Leukemia (2015), 1–7 © 2015 Macmillan Publishers Limited All rights reserved 0887-6924/15 www.nature.com/leu
ORIGINAL ARTICLE
Increased IL6 plasma levels in indolent systemic mastocytosis patients are associated with high risk of disease progression A Mayado1, C Teodosio2, AC Garcia-Montero1, A Matito3, A Rodriguez-Caballero1, JM Morgado3, C Muñiz1,4, M Jara-Acevedo1, I Álvarez-Twose3, L Sanchez-Muñoz3, S Matarraz1, C Caldas1, JI Muñoz-González1, L Escribano1 and A Orfao1 Systemic mastocytosis (SM) is a heterogeneous disease with altered interleukin (IL)-6 and IL13 plasma levels. However, no study has simultaneously investigated the plasma levels of IL1β, IL6, IL13, CCL23 and clusterin in SM at diagnosis and correlated them with disease outcome. Here we investigated IL1β, IL6, IL13, CCL23 and clusterin plasma levels in 75 SM patients—66 indolent SM (ISM) and 9 aggressive SM—and analyzed their prognostic impact among ISM cases grouped according to the extent of hematopoietic involvement of the bone marrow cells by the KIT D816V mutation. Although increased IL1β, IL6 and CCL23 levels were detected in SM patients versus healthy controls, only IL6 and CCL23 levels gradually increased with disease severity. Moreover, increased IL6 plasma levels were associated with ISM progression to more aggressive disease, in particular among ISM patients with multilineal KIT mutation (ISM-ML), these patients also showing a higher frequency of organomegalies, versus other ISM-ML patients. Of note, all ISM patients who progressed had increased IL6 plasma levels already at diagnosis. Our results indicate that SM patients display an altered plasma cytokine profile already at diagnosis, increased IL6 plasma levels emerging as an early marker for disease progression among ISM cases, in particular among high-risk ISM patients who carry multilineage KIT mutation. Leukemia advance online publication, 31 July 2015; doi:10.1038/leu.2015.176
INTRODUCTION Systemic mastocytosis (SM) consists of a heterogeneous group of disorders characterized by an abnormal expansion and accumulation of pathological mast cells (MCs) in the bone marrow (BM) and other organs such as the skin, liver, spleen and/or lymph nodes.1–3 At presentation, clinical variants of SM include both indolent, for example, indolent SM (ISM), and aggressive, even life-threatening, forms of the disease, for example, aggressive SM (ASM) and MC leukemia (MCL), classified by the World Health Organization4 into seven distinct subtypes plus one provisional subvariant (smoldering SM (SSM)). In the vast majority of cases (490%), the clonal nature of pathological MC can be demonstrated by the presence of mutations in the tyrosine kinase domain of the stem cell growth factor receptor gene (KIT),5,6 except for a rare subset of welldifferentiated SM.7,8 Most ISM cases typically show stable disease for decades; in contrast, a smaller fraction of ISM patients display progression to more aggressive variants of the disease, for example, SSM, ASM, SM-associated with a clonal non-mast lineage hematopoietic disease and MCL, to a myeloproliferative neoplasm, myelodysplastic syndrome and/or acute myeloid leukemia.9,10 Currently, the most powerful independent prognostic factor for progression-free survival (PFS) among ISM patients relies on the presence of multilineage involvement of myeloid or myeloid plus lymphoid hematopoietic cells other than BM MC by the KIT mutation.10,11 Nowadays, evaluation of multilineage involvement of BM cells by the KIT mutation requires a BM study based on complex cell-purification procedures followed by molecular analysis of highly purified BM cell populations;5 because of this,
routine assessment of multilineage involvement of hematopoiesis by the KIT mutation remains restricted to a few specialized reference centers.5,10,12 Alternatively, it has been proposed that frequent monitoring of serum baseline tryptase (sBT) levels could be a surrogate marker for disease progression.13 However, sBT has a lower predictive value than the degree of involvement of hematopoiesis by the KIT mutation; at the same time, reliable assessment of increased sBT levels appears to require serial measurements for a relatively long period of time. In recent years, several groups have reported altered plasma levels of cytokines (for example, interleukin (IL)-IL6, IL13 and IL31) in SM patients.14–16 In addition, some of these cytokines (for example, IL6 and IL31)14,16 have been associated with a greater severity of mastocytosis and more extended disease, with greater levels in aggressive versus indolent variants of SM. In turn, it is well known that several of those cytokines and chemokines reported to be altered in SM have a critical role in MC proliferation and tissue migration.17 In line with these observations, we have recently reported that the distinct gene expression profile of purified BM MC from ISM versus ASM patients include differential expression of genes encoding for several cytokines (for example, IL-1β and IL13) and chemokines (for example, CCL23), as well as for other plasma proteins, such as the clusterin complement regulatory protein.18 Altogether, these findings suggest that the plasma levels of the above proteins might potentially provide prognostic information about disease outcome in SM patients. Despite this, to the best of our knowledge, no study has been reported so far in which the plasma levels of IL1β, IL6, IL13, CCL23
1 Servicio General de Citometría (NUCLEUS), Centro de Investigación del Cáncer (IBMCC-CSIC/USAL and IBSAL) and Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain; 2Department of Immunology, Erasmus MC University Medical Center Rotterdam, Rotterdam, The Netherlands; 3Centro de Estudios de Mastocitosis de Castilla La Mancha (CLMast), Hospital Virgen del Valle, Toledo, Spain and 4Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF) Instituto de Salud Carlos III, Madrid, Spain. Correspondence: Professor A Orfao, Servicio General de Citometría (NUCLEUS), Centro de Investigación del Cáncer (IBMCC-CSIC/USAL and IBSAL) and Departamento de Medicina, Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain. E-mail: [email protected] Received 24 March 2015; revised 15 May 2015; accepted 16 June 2015; accepted article preview online 8 July 2015
Prognostic value of IL6 in systemic mastocytosis A Mayado et al
2 and clusterin have been simultaneously assessed at diagnosis in SM patients and correlated with the outcome of the disease. Here we analyzed the IL1β, IL6, IL13, CCL23 and clusterin plasma levels versus sBT at diagnosis, in a large cohort (n = 75) of SM patients, including 66 ISM cases. Our major goal was to investigate the relationship between the plasma levels of these proteins and the outcome of ISM as reflected by progression to more advanced forms of the disease. MATERIALS AND METHODS Patients, controls and samples A total of 75 adult SM patients diagnosed at the Reference Centers of the Spanish Network on Mastocytosis (Mast Cell Unit, Hospital del Valle, Toledo, and Cytometry Service, Cancer Research Center, Salamanca, Spain), were studied. Patients were classified according to the World Health Organization criteria4 and more recent consensus recommendations.19,20 All patients had the D816V KIT mutation; in 38 ISM cases the KIT mutation was restricted to the MC compartment in the BM (ISM-MC), whereas the other 28 ISM patients and the 9 ASM cases studied had multilineage KIT mutation (ISM-ML) as defined elsewhere.10 Fifty-one healthy subjects (excluding individuals with a past or present history of allergy) were studied in parallel as controls. The demographic and biological characteristics of the patients analyzed are shown in Supplementary Table S1. Plasma and serum samples, as well as BM aspirated samples, were obtained and assessed at diagnosis and stored at the National DNA Bank Carlos III (www.bancoadn.org; University of Salamanca, Spain) and/or the Reference Centers of the Spanish Network on Mastocytosis, after written informed consent was given by each donor, according to the guidelines of the local Ethics Committee. Of note, in every patient, including those who progressed, plasma samples were obtained before any cytotoxic therapy, for example, interferon, hydroxyurea and cladribine (2CDA), had been administrered. At the moment of closing the study, the median follow-up of all SM patients was 36 months. ISM patients showed an overall rate of progression of ISM to SSM, SM-associated with a clonal non-mast lineage hematopoietic disease, ASM or MCL of 6% (4/66 ISM cases), after a median PFS of 35 ± 37 months; the criteria used to define progression from ISM to SSM included: coexistence of two B (Borderline–Benign) findings, for example, (a) massive MC burden (huge MC marrow infiltration, serum tryptase 4200 ng/ml), (b) organomegaly (spleen, liver and lymph node involvement) without impairment of organ function and (c) dysmyelopoiesis (hypercellular marrow with signs of myelodysplasia or myeloproliferation, but no criteria for myelodysplastic syndrome or myeloproliferative neoplasm).21 Of note, all nine ASM cases were excluded from PFS analyses.
Detection of the KIT mutation The KIT D816V mutation was investigated in fluorescence-activated cell sorting-purified (⩾97% purity) BM MC, eosinophils, monocytes, neutrophils, CD34+ hematopoietic precursor cells and T cells (FACSAria, Becton/ Dickinson Biosciences (BD), San José, CA, USA), using a peptide nucleic acid-mediated PCR-clamping technique, as previously described in detail.5
Serum tryptase levels sBT levels were measured using the Phadia ImmunoCAP Tryptase System (Phadia, Uppsala, Sweden; Thermo Fisher Scientific Inc., Waltham, MA, USA), according to the manufacturer’s instructions.
Quantification of CCL23 and clusterin plasma levels Peripheral blood samples were collected using EDTA-coated tubes. Briefly, fresh samples were centrifuged (10 min at 800 g), the platelet-rich plasma was collected and centrifuged again (5 min at 2000 g); afterward, the plasma was collected and stored at − 80 °C. The CCL23 and clusterin plasma levels were measured using commercially available sandwich enzyme-linked immunosorbent assays (ELISA): the CCL23 (Human) ELISA Kit (Abnova, Heidelberg, Germany) and the Human Clusterin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA), respectively.
Quantification of IL1β, IL6 and IL13 plasma levels Evaluation of IL1β, IL6 and IL13 plasma levels was performed in the same plasma samples obtained as described above, using the Cytometric Bead Leukemia (2015) 1 – 7
Array immunoassay system (CBA) according to the manufacturer’s instructions: human IL-1β CBA Flex Set, Human IL-6 CBA Flex Set and Human IL-13 CBA Flex Set (BD). Briefly, plasma samples were diluted 1:4 (vol:vol) and incubated for 1 h at room temperature, with 50 μl of each of the anti-cytokine antibody-coated beads. Afterward, 50 μl of the phycoerythrin-conjugated detection antibody reagents was added to each sample and a further incubation for 2 h at room temperature was performed. Then, the unbound antibody was washed out (1 × ) and the beads were measured in a FACSCalibur flow cytometer (BD) using the CellQUEST software program (BD). During acquisition, information was stored for 3000 events corresponding to each bead population analyzed per sample (total number of beads 49000). For data analysis, the Paint-AGate PRO (BD) and the CBA (BD) software programs were used, as described elsewhere.22
Statistical methods Median, range, mean and s.d. values, as well as the 10th, 25th, 75th and 90th percentiles, were calculated for all continuous variables; for categorical variables, frequencies were reported. IL6, IL13, CCL23 and clusterin protein levels were considered to be altered in a plasma sample, when they were higher or lower than the mean ± 2 s.d. of those values obtained for the plasma samples from healthy controls analyzed. In turn, cutoff values for IL1β were established at the limit of detection of the technique; for sBT, cutoff values were established at previously defined normal values ( o11.4 ng/ml).23 In order to determine the statistical significance of the differences observed among groups, the Kruskal–Wallis and the Mann–Whitney U-tests (for continuous variables), as well as the χ2-test (for categorical variables), were used. The degree of correlation between different variables was assessed by the Spearman’s test. PFS curves were plotted according to the method of Kaplan–Meier and they were compared by the (one-sided) log-rank test. P-values o 0.05 with a false discovery rate correction for multiple comparisons of o5% were considered to be associated with statistical significance. For all statistical analyses, the SPSS 18.0 software (SPSS, Chicago, IL, USA) was used.
RESULTS Disease progression among ISM cases From all 66 ISM patients studied, 4 (6%) showed progression to more advanced forms of the disease: 1 patient progressed to SSM, 1 evolved to an ASM, 1 progressed to SM-associated with a clonal non-mast lineage hematopoietic disease and the fourth case to MCL. Of note, all 4 ISM cases that progressed had multilineage KIT mutation (ISM-ML) at diagnosis involving the myeloid (n = 1) or the myeloid plus lymphoid (n = 3) hematopoietic cells, other than BM MC, whereas this was present in only 24 of the 62 ISM (39%) patients who did not show disease progression. IL1β, IL6, IL13, CCL23 and clusterin plasma levels in SM patients Overall, SM patients showed increased IL1β (P = 0.001), IL6 (P = 0.001) and CCL23 (P = 0.005) plasma levels versus healthy controls (Figures 1a–d). In contrast, similar IL13 and clusterin plasma levels were found in SM patients and healthy controls. Despite this, variable levels and distinct patterns of alteration were observed for the different cytokines, among the different diagnostic and prognostic subtypes of SM. Thus, IL1β was of significantly increased (at similar) levels in all diagnostic and prognostic subtypes of SM (P ⩽ 0.04; Figure 1a), although ASM cases had increased IL1β plasma levels more frequently than ISMMC and ISM-ML patients (Table 1). In contrast, IL-6 and CCL23 plasma levels gradually increased (versus controls) from ISM-MC (P o 0.001 and P40.05, respectively) to ISM-ML (P o 0.001 and Po 0.001, respectively) and ASM cases (P o0.001 and P = 0.03, respectively) (Figures 1b and d); interestingly, both IL6 and CCL23 plasma levels were also significantly higher in ISM-ML versus ISMMC patients (P = 0.007 and P = 0.003, respectively) (Figures 1b and d). Despite a similar altered profile observed among the different patient groups for IL6 and CCL23 plasma levels, no significant correlation was found between the plasma levels of both © 2015 Macmillan Publishers Limited
Prognostic value of IL6 in systemic mastocytosis A Mayado et al
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Figure 1. IL1β (a), IL6 (b), IL13 (c), CCL23 (d), clusterin (e) and sBT (f) plasma levels among adults with different subtypes of ISM and with ASM versus healthy controls. Notched boxes extend from the 25th to the 75th percentile values; the lines in the middle and vertical lines correspond to median values and the 10th and 90th percentiles, respectively. P-values o0.05 with false discovery rate (FDR) correction for multiple comparisons of o5% were considered to be associated with statistical significance versus the *control group, †ISM-MC and ‡ ISM-ML cases.
Table 1.
Frequency of systemic mastocytosis patients with different clinical and prognostic subtypes of the disease (n = 75) with altered IL-1β, IL6, IL13, CCL23 and clusterin plasma levels Cytokine/protein IL1β IL6 IL13 CCL23 Clusterin sBT
Cutoff for altered levels
ISM-MC ISM-ML ASM (n = 38) (n = 28) (n = 9)
⩾ 2.3 pg/ml ⩾ 1.6 pg/ml ⩾ 5.4 pg/ml ⩾ 1014.6 pg/ml ⩽ 218.5 ng/ml ⩾ 346.3 ng/ml ⩾ 15 ng/ml
3% 5% 3% 11% 9% 18% 79%
4% 40% 20% 62% 6% 19% 100%
P-values
25% 0.02a 100% ⩽ 0.003b 75% ⩽ 0.02b 100% 0.002a,c 0% 0.962 0% 0.40 100% 0.01c
Abbreviations: ASM, aggressive systemic mastocytosis; IL, interleukin; ISMMC, indolent systemic mastocytosis with KIT mutation restricted to bone marrow mast cells; ISM-ML, indolent systemic mastocytosis with multilineage KIT mutation; sBT, serum baseline tryptase levels. Results expressed as percentage of cases showing increased or decreased cytokine/protein levels. aISM-MC versus ASM. bPo0.05 versus all others groups. cISM-MC versus ISM-ML.
cytokines (r = 0.01; P = 0.95). Although similar IL13 plasma levels were found in SM patients versus healthy controls, higher IL13 plasma levels were observed among ASM patients versus both the controls (P = 0.001) and the two ISM (P ⩽ 0.003) patient groups (P o 0.001 versus ISM-MC and P = 0.003 versus ISM-ML patients, respectively). Of note, ISM-ML patients also displayed higher IL13 plasma levels than ISM-MC cases (P = 0.02). sBT showed a similar profile to that observed for IL6 within the different subgroups of SM patients, (Figure 1f). Furthermore, a significant correlation was found between sBT levels and the © 2015 Macmillan Publishers Limited
amount of most plasma proteins investigated here, that is, IL6 (r = 0.47; P o 0.001), IL13 (r = 0.31; P = 0.009) and CCL23 (r = 0.63; P o0.001), except for IL1β (r = 0.02; P = 0.90). Interestingly, the above described differences among the distinct groups of individuals in their IL1β, IL6, IL13 and CCL23 plasma levels, as well as in sBT, were typically due to a higher percentage of cases with altered plasma levels for these cytokines from ISM-MC to ISM-ML and ASM patients. Briefly, a greater frequency of patients with increased IL1β was found among ASM versus ISM-MC cases (25% versus 3%; P = 0.02); similarly, the frequency of SM patients displaying abnormally increased IL6, IL13, CCL23 and sBT levels progressively increased from ISM-MC (5%, 3%, 11% and 79%, respectively) to ISM-ML (40%, 20%, 62% and 100%, respectively) and ASM (100%, 75%, 100% and 100%, respectively; P ⩽ 0.003, P ⩽ 0.02, P = 0.002 and P = 0.01, respectively) (Table 1). Impact of cytokine plasma levels on PFS of ISM patients From all cytokines evaluated, only IL6 (and to a lesser extent also IL6 in combination with CCL23) emerged as being significantly associated with progression of ISM to more aggressive forms of the disease (P o 0.001 and P = 0.02, respectively; Figures 2a and c). Most interestingly, such prognostic value of IL6 was restricted to the subgroup of ISM cases who had multilineage KIT mutation (ISM-ML), whereas it showed no significant impact among the other ISM cases who had an MC-restricted KIT mutation profile (ISM-MC) and normal IL6 levels. Thus, ISM-ML patients with altered IL6 plasma levels showed a median PFS of 42 months (range: 24–61 months) versus not reached for the other ISM-ML cases (P = 0.006), with 3-year PFS rates of 33 ± 31% versus 0 ± 0%, respectively (Figure 2b). Of note, 4 of 11 cases that had increased IL6 levels showed multilineage KIT mutation in association with Leukemia (2015) 1 – 7
Prognostic value of IL6 in systemic mastocytosis A Mayado et al
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Figure 2. Impact of high IL6 plasma levels alone (a and b) and in combination with CCL23 (c and d) as well as sBT levels (e) on PFS of ISM patients (a, c and e) and ISM cases with multilineage KIT mutation at diagnosis (ISM-ML; b and d).
disease progression; in turn, most (5 of 7) of the other ISM cases showing increased IL6 plasma levels also displayed multilineage KIT mutation but they had stable disease until the moment of closing this study. In our cohort, sBT levels at diagnosis did not show a significant impact on PFS of ISM patients (P40.05), despite cases showing increased sBT levels had a slightly poorer outcome (3-year PFS rates of 0 ± 0% and of 6 ± 6%, respectively; Figure 2e). Although, cases with increased IL6 and/or CCL23 plasma levels also showed lower PFS rates, combined assessment of both cytokines was Leukemia (2015) 1 – 7
associated with a lower prognostic impact than that observed for IL6 alone (Figures 2c and d versus Figures 2a and b, respectively). Association between increased IL6 plasma levels and adverse clinical features of the disease On comparing the clinical features of ISM-ML patients presenting with increased versus normal IL6 plasma levels (Table 2), the former cases showed a higher frequency of organomegalies at diagnosis, including both lymphadenopathies (33% versus 0%, © 2015 Macmillan Publishers Limited
Prognostic value of IL6 in systemic mastocytosis A Mayado et al
5 Table 2.
Relationship between increased IL6 plasma levels and the clinical characteristics of ISM-ML versus other ISM (ISM-MC) and ASM cases
Disease features
ISM-MC (n = 38)
ISM-ML IL6 ( o1.6 pg/ml) n = 15
IL6 (⩾1.6 pg/ml) n = 9
P-values
ASM (n = 9)
MC mediators release-associated symptoms Abdominal pain Anaphylaxis Flushing GI symptoms Pruritus Signs of hemolysis
87% 27% 58% 29% 40% 41% 3%
100% 20% 20% 53% 40% 73% 13%
79% 11% 22% 22% 22% 67% 11%
0.06 0.57 0.90 0.13 0.37 0.73 0.87
100% 67% 22% 11% 67% 22% 67%
Others symptoms Constitutional symptoms
0%
0%
0%
0.99
67%
Organomegalies Lymphadenopathies Hepatomegaly Splenomegaly
0% 6% 3%
0% 20% 20%
33% 56% 67%
0.01 0.07 0.02
44% 67% 78%
Bone disease Fibrosis Pathologic fractures Lytic lesions Disease progression
29% 3% 0% 0%
55% 0% 7% 0%
56% 22% 11% 44%
0.82 0.06 0.70 0.005
100% 22% 11% NA
Abbreviations: ASM, aggressive systemic mastocytosis; GI, gastrointestinal; IL, interleukin; ISM-MC, indolent systemic mastocytosis with KIT mutation restricted to bone marrow mast cells; ISM-ML, indolent systemic mastocytosis with multilineage KIT mutation; MC, mast cell; NA, not applicable; sBT, serum baseline tryptase levels. Results expressed as percentage of cases. P-values are specifically shown for the ISM-ML group of patients for the comparison between ISM-ML cases with normal versus increased (⩾1.6 pg/ml) IL6 plasma levels.
P = 0.01) and splenomegaly (67% versus 20%, P = 0.02), together with a higher rate of disease progression (44% versus 0%, P = 0.005) versus other ISM-ML patients (Table 2). In turn, ISM-ML patients with higher CCL23 plasma levels displayed a lower frequency of anaphylaxis than ISM-ML cases with normal CCL23 levels (20% versus 13%, P = 0.03), whereas cases with greater IL6 and/or CCL23 more frequently had BM fibrosis (60% versus 33%, P = 0.047). Sequential assessment of IL6 plasma levels at diagnosis and at disease progression In the four ISM patients who progressed to more advanced forms of the disease, paired IL6 plasma levels and serum tryptase levels were studied at diagnosis and, subsequently, at disease progression (Figures 3a and b), after a median time to progression of 15 months (range: 0–39 months). Of note, all four patients had increased baseline IL6 and sBT levels already at diagnosis. For IL6, the majority of the patients (three of four cases) showed increased IL6 plasma levels at disease progression versus those found at diagnosis, the highest level of increase corresponding to an ISM patient who progressed to MCL (Figure 3a). Similarly, two of four patients had higher sBT levels at disease progression versus those detected at diagnosis, the other two cases showing levels at progression that were even lower than those detected at diagnosis (Figure 3b). DISCUSSION Current World Health Organization criteria for the classification of SM clearly distinguishes among cases with indolent (for example, ISM) versus aggressive disease (for example, ASM and MCL), the two groups of patients showing distinct clinical outcomes.19,21,24 Despite this, among ISM there is still a subset of patients who show progression to more aggressive disease (for example, SSM, ASM and MCL) including the development of an associated hematological malignancy (for example, ISM-associated with a © 2015 Macmillan Publishers Limited
clonal non-mast lineage hematopoietic disease) such as myelodysplastic syndrome, myeloproliferative neoplasm and/or acute myeloid leukemia.10 In the past, several different prognostic factors have been proposed to help in identifying ISM patients at greater risk of disease progression.5,9,13 Among others, the presence of multilineage involvement of hematopoiesis by the KIT mutation and increasing sBT levels have emerged as being among the most relevant ones.5,13 In addition, soluble CD25 and sCD117 have also been long described as markers of disease severity.25 In line with these latter findings, Pardanani et al.26 have also reported in a relatively limited number of ISM cases (n = 23), which had increased soluble levels of the IL2 receptor α-chain (soluble CD25) to be associated with a poorer overall survival in both advanced (n = 8) and ISM (n = 4); nevertheless, in this latter study, data about soluble CD25 levels were missing at diagnosis in a substantial proportion of cases, the prognostic value of soluble CD25, as well as of sCD117, deserving further prospective validation. In recent years, several evidences have accumulated, which point out for a potential association between cytokine levels and the pathophysiology of SM.15,27,28 Thus, several studies reported increased IL6 levels in patients with mastocytosis, such increased IL6 levels being correlated with the extent of tissue involvement.14 Another cytokine that has emerged as potentially relevant in mastocytosis is IL13; thus, higher IL13 serum levels, as well as an increased prevalence of the − 1112C/T single-nucleotide polymorphism allele variant of the IL13 promoter gene, have been more frequently found in SM patients than in cutaneous mastocytosis patients and in healthy controls.15 More recently, we also showed that highly purified BM MC from ISM and ASM patients display distinct gene expression profiles, which include greater levels of CCL23, IL13 and IL1β mRNAs in ASM versus ISM cases, supporting the notion that such cytokines could contribute to discriminate between indolent and aggressive forms of SM.18 Despite this, no study has been reported so far, in which the relationship between the cytokine plasma levels at diagnosis have Leukemia (2015) 1 – 7
Prognostic value of IL6 in systemic mastocytosis A Mayado et al
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Figure 3. Paired IL6 plasma levels (a) and tryptase serum levels (b) from four patients studied both at diagnosis and subsequently at disease progression. ISM, indolent systemic mastocytosis; ISM-associated with a clonal non-mast lineage hematopoietic disease, indolent systemic mastocytosis associated with a clonal non-mast cell lineage hematopoietic disease; SSM, smouldering systemic mastocytosis; ASM, aggressive systemic mastocytosis; MCL, mast cell leukemia.
neither been investigated in a large series of ISM patients in parallel to sBT and the presence of multilineage KIT mutation nor they have been related to disease progression. Overall, our results confirm and extend on previous observations18 regarding the presence of altered (for example, increased) levels of IL1β, IL6, IL13 and CCL23 at diagnosis in SM patients. Of note, different cytokine profiles were observed at diagnosis in the plasma of ISM versus ASM cases, as well as in ISM cases with MC-restricted versus ISM patients with multilineage involvement of hematopoiesis by the KIT mutation. Thus, although IL1β and IL6 were increased in all SM patient subgroups, increased IL13 levels were restricted to ASM and increased CCL23 was only found among ISM-ML and ASM patients. Most interestingly, IL6 plasma levels (alone or in combination with CCL23), but no other cytokine nor the sBT levels, were significantly associated with disease progression and PFS among our ISM patients, in particular among ISM cases who had multilineage involvement of BM hematopoiesis by the KIT mutation. As expected, an extended pattern of involvement of BM hematopoiesis by the KIT mutation, as well as increased sBT levels, were also associated with a greater rate of disease progression, although for sBT the differences did not reach statistical significance. Of note, the prognostic impact of IL6 plasma levels was particularly evident among ISM-ML and, to a less extent also, among patients with increased sBT (data not shown). Most interestingly, those four ISM patients in whom serial determinations of IL6 plasma levels were performed at diagnosis and at disease progression showed levels of this cytokine, which were systematically increased already at diagnosis; despite this, in most cases they increased even further at disease progression. Previous studies have suggested that IL6 plasma levels correlate with sBT levels and adverse clinical features of SM, such as the presence of organomegalies.14 In line with these results, ISM-ML patients who had increased IL6 plasma levels at diagnosis also displayed a higher frequency of organomegalies (lymphadenopathies and splenomegaly) versus ISM-ML patients with normal IL6 plasma levels. These observations further support the notion that among ISM patients, increased IL6 plasma levels at diagnosis are associated with both a higher risk of disease progression and more adverse clinical features. Although increased sBT levels were also observed at diagnosis, they frequently remained relatively constant (or they even decreased) at disease progression, except for the patient who progressed to MCL; this could be due to the fact that most of our patients, including this small group of patients showing disease progression, received therapy with sodium cromolyn, which decreases the release of MC mediators Leukemia (2015) 1 – 7
into the extracellular medium, potentially also contributing to modulate sBT levels. Altogether, these results suggest that IL6 could have an important role in the pathogenesis of ISM, and that determination of the plasma levels of this cytokine could contribute to identify already at diagnosis, a small subset of ISM patients within those cases that carry multilineage KIT mutation, who are at higher risk for disease progression, independently of sBT levels. Despite this, the specific cell compartment contributing to such increased IL6 plasma levels still remains to be identified, as BM MC from ASM patients appear to produce less IL6 mRNA than BM MC from ISM cases.18 In this regard, it may be speculated that other myeloid cells such as the monocytic-macrophage cells and the dendritic cells could be involved in the increased IL6 production; however, further studies are required. Regarding the plasma levels of IL1β and CCL23, our results confirmed previous observations showing that both IL1β and CCL23 are increased in SM patients; 18 whether or not such increased plasma levels are directly associated with a greater production of both cytokines by pathological MC18 or other immune cells29,30 remains to be determined. In any case, only CCL23 (but not IL1β plasma levels) appear to gradually increase from ISM-MC to ISM-ML and ASM. These results indicate that CCL23 and IL6, but not IL1β, show a similar profile among the different subtypes of SM patients analyzed here; despite this, upregulation of both CCL23 and IL6 might reflect unrelated mechanisms (for example, inflammation versus angiogenesis), as no direct correlation was found between them for individual patients. Further studies are necessary to elucidate the precise mechanisms involved. Overall, similar IL13 and clusterin plasma levels were observed in SM patients versus healthy subjects; however, increased IL13 was more frequently detected among ASM and ISM-ML patients than in ISM-MC cases, whereas no significant differences were found between ISM and ASM for clusterin plasma levels. The IL13 plasma profile is consistent with previous observations18 that showed significant upregulation of this cytokine on BM MC from ASM patients versus both ISM patients and healthy controls. In contrast, no differences were observed in clusterin plasma levels between the distinct subgroups of SM patients and healthy controls, whereas upregulation of clusterin gene expression by BM MC from SM patients versus healthy controls has been previously reported.18 These apparently contradictory results might be owing to the fact that clusterin has two isoforms, which differ in both their structure and subcellular localization (that is, nuclear and secretory clusterin),31,32 and, therefore, assessment of clusterin plasma levels only reflects the levels of secretory clusterin. © 2015 Macmillan Publishers Limited
Prognostic value of IL6 in systemic mastocytosis A Mayado et al
7 In summary, here we show that SM patients display an altered cytokine/chemokine profile in plasma; although some cytokines (for example, IL6, CCL23 and IL1β) appear to be frequently increased already at diagnosis, others (for example, IL13) would only increase in advanced forms of the disease in parallel to (even) greater levels of IL6. Most interestingly, our results showed a clear association between IL6 plasma levels at diagnosis and disease progression among ISM cases, in particular for the subgroup of ISM-ML cases that has previously been shown to have higher risk of progression.10 These results suggest that an increase in IL6 plasma levels is an early event among ISM-ML patients who are more likely to undergo progression of the disease in the first years after diagnosis. Further studies in which the molecular mechanisms underlying deregulation of IL6 production and its source are investigated are required to better understand the physiopathological mechanisms of disease progression in ISM patients and to establish new targeted therapies that might prevent clinical transformation of ISM into more advanced forms of the disease. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This work was supported by Fondo de Investigaciones Sanitarias (FIS) of the Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain (grant PI11/02399, FEDER), and Fundacion Ramon Areces, Madrid, Spain (grant CIVP16A1806). AM and AR-C were supported by a grant RTICC (Red Tematica de Investigacion Cooperativa en Cancer, grant RD12/0036/0048, FIS, FEDER). SM was supported by Fundación Científica de la Asociación Española contra el Cáncer. We gratefully acknowledge Juan Alejandro Flores Montero for his contribution in the recruitment of the controls.
REFERENCES 1 Lennert K, Parwaresch MR. Mast cells and mast cell neoplasia: a review. Histopathology 1979; 3: 349–365. 2 Valent P, Sperr WR, Schwartz LB, Horny HP. Diagnosis and classification of mast cell proliferative disorders: delineation from immunologic diseases and non-mast cell hematopoietic neoplasms. J Allergy Clin Immunol 2004; 114: 3–11, quiz 2. 3 Metcalfe DD. The liver, spleen, and lymph nodes in mastocytosis. J Invest Dermatol 1991; 96: 45S–46S. 4 Horny HP, Metcalfe DD, Bennet JM, Bain BJ, Akin C, Escribano L et al. Mastocytosis. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H et al. (eds). World Health Organization (WHO) Classification of Tumours Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, 4th edn. International Agency for Research on Cancer (IARC): Lyon, 2008; pp 54–63. 5 Garcia-Montero AC, Jara-Acevedo M, Teodosio C, Sanchez ML, Nunez R, Prados A et al. KIT mutation in mast cells and other bone marrow hematopoietic cell lineages in systemic mast cell disorders: a prospective study of the Spanish Network on Mastocytosis (REMA) in a series of 113 patients. Blood 2006; 108: 2366–2372. 6 Orfao A, Garcia-Montero AC, Sanchez L, Escribano L. Recent advances in the understanding of mastocytosis: the role of KIT mutations. Br J Haematol 2007; 138: 12–30. 7 Akin C, Metcalfe DD. The biology of Kit in disease and the application of pharmacogenetics. J Allergy Clin Immunol 2004; 114: 13–19, quiz 20. 8 Alvarez-Twose I, Jara-Acevedo M, Morgado JM, García-Montero AC, Sánchez-Muñoz L, Teodósio C et al. Clinical, immunophenotypical and molecular characteristics of well-differentiated systemic mastocytosis. J Allergy Clin Immunol 2015; e-pub ahead of print 19 June 2015; doi:10.1016/j.jaci.2015.05.008. 9 Lim KH, Tefferi A, Lasho TL, Finke C, Patnaik M, Butterfield JH et al. Systemic mastocytosis in 342 consecutive adults: survival studies and prognostic factors. Blood 2009; 113: 5727–5736. 10 Escribano L, Alvarez-Twose I, Sanchez-Munoz L, Garcia-Montero A, Nunez R, Almeida J et al. Prognosis in adult indolent systemic mastocytosis: a long-term study of the Spanish Network on Mastocytosis in a series of 145 patients. J Allergy Clin Immunol 2009; 124: 514–521.
11 Teodosio C, Garcia-Montero AC, Jara-Acevedo M, Alvarez-Twose I, Sanchez-Munoz L, Almeida J et al. An immature immunophenotype of bone marrow mast cells predicts for multilineage D816V KIT mutation in systemic mastocytosis. Leukemia 2012; 26: 951–958. 12 Jara-Acevedo M, Teodósio C, Sánchez-Muñoz L, Alvarez-Twose I, Mayado A, Caldas C et al. Detection of the KIT D816V mutation in peripheral blood of systemic mastocytosis: diagnostic implications. Mod Pathol 2015; e-pub ahead of print 12 January 2015; doi:10.1038/modpathol.2015.72. 13 Matito A, Morgado JM, Alvarez-Twose I, Sanchez-Munoz L, Pedreira CE, JaraAcevedo M et al. Serum tryptase monitoring in indolent systemic mastocytosis: association with disease features and patient outcome. PLoS One 2013; 8: e76116. 14 Brockow K, Akin C, Huber M, Metcalfe DD. IL-6 levels predict disease variant and extent of organ involvement in patients with mastocytosis. Clin Immunol 2005; 115: 216–223. 15 Nedoszytko B, Niedoszytko M, Lange M, van Doormaal J, Glen J, Zablotna M et al. Interleukin-13 promoter gene polymorphism -1112C/T is associated with the systemic form of mastocytosis. Allergy 2009; 64: 287–294. 16 Hartmann K, Wagner N, Rabenhorst A, Pflanz L, Leja S, Forster A et al. Serum IL-31 levels are increased in a subset of patients with mastocytosis and correlate with disease severity in adult patients. J Allergy Clin Immunol 2013; 132: 232–235. 17 Adams DH, Lloyd AR. Chemokines: leucocyte recruitment and activation cytokines. Lancet 1997; 349: 490–495. 18 Teodosio C, Garcia-Montero AC, Jara-Acevedo M, Sanchez-Munoz L, Pedreira CE, Alvarez-Twose I et al. Gene expression profile of highly purified bone marrow mast cells in systemic mastocytosis. J Allergy Clin Immunol 2013; 131: 1213–1224, e1–4. 19 Valent P, Horny HP, Escribano L, Longley BJ, Li CY, Schwartz LB et al. Diagnostic criteria and classification of mastocytosis: a consensus proposal. Leuk Res 2001; 25: 603–625. 20 Horny HP, Sotlar K, Valent P, Hartmann K. Mastocytosis: a disease of the hematopoietic stem cell. Dtsch Arztebl Int 2008; 105: 686–692. 21 Valent P, Akin C, Escribano L, Fodinger M, Hartmann K, Brockow K et al. Standards and standardization in mastocytosis: consensus statements on diagnostics, treatment recommendations and response criteria. Eur J Clin Invest 2007; 37: 435–453. 22 Rodriguez-Caballero A, Garcia-Montero AC, Bueno C, Almeida J, Varro R, Chen R et al. A new simple whole blood flow cytometry-based method for simultaneous identification of activated cells and quantitative evaluation of cytokines released during activation. Lab Invest 2004; 84: 1387–1398. 23 Rueff F, Przybilla B, Bilo MB, Muller U, Scheipl F, Aberer W et al. Predictors of severe systemic anaphylactic reactions in patients with Hymenoptera venom allergy: importance of baseline serum tryptase-a study of the European Academy of Allergology and Clinical Immunology Interest Group on Insect Venom Hypersensitivity. J Allergy Clin Immunol 2009; 124: 1047–1054. 24 Akin C, Valent P. Diagnostic criteria and classification of mastocytosis in 2014. Immunol Allergy Clin North Am 2014; 34: 207–218. 25 Akin C, Schwartz LB, Kitoh T, Obayashi H, Worobec AS, Scott LM et al. Soluble stem cell factor receptor (CD117) and IL-2 receptor alpha chain (CD25) levels in the plasma of patients with mastocytosis: relationships to disease severity and bone marrow pathology. Blood 2000; 96: 1267–1273. 26 Pardanani A, Finke C, Abdelrahman RA, Lasho TL, Hanson CA, Tefferi A. Increased circulating IL-2Ralpha (CD25) predicts poor outcome in both indolent and aggressive forms of mastocytosis: a comprehensive cytokine-phenotype study. Leukemia 2013; 27: 1430–1433. 27 Theoharides TC, Boucher W, Spear K. Serum interleukin-6 reflects disease severity and osteoporosis in mastocytosis patients. Int Arch Allergy Immunol 2002; 128: 344–350. 28 Rabenhorst A, Christopeit B, Leja S, Gerbaulet A, Kleiner S, Forster A et al. Serum levels of bone cytokines are increased in indolent systemic mastocytosis associated with osteopenia or osteoporosis. J Allergy Clin Immunol 2013; 132: 1234–7 e7. 29 Nardelli B, Tiffany HL, Bong GW, Yourey PA, Morahan DK, Li Y et al. Characterization of the signal transduction pathway activated in human monocytes and dendritic cells by MPIF-1, a specific ligand for CC chemokine receptor 1. J Immunol 1999; 162: 435–444. 30 Sims JE, Smith DE. The IL-1 family: regulators of immunity. Nat Rev Immunol 2010; 10: 89–102. 31 Essabbani A, Garcia L, Zonetti MJ, Fisco T, Pucci S, Chiocchia G. Exon-skipping strategy by ratio modulation between cytoprotective versus pro-apoptotic clusterin forms increased sensitivity of LNCaP to cell death. PLoS One 2013; 8: e54920. 32 Trougakos IP, Djeu JY, Gonos ES, Boothman DA. Advances and challenges in basic and translational research on clusterin. Cancer Res 2009; 69: 403–406.
Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)
© 2015 Macmillan Publishers Limited
Leukemia (2015) 1 – 7
ORIGINAL ARTICLE
Increased IL6 plasma levels in indolent systemic mastocytosis patients are associated with high risk of disease progression A Mayado1, C Teodosio2, AC Garcia-Montero1, A Matito3, A Rodriguez-Caballero1, JM Morgado3, C Muñiz1,4, M Jara-Acevedo1, I Álvarez-Twose3, L Sanchez-Muñoz3, S Matarraz1, C Caldas1, JI Muñoz-González1, L Escribano1 and A Orfao1 Systemic mastocytosis (SM) is a heterogeneous disease with altered interleukin (IL)-6 and IL13 plasma levels. However, no study has simultaneously investigated the plasma levels of IL1β, IL6, IL13, CCL23 and clusterin in SM at diagnosis and correlated them with disease outcome. Here we investigated IL1β, IL6, IL13, CCL23 and clusterin plasma levels in 75 SM patients—66 indolent SM (ISM) and 9 aggressive SM—and analyzed their prognostic impact among ISM cases grouped according to the extent of hematopoietic involvement of the bone marrow cells by the KIT D816V mutation. Although increased IL1β, IL6 and CCL23 levels were detected in SM patients versus healthy controls, only IL6 and CCL23 levels gradually increased with disease severity. Moreover, increased IL6 plasma levels were associated with ISM progression to more aggressive disease, in particular among ISM patients with multilineal KIT mutation (ISM-ML), these patients also showing a higher frequency of organomegalies, versus other ISM-ML patients. Of note, all ISM patients who progressed had increased IL6 plasma levels already at diagnosis. Our results indicate that SM patients display an altered plasma cytokine profile already at diagnosis, increased IL6 plasma levels emerging as an early marker for disease progression among ISM cases, in particular among high-risk ISM patients who carry multilineage KIT mutation. Leukemia advance online publication, 31 July 2015; doi:10.1038/leu.2015.176
INTRODUCTION Systemic mastocytosis (SM) consists of a heterogeneous group of disorders characterized by an abnormal expansion and accumulation of pathological mast cells (MCs) in the bone marrow (BM) and other organs such as the skin, liver, spleen and/or lymph nodes.1–3 At presentation, clinical variants of SM include both indolent, for example, indolent SM (ISM), and aggressive, even life-threatening, forms of the disease, for example, aggressive SM (ASM) and MC leukemia (MCL), classified by the World Health Organization4 into seven distinct subtypes plus one provisional subvariant (smoldering SM (SSM)). In the vast majority of cases (490%), the clonal nature of pathological MC can be demonstrated by the presence of mutations in the tyrosine kinase domain of the stem cell growth factor receptor gene (KIT),5,6 except for a rare subset of welldifferentiated SM.7,8 Most ISM cases typically show stable disease for decades; in contrast, a smaller fraction of ISM patients display progression to more aggressive variants of the disease, for example, SSM, ASM, SM-associated with a clonal non-mast lineage hematopoietic disease and MCL, to a myeloproliferative neoplasm, myelodysplastic syndrome and/or acute myeloid leukemia.9,10 Currently, the most powerful independent prognostic factor for progression-free survival (PFS) among ISM patients relies on the presence of multilineage involvement of myeloid or myeloid plus lymphoid hematopoietic cells other than BM MC by the KIT mutation.10,11 Nowadays, evaluation of multilineage involvement of BM cells by the KIT mutation requires a BM study based on complex cell-purification procedures followed by molecular analysis of highly purified BM cell populations;5 because of this,
routine assessment of multilineage involvement of hematopoiesis by the KIT mutation remains restricted to a few specialized reference centers.5,10,12 Alternatively, it has been proposed that frequent monitoring of serum baseline tryptase (sBT) levels could be a surrogate marker for disease progression.13 However, sBT has a lower predictive value than the degree of involvement of hematopoiesis by the KIT mutation; at the same time, reliable assessment of increased sBT levels appears to require serial measurements for a relatively long period of time. In recent years, several groups have reported altered plasma levels of cytokines (for example, interleukin (IL)-IL6, IL13 and IL31) in SM patients.14–16 In addition, some of these cytokines (for example, IL6 and IL31)14,16 have been associated with a greater severity of mastocytosis and more extended disease, with greater levels in aggressive versus indolent variants of SM. In turn, it is well known that several of those cytokines and chemokines reported to be altered in SM have a critical role in MC proliferation and tissue migration.17 In line with these observations, we have recently reported that the distinct gene expression profile of purified BM MC from ISM versus ASM patients include differential expression of genes encoding for several cytokines (for example, IL-1β and IL13) and chemokines (for example, CCL23), as well as for other plasma proteins, such as the clusterin complement regulatory protein.18 Altogether, these findings suggest that the plasma levels of the above proteins might potentially provide prognostic information about disease outcome in SM patients. Despite this, to the best of our knowledge, no study has been reported so far in which the plasma levels of IL1β, IL6, IL13, CCL23
1 Servicio General de Citometría (NUCLEUS), Centro de Investigación del Cáncer (IBMCC-CSIC/USAL and IBSAL) and Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain; 2Department of Immunology, Erasmus MC University Medical Center Rotterdam, Rotterdam, The Netherlands; 3Centro de Estudios de Mastocitosis de Castilla La Mancha (CLMast), Hospital Virgen del Valle, Toledo, Spain and 4Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF) Instituto de Salud Carlos III, Madrid, Spain. Correspondence: Professor A Orfao, Servicio General de Citometría (NUCLEUS), Centro de Investigación del Cáncer (IBMCC-CSIC/USAL and IBSAL) and Departamento de Medicina, Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain. E-mail: [email protected] Received 24 March 2015; revised 15 May 2015; accepted 16 June 2015; accepted article preview online 8 July 2015
Prognostic value of IL6 in systemic mastocytosis A Mayado et al
2 and clusterin have been simultaneously assessed at diagnosis in SM patients and correlated with the outcome of the disease. Here we analyzed the IL1β, IL6, IL13, CCL23 and clusterin plasma levels versus sBT at diagnosis, in a large cohort (n = 75) of SM patients, including 66 ISM cases. Our major goal was to investigate the relationship between the plasma levels of these proteins and the outcome of ISM as reflected by progression to more advanced forms of the disease. MATERIALS AND METHODS Patients, controls and samples A total of 75 adult SM patients diagnosed at the Reference Centers of the Spanish Network on Mastocytosis (Mast Cell Unit, Hospital del Valle, Toledo, and Cytometry Service, Cancer Research Center, Salamanca, Spain), were studied. Patients were classified according to the World Health Organization criteria4 and more recent consensus recommendations.19,20 All patients had the D816V KIT mutation; in 38 ISM cases the KIT mutation was restricted to the MC compartment in the BM (ISM-MC), whereas the other 28 ISM patients and the 9 ASM cases studied had multilineage KIT mutation (ISM-ML) as defined elsewhere.10 Fifty-one healthy subjects (excluding individuals with a past or present history of allergy) were studied in parallel as controls. The demographic and biological characteristics of the patients analyzed are shown in Supplementary Table S1. Plasma and serum samples, as well as BM aspirated samples, were obtained and assessed at diagnosis and stored at the National DNA Bank Carlos III (www.bancoadn.org; University of Salamanca, Spain) and/or the Reference Centers of the Spanish Network on Mastocytosis, after written informed consent was given by each donor, according to the guidelines of the local Ethics Committee. Of note, in every patient, including those who progressed, plasma samples were obtained before any cytotoxic therapy, for example, interferon, hydroxyurea and cladribine (2CDA), had been administrered. At the moment of closing the study, the median follow-up of all SM patients was 36 months. ISM patients showed an overall rate of progression of ISM to SSM, SM-associated with a clonal non-mast lineage hematopoietic disease, ASM or MCL of 6% (4/66 ISM cases), after a median PFS of 35 ± 37 months; the criteria used to define progression from ISM to SSM included: coexistence of two B (Borderline–Benign) findings, for example, (a) massive MC burden (huge MC marrow infiltration, serum tryptase 4200 ng/ml), (b) organomegaly (spleen, liver and lymph node involvement) without impairment of organ function and (c) dysmyelopoiesis (hypercellular marrow with signs of myelodysplasia or myeloproliferation, but no criteria for myelodysplastic syndrome or myeloproliferative neoplasm).21 Of note, all nine ASM cases were excluded from PFS analyses.
Detection of the KIT mutation The KIT D816V mutation was investigated in fluorescence-activated cell sorting-purified (⩾97% purity) BM MC, eosinophils, monocytes, neutrophils, CD34+ hematopoietic precursor cells and T cells (FACSAria, Becton/ Dickinson Biosciences (BD), San José, CA, USA), using a peptide nucleic acid-mediated PCR-clamping technique, as previously described in detail.5
Serum tryptase levels sBT levels were measured using the Phadia ImmunoCAP Tryptase System (Phadia, Uppsala, Sweden; Thermo Fisher Scientific Inc., Waltham, MA, USA), according to the manufacturer’s instructions.
Quantification of CCL23 and clusterin plasma levels Peripheral blood samples were collected using EDTA-coated tubes. Briefly, fresh samples were centrifuged (10 min at 800 g), the platelet-rich plasma was collected and centrifuged again (5 min at 2000 g); afterward, the plasma was collected and stored at − 80 °C. The CCL23 and clusterin plasma levels were measured using commercially available sandwich enzyme-linked immunosorbent assays (ELISA): the CCL23 (Human) ELISA Kit (Abnova, Heidelberg, Germany) and the Human Clusterin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA), respectively.
Quantification of IL1β, IL6 and IL13 plasma levels Evaluation of IL1β, IL6 and IL13 plasma levels was performed in the same plasma samples obtained as described above, using the Cytometric Bead Leukemia (2015) 1 – 7
Array immunoassay system (CBA) according to the manufacturer’s instructions: human IL-1β CBA Flex Set, Human IL-6 CBA Flex Set and Human IL-13 CBA Flex Set (BD). Briefly, plasma samples were diluted 1:4 (vol:vol) and incubated for 1 h at room temperature, with 50 μl of each of the anti-cytokine antibody-coated beads. Afterward, 50 μl of the phycoerythrin-conjugated detection antibody reagents was added to each sample and a further incubation for 2 h at room temperature was performed. Then, the unbound antibody was washed out (1 × ) and the beads were measured in a FACSCalibur flow cytometer (BD) using the CellQUEST software program (BD). During acquisition, information was stored for 3000 events corresponding to each bead population analyzed per sample (total number of beads 49000). For data analysis, the Paint-AGate PRO (BD) and the CBA (BD) software programs were used, as described elsewhere.22
Statistical methods Median, range, mean and s.d. values, as well as the 10th, 25th, 75th and 90th percentiles, were calculated for all continuous variables; for categorical variables, frequencies were reported. IL6, IL13, CCL23 and clusterin protein levels were considered to be altered in a plasma sample, when they were higher or lower than the mean ± 2 s.d. of those values obtained for the plasma samples from healthy controls analyzed. In turn, cutoff values for IL1β were established at the limit of detection of the technique; for sBT, cutoff values were established at previously defined normal values ( o11.4 ng/ml).23 In order to determine the statistical significance of the differences observed among groups, the Kruskal–Wallis and the Mann–Whitney U-tests (for continuous variables), as well as the χ2-test (for categorical variables), were used. The degree of correlation between different variables was assessed by the Spearman’s test. PFS curves were plotted according to the method of Kaplan–Meier and they were compared by the (one-sided) log-rank test. P-values o 0.05 with a false discovery rate correction for multiple comparisons of o5% were considered to be associated with statistical significance. For all statistical analyses, the SPSS 18.0 software (SPSS, Chicago, IL, USA) was used.
RESULTS Disease progression among ISM cases From all 66 ISM patients studied, 4 (6%) showed progression to more advanced forms of the disease: 1 patient progressed to SSM, 1 evolved to an ASM, 1 progressed to SM-associated with a clonal non-mast lineage hematopoietic disease and the fourth case to MCL. Of note, all 4 ISM cases that progressed had multilineage KIT mutation (ISM-ML) at diagnosis involving the myeloid (n = 1) or the myeloid plus lymphoid (n = 3) hematopoietic cells, other than BM MC, whereas this was present in only 24 of the 62 ISM (39%) patients who did not show disease progression. IL1β, IL6, IL13, CCL23 and clusterin plasma levels in SM patients Overall, SM patients showed increased IL1β (P = 0.001), IL6 (P = 0.001) and CCL23 (P = 0.005) plasma levels versus healthy controls (Figures 1a–d). In contrast, similar IL13 and clusterin plasma levels were found in SM patients and healthy controls. Despite this, variable levels and distinct patterns of alteration were observed for the different cytokines, among the different diagnostic and prognostic subtypes of SM. Thus, IL1β was of significantly increased (at similar) levels in all diagnostic and prognostic subtypes of SM (P ⩽ 0.04; Figure 1a), although ASM cases had increased IL1β plasma levels more frequently than ISMMC and ISM-ML patients (Table 1). In contrast, IL-6 and CCL23 plasma levels gradually increased (versus controls) from ISM-MC (P o 0.001 and P40.05, respectively) to ISM-ML (P o 0.001 and Po 0.001, respectively) and ASM cases (P o0.001 and P = 0.03, respectively) (Figures 1b and d); interestingly, both IL6 and CCL23 plasma levels were also significantly higher in ISM-ML versus ISMMC patients (P = 0.007 and P = 0.003, respectively) (Figures 1b and d). Despite a similar altered profile observed among the different patient groups for IL6 and CCL23 plasma levels, no significant correlation was found between the plasma levels of both © 2015 Macmillan Publishers Limited
Prognostic value of IL6 in systemic mastocytosis A Mayado et al
3
Figure 1. IL1β (a), IL6 (b), IL13 (c), CCL23 (d), clusterin (e) and sBT (f) plasma levels among adults with different subtypes of ISM and with ASM versus healthy controls. Notched boxes extend from the 25th to the 75th percentile values; the lines in the middle and vertical lines correspond to median values and the 10th and 90th percentiles, respectively. P-values o0.05 with false discovery rate (FDR) correction for multiple comparisons of o5% were considered to be associated with statistical significance versus the *control group, †ISM-MC and ‡ ISM-ML cases.
Table 1.
Frequency of systemic mastocytosis patients with different clinical and prognostic subtypes of the disease (n = 75) with altered IL-1β, IL6, IL13, CCL23 and clusterin plasma levels Cytokine/protein IL1β IL6 IL13 CCL23 Clusterin sBT
Cutoff for altered levels
ISM-MC ISM-ML ASM (n = 38) (n = 28) (n = 9)
⩾ 2.3 pg/ml ⩾ 1.6 pg/ml ⩾ 5.4 pg/ml ⩾ 1014.6 pg/ml ⩽ 218.5 ng/ml ⩾ 346.3 ng/ml ⩾ 15 ng/ml
3% 5% 3% 11% 9% 18% 79%
4% 40% 20% 62% 6% 19% 100%
P-values
25% 0.02a 100% ⩽ 0.003b 75% ⩽ 0.02b 100% 0.002a,c 0% 0.962 0% 0.40 100% 0.01c
Abbreviations: ASM, aggressive systemic mastocytosis; IL, interleukin; ISMMC, indolent systemic mastocytosis with KIT mutation restricted to bone marrow mast cells; ISM-ML, indolent systemic mastocytosis with multilineage KIT mutation; sBT, serum baseline tryptase levels. Results expressed as percentage of cases showing increased or decreased cytokine/protein levels. aISM-MC versus ASM. bPo0.05 versus all others groups. cISM-MC versus ISM-ML.
cytokines (r = 0.01; P = 0.95). Although similar IL13 plasma levels were found in SM patients versus healthy controls, higher IL13 plasma levels were observed among ASM patients versus both the controls (P = 0.001) and the two ISM (P ⩽ 0.003) patient groups (P o 0.001 versus ISM-MC and P = 0.003 versus ISM-ML patients, respectively). Of note, ISM-ML patients also displayed higher IL13 plasma levels than ISM-MC cases (P = 0.02). sBT showed a similar profile to that observed for IL6 within the different subgroups of SM patients, (Figure 1f). Furthermore, a significant correlation was found between sBT levels and the © 2015 Macmillan Publishers Limited
amount of most plasma proteins investigated here, that is, IL6 (r = 0.47; P o 0.001), IL13 (r = 0.31; P = 0.009) and CCL23 (r = 0.63; P o0.001), except for IL1β (r = 0.02; P = 0.90). Interestingly, the above described differences among the distinct groups of individuals in their IL1β, IL6, IL13 and CCL23 plasma levels, as well as in sBT, were typically due to a higher percentage of cases with altered plasma levels for these cytokines from ISM-MC to ISM-ML and ASM patients. Briefly, a greater frequency of patients with increased IL1β was found among ASM versus ISM-MC cases (25% versus 3%; P = 0.02); similarly, the frequency of SM patients displaying abnormally increased IL6, IL13, CCL23 and sBT levels progressively increased from ISM-MC (5%, 3%, 11% and 79%, respectively) to ISM-ML (40%, 20%, 62% and 100%, respectively) and ASM (100%, 75%, 100% and 100%, respectively; P ⩽ 0.003, P ⩽ 0.02, P = 0.002 and P = 0.01, respectively) (Table 1). Impact of cytokine plasma levels on PFS of ISM patients From all cytokines evaluated, only IL6 (and to a lesser extent also IL6 in combination with CCL23) emerged as being significantly associated with progression of ISM to more aggressive forms of the disease (P o 0.001 and P = 0.02, respectively; Figures 2a and c). Most interestingly, such prognostic value of IL6 was restricted to the subgroup of ISM cases who had multilineage KIT mutation (ISM-ML), whereas it showed no significant impact among the other ISM cases who had an MC-restricted KIT mutation profile (ISM-MC) and normal IL6 levels. Thus, ISM-ML patients with altered IL6 plasma levels showed a median PFS of 42 months (range: 24–61 months) versus not reached for the other ISM-ML cases (P = 0.006), with 3-year PFS rates of 33 ± 31% versus 0 ± 0%, respectively (Figure 2b). Of note, 4 of 11 cases that had increased IL6 levels showed multilineage KIT mutation in association with Leukemia (2015) 1 – 7
Prognostic value of IL6 in systemic mastocytosis A Mayado et al
4
Figure 2. Impact of high IL6 plasma levels alone (a and b) and in combination with CCL23 (c and d) as well as sBT levels (e) on PFS of ISM patients (a, c and e) and ISM cases with multilineage KIT mutation at diagnosis (ISM-ML; b and d).
disease progression; in turn, most (5 of 7) of the other ISM cases showing increased IL6 plasma levels also displayed multilineage KIT mutation but they had stable disease until the moment of closing this study. In our cohort, sBT levels at diagnosis did not show a significant impact on PFS of ISM patients (P40.05), despite cases showing increased sBT levels had a slightly poorer outcome (3-year PFS rates of 0 ± 0% and of 6 ± 6%, respectively; Figure 2e). Although, cases with increased IL6 and/or CCL23 plasma levels also showed lower PFS rates, combined assessment of both cytokines was Leukemia (2015) 1 – 7
associated with a lower prognostic impact than that observed for IL6 alone (Figures 2c and d versus Figures 2a and b, respectively). Association between increased IL6 plasma levels and adverse clinical features of the disease On comparing the clinical features of ISM-ML patients presenting with increased versus normal IL6 plasma levels (Table 2), the former cases showed a higher frequency of organomegalies at diagnosis, including both lymphadenopathies (33% versus 0%, © 2015 Macmillan Publishers Limited
Prognostic value of IL6 in systemic mastocytosis A Mayado et al
5 Table 2.
Relationship between increased IL6 plasma levels and the clinical characteristics of ISM-ML versus other ISM (ISM-MC) and ASM cases
Disease features
ISM-MC (n = 38)
ISM-ML IL6 ( o1.6 pg/ml) n = 15
IL6 (⩾1.6 pg/ml) n = 9
P-values
ASM (n = 9)
MC mediators release-associated symptoms Abdominal pain Anaphylaxis Flushing GI symptoms Pruritus Signs of hemolysis
87% 27% 58% 29% 40% 41% 3%
100% 20% 20% 53% 40% 73% 13%
79% 11% 22% 22% 22% 67% 11%
0.06 0.57 0.90 0.13 0.37 0.73 0.87
100% 67% 22% 11% 67% 22% 67%
Others symptoms Constitutional symptoms
0%
0%
0%
0.99
67%
Organomegalies Lymphadenopathies Hepatomegaly Splenomegaly
0% 6% 3%
0% 20% 20%
33% 56% 67%
0.01 0.07 0.02
44% 67% 78%
Bone disease Fibrosis Pathologic fractures Lytic lesions Disease progression
29% 3% 0% 0%
55% 0% 7% 0%
56% 22% 11% 44%
0.82 0.06 0.70 0.005
100% 22% 11% NA
Abbreviations: ASM, aggressive systemic mastocytosis; GI, gastrointestinal; IL, interleukin; ISM-MC, indolent systemic mastocytosis with KIT mutation restricted to bone marrow mast cells; ISM-ML, indolent systemic mastocytosis with multilineage KIT mutation; MC, mast cell; NA, not applicable; sBT, serum baseline tryptase levels. Results expressed as percentage of cases. P-values are specifically shown for the ISM-ML group of patients for the comparison between ISM-ML cases with normal versus increased (⩾1.6 pg/ml) IL6 plasma levels.
P = 0.01) and splenomegaly (67% versus 20%, P = 0.02), together with a higher rate of disease progression (44% versus 0%, P = 0.005) versus other ISM-ML patients (Table 2). In turn, ISM-ML patients with higher CCL23 plasma levels displayed a lower frequency of anaphylaxis than ISM-ML cases with normal CCL23 levels (20% versus 13%, P = 0.03), whereas cases with greater IL6 and/or CCL23 more frequently had BM fibrosis (60% versus 33%, P = 0.047). Sequential assessment of IL6 plasma levels at diagnosis and at disease progression In the four ISM patients who progressed to more advanced forms of the disease, paired IL6 plasma levels and serum tryptase levels were studied at diagnosis and, subsequently, at disease progression (Figures 3a and b), after a median time to progression of 15 months (range: 0–39 months). Of note, all four patients had increased baseline IL6 and sBT levels already at diagnosis. For IL6, the majority of the patients (three of four cases) showed increased IL6 plasma levels at disease progression versus those found at diagnosis, the highest level of increase corresponding to an ISM patient who progressed to MCL (Figure 3a). Similarly, two of four patients had higher sBT levels at disease progression versus those detected at diagnosis, the other two cases showing levels at progression that were even lower than those detected at diagnosis (Figure 3b). DISCUSSION Current World Health Organization criteria for the classification of SM clearly distinguishes among cases with indolent (for example, ISM) versus aggressive disease (for example, ASM and MCL), the two groups of patients showing distinct clinical outcomes.19,21,24 Despite this, among ISM there is still a subset of patients who show progression to more aggressive disease (for example, SSM, ASM and MCL) including the development of an associated hematological malignancy (for example, ISM-associated with a © 2015 Macmillan Publishers Limited
clonal non-mast lineage hematopoietic disease) such as myelodysplastic syndrome, myeloproliferative neoplasm and/or acute myeloid leukemia.10 In the past, several different prognostic factors have been proposed to help in identifying ISM patients at greater risk of disease progression.5,9,13 Among others, the presence of multilineage involvement of hematopoiesis by the KIT mutation and increasing sBT levels have emerged as being among the most relevant ones.5,13 In addition, soluble CD25 and sCD117 have also been long described as markers of disease severity.25 In line with these latter findings, Pardanani et al.26 have also reported in a relatively limited number of ISM cases (n = 23), which had increased soluble levels of the IL2 receptor α-chain (soluble CD25) to be associated with a poorer overall survival in both advanced (n = 8) and ISM (n = 4); nevertheless, in this latter study, data about soluble CD25 levels were missing at diagnosis in a substantial proportion of cases, the prognostic value of soluble CD25, as well as of sCD117, deserving further prospective validation. In recent years, several evidences have accumulated, which point out for a potential association between cytokine levels and the pathophysiology of SM.15,27,28 Thus, several studies reported increased IL6 levels in patients with mastocytosis, such increased IL6 levels being correlated with the extent of tissue involvement.14 Another cytokine that has emerged as potentially relevant in mastocytosis is IL13; thus, higher IL13 serum levels, as well as an increased prevalence of the − 1112C/T single-nucleotide polymorphism allele variant of the IL13 promoter gene, have been more frequently found in SM patients than in cutaneous mastocytosis patients and in healthy controls.15 More recently, we also showed that highly purified BM MC from ISM and ASM patients display distinct gene expression profiles, which include greater levels of CCL23, IL13 and IL1β mRNAs in ASM versus ISM cases, supporting the notion that such cytokines could contribute to discriminate between indolent and aggressive forms of SM.18 Despite this, no study has been reported so far, in which the relationship between the cytokine plasma levels at diagnosis have Leukemia (2015) 1 – 7
Prognostic value of IL6 in systemic mastocytosis A Mayado et al
6
Figure 3. Paired IL6 plasma levels (a) and tryptase serum levels (b) from four patients studied both at diagnosis and subsequently at disease progression. ISM, indolent systemic mastocytosis; ISM-associated with a clonal non-mast lineage hematopoietic disease, indolent systemic mastocytosis associated with a clonal non-mast cell lineage hematopoietic disease; SSM, smouldering systemic mastocytosis; ASM, aggressive systemic mastocytosis; MCL, mast cell leukemia.
neither been investigated in a large series of ISM patients in parallel to sBT and the presence of multilineage KIT mutation nor they have been related to disease progression. Overall, our results confirm and extend on previous observations18 regarding the presence of altered (for example, increased) levels of IL1β, IL6, IL13 and CCL23 at diagnosis in SM patients. Of note, different cytokine profiles were observed at diagnosis in the plasma of ISM versus ASM cases, as well as in ISM cases with MC-restricted versus ISM patients with multilineage involvement of hematopoiesis by the KIT mutation. Thus, although IL1β and IL6 were increased in all SM patient subgroups, increased IL13 levels were restricted to ASM and increased CCL23 was only found among ISM-ML and ASM patients. Most interestingly, IL6 plasma levels (alone or in combination with CCL23), but no other cytokine nor the sBT levels, were significantly associated with disease progression and PFS among our ISM patients, in particular among ISM cases who had multilineage involvement of BM hematopoiesis by the KIT mutation. As expected, an extended pattern of involvement of BM hematopoiesis by the KIT mutation, as well as increased sBT levels, were also associated with a greater rate of disease progression, although for sBT the differences did not reach statistical significance. Of note, the prognostic impact of IL6 plasma levels was particularly evident among ISM-ML and, to a less extent also, among patients with increased sBT (data not shown). Most interestingly, those four ISM patients in whom serial determinations of IL6 plasma levels were performed at diagnosis and at disease progression showed levels of this cytokine, which were systematically increased already at diagnosis; despite this, in most cases they increased even further at disease progression. Previous studies have suggested that IL6 plasma levels correlate with sBT levels and adverse clinical features of SM, such as the presence of organomegalies.14 In line with these results, ISM-ML patients who had increased IL6 plasma levels at diagnosis also displayed a higher frequency of organomegalies (lymphadenopathies and splenomegaly) versus ISM-ML patients with normal IL6 plasma levels. These observations further support the notion that among ISM patients, increased IL6 plasma levels at diagnosis are associated with both a higher risk of disease progression and more adverse clinical features. Although increased sBT levels were also observed at diagnosis, they frequently remained relatively constant (or they even decreased) at disease progression, except for the patient who progressed to MCL; this could be due to the fact that most of our patients, including this small group of patients showing disease progression, received therapy with sodium cromolyn, which decreases the release of MC mediators Leukemia (2015) 1 – 7
into the extracellular medium, potentially also contributing to modulate sBT levels. Altogether, these results suggest that IL6 could have an important role in the pathogenesis of ISM, and that determination of the plasma levels of this cytokine could contribute to identify already at diagnosis, a small subset of ISM patients within those cases that carry multilineage KIT mutation, who are at higher risk for disease progression, independently of sBT levels. Despite this, the specific cell compartment contributing to such increased IL6 plasma levels still remains to be identified, as BM MC from ASM patients appear to produce less IL6 mRNA than BM MC from ISM cases.18 In this regard, it may be speculated that other myeloid cells such as the monocytic-macrophage cells and the dendritic cells could be involved in the increased IL6 production; however, further studies are required. Regarding the plasma levels of IL1β and CCL23, our results confirmed previous observations showing that both IL1β and CCL23 are increased in SM patients; 18 whether or not such increased plasma levels are directly associated with a greater production of both cytokines by pathological MC18 or other immune cells29,30 remains to be determined. In any case, only CCL23 (but not IL1β plasma levels) appear to gradually increase from ISM-MC to ISM-ML and ASM. These results indicate that CCL23 and IL6, but not IL1β, show a similar profile among the different subtypes of SM patients analyzed here; despite this, upregulation of both CCL23 and IL6 might reflect unrelated mechanisms (for example, inflammation versus angiogenesis), as no direct correlation was found between them for individual patients. Further studies are necessary to elucidate the precise mechanisms involved. Overall, similar IL13 and clusterin plasma levels were observed in SM patients versus healthy subjects; however, increased IL13 was more frequently detected among ASM and ISM-ML patients than in ISM-MC cases, whereas no significant differences were found between ISM and ASM for clusterin plasma levels. The IL13 plasma profile is consistent with previous observations18 that showed significant upregulation of this cytokine on BM MC from ASM patients versus both ISM patients and healthy controls. In contrast, no differences were observed in clusterin plasma levels between the distinct subgroups of SM patients and healthy controls, whereas upregulation of clusterin gene expression by BM MC from SM patients versus healthy controls has been previously reported.18 These apparently contradictory results might be owing to the fact that clusterin has two isoforms, which differ in both their structure and subcellular localization (that is, nuclear and secretory clusterin),31,32 and, therefore, assessment of clusterin plasma levels only reflects the levels of secretory clusterin. © 2015 Macmillan Publishers Limited
Prognostic value of IL6 in systemic mastocytosis A Mayado et al
7 In summary, here we show that SM patients display an altered cytokine/chemokine profile in plasma; although some cytokines (for example, IL6, CCL23 and IL1β) appear to be frequently increased already at diagnosis, others (for example, IL13) would only increase in advanced forms of the disease in parallel to (even) greater levels of IL6. Most interestingly, our results showed a clear association between IL6 plasma levels at diagnosis and disease progression among ISM cases, in particular for the subgroup of ISM-ML cases that has previously been shown to have higher risk of progression.10 These results suggest that an increase in IL6 plasma levels is an early event among ISM-ML patients who are more likely to undergo progression of the disease in the first years after diagnosis. Further studies in which the molecular mechanisms underlying deregulation of IL6 production and its source are investigated are required to better understand the physiopathological mechanisms of disease progression in ISM patients and to establish new targeted therapies that might prevent clinical transformation of ISM into more advanced forms of the disease. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This work was supported by Fondo de Investigaciones Sanitarias (FIS) of the Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain (grant PI11/02399, FEDER), and Fundacion Ramon Areces, Madrid, Spain (grant CIVP16A1806). AM and AR-C were supported by a grant RTICC (Red Tematica de Investigacion Cooperativa en Cancer, grant RD12/0036/0048, FIS, FEDER). SM was supported by Fundación Científica de la Asociación Española contra el Cáncer. We gratefully acknowledge Juan Alejandro Flores Montero for his contribution in the recruitment of the controls.
REFERENCES 1 Lennert K, Parwaresch MR. Mast cells and mast cell neoplasia: a review. Histopathology 1979; 3: 349–365. 2 Valent P, Sperr WR, Schwartz LB, Horny HP. Diagnosis and classification of mast cell proliferative disorders: delineation from immunologic diseases and non-mast cell hematopoietic neoplasms. J Allergy Clin Immunol 2004; 114: 3–11, quiz 2. 3 Metcalfe DD. The liver, spleen, and lymph nodes in mastocytosis. J Invest Dermatol 1991; 96: 45S–46S. 4 Horny HP, Metcalfe DD, Bennet JM, Bain BJ, Akin C, Escribano L et al. Mastocytosis. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H et al. (eds). World Health Organization (WHO) Classification of Tumours Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, 4th edn. International Agency for Research on Cancer (IARC): Lyon, 2008; pp 54–63. 5 Garcia-Montero AC, Jara-Acevedo M, Teodosio C, Sanchez ML, Nunez R, Prados A et al. KIT mutation in mast cells and other bone marrow hematopoietic cell lineages in systemic mast cell disorders: a prospective study of the Spanish Network on Mastocytosis (REMA) in a series of 113 patients. Blood 2006; 108: 2366–2372. 6 Orfao A, Garcia-Montero AC, Sanchez L, Escribano L. Recent advances in the understanding of mastocytosis: the role of KIT mutations. Br J Haematol 2007; 138: 12–30. 7 Akin C, Metcalfe DD. The biology of Kit in disease and the application of pharmacogenetics. J Allergy Clin Immunol 2004; 114: 13–19, quiz 20. 8 Alvarez-Twose I, Jara-Acevedo M, Morgado JM, García-Montero AC, Sánchez-Muñoz L, Teodósio C et al. Clinical, immunophenotypical and molecular characteristics of well-differentiated systemic mastocytosis. J Allergy Clin Immunol 2015; e-pub ahead of print 19 June 2015; doi:10.1016/j.jaci.2015.05.008. 9 Lim KH, Tefferi A, Lasho TL, Finke C, Patnaik M, Butterfield JH et al. Systemic mastocytosis in 342 consecutive adults: survival studies and prognostic factors. Blood 2009; 113: 5727–5736. 10 Escribano L, Alvarez-Twose I, Sanchez-Munoz L, Garcia-Montero A, Nunez R, Almeida J et al. Prognosis in adult indolent systemic mastocytosis: a long-term study of the Spanish Network on Mastocytosis in a series of 145 patients. J Allergy Clin Immunol 2009; 124: 514–521.
11 Teodosio C, Garcia-Montero AC, Jara-Acevedo M, Alvarez-Twose I, Sanchez-Munoz L, Almeida J et al. An immature immunophenotype of bone marrow mast cells predicts for multilineage D816V KIT mutation in systemic mastocytosis. Leukemia 2012; 26: 951–958. 12 Jara-Acevedo M, Teodósio C, Sánchez-Muñoz L, Alvarez-Twose I, Mayado A, Caldas C et al. Detection of the KIT D816V mutation in peripheral blood of systemic mastocytosis: diagnostic implications. Mod Pathol 2015; e-pub ahead of print 12 January 2015; doi:10.1038/modpathol.2015.72. 13 Matito A, Morgado JM, Alvarez-Twose I, Sanchez-Munoz L, Pedreira CE, JaraAcevedo M et al. Serum tryptase monitoring in indolent systemic mastocytosis: association with disease features and patient outcome. PLoS One 2013; 8: e76116. 14 Brockow K, Akin C, Huber M, Metcalfe DD. IL-6 levels predict disease variant and extent of organ involvement in patients with mastocytosis. Clin Immunol 2005; 115: 216–223. 15 Nedoszytko B, Niedoszytko M, Lange M, van Doormaal J, Glen J, Zablotna M et al. Interleukin-13 promoter gene polymorphism -1112C/T is associated with the systemic form of mastocytosis. Allergy 2009; 64: 287–294. 16 Hartmann K, Wagner N, Rabenhorst A, Pflanz L, Leja S, Forster A et al. Serum IL-31 levels are increased in a subset of patients with mastocytosis and correlate with disease severity in adult patients. J Allergy Clin Immunol 2013; 132: 232–235. 17 Adams DH, Lloyd AR. Chemokines: leucocyte recruitment and activation cytokines. Lancet 1997; 349: 490–495. 18 Teodosio C, Garcia-Montero AC, Jara-Acevedo M, Sanchez-Munoz L, Pedreira CE, Alvarez-Twose I et al. Gene expression profile of highly purified bone marrow mast cells in systemic mastocytosis. J Allergy Clin Immunol 2013; 131: 1213–1224, e1–4. 19 Valent P, Horny HP, Escribano L, Longley BJ, Li CY, Schwartz LB et al. Diagnostic criteria and classification of mastocytosis: a consensus proposal. Leuk Res 2001; 25: 603–625. 20 Horny HP, Sotlar K, Valent P, Hartmann K. Mastocytosis: a disease of the hematopoietic stem cell. Dtsch Arztebl Int 2008; 105: 686–692. 21 Valent P, Akin C, Escribano L, Fodinger M, Hartmann K, Brockow K et al. Standards and standardization in mastocytosis: consensus statements on diagnostics, treatment recommendations and response criteria. Eur J Clin Invest 2007; 37: 435–453. 22 Rodriguez-Caballero A, Garcia-Montero AC, Bueno C, Almeida J, Varro R, Chen R et al. A new simple whole blood flow cytometry-based method for simultaneous identification of activated cells and quantitative evaluation of cytokines released during activation. Lab Invest 2004; 84: 1387–1398. 23 Rueff F, Przybilla B, Bilo MB, Muller U, Scheipl F, Aberer W et al. Predictors of severe systemic anaphylactic reactions in patients with Hymenoptera venom allergy: importance of baseline serum tryptase-a study of the European Academy of Allergology and Clinical Immunology Interest Group on Insect Venom Hypersensitivity. J Allergy Clin Immunol 2009; 124: 1047–1054. 24 Akin C, Valent P. Diagnostic criteria and classification of mastocytosis in 2014. Immunol Allergy Clin North Am 2014; 34: 207–218. 25 Akin C, Schwartz LB, Kitoh T, Obayashi H, Worobec AS, Scott LM et al. Soluble stem cell factor receptor (CD117) and IL-2 receptor alpha chain (CD25) levels in the plasma of patients with mastocytosis: relationships to disease severity and bone marrow pathology. Blood 2000; 96: 1267–1273. 26 Pardanani A, Finke C, Abdelrahman RA, Lasho TL, Hanson CA, Tefferi A. Increased circulating IL-2Ralpha (CD25) predicts poor outcome in both indolent and aggressive forms of mastocytosis: a comprehensive cytokine-phenotype study. Leukemia 2013; 27: 1430–1433. 27 Theoharides TC, Boucher W, Spear K. Serum interleukin-6 reflects disease severity and osteoporosis in mastocytosis patients. Int Arch Allergy Immunol 2002; 128: 344–350. 28 Rabenhorst A, Christopeit B, Leja S, Gerbaulet A, Kleiner S, Forster A et al. Serum levels of bone cytokines are increased in indolent systemic mastocytosis associated with osteopenia or osteoporosis. J Allergy Clin Immunol 2013; 132: 1234–7 e7. 29 Nardelli B, Tiffany HL, Bong GW, Yourey PA, Morahan DK, Li Y et al. Characterization of the signal transduction pathway activated in human monocytes and dendritic cells by MPIF-1, a specific ligand for CC chemokine receptor 1. J Immunol 1999; 162: 435–444. 30 Sims JE, Smith DE. The IL-1 family: regulators of immunity. Nat Rev Immunol 2010; 10: 89–102. 31 Essabbani A, Garcia L, Zonetti MJ, Fisco T, Pucci S, Chiocchia G. Exon-skipping strategy by ratio modulation between cytoprotective versus pro-apoptotic clusterin forms increased sensitivity of LNCaP to cell death. PLoS One 2013; 8: e54920. 32 Trougakos IP, Djeu JY, Gonos ES, Boothman DA. Advances and challenges in basic and translational research on clusterin. Cancer Res 2009; 69: 403–406.
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