) belongs to the TNF superfamily produces an effect opposite to that of BAFF and APRIL and participates in the elimination of neoplastic cells including leukaemic cells [18,19]. TRAIL, also known as Apo-2L, can be biologically effective as an integral membrane protein (mTRAIL, 32 kDa) as well as a soluble cytokine (sTRAIL, 24 kDa) [19]. To date, several important functions of TRAIL have been reported. Firstly, TRAIL-mediated cytotoxicity plays an important role in innate and adaptive immune responses [20]. Secondly, TRAIL exerts a regulatory function on erythroid and myeloid maturation in normal haematopoiesis [21–24]. Moreover, senescent neutrophils can be eliminated by TRAIL-induced apoptosis upon their return to the bone marrow [25]. Importantly, TRAIL was demonstrated to induce apoptosis of leukaemic cells in the course of haematological malignancies, including multiple myeloma cells and Philadelphia chromosome-positive leukaemia [26,27]. In contrast, AML blasts were shown to exhibit a very low sensitivity to the pro-apoptotic effects of TRAIL [28]. The principal objective of the present study was to evaluate serum levels of BAFF, APRIL and TRAIL in healthy volunteers and in ALL patients with varying severity of the disease. In addition, we intended to analyze mutual relationships among baseline BAFF, APRIL and TRAIL levels and other known prognostic parameters in the cohort of ALL patients. Finally, we aimed to evaluate whether measuring serum APRIL, BAFF and TRAIL concentrations could improve prognostication of ALL patients. 2. Patients Forty-eight patients with newly diagnosed acute lymphoblastic leukaemia B linage were enrolled in the study. Their median age at the time of sample collection was 31.6 and the range was 18–56. Twenty-three subjects were male and 25 female. Patients who received corticosteroids before the beginning of the treatment course had been excluded from the study. Diagnoses were established according to the 2008 WHO recommendation [29]. Blood counts, flow cytometry, molecular study, FISH and cytogenetic

analysis were performed, reviewed, and classified. Patients were treated in the Haematology Department of the Medical University of Bialystok from 2007 to 2013 with induction chemotherapy regimens corresponding to the standard therapy based on the Polish Adult Leukaemia Group: therapy consisted of prednisone pretreatment p.o. (PDN) 60/40* mg/m2 (* for patients over 40 years old) for 7 days followed by 4 weeks induction therapy: prednisone for 28 days with the same dose as pretreatment with daunorubicine i.v. 50/40* mg/m2 and vincristine i.v. 2 mg days 1, 8, 15 and 22, together with Peg-Asparginase i.v. 1000 IE/m2 on day 13. After induction, the response was evaluated following the recommendation by NCCN Guidelines. Thirty-eight patients after 1st induction achieved complete remission (CR), which was defined as the absence of physical signs of leukaemia or detectable leukaemia cells on blood smears, a bone marrow with active haematopoiesis and <5% leukaemia blast cells, and normal cerebrospinal fluid. Eight patients were non-responders with the levels of blastic cells at least 20%, [4 patients with normal karyotype, 2 with t(v;11q23) 1 with hypodiploidy and 1 with t(12;21)(p13;q22)]. Patients under 40 who achieved CR but were MRD positive (≥0.1%) and all non-responders were administered a second-line induction, FLAM (fludarabine, cytarabine and mitoxantrone). For all patients who achieved complete remission after one or two induction therapies consolidation therapy included a course of high-dose cytarabine plus cyclophosphamide and a cycle of high-dose methotrexate plus etoposide and dexamethasone. Central nervous system (CNS) prophylaxis consisted of intrathecal therapy with Depocyte and prednisolone administered twice during the induction therapy (during pretreatment −7 to −3 days) and on day 10 as well as once during each consolidation. For all patients with t(9;22)(q34;q11.2), (BCR-ABL positive gene fusion) the standard treatment was supplemented with Glivec at 600 mg/per day. Upon the termination of consolidation, patients were stratified into either a standard risk (SR) or a high-risk (HR) group. HR was defined as the presence of at least one of the following factors: age ≥35 years, WBC at diagnosis ≥30 × 109 /L, adverse immunophenotype, cytogenetic and molecular abnormalities defined by the presence of at least one of the following parameters: pro-B, early-T or mature-T phenotype, two courses of induction required to achieve CR, t(9;22) and t(4:11) [BCR-ABL and MLL] and low hypodiploidy, or CR achieved after two induction cycles. Patients with none of the above features were assigned to the SR group. Subjects from the HR group were intended for either alloHSCT or autologous (autoHSCT) haematopoietic stem cell transplantation, depending on the availability of an appropriate donor. A search for a matched unrelated volunteer for patients without a human leukocyte antigen identical sibling was initiated. Patients allocated to the SR group were given 2-year maintenance therapy consisting of epirubicin, vincristine, prednisone, mercaptopurine and methotrexate. The characteristics of ALL patients are listed in Table 1. The control group was population-based and comprised fortyfour age-matched healthy volunteers (22 males, 22 females, median of age 32, range: 20–57), who had no history of acute or chronic diseases and received no medications. The analysis of their complete blood counts revealed no abnormalities, either. All patients’ samples as well as the samples from healthy volunteers were collected under the Ethics Committee of the Medical University of Bialystok upon signing an approved protocol and a written informed consent form in accordance with the Declaration of Helsinki, No. R-I-002/218/2007. 3. Methods The cytokine measurement was done following the manufacturers’ instructions. Three independent sets of experiments were performed. Each experiment included the kit’s standards and samples both from the ALL patients and healthy controls run in triplicate. No significant variations were observed among the experiments.

Please cite this article in press as: Bolkun L, et al. Prognostic significance of ligands belonging to tumour necrosis factor superfamily in acute lymphoblastic leukaemia. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2014.12.012

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L. Bolkun et al. / Leukemia Research xxx (2015) xxx–xxx Table 1 Characteristics of acute lymphoblastic leukaemia patients. Characteristic

ALL

Number of patients Male/female Mean (range) age, year Age > 35 years, n (%) Mean (±SD) white blood cell count (×109 /L) WBC >30 × 109 /L, n (%)

48 23/25 31.6 (18–56) 17 (35.4) 79.09 ± 34.22 19 (39.5)

Immunophenotype, n (%) Pro-B Common/pre-B

12 (25.0) 36 (65.0)

Cytogenetic and molecular classification of ALL, n (%) Normal (diploidy) t(9;22)(q34;q11.2); BCR-ABL t(v;11q23); MLL t(12;21)(p13;q22); TEL-AML 1 Hyperdiploidy Hypodiploidy Unclassified

12 (25.0) 11 (22.9) 6 (12.5) 2 (4.2) 3 (6.25) 3 (6.25) 11 (22.9)

Outcome of induction therapy, n CR achieved after 1st induction CR achieved after 2nd induction NR Mortality during 1st/2nd induction/consolidation

38 2 2 2/4/0

Risk group, n (%) Standard Higha

14 (29.2) 34 (70.8)

Post-consolidation treatmentb Maintenance AutoHSCT AlloHSCT

18 5 17

ALL, acute lymphoblastic leukaemia; MRD, minimal residual disease; WBC, white blood cell; CR, complete remission; NR, non-responder; AutoHSCT, autologous haematopoietic stem cell transplantation; AlloHSCT, allogeneic haematopoietic stem cell transplantation. a High-risk group was defined by the presence of at least one of the following parameters: age > 35 years, WBC count >30 × 109 /L, pro-B, early-T or mature-T phenotype, two courses of induction required to achieve CR, high cytogenetic and molecular risk patients: t(9;22) and t(4:11)/BCR-ABL and MLL and low hypodiploidy. b Only patients who completed consolidation in CR are considered.

Quantitative assessments of cytokines were performed by means of ELISA assays. Soluble APRIL concentrations were measured in the serum with Human APRIL (Platinum ELISA, eBioscience Austria). Soluble TRAIL and BAFF were evaluated in the serum using commercially available test kits (Quantikine® , R&D kits, Minneapolis, MN, USA).

4. Statistics Results were expressed as means ± SD. The chi-square or, alternatively, the Kruskal–Wallis test was used to assess relationships between categorical variables. Comparisons between the ALL and the control groups were made using the non-parametric Mann–Whitney test. The Spearman’s order correlation coefficient was applied to determine correlations between the measured parameters. Survival times were compared by means of the

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log-rank test. Multivariate models were reduced by one factor at a time to ensure that all the factors remaining in the model were statistically significant at the 5% level. P-values below 0.05 were considered to be statistically significant. Descriptive statistics were analyzed and univariate analyses were performed.

5. Results Mean (±SD) serum concentrations of BAFF, APRIL and TRAIL in ALL patients and healthy volunteers are listed in Table 2. At the time of diagnosis, ALL patients had significantly higher serum concentrations of BAFF, APRIL and lower concentrations of TRAIL as compared to healthy volunteers (p < 0.0001 for all above-mentioned comparisons). In addition, the concentrations of APRIL and TRAIL in newly diagnosed ALL patients were significantly different from measurements performed in patients after induction therapy (p = 0.02 and p = 0.008, for APRIL and TRAIL, respectively). However, no significant differences were found between the concentrations of BAFF before and after chemotherapy. Notably, we demonstrated differences between patients with complete remission (CR) and non-respondent patients (NR). NR patients presented significantly lower concentrations of APRIL (p = 0.03), but not BAFF (p = 0.32). In some contrast, we did not find significant decrease in the concentration of TRAIL in NR patients (as compared to the CR patients, p = 0.72; Table 3). Significant differences in APRIL levels were found between these ALL patients who achieved CR and had negative MRD (minimal residual disease) status and those who achieved CR but had positive MRD status after the induction (p = 0.04), Table 3. In order to assess the prognostic value of the concentration of above-mentioned ligands, we performed a correlative analysis of serum APRIL, BAFF and TRAIL levels with variables bearing prognostic significance, including age at diagnosis, sex, WBC count at diagnosis, BCR/ABL and risk group. We demonstrated no significant relationships of APRIL, BAFF and TRAIL levels with age, sex and BCR/ABL fusion gene in any studied ligands (p > 0.05). Furthermore, we did not demonstrate significant correlations between C-reactive protein (CRP) and concentration of studied cytokines: for APRIL (rho = 0.09, p = 0.81), BAFF (rho = 0. 12, p = 0.72) and TRAIL (TRAIL rho = −0.19, p = 0.62). However, we demonstrated statistically significant correlations between APRIL levels and peripheral blood blastic cell counts (rho = 0.33, p = 0.04). Moreover, BAFF levels correlated positively with APRIL levels (rho = 0.42, p = 0.001) and negatively with TRAIL levels (rho = −0.45, p = 0.0009). Statistically significant negative correlation was also found for APRIL and TRAIL levels (rho = −0.4, p = 0.002). Additionally, we observed that pre-treatment ALL patients with serum APRIL values higher than the median (5.51 ng/ml) presented with significantly shorter leukaemia-free survival (LFS) and overall survival (OS) as compared to patients with lower APRIL values, (p = 0.03 and p = 0.04), respectively (Fig. 1). On the other hand, there were no statistically significant differences between LFS and OS in the subgroups of ALL patients with regard to the

Table 2 The mean ± SD values of chosen parameters of all ALL patients and healthy volunteers. Parameters

TRAIL (pg/ml) BAFF (pg/ml) APRIL (ng/ml)

No. of patients Healthy volunteers n = 44

New diagnosed ALL patients n = 48

ALL Ph positive n = 11

ALL Ph negative n = 37

80.33 ± 15.21 733.4 ± 214.7 2.04 ± 1.42

51.73 ± 26.75* 2074.1 ± 1820.2* 10.57 ± 9.32*

41.78 ± 20.5 2349.76 ± 1399.4 10.84 ± 9.44

54.12 ± 31.08 1890.5 ± 1643.1 7.61 ± 6.64

The values are presented as mean ± SD. ALL, acute lymphoblastic leukaemia; BAFF, B-cell activating factor; APRIL, a proliferation-inducing ligand; TRAIL, TNF-related apoptosis inducing ligand. * p < 0.05 between ALL patients and healthy volunteers.

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Table 3 The differences (mean ± SD) of chosen parameters between ALL patients before and after treatment. No. of patients New diagnosed patients n = 48

TRAIL (pg/ml) BAFF (pg/ml) APRIL (ng/ml)

51.73 ± 26.75 2074.1 ± 1820.2 10.57 ± 9.32

After the treatment n = 48

30.17 ± 16.72* 1821.9 ± 1243.4 17.232 ± 19.65*

Patients after a induction treatment

With NR n=8

With CR n = 38

With CR MRD negative n = 30

With CR MRD positive n=8

28.27 ± 8.95 2279.9 ± 2499.8 38.87 ± 20.4

32.6 ± 19.58 1712.7 ± 852.8 7.23 ± 2.32**

37.62 ± 25.7 1717.6 ± 1212.8 4.28 ± 1.6

28.8 ± 18.01 2141.1 ± 1662.8 14.25 ± 9.47***

The values are presented as mean ± SD. ALL, acute lymphoblastic leukaemia; BAFF, B-cell activating factor; APRIL, a proliferation-inducing ligand; TRAIL, TNF-related apoptosis inducing ligand; CR, complete remission; NR, non-responders; MRD, minimal residual disease. * p < 0.05 between ALL patients between and after treatment. ** p < 0.05 between ALL patients with CR and NR. *** p < 0.05 between CR MRD positive vs CR MRD negative.

Fig. 1. Kaplan–Meier curves of leukaemia-free survival (LFS) and overall survival (OS) estimates according to APRIL serum levels, in patients with newly diagnosed acute lymphoblastic leukaemia. Patients with APRIL values higher (bottom or blue line), than median (5.51 ng/ml) have a significant shorter RFS and OS than patients with lower APRIL value (upper line or red line). The two curves are significantly different for RFS, p = 0.03 and OS, p = 0.01.

median values of BAFF (1199.8 pg/ml) and TRAIL (50.5 pg/ml), p = 0.31 and p = 0.4, respectively. As expected, age ≥35 years, WBC at diagnosis ≥30 × 109 /L and performance status turned out to be strong predictors of LFS and OS in the group of ALL patients (p < 0.05). However, with the use of multivariate Cox proportional hazard model, which incorporated all significant factors along with the studied ligands, we demonstrated that only APRIL can be considered an independent risk factor (Table 4).

6. Discussion The last few decades have witnessed intense efforts directed at establishing prognostic markers capable of discriminating highrisk patients, identifying a new complex network of cytokines that either promote or inhibit cell growth. The altered anti-apoptotic activities of APRIL and BAFF have so far been demonstrated in Blymphoma, multiple myeloma and B-CLL, suggesting that these

Table 4 Univariate and multivariate analysis of concentrations of TNF-␣ superfamily associated with leukaemia free survival and overall survival of ALL patients. LFS HR

Overall survival Risk ratio (95% CI)

p

HR

Risk ratio (95% CI)

p

Univariate analysis Sex (male vs female) WBC (>30 × 109 /L vs <30 × 109 /L) Age (<35 vs 35 > ) Standard Risk vs High Riska Maintenance vs Auto vs AlloHSCT APRIL (low vs high) BAFF (low vs high) TRAIL (low vs high)

1.36 1.73 1.93 2.01 3.21 2.81 1.18 0.98

0.51–2.25 0.72–3.88 0.59–3.89 1.02–3.98 2.02–6.44 0.93–5.73 0.48–2.92 0.46–1.79

0.11 0.07 0.06 0.02 0.001 0.03 0.72 0.78

1.44 1.85 2.01 2.16 3.55 3.22 1.21 0.88

0.44–1.98 0.98–4.01 1.11–3.92 1.23–4.12 2.34–6.87 1.34–5.82 0.52–2.88 0.32–1.65

0.21 0.05 0.04 0.03 0.001 0.01 0.54 0.81

Multivariate analysis APRIL (low vs high)

2.69

0.92–5.79

0.04

2.77

1.04–5.88

0.03

Bold values are statistically significant. ALL, acute lymphoblastic leukaemia; BAFF, B-cell activating factor; APRIL, a proliferation-inducing ligand; TRAIL, TNF-related apoptosis inducing ligand; LFS, leukaemiafree survival; OS, overall survival; WBC, white blood cell; AutoHSCT, autologous haematopoietic stem cell transplantation; AlloHSCT, allogeneic haematopoietic stem cell transplantation. a High-risk group was defined by the presence of at least one of the following parameters: age > 35 years, WBC count >30 × 109 /L, pro-B, early-T or mature-T phenotype, two courses of induction required to achieve CR, high cytogenetic and molecular risk patients: t(9;22) and t(4:11)/BCR-ABL and MLL and low hypodiploidy.

Please cite this article in press as: Bolkun L, et al. Prognostic significance of ligands belonging to tumour necrosis factor superfamily in acute lymphoblastic leukaemia. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2014.12.012

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ligands could play an important pathogenic role in haematological disorders [30]. The expression of BAFF-R was reported to be restricted to more mature B cells, starting at the T1 transitional B-cell stage [31]. The implied lack of significance of this ligand/receptor axis in the early B-cell development was supported by observations of normal pro- and pre-B-cell compartments in mice lacking in BAFF-R or BAFF [32,33]. Surprisingly, Parameswaran et al. confirmed that all cells in the primary Ph-positive and Phnegative ALL samples were highly positive for BAFF-R and TACI cell surface expression. In contrast, the expression of BCMA was either very low or absent [34]. In addition, recombinant BAFF was shown to support the survival of ALL cells in the absence of stroma and to significantly attenuate the rate of apoptosis caused by an exposure to nilotinib, a drug used therapeutically to treat Ph-positive ALLs. Interestingly, despite identifying detectable BAFF levels in pre-B ALL cell lysates, the soluble human BAFF in conditioned ALL cell culture medium was not detected [34]. In addition, Sun et al. confirmed the up-regulation of the APRIL mRNA expression in BMCs of ALL patients and demonstrated that BMCs constituted a cellular source of plasma APRIL in ALL. Even more importantly, their study evidenced lack of significant changes in the expression of TACI or elevation of BCMA mRNA expression in childhood ALL patients [35]. No study to date analyzed simultaneously concentrations of BAFF and APRIL in ALL patients. Notably, our data did not reveal differences in concentrations of BAFF after induction therapy or significant differences between CR patients and NR. Similarly, we did not find any correlations of BAFF concentrations with WBC or blastic cell counts, suggesting that the latter could rather not be the source of BAFF. Indeed, BAFF was also detected in monocytes/macrophages, dendrite cells and activated T lymphocytes [30]. Interestingly, competitive inhibition with a fusion protein comprising BAFF and the toxin gelonin has shown promising efficacy in promoting tumour clearance in childhood ALL when combined with mobilizing agents such as a CXCR4 antagonist [36]. In this study we not only found higher APRIL levels in ALL patients compared to healthy volunteers, but also demonstrated significant differences in APRIL levels between CR patients and NR, which tallies with the previous reports [35]. We further extended this observation by showing the concentrations of APRIL to be significantly lower in the subgroup of CR patients with a negative MRD status compared to our CR patients with a positive MRD status after induction therapy. Our results indicated that baseline concentrations of APRIL could be used in ALL prognosing, since MRD evaluation after induction therapy is considered a high risk factor of relapse [37]. Thus, inhibition of APRIL could potentially become a therapeutic tool increasing efficacy of tumour clearance. Furthermore, in the current study, we found significant correlations between the levels of BAFF and APRIL, and more importantly, between APRIL levels and blastic cells counts, which likewise suggests that APRIL is closely related to the severity and prognosis of the disease. On the other hand, one needs to take into consideration that our finding concerning the elevated APRIL concentrations can be associated with some other confounding variables present in ALL patients. In order to address some of these possibilities, we analyzed APRIL levels in the context of possible on-going infections and other factors accounting for a worse prognosis in ALL patients. For example, we demonstrated that APRIL levels were related neither to CRP status nor BCR/ABL status. Nevertheless, our data indicate that the exact role of APRIL (and other ligands belonging to the tumour necrosis factor family) in the pathogenesis of ALL deserves further investigation in more detailed experimental studies. Importantly, our study revealed clinically important differences in LFS and OS between subgroups of patients with different levels of APRIL, but not BAFF: patients with higher APRIL levels presented significantly shorter OS and LFS. These results are in concert with our previous study that showed, that serum BAFF and APRIL levels can be used

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as prognostic factors for progression-free survival (PFS) in multiple myeloma patients [16]. On the other hand, Kim et al. demonstrated in multivariate analysis that serum BAFF, but not APRIL, level was an independent prognostic factor for OS and PFS in NHL patients [38]. Therefore, it is reasonable to presume that APRIL and BAFF may play some overlapping and synergetic roles in the pathogenesis of ALL and they can constitute promising targets for novel therapeutic strategies. Recently, studies on TRAIL have raised hopes that this ligand can be used as an anti-neoplastic agent in different types of tumours, including haematological malignancies such as ALL [39]. In in vivo study, Ph-positive ALL cell lines were highly or moderately sensitive to TRAIL-induced apoptosis [27]. These findings are in concert with study of Plasilova et al., who recently reported that the growth of CML progenitors in the chronic, accelerated and blastic phases was significantly suppressed by recombinant TRAIL [40]. TRAILsensitive cell lines, unlike the TRAIL-resistant ones, were further confirmed to express the death-inducing receptors DR4 and/or DR5 on their surface [27]. Importantly, it was demonstrated that imatinib mesylate efficiently repressed most of the TRAIL-resistant cell lines while TRAIL repressed the majority of the imatinib mesylateresistant cell lines. This suggests a potential clinical usefulness of TRAIL, particularly in patients with imatinib mesylate-resistant CML and Ph1-positive ALL [40]. Previous studies have also demonstrated that the binding of TRAIL to DR4 and DR5 induces the NF-B activation [41] and that the increased NF-B activity can protect tumour cells from various pro-apoptotic stimuli [30]. Most importantly from clinical perspective, tumour cells from three children with B precursor or T cell acute lymphoblastic leukaemia exhibited an increased TRAIL-induced apoptosis upon the knockdown of either cyclinB or cyclinE that arrested the cell cycle in G2 or G1 phase, respectively [39]. Therefore, TRAIL might represent an interesting therapeutic agent that can be used for treatment of static-tumour disease (e.g. during the minimal residual disease). In the current study, we found significantly lower concentrations of soluble TRAIL in ALL patients than in healthy individuals. However, we did not find any significant correlations between TRAIL levels and other important prognostic variables, such as WBC, age or prognostic status. Furthermore, despite a significant decrease in the concentration of TRAIL observed after treatment, we did not demonstrate significant differences between concentrations of this ligand in CR patients and NR. Moreover, our study did not show important differences with regard to LFS and OS in subgroups with TRAIL levels. Thus, based on our findings, we suggest that analysis of TRAIL will not contribute to better prognosing of ALL patients. In conclusion, our results have indicated that analysis of serum concentrations of APRIL, but not BAFF or TRAIL, could become a useful tool for assessment of ALL disease activity and progression. Therefore, quantification of pre-treatment concentrations of APRIL could significantly improve prognostication of leukeamia-free survival and overall survival in ALL patients.

Conflict of interest The authors declare that they have no conflict of interest.

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Please cite this article in press as: Bolkun L, et al. Prognostic significance of ligands belonging to tumour necrosis factor superfamily in acute lymphoblastic leukaemia. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2014.12.012