Int J Hematol DOI 10.1007/s12185-015-1775-3

CASE REPORT

Successful treatment of refractory cold hemagglutinemia in MYD88 L265P mutation‑negative Waldenström’s macroglobulinemia with bortezomib Mayuko Izumi1 · Hiroko Tsunemine1 · Yasuhiro Suzuki2 · Akihiro Tomita2 · Toshiko Kusumoto3 · Taiichi Kodaka1 · Kiminari Itoh1 · Takayuki Takahashi1 

Received: 18 August 2014 / Revised: 27 February 2015 / Accepted: 2 March 2015 © The Japanese Society of Hematology 2015

Abstract  We report here the successful treatment of cold agglutinin-associated refractory hemolysis with bortezomib in a patient with Waldenström’s macroglobulinemia (WM). A 78-year-old man was referred to our hospital with cold hemagglutinemia of unknown cause. Laboratory examination revealed a hemoglobin concentration of 6.9 g/dL, serum IgM concentration of 1904 mg/dL, and a titer of cold hemagglutinin of over ×8192. Serum immunoelectrophoresis demonstrated monoclonal protein of the IgM-κ type. A bone marrow aspirate showed many lymphoplasmacytic cells, which were positive for CD19, CD20, CD38, and cytoplasmic μ and κ light chains. A diagnosis of WMassociated cold hemagglutinemia was made. Because of red blood cell transfusion-dependency, we treated him with intravenous fludarabine, oral melphalan–prednisolone, cyclophosphamide, and melphalan, and two courses of R-CHOP in sequence with a marked decrease of serum IgM (928 mg). We then started weekly bortezomib plus dexamethasone (BD) therapy, as he was still transfusiondependent. Soon after the initiation of BD, he achieved transfusion independence, with a further decrease in serum levels of IgM and marked improvement of anemia. Interestingly, his marrow abnormal lymphocytes were later found not to carry the MYD88 L265P mutation. The successful

* Takayuki Takahashi [email protected] 1

Department of Hematology, Shinko Hospital, 4‑47 Wakihama‑cho, 1‑chome, Chuo‑ku, Kobe 651‑0072, Japan

2

Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan

3

Department of Laboratory Medicine, Kobe Medical Center General Hospital, Kobe, Japan



treatment with bortezomib for WM lacking this mutation is discussed. Keywords  Waldenström’s macroglobulinemia · Cold hemagglutinemia · Hemolytic anemia · MYD88 L265P mutation · Bortezomib

Introduction Waldenström’s macroglobulinemia (WM) is an uncommon lymphoproliferative disorder characterized by the neoplastic proliferation of lymphoplasmacytic cells which produce IgM-type monoclonal protein [1]. Histopathologically, WM is classified as an IgM-producing subtype of lymphoplasmacytic lymphoma (LPL) [2], and it mainly affects the bone marrow, spleen, and liver. Asymptomatic patients with WM do not always require chemotherapeutic intervention because of its slow growth and indolent clinical picture. In contrast, we should consider chemotherapies for symptomatic WM patients, because their median survival is 5–6 years without treatment [3]. One of the symptoms or complications which require therapeutic intervention is cold hemagglutinemia, in which IgM M-protein has cold hemagglutinin activity as the antibody for the I or i antigen of red blood cells (RBC), although this complication is rare in WM [4–6]. As the therapeutic modality for WM, purine analogs (PA) such as fludarabine with or without rituximab have been recommended [3, 7, 8]. In recent years, R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone) as the standard regimen for B cell lymphomas and novel agents for multiple myeloma, such as bortezomib, thalidomide, or lenalidomide, have been employed for the treatment of WM [9, 10]. More recently,

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BDR therapy (bortezomib, dexamethasone, and rituximab) was reported to be highly effective for WM [11]. We encountered a red cell transfusion-dependent WM patient in whom M-protein exhibited potent cold hemagglutinin activity. To achieve RBC transfusion-independency, we attempted to reduce the number of WM lymphoplasmacytic cells and the amount of IgM M-protein in this patient. Standard treatments including fludarabine failed to improve the cold hemagglutinemia; however, the combination with bortezomib and dexamethasone (BD) improved the hemolysis and freed the patient from RBC transfusion. To our knowledge, there has been only one report of successful BD therapy for WM complicated by cold hemagglutinemia [12]. Interestingly, his marrow abnormal lymphocytes later revealed not to carry the MYD88 L265P mutation. The successful treatment with bortezomib for WM without this mutation may be novel and the mechanism of the efficacy is discussed.

Case report A 78-year-old man was referred to our hospital because of exertional dyspnea and general fatigue. As past history, he had had pulmonary tuberculosis and undergone partial gastrectomy because of a gastric ulcer at the ages of 28 and 43, respectively. In addition, he routinely visited a clinic for aortic valve insufficiency. In February 2009, he was diagnosed with idiopathic cold hemagglutinemia in another hospital based on hemolytic anemia with a hemoglobin (Hb) concentration of 8.9 g/dL and a high titer of cold hemagglutinin of over ×8192, without subsequent specific treatment. He did not have numbness or cyanosis of the distal extremities on exposure to cold. He was referred to our hospital because of the exacerbation of hemolytic anemia in November 2011. Physically, the spleen was palpable 3 cm below the left costal margin, and mild pretibial pitting edema was observed. Neurological examination was unremarkable. Hematologic tests revealed a white blood cell (WBC) count of 5.8 × 109/L with 57.6 % neutrophils, 0.8 % eosinophils, 0.8 % basophils, 5.2 % monocytes, and 35.6 % lymphocytes, a hemoglobin concentration of 6.9 g/dL, a reticulocyte percentage of 13.4 %, and a platelet count of 308 × 109/L. Serum serological and biochemical examinations showed that the IgM concentration had increased to 1904 mg/dL, and an M-peak was observed on serum electrophoresis, which was revealed to be the IgM-κ type by serum immunoelectrophoresis. However, serum concentrations of IgG and IgA had decreased to 278 mg/dL (normally 870–1700 mg) and 43 mg/dL (normally 110–410 mg/dL), respectively. The titer of cold hemagglutinin was over ×8192. Serum haptoglobin concentration was decreased to below the

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detection limit. A bone marrow aspirate showed many small lymphoplasmacytic cells comprising 20.8 % of nucleated marrow cells. Regarding pathological immunostaining, we could not perform this, because we did not make paraffin-embedded bone marrow clot preparation; however, bone marrow involvement was characterized by a diffuse infiltrate composed predominantly of smallsized lymphoplasmacytic cells (data not shown). Flow cytometric analysis showed that these lymphoplasmacytic cells were positive for CD19 (81.9 %), CD20 (63.9 %), cyκ-light chain (76.7 %), and cyλ-light chain (22.7 %) in CD38-positive cells. This analysis was performed in SRL (Hachioji, Tokyo, Japan) and was done based on CD38gated cells; therefore, we performed again this analysis by CD45-gating in our institution to obtain further phenotypic information using cryopreserved bone marrow mononuclear cells collected at the same occasion. As shown in Fig.  1, marrow abnormal lymphocytes were positive for CD19 (58.6 %), CD20 (65.7 %), CD38 (44.4 % in cyIgMpositive cells), cyIgM (58.6 %), and cyκ-chain (56.4 %), but negative for CD5, CD10, CD56 (data not shown), CD138 (data not shown), and cyλ-chain in CD45-positive cells. Multiplex PCR analysis of bone marrow cells demonstrated monoclonal rearranged band of immunoglobulin heavy chain but not that of T-cell receptor γ-chain genes (data not shown). From these findings, a diagnosis of WM complicated by cold hemagglutinemia was made. We conducted chemotherapies for WM itself, because of the RBC transfusion-dependency of this patient. The clinical course is shown in Fig. 2a. Firstly, we treated him with 2 courses of intravenous fludarabine (25 mg/body, for 5 days) from February 2011, without the improvement of hemolytic anemia or reduction of serum IgM levels, although the spleen became impalpable. Next, we treated him in sequence with MP chemotherapy from January 2012, oral cyclophosphamide (50 mg/day, daily) from May 2012, oral melphalan (2 mg/day, daily) from August 2012, and two courses of R-CHOP (500 mg of rituximab, 500 mg of cyclophosphamide, 50 mg of doxorubicin, 1.4 mg of vincristine, and 100 mg of oral prednisolone for 5 days) in October 2012, with a marked decrease of serum IgM levels (around 950 mg/dL). Although the frequency of RBC transfusion was decreased, he was still transfusion dependent, presumably due to potent cold hemagglutinin activity of the IgM M-protein; indeed, the titer of serum cold hemagglutinin was still over ×8192 after the series of chemotherapies (Fig. 2a). As for the R-CHOP, this procedure caused significant myelosuppression (WBC 0.9 × 109/L, platelet 68 × 109/L, hemoglobin 5.0 g/dL) and peripheral neuropathy; therefore, the patient did not agree to receive further courses of R-CHOP, despite CD20 expression by marrow lymphoplasmacytic cells after 2 courses of this chemotherapy (data not shown). Regarding sole rituximab

Successful treatment of refractory cold hemagglutinemia

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therapy, we considered not to employ this agent, because 2 courses of R-CHOP did not make the patient free from RBC transfusion. At the same time, L265P somatic mutation in MYD88, which is an important mediator in signal transduction from Toll-like receptor to NF-κB, was identified and demonstrated to be a constitutional stimulator for NF-κB in WM [13]. Based on this investigation, we thought that bortezomib could be a potent therapeutic agent for WM through its activity against NF-κB. However, bortezomib was not available for WM under the Japanese health insurance system. Therefore, we obtained written informed consent from the patient, and treatment with bortezomib was approved by the review board of Shinko Hospital. We started to treat him with weekly BD therapy (subcutaneous infusion of 1.8 mg of bortezomib and 12 mg of oral dexamethasone) in February 2013. As shown in Fig. 2b, he became free from RBC transfusion soon after the initiation of BD therapy, with an increase of the Hb concentration to as high as over 11 g/dL and decrease of the serum IgM concentration to as low as below 700 mg/dL from June 2013. Serum concentrations of LDH and indirect bilirubin were also decreased and maintained around normal levels (normal levels: below 230 IU/L and 0.9 mg/dL, respectively), while the titer of cold hemagglutinin remained at over ×8192 with persisting minimum hemolysis. We, therefore, performed the

thermal amplitude test according to the method by Dacie [14] in October 2014, without agglutination activity at temperatures over 24 °C (data not shown), presumably due to reduced hemolytic activity of the cold agglutinin after successful bortezomib treatment. Interestingly, the Hb concentration decreased to below 9 g/dL with the mild elevation of indirect bilirubin from November 2013, which then recovered to 11–12 g/dL from March 2014 (data not shown), indicating the exacerbation of cold hemagglutinemia in the cold season. Currently (December 2014), he shows no adverse effects of BD therapy such as neuropathy of the extremities. Because bortezomib brought about marked improvement of both cold agglutinin disease and WM, we examined the presence or absence of the MYD88 L265P somatic mutation in bone marrow lymphoplasmacytic cells using cryopreserved marrow mononuclear cells (collected in November 2011) to verify the exact mechanism of the efficacy of this agent. As shown in Fig. 3, the MYD88 L265P mutation was absent in the present patient in accordance with a study describing that cold agglutinin-associated B cell lymphoproliferative disease lacks this mutation [15]. The MYD88 L265P mutation was found in about 90 % WM/lymphoplasmacytic lymphoma (LPL) [13] but, of interest, it was not found in almost all cases of LPL with IgM cold agglutinin such as our case [15]. The absence of the MYD88 L265P

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Fig. 2  Clinical course of the present patient. a Clinical course of the patient from December 2011 to January 2013. MP oral melphalan and prednisolone, R-CHOP: rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone, CHAT: cold hemagglutinin. The

titer of CHAT was over ×8192 throughout the course. b The clinical course from February 2013 to June 2013. BD: bortezomib and dexamethasone

mutation suggests that cold agglutinin-associated LPL is distinct from typical LPL. In addition, CXCR4 mutation which is observed in about one-third of WM patient [16] was also negative in the present patient (Fig. 3).

hemagglutinin, with intravenous fludarabine, then with MP, oral cyclophosphamide, and oral melphalan, which are employed for the treatment of multiple myeloma, with insufficient effects for the control of M-protein. Next, we treated the patient with 2 courses of R-CHOP, with a moderate reduction of M-protein; however, the patient did not become free from RBC transfusion. As the next option, we treated the patient with bortezomib based on the rationale that this agent could be a causal treatment for WM [13] with a marked reduction of M-protein and the patient

Discussion In the present patient, we first tried to reduce the amount of IgM-type M-protein, which shows potent activity of cold

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Successful treatment of refractory cold hemagglutinemia Fig. 3  Analysis of MYD88 L265P and CXCR4 mutations on cryopreserved bone marrow mononuclear cells collected in November 2011. The analysis was performed by Sanger sequencing [26] in the laboratory of Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan. The marrow mononuclear cells of the present patient did not carry the MYD88 L265P and CXCR4 mutations. Circulating mononuclear cells collected in October 2014 was used as negative control

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finally became free of RBC transfusion. This clinical course clearly shows the definite effect of bortezomib as the WM-molecular targeting agent. In recent years, bortezomib has been recommended as one of the principal agents for WM by a number of therapeutic guidelines [17–20]. The rationale of the recommendation may have been based on the close relationship between WM and multiple myeloma, and the recommendation was theoretically supported by MYD88 L265P somatic mutation in WM [13] in 2012. However, recently, Randen et al. reported that cold agglutinin-associated B cell lymphoproliferative disease lacks the MYD88 L265P mutation [15]. The neoplastic lymphocytes of the present patient subsequently revealed not to carry this mutation. Three mechanisms regarding the efficacy of bortezomib in the present patient could be possible. First, activated signaling from B cell receptor (BCR) to NF-κB, which is typically observed in chronic lymphocytic leukemia [21], may be enhancing the neoplastic proliferation of lymphoplasmacytic cells. Second, constitutive activation of Bruton tyrosine kinase (BTK) on the way of signaling from BCR to NF-κB [22], which is typically observed in mantle cell lymphoma, may be acting. In these 2 mechanisms, bortezomib may have acted as proteasome inhibitor and subsequently caused NF-κB inhibition in the present patient. As the third possibility, bortezomib may have acted as histone deacetylase (HDAC) inhibitor as reported in the treatment of multiple myeloma [23]. Although the successful treatment of WM without the MYD88 L265P mutation appears to be novel, its precise mechanism should be elucidated and more effective treatment established in future, because Randen et al. stated that hemolysis in cold agglutinin-associated lymphoproliferative disease is refractory and about 50 % of such patients become transfusion-dependent [15].

In the present patient, the titer of cold hemagglutinin remains at over ×8192 regardless of the decrease of serum IgM and improvement of hemolysis (Fig. 2a, b). Although the cold hemagglutinin assay system employed in our hospital is incapable of showing the real quantity of the cold hemagglutinin titer when it exceeds ×8192, the titer may have significantly decreased in the high area over ×8192, considering the improvement of hemolysis and anemia in the present patient. Among B cell neoplasms which produce IgM M-protein with an activity of cold hemagglutinin, the κ-light chain of the M-protein predominates in WM [4]. Furthermore, the cold hemagglutinin mostly directs to I antigen on red blood cells and exhibits potent hemolytic activity, when the light chain is of the κ type [4]. The potent cold hemagglutinin activity in the present patient appears to be attributable to the κ-light chain of M-protein. On the other hand, the λ-light chain predominates in malignant lymphomas other than WM and chronic lymphocytic leukemia, and appears to direct to antigens other than I antigen on red blood cells [4]. The thermal amplitude in the present patient before treatment is unclear, because we performed this test in the remission period of cold agglutinin disease. In addition, recently, Shi et al. reported that a mouse monoclonal antibody against complement C1s inhibits in vitro the induction of hemolysis by cold agglutinins [24]. This immunotherapy would be a potent tool to treat refractory cold hemagglutinin disease apart from the control of WM. Furthermore, Tanaka et al. [25] reported successful treatment of idiopathic cold agglutinin disease with rituximab in a patient who developed later lymphoplasmacytic lymphoma. The present patient, however, did not achieve a complete remission of WM, but did achieve a partial one of the BD therapy regardless of being freed from RBC transfusion.

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Considering the possible mechanism of successful bortezomib treatment stated above, additional targeting of signaling from BCR to NF-κB would bring about a better condition. Among molecular targeting agents, BTK inhibitor [22] may be promising to obtain a complete or near complete remission of both cold hemagglutinin disease and WM in the present patient. Conflict of interest  The authors disclose that they have no conflicts of interest with any individuals or companies.

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