An IgM Pyroglobulin Associated with Lymphosarcoma%

ROY PATTERSON, M.D.

MARY ROBERTS,

WALTER RAMBACH, M.D.

ANGELO FALLERONI, M.D.

Chicago, Illinois

* From the Department of Medicine, Sections

of Allergy-Immunology and Hematology, North-

western University Medical School and the De-

partment of Research, Chicago Wesley Memorial

Hospital. This study was supported by U. S.

Public Health Service Research Grant 5 TO1 Al

00057 from the National Institute of Allergy and

InfectiokDiseases and in part by an Ernest S.

Bazley Research Grant to Chicago Wesley Me-

morial Hospital and Northwestern University,

Chicago, Illinois. Requests for reprints should

be addressed to Dr. Roy Patterson, Allergy Re-

search Laboratory, Northwestern University

Medical School, 303 East Chicago Avenue, Chi-

cago, Illinois 60611. Manuscript received July

11, 1969.

A pyroglobulin found in the serum of a patient with lymphosarcoma and pancytopenia proved to be a 17s immunoglobulin identified as an IgM type. The thermolability of the IgM pyroglobulin was dependent on the intact molecule since the 7s monomers of the macroglobulin pentamer were not heat precipitable. Reconstruction of the whole molecule by reassociation of the reduced 7S subunits restored the thermoprecipitability. The serum concentration of the IgM pyroglobulin was not substantially altered by treatment of the patient with cyclophosphamide, despite some clinical response evidenced by a reversal of the patient’s pancytopenia.

Heat-precipitable immunoglobulins have been associated not only with plasma cell myeloma [l] but also with lymphoproliferative neoplasia [2,3]. These myeloma immunoglobulins, which have been termed pyroglobulins

[l], have all been of the IgG type with the exception of a thermolabile IgM described by Meltzer and Franklin 131 which manifested both heat and cold precipitability. We describe a pyroglobulin of the IgM type asso- ciated with a small cell lymphosarcoma. Studies of the pyroglobulin demonstrated that the heat precipitability was dependent on the molecule’s existing as an intact polymer since 7’S subunits were not thermolabile.

CASE REPORT

A fifty-two year old man was hospitalized for diagnostic evaluation because of the results of a health evaluation examination which demonstrated anemia, leukopenia and thrombocytopenia. He had a thirty year history of bruising following minor trauma and a two year history of progressive anemia which had not been characterized. He had no specific complaints. The findings on physical examination were normal except for moderate pallor and two ecchy- moses on the extremities.

Laboratory examinations revealed a hemoglobin of 7.6 gm per cent, hema-

tocrit 26 per cent, red blood ceil count 2,900,OOO per cu mm, reticuloctye count 1.7 per cent and white blood cells 1,850 per cu mm with 59 per cent polymorphonuclears, 26 per cent lymphocytes, 7 per cent eosinophils, 4 per cent monocytes and 2 per cent metamyelocytes. The platelet count was 95,000 per cu mm. Blood urea nitrogen was 18 mg per cent and serum total protein was 6.7 gm per cent. Paper electrophoresis of serum revealed a monoclonal peak migrating in the bsta, region. This protein was characterized as an IgM pyroglobulin. The urinalysis was normal, and no Bence Jones protein was detected. Roentgenographic studies, including chest examination, an x-ray series of the gastrointestinal tract and an intravenous pyelogram, were normal. A Schilling test was normal. Bone marrow examination revealed a preponderance of mature lymphocytes (Fig. 1) with an occasional prolym- phocyte. A few mature granulocytes, rare megakaryocytes and scattered normoblasts were present. A diagnosis of lymphosarcoma was made, and treatment was initiated with 20 mg of nitrogen mustard in two divided doses. Four blood transfusions were administered. Following discharge from the hospital, treatment was continued with cyclophosphamide, 50 mg daily, prednisone, 30 mg daily, and fluoxymesterone, 30 mg daily. The patient re- mained asymptomatic on this regimen, and the peripheral blood picture returned to normal. Thirteen weeks after the patient was discharged jaundice developed, and the chief physical finding was tender hepatomegaly. Abnormal

Volume 48, April 1970 503

AN IGM PYROGLOBlJLlN ASSOCIATED WITH LYMPHOSARCOMA - PATTERSON ET AL.

Fig. 1. Bone marrow specimen from patient with lymphosarcoma

and IgM pyroi:lobulin. Mature lymphocytes are present.

laboratory flndings included serum total bilirubin 5.5 mg

per cent (direct fraction 4.5 mg per cent), serum glutamic

oxalacetic transaminase (SGOT) 1,500 units per ml, serum

glutamic pyruvic transaminase (SGPT) 1,980 units per ml

and lactic dehydrogenase 840 units per ml. The diagnosis

was considered to be serum hepatitis. Over a period of

several days the patient showed no improvement; suddenly

severe abdominal pain and shock developed and the patient

died. Postmortem examination disclosed retroperitoneal

hemorrhage of the mesenteric root and pancreas, massive

necrosis of the liver, and ascites and atelectasis of both

lower lungs.

METHODS Measurement of Pyroglobulin. Serum samples, 0.1 ml of

each, were heated at 60°C for one hour. The precipitate which formed was removed by centrifugation at 5,000 rpm and washed three times with 0.15 M sodium chloride. The precipitates were then dissolved in 0.1 N sodium hydroxide, and the optical density at 280 rnfi for each sample was

determined.

Electrophoresis. Zone electrophoresis of serum was per- formed in agar gel using Lonagar No. 2 at pH 8.2 in 0.05 ionic strength barbital buffer. Following electrophoresis, slides were heated to 6O”C, photographed, stained with thiazine red R [4] and photographed again. Paper electro-

phoresis was carried out by standard procedures with a

Beckman Model R apparatus.

Dextran Gel Chromatography. Serum was fractionated

by passage through a Sephadex@ G-200 (Pharmacia) 2.5 x

100 cm column using 1 M sodium chloride in 0.1 M Tris-

HCl, pH 8.0 [5]. Reduction and Alkylation. The IgM of the serum was

contained in peak I eluate after Sephadex G-200 chroma- tography. Six milliliters of IgM solution containing 16 mg

protein per ml was dissociated using the method of Schro-

henloher et al. 161. Following treatment with 0.1 M 2-mer-

captoethanol, the reduced subunits of IgM pyroglobulin were

allowed to reassociate by removal of the mercaptoethanol

by dialysis [6] or were alkylated with 0.02 M iodoacetamide. Samples of the reduced and alkylated IgM subunits were

passed through a Sephadex G-200 column in 0.2 ionic

sodium phosphate buffer, pH 7.5.

Analytical Ultracentrifugation. Samples of whole IgM py-

roglobulin obtained from the peak I eluates of the Sephadex

G-200 chromatograph and reduced and alkylated samples of

the IgM pyroglobulin were analyzed by standard technics

using the Beckman Model E analytical ultracentrifuge 171

(The analyses were performed by Dr. Burton Andersen).

Determination of Immunoglobulins. Concentrations of

IgG, IgA and IgM in whole serum or in eluates obtained

from Sephadex G-200 chromatographic preparations were

estimated by means of radial diffusion plates (Hyland)

using antiserum specific for G, A and M heavy chains. A

test for the presence of. IgD was performed qualitatively by

double gel diffusion precipitin technics [8], using precipitat-

ing anti-IgD-serum (Hoechst).

RESULTS Demonstration of IgM as a Pyroglobulin. The patient’s serum was initially studied for the presence of cryoglo-

bulins and pyroglobulins by adjustment to 4” and 60°C. No precipitation occurred at 4”C, but a dense white pre- cipitate occurred at 60°C. The serum after heating had the appearance of a “white clot,” as previously seen with other pyroglobulin precipitates [2]. The determination of the serum immunoglobulins in the patient’s serum by radial diffusion technic demonstrated 950 mg per cent of IgM, 35 mg per cent of IgA and 365 mg per cent of IgG. No IgD was detected.

Separate e,lectrophoretic preparations of whole serum, which were either stained for protein or heated to 6O”C, demonstrated that the thermolabile protein migrated elec- trophoretically as a beta globulin (Fig. 2). These results suggested that the M peak which migrated as a beta globulin on paper electrophoresis was a pyroglobulin.

Separation of the immunoglobulins by Sephadex G-200 (Fig. 3) demonstrated a much higher protein concen- tration in peak I, as compared with a similar separation of normal human serum. Correspondingly there was a marked increase in the IgM content of the peak I frac- tions in which the thermoprecipitable protein was local- ized. No other fractions were precipitated by heat. The results of these experiments showed that the thermolabile protein was an IgM pyroglobulin. The temperature re- quirements for precipitation of the protein are shown in Figure 4. Characteristics of IgM Monomers. The IgM pyroglobulin was reduced with mercaptoethanol and divided into two aliquots. One aliquot was allowed to reassociate by dialysing in phosphate buffer, which removed the mer- captoethanol; the other aliquot was alkylated with iodo- acetamine. The products of these procedures were fractionated by gel filtration on Sephadex G-200. The IgM polymer formed by reassociation of the subunits was located in peak I (Fig. 5). Following reduction and alky lation, the IgM determinant was detected largely in peak II (Fig. 5).

Heat precipitability of the IgM whole molecule and its subunits was tested in the following manner. Samples containing the same concentration of protein (7 mg per ml) were prepared. Samples of unreduced IgM, reduced

504 The American Journal of Medicine

AN IGM PYROGLOBULIN ASSOCIATED WITH LYMPHOSARCOMA - PA7TERSON ET AL.

4.

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D-

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I , I I 30 40 50 60 70 30 40 50 60

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Fig. 2. A, agar electrophoresis slide stained with

thiazine red R. B, identical preparation as A heated

to 60°C without staining. PS, patient’s serum; SP,

Sephadex G-200 peak I; NS, normal human serum.

d Fig. 3. Sephadex G-200 fractionation of patient’s serum (left) and normal human

serum (right). IgG,lgA,lgM and P, thermoprecipitable proteins are localized frac-

tions of each serum.

and alkylated IgM, reduced and reassociated IgM and reduced, alkylated and separated IgM subunits were heated to 60°C for one hour and the protein content of the washed precipitate was measured. Eighty-two per cent of the whole IgM was precipitated by heat. None of the fractionated peak II 7S subunits of IgM or the re- duced, alkylated and unfractionated IgM was precipitated by heat. The results demonstrated that the thermolabil-

ity of the IgM pyroglobulin was dependent upon the intact molecule and that the monomeric subunits of the IgM pentamer were not heat labile. Examination by ultra- centrifugal analysis of the whole IgM molecule and its subunits demonstrated that the whole IgM molecule had an s20v of 17.2, whereas the SzoW for the reduced and alkylated subunits was 7 (Fig. 6). Clinical Response to Treatment. The therapeutic reg-

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Fiu. 4. Effect of tamDerature and dilution 0 t05-

on precipitation of IgM pyroglobulin. Left,

precipitate resulting from heating whole

serum at various temperatures. Right, effect

of dilution on amount of protein precipitated e,,. 58’ 56’ 54. 52.0 by heating to 60°C for thirty minutes. SERUM ‘:’ DILUTION

I:4 TEMPERATURE CC.1

Volume 48, April 1970 505

AN IGM PYROGLOBULIN ASSOCIATED WITH LYMPHOSARCOMA - PATTERSON ET AL.

x

x

I

’ :, , I

a I\ Fig. 5. Thermoprecipitability of dissociated

and reassociated subunits of IgM pyroglobu-

lin. Top, Sephadex G-200 fractionation. Bot-

tom, localization of IgM in Sephadex frac-

tions by radial diffusion plates. X-X,

reassociated IgM subunits; O----O, dis-

sociated IgM subunits; P. fractions pre-

cipitated at 60°C.

imen employed was followed by an increase in hemoglobin, hematocrit and platelet count (Fig. 7). The total leukocyte count fluctuated but was generally higher than pretreat-

ment levels. The IgM pyroglobulin level did not appear to be altered by prolonged chemotherapy (Fig. 7).

COMMENTS The hematologic abnormalities of the patient consisted of

pancytopenia and infiltration of the bone marrow with lymphocytes. These were small, mature lymphocytes with an occasional prolymphocyte. There was no associated

lymphadenopathy or hepatosplenomegaly. The serum portein abnormalities found were a reduction in normal levels of IgA and IgG and a monoclonal IgM peak. The

cytologic changes, consistent with a lymphosarcoma, and the monoclonal IgM peak suggest more than a coincidental relationship between these two abnormalities. Definitive

evidence that the small lymphocytes were involved in production of the IgM protein was not established by immunofluorescent or tissue culture technics. No cells of the lymphocytoid plasma cell type seen in patients with Waldenstrom’s macroglobulinemia were noted. The possibility that the lymphocytes were producing the IgM protein must be considered because sufficient num- bers of other cell types which could be responsible for the monoclonal immunoglobulin could not be demon-

strated. A previous case records an IgG pyroglobulin associated with an infiltrate of lymphocytic type cells in the bone marrow; in that case, culture of lymphocytic cells

from a lymph node was followed by a morphologic altera-

tion from lymphocytic to plasma cell types [2]. The IgM pyroglobulin did not appear to result in any

clinical abnormalities, such as symptoms of vascular in- sufficiency, which may be associated with cryoglobulins

Fig. 6. Ultracentrifuge patterns of whole

IgM pyroglcbulin and the reduced and

alkylated subunits. W, whole IgM; R. reduced

to subunits. The photographs were taken

after eight, twenty-four, forty and fifty-six

minutes at 59 and 780 rpm

506 The American Journal of Medicine

AN IGM PYROGLOBULIN ASSOCIATED WITH LYMPHOSARCOMA - PATTERSON ET AL.

I WK.

ULCTRO- PMORESIS

n

+ 14 WK.

PREoNlSoNE 30 MG DAILY 20 W a.0.d.

FLUOXYMESTERONE 30 MC DAILY ..__.~~~ ____~ f I 2 3 + 4 5 6 7 I

DlSb AKGED

6 I 9 , lo ll I2 , I3 I 14 I l5 1 I6 17 !

ADMITTED WEEKS

Fig. 7. Clinical course of patient with IgM pyroglobulinemia and lymphasarcoma.

[9]. The lack of symptoms due to the IgM pyroglobulin is similar to observations noted in patients with IgG pyroglobulins in whom there were no apparent clinical manifestations resulting from the presence of the ther-

molabile protein [2]. A comparison of certain characteristics of the IgM

pyroglobulin and two IgG pryoglobulins (Table I) showed similarities in that none of the pyroprecipitates dissolved in temperatures beneath the level required to precipitate the proteins. However, a difference between the thermo- precipitability of the molecular subunits was observed; the IgM pyroglobulin required the intact molecule for thermoprecipitation but the 7s subunits remained soluble after heating. Both 7s IgG pyroglobulins tested in our laboratory had thermoprecipitable heavy chains [2].

All thermoprecipitable immunoglobulins which have been described previously have been of the IgG type, with the exception of an IgM paraprotein which was both a cryoglobulin and a pyroglobulin [3].

TABLE I Comparison of IgM Pyroglobulin with Two IgG Pyroglobulins

- Pyroglobulin

-

Data tgM IgG* IgG*

Temperature require- 60°C 56°C 60°C ment for precipitation

L chain type Lambda Kappa Kappa Sedimentation char- 17.2s 7s 7s

acteristics (SW)

Heat precipitability 7s subunits H chain H chain of molecular do not precipi- precipi- subunits precipitate tates tates

Reversibility of heat No No No precjpitability on cooling

* Characteristics reported previously [2].

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AN IGM PYROGLOBULIN ASSOCIATED WITH LYMPHOSARCOMA - PATTERSON ET AL

The studies of Meltzer and Franklin [3] on the char-

acteristics of cryoglobulins demonstrate certain compar-

ative aspects of these thermolabile proteins. The cold precipitability of the cryoglobulins was readily reversible

with warming, in contrast to the irreversible heat pre- cipitation of the IgM pyroglobulin. Both the IgM cryo- globulins and IgM pyroglobulin, after reduction with mercaptoethanol, lost their thermolability. After the dis- sociated fragments were reassociated, thermolability was restored.

The studies of cryoglobulins were interpreted as a demonstration that the mechanism of cryoprecipitation is the result of protein-protein interaction at low temper-

ature through a weak, noncovalent bond. The mechanism of pyroprecipitation may also be on the basis of protein- protein interaction. The irreversible reaction of the pyro-

precipitate of the IgM, the IgG and the IgM cryopyroglo.

bulin [3] indicates a different type of protein-protein

bonding. In an analysis of seven IgG myeloma cryoglobulins by

Grey et al., six showed anti-IgG activity directed against the Fc fragment of primate immunoglobulins [lo]. The demonstration of any specificity in the pyroglobulins has not yet been possible, partially because of the apparently complete irreversibility of the pyroglobulin precipitate reaction.

The good hematologic response of our patrent to ther-

apy with nitrogen mustard, cyclophosphamide, fluoxyme- sterone and prednisone, as evidenced by the reversal of the pancytopenia, was not accompanied by a decrease in the level of the circulating IgM pyroglobulin during the period treatment was given.

REFERENCES

1. Martin WJ. Mathieson DR: Pyroglobulinemia. Heat coagulable

globulin in the blood. Proc Mayo Clin 28: 545, 1953.

2. Patterson R, Weiszer I, Rambach W, Roberts M, Suszko IM:

Comparative cellular and immunochemical studies of two

cases of pyroglobulinemia. Amer J Med 44: 147, 1968.

3. Meltzer MD, Franklin EC: Cryoglobulinemia. A study of twenty-

nine patients. Amer J Med 40: 828, 1966.

4. Crowle AJ: Immunodiffusion, New York, Academic Press, 1961,

p 268

5 Flodin P. Killander J: Fractaonation of human serum proteins by

gel filtration. B&him Biophys Acta 63: 403, 1962.

6. Schrohenloher RE. Kunkel HG. Tomasi TB: Activity of dissociated

and reassociated 19s anti-?-globulin, J Exp Med 120: 1215,

1964.

7. Schachman HK: Methods in Enzymology, vol 4 New York.

Academic Press Inc. 1957, p 32.

8. Ouchterlony 0: Antigen antibody reactions in gels. Acta Path

Microbial Stand 32: 231, 1953.

9. Ritzmann SE, Lewin WC: Cryopathies. A review. Arch Intern Med

107: 754, 1961.

10. Grey HM. Kohler PF, Terry WD, Franklin EC: Human monoclonal

IgG-cryoglobulins with anti-r-globulin activity. J Clin Invest

47: 1875, 1968.

508 The American Journal of Medlclne