International Journal of Myeloma 7(1): 1–5, 2017
©Japanese Society of Myeloma

CASE REPORT

Multiple myeloma with high adenosine deaminase expression
Shingen NAKAMURA1, Hirokazu MIKI2, Masami IWASA1, Akishige IKEGAME3,
Tomoko MARUHASHI4, Ryohei SUMITANI1, Munenori UEMURA1, Mamiko TAKAHASHI1,
Asuka ODA1, Shiro FUJII1, Kumiko KAGAWA1 and Masahiro ABE1
A 50-year-old man with immunoglobulin A type multiple myeloma (MM) was referred to our hospital after bor­
tezomib therapy. He had high alkaline phosphatase and lactate dehydrogenase levels. Computed tomography
showed osteolytic and osteoblastic bone lesions. Response to salvage chemotherapy was temporary, and hedeveloped a right pleural effusion with high adenosine deaminase (ADA) levels. He died from bleeding associatedwith a pelvic bone fracture 9 months later. ADA mRNA expression and ADA secretion of the MM cells from thepatient were higher than those from myeloma cell lines tested. Clinical relevance of high ADA expression in MM
cells is warranted.
Key words: adenosine deaminase, multiple myeloma, pleural effusion

Introduction

Here, we report a patient with MM exhibiting an aggressive
clinical course with a pleural infiltration of MM cells expressing

Multiple myeloma (MM) is characterized by the accumula-high levels of ADA.

tion of neoplastic plasma cells in the bone marrow which
generates multiple bone lesions through enhanced osteo­­

Case Reportclastogenesis and concomitant suppression of osteoblastic
differentiation from the bone marrow stromal cells. MM is still

A 50-year-old man presented with general fatigue, appetite

difficult to be cured; therefore, additional novel treatment

loss, and pain in shoulders, back, and ribs from December

strategies are needed.

20XY. He was diagnosed with MM in March next year. He had

Adenosine deaminase (ADA) is an important enzyme in thehypercalcemia, renal insufficiency, and serum immunoglobulin

purine metabolism that catalyzes the deamination of bothA (IgA)-λ type M-protein at 2420 mg/dl with multiple osteo-

adenosine and 2’-deoxyadenosine to inosine and 2’-deoxyinosine,

lytic and osteoblastic lesions. In addition, 88% of bone

respectively [1]. However, limited information is available­m arrow cells were occupied by MM cells. After 2 courses

regarding ADA production and its role in MM.

of bortezomib, dexamethasone, and cyclophosphamide­combination therapy, he was referred to our hospital for subsequent treatment. Urinalysis revealed proteinuria, whilst

Received: April 20, 2017, accepted: June 5, 2017
1
Department of Hematology, Endocrinology and Metabolism,
Institute of Biomedical Sciences, Tokushima University Graduate
School, Tokushima, Japan
2
Division of Transfusion Medicine and Cell Therapy, Tokushima
University Hospital, Tokushima, Japan
3
Division of Medical Technology, Tokushima University Hospital,
Tokushima, Japan
4
Department of Internal Medicine, Local Incorporated Administrative
Agency Tokushima prefecture Naruto hospital, Tokushima, Japan
Corresponding author: Shingen NAKAMURA
Department of Hematology, Endocrinology and Metabolism,
Institute of Biomedical Sciences, Tokushima University Graduate
School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
E-mail: shingen@tokushima-u.ac.jp­peripheral blood tests showed normocytic anemia, high alkaline phosphatase (ALP), high lactate dehydrogenase (LDH) and
IgA level of 608 mg/dl. Bone metabolic markers revealed
high levels of bone resorption and formation activity (Table 1).
A chest X-ray scan showed multiple extramedullary tumors
in both lungs. Computed tomography revealed systemicbone lesions. Most notably, osteolytic and osteoblastic lesions
were intermingled in spinal bones and the sternum (Fig. 1a,
1b, and 1c). Bone marrow MM cells were immature in appearance with a CD38 (+), CD138 (+), CD19 (–), CD56 (+), CD20 (–),
CD33 (–), CD49e (–), and MPC-1 (–) phenotypes (Fig. 1d).
­Fluorescent in situ h­ ybridization analysis revealed complicatedkaryotypes with deletion of chromosomes 1,13q and 17
together with (4;14) translocation in the bone marrow cells.

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vol. 7 no. 1 (2017)

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NAKAMURA et al.

Table 1.
<Peripheral blood>
WBC
stab
seg
baso
eosino
mono
lymph
other
RBC
Hb
Ht
MCV
MCH
PLT

4000 /μl
1.0 %75.0 %
1.0 %
2.0 %
5.0 %
16.0 %
0.0 %341 × 104 /μl
10.9 g/dl
32.9 %
96.4 fL
32.3 pg22.2 × 104 /μl

Laboratory findings on admission<Blood chemistry><Immune serology>

T-Bil
AST
ALT
ALP
γ-GTP
LDH
TP
Alb
BUN
Crn
UA
Na
K
Cl
Ca
IP
PG

IgG
IgA
IgM
CRP
β2MG
FLC-κ
FLC-λ
κ/λ

0.4 mg/dl
34 U/l
14 U/l1831 U/l
26 U/l706 U/l
7.5 g/dl
4.1 g/dl
9 mg/dl
0.75 mg/dl
5.9 mg/dl
142 mEq/l
3.5 mEq/l
101 mEq/l
9.1 mg/dl
3.3 mg/dl
95 mg/dl

278 mg/dl
608 mg/dl
27 mg/dl<0.05 mg/dl
3.86 mg/l
12.8 mg/l
315 mg/l
0.04<Bone metabolism markers>
BAP
Osteocalcin
TRACP-5b
uDPD
1CTP
NTX
uNTX

173.0 μg/l
32.0 ng/ml
1180 mU/dl
602.0 nmol/mmolCr
41.6 ng/ml
84.5 nmolBCE/l
480.0 nmolBCE/mmolCr

Figure 1. Myeloma bone lesions and morphology of myeloma cells. a: Multiple punched out lesions are observed in an X-ray scan of the skull(red arrow). b: Osteolytic myeloma bone disease in the right upper arm (red arrow). c: Osteogenic myeloma bone disease in the vertebral
body (blue arrow), osteolytic bone disease in the vertebrae and sternum (red arrow). d: Myeloma cells observed in the bone marrow.

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International Journal of Myeloma

vol. 7 no. 1 (2017)

Multiple myeloma with high adenosine deaminase expression

Figure 2. Clinical course. After hyper-CVAD therapy as a salvage therapy, a right pleural effusion with high ADA expressive myeloma cells
developed. Patient underwent talc pleurodesis to palliate his respiratory symptoms. CHOP, DeVIC and D-PACE salvage chemotherapy
induced repetitive TLS, however, anti-tumor effect was temporary. The patient died from a pelvic bone fracture after 13 months after
diagnosis of myeloma.
Abbreviations: ADA = adenosine deaminase; CHOP = cyclophosphamide, doxorubicin, vincristine, and prednisone; DeVIC = dexamethasone,
etoposide, ifosfamide and carboplatin; D-PACE = dexamethasone, cisplatin, doxorubicin, cyclophosphamide and etoposide; hyper-CVAD =hyperfractionated cyclophosphamide, vincristine, doxorubicin (Adriamycin), and dexamethasone; IgA = immunoglobulin A; LDH = lactate
dehydrogenase; TLS = tumor lysis syndrome.

Bortezomib, lenalidomide, and dexamethasone combination­pelvic bone fracture (Fig. 2), only 13 months after the diag­chemotherapy was not effective; however, two salvage courses

nosis and 9 months after the emergence of the pleural effu-of hyperfractionated cyclophosphamide, vincristine, doxo­

sion. Serum levels of LDH and ADA were found to reflect a

rubicin, and dexamethasone (hyper-CVAD) therapy followed

MM tumor ­expansion.

by thalidomide monotherapy appeared effective; and the­patient achieved a partial response. ALP was lowered to nor-

Discussion

mal range after ­hyper-CVAD therapy; however, LDH and IgAlevels resumed to increase immediately followed by rapid ex-

Herein, we described an MM patient with poor risk chro-pansion of a right pleural effusion. Examination of the pleuralmosomal abnormality and severe bone lesions who quickly

effusion revealed a high level of ADA (453 U/L) with abun-developed a right pleural effusion with high ADA mRNAdant myeloma cells. Mycobacterium species were not detected

expression and secretion from MM cells (Fig. 3). The progres-in the pleural effusion. Computed tomography scans showed

sive nature of his condition and his resistance to chemo­

multiple ex­tramedullary expansion of tumors with a bulky

therapy eventually lead to his death.

mass. Com­
bination chemotherapy of cyclophosphamide,

Pleural effusion in myeloma is not uncommon; Kintzer et al.

doxorubicin, ­vincristine, and prednisone was temporarily effec­

reported that 6% of MM cases develop pleural effusion [2].

tive as subsequent salvage chemotherapy although a tumor

Most cases of effusion are derived from renal failure, heart

lysis syndrome emerged. He underwent a talc pleurodesis­failure, hypoalbuminemia, and infection. However, malignant

to palliate his respiratory ­distress. Salvage chemotherapy

pleural effusion only occurs in 1% or less of MM cases, making

regimens of dexamethasone, etoposide, ifosfamide and­it relatively rare [2]. In our case, the patient developed unilat-­carboplatin; ­fludarabine; dexamethasone, cisplatin, doxorubi-

eral pleural effusion with MM cell infiltration and high ADA in

cin, cyclophosphamide and etoposide were only temporarily

the supernatant of the effusion. Repeated examination ruled

effective. The patient died from bleeding, resulting from aout tuberculosis, and we verified in vitro that ADA was pro-International Journal of Myeloma

vol. 7 no. 1 (2017)

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NAKAMURA et al.

duced by MM cells in the pleural effusion because both transcriptional and protein levels were much higher than in otherMM cell lines (Fig. 3). In a clinical setting, ADA is used as adiagnostic marker for tuberculous pleuritis, peritonitis, andmeningitis because ADA protein expression in these effusions
is known to be positively correlated with tuberculosis [3–5].
The role of ADA in MM is unclear, and to date, only 7 patientswith MM and high ADA expression have been reported [6–8].
All of them developed pleural effusion during their treatment.
Six of seven patients were under sixty-year-old, markedly
young. The IgD isotype was seen in 3 cases, and the IgG and
IgA isotypes were in 2 cases respectively. Bone disease andmonosomy 13 or del(13q) were seen in three cases (Table 2).
In our case, the ADA level from the effusion was the highest
Figure 3. Expression of ADA in MM cells. MM cells from his pleural
effusion were purified with positive selection using CD138

among all previously reported cases. Period of their survival
was reported to be between only a month and 1 year.

(Syndecan-1) microbeads and the Miltenyi magnetic cell-sorting

ADA is a critical enzyme expressed in all tissues of humansystem (Miltenyi Biotec, Auburn, CA), according to the manufac-

body that catalyzes the deamination of both adenosine andturer’s instructions. The MM cells were then cultured, and total
RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad,
CA). For reverse transcription–polymerase chain reaction as

2’-deoxyadenosine to inosine and 2’-deoxyinosine, respectively [1]. ADA is also regarded as an activation marker of­previously described. The specific primers used were forward

lymphoid cells, and the absence of ADA results in both intra-

5’-TTCCTTCCAAGAAGACCATGA-3’, reverse 5’-GGTTTCAGATTCA

and extracellular accumulation of adenosine, which leads to

ACCATGC-3’, for human ADA, forward 5’-TGTCTTCACCACCATG

lymphocyte apoptosis and immune deficiency [9]. High ADAGAGAAGG-3’ and reverse 5’-GTGGATGCAGGGATGATGTTCTG-3’

expression reflects high purine metabolism activity; therefore,

for human glyceraldehyde-3-phosphate dehydrogenase. MM
cells were cultured for two days at 1 × 106 cells/ml in RPMI 1640
medium with 5% fetal bovine serum. The supernatants werecollected and their ADA levels were measured. All procedures

ADA is thought to be a probable target for the inhibiting­cancer metabolism. A number of ADA inhibitors have beensynthesized [10], and some are approved for their clinical use,

involving human specimens were performed with written

including cladribine for hairy cell leukemia and relapsed/

informed consent according to the Declaration of Helsinki, andrefractory follicular or mantle cell lymphoma as well as

the protocol was approved by the Institutional Review Board for­pen­tostatin for hairy cell leukemia and adult T-cell leukemia.

human protection in Tokushima University hospital (approval­However, ADA inhibitors has not yet been studied in MM.

No. 240-2). ADA mRNA expression and production of thepatient’s MM cells from the pleural effusion were significantly
higher compared to those in peripheral blood mononuclear
cells (PBMCs) from a healthy donor and MM cell lines tested.
Abbreviations: ADA = adenosine deaminase; PBMCs = peripheral
blood mononuclear cells.

Table 2.

Adenosine, a substrate of ADA, has drawn considerable
attention for its association with cancer. Adenosine activatesthe adenosine receptors on the target cell, which generatevarious cellular responses, including immune cell regulationand tumor cell proliferation that result in alteration of theReported myeloma cases with high ADA expression in the pleural effusion

Ref.

Age

GenderType

ADA (U/l)

Bone disease

Monosomy13
or del (13q)

Survival after pleural effusion
appearance (month)

6

49

M

IgA-κ

61

ND

ND

11

7

58

M

IgD

50

ND

ND

1

8

53

M

IgD-λ

68.4

osteolytic

+

3.7

8

53

F

IgA-κ

84.2

—

ND

0.7

8

53

M

IgG-κ

108.5

osteolytic

+

0.8

8

67

M

IgD-λ

117.8

osteolytic

+

15.6

8

48

F

IgG-κ

60.6

—

–

4.1

present case

55

M

IgA-λ

453.0

osteolytic andosteoblastic+

9

ND: not described

4International Journal of Myeloma

vol. 7 no. 1 (2017)

Multiple myeloma with high adenosine deaminase expression

tumor microenvironment [11]. On the other hand, deaminase
activity also seems to be important in cancer. The adenosine
deaminase acting on RNA (ADAR) family genes and proteins4.

deaminate adenosine to inosine in double-stranded RNA.
They were discovered through the purification of Xenopus,
bovine liver, and calf thymus [12–14] . This reaction is5.

­commonly referred to as Adenosine-to-Inosine RNA editing,a highly conserved mechanism in mammals [15]. ADAR isreported to affect various human biochemical activities, such
as mRNA translation, RNA stability, RNA replication, and RNA
silencing, which involves hematopoiesis and pluripotency of

6.
7.

hematopoietic stem cells [16]. Although it is unclear whether
deamination activity influences on MM progression, we needfurther studies on adenosine or its deamination-related

8.

pathophysiology and tumor pathophysiology in MM.
Bone disease in our case showed a characteristic feature
with simultaneous osteoblastic and osteoclastic bone lesions
when he was referred to our hospital. In humans, osteoblast

9.
10.

precursor produce adenosine that has a potent stimulatory
action on IL-6 secretion but an inhibitory action on osteo­
protegerin [17]. In addition, adenosine signaling is reported

11.

to stimulate human osteoblastogenesis [18] and osteo­
clastogenesis [19]. Therefore, adenosine or ADA activity

12.

might affect the characteristic phenotype of bone diseasein our patient.
In summary, we described a patient with MM showing high13.

ADA expression with bone disease mixed with osteolytic andosteoblastic lesions and unilateral pleural effusion. Because
the role of ADA in MM pathophysiology and MM bone lesions

14.

remains unknown, further clinical experiences and laboratory
investigations are warranted.
15.

Conflicts of Interest Disclosures
The authors declare no competing financial interests related

16.
17.

to this work.

References18.
1. Cortes A, Gracia E, Moreno E, Mallol J, Lluis C, Canela EI, et al.
Moonlighting adenosine deaminase: a target protein for drug
development. Med Res Rev. 2015;35(1):85–125.
2. Kintzer JS, Jr., Rosenow EC, 3rd, Kyle RA. Thoracic and pulmonary
abnormalities in multiple myeloma. A review of 958 cases. Arch
Intern Med. 1978;138(5):727–30.
3. Liang QL, Shi HZ, Wang K, Qin SM, Qin XJ. Diagnostic accuracy of

19.

adenosine deaminase in tuberculous pleurisy: a meta-analysis.
Respir Med. 2008;102(5):744–54.
Riquelme A, Calvo M, Salech F, Valderrama S, Pattillo A, Arellano
M, et al. Value of adenosine deaminase (ADA) in ascitic fluid forthe diagnosis of tuberculous peritonitis: a meta-analysis. J Clin
Gastroenterol. 2006;40(8):705–10.
Tuon FF, Higashino HR, Lopes MI, Litvoc MN, Atomiya AN,
Antonangelo L, et al. Adenosine deaminase and tuberculous
meningitis—a systematic review with meta-analysis. Scand J
Infect Dis. 2010;42(3):198–207.
Rodriguez JN, Pereira A, Martinez JC, Conde J, Pujol E. Pleural­effusion in multiple myeloma. Chest. 1994;105(2):622–4.
Yokoyama T, Tanaka A, Kato S, Aizawa H. Multiple myeloma­presenting initially with pleural effusion and a unique paraspinal
tumor in the thorax. Intern Med. 2008;47(21):1917–20.
Cho YU, Chi HS, Park CJ, Jang S, Seo EJ, Suh C. Myelomatous­pleural effusion: a case series in a single institution and literature review. Korean J Lab Med. 2011;31(4):225–30.
Gaspar HB, Aiuti A, Porta F, Candotti F, Hershfield MS, Notarangelo
LD. How I treat ADA deficiency. Blood. 2009;114(17):3524–32.
Cristalli G, Costanzi S, Lambertucci C, Lupidi G, Vittori S, Volpini R,
et al. Adenosine deaminase: functional implications and different
classes of inhibitors. Med Res Rev. 2001;21(2):105–28.
Antonioli L, Blandizzi C, Pacher P, Hasko G. Immunity, inflammation and cancer: a leading role for adenosine. Nat Rev Cancer.
2013;13(12):842–57.
Hough RF, Bass BL. Purification of the Xenopus laevis doublestranded RNA adenosine deaminase. J Biol Chem. 1994;
269(13):9933–39.
Kim U, Garner TL, Sanford T, Speicher D, Murray JM, Nishikura K.
Purification and characterization of double-stranded RNA­adenosine deaminase from bovine nuclear extracts. J Biol Chem.
1994;269(18):13480–9.
O’Connell MA, Keller W. Purification and properties of doublestranded RNA-specific adenosine deaminase from calf thymus.
Proc Natl Acad Sci U S A. 1994;91(22):10596–600.
Zinshteyn B, Nishikura K. Adenosine-to-inosine RNA editing. Wiley
Interdiscip Rev Syst Biol Med. 2009;1(2):202–9.
Avesson L, Barry G. The emerging role of RNA and DNA editing
in cancer. Biochim Biophys Acta. 2014;1845(2):308–16.
Evans BA, Elford C, Pexa A, Francis K, Hughes AC, Deussen A, et al.
Human osteoblast precursors produce extracellular adenosine,
which modulates their secretion of IL-6 and osteoprotegerin. J
Bone Miner Res. 2006;21(2):228–36.
Costa MA, Barbosa A, Neto E, Sa-e-Sousa A, Freitas R, Neves JM,
et al. On the role of subtype selective adenosine receptor agonists
during proliferation and osteogenic differentiation of human primary bone marrow stromal cells. J Cell Physiol. 2011;226(5):1353–
66.
Pellegatti P, Falzoni S, Donvito G, Lemaire I, Di Virgilio F. P2X7
receptor drives osteoclast fusion by increasing the extracellularadenosine concentration. Faseb J. 2011;25(4):1264–74.

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