ORIGINAL ARTICLE: CLINICAL

Immune parameters in multiple myeloma patients: Influenceof treatment and correlation with opportunistic infections

PHILIPP SCHUTT1, DIETER BRANDHORST1, WERNER STELLBERG1, MIRIAM POSER1,

PETER EBELING1, SIEMKE MULLER1, ULRIKE BUTTKEREIT1, BERTRAM OPALKA1,

MONIKA LINDEMANN2, HANS GROSSE-WILDE2, SIEGFRIED SEEBER1,

THOMAS MORITZ1, & MOHAMMAD R. NOWROUSIAN1

1Department of Internal Medicine (Cancer Research) and 2Institute of Immunology, West German Cancer Center, University

of Duisburg-Essen Medical School, Essen, Germany

(Received 22 August 2005; accepted 2 November 2005)

AbstractThe present study evaluated cellular and humoral immune parameters in myeloma patients, focusing on the effect oftreatment and the risk of opportunistic infections. Peripheral blood lymphocyte subsets and serum levels of nonmyelomaimmunoglobulins (Ig) were analysed in 480 blood samples from 77 myeloma patients. Untreated myeloma patients exhibitedsignificantly reduced CD4þ/45ROþ, CD19þ, CD3þ/HLA-DRþ, and natural killer (NK) cells, as well as nonmyelomaIgA, IgG and IgM. Conventional-dose chemotherapy resulted in significantly reduced CD4þ and even further decline ofCD4þ/CD45ROþ and CD19þ cells, most notably in relapsed patients. Additional thalidomide treatment had no significanteffects on these parameters. Following high-dose chemotherapy (HD-CTX), prolonged immunosuppression was observed.Although CD8þ, NK, CD19þ and CDþ/CD45ROþ cells recovered to normal values within 60, 90, 360 and 720 days,respectively, CD4þ counts remained reduced even thereafter. Nine opportunistic infections were observed, including fivecytomegalovirus (CMV) diseases, one Pneumocystis carinii pneumonia (PCP) and three varicella zoster virus infections withCMV diseases and PCP occurring exclusively after HD-CTX. Opportunistic infections were correlated with severely reducedCD4þ, as well as CD4þ/CD45ROþ and CD19þ counts. Thus, myeloma patients display cellular and humoralimmunodeficiencies, which increase following conventional as well as HD-CTX, and constitute an important predisposingfactor for opportunistic infections.

Keywords: Multiple myeloma, thalidomide, lymphocyte recovery, opportunistic infections, cytomegalovirus

Introduction

Multiple myeloma repeatedly has been associated

with an immunodeficient state, and decreased serum

levels of nonmyeloma immunoglobulins, as well as

substantial impairment of the cellular immune

system, have been described [1,2]. In untreated

patients reduced CD4þ and CD19þ cell counts have

been shown to be linked to a poor prognosis [2,3].

Chemotherapy has additional suppressive effects on

the immune system and significant reductions in

the numbers of total leukocytes and lymphocytes,

T lymphocytes, CD4þ lymphocytes and CD8þlymphocytes in the peripheral blood have been

reported following conventional-dose regimens

(conv-CTX) [3,4]. However, most of these studies

have been performed in the context of first-line

treatment [4], and much less is known about the

effects of prolonged and repetitive chemotherapeutic

treatment on the immune system in patients with

advanced or relapsed disease. High-dose chemother-

apy (HD-CTX), despite the use of peripheral blood

stem cell transplantation, clearly induces a profound

reduction of peripheral blood lymphocytes and

polyclonal immunoglobulins [3,5,6]. Although some

parameters such as CD8þ lymphocytes recover as

early as 6 weeks after HD-CTX, CD4þ lymphocytes

remain diminished for a prolonged period of time,

Correspondence: Philipp Schutt, Department of Internal Medicine (Cancer Research), West German Cancer Center, University of Essen Medical School,

Hufelandstraße 55, 45122 Essen, Germany. E-mail: philipp.schuett@uni-essen.de

Leukemia & Lymphoma, August 2006; 47(8): 1570 – 1582

ISSN 1042-8194 print/ISSN 1029-2403 online � 2006 Informa UK Ltd.

DOI: 10.1080/10428190500472503

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at least several months [7,8] and, according to one

recently published study, normalization of CD4þlymphocytes after HD-CTX may require up to 6

years [5]. Thalidomide is a recently introduced

therapeutic option for myeloma patients and re-

sponse rates of up to 35% and 70% have been

described for use as single agent or in combination

with dexamethasone, respectively [9 – 11]. To ex-

plain the antimyeloma activity of thalidomide, anti-

angiogenic as well as direct cytotoxic effects have

been presumed [12 – 14], but also a variety of

immunomodulatory properties have been described.

This includes activation of CD8þ lymphocytes [15],

augmentation of the interleukin-2 production of

mononuclear cells [16], induction of T-helper cell

type 2 cytokine production [17] and enhancement of

the natural killer (NK) cell activity [18] in vitro, as

well as stimulation of humoral and cellular immune

effector mechanisms in mice [19]. In addition to

these immunostimulatory properties, an immuno-

suppressive activity of thalidomide by inhibiting the

tumor necrosis factor alpha production of activated

monocytes [20] has been described and, in some

instances, serious infectious complications have been

reported [21,22]. Because myeloma patients are

generally prone to infectious complications, a causal

relationship between thalidomide treatment and

opportunistic infections in myeloma patients is not

compellingly conclusive [23,24]. Given the multi-

tude of factors affecting the immune system of

myeloma patients and the susceptibility to infections

of these patients, the present study aimed to define

the immunologic deficits in myeloma patients in the

context of disease course and treatment and to

analyse their relevance for the occurrence of oppor-

tunistic infections.

Materials and methods

Patients and treatment schedules

Between January 1999 and February 2004, 480

peripheral blood samples from 77 myeloma patients

were investigated. Samples were obtained from

previously untreated patients (45 samples, 32 pa-

tients), patients after conv-CTX (276 samples, 46

patients), and patients after HD-CTX (159 samples,

32 patients), with some patients contributing to more

than one group. Of the untreated group, six patients

(19%) had stage 1A, six (19%) stage 2A, 19 (59%)

stage 3A and one patient (3%) stage 3B disease

according to the classification of Durie and Salmon

[25]. Conv-CTX consisted of vincristine, epirubicin

and dexamethasone combined with thalidomide

(VED-T) [26]. Further conv-CTX were combina-

tions of bendamustine and prednisone (with or

without concomitant thalidomide); melphalan and

prednisone; and cisplatin, etoposide, dexamethasone

and thalidomide. Before HD-CTX, remission of

myeloma was induced with 3-weekly cycles of VED-

T, which were repeated until maximal response of

myeloma was obtained. Thereafter, peripheral blood

stem cell harvesting was performed after one cycle of

cyclophosphamide followed by daily injection of

granulocyte colony-stimulating factor. High-dose

chemotherapy consisted of one or two cycles of

high-dose melphalan with retransfusion of at least

2.56106 CD34þ cells/kg bodyweight on day 4.

Thalidomide (up to 400 mg daily) was administered

in addition to conv-CTX in 40 patients (215

samples) and, in these patients, thalidomide treat-

ment was continued until progression of myeloma

was noted. Age-matched healthy volunteers (n¼ 30)

served as controls.

Analysis of lymphocyte subsets by flow cytometry

Lymphocyte subsets in the peripheral blood were

assessed by flow cytometry using four-color immuno-

fluorescence analysis on a Coulter EPICS XL

(Beckman Coulter, Krefeld, Germany). In brief,

2.7 ml of peripheral blood were collected in EDTA-

treated tubes. Samples of 100 ml were incubated with

combinations of FITC-, PE-, ECD- (PE-Texas Red)

and PC5- (PE-Cy5.1) labeled monoclonal antibodies

specific for CD45/CD4/CD8/CD3, CD45/CD56/

CD19/CD3, CD4/CD45RO/CD8 or CD23/CD19/

CD3/HLA-DR (Beckman Coulter). After 15 min of

incubation in the dark, red cells were lysed and the

samples were fixed using the Multi-Q-Prep device

and the Immunoprep reagent kit (both Beckman

Coulter). At least 56103 lymphocytes were analysed

for each marker combination. Lymphocytes were

gated according to their typical forward and side

scatter and lymphocyte subsets were classified ac-

cording to the expression of typical marker

constellation as: CD3þ (T lymphocytes); CD3þ/

CD4þ; CD3þ/CD8þ; CD3þ/HLA-DRþ; CD4þ/

CD45ROþ; CD19þ (B lymphocytes); and CD3-

CD16/56þ (NK cells).

Assessment of immunoglobulins IgG, IgA and IgM

Serum IgG, IgA and IgM levels were quantified by

immunonephelometry (IMMAGE System; Beckman

Coulter). Reference intervals, as published elsewhere

[27], were used for comparison with untreated

patients. Immunoglobulin heterogeneity of all serum

samples was determined by agarose gel electrophor-

esis and immunofixation using IgG, IgA, IgM, kappa

and lambda-specific antisera (Beckman Coulter).

Because we were primarily interested in the amount

Immune parameters in multiple myeloma patients 1571

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of nonmyeloma IgG or IgA, the respective values of

either IgG or IgA were only included in the analysis

when the myeloma-specific paraprotein was unde-

tectable in immunofixation.

Statistical analysis

Statistical analysis was performed using SPSS 12.0

for Windows (SPSS Inc., Chicago, IL, USA).

Differences between groups were assessed using the

Mann–Whitney test or the chi-square test as indicated.

To test for the relationship between variables,

Spearman’s rank correlation coefficients were calcu-

lated. When two or more samples from one patient

were available within one specific subgroup, the medi-

an value of these samples was used for data analysis.

P50.05 was considered statistically significant.

Results

Immune parameters in untreated myeloma patients

Compared with healthy controls, myeloma patients

showed significantly reduced median values of

CD19þ lymphocytes, CD4þ/CD45ROþ lympho-

cytes, NK cells and CD3þ/HLA-DRþ lymphocytes

in the peripheral blood, although no significant

differences were observed for median values of

CD4þ or CD8þ lymphocytes, granulocytes and

monocytes. With regard to serum levels of nonmye-

loma IgA, IgG and IgM, myeloma patients exhibited

values below physiological ranges in 58%, 87%, and

75% of patients, respectively (Table I).

Impact of conventional-dose chemotherapy

To assess the impact of conv-CTX on the immune

system, peripheral blood cell counts and levels of

nonmyeloma immunoglobulins were determined

at a median of 21 days (range 18 – 42 days) after

application of chemotherapy (i.e. when patients

routinely returned to the clinic for continuation of

treatment). Compared with untreated patients,

counts of CD4þ lymphocytes and CD4þ/CD45RO –

lymphocytes were significantly decreased and CD4þ/

CD45ROþ as well as CD19þ lymphocytes were even

further reduced. On the other hand, values for

CD3þ/HLA-DRþ lymphocytes and monocytes were

significantly increased following conv-CTX. No

significant differences were observed for CD8þlymphocytes, NK cells, granulocytes and serum levels

of nonmyeloma IgG, IgA and IgM (Table I).

Patients were further stratified according to

whether they received treatment for myeloma for

the first time (‘first-line CTX’), in first relapse

(‘second-line CTX’), or in more advanced disease

(i.e. second or higher relapse; ‘� third-line CTX’).

As shown in Figure 1, counts of CD4þ, CD4þ/

CD45ROþ, CD4þ/CD45RO7 and CD19þ lympho-

cytes, as well as CD4þ to CD8þ and CD45RO – to

Table I. Immune parameters of multiple myeloma patients.

Healthy controls (n¼ 30) MM (untreated) (n¼ 32) MM (conv-CTX) (n¼46)

Cellular parameters

CD4þ (/ml) 790 (430 – 1440) 800 (290 – 1640) 450 (21 – 1580)*,**

CD4þ/CD45ROþ (/ml) 430 (170 – 940) 340 (130 – 880)* 250 (21 – 1020)*,**

CD4þ/CD45RO– (/ml) 400 (79 – 750) 410 (77 – 890) 140 (1 – 670)*,**

CD45RO– to CD45ROþ cell

ratio

0.85 (0.23 – 2.74) 1.1 (0.22 – 3.42) 0.50 (0.01 – 2.57)*,**

CD8þ (/ml) 400 (110 – 1250) 406 (81 – 1250) 380 (16 – 2180)

CD3þ/HLA-DRþ (/ml) 180 (47 – 440) 88 (7 – 610)* 180 (24 – 1920)**

CD4þ to CD8þ cell ratio 1.95 (0.8 – 7.2) 1.90 (0.8 – 5.3) 1.3 (0.1 – 5.3)*,**

CD19þ (/ml) 200 (54 – 390) 120 (18 – 740)* 19 (1 – 170)*,**

NK cells (/ml) 350 (59 – 1120) 160 (49 – 560)* 180 (58 – 730)*

Granulocytes (/ml) 4010 (1760 – 7890) 3750 (1320 – 11500) 3890 (1070 – 19600)

Monocytes (/ml) 519 (280 – 910) 490 (110 – 1450) 850 (260 – 3180)*,**

Nonmyeloma immunoglobulins

Immunoglobulin G (g/l){ [6.4 – 13.5]x 5.4 (1.96 – 16.1) (n¼ 15) 5.7 (1.4 – 11.9) (n¼38)

Immunoglobulin A (g/l){ [0.70 – 3.1]x 0.58 (0.04 – 3.04) (n¼ 26) 0.70 (0.11 – 5.9) (n¼41)

Immunoglobulin M (g/l) [0.56 – 3.5]x 0.31 (0.04 – 3.04) (n¼ 32) 0.29 (0.09 – 1.3) (n¼45)

*Significant (P 50.05) differences compared to healthy controls.

**Significant (P 50.05) differences compared to untreated myeloma patients.{Values from patients with detectable amounts of myeloma paraprotein of the respective immunoglobulin type were excluded.xNormal reference intervals of serum immunoglobulin levels are given ( Jolliff et al. [27]).

MM, Multiple myeloma; NK cells, natural killer cells.

Median values (range) in peripheral blood are given. Conv-CTX denotes values within 6 weeks after conventional-dose chemotherapy.

1572 P. Schutt et al.

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Figure 1. Cellular parameters following conventional-dose chemotherapy. Median values (bold lines) and interquartile ranges (boxes) of

peripheral blood leukocyte subset cell counts from previously untreated myeloma patients and from patients on treatment with

conventional-dose chemotherapy (within 6 weeks) are depicted. Whiskers shown above and below the boxes represent the largest and

smallest scores observed that are less than 1.5 box length from the end of the box. Treated patients were stratified according to whether

they received treatment for previously untreated myeloma (‘first-line CTX’), first relapse (‘second-line CTX’), or more advanced disease

(i.e. second or higher relapse; ‘� third-line CTX’). NK cells, NK cells. *Significant differences (P5 0.05) compared to untreated

patients, {Significant differences (P5 0.05) compared to ‘first-line CTX’. {Significant differences (P50.05) compared to ‘second-line

CTX’.

Immune parameters in multiple myeloma patients 1573

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CD45ROþ cell ratios, significantly declined along

with the number of relapses, while no significant

suppressive effects were observed on CD8þ and

CD3þ/HLA-DRþ lymphocytes or NK cells. Fur-

thermore, monocyte counts and nonmyeloma IgG

and IgA levels remained unchanged (data not shown).

Granulocyte counts [2440 (490 – 4620)/ml vs. 3750

(1320 – 11500)/ml or 4090 (1430 – 12800)/ml, respec-

tively] and serum levels of IgM [0.14 (0.09 – 0.75) g/l

vs. 0.31 (0.11 – 1.81) g/l or 0.29 (0.09 – 1.28) g/l,

respectively] were significantly decreased in � third-

line CTX vs. untreated patients or in first-line CTX,

respectively.

Impact of high-dose chemotherapy

Following HD-CTX, CD4þ and in particular

CD4þ/CD45RO – , lymphocyte counts were signifi-

cantly decreased and, despite some degree of

reconstitution, remained significantly reduced com-

pared to controls but also untreated patients for more

than 2 years (Figure 2A,C). CD4þ/CD45ROþlymphocyte numbers (Figure 2B) showed a similar

course of regeneration, whereas CD8þ and CD3þ/

HLA-DRþ lymphocytes (Figure 2E,H) and mono-

cytes (Figure 2K) regenerated within 60 days

and NK cells within 90 days after transplantation

(Figure 2I). CD45RO – to CD45ROþ and CD4þ to

CD8þ cell ratios were significantly reduced for more

than 2 years after HD-CTX (2D,F). CD19þlymphocytes (Figure 2G) were the second important

group of immune effector cells, which showed

significant reduction over an extended time period.

However, compared with CD4 cells, recovery was

more rapid and levels comparable with untreated

patients or healthy controls were observed within 180

and 360 days, respectively. For peripheral blood

granulocytes, there was an initial recovery to

normal levels within the first 60 days after HD-

CTX, but the counts of these cells decreased

again and remained significantly reduced for up to

180 days (Figure 2J). Serum levels of nonmyeloma

IgG were significantly increased within the first year

after HD-CTX compared to untreated patients,

whereas no significant differences in serum levels of

IgA were observed (Figure 2L,M). Compared with

Figure 1. (Continued ).

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untreated patients, serum levels of IgM appeared to

be increased after 90 days of HD-CTX with

significant differences after 91 – 180 days and 361 –

720 days (Figure 2N).

Impact of thalidomide treatment

A possible effect of thalidomide on immune para-

meters was assessed by comparing patients receiving

Figure 2. Immune reconstitution following high-dose chemotherapy/peripheral blood stem cell transplantation. Boxplots of numbers of

leukocyte subsets and serum levels of nonmyeloma immunoglobulins from previously untreated myeloma patients and patients receiving

high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation are depicted. Days (d) after transplantation are

given on the x-axis. Patients on treatment for relapsed disease after transplantation were excluded. NK cells, NK cells. *Significant

differences (P 50.05) compared to untreated patients.

Immune parameters in multiple myeloma patients 1575

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thalidomide treatment in addition to conv-CTX with

patients receiving conv-CTX only (Table II). In our

patients, no significant differences between the two

groups were detected with regard to the numbers

of major immune effector cells such as CD4þ,

CD4þ/45ROþ, CD4þ/CD45RO – and CD19þlymphocytes, as well as NK cell numbers. Further-

more, counts of peripheral blood granulocytes

showed no differences between the groups. Interest-

ingly, monocyte counts, as well as the levels of

nonmyeloma IgA and IgM, were significantly in-

creased in patients receiving additional thalidomide

therapy (Table II).

Opportunistic infections

Nine opportunistic infections were observed in

eight patients, including five CMV diseases, one

Figure 2. (Continued ).

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Figure 2. (Continued ).

Table II. Influence of thalidomide on immune parameters of multiple myeloma patients treated with conventional-dose chemotherapy.

Conv-CTX (þ thal) (n¼ 40) Conv-CTX (– thal) (n¼15) P

Cellular parameters

CD4þ (/ml) 460 (21 – 1580) 430 (19 – 1430) 0.49

CD4þ/CD45ROþ (/ml) 260 (21 – 1000) 210 (8 – 460) 0.27

CD4þ/CD45RO7 (/ml) 110 (1 – 670) 165 (1 – 970) 0.96

CD45RO7 to CD45ROþ ratio 0.46 (0.01 – 2.57) 0.63 (0.01 – 2.15) 0.33

CD8þ (/ml) 380 (16 – 2180) 300 (110 – 890) 0.21

CD3þ/HLA-DRþ (/ml) 190 (24 – 1920) 140 (38 – 500) 0.29

CD4þ to CD8þ ratio 1.2 (0.05 – 5.3) 0.8 (0.2 – 4.1) 1.0

CD19þ (/ml) 16 (1 – 160) 12 (3 – 100) 0.50

NK cells (/ml) 210 (58 – 730) 140 (16 – 450) 0.42

Granulocytes (/ml) 3680 (1070 – 18000) 3160 (880 – 21100) 0.56

Monocytes (/ml) 840 (260 – 2290) 450 (179 – 5820) 0.027*

Nonmyeloma immunoglobulins

Immunoglobulin G (g/l){ 5.3 (0.86 – 11.9) n¼ 38 2.36 (0.82 – 9.57) n¼ 9 0.17

Immunoglobulin A (g/l){ 0.82 (0.12 – 5.85) n¼ 35 0.31 (0.08 – 1.04) n¼ 12 0.002**

Immunoglobulin M (g/l) 0.31 (0.09 – 1.28) 0.20 (0.09 – 0.70) 0.017*

*,**Significant differences (P 50.05 and P 50.01, respectively) between (þ thal) and (7 thal) groups.{Values from patients with detectable amounts of myeloma paraprotein of the respective immunoglobulin type were excluded.

The median values (range) of peripheral blood cell counts and of nonmyeloma immunoglobulin serum levels from myeloma patients within 6

weeks of conventional-dose chemotherapy (conv-CTX) are given. Patients were stratified as to whether they received additionally

thalidomide (þ thal) or not (– thal). NK cells, natural killer cells.

Immune parameters in multiple myeloma patients 1577

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Pneumocystis carinii pneumonia (PCP) and three

varicella zoster virus (VZV) infections, with one

patient infected consecutively with CMV first

and with VZV thereafter (Table III). Although

CMV disease and PCP occurred exclusively after

HD-CTX, VZV infections were also observed in the

context of conv-CTX. No stringent association was

observed in the context of thalidomide treatment.

Two of five patients with CMV diseases died of

their infection and one additional CMV patient as

well as the PCP patient required mechanical

ventilation for 10 and 14 days, respectively.

However, the latter two patients were successfully

treated with ganciclovir for CMV or trimethoprim-

sulfamethoxazole and prednisone for PCP. The

VZV infections were less severe and all three

patients presented with characteristical skin lesions

localized unilaterally along a dermatome. Antiviral

therapy consisted of acyclovir for 1 – 2 weeks until

vesicular skin lesions were completely healed. The

incidence of opportunistic infections within the first

6 months of HD-CTX was 13% for CMV disease

and 3.2% for each VZV infections and PCP.

Patients who developed opportunistic infections

were characterized by significantly reduced counts

of CD4þ, CD4þ/CD45ROþ, CD4þ/CD45RO –

and CD19þ lymphocytes, and significantly increased

counts of CD3þ/HLA-DRþ lymphocytes compared

to patients without infections (Table IV). Also,

CD4þ to CD8þ and CD45RO – to CD45ROþ cell

ratios were significantly reduced in patients with an

opportunistic infection. No significant differences

were observed with respect to CD8þ lymphocytes,

NK cells, granulocytes, monocytes and serum levels

of nonmyeloma IgG, IgA and IgM between the two

groups of patients. A similar picture was observed

when only the subgroup of patients after HD-CTX

was analysed (Table IV). Of note, also baseline

values of CD4þ and CD45/ROþ immediately prior

to HD-CTX were significantly reduced when the

groups of patients with and without subsequent

opportunistic infections were compared [CD4þlymphocytes: 110 (39 – 750)/ml vs. 260 (83 – 564)/ml;

CD4þ/CD45ROþ lymphocytes: 91 (8 – 560)/ml vs.

220 (53 – 470)/ml]. However, no significant differ-

ences between these two groups were observed with

regard to CD4þ/CD45RO – , CD8þ, CD19þ and

CD3þ/HLA-DRþ lymphocytes, NK cells, granulo-

cytes and monocytes. The median values (range) of

CD4þ, CD4þ/CD45ROþ, CD8þ and CD19þlymphocytes for patients with CMV disease were

51 (39 – 150)/ml; 51 (8 – 120)/ml; 670 (310 – 960)/ml;

and 4 (1 – 130)/ml, respectively, and, for patients

with VZV infections, 320 (170 – 470)/ml; 280 (120 –

440)/ml; 820 (360 – 1130)/ml; and 29 (4 – 320)/ml,

respectively.

Discussion

Because reduced immune function in multiple

myeloma can facilitate severe infective periods or

compromise immunotherapeutic interventions, we

systematically assessed immune function in a

cohort of 77 myeloma patients presenting to our

institution between 1999 and 2004. In accordance

with previous studies [1,2,28,29], substantial im-

pairment of the immune system with significantly

reduced counts of CD19þ and NK cells, as well as

nonmyeloma immunoglobulin levels in the periph-

eral blood, was observed even in untreated

myeloma patients. Although other studies also have

described significantly reduced numbers of CD4þlymphocytes [1,28,29], in the present study, med-

ian values for CD4þ lymphocytes did not

Table III. Characteristics of myeloma patients with opportunistic infections.

Patient

Age

(years)

Type of

infection

Myeloma

stage*

Number

of

relapses

Remission

status HD-CTX

Days

after

CTX{CD4þ(/ml)

CD4þ/

CD45þ(/ml)

CD8þ(/ml)

CD19þ(/ml)

1 52 CMV 3B 0 CR Yes 22 39 8 960 1

2 57 CMV 2A 1 PR Yes 22 51 51 360 4

3 65 CMV 3A 0 CR Yes 35 71 67 670 25

4 45 CMV 3A 1 PR Yes 23 48 41 310 1

5 64 CMV 3A 0 CR Yes 42 150 120 870 130

VZV 3A 0 CR Yes 119 320 280 820 320

6 63 VZV 3A 0 PR No 21 170 120 360 4

7 69 VZV 3A 2 PR No 20 470 440 1130 29

8 45 PCP 3A 2 CR Yes 24 69 60 670 29

*According to Durie and Salmon [25].{Days from application of the last chemotherapy (CTX) to diagnosis of opportunistic infection.

HD-CTX, High-dose chemotherapy/peripheral stem cell transplantation; CMV, cytomegalovirus disease; VZV, varicella zoster virus

infection; PCP, Pneumocystis carinii pneumonia; CR, complete remission; PR, partial remission. Patient number 5 had infections caused by

CMV and VZV at day 42 and at day 119 after HD-CTX, respectively.

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significantly differ between healthy controls and

untreated myeloma patients. However, in three

untreated myeloma patients, counts of CD4þcells below 400/ml (290/ml, 350/ml and 350/ml,

respectively) were found, consistent with a sup-

pressive effect of multiple myeloma on CD4þ cells

at least in some patients. It has been suggested that

this reduction of CD4þ lymphocytes in untreated

patients is mainly due to a reduction of CD4þ/

CD45ROþ cells [3].

The immunodeficient state of myeloma patients

was aggravated by chemotherapy given at conven-

tional doses, as well as by HD-CTX. Both forms of

therapies resulted in a significant reduction of CD4þas well as CD4þ CD45ROþ cells and, following

conv-CTX, this reduction was more pronounced in

patients treated for relapsed diseases. In these

patients, peripheral blood counts of CD4þ and

CD4þ/CD45ROþ lymphocyte counts were in the

same range as in patients within the first months after

HD-CTX. For both parameters, a long-lasting

suppression was observed, which was more promi-

nent for total CD4þ lymphocytes. In accordance

with other studies reporting reduced CD4þ lympho-

cyte counts for up to 6 years after HD-CTX [5], in

our hands, suppression of total CD4þ cells still

persisted after 2 years. Reconstitution of the CD4þcompartment within the first year after HD-CTX

was mainly due to recovery of CD4þ CD45ROþlymphocytes, as previously reported [8,30,31]. A

possible explanation for this observation is that

expression of the CD45RO antigen characterizes

memory-type T lymphocytes. The regeneration of

these cells has been shown to be thymus-indepen-

dent, whereas CD4þ/CD45RO – lymphocytes are

considered to be naıve T cells whose restitution is

thymus-dependent. Because the thymus is involuted

in adult patients, the reconstitution of CD4þlymphocytes after HD-CTX mainly follows an

extra-thymic pathway resulting predominantly in

CD4þ/CD45ROþ cells [32], a hypothesis which

also is supported by our data demonstrating severe

and prolonged suppression, particularly of CD4/

CD45RO – cells. The suppressed cell count of

CD4þ lymphocytes also has functional conse-

quences because antigen- and mitogen-induced

T-cell proliferation is significantly reduced within

the first year after HD-CTX [33].

Profound depletion of CD19þ lymphocytes was

observed with conventional-dose as well as HD-

CTX. Following HD-CTX, CD19þ cells recovered

to normal values within 180 days and a similar time

course was observed for nonmyeloma immunoglo-

bulin levels. For CD19þ lymphocytes, this time

pattern has already been described following HD-

CTX administered in the context of other disease

Table IV. Immune parameters of myeloma patients with and without an opportunistic infection.

All patients (n¼ 77) Only patients after HD-CTX{ (n¼ 18)

Yes (n¼8) No (n¼69) P Yes (n¼ 6) No (n¼12) P

Cellular parameters

CD4þ (/ml) 58 (39 – 470) 430 (29 – 1530) 0.001** 48 (39 – 84) 167 (92 – 630) 0.001**

CD4þ/CD45ROþ (/ml) 64 (8 – 440) 240 (29 – 880) 0.007** 45 (8 – 73) 150 (88 – 510) 0.001**

CD4þ/CD45RO– (/ml) 20 (1 – 44) 166 (1 – 890) 0.001** 6 (1 – 31) 18 (3 – 130) 0.036*

CD45RO– to CD45ROþratio

0.15 (0.01 – 3.85) 0.60 (0.01 – 3.42) 0.03* 0.13 (0.01 – 3.85) 0.13 (0.02 – 0.50) 0.62

CD8þ (/ml) 670 (310 – 1130) 430 (30 – 1250) 0.15 590 (310 – 960) 770 (130 – 2200) 0.42

CD3þ/HLA-DRþ (/ml) 400 (190 – 1140) 190 (7 – 1120) 0.005** 290 (130 – 870) 810 (95 – 1750) 0.18

CD4þ to CD8þ ratio 0.18 (0.10 – 0.40) 1.25 (0.10 – 5.2) 0.001** 0.10 (0.04 – 0.17) 0.33 (0.09 – 2.16) 0.04*

CD19þ (/ml) 14 (1 – 250) 64 (2 – 760) 0.01* 5 (1 – 51) 10 (2 – 83) 0.30

NK cells (/ml) 150 (30 – 560) 180 (49 – 540) 0.16 93 (30 – 180) 130 (37 – 580) 0.21

Granulocytes (/ml) 3420 (1180 – 5730) 2840 (600 – 19550) 0.78 3160 (1180 – 5730) 2400 (690 – 5980) 0.62

Monocytes (/ml) 710 (360 – 1020) 580 (110 – 3180) 0.37 670 (140 – 1020) 770 (180 – 1600) 0.38

Nonmyeloma immunoglobulins

Immunoglobulin G (g/l){ 6.51 (3.96 – 12.4) 6.30 (1.42 – 16.1) 0.87

Immunoglobulin A (g/l){ 0.58 (0.12 – 1.76) 0.80 (0.04 – 4.26) 0.45

Immunoglobulin M (g/l) 0.40 (0.19 – 0.66) 0.33 (0.09 – 1.56) 0.63

*,**Significant differences (P 50.05 and P 50.01, respectively) between patients with and without an opportunistic infection.{Values from patients with detectable amounts of myeloma paraprotein of the respective immunoglobulin type were excluded.{For this comparison, a time limit of 2 months after HD-CTX was applied because most of the opportunistic infections occurred within this

time frame.

NK cells, Natural killer cells.

The median values (range) of peripheral blood cell counts and nonmyeloma immunoglobulin serum levels of myeloma patients stratified

according to the existence of an opportunistic infection (‘Yes’ and ‘No’) caused by cytomegalovirus, varicella zoster virus, or pneumocystis

carinii are given.

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entities [31]. Of note, CD19þ lymphocytes were

significantly increased in patients with more than 180

days after HD-CTX compared to untreated mye-

loma patients, and this might reflect the therapeutic

efficacy of HD-CTX and humoral reconstitution in

the absence of myeloma disease.

Interestingly, counts of monocytes were increased

during the second month after HD-CTX, an

observation with some relevance for immunother-

apeutic strategies using monocyte-derived dendritic

cells. Along this line, we have recently demonstrated

high yields of ex-vivo generated monocyte-derived

dendritic cells within the first 6 months after HD-

CTX [34].

In experimental systems, various immunostimula-

tory effects have been described for thalidomide

[15 – 19]. In clinical studies, thalidomide has been

shown to increase the serum levels of soluble

interleukin-2 receptor in myeloma as well as HIV-

infected patients, probably reflecting lymphocyte

activation [11,35]. Despite these immunostimulatory

properties, treatment with thalidomide has been

reported to be associated with severe infections and

neutropenia in myeloma patients [10,21,22,36]. In

the present study, we were unable to detect any

significant humoral or cellular immunodeficiencies

associated with thalidomide treatment when we

compared myeloma patients receiving thalidomide

in combination with conv-CTX with patients receiv-

ing chemotherapy only. In particular, and in

accordance with several other studies [9 – 11,37],

no significant effect on granulocyte counts was

observed, although some studies have reported the

occurrence of neutropenia [36,38]. Furthermore, we

did not observe any significant differences in the

CD4þ and CD8þ lymphocyte counts with respect to

thalidomide treatment. Although this finding recapi-

tulates data by Strupp et al. [39], other studies have

reported significant increases in CD4þ and CD8þlymphocyte counts during thalidomide therapy in

patients responding to treatment [40]. Interestingly,

we observed significantly increased levels of non-

myeloma IgA and IgG in thalidomide-treated

patients. One way to explain this observation is a

therapeutic response to the thalidomide, which in

turn leads to amelioration of the myeloma-associated

hypoimmunoglobulinemia. However, another expla-

nation might be the induction of T-helper cell type 2

cytokine production by thalidomide [17], leading to

stimulation of B lymphocyte differentiation and,

finally, antibody production [41].

The immunodeficiency associated with multiple

myeloma and its therapy is reflected by the

occurrence of opportunistic infections [7,23,42]

and infections with CMV, pneumocystis carinii,

VZV, herpes simplex virus (HSV), aspergillus, or

other pathogens have been described [21,24,42,43].

Although CMV disease usually is observed after

stem cell transplantation, infections due to HSV

and VZV also occur after conv-CTX or even

spontaneously [44]. In our cohort of patients, an

infection with CMV or with Pneumocystis carinii was

only observed within 3 months after HD-CTX, and

only in patients with significantly decreased CD4þlymphocytes. These results are in accordance with

Einsele et al. [45] who reported substantially

reduced CD4þ lymphocytes as a significant pre-

disposing factor for the occurrence of CMV

disease. Because CMV disease is less common in

autologous transplant recipients than allograft

recipients, CMV prevention strategies are not

generally recommended in autologous transplanta-

tion using unselected grafts. Nevertheless, CMV

surveillance and pre-emptive therapy with ganciclo-

vir is recommended if antigenemia levels of 45

pp65þ cells per slide are detected [46]. Pneumo-

cystis carinii is another pathogen causing

opportunistic infections in immunocompromised

patients suffering from AIDS as well as solid or

hematologic malignancies, including multiple mye-

loma [47 – 49]. Here again, depletion of CD4þlymphocytes is an important predisposing factor

[50]. In the present study, the only PCP infection

occurred in a patient with profoundly reduced CD4þlymphocytes. PCP is relatively rare in adult cancer

patients receiving autologous stem cell transplanta-

tion. Thus, no recommendations for prophylactic

treatment with trimethoprim-sulfamethoxazole can

be given on the basis of controlled clinical trials,

although prophylaxis is recommended for patients

with hematologic malignancies receiving intense

conditioning regimens, especially in the context of

suppressed CD4þ lymphocytes [46,50]. Certainly,

the severe outcome of CMV disease or PCP in some

of the patients with hematologic malignancies sup-

ports pre-emptive or prophylactic treatment and the

CD4þ cell count might serve as a useful parameter to

guide these strategies.

In our small cohort, the three patients with a

localized VZV infection demonstrated only minor, if

any, changes in CD4þ lymphocyte counts compared

to overall patients treated with conv-CTX. On the

other hand, evidence exists demonstrating that the

CD4þ lymphocyte compartment may be crucial for

providing VZV-specific immunity because localized

and generalized VZV infections have been reported

in patients with suppressed CD4þ lymphocyte

counts such as those with malignant lymphoma and

those infected by HIV [51 – 53].

In summary, patients with multiple myeloma

display multiple cellular and humoral immunodefi-

ciencies, which increase with both conv-CTX and

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HD-CTX, and constitute an important predisposing

factor for opportunistic infections. These are parti-

cularly observed in the context of low CD4þlymphocyte counts and during the first 3 months

after HD-CTX. For these patients, prophylactic and/

or pre-emptive anti-infectious therapies might be

beneficial. The disease- and treatment-associated

immunodeficiency should also be taken into account

when considering immunotherapeutic interventions

such as antitumor vaccination strategies in myeloma

patients.

Acknowledgment

We thank Mrs C. Wartchow for her help in preparing

the manuscript.

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