Medical and Pediatric Oncology 7: 181-190 (1979)
Successful Therapy of Crystalcryoglobulinemia: A Case Report F. J. Cummings, MD, C. H. Park, MD, H. A. Bogaars, MD, A. E. Kalderon, MD, I. Melnicoff, DO, S.R. Kaplan, MD, 1. Diamond, MD, and P. Calabresi, MD Departmentsof Medicine (F.J.C., C.H.P., l.M.,S.R.K., P.C.) and Pathology (H.A.B., A.E.K., I. 0.1, Roger Williams General Hospital, Providence, and Program in Medicine, Brown University, Providence
Successful therapy for a case of multiple myeloma with a spontaneously crystallizing cryoglobulin of the IgG2-kappa light chain variety was achieved, using both continuous-flow cell centrifugation plasmapheresis to rapidly lower the M component and combination chemotherapy with phenylalanine mustard, prednisone, procarbazine, and vincristine to control the myeloma process. This resulted in resolution of incapacitating large and small necrotic cutaneous ulcerations of the extremities. Physicochemical studies of the crystalcryoprotein demonstrated that cryoprecipitation was rapid and accompanied by the formation of needleshaped crystals, yet was completely reversible at 37'C. Cryocrit determinations varied depending upon relative centrifugal forces and temperature and did not always relate linearly to the amount of abnormal protein, thus making these alone unreliable in assessing response to therapy. Key words: myeloma, IgC2-kappa, chemotherapy, plasmapheresis, necrotic cutaneous ulcerations, crystalcryoprotein
The occurrence of a spontaneously crystallizable human immunoglobulin was first noted in 1938 by von Bonsdorff et a l in a patient with multiple myeloma [ 13. Cryocrystalline proteins have been identified only rarely since that report in patients with multiple myeloma or in association with poorly defined clinical syndromes with symptoms secondary to vascular insufficiency.
Dr. Park is now at the University of Kansas Medical Center, Kansas City, KS 66 103; Dr. Kalderon is at the University of Arkansas Medical Center, Little Rock, AR 77201; Dr. Melnicoff is at the Chicago College of Osteopathic Medicine, Chicago, IL 60615. Address reprint requests to F. J. Cummings, MD, Division of Oncology-Hematology, Department of Medicine, Roger Williams General Hospital-Brown University, Providence, RI 02912.
0098-1532/79/0702-0lS1$02.00 @ 1979 Alan R. Liss, Inc.
Cummings e t a1
We have achieved an excellent response to therapy in a patient who presented with many incapacitating large and small necrotic cutaneous ulcerations of the extremities. He was found to have overt multiple myeloma, and his serum contained a spontaneoulsy crystallizing cryoglobulin. The previously undescribed tubular crystal structure of this IgG cryoglobulin has been noted . This report details his clinical presentation and successful therapy with continuous-flow cell centrifugation-plasmapheresisand combination chemotherapy. CASE REPORT
A 53-year-old white used-car salesman was admitted to Roger Williams General Hospital because of ulcerating lesions and hard eschars of both lower extremities. Coinsized brownish indurated skin lesions of both lower legs appeared approximately four months prior to admission and became larger three to four weeks later. New smaller lesions also appeared on other parts of the legs, ears, arms, and chest at this time and they continued to enlarge, until one week prior to admission he experienced burning, numbness, and dull pain over both lower legs to the extent that he was unable to straighten his knees. He had anorexia and lost approximately 18 pounds over this four-month period. On physical examination, he was found to be emaciated, lying in bed with his knees flexed. He had multiple ulcerations covered with hard brownish eschars surrounded by a thin rim of erythema located mostly on the legs, but also on the chest, elbows, and ear lobes. These ranged in size from less than 1 cm to greater than 10 cm in diameter (Fig. 5). The remainder of the physical exam was noncontributory. Initial laboratory evaluations revealed a hemoglobin of 8.8 gm%, hematocrit 25%, and normal platelets, leukocyte count, and differential. Reticulocytes were 1.5%. Total serum protein was 12.4 gm% with albumin 1.7 gm% and globulin 10.7 gm%. Serum sodium was 124 mEq/liter, chloride 88 mEq/liter, and potassium 3.1 mEq/liter. Serum calcium was 7.9 mg%. Serum cholesterol was 91 mg%. All other SMA-12 values were within normal limits. Serum protein electrophoresis revealed an M component in the fast gamma range. Quantitative immunodiffusion showed IgG 12.23 gm%, IgM 0.03 gm%, IgA 0.05 gm%, and IgD was not measurable. Immunoelectrophoresis on agarose gel (Scheidegger), using heavy- and light-chain-specific antisera (Hyland), identified the M component as IgG with kappa light chains. The heavy-chain subclass was determined to be IgG2.* A 24-hour collection of urine contained 420 mg protein, and electrophoresis after 150-fold concentration demonstrated a narrow, tall peak in the same region as the serum peak. Venereal Disease Research Laboratory test (VDRL), latex fixation, LE test, antinuclear antibody, prothrombin time, partial thromboplastin time, bleeding time, and fibrinogen level were normal. Tests for cold hemolysins and cold agglutinins were negative and urine hemosiderin was positive. Serum C3 was 31 mg% and 50 mg% (normal 121-167 mg%) on two determinations. Serum haptoglobin and free hemoglobin were normal and urine hemoglobin was negative. On skeletal films, osteolytic lesions were noted in both tibiae, the pelvis, and the neck of the left femur, and there was collapse of the seventh thoracic vertebra. A posterior iliac crest bone marrow biopsy revealed complete marrow replacement by sheets of well-differentiated plasma cells. On immunofluorescence staining with fluorescein-labeled heavychain-specific antisera against human IgG, IgA, IgM, and IgD, only IgG could be demonstrated in the cytoplasm of these plasma cells. A skin biopsy at the *Performed in the laboratory of Peter Schur, MD, Robert Breck Brigham Hospital, Boston.
edge of one of the necrotic leg lesions revealed inflammation of arterial and venous blood vessels in dermis and subcutaneous tissue. Both acute lesions with fibrinoid necrosis and acute inflammatory cells in vessel walls and chronic lesions with organized thrombi were present. In some of the vessel lumina there was an amorphous eosinophilic proteinaceous material present. Immunofluorescent staining of a biopsy of grossly uninvolved skin failed to demonstrate immunoglobulins or complement deposition in vessel walls. Studies of the Cryoprotein
The serum showed a milky turbidity when examined at room temperature and formed a solid gel after being placed at 4°C in the refrigerator. The turbidity cleared completely by warming the serum to 37°C. Measurement of the serum turbidity was carried out in a spectrophotometer at 430 nm, while gradually decreasing the temperature from 37°C. Initiation of turbidity occurred at 31°C and thereafter progressed rapidly until gelling made further measurements impossible. In order to isolate the cryoprotein, the cryoprecipitate was washed repeatedly in small volumes of ice-cold phosphate-buffered saline, pH 7.2, or the serum was subjected to DEAE cellulose ion exchange chromatography at room temperature. Both methods resulted in the recovery of a protein fraction which formed a single precipitation line by Ouchterlony double diffusion against anti-whole human serum and anti-IgG. No precipitation lines were demonstrable when diffused against anti-IgA, -IgM, and - 1 0 complement components, antifibrin, and antialbumin. Cryoprecipitation in the serum and of the isolated protein was completely and repeatedly reversible by heating to 37°C. This was not affected by heating for 30 min at 56°C nor by adding heparin to a concentration of 500 units/ml but was abolished by acidification to pH 4.0, dilution with physiologic saline by a factor of more than eight, and increasing the sodium chloride concentration to 4 gm/lOO ml. Dialysis against phosphate buffered saline promptly restored the precipitability. The cold precipitation in the serum, and of the isolated protein, was accompanied by the formation of needle-shaped crystals varying in length from 10 I.( to 50 p and increasing in size on cooling. The crystals were easily demonstrable by light microscope examination at room and lower temperatures and they rendered a silvery shine to the turbid serum in reflected light. Birefringence and dichroism of the crystals were demonstrated by polarized light microscopy (Fig. 1). Their unique ultrastructural appearance has been the subject of a previous report . 1n.order to obtain serum for cryocrit determinations, whole blood was drawn and immediately incubated at 37°C and stored for 24 hours. After centrifugation with a desktop centrifuge in a warm room at 37"C, serum was pipetted off. For cryocrit determinations, serum was stored in a refrigerator (4°C) for five days (unless stated otherwise) before centrifugation in a refrigerated centrifuge (International Centrifuge, Model PR-2 for relative centrifugal forces less than 2,20Og, or Sorvall Centrifuge Model RC2-B for relative centrifugal forces greater than 2,200g). The volume of packed precipitate was determined by subtracting the volume of supernatant from the total volume of serum, and the cryocrit was calculated by dividing the volume of packed precipitate by the total volume of serum. Dilutions of sera were made in phosphate-buffered saline. Cryocrit values were dependent on temperature. Centrifugation at 2,OOOg for 20 minutes yielded a cryocrit of 50% for serum stored at 4"C, 20%for serum stored at 22"C, and 0% for serum stored at 37°C. Cryocrit levels varied markedly depending on g forces, as shown in Figure 2. It was determined that cryocrit values appeared to level off at approx-
Cummings et a1
Fig. 1. A) Crystals noted in a drop of serum examined by llght microscopy at room temperature. An erythrocyte and lymphocyte are seen in the center of the photograph for size comparison. Other normal leukocytes and erythrocytes are seen at the periphery (X 400). B) Ultrastructural analysis of crystals in serum. Note the uniformly circular tubular unit structure on cross-sectional view and the parallel nonbranching units on longitudinal section (X 76,000).
Fig. 2. Changes in cryocrit values depending upon relative centrifugal forces (g). Cryoprecipitate was obtained at 4°C by centrifugation of initial pretreatment serum for 20 minutes at various g forces. A marked reduction in cryocrit values is noted with increasing g forces up to 10,000 g, whereupon cryocrit values level off.
imately 10,000 g, and this g force was used in all subsequent studies. The cryocrit value did not necessarily represent the total amount of abnormal protein, since several dilutions of serum gave a nonlinear relationship with cryocrit values (Fig. 3). At both extremes of serum dilutions, cryocrit values were less than expected. The supernatant removed after centrifugation of undiluated serum could still form a small amount of cryoprecipitate. Cryoprecipitation appeared promptly, since serum stored for one hour yielded the same cryocrit value as that stored for one week. Cryoprecipitation was completely reversible, and rewarming and recooling up to four times gave similar cryocrit values. Therapy and Course
Initially the patient received 2 units of packed red blood cells to improve his anemia. Since an immediate reduction in the level of his abnormal serum crystalcryoprotein would not be anticipated with systemic chemotherapy, it was felt that this man was at considerable risk of further progression of his skin lesions and of the development of new ones. Accordingly, we attempted to lower the level of his abnormal globulin more rapidly with plasmapheresis performed on a continuous-flow cell centrifuge (Celltrifuge, American Instrument Company). A total of 1,560 ml of plasma was removed over a six-hour period at a blood flow rate of 25 ml/min and centrifugation at 700 rpm. During this time, his volume replacement consisted of 1,000 ml normal saline and 2 units of whole blood. He tolerated the procedure well. No special precautions were needed to prevent cryoprecipitation of his abnormal protein in the tubing or centrifuge bowl, where the temperature was slightly less than 37'C. This procedure was repeated on two subsequent days. Prior to the initial procedure, total protein was 12.4 gm% with 8.5 gm% gamma-globulin. This decreased to 10.3 gm% and 6.8 gm%, respectively, following the third plasmapheresis.
Cummings et a1 /
/ / /
Fig. 3. Cryocrit values in serially diluted patient serum. The initial pretreatment serum was diluted serially with phosphate-buffered saline. The original serum and each diluted one was stored at 4OC and centrifuged for 20 minutes at 13,000 g to obtain a cryoprecipitate. Cryocrit values and serum dilutions are plotted on a linear scale. There is a nonlinear relationship between the cryocrit and serum dilutions. The values of cryocrit are less than expected at both extremes of dilution.
The patient was started on systemic chemotherapy, since it was felt that the decrease in serum M protein levels achieved with plasmapheresis would be transient. He received oral phenylalanine mustard (0.2 mg/kg/day X 4), prednisone (2 mg/kg/day X 4), and procarbazine (1 50 mg/day X 4), and intravenous vincristine (1 mg on day 1 and day 8); this regimen was repeated at six-week intervals. His total protein was still in the range of 10.5-1 1.8 gm% and his M protein was 5.3-6.0 gm%, after the first three courses of chemotherapy. Because of this he was plasmapheresed on three subsequent occasions and his total protein fell to 6.6 gm% and his M protein to 3.3 gm%. With continued combination chemotherapy, total protein leveled off at 8.0-9.0 gm% and M protein at 2.1 gm% (Fig. 4). The patient continued on this chemotherapy for 33 months until, of his own accord, he stopped taking phenylalanine mustard, prednisone, and procarbazine because he felt so well. He did not bring this to the attention of his physicians for one year. Over this 12-month period his M protein had gradually increased from 2.1 gm% to 6.1 gm%, while he was receiving only vincristine. In June 1976 he agreed to resume his previous chemotherapy with phenylalanine mustard, prednisone, and procarbazine, and vincristine was discontinued. His abnormal immunoglobulin again decreased to 2.4 gm%. His bone marrow plasmacytosis was originally 95% and this decreased to 5% after three courses of chemotherapy and subsequently to 1%, where it remained. The patient tolerated his therapy. Although he was unable to straighten his knees on his initial admission to the hospital, he gradually improved to the point that he is fully ambulatory without assistance and is working. The condition of his skin lesions after 24 months of therapy is shown in Figure 5. This picture has remained the same, even though he stopped taking combination chemotherapy for 12 months. During this time, however, he developed one new osteolytic lesion in this occipital bone.
M- protein (gmr X )
LPAM Prdrrme Roearbazm Vmcrirlw
Fig. 4. Clinical course depicting sequential determinations of serum M protein, and cryocrit and response to continuous-flow cell centrifugation plasmapheresis and combination chemotherapy.
Cummings et a1
This patient had multiple myeloma, with large ulcers and lamellar eschars of the extremities and a spontaneously crystallizing IgC2-kappa cryoglobulin. Electron microscope studies of the crystals demonstrated a structure previously undescribed. In view of two reports of necrotizing cutaneous ulcerations [4,5], one associated with crystalglobulinemia and the other with a monoclonal cryoparaprotein but neither with overt myeloma, it is important to emphasize that myeloma must be considered in the diagnostic evaluation of all patients who present with such a clinical picture and that aggressive treatment can be very successful. The crystalline cryoglobulin in this patient was shown to be immunoglobulin IgG2kappa. Only several other crystalline cryoglobulins have been previously identified with regard to IgG subclass. Abraham et al demonstrated the Fc fragment (of the cryoglobulin) to be an essential prerequisite for the formation of the crystalline structure , while
Fig. 5. Photographs of selected skin lesions taken before and after dbbridement prior to (A, B, C) and after 24 months of therapy (D).
studies by Grossman et al suggest the functional specificity of the cryoglobulin for IgCl and IgG3 determinants . Wang and Fudenberg  studied, in a patient with very primitive bone marrow cells, a crystalcryoglobulin whose amino acid sequence resembled that of mouse immunoglobulin more than that of humans. Ultrastructural analysis of the crystalline material in bone marrow cells from our patient has recently been reported , and studies of amino acid sequencing have not been completed. Attempts to demonstrate an immune mechanism for the vascular lesions, as was suggested by the low serum complement levels, were not successful. Deposition of immunoglobulins or complement in blood vessel walls in nonlesional skin, such as has been shown in mixed essential cryoglobulinemia [ 101, was not found in our case. The skin lesions, being of considerable chronicity, did not and were not expected to show the presence of immune complexes, which are only demonstrable in early lesions of less than 24 hours duration, as has been shown in patients with leukocytoclastic vasculitis involving the skin [ 111. In the absence of demonstrable immune complex deposition with complement activation as a causative factor in the vascular injury, no satisfactory explanation can be offered for the lowering of the serum complement in our case. In cases of mixed essential cryoglobulinemia, reduction of serum complement is a common occurrence due to the antigen-antibody nature of the protein complexes . The question whether the unusual crystalline structure of the monoclonal cryoglobulin in our patient enabled it to behave like aggregated human gamma-globulin and fix complement [ 131 will remain unanswered. It was noted that cryocrit values depended upon relative centrifugal forces and vaned with changes in temperature. The amount of cryocrit was not consistently linear in relation to the amount of abnormal M protein, since it was observed that at very high or low concentrations the value of the cryocrit was less than expected. Supernatant from
Cummings et a1
which cryoprecipitant had been removed still contained considerable amounts of abnormal M protein. For these reasons, it appears that the measurement of cryocrit levels would not be a reliable method for assessing the response of the myeloma process to therapy. The combination chemotherapy program we employed was a modification of one designed by Alexanian and colleagues . Our patient tolerated this regimen without difficulty. His cutaneous lesions healed with residual scarring, but this did not prevent him from returning t o full ambulation and normal activity. The value to this patient of procarbazine and vincristine in this combination program is uncertain, since it was thought that neither agent added to the responses seen with melphalan-prednisone combinations . There has been a suggestion, however, that drug combinations for myeloma that included vincristine were associated with higher response rates and longer survival if given at shorter (three-week) intervals  . Dramatic results of aggressive therapy for typical myeloma patients have been noted recently [ 141. Our patient demonstrates that excellent long-lasting results can be achieved in this rare clinical entity as well as with aggressive therapy, even though the presenting manifestations appear ominous. ACKNOWLEDGMENTS
This work was supported by US Public Health Service grants GM 16538-04 and -05 and CA 13943-01, -02, and -04. The authors wish to express our thanks to the late Dr. Alden Blackman and Dr. Herbert Rakatansky who referred this patient to us for evaluation and to Mrs. Cathy Prest for her secretarial assistance in the preparation of t h s manuscript. REFERENCES 1. Von Bonsdorff B, Grogy H, Packaleu T: On the presence of a high molecular crystallizable protein in blood serum in myeloma. Folia Haemotol (Leip) 59:184, 1938. 2. Bogaars HA, Kalderon AE, Cummings FJ, Kaplan S, Melnicoff I, Park C, Diamond I, Calabresi P: Human IgG cryoglobulin with tubular crystal structure. Nature New Biol 245: 117, 1973. 3. Kalderon AE, Bogaars HA, Diamond I, Cummings FJ, Kaplan SR, Calabresi P: Ultrastructure of myeloma cells in a case with crystalcryoglobulinemia. Cancer 39: 1475, 1977. 4. Grossman J, Abraham GN, Leddy JP, Condemi J: Crystalglobulinemia. Ann Intern Med 77:395, 1972. 5 . Brody J , Samitz MH: Cutaneous signs of cryoparaproteinemia: Control with burst alkeran and prednisone. Am J Med 55:211,1973. 6. Wang AC, Fudenberg HH: Gene expansion and antibody variability. J lmmunogenetics 1:303314,1974. 7. Abraham GN, Leddy JP, Grossman J: Properties of crystalline lgG3 globulin. Biochem Biophys Res Commun 46:162,1972. 8. Alexanian R, Balcerzak S, Haut A, Hewlett J , Gehan E: Remission maintenance therapy for multiple myeloma. Arch Intern Med 135:147, 1975. 9. Alexanian R, Salmon S, Bonnet J , Gehan E, Haut A, Weick J: Combination therapy for multiple myeloma. Cancer 40:2765,1977. 10. Giannetti A, Serri F, Bernasconi C: Immunofluorescent studies of the skin in mixed cryoglobulinemia and Schoenlein-Henoch purpura. Acta Dermatol Veneral56:211, 1976. 11. Braverman IM, Yen A: Demonstration of immune complexes in spontaneous and histamine-induced lesions and in normal skin of patients with leukocytoclastic angiitis. J Invest Dermatol64: 105, 1975. 12. Riethmuller G, Meltzer M, Franklin E, et al: Serum complement levels in patients with mixed cryoglobulinemia. Clin Exp lmmunol 1:337,1966. 13. lshizaka T, lshizaka K: Biological activities of aggregated gamma globulin. Proc SOCExp Biol Med 101:845,1959. 14. Case D, Lee B, Clarkson B: Improved survival times in multiple myeloma treated with melphalan, prednisone, cyclophosphamide, vincristine and BCNU: M-2 protocol. Am J Med 63:897-903, 1977.