Ann Hematol (2014) 93:107–111 DOI 10.1007/s00277-013-1942-7
ORIGINAL ARTICLE
Differential humoral responses against heat-shock proteins after autologous stem cell transplantation in multiple myeloma Natalia Tovar & Carlos Fernández de Larrea & Fabiola Pedrosa & Juan I. Aróstegui & Ma. Teresa Cibeira & Laura Rosiñol & Montserrat Elena & Xavier Filella & Jordi Yagüe & Joan Bladé
Received: 19 July 2013 / Accepted: 15 October 2013 / Published online: 12 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Heat-shock proteins (HSP) are important molecules in the pathogenesis of multiple myeloma (MM). Their blockages by drugs or cellular immune response have been investigated, and a possible association with the presence of oligoclonal bands (OB) has been postulated in patients with MM after allogenic stem cell transplantation. The aim of the present study was to ascertain the serum antibody levels against three HSP (60, 70 and 90) by ELISA in patients with MM in complete remission after autologous stem cell transplantation (ASCT), with or without OB, and compare them with those patients with stable gammopathy of undetermined significance (MGUS) and healthy controls. Our results in samples after ASCT showed no differential levels of anti-HSP according to the presence or absence of Natalia Tovar and Carlos Fernández de Larrea equally contributed in this article N. Tovar : C. Fernández de Larrea : F. Pedrosa : M. T. Cibeira : L. Rosiñol : J. Bladé Department of Hematology, Amyloidosis and Myeloma Unit, Hospital Clínic, Barcelona, Institut d’Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain J. I. Aróstegui : J. Yagüe Department of Immunology, Amyloidosis and Myeloma Unit, Hospital Clínic, Barcelona, Institut d’Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain M. Elena : X. Filella Department of Biochemistry, Amyloidosis and Myeloma Unit, Hospital Clínic, Barcelona, Institut d’Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain J. Bladé (*) Servei d’Hematologia, Hospital Clínic de Barcelona, Villarroel 170, 08036 Barcelona, Spain e-mail: [email protected]
the oligoclonal response. However, higher levels of antiHSP90 were found in patients with stable MGUS in comparison with MM patients (p =0.004). In the same line, a longer progression-free survival was observed in those patients who presented higher anti-HSP90 levels after ASCT (p =0.042). These results suggest, for first time, the potential of anti-HSP90 humoral immune response for long-term control of malignant plasma cell disorders. Keywords Heat-shock proteins . Multiple myeloma . Autologous stem cell transplantation . Oligoclonal bands
Introduction The achievement of complete remission (CR) is the crucial step for a long-lasting response and prolonged survival after autologous stem cell transplantation (ASCT) in patients with multiple myeloma (MM) [1, 2] The emergence of an oligoclonal humoral response, different from the original malignant paraprotein, is a well-recognized event after ASCT in MM [3]. This humoral response has been associated with a favourable prognosis, probably due to a more durable immune reconstitution [4]. However, the precise mechanism associated with the emergence of these oligoclonal bands (OB) as well as their precise prognostic significance is still largely unknown [5]. It has been recently described that the immunoglobulins of these oligoclonal bands interacted with different heat-shock proteins (HSP) on the surface of malignant plasma cell [6]. These proteins become thus accessible to the immune system after recurrent and aberrant membrane exposition, and the response against HSP may contribute to the more favourable survival impact of patients with OB. The aim of the present study was to determine the specific prognostic role of the levels of serum antibodies against three HSP (HSP60, HSP70 and HSP90) in patients with MM in CR after ASCT.
108
Materials and methods We collected serum obtained from 76 individuals: 37 from patients with MM in CR after melphalan-based ASCT, 19 from patients with MM at diagnosis, 8 cases with stable monoclonal gammopathy of undetermined significance (MGUS) for more than 5 years with yearly evaluation at our institution and 12 healthy controls. The definition of CR was based on the European Blood and Marrow Transplantation (EBMT) group criteria requiring a negative immunofixation electrophoresis (IFE) in serum and urine for the original paraprotein, and the absence of increased bone marrow plasma cells [7]. An OB response was defined as the presence of a serum and/or urine IFE monoclonal spike different from the original myeloma protein either in heavy and/or light chains as well as at IFE migration pattern. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (Ethics Committee of Hospital Clínic of Barcelona) and with the Helsinki Declaration of 1975, as revised in 2008. Informed consent was obtained from all patients for being included in the study. We employed a commercial ELISA to measure the circulating levels of anti-HSP60 and anti-HSP70 antibodies (anti-HSP60 and anti-HSP70 IgG/A/M ELISA Kit; Enzo®, Farmingdale, NY, USA). Briefly, 100 μL of 1:500 diluted serum samples was added to a well in a pre-coated plate with either HSP60 or HSP70 and left for 2 h in constant lowintensity agitation followed by four sequential wash. After, 100 μL of horseradish peroxidase conjugated polyclonal antibody specific for human IgA/IgG/IgM antibodies was added (1:5,000 diluted in blocking solution) and left 1 h in constant low-intensity agitation. A second wash was performed previous to incubation with 100 μL of tetramethyl benzidine (TMB) as substrate for 20 min, followed by stop solution (all reactives from Life Technologies ®, Paiseley, Scotland/UK) and reading in a spectrophotometer at 450 nm. Values were expressed in optical density (OD). The anti-HSP90 assay was developed and standardized at our laboratory based on the technical principles and similar reagents described in previous reports [8, 9] and validated in a healthy control population. One hundred microlitres of human HSP90 (Merck®, Whitehouse Station, NJ, USA) with 50 ng/ well concentration was incubated in blocking solution (Life Technologies ® Paiseley, Scotland/UK) overnight at 20 °C. Two hundred microlitres of this blocking solution was added and mixed for 1 h in constant low-intensity agitation. The ELISA plate (96-well RIA/EIA plate, flat bottom, medium binding; Corning®, NY, USA) was then washed and mixed with 100 μL of 1:50 diluted serum sample with blocking solution for 2 h at 37 °C. After a second wash, 100 μL of
Ann Hematol (2014) 93:107–111
anti-human polyvalent immunoglobulin (IgG, IgA, IgM) peroxidase antibodies (Sigma-Aldrich®, MO, USA; diluted 1:1,000 in blocking solution) was placed in each well and left for 1 h at 37 °C. Finally, 100 μL of TMB substrate (Life Technologies ® Paiseley, Scotland/UK) was added after a third wash and left for 30 min before adding stop solution (Life Technologies ® Paiseley, Scotland/UK) and reading in spectrophotometer at 450 nm. Statistical analyses were performed with PAWS statistics software 18.0 for Windows® and GraphPad 5. Mann– Whitney and Kruskal–Wallis tests were used to compare differential levels of the proteins across the groups. Progression-free survival (PFS) was defined as survival from ASCT until relapse or dead from any cause. Overall survival (OS) was calculated from the time of ASCT to the last followup or death. Cox proportional hazard models were used to estimate significance of anti-HSP levels with PFS and OS. Survival probabilities were estimated using the Kaplan–Meier method and analysed by means of the log-rank test. Statistical significance was defined as p ≤0.05.
Results Thirty-seven patients with MM in CR were analysed (14 males/23 females; median age, 56 years; range, 25–66). Patients underwent ASCT after one (32) or two (4) induction regimens, based on combinations of polychemotherapy and glucocorticoids (19), bortezomib (12) and thalidomide (6). One patient received a high-dose therapy, followed by autologous peripheral blood stem cells, with no induction therapy. Thirty (81 %) patients showed oligoclonal bands in serum and/or urine after ASCT that lasted for a median of 2 years (range, 2 months to 9 years). PFS and OS at 5 years in this group were 69 and 88 %, respectively. Levels of antibodies against HSP 60, 70 and 90 were not significantly different in patients with MM at diagnosis versus at CR. Only a trend was found in serum anti-HSP60 measurements (median values, 0.13 vs. 0.2 OD, respectively; p =0.086). In the 37 patients in CR after ASCT, we also found no difference among all three measured antibody levels according to the presence or absence of OB, except for a trend towards anti-HSP70 (p =0.116) (Fig. 1). There were also no differences regarding initial clinical and biological characteristics and in the induction treatment received with or without novel agents between the two groups. There was only a trend for lower bone marrow plasma cell infiltration at diagnosis (28.8 vs. 18 %; p =0.057) in the group with higher anti-HSP90 levels.
Ann Hematol (2014) 93:107–111
109
a p=0.505
p=0.004
b
Fig. 2 Serum levels of total anti-HSP90 in patients with MM in CR or at diagnosis, stable MGUS and healthy controls
p=0.116
When data from healthy controls and patients with MGUS were included in the analysis, anti-HSP90 antibodies showed higher levels in patients with MGUS than in patients with MM (p =0.004; Fig. 2) and healthy controls (p =0.007). Overall, asymptomatic individuals (MGUS and controls) had significantly higher levels of anti-HSP90 than MM patients, whenever the samples were obtained at diagnosis or at the time of CR (p =0.007).
anti-HSP90≥0.23
c p=0.373
anti-HSP90
ORIGINAL ARTICLE
Differential humoral responses against heat-shock proteins after autologous stem cell transplantation in multiple myeloma Natalia Tovar & Carlos Fernández de Larrea & Fabiola Pedrosa & Juan I. Aróstegui & Ma. Teresa Cibeira & Laura Rosiñol & Montserrat Elena & Xavier Filella & Jordi Yagüe & Joan Bladé
Received: 19 July 2013 / Accepted: 15 October 2013 / Published online: 12 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Heat-shock proteins (HSP) are important molecules in the pathogenesis of multiple myeloma (MM). Their blockages by drugs or cellular immune response have been investigated, and a possible association with the presence of oligoclonal bands (OB) has been postulated in patients with MM after allogenic stem cell transplantation. The aim of the present study was to ascertain the serum antibody levels against three HSP (60, 70 and 90) by ELISA in patients with MM in complete remission after autologous stem cell transplantation (ASCT), with or without OB, and compare them with those patients with stable gammopathy of undetermined significance (MGUS) and healthy controls. Our results in samples after ASCT showed no differential levels of anti-HSP according to the presence or absence of Natalia Tovar and Carlos Fernández de Larrea equally contributed in this article N. Tovar : C. Fernández de Larrea : F. Pedrosa : M. T. Cibeira : L. Rosiñol : J. Bladé Department of Hematology, Amyloidosis and Myeloma Unit, Hospital Clínic, Barcelona, Institut d’Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain J. I. Aróstegui : J. Yagüe Department of Immunology, Amyloidosis and Myeloma Unit, Hospital Clínic, Barcelona, Institut d’Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain M. Elena : X. Filella Department of Biochemistry, Amyloidosis and Myeloma Unit, Hospital Clínic, Barcelona, Institut d’Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain J. Bladé (*) Servei d’Hematologia, Hospital Clínic de Barcelona, Villarroel 170, 08036 Barcelona, Spain e-mail: [email protected]
the oligoclonal response. However, higher levels of antiHSP90 were found in patients with stable MGUS in comparison with MM patients (p =0.004). In the same line, a longer progression-free survival was observed in those patients who presented higher anti-HSP90 levels after ASCT (p =0.042). These results suggest, for first time, the potential of anti-HSP90 humoral immune response for long-term control of malignant plasma cell disorders. Keywords Heat-shock proteins . Multiple myeloma . Autologous stem cell transplantation . Oligoclonal bands
Introduction The achievement of complete remission (CR) is the crucial step for a long-lasting response and prolonged survival after autologous stem cell transplantation (ASCT) in patients with multiple myeloma (MM) [1, 2] The emergence of an oligoclonal humoral response, different from the original malignant paraprotein, is a well-recognized event after ASCT in MM [3]. This humoral response has been associated with a favourable prognosis, probably due to a more durable immune reconstitution [4]. However, the precise mechanism associated with the emergence of these oligoclonal bands (OB) as well as their precise prognostic significance is still largely unknown [5]. It has been recently described that the immunoglobulins of these oligoclonal bands interacted with different heat-shock proteins (HSP) on the surface of malignant plasma cell [6]. These proteins become thus accessible to the immune system after recurrent and aberrant membrane exposition, and the response against HSP may contribute to the more favourable survival impact of patients with OB. The aim of the present study was to determine the specific prognostic role of the levels of serum antibodies against three HSP (HSP60, HSP70 and HSP90) in patients with MM in CR after ASCT.
108
Materials and methods We collected serum obtained from 76 individuals: 37 from patients with MM in CR after melphalan-based ASCT, 19 from patients with MM at diagnosis, 8 cases with stable monoclonal gammopathy of undetermined significance (MGUS) for more than 5 years with yearly evaluation at our institution and 12 healthy controls. The definition of CR was based on the European Blood and Marrow Transplantation (EBMT) group criteria requiring a negative immunofixation electrophoresis (IFE) in serum and urine for the original paraprotein, and the absence of increased bone marrow plasma cells [7]. An OB response was defined as the presence of a serum and/or urine IFE monoclonal spike different from the original myeloma protein either in heavy and/or light chains as well as at IFE migration pattern. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (Ethics Committee of Hospital Clínic of Barcelona) and with the Helsinki Declaration of 1975, as revised in 2008. Informed consent was obtained from all patients for being included in the study. We employed a commercial ELISA to measure the circulating levels of anti-HSP60 and anti-HSP70 antibodies (anti-HSP60 and anti-HSP70 IgG/A/M ELISA Kit; Enzo®, Farmingdale, NY, USA). Briefly, 100 μL of 1:500 diluted serum samples was added to a well in a pre-coated plate with either HSP60 or HSP70 and left for 2 h in constant lowintensity agitation followed by four sequential wash. After, 100 μL of horseradish peroxidase conjugated polyclonal antibody specific for human IgA/IgG/IgM antibodies was added (1:5,000 diluted in blocking solution) and left 1 h in constant low-intensity agitation. A second wash was performed previous to incubation with 100 μL of tetramethyl benzidine (TMB) as substrate for 20 min, followed by stop solution (all reactives from Life Technologies ®, Paiseley, Scotland/UK) and reading in a spectrophotometer at 450 nm. Values were expressed in optical density (OD). The anti-HSP90 assay was developed and standardized at our laboratory based on the technical principles and similar reagents described in previous reports [8, 9] and validated in a healthy control population. One hundred microlitres of human HSP90 (Merck®, Whitehouse Station, NJ, USA) with 50 ng/ well concentration was incubated in blocking solution (Life Technologies ® Paiseley, Scotland/UK) overnight at 20 °C. Two hundred microlitres of this blocking solution was added and mixed for 1 h in constant low-intensity agitation. The ELISA plate (96-well RIA/EIA plate, flat bottom, medium binding; Corning®, NY, USA) was then washed and mixed with 100 μL of 1:50 diluted serum sample with blocking solution for 2 h at 37 °C. After a second wash, 100 μL of
Ann Hematol (2014) 93:107–111
anti-human polyvalent immunoglobulin (IgG, IgA, IgM) peroxidase antibodies (Sigma-Aldrich®, MO, USA; diluted 1:1,000 in blocking solution) was placed in each well and left for 1 h at 37 °C. Finally, 100 μL of TMB substrate (Life Technologies ® Paiseley, Scotland/UK) was added after a third wash and left for 30 min before adding stop solution (Life Technologies ® Paiseley, Scotland/UK) and reading in spectrophotometer at 450 nm. Statistical analyses were performed with PAWS statistics software 18.0 for Windows® and GraphPad 5. Mann– Whitney and Kruskal–Wallis tests were used to compare differential levels of the proteins across the groups. Progression-free survival (PFS) was defined as survival from ASCT until relapse or dead from any cause. Overall survival (OS) was calculated from the time of ASCT to the last followup or death. Cox proportional hazard models were used to estimate significance of anti-HSP levels with PFS and OS. Survival probabilities were estimated using the Kaplan–Meier method and analysed by means of the log-rank test. Statistical significance was defined as p ≤0.05.
Results Thirty-seven patients with MM in CR were analysed (14 males/23 females; median age, 56 years; range, 25–66). Patients underwent ASCT after one (32) or two (4) induction regimens, based on combinations of polychemotherapy and glucocorticoids (19), bortezomib (12) and thalidomide (6). One patient received a high-dose therapy, followed by autologous peripheral blood stem cells, with no induction therapy. Thirty (81 %) patients showed oligoclonal bands in serum and/or urine after ASCT that lasted for a median of 2 years (range, 2 months to 9 years). PFS and OS at 5 years in this group were 69 and 88 %, respectively. Levels of antibodies against HSP 60, 70 and 90 were not significantly different in patients with MM at diagnosis versus at CR. Only a trend was found in serum anti-HSP60 measurements (median values, 0.13 vs. 0.2 OD, respectively; p =0.086). In the 37 patients in CR after ASCT, we also found no difference among all three measured antibody levels according to the presence or absence of OB, except for a trend towards anti-HSP70 (p =0.116) (Fig. 1). There were also no differences regarding initial clinical and biological characteristics and in the induction treatment received with or without novel agents between the two groups. There was only a trend for lower bone marrow plasma cell infiltration at diagnosis (28.8 vs. 18 %; p =0.057) in the group with higher anti-HSP90 levels.
Ann Hematol (2014) 93:107–111
109
a p=0.505
p=0.004
b
Fig. 2 Serum levels of total anti-HSP90 in patients with MM in CR or at diagnosis, stable MGUS and healthy controls
p=0.116
When data from healthy controls and patients with MGUS were included in the analysis, anti-HSP90 antibodies showed higher levels in patients with MGUS than in patients with MM (p =0.004; Fig. 2) and healthy controls (p =0.007). Overall, asymptomatic individuals (MGUS and controls) had significantly higher levels of anti-HSP90 than MM patients, whenever the samples were obtained at diagnosis or at the time of CR (p =0.007).
anti-HSP90≥0.23
c p=0.373
anti-HSP90
