References
1. Pepys MB. Amyloidosis. Annu Rev Med. 2006;57:223-241.
2. Benson MD, Uemichi T. Transthyretin amyloidosis. Amyloid: Int J Exp Clin Invest. 1996;3:44-56.
3. Merlini G, Seldin DC, Gertz MA. Amyloidosis: pathogenesis and new therapeutic options. J Clin Oncol. 2011;29(14):1924-1933.
4. Picken MM. Amyloidosis-where are we now and where are we heading? Arch Pathol Lab Med. 2010;134(4):545-551.
5. Picken MM, Dogan A, Herrera AG, et al. Amyloid Typing: Experience from a Large Referral Centre. Amyloid and Related Disorders. Totowa, NJ: Humana Press; 2012:231-238.
6. Loo D, Mollee PN, Renaut P, Hill, MM, Proteomics in molecular diagnosis: typing of amyloidosis. J Biomed Biotechnol. 2011;2011:754109.
7. Hawkins PN, Lavender JP, Pepys MB. Evaluation of systemic amyloidosis by scintigraphy with123I-labeled serum amyloid P component. N Engl J Med. 1990; 323(8):508-513.
8. Rapezzi C, Quarta CC, Guidalotti PL, et al. Usefulness and limitations of 99mTc-3,3-diphosphono-1,2-propanodicarboxylic acid scintigraphy in the aetiological diagnosis of amyloidotic cardiomyopathy. Eur J Nucl Med Mol Imaging. 2011;38(3):470-478.
9. Bokhari S, Castan˜o A, Pozniakoff T, Deslisle S, Latif F, Maurer MS. (99m)Tc-pyrophosphate scintigraphy for differentiating light-chain cardiac amyloidosis from the transthyretin-related familial and senile cardiac amyloidoses. Circ Cardiovasc Imaging. 2013;6(2):195-201.
© 2014 by The American Society of Hematology
To the editor:
A germ line mutation in cathepsin B points toward a role in asparaginase pharmacokinetics
L-Asparaginase (ASNase) is a key component of protocols used to treat acute lymphoblastic leukemia (ALL). Poorly understood interpatient differences in ASNase pharmacokinetics demand thera-peutic drug monitoring to prevent patients from receiving an inadequate dose.1Although it is unclear whether there is a causal
relation between elevated ASNase levels and toxicities, under-exposure compromises therapeutic benefits. A recent report demon-strated that lysosomal proteases degrade ASNase in vitro.2However, to which extent these proteases contribute to ASNase clearance in patients remains unclear. Here we link a strongly prolonged ASNase turnover to a germ line mutation in the gene encoding cathepsin B.
A pediatric patient, treated for common B-cell progenitor ALL, developed encephalopathy associated with hyperammone-mia (354mmol/L) after the sixth dose of Erwinase. She required plasmapheresis to reduce serum ammonia levels. Serum analysis revealed abnormally high concentrations of Erwinase at the time of plasmapheresis (2167 IU/mL, 2 days after the last dose). After recovery, Erwinase treatment was restarted under therapeutic drug monitoring. This revealed a strongly increased half-life of Erwinase (Figure 1A), which could explain the high Erwinase serum concen-trations (see supplemental Case Report for detailed information; available on the Blood Web site).
We hypothesized that a defect in one of the lysosomal proteases, previously reported to be capable of degrading asparaginase in vitro,2 might be responsible for the prolonged half-life observed in this patient. Sequencing of DNA isolated from peripheral blood mono-nuclear cells (PBMCs) and from buccal cells revealed a hetero-zygous single codon deletion (c.709_711delAAG) in the gene encoding cathepsin B in the germ line of the patient, which is not listed in the Database of Single Nucleotide Polymorphisms (dbSNP; National Center for Biotechnology Information, Bethesda, MD) or in our in-house database containing the exome sequence data of 1154 individuals (Figure 1B). This mutation results in a deletion of a highly conserved lysine residue in the C terminus of the protein (p.K237del), which is predicted3to lead to a loss of structural integrity of the protein (supplemental Figure 1A-B).
Cathepsin B is synthesized as a 44-kDa pre-proenzyme that is processed in late endosomes to a 33-kDa active single chain and matured into an active 2-chain form consisting of a 24-kDa heavy chain (and a 27-kDa glycosylated form) and a 5-kDa light chain.4 We expressed both wild-type and mutant cathepsin B, cloned from RNA extracted from patient PBMCs, in HEK293 cells to follow the maturation process. Biochemical analysis revealed defective maturation
of the mutant cathepsin B, which was confirmed by an aberrant subcellular localization (supplemental Figure 1C-F).
To test whether this mutation affects the protease activity, we assessed cathepsin B activity in Epstein-Barr virus (EBV)– immortalized B cells obtained from the patient. Indeed, cathepsin B activity in the B cells obtained from the patient was strongly reduced as compared with B cells from age-matched donors (Figure 1C), indicating that deletion of residue K237 results in a loss of function. Next, we determined whether the reduced protease activity of the mutant cathepsin B would result in a diminished degradation of ASNase. Therefore, we incubated Erwinase in lysates of cells expressing either wild-type or mutant cathepsin B and analyzed samples taken at the indicated time points for residual ASNase activity (Figure 1D). Mock transfected cells showed limited protease activity and cleared the Erwinase after 20 hours of incubation. Of note, this degradation was inhibited by the addition of a specific cathepsin B inhibitor, CA-074 (color marked data points, Figure 1D), indicating that endogenous cathepsin B is responsible for ASNase degradation in these lysates. Overexpression of the wild-type cathepsin B resulted in a rapid clearance of the Erwinase from the lysate, which again was fully inhibited by the addition of the cathepsin B inhibitor CA-074. Expression of the K237del mutant cathepsin B protein was still capable of degrading ASNase, but the rate of clearance was significantly reduced (P , .05) in com-parison with the wild-type protein, consistent with the delayed ASNase clearance observed in this patient. Western blot analysis of asparaginase incubated in these cell lysates confirmed that degradation of asparaginase protein rather than the inhibition of enzymatic activity causes the decrease in asparaginase activity that we measured in the previous assay (supplemental Figure 1G-H).
It is unknown where degradation of ASNase occurs. Low levels of cathepsin B activity are detected in human serum (data not shown), but these amounts are insufficient to degrade Erwinase or Escherichia coli ASNase in vitro (supplemental Figure 1). Instead, it appears that cathepsin B–mediated degradation of ASNase occurs intracellularly, after being removed from the blood by phagocytic cells, which is consistent with the fact that pegylated forms of this protein show an increased serum half-life.5
Current knowledge of factors influencing ASNase pharmacoki-netics is limited.5,6Only the presence of inhibitory antibodies that bind asparaginase is known to significantly shorten the half-life of ASNase.7In our patient, we found no evidence of an immune response targeting the ASNase. Our observations support the
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hypothesis that the lysosomal protease cathepsin B is an essential component in the regulation of ASNase turnover. Mutations in the cathepsin B gene, as observed in our patient, are rare events. However, because cathepsin B activity is tightly regulated, not only at the level of gene expression, but also by protein maturation and enzyme activity,8,9many additional factors may contribute to
variations in cathepsin B activity and can therefore contribute to the interpatient differences in ASNase kinetics.
Laurens T. van der Meer Laboratory of Pediatric Oncology, Department of Pediatrics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands Esm ´e Waanders Department of Human Genetics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands Marloes Levers Laboratory of Pediatric Oncology, Department of Pediatrics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands Hanka Venselaar Center for Molecular and Biomolecular Informatics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands
Debbie Roeleveld Laboratory of Pediatric Oncology, Department of Pediatrics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands Joachim Boos Department of Pediatric Hematology and Oncology, University Children’s Hospital of M ¨unster, M ¨unster, Germany Claudia Lanvers Department of Pediatric Hematology and Oncology, University Children’s Hospital of M ¨unster, M ¨unster, Germany Roger J. Br ¨uggemann Department of Pharmacy, Radboud University Medical Center, Nijmegen, The Netherlands Roland P. Kuiper Department of Human Genetics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands Peter M. Hoogerbrugge Laboratory of Pediatric Oncology, Department of Pediatrics, Radboud University Medical Center, Nijmegen, The Netherlands Frank N. van Leeuwen Laboratory of Pediatric Oncology, Department of Pediatrics, Radboud Institute for Molecular Life Sciences,
Figure 1. A mutation in the gene encoding cathep-sin B in a patient with strongly reduced metabolism asparaginase. (A) Pharmacokinetics of Erwinase serum levels indicate a strongly delayed metabolism of ASNase in this patient. The half-life (ln2/Kel) of 28.5 hours (normal 7-15 hours5) was calculated using best-fit nonparametric modeling of the data points. (B) DNA sequencing of the cathepsin B gene reveals a heterozygous deletion of a single codon (c.709_711delAAG) in DNA isolated from PBMCs and buccal cells of the patient. (C) Cathepsin B activity in lysates of EBV-transformed B cells of the patient (carrying the mutation) and age-matched con-trols (N5 5) was determined by measuring cleavage of the fluorescent substrate Ac-RR-AFC. The plot shows an average of 2 experiments with standard deviation. One of the control samples was set to 1, and all samples were correlated to this sample. Unpaired 2-tailed t test was used to determine significance. (D) ASNase was incubated in lysate of HEK293 cells expressing wild-type or mutant cathepsin B. After incubation, residual ASNase activity was assayed as described in the supplemental Methods section. Cathepsin inhibitor CA-074 was included in selected samples to confirm the contribution of cathepsin B in this degradation. The plot shows an average of 3 independent experiments with standard error of the mean. Analysis of variance statistical analysis was applied to test for significance.
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Radboud University Medical Center, Nijmegen, The Netherlands D. Maroeska te Loo Pediatric Hemato-Oncology, Department of Pediatrics, Radboud University Medical Center, Nijmegen, The Netherlands F.N.v.L. and D.M.t.L. contributed equally to this study.
The online version of this article contains a data supplement.
Acknowledgments: The authors thank Dr Jeroen Middelbeek for help with microscopy imaging and Simon van Reijmersdal for technical assistance. This work was supported in part by research funding from KiKa (grant #134) (L.T.v.d.M.).
Contribution: L.T.v.d.M. performed experiments, analyzed and interpreted data, and wrote the manuscript; E.W. designed and performed the genomic analysis; M.L. performed research; H.V. performed in silico structure analysis; D.R. performed research; J.B. and C.L. performed drug monitoring and designed research; R.J.B. performed the pharmacokinetic studies; R.P.K. analyzed and interpreted the genomic analysis; P.H. designed the research and interpreted the data; F.N.v.L. and D.M.t.L. designed and supervised the research; and all authors edited and approved the manuscript.
Conflict-of-interest disclosure: J.B. served personally as a consultant and participated in advisory boards and safety boards for different asparaginase-selling companies including EUSA Pharma and former Erwinase license holders, Bayer and Medac GmbH. The laboratory at the University of M ¨unster is also involved in scientific collaborations with EUSA, Medac, and Sigma-Tau, which to some extent contributed financially to the analytical procedures. The remaining authors declare no competing financial interests.
Current affiliation for D.R. is Department of Experimental Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands. Correspondence: Frank N. van Leeuwen, 412 Laboratory of Pediatric Oncology, Department of Pediatrics, Radboud University Medical Centre, PO
Box 9101, 6500 HB Nijmegen, The Netherlands; e-mail: frankn.vanleeuwen@ radboudumc.nl.
References
1. Tong WH, Pieters R, Kaspers GJ, et al. A prospective study on drug monitoring of PEGasparaginase and Erwinia asparaginase and
asparaginase antibodies in pediatric acute lymphoblastic leukemia. Blood. 2014;123(13):2026-2033.
2. Patel N, Krishnan S, Offman MN, et al. A dyad of lymphoblastic lysosomal cysteine proteases degrades the antileukemic drug L-asparaginase. J Clin Invest. 2009;119(7):1964-1973.
3. Venselaar H, Te Beek TA, Kuipers RK, Hekkelman ML, Vriend G. Protein structure analysis of mutations causing inheritable diseases. An e-Science approach with life scientist friendly interfaces. BMC Bioinformatics. 2010; 11:548.
4. Mach L, St ¨uwe K, Hagen A, Ballaun C, Gl ¨ossl J. Proteolytic processing and glycosylation of cathepsin B. The role of the primary structure of the latent precursor and of the carbohydrate moiety for cell-type-specific molecular forms of the enzyme. Biochem J. 1992;282(pt 2):577-582.
5. Asselin BL, Whitin JC, Coppola DJ, Rupp IP, Sallan SE, Cohen HJ. Comparative pharmacokinetic studies of three asparaginase preparations. J Clin Oncol. 1993; 11(9):1780-1786.
6. Avramis VI, Panosyan EH. Pharmacokinetic/pharmacodynamic relationships of asparaginase formulations: the past, the present and recommendations for the future. Clin Pharmacokinet. 2005;44(4):367-393.
7. Liu C, Kawedia JD, Cheng C, et al. Clinical utility and implications of asparaginase antibodies in acute lymphoblastic leukemia. Leukemia. 2012; 26(11):2303-2309.
8. Sloane BF, Moin K, Krepela E, Rozhin J. Cathepsin B and its endogenous inhibitors: the role in tumor malignancy. Cancer Metastasis Rev. 1990;9(4): 333-352.
9. Yan S, Sloane BF. Molecular regulation of human cathepsin B: implication in pathologies. Biol Chem. 2003;384(6):845-854.
© 2014 by The American Society of Hematology
To the editor:
Bendamustine, etoposide, cytarabine, melphalan, and autologous stem cell rescue produce
a 72% 3-year PFS in resistant lymphoma
Based on the results of the PARMA and CORAL studies, high-dose chemotherapy (HDT) followed by autologous stem cell rescue has become the standard of care for patients with relapsed, chemo-sensitive aggressive non-Hodgkin lymphoma (NHL).1,2Moreover, HDT/autologous stem cell rescue is considered the therapy of choice for Hodgkin lymphoma (HL) patients in chemosensitive relapse.3 However, HDT/autologous stem cell rescue is hampered by several major pitfalls, namely the toxicity related to the procedure and the lack of efficacy in chemoresistant patients.
We previously demonstrated the safety of a new HDT regimen with bendamustine, etoposide, cytarabine, and melphalan (BeEAM) prior to autologous stem cell rescue in 43 patients with resistant/ relapsed lymphoma.4The characteristics of patients are reported in the previously published paper.4The study was conducted in accordance with the principles of the Declaration of Helsinki, Good Clinical Practice (ICH-GCP), and the current national guidelines for conducting clinical studies. The protocol was approved by the Institutional Ethics Committee. All subjects gave written informed consent. The study was registered at the European Medicines Agency with the European Clinical Trials Database number 2008-002736-15. Transplant-related mortality was 0%. The cumulative
incidence of infectious complications was approximately 60%, without any serious adverse events (grades 3-4). Furthermore, this regimen showed significant antilymphoma activity, with 80% of patients being in complete remission after transplant.5Disease type (NHL vs HL) and disease status at transplant (chemosensitive vs chemoresistant) were the only statistically significant variables influencing progression-free survival (PFS), whereas disease status at transplant was the only variable affecting overall survival (OS) at the time of writing.4However, the primary objective of the study was to determine the 36-month PFS rate, according to Fleming’s method ([p0]5 40%, [p1] 5 60%, a 5 0.05, and 1-b 5 0.80). At the time of publication, the median follow-up for surviving patients was short (18 months), and therefore, it was not possible either to establish if we had met the primary end point of the study or to draw final conclusions on the efficacy of this regimen.
With this in mind, we updated the follow-up at 41 months after transplant in order to evaluate the midtime efficacy of the BeEAM regimen in terms of PFS and OS. Responses were again evaluated ac-cording to the criteria reported elsewhere by Cheson et al.5At present, 31/43 patients are still in complete remission (72%), as documented by both PET and CT scans. Two patients (4.6%) were refractory to
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doi:10.1182/blood-2014-06-582627
2014 124: 3027-3029
Frank N. van Leeuwen and D. Maroeska te Loo
Joachim Boos, Claudia Lanvers, Roger J. Brüggemann, Roland P. Kuiper, Peter M. Hoogerbrugge,
Laurens T. van der Meer, Esmé Waanders, Marloes Levers, Hanka Venselaar, Debbie Roeleveld,
asparaginase pharmacokinetics
A germ line mutation in cathepsin B points toward a role in
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