American Journal of Hematology 23:81-87(1986)

Quantitation of the Monoclonal Plasma Cell Component in Bone Marrow From Patients With Serum Paraproteinemia and Nondiagnostic Marrow Morphology Vanessa Gordon, Werner Bezwoda, Dennis Derman, Sydney Kramer, and Barry Mendelow

Department of Hematology, School of Pathology of the South African Institute for Medical Research (VG., S.K., B.M.) and Department of Medicine, University of the Witwatersrand (WS., D.D.), Johannesburg, South Africa

Twenty-one patients with serum monoclonal gammopathy but lacking acceptable morphological evidence of myelomatosis were studied with reference to the degree, if any, of monoclonal plasma cell expansion in aspirated marrow samples, enriched for plasma cells and analysed with respect to light chain distribution. Four of these patients had a biopsy-proven plasmacytoma of bone. Bone marrow aspirated from sites distant to the tumor showed clear evidence of infiltration by monoclonal plasma cells in two of the cases studied; the other two patients had normal results. Of the 17 other cases, 14 showed evidence of a monoclonal plasma cell component qualitatively concordant with the serum paraprotein as one would expect. These cases could be subdivided into those with myeloma (six cases) and those with monoclonal gammo- pathy of undetermined significance (eight cases) on the basis of conventional biochem- ical and radiological criteria. Three of the 17 patients, however, did not show evidence of monoclonal plasma cell infiltration, despite the presence of lytic lesions. It is important to recognize this minority group that simulates myeloma but that may well reflect alternative pathology that has not been identified.

Key words: myeloma, plasma cells, monoclonal gammopathy

INTRODUCTION

Current criteria for the diagnosis of multiple myeloma include demonstration of a clear increase (> 10%) in plasma and/or myeloma cells in the marrow and/or biopsy-proven plasmacytoma as essential features without which the diagnosis cannot be made [ 1,2]. However, failure to demonstrate infiltration by morphological exami- nation of single samples occurs not uncommonly owing to the focal nature of the disease. In addition, there remains a group of patients in whom repeated marrow examination fails to establish the presence of plasma cell infiltration.

We have previously described a technique for the separation of normal bone marrow plasma cells, based on a selective affinity of plasma cells for elements of the bone marrow stroma in culture [3]. This technique was shown to be nonselective for plasma cell isotype since the secreted 1gG:IgA:IgM ratios were maintained at constant

Received for publication April 23, 1985; accepted March 6, 1986. Address reprint requests to V. Gordon, Department of Hematology, The South African Institute for Medical Research, P.O. Box 1038, Johannesburg, 2000, South Africa.

0 1986 Alan R. Liss, Inc.

82 Gordon et a1

levels throughout the culture period. This suggested a possible application in refining the detection of monoclonal bone marrow plasma cells in patients with serum para- proteinemia but lacking morphological evidence of marrow infiltration.

The present study was undertaken to establish whether or not objective evidence of monoclonal plasma cell expansion may be found and defined in qualitative terms in the bone marrow of these patients, using light chain restriction analysis of enriched plasma cell fractions. Such information would be potentially useful in determining evidence of dissemination in patients with “solitary” plasmacytoma, and also diag- nostically in other patients in whom current criteria for the diagnosis of multiple myeloma are not fulfilled, because visible evidence of tumor cells is not available from conventional investigation.

PATIENTS AND METHODS Patients

Plasma cell analysis was performed on routine bone marrow aspirate samples obtained from 21 patients with paraproteinemia but lacking morphological evidence of bone marrow infiltration, ie, < lo% bone marrow plasma cells. Four of these patients had biopsy-proven plasmacytomas of bone. Of the remaining 17 patients, eight had bone lesions, and one case had a pathological fracture of a rib, which could have been due to his squamous cell carcinoma of the lung. Only three of the 21 cases had > 3 g% of paraprotein, whereas five had Bence Jones protein in the urine. Concomitant pathologies included one case each of carcinoma, lymphoma, cryoglob- ulinemia, amyloidosis, and polycythemia Vera. Two patients were found to have hepatomegaly, while another had alcoholic liver damage. Information pertinent to each case is included in Table I.

An overt myeloma control group consisting of 31 patients had plasma cell analysis performed on their bone marrow aspirates. All patients in this group had overt myelomatosis, ie, > 10% plasma cells in the bone marrow aspirate, a monoclo- nal gammopathy in the serum and/or urine, and, in the majority of cases, osteolytic lesions on X-ray examination of the skeleton.

Normal values were obtained by immunofluorescent analysis performed on bone marrow plasma cells from 23 patients without primary involvement of the immune system or paraproteinemia/uria.

Culture Technique Bone marrow aspirated at the time of the routine morphological examination

was collected into McCartney’s bottles containing 10 ml of Modified McCoy’s 5A medium (Flow Laboratories) and 100 units of preservative-free Heparin (Pularin, Evans Medical). The bone marrow was layered over Hypaque Ficoll (Protea Medical Surgical, South Africa) and centrifuged at 400g for 30 min. The bone marrow particles and the mononuclear cells at the interface were harvested for analysis. Cultures of bone marrow fragments were performed by the method of Mendelow et a1 131. The particles were cultured in 3-ml plastic petri dishes in Modified McCoy’s 5A medium supplemented with 10% heat-inactivated foetal calf serum. These were incubated at 37°C in a 5% CO2 humidified incubator for 7 days. Postculture frag- ments were then disaggregated with trypsin at 37°C for 10-15 min. The separated cells were then centrifuged gently for 15 min to yield a cell pellet that normally

TA

BL

E I.

Clin

ical

Det

ails

. Im

mun

oflu

ores

cent

Par

anro

tein

Ana

lvsi

s. an

d M

onoc

lona

l Com

mne

nt E

valu

atio

ns*

Perc

enta

ge

Para

prot

ein

Perc

enta

ge o

f of

pla

sma

Para

prot

ein

conc

entra

tion

Bon

e ka

ppa:

m

onoc

lona

l Su

rviv

al

Patie

nt

cells

cl

ass

(g%

) le

sion

s C

linic

al d

etai

ls

1gG

:IgA

la

mbd

a co

mpo

nent

St

atus

(m

onth

s)

GL

1

IgG

K

1.8

Loca

l Pl

asm

acyt

oma

2.60

2.

70

19

A

JM

3 Ig

GK

0.

9 Lo

cal

Plas

mac

ytom

a 1.

30

1.59

8

D

0.2

AM

<

10

IgG

K

1.7

Loca

l Pl

asm

acyt

oma

8 .oO

9.10

70

D

18

JD

3

IgA

1.

2 Lo

cal

Plas

mac

ytom

a 0.

11

0.10

86

LT

F

AK

<

5

IgG

K

1.7

+ M

ultip

le p

lasm

a-

5.00

5.

00

50

D

6.1

cyto

mas

of

bone

cw

1

IgG

K

1.2

+ Pn

eum

onia

N

D

0.14

82

D

7.

0 Ig

GL

1 .o

DD

4

IgG

L 1.

5 +

5.25

0.

63

42

D

5.9

EB

5 Ig

GK

3.

7 +

Bon

e pa

in

> 10

0 >

100

> 97

D

10

.2

EZ

<5

Ig

GL

2.3

+ C

a lu

ng

11.2

0 0.

19

76

D

19.5

M

R 10

Ig

GK

4.

1 +

Para

pleg

ia, b

one

> 10

0 >

100

> 97

D

17

.0

AN

4

IgG

L I .

6 N

D

1.07

22

A

>3

0.9

AP

4 Ig

GK

0.

9 -

Bon

e pa

in

5.75

7.

10

63

A

>23.

7

pain

-

IgG

K

0.7

BM

2 Ig

GK

1.

8 -

Lym

phom

a >

100

> 10

0 >

97

A

>21.

1 D

W

10

IgA

L 1.

2 Pa

ncre

atiti

s 0.

14

0.37

59

D

20

.6

FS

3 Ig

GL

1.1

-

Rep

eate

d in

fec-

46

0.

02

97

LTF

HW

2

IgG

L 0.

6 -

Cry

oglo

bulin

emia

9.

50

0.50

50

A

>6

1.6

MK

0.

5 Ig

GK

0.

9 -

Hep

atom

egal

y N

D

3 .oo

25

A

> 12

.7

PR

7 Ig

GK

2.

2 -

Poly

cyth

emia

Ver

a >

100

> 10

0 >

97

A

>42.

7

PH

5 Ig

GK

1.

2 +

Alc

ohol

ic li

ver

0.85

1.

60

8 A

>

15.4

RG

6

IgG

L 3.

1 +

Hep

atom

egal

y ,

1.56

1.

31

15

LTF

-

tions

dam

age

rheu

mat

oid

arth

ritis

SM

8

IgG

K

0.6

+ Ev

iden

ce of

1.

35

1.48

10

D

17

.8

amy l

oido

sis

*+, p

rese

nt; -,

abs

ent;

ND

, not

don

e; A

, aliv

e; D

, dea

d; L

TF, l

ost t

o fo

llow

-up;

Ca,

car

cino

ma.

84 Gordon et a1

contained a nonadherent fraction consisting of 60-95 % plasma cells. This technique was useful in obtaining enriched plasma cell suspensions from these patients with fewer than 10% plasma cells in unfractionated marrow samples.

Heavy-Chain lsotype and Light-Chain Distribution Cytocentrifuge preparations of the cells were stained with commercially ob-

tained FITC-conjugated anti-human IgG, IgA, IgM, IgD, kappa, or lambda antisera (Hyland Laboratories, Costa Mesa, CA) using the method of Hijmans et a1 [4]. Preparations were examined under a Reichert Zetopan fluorescence microscope, using transmitted illumination. The percentages of cells containing cytoplasmic IgG, IgA, and where necessary (when a serum paraprotein was present and the combined IgG and IgA percentages were less than the percentage of plasma cells in the enriched sample analyzed) IgM and IgD, were counted on each slide, and the results were expressed as ratios. Similar calculations were applied to the quantitation of lambda and kappa light-chain-containing cells.

Calculation of the Monoclonal Plasma Cell Component The mean kappa: lambda ratio in the 23 negative control patients was found to

be 2.0 -t 0.4 (SD):l. This is similar to the results of 1.6 and 2.2 obtained by Morell et a1 [5] and Hijmans et a1 [6]. This figure of 2: 1 was used as the basis for calculation of the monoclonal plasma cell component in the other patients in this study.

For the purpose of calculation, patients with observed kappa:lambda ratios of > 2: 1 (kappa clones) were treated differently from those with observed kappa:lambda ratios of < 2: 1 (lambda clones), in that for the former group the normal kappa:lambda ratio of 2: 1 was used in the calculation, while in the latter the reciprocal 1ambda:kappa ratio of 0.5: 1 was employed.

Thus for kappa clones,

Normal K:X + 1 Patient K:X + 1

% normal plasma cells = x 100,

eg, for a hypothetical patient with an observed K:X ratio of 4:1, % normal plasma cells = (2 + 1/4 + 1) x 100 = 60%.

For lambda clones,

Normal X:K + 1 Patient X:K + 1

% normal plasma cells = x 100,

eg, for a hypothetical patient with an observed K:X ratio of 0.6, the reciprocal h : ~ ratio = 1/0.6 = 1.7. Applying the formula:

0.5 + 1 1.7 + 1

% normal plasma cells = ~ x 100 = 56%, (3)

for both groups, % monoclonal plasma cells = 100 - % normal plasma cells. These calculations are based on the assumption that the degree of suppression

of normal kappa and lambda light-chain-producing cells is equivalent. It should be

Quantitation of Monoclonal Plasma Cells 85

noted, however, that there is evidence to suggest that normal peripheral blood lymphocytes and gut-related plasma cells may exhibit a degree of selective light-chain suppression of the isotype involved in the paraprotein [7,8]. This phenomenon is related to the level of the paraprotein and was virtually negligible in patients with benign monoclonal gammopathy [8]. Whether or not it applies to bone marrow plasma cells is unclear, but as the patients in the present study generally had low levels of paraprotein, its effect is likely to have been minimal in any event. Nevertheless, the calculated monoclonal components should be regarded as conservative or minimum values, because if selective light-chain inhibition of the type described was operative, it would have had the effect of depressing the calculated monoclonal component.

RESULTS

All the cases in the overt myeloma control group displayed clonal expansion of plasma cells in the bone marrow as evidenced by disturbed 1gG:IgA and/or kappa:lambda ratios compatible with the serum paraprotein class. The ratios were commonly grossly abnormal, with calculated percentage monoclonal plasma cell components ranging from 32 % to > 99 % .

Details of the 21 patients under study, their 1gG:IgA and kappa:lambda ratios, and calculated monoclonal components are presented in Table I. The patients have been grouped into those with “solitary” plasmacytomas, and those with other forms of presentation.

Of the four patients with “solitary” plasmacytoma, two were found to have plasma cell kappa:lambda ratios more than 2 standard deviations beyond the normal mean in marrow samples from sites far removed from their plasmacytoma. The calculated monoclonal components were 70% and 86% of the total marrow plasma cells. Furthermore, the class of clone inferred by the kappa:lambda and/or 1gG:IgA ratios were concordant with available data on the serum paraprotein in these two cases. In patient J.D., light-chain concordance could not be assessed because no data is available on the light-chain analysis of the serum paraprotein. In contrast, the other two patients had ratios within 2 SD of the normal mean and minor inferred clones (8% and 19%), which were not concordant with the serum paraprotein class and therefore probably representative of normal variation and/or limits of precision of the technique. It was concluded that the results indicated evidence of monoclonal plasma cell dissemination in the former two cases only.

The remaining 17 patients again showed one of two patterns of results. Fourteen had kappa:lambda ratios outside the normal range and inferred plasma cell clones that were fully concordant with the serum and/or urine paraprotein or Bence Jones protein class, comprising between 22 % and > 97 % of the total marrow plasma cell fraction. In patient M.K., concordance could not be assessed because the serum paraprotein was biclonal, although the marrow plasma cells appeared to reflect predominantly the IgG (lambda) clone. These 14 patients with evidence of monoclonal plasma cell dissemination were now readily subdivisible into those with myeloma (A.K., C . W., D.D., E.B., E.Z., and M.R.) and those with monoclonal gammopathy of undeter- mined significance (A.N., A.P., B.M., D.W., F.S., H.W., M.K., and P.R.) on the basis of whether or not they had lytic lesions and/or serum paraprotein levels > 3 g % . The precision of these prognostic variables is reflected in the striking differences in survival time of these two groups since investigation. While all six patients with

86 Gordon et a1

myeloma were dead at that time (survival times all <20 months), six of the eight MGUS patients were alive with a mean follow-up time of 32.1 months (range 12.7- 61.6 months).

Three of the 17 patients (P.H., R.G., and S.M.) had kappa:lambda ratios within the normal range, and minimal clones (< 20%) that were not concordant with the serum paraprotein class. These cases were interpreted as lacking evidence of mono- clonal plasma cell expansion in the bone marrow, despite their resemblance to myelomatosis. One of these patients (S.M.) had lytic lesions and evidence of amyloi- dosis. He then received two courses of melphalan and prednisone and was reinvesti- gated 17.4 months later. At this time the monoclonal plasma cell component was still shown to be < 20%. This patient died of renal failure as a result of amyloidosis 17.8 months after initial investigation. Patient R.G. was noted to have hepatomegaly and anemia of chronic disorder. He was also shown to be positive for rheumatoid factor and serum electrophoresis, and immunoelectrophoresis revealed a polyclonal gam- mopathy with monoclonal and oligoclonal elements. This patient was lost to follow- up. Patient P.H., who had alcoholic liver damage, had multiple lytic lesions, a small IgG paraprotein, and a polyclonal increase in all immunoglobulin classes. The bone marrow picture was suggestive of megaloblastic anemia, and plasma cells constituted 5 % of the myelogram. This patient is still alive at 15.4 months after investigation.

DISCUSSION

In patients with overt myelomatosis, characterized by morphologically obvious bone marrow plasma cell infiltration, osteolytic lesions, and significant paraproteine- mia or Bence Jones proteinuria, the diagnosis and decision to treat accordingly present little difficulty. However, there remains a group of patients in whom these features are insufficiently developed to allow a confident diagnosis to be made. This problem has relevance in assessing the degree of dissemination in patients with “solitary” plasmacytoma, and also in those patients with paraproteinemia but who lack morpho- logical evidence of bone marrow plasma cell infiltration.

Two of the patients who had “solitary” plasmacytoma of bone were shown to have definite evidence of dissemination. In contrast, the other two patients in this group had no such evidence. Since the bone marrow investigated here was aspirated from a distant site, the lesion could now be diagnosed as being truly solitary or at least as lacking evidence of widespread dissemination.

Fourteen of the 17 patients without obvious solitary plasmacytomas showed evidence of monoclonal plasma cell expansion as would be expected. Of these 14 cases, six had, in addition to evidence of disseminated monoclonal bone marrow involvement, high serum globulin peaks and/or multiple lytic lesions. In these six patients (all deceased), a confident diagnosis of myeloma could now be made. The remaining eight patients had neither bone lesions nor high serum peaks and could now be diagnosed as having monoclonal gammopathy of undetermined significance (MGUS) with disseminated bone marrow involvement. The presence of monoclonal plasma cells in benign gammopathy is consistent with other studies showing a single clone of cells by flow cytometry [9].

Of particular interest are the three patients in the latter group who lack evidence of monoclonal plasma cell expansion. They can be said to have a monoclonal gammopathy of undetermined significance without evidence of disseminated bone

Quantitation of Monoclonal Plasma Cells 87

marrow involvement. It is therefore difficult to justify a diagnosis of multiple mye- loma in these cases despite their radiological and biochemical mimicry of this condition.

In conclusion, our results indicate that monoclonal gammopathy does not nec- essarily denote monoclonal plasma cell infiltration of bone marrow even when using refined techniques capable of detecting monoclonality in marrow samples containing as few as 0.5% total plasma cells (patient M.K.).

ACKNOWLEDGMENTS

This study was supported by grants from the National Cancer Association and the Medical Research Council of South Africa.

REFERENCES

1. Costa G , Engle RL, Schilling A, Carbone P, Kochwa S, Nachman RL, Glidewell 0: Melphalan and prednisone: An effective combination for the treatment of multiple myeloma. Am J Med 54:589, 1973.

2. Bergsagel DE, Carbone PP, Costa G, Engle RL, Gehan E, Griffith K, Huguley CM, Steinfeld JL, Weiner L, Jones BL: Proposed guidelines for protocol studies. II. Plasma cell myeloma. Cancer Chemother Rep 4: 145, 1973.

3. Mendelow B, Grobicki D, Katz J, Metz J: Separation of normal mature bone marrow plasma cells. Br J Haematol45:251, 1980.

4. Hijmans W, Schuit HRE, Klein F: An immunofluorescence procedure for the detection of intracel- lular immunoglobulins. Clin Exp Immunol4:457, 1969.

5. Morel1 A, Skvaril F, Huser HJ, Splengler G, Barandun S: Cytoplasmic immunoglobulins in bone marrow cells of polyclonal and of monoclonal origin. Acta Haematol60:137, 1978.

6. Hijmans W, Schuit HRE, Hulsing-Hesselink E: An immunofluorescence study on intracellular immunoglobulins in human bone marrow cells. Ann NY Acad Sci 177:290, 1971.

7. Leonard RCF, MacLennan ICM, Smart Y, Vanhegan RI, Cuzick J: Light chain isotype-associated suppression of normal plasma cell numbers in patients with multiple myeloma. Int J Cancer 24:385, 1979.

8. Wearne A, Joshua DE, Kronenberg H: Light chain isotype associated suppression of surface immunoglobulin expression on peripheral blood lymphocytes in myeloma during plateau phase. Br J Haematol58:483, 1984.

9. Latreille J, Barlogie B, Johnston D, Drewinko B, Alexanian R: Ploidy and proliferative character- istics in monoclonal gammopathies. Blood 59:43, 1982.