Leukemia Research 50 (2016) 78–84
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Research paper
Remarkable expression of vascular endothelial growth factor in bone marrow plasma cells of patients with POEMS syndrome Chen Wang, Xu-Fei Huang, Qian-Qian Cai, Xin-Xin Cao, Hao Cai, Daobin Zhou, Jian Li (MD) ∗ Department of Hematology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
a r t i c l e
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Article history: Received 20 June 2016 Received in revised form 11 August 2016 Accepted 26 September 2016 Available online 26 September 2016 Keywords: POEMS syndrome Vascular endothelial growth factor Bone marrow plasma cell Interleukin-6
a b s t r a c t Vascular endothelial growth factor (VEGF) is pathognomonically elevated in patients with POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes) syndrome. However, its source of overproduction is unclear. As clinical improvement is almost always associated with VEGF reduction after anti-plasma cell therapy, its increase at diagnosis has been attributed to the underlying monoclonal gammopathy, although direct evidence is still lacking. In the current study, we systemically measured VEGF levels in POEMS patients, before and after treatment. Bone marrow plasma cells showed remarkable VEGF expression, in both mRNA and protein levels, which decreased gradually in response to therapy. Of note, statistically linear correlations were observed between serum and bone marrow plasma cell VEGF levels (mRNA vs. serum, rho 0.343, p = 0.003; protein vs. serum, rho 0.644, p < 0.0001), supporting bone marrow plasma cells as the main source of circulating VEGF. Intriguingly, immunophenotyping revealed that bone marrow plasma cells were polyclonal in most patients at diagnosis. A clear monoclonal population, coexistent with polytypic cells, was only detectable in 11 cases (18%), in which comparable intracellular VEGF expression was observed between these two plasma cell populations (p = 0.594), while monoclonal cells showed higher intracellular interleukin-6 expression (p = 0.006). These patients had more serum monoclonal protein, less post-therapeutic complete remission, and inferior overall (p = 0.027) and progression-free survival (p = 0.002). Collectively, bone marrow plasma cells, mainly polyclonal population, are the major source of VEGF overproduction in POEMS patients. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes) syndrome is a rare plasma cell dyscrasia. As an illustrative example of paraneoplastic disorders, most of its featured manifestations are attributed to markedly elevated cytokines in the circulation, rather than the direct invasion by underlying clonal plasma cells [1,2]. Among the several proinflammatory and angiogenic cytokines studied so far, vascular endothelial growth factor (VEGF), a multifunctional factor inducing both neovascularization and microvascular hyperpermeability, correlates best with disease activity, and therefore utilized as a biomarker in clinical practice [3,4]. To date, the cellular source of VEGF overproduction in this disorder is still obscure. Several case reports, with the use of immunostaining, identified strong VEGF expression in tumor cells
of the patients with solitary plasmacytoma. And surgical resection did achieve symptomatic relief, as well as VEGF reduction, supporting these clonal plasma cells as the source of VEGF [5–7]. Given the fact that most patients do not have plasmacytoma, and successful outcomes are always observed after therapies against the underlying monoclonal gammopathy, plasma cells in the bone marrow are attractive and plausible source, although direct evidence from a series of patients is still lacking. To dissect the real contribution of bone marrow plasma cells, we systemically measured VEGF in 62 patients with newly diagnosed POEMS syndrome, and investigate their changes after therapy, using several methodologies. In addition, the possible regulatory mechanism governing VEGF production was also attempted. 2. Methods 2.1. Patients and clinical management
∗ Corresponding author. E-mail address:
[email protected] (J. Li). http://dx.doi.org/10.1016/j.leukres.2016.09.017 0145-2126/© 2016 Elsevier Ltd. All rights reserved.
Sixty-two patients with newly diagnosed POEMS syndrome, seen consecutively at Peking Union Medical College Hospital
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between February 2014 and April 2015, were included in the current study. All patients met the diagnostic criteria proposed by Dispenzieri [2]. Detailed clinical features and laboratory information were acquired at diagnosis, as described previously [8,9]. All patients received lenalidomide plus dexamethasone regimen (Rdex) [10]. Thirty-five of them completed at least 6 cycles, and 11 patients finished 12 cycles of treatment during the study period. Bone marrow and peripheral blood samples were collected at diagnosis and after treatment. Serum VEGF was measured with a human Quantikine ELISA Kit (R&D Systems, MN, USA; normal <600 pg/mL) at diagnosis and after treatment, as described elsewhere [8]. Hematological complete response was defined as the disappearance of monoclonal protein in both serum and urine specimens. Complete response for serum VEGF was the normalization of its level after treatment (<600 pg/mL) [8,11]. No other levels of response were specified. All patients gave their informed consent, and the study was approved by the Institutional Review Board of Peking Union Medical College Hospital, in accordance with the Declaration of Helsinki. 2.2. Bone marrow plasma cells purification Bone marrow mononuclear cells from POEMS patients and those who with other plasma cell dyscrasias (multiple myeloma, N = 10; primary plasma cell leukemia, N = 6) were first isolated using Ficoll-Hypaque density gradient centrifugation (GE Healthcare, Uppsala, Sweden). Immunomagnetic separation of plasma cells was then performed using CD138 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s protocol. The purity of plasma cells obtained was >90%, as confirmed by flow cytometry [12].
data to ensure that all Ig-positive plasma cells were included. Monoclonal population was identified when an unequivocal cytoplasmic light chain restriction was demonstrated (Ig/Ig > 10:1 or <1:10) [14]. Expression levels of VEGF and IL-6 were quantified by mean fluorescence intensity for each sample without knowledge of other clinical information. All reagents were from BD Biosciences, except VEGF (R&D Systems, MN, USA) and IL-6 (eBioscience, CA, USA) APC-conjugated monoclonal antibodies. 2.5. Immunohistochemical staining Immunohistochemical staining of bone marrow trephine biopsies (DAB peroxidase method) used antibodies against the following antigens: CD138 (clone MI15, Dako, Denmark), immunoglobulin light chains (rabbit polyclonal, Dako, Denmark), VEGFA (clone VG1, Dako, Denmark), IL-6 (rabbit polyclonal, Abcam, USA) and HIF-1␣ (clone MGC3, Abcam, USA). 2.6. Statistical analysis Analyses were performed with SPSS 23 (SPSS Inc., IL, USA). The Fisher exact test and the Kruskal-Wallis method were used to ascertain differences between categorical and continuous variables, respectively. Relationship between continuous variables was detected using Spearman’s correlation analysis. Overall survival (OS) and progression free survival (PFS) were calculated from the date of treatment. And the cutoff date for survival analysis was May 1, 2016. For PFS analyses, death or progression were considered as events. Survival curves were plotted with the Kaplan–Meier method and compared with a log-rank test. All data were considered statistically significant at p < 0.05.
2.3. Real-time quantitative polymerase chain reaction (RQ-PCR)
3. Results
Total RNA was extracted from purified bone marrow plasma cells using TRIzol, and those with sufficient quantity and quality (46 of 62 newly diagnosed, 23 of 35 after 6 cycles, and 8 of 11 after 12 cycles of Rdex; in total, 77 of 108 patients) were reverse transcribed into cDNA (Life Technologies, CA, USA). VEGFA RQ-PCR was performed using SYBR Green PCR master-mix (Roche Diagnostics, Mannheim, Germany), with each sample measured in triplicate. -actin was used as the internal control. Relative expression levels of each clinical sample were determined by 2−Ct , normalized to the level of U266 cell line with stable expression (ATCC, VA, USA). Primers (5 -3 ) were listed as follow: VEGFA forward ATCTTCAAGCCATCCTGTGTGC, reverse GCTCACCGCCTCGGCTTGT [13]; -actin forward AGAGCTACGAGCTGCCTGAC, reverse AGCACTGTGTTGGCGTACAG.
3.1. Patients’ characteristics
2.4. Flow cytometry Approximately 1 × 106 bone marrow mononuclear cells were immunophenotyped using three different 6-color combinations of monoclonal antibodies (tube 1: CD138 APC/CD38 PE-Cy7/CD45 PerCP-Cy5.5/CD19 APC-H7/Ig- FITC/Ig- PE; tube 2: VEGF APC/CD38 PE-Cy7/CD138 PerCP-Cy5.5/CD45 APC-H7/Ig- FITC/Ig PE; tube 3: IL-6 APC/CD38 PE-Cy7/CD138 PerCP-Cy5.5/CD45 APC-H7/Ig- FITC/Ig- PE). Surface antigens were stained first, and then cells were fixed, permeabilized for subsequent intracellular antigen staining (Invitrogen, CA, USA). Events (1.5-2 × 105 ) were acquired using FACSCantoII flow cytometer (BD Bioscience, CA, USA), and analyzed with FACSDiva software. The instrument was standardized with multicolor setup beads before measurement. Plasma cells were identified by gating on cells with moderateto-bright co-expression of CD138 and CD38 with review of ungated
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Baseline characteristics are shown in Table 1. The median age at diagnosis was 48 years (21–74 years), and 44 of them (71.0%) were male. All had monoclonal gammopathy (56.6% IgA heavy chain use) and polyneuropathy (median ONLS score, 4). Fortyfive patients (72.6%) showed osteosclerosis and five of six (83.3%) who underwent lymph-node biopsy with co-existent Castleman’s disease. Serum VEGF levels were dramatically elevated (median, 5958; range, 810–23728 pg/mL). Other relevant manifestations were observed in various proportions of patients, as shown in Table 1. After a median follow-up of 21.5 months (13.3–26.6 months), six patients died, and another four showed disease progression, with estimated 2-year OS and PFS as 90.3% and 83.2%, respectively. In terms of best therapeutic responses before salvage, 27 of 62 (43.5%) and 29 of 59 (49.2%) achieved hematological and VEGF complete remission, respectively. Of the four patients with progression, three received autotransplants, and one is under close monitoring. 3.2. VEGFA expression in bone marrow plasma cells VEGFA mRNA levels were markedly elevated in bone marrow plasma cells from POEMS patients at diagnosis, when compared to plasma cells sorted from myeloma (p = 0.004) and plasma cell leukemia patients (p = 0.028). Their levels were also significantly higher than CD138 negative cells from POEMS patients (p < 0.0001) (Fig. 1A). In response to treatment, its levels reduced gradually (Fig. 1B). VEGF protein expression in plasma cells quantified by flow cytometry correlated with its mRNA levels, in the 77 patients with
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Table 1 Patients’ characteristics. All patients (N = 62)
Monoclonal PC (+) (N = 11)
Monoclonal PC (−) (N = 51)
p valuea
Demographic information Age (median, range) (years) Male (N, %)
49 (21–70) 44 (71)
52 (31–65) 9 (82)
48 (21–70) 35 (69)
0.545 0.485
POEMS features Polyneuropathy: ONLS score (median, range)b
4 (0–12)
4 (1–10)
4 (0–12)
0.491
Monoclonal gammopathy Measurable M spike on SPE (N, %) M-spike (median, range) (g/L)c BMPC (median, range) (%) Heavy chain use: IgA (N, %) Castleman’s disease (N, %)d Osteosclerosis (N, %) Serum VEGF (median, range) (pg/mL)
33 (53) 2.9 (0.3–13.5) 2.0 (0–24.0) 35 (56) 5 (83) (N = 6) 45 (73) 5958 (810-23728)
9 (82) 4.4 (0.4–9.0) 2.5 (0.5–24.0) 5 (45) 1 (100) (N = 1) 6 (55) 8648 (1533-13189)
24 (47) 2.5 (0.3–13.5) 1.5 (0–13.0) 30 (59) 4 (80) (N = 5) 39 (76) 5632 (810-23728)
0.048 0.093 0.159 0.510 1.000 0.155 0.047
Organomegaly Hepatomegaly (N, %) Splenomegaly (N, %) Lymphadenopathy (N, %)
20 (32) 27 (44) 45 (73)
2 (18) 7 (64) 6 (55)
18 (35) 20 (39) 39 (76)
0.478 0.185 0.155
Endocrinopathy Hypothyroidism (N, %) Hypoadrenalism (N, %) Diabetes (N, %)
40 (65) 36 (58) 8 (13)
6 (55) 7 (64) 1 (9)
34 (67) 29 (57) 7 (14)
0.499 0.748 1.000
Skin changes Hyperpigmentation (N, %) Hemangioma (N, %)
58 (94) 41 (66)
9 (82) 7 (64)
49 (96) 34 (67)
0.141 1.000
Extravascular volume overload Edema (N, %) Pleural effusion (N, %) Ascites (N, %) Papilledema (N, %) Pulmonary hypertension (N, %) Thrombocytosis (N, %)
57 (92) 17 (27) 23 (37) 33 (53) 5 (8) 32 (52)
9 (82) 2 (18) 3 (27) 6 (55) 1 (9) 6 (55)
48 (94) 15 (29) 20 (39) 27 (53) 4 (8) 26 (51)
0.212 0.712 0.516 1.000 1.000 1.000
Therapeutic complete responses Hematological response (N, %) VEGF response (N, %)
27 (44) 29 (49) (N = 59)
1 (11) 1 (10) (N = 10)
26 (51) 28 (57) (N = 49)
0.017 0.012
ONLS, overall neuropathy limitation scale; SPE, serum protein electrophoresis; BMPC, bone marrow plasma cell; VEGF, vascular endothelial growth factor. a Comparisons between patients with and without flow cytometry-identifiable monoclonal plasma cells in bone marrow at baseline. b Nerve conduction study and electromyography were performed in patients with an ONLS score of 0 to ascertain the presence of neuropathy. c Serum protein electrophoresis was performed in all patients, and measurable M-protein was identified in 33 patients. Median values were calculated for patients with measurable M-protein. d Castleman’s disease was diagnosed in five of six patients underwent lymph-node biopsies.
Fig. 1. VEGF measurements in patients with POEMS syndrome. (A) Comparison of VEGFA mRNA expression in bone marrow plasma cells of POEMS patients with those from other plasma cell dyscrasias, and CD138 negative cells of POEMS patients. (B) Changes of VEGFA mRNA expression in bone marrow plasma cells of POEMS patients in response to treatment. (C) Correlation between levels of serum VEGF and cytoplasmic VEGF expression of bone marrow plasma cells (BMPCs).
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Fig. 2. Bone marrow flow cytometry of POEMS patients. (A) Representative results of a POEMS patient at diagnosis and after 12 cycles of Rdex treatment. (B) Presence of both light chain-restricted monoclonal plasma cells, and normal, polytypic plasma cells. (C) Comparison of intracellular VEGF and IL-6 in polyclonal and monoclonal plasma cells.
paired measurement (Spearman rho 0.320, p = 0.008). Of note, linear correlations were further observed between VEGF expression of plasma cells and the serum VEGF levels (mRNA vs. serum VEGF, Spearman rho 0.343, p = 0.003; protein vs. serum VEGF, Spearman rho 0.644, p < 0.0001) (Fig. 1C). As VEGF has proven to be present in platelet, and blood coagulation could trigger its release, the linear correlation was further analyzed with the use of serum VEGF to platelet count ratio. After such a normalization, similar results were still observed (mRNA vs. serum VEGF/platelet ratio, Spearman rho 0.357, p = 0.003; protein vs. serum VEGF/platelet ratio, Spearman rho 0.589, p < 0.0001)
3.3. Monoclonal plasma cell population in bone marrow Eleven of the 62 newly diagnosed cases (17.7%) identified a clear monoclonal plasma cell population within a background of polyclonal plasma cells by flow cytometry. Others only had polyclonal plasma cells detectable (Fig. 2A). Compared to polyclonal population, monoclonal plasma cells showed diminished CD45 and CD19 expressions, and a definitive monotypic staining of cytoplasmic light chains (Fig. 2B). After treatment, three of the 35 (8.6%) and one of the 11 (9.1%) samples collected after 6 and 12 cycles of Rdex, respectively, showed monoclonal plasma cells.
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Fig. 3. Bone marrow immunohistochemical staining of POEMS patients. (A) Immunoglobulin and light chain staining revealed two patterns (left panel, scattered polytypic plasma cells; right panel, focal aggregates of monoclonal plasma cells). (B) Immunostaining of VEGF, HIF-1␣ and IL-6 in monoclonal plasma cell aggregates. Scale bar, 100 m.
Of interest, when we compared cytoplasmic VEGFA levels in the 11 cases with two plasma cell populations, similar levels were revealed (p = 0.594). In contrast, in the six cases with cytoplasmic IL-6 staining, a higher expression was observed in the monoclonal population (p = 0.006) (Fig. 2C).
Forty-six patients had bone marrow trephine biopsies at diagnosis, and monoclonal plasma cells could be detected in 21 of them (45.7%) by immunohistochemical staining. Typically, light chain-restricted plasma cells distributed as focal aggregates in a background of polyclonal plasma cells (Fig. 3A). In addition, cyto-
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Fig. 4. Survival of patients with and without flow cytometry-identifiable monoclonal plasma cells at diagnosis. (A) Overall survival. (B) Progression-free survival.
plasmic VEGF and nuclear HIF-1␣ expressions were also observed in the monoclonal plasma cell aggregates (Fig. 3B). 3.4. Clinical features of patients with flow cytometry-identifiable monoclonal plasma cells Regarding clinical features at baseline, the 11 patients with flow cytometry-identifiable monoclonal plasma cells showed higher prevalence of monoclonal protein detectable on serum protein electrophoresis (82 vs. 47%, p = 0.048), and a trend of elevated monoclonal protein concentration (4.4 vs. 2.5 g/L, p = 0.093). Serum VEGF levels also increased more dramatically (8648 vs. 5632 pg/mL, p = 0.047). Moreover, these cases had less complete remission in the categories of hematology (1/11 [9.1%] vs. 26/51 [51.0%], p = 0.017) and VEGF (1/10 [10.0%] vs. 28/49 [57.1%], p = 0.012). When looking at the survival curves, these patients also showed inferior OS (estimated 2-year 72.7 vs. 94.1%, p = 0.027) and PFS (estimated 2-year 50.9 vs. 90.2%, p = 0.002) (Fig. 4). 4. Discussion As reported repeatedly, POEMS patients have markedly increased levels of serum VEGF, and its decrease is associated with symptomatic improvement after treatment. These observations provide mechanistic insights that most manifestations of this multisystem disorder are due to the paraneoplastic effects of VEGF, a potent cytokine resulting in vessel proliferation and functional alterations [15]. Source of VEGF overproduction is a crucial question, and if answered, could further deepen our knowledge of this disorder. Except a single case reporting bone marrow stromal cells as the main VEGF source through in vitro culture assessment [16], most studies attributed VEGF elevation to the underlying monoclonal gammopathy, given the excellent VEGF reduction after anti-plasma cell therapies [3,8,11]. However, besides the several aforementioned cases showing strong VEGF expression by immunohistochemical staining in plasmacytoma, there is actually lacking of direct evidence to demonstrate high VEGF expression in bone marrow plasma cells. Trying to fill this gap, we measured VEGF levels in a series of POEMS patients before and after therapy, by several methodologies. As expected, VEGFA mRNA levels were significantly higher in bone marrow plasma cells isolated from POEMS patients, than those from other plasma cell dyscrasias. In addition, similar drastic difference was also observed between CD138 positive and negative populations from POEMS patients, diminishing the concern of stromal cells. It is noteworthy that a linear correlation was observed between levels of bone marrow plasma cell VEGFA mRNA and serum VEGF, corroborating the role of bone marrow plasma cells in VEGF
overproduction. These findings were further justified through normalization of serum VEGF levels by platelet count, as VEGF is also released during platelet aggregation and POEMS patients always show thrombocytosis [17,18]. We further proceeded to cytoplasmic VEGF detection via flow cytometry, and revealed correlated VEGF expression in plasma cells gated by CD138 and CD38 expression to those serum measurements. Of note, we found that bone marrow plasma cells in most POEMS patients at diagnosis were actually polyclonal, and only 11 cases had clear monoclonal populations, still in an obvious background of polyclonal cells, when examined by intracellular immunoglobulin light chain staining. Indeed, similar observations were also made in the well-characterized Mayo series, i.e., 25 of 67 pretreatment bone marrow identified clonal plasma cells by flow cytometry [19]. Intriguingly, nearly half of our cases, when studied by immunohistochemical staining, had monoclonal plasma cells, consistent with the notion that trephine biopsy is more sensitive, as it provides key information of bone marrow architecture and clonal plasma cells always distribute as focal aggregates, in the polytypic background [2]. These findings let us rethink the pathogenic roles of different bone marrow plasma cell populations in POEMS patients. On one hand, polyclonal populations were observed in all patients and did express high levels of VEGF, which correlated with serum measurements, supporting them as main source of VEGF overproduction. Furthermore, these cells are supposed to be more sensitive to chemotherapy [20], explaining why most POEMS patients have substantial reduction of serum VEGF after treatment, while only part of them could achieve hematological complete remission, i.e., clonal plasma cell eradication. On the other hand, monoclonal plasma cells, variably detected by different methods, only composed a minor population, in line with the small size of monoclonal protein in most POEMS patients. However, the fundamental position of monoclonal gammopathy could not be denied. In addition to its similar secretion of VEGF compared to polyclonal population, these cells seemed to have high intracellular IL-6 expression. Given the stimulatory effect of IL-6 on reactive plasmacytosis [21], clonal plasma cells could play a critical role of disease initiation by expanding VEGF-producing polyclonal plasma cells in bone marrow microenvironment. In fact, for POEMS patients with significant VEGF reduction and clinical improvement, those who with monoclonal protein remained have higher risk of relapse. This was also observed when we compared patients with and without flow cytometry identifiable monoclonal population. Moreover, the ancillary pathogenic contribution of IL-6 has also been reported in POEMS patients [22,23]. The present study has several limitations. IL-6 staining was only performed in small number of cases by flow cytometry, and its stimulatory effect on local reactive plasmacytosis in POEMS syndrome is still speculative based on literature. Although we tried to use
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immunohistochemical staining to clarify this concern, the mixed cell distribution and semi-quantitative nature of this technique made interpretation uneasy. Therefore, further studies, including flow cytometry and even in vitro culture, are certainly required. In addition, we notice the recent advances of next generation flow cytometry, with the use of well-optimized antibody combination and analytical algorithm, to detect minimal residual clonal plasma cells in myeloma [24]. This technique, if used in POEMS patients, may make more of them with detectable clonal plasma cells in bone marrow, and extend our knowledge. However, it application is only available in several specialized centers. To conclude, bone marrow plasma cells, mainly polyclonal population, are the source of VEGF overproduction in POEMS patients. Role of the minor clonal plasma cells need further clarifications. Author contributions C.W. was involved in study design, data collection, results interpretation and wrote the manuscript. X.-F.H, Q.-Q.C. and X.-X.C. participated in patient recruitment. H.C. measured the serum VEGF. D.-B.Z. supervised the study and interpreted the data. J.L. designed the study, recruited the patients, analyzed the data, wrote the manuscript and critically revised the manuscript. All authors have approved the final manuscript. Conflict of interest The authors declare no conflict of interest. Acknowledgments The authors would like to thank all the patients who participated in this study. The National Natural Science Foundation of China (grant No. 81570195), the Beijing Natural Science Foundation (grant No. 7142130), the Specialized Research Fund for the Doctoral Program of Higher Education (grant No. 2013110611000), the Peking Union Medical College Youth Fund (grant No. 3332015093) and the Peking Union Medical College New Star (2011, for LJ) supported this research. References [1] J. Li, D.B. Zhou, New advances in the diagnosis and treatment of POEMS syndrome, Br. J. Haematol. 161 (3) (2013) 303–315. [2] A. Dispenzieri, POEMS syndrome: update on diagnosis, risk-stratification, and management, Am. J. Hematol. 90 (10) (2015) 951–962. [3] A. D’Souza, S.R. Hayman, F. Buadi, M. Mauermann, M.Q. Lacy, M.A. Gertz, The utility of plasma vascular endothelial growth factor levels in the diagnosis and follow-up of patients with POEMS syndrome, Blood 118 (17) (2011) 4663–4665. [4] C. Wang, Y.L. Zhou, H. Cai, X.Q. Cheng, W. Zhang, W.Y. Kang, Markedly elevated serum total N-terminal propeptide of type I collagen is a novel marker for the diagnosis and follow up of patients with POEMS syndrome, Haematologica 99 (6) (2014) e78–80. [5] A. Nakano, T. Mitsui, I. Endo, Y. Takeda, S. Ozaki, T. Matsumoto, Solitary plasmacytoma with VEGF overproduction: report of a patient with polyneuropathy, Neurology 56 (6) (2001) 818–819.
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