Nonsecretory Multiple Myeloma lmmunohistologic and Ultrastructural Observations on Two Patients
RAUL MANCILLA.
M.D.
GUSTAVE L. DAVIS,
M.D.
St. Louis, Missouri
From the Division of Surgical Pathology. Barnes Hospital, and the Department of Pathology and Laboratory Medicine, The Jewish Hospital of St. Louis, Washington University Medical Center, St. Louis, Missouri. Requests for reprints should be addressed to Dr. Gustave L. Davis, Department of Pathology and Laboratory Medicine, The Jewish Hospital of St. Louis, 216 South Kingshighway Boulevard, St. Louis, Missouri 63110. Manuscript accepted November 19, 1976.
Two well documented examples of nonsecretory multiple myeloma were studied by electron microscopic and immunohistologic methods. In one case, repeat studies revealed no intracytoplasmic immunoglobulins, and the cells displayed a “plasmacytoid” appearance with poor development of rough endoplasmic reticulum and Golgi regions. In the other case, most cells contained intracytoplasmic immunoglobulins of a monoclonal type and the ultrastructural appearance was that of cells actively engaged in protein synthesis. These findings and others in the literature suggest that myelomas without an M component can be separated into nonproducers and true nonsecretors of immunoglobulins. In one case, immunofluorescence of bone marrow smears with double labels demonstrated three different plasma cell populations: those producing either monoclonal immunoglobulins M ( IgM) or A (IgA) and those synthesizing simultaneously IgM and IgA. Dual immunoglobulin production, although known to occur in myelomas with paraproteinemia, has not been previously documented in the nonsecretory variety. The presence of a homogeneous immunoglobulin in serum and/or urine is a cardinal point in the diagnosis of multiple myeloma. However, in an estimated 1 to 5 per cent of cases [l-4], a monoclonal spike cannot be identified. The term nonsecretory, coined for this variety of myeloma, implies that a block in release rather than synthesis accounts for the absence of paraproteinemia. This is probably true for the cases in which abnormal cytoplasmic immunoglobulins are demonstrated by immunofluorescence methods [5-81. However, the absence of intracellular immunoglobulins in some cases [9-l l] suggests an actual lack of production. These concepts are supported by electromicroscopic and immunohistologic correlations we have made in two cases of nonsecretory multiple myeloma. One case was of particular interest in that immunohistologic studies documented the production of two abnormal immunoglobulins by the myeloma cells. A description of our findings and a review of the pertinent literature are the bases of this report. CASES REPORTS Patient 1. In January 1975, a 62 year old white man was admitted to the hospital with a five week history of anorexia, weight loss and pain aggravated by movement in the anterior and lateral areas of the chest and the left hip. Physical examination revealed only bilateral chest and lumbar tenderness to percussion. A roentgenographic bone survey showecl destruction and
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collapse of the fourth, sixth and seventh thoracic vertebrae, and multiple lytic lesions in the mandible and intertrochanteric area. Laboratory data included a hemoglobin level of 11.2 g/l00 ml, a hematocrit of 34 mm/100 ml, a white blood cell count of 2,25O/ml with a normal differential and a platelet count of 420,00O/ml. Blood urea nitrogen, serum creatinine, uric acid, calcium, phosphorus, alkaline phosphatase and urinalysis were within normal limits. Cellulose acetate serum electrophoresis was normal showing 4.02 g/ 100 ml albumin, 2.88 g/ml globulins (alpha, globulin 0.20 g, alphas globulin 0.73 g. beta globulin 1.10 g and gamma globulin 0.84 g). Urine electrophoresis was also within normal limits. Immunoelectrophoresis of serum and concentrated urine showed no monoclonal immunoglobulins. Quantification of serum immunoglobulins showed normal levels of IgG (804 mg/lOO ml) and IgA (121 mg/lOO ml), and slightly low levels of IgM (49 mg/lOO ml). A bone marrow biopsy specimen and aspirate were diagnostic of multiple myeloma showing 32 per cent plasma cells in different stages of maturation (Figure 1). The patient was given 2,150 rads to the lumbar spine with control of the pain. Treatment with melphalan and prednisone was also started with good subjective response. On his last admission (December 1975) a marked increase in the number of osteolytic lesions was noticed. Case 2. In 1965, a 46 year old man was admitted with signs and symptoms of spinal cord compression. An x-ray examination showed destruction of the fourth, fifth and sixth thoracic vertebrae. A laminectomy was performed, and a diagnosis of plasmacytoma was made (Figure 2). Radiation was given to the tumor area and the patient did well until 1969, when he was readmitted with a pathologic fracture of the left femur. A needle biopsy specimen from this area was diagnosed as plasmacytoma. The lesion was pinned, and a new
course of local radiotherapy was started. One year later the patient was readmitted because of pain in the chest and extremities. A roentgenographic bone survey disclosed multiple lytic lesions in the spine, radii, ribs and hips. A diagnosis of multiple myeloma was established, and treatment with melphalan and prednisone was started with good response. In July 1974, the patient was readmitted with pain in the ankles, left forearm and knee. Roentgenographic bone survey showed an increased number of lytic lesions. In a surgical biopsy specimen of one of the lytic lesions, the diagnosis of multiple myeloma was confirmed. Three months later he was admitted for the last time. On this admission the skeletal destructive process was more marked; lytic lesions were now present in the skull, humeri, radii, femurs and left ulna. The ulnar lesion was biopsied primarily to obtain tissue for immunofluorescence and electronmicroscopic studies. Laboratory examination disclosed a hemoglobin level, hematocrit value and white blood cell counts within normal limits. Other laboratory studies, including urinalysis, blood urea nitrogen, serum creatinine, calcium and phosphorus, have also yielded normal results. Since 1969 several serum and urine electrophoreses have failed to disclose a monoclonal spike. Examinations for Bence Jones protein have also been negative. On the patient’s last admission, serum and urine immunoelectrophoresis showed no abnormal immunoglobulins. Quantification of serum immunoglobulins revealed immunoglobulin G (IgG) and M IgM levels within the lower normal limits (727 and 63 mg/lOO ml, respectively) and also normal IgA levels (94 mg/lOO ml). MATERIAL
AND METHODS
Electronmicroscopy. Small blocks of biopsy specimens and bone marrow aspirates were fixed for 1 hour in phosphate buffered 2.5 per cent glutaraldehyde solution, pH 7.2, and
Figure 2. Case 2. Immature plasma cells with abundant granular cytoplasm and round nuclei. A trinucleated cell is seen at the top. Hematoxylin and eosin stain; magnification X600, reduced by 10 per cent.
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FlguIre 3. Two myeloma cells of patient (Case 1) showing immature nuclei and abundant r(,ugh end ?plasmic reticulum (RER). The cell at the right shows dilated RER sacs filled with gr: 3r)u/ar and osmiophilic material. ( X 12,000). Original magnification X 72,000, reduced by 14 per cent
Figure 4. Case 1. Myeloma cell with very atypical nucleus. In contrast the cytoplasm is highly developed showing prominent Golgi apparatus, abundant rough endoplasmic reticulum and numerous round electrodense bodies. (X 75,500). Some dense bodies are surrounded by double membranes and others are probably located within rough endoplasmic reticulum cysternae. Original magnification X 15,500 (insert X 56,500); reduced by 4 per cent.
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Case 2. Two plasmacytoid ceils showing poor RER development Original magnification X 7,000; reduced by 14 per cent.
and no Golgi.
Figure 6. Case 2. This myeloma cell shows more RER, although the cysternae are small, flattened and contain no electrodense materials. A single, small dense body is seen near the Golgi area. Original magnification X 76,000; reduced by 14 per cent.
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Figure 7. Case 1. A, bone marrow smear stained with fluorescent antiserum to lambda light chains. Many cells contain bright, intracytoplasmic fluorescent granules. B, myeloma cell showing bright cyfoplasmic fluorescence with antiserum to IgA. An intranuclear fluorescent inclusion body is seen. postfixed in phosphate-buffered 1 per cent osmium tetroxide solution. Blocks were dehydrated in graded concentrations of ethanol and embedded in epoxy resin. Ultrathin sections were double stained with uranyl acetate and lead citrate, and examined with a Phillips EM-300 or 201C electron microscope. lmmunohistopathology. Cryostat sections of rapidly frozen tissue and frozen marrow smears were rinsed in phosphate buffer solution, pH 7.2, briefly fixed in cold acetone or absolute alcohol and incubated for 30 minutes with commercially purchased heavy chain specific fluorescein-labelled goat antiserums to human IgG, IgM, IgA, IgE, IgD, and kappa and lambda light chains (Hyland Laboratories, Costa Mesa, Calif.; Meloy Laboratories, Springfield, Va.). In addition, some smears from one patient (Case 1) were double stained in succession or with mixtures of fluorescein-labelled antiserum to IgM and rhodamine-labelled antiserum to IgA (Cappel Laboratories, Downington, Pa.). The monospecificity of the antiserums was previously assessed by immunoelectrophoresis. Inhibition controls were carried out by overlying slides with unlabelled antiserum and then with the corresponding labelled antiserum. Fluorescent microscopy was performed with a Leitz Ortholux microscope equipped with a vertical illuminator. The light source was an Osram HBO mercury lamp. A filter system as described by Bouvet et al. 1121was used. PATHOLOGIC OBSERVATIONS
Electronmicroscopy. In Case 1 all examined cells asynshowed varying degrees of nucleus-cytoplasm chrony, with primitive nuclei. The nucleus to cytoplasm ratio was increased in most cells, and infoldings and other irregularities of nuclear contour were common. The chromatin pattern varied from cell to cell, from fine granules randomly distributed throughout the nucleus to scattered coarser granules peripherally located. The margination of the chromatin never reached the proportions seen in normal plasma cells. Some nuclei
displayed 1 or 2 prominent nucleoli. Nuclear inclusion bodies were not seen. In contrast to the nuclear immaturity, the cytoplasm was highly differentiated showing very prominent Golgi apparatus and large amounts of ribosome-studded endoplasmic reticulum (RER) (Figure 3). In some cells, the cysternae were dilated and filled with finely granular, osmiophilic material (Figure 3). In many cells, moderately sized, round dense bodies were seen within the RER sacs (Russell bodies) (Figure 4). Extracysternal dense bodies were also common, particularly in the Golgi regions. In Case 2 a different electronmicroscopic picture was observed. The nucleus-cytoplasm asynchrony was reversed with the nucleus appearing more mature than the cytoplasm. In many cells, the chromatin pattern was similar to that observed in normal plasma cells and nucleoli were not prominent. Most cells had a “plasmacytoid” appearance as the RER was scanty and flattened, and the Golgi apparatus was poorly developed (Figures 5 and 6). In a few cells the cysternae were moderately dilated but they did not c’ontain granular material or Russell bodies. Few, small, dense bodies were seen in the intercysternal cytoplasm of some cells. Immunohistology. In Case 2, immunofluorescent examination of two different biopsy specimens gave consistently negative results. In Case 1, exposure of bone marrow smears to fluorescein-labelled antiserum to lambda light chains resulted in bright cytoplasmic fluorescence in approximately 30 per cent of the nucleated cells (Figure 7A). Their morphology was consistent with immature plasma cells. In some cells, intranuclear fluorescent inclusion bodies were seen (Figure 78). Smears stained with antiserum to IgM showed many fluorescent cells in slightly fewer numbers than those stained with antiserum to lambda light
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Figure 8. Case 1. Composite picture of plasma cells stained with a mixture of fluorescein-labelled anti-IgM and rhodamin-labelled anti-IgA antiserums. At the left, the cells stain green for IgM. The same cells viewed with a different filter system also stain red for IgA (right). One cell contains only IgA (arrow)
chains. Cells containing IgA were also found but in fewer numbers than those containing IgM and lambda chains. Staining with antiserums to IgG, IgD, IgE and kappa chains was negative except by an exceptional IgG- or kappa-positive cell. On marrow smears stained sequentially or with mixtures of fluorescein-labelled antiserum to IgM and rhodamine-labelled antiserum to IgA, the use of suitable filter combinations [ 121 allowed the characterization of three different cell populations. Approximately 40 per cent of the positive plasma cells displayed exclusively green fluorescence (IgM), 40 per cent had only red fluorescence (IgA) and the remaining 20 per cent of the cells displayed both green and red cytoplasmic fluorescence (Figure 8), indicating the simultaneous production of IgM and IgA. Appropriate filters revealed a weak yellow fluorescence on these cells. COMMENTS Other than the absence of a monoclonal spike, few features differentiate secretory from nonsecretory multiple myeloma. The levels of normal immunoglobulins in nonsecretory multiple myeloma may be severely depressed [ 2,131. However, in our two patients and in other cases, normal or near normal values were found [3,9,11,14]. The group of patients studied by Azar et al. [ 131 had a median survival of only 7.5 months in contrast with the 23 months median survival in patients
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with myelomas of all types which suggests that patients with nonsecretory multiple myeloma have shorter life spans [2,15]. More recent data, however, indicate that the prognosis is not necessarily dismal. Forssman and Nilsson [ 161, and Kim et al. [3] have observed patients who survived several years, and one of our patients (Case 2) is alive 10 years after diagnosis. The clinical course, however, seems to differ in that renal complications, which are frequent and severe in secretory myeloma [4], occur rarely in nonsecretory multiple myeloma [ 131. The phenomenon of “nonsecretion” in multiple myeloma remains speculative. Most electronmicrostopic studies have failed to reveal abnormalities which explain the lack of a monoclonal spike. Indeed, as in multiple myeloma with an M component, nonsecretory multiple myeloma cells possess well developed RER and Golgi regions, electrodense material within RER sacs and dense bodies in the Golgi region [ 13,17,18]. This impression, however, is not unanimous. In a case studied by Gach et al. [9] the cells had a “plasmacytoid” appearance, the RER was scanty and the Golgi apparatus inconspicuous. The functional status of nonsecretory multiple myeloma cells has been evaluated at the cellular level by immunofluorescence in 10 instances. In three cases [9-l I] neither heavy nor light chains were demonstrated, and in the remaining cases the cells contained intracy-toplasmic immunoglobulins of a monoclonal type [5-81. A combined ultrastructural and immunohistologic approach, as in the present study, has been undertaken only twice before [8,9]. In Case 1, as in that studied by Whither et al. [ 81, most cells contained an abnormal immunoglobulin; ultrastructurally, the cells appear to be actively engaged in protein synthesis as seen in multiple myeloma with a monoclonal spike [ 18,191. In contrast, the cells of our patient (Case 2) displayed a paucity of RER and Glogi regions. It is interesting that, as in Gach’s case [9], the immaturity of organelles known to participate in protein production correlated with an absence of intracellular immunoglobulins as revealed by the immunofluorescent method. From these data two possible explanations emerge for the absence of paraproteinemia in multiple myeloma. First, a lack of synthetic activity when immunohistologic methods fail to uncover intracytoplasmic immunoglobulins. The term nonproducer is tentatively applied to this type of myeloma. However, it is possible that, as seen in mouse myeloma [20], more refined technics of protein analysis could eventually demonstrate immunoglobulin synthesis in apparently nonproducing cells. A second possibility is that nonsecretory multiple myeloma cells yet capable of immunoglobulin synthesis are unable to release their product. That this is probably the most common situation is strongly suggested by the
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presence of intracellular immunoglobulins in most cases to which immunohistologic methods have been applied. These will be true nonsecretory myelomas. To explain a block in the secretory process, several possibilities are entertained. Prior to secretion the assembled immunoglobulin molecule is carried through the endoplasmic reticulum by a transport protein which recognizes light chains, the recognition site probably being located at the constant region [ 211. It is conceivable, therefore, that abnormal light chains or absence of the transport protein could result in block of this initial secretory step. Because attachment of carbohydrate has been considered a prerequisite for the release [22] of the immunoglobulin molecule, a block in this step may account for a lack of secretion in nonsecretory myelomas [ 131. However, this possibility is negated by the fact that myelomas secreting immunoglobulins devoid of carbohydrate are known to occur [ 231. An increased intracellular breakdown [6] and a faulty heavy and/or light chain synthesis prior to assembly [5] have also been considered. The latter possibility is supported by Cowan’s [20] observations documenting a defective heavy chain synthesis in nonsecreting clones of mouse myeloma. The immunohistologic findings in Case 1 are, to our knowledge, previously unreported. By double label technics we documented the presence of three functionally different plasma cell populations. Approximately 80 per cent of the positive cells produced either IgM and IgA. The possibility that some of the positive cells were residual normal plasma cells is negated by the fact that only lambda light chains were found and that in multiple blocks examined by electron microscopy virtually all cells showed nucleus-cytoplasm asynchrony indicative of a neoplastic transformation [ 181. Our immunofluorescent findings suggest that nonsecretory variants may share, at the cellular level, the
DAVIS
functional diversity of secretory myelomas. Indeed, two abnormal immunoglobulins belonging to different classes may be rarely found in the serum of patients with multiple myeloma. The most common is a combination of IgG and IgA [24-321 or IgG and IgM [33-361, generally sharing the same type of light chain. To our knowledge, double IgM-IgA paraproteinemia has not been documented in patient with classic multiple myeloma. Such combination, however, has been detected in patients with lymphosarcoma [37], WaldenStrom’s macroglobulinemia [38] and other plasma cell disorders [39]. In six patients with double myelomas the dual immunoglobulin production was documented at the cellular level by immunofluorescence of bone marrow smears. In four cases it was found that each immunoglobulin was produced by different cell lines [27,28,33,34], and in two instances double label technics revealed the two myeloma Iproteins being produced simultaneously by single cells [24,25]. Thus, the first case reported herein appears to be the nonsecretory counterpart of the latter type of double myeloma. The existence of myelomas with a double M component has raised questions concerning their monoclonal origin. However, detailed immunologic studies in some cases have shown that both myeloma proteins share similar if not identical idiotypic determinations [24-26,33,35,36] thus indicating a common origin from a single ancestor cell. Since during the antibody response a clone of cells may initially produce IgM and switch over to IgG synthesis, Levin et al. [36] proposed that a malignant transformation occurring during the switch over phase may result in myelomas capable of producing IgM and IgG sharing the same antigenic determinants. A similar sequence of events has been suggested to explain the existence of IgGlgA myelomas [25]. Whether this also applies to myelomas producing other immunoglobulin combinations has yet to be documented.
REFERENCES 1.
2. 3. 4.
5.
6.
7.
DiGuglielmo R: Unusual morphologic and humoral conditions in the field of plasmocytoms and Mdysproteinemia. Acta Med Stand 445 (suppl): 260. 1966. Hobbs JR: lmmunochemical classes of myelomatosis. Br J Haematol 16: 599, 1969. Kim I, Harley JB, Weksler B: Multiple myeloma without initial paraproteins. Am J Med Sci 264: 267, 1972. Osserman EF, Takatsuki K: Plasma cell myeloma-gamma globulin synthesis and structure. Medicine (Baltimore) 42: 357, 1963. Arend WP, Adamson JW: Nonsecretory myeloma-immunofluorescent demonstration of paraprotein within bone marrow plasma cells. Cancer 33: 721, 1974. Hurez D, Preud’Homme JL, Seligmann M: Intracellular “monoclonal” immunoglobulin in nonsecretory human myeloma. J lmmunol 104: 263, 1970. Menkes CJ, Herreman G, Preud’Homme JL, et al.: Myeloma a plasmocytes non excretants. Nouv Presse Med 1: 309, 1972.
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11. 12.
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Whither JT. Davies JD, Grayburn JA: Intact and fragmented intracellular immunoglobulin in a case of non-secretory myeloma. J Clin Pathol 28: 54, 1975. Gach J, Simar L, Salmon J: Multiple myeloma without M-type proteinemia. Report of a case with immunologic and ultrastructure studies. Am J Med 50: 83’5, 1971. lndiveri F, Barabino A, Santolini ME, et al.: Nonsecretory multiple myeloma. Report of a case. Acta Haematol 51: 302, 1974. River GL, Tewksbury DA, Fudenberg HH: “Nonsecretory” multiple myeloma. Blood 40: 204, 1972. Bouvet JP, Buffe D. Liacopoulos P: Two myeloma globulins IgGt-K and IgGl-X, from a single patient (lm). II. Their common cellular origin as revealed by lmmunofluorescence studies. Immunology 27: 1095, 1974. Azar HR. Zaino EC, Pham TC. et al.: “Non-secretory” plasma cell myeloma. Observations on 7 cases with electron microscopic studies. Am J Clin Pathol 58: 618, 1972. Coltman CA Jr: Multiple myeloma without a paraprotein. Arch
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15. 16.
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Intern Med 120: 687, 1967. Alexanian R, Balcerzak S, Bonnet J, et al.: Prognostic factors in multiple myelomas. Cancer 36: 1192, 1975. Forssman 0, Nilsson G: A case of myeloma with flaming plasma cells but no significant M-compound in serum or urine. Acta Med Stand 181: 33, 1967. Guillan RA, Ranjini R, Zelman S, et al.: Multiple myeloma with hypogammaglobulinemia-electron microscopic and chromosome studies. Cancer 25: 1187, 1970. Graham RC. Bernier GM: The bone marrow in multiple myeloma: a correlation of plasma cell ultrastructure and clinical state. Medicine (Baltimore) 54: 225, 1975. Maldonado JE, Brown AL Jr, Bayrd ED, et al.: Ultrastructure of the myeloma cell. Cancer 19: 1613. 1966. Cowan NJ, Secher DS, Milstein C: Intracellular immunoglobulin synthesis in non-secreting variants of mouse myeloma: detection of inactive light chain messenger RNA. J Mel Biol 90: 691, 1974. Lennox ES, Cohn M: Immunoglobulins. Ann Rev Biochem 36: 365, 1967. Melchers F: Biosynthesis of the carbohydrate portion of immunoglobulins. Biochem J 119: 765, 1970. Abel CA, Spiegelberg HL, Grey HM: The carbohydrate content of fragments and polypeptide chains of human yGmyeloma proteins of different heavy chain subclasses. Biochemistry 7: 1271, 1968. Costea N, Yakulis VJ, Libnoch JA, et al.: Two myeloma globulins (IgG and IgA) in one subject and one cell line. Am J Med 42: 630. 1967. Rudders RA, Yakulis V, Heller P: Double myeloma. Production of both IgG type lambda and IgA type lambda myeloma proteins by a single plasma cell line. Am J Med 55: 215, 1973. Prendergast RA. Grey HM, Kunkel HG: Recombination of heavy and light chains of human myeloma proteins. J Exp Med 114: 185. 1966. Dittmar K, Kochwa S, Zucker-Franklin D, et al.: Coexistence of polycythemia vera and biclonal gammopathy (-yGK and yAL) with two Bence Jones proteins (BJK and BJL). Blood 31: 81, 1968.
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Rosen BJ, Smith TW, Block KJ: Multiple myeloma associated with two serum M components; yG type K and yA type L. N Engl J Med 227: 902. 1967. Bachmann R: Simultaneous occurrence of two immunologically different M-components in serum. Acta Med Stand 177: 593, 1965. Kistner S, Norberg R: Simultaneous occurrence of two different myeloma proteins. Stand J Clin Lab Invest 17: 32 1, 1965. Radl J. Chott L, Richova D: Ein Fall von myelom und bildung zweier in antigener hinsicht unterschiedlicher paraprotein. Klin Wochenschr 44: 1117, 1966. Wetter 0, Hertenstein C: Die optiscne rotationsdispersion isolierter paraproteine. IV. Untersuchungen bei multiplem myelom mit doppelter proteinanomalie. Klin Wochenschr 44: 1111, 1966. Wang AC, Wang IYF, McCormack JN, et al.: The identity of the light chains of monoclonal IgG and monoclonal IgM in one patient. Immunochemistry 6: 451, 1969. Curtain CC: Immuno-cytochemical localization of two abnormal serum globulins in the one bone marrow smear. Australs Ann Med 13: 136, 1964. Penn GM, Kunkel GH, Grey HM: Sharing of individual antigenic determinants between a yo and a yu protein in the myeloma serum. Proc Sot Exp Biol Med 135: 660, 1970. Levin AC, Fudenberg HH, Hooper JE, et al.: Immunofluorescence evidence for cellular control of synthesis of variable regions of light and heavy chains of immunoglobulins G and M by the same gene. Proc Natl Acad Sci USA 68: 169, 1971. Yagi Y, Pressman D: Monoclonal IgA and IgM in the serum of a single patient (SC). I. Sharing of individually specific determinants between IgA and IgM. J lmmunol 110: 335, 1973. Fateh-Moghadam A, Beil E, Borchers H, et al.: Plasmozytom. Makroglobulinamie Waldenstrcm und Morbus Paget bei einem Patienten mit IgAK+lgMK-Doppelparaproteinamie und Bence-Jones-Protein (Typ K). Blut 21: 146, 1970. lmhof JW, Ballieux RE, Mul NA, et al.: Monoclonal and biclonal gammopathies. Acta Med Stand 445 (suppl): 102, 1966.
RAUL MANCILLA.
M.D.
GUSTAVE L. DAVIS,
M.D.
St. Louis, Missouri
From the Division of Surgical Pathology. Barnes Hospital, and the Department of Pathology and Laboratory Medicine, The Jewish Hospital of St. Louis, Washington University Medical Center, St. Louis, Missouri. Requests for reprints should be addressed to Dr. Gustave L. Davis, Department of Pathology and Laboratory Medicine, The Jewish Hospital of St. Louis, 216 South Kingshighway Boulevard, St. Louis, Missouri 63110. Manuscript accepted November 19, 1976.
Two well documented examples of nonsecretory multiple myeloma were studied by electron microscopic and immunohistologic methods. In one case, repeat studies revealed no intracytoplasmic immunoglobulins, and the cells displayed a “plasmacytoid” appearance with poor development of rough endoplasmic reticulum and Golgi regions. In the other case, most cells contained intracytoplasmic immunoglobulins of a monoclonal type and the ultrastructural appearance was that of cells actively engaged in protein synthesis. These findings and others in the literature suggest that myelomas without an M component can be separated into nonproducers and true nonsecretors of immunoglobulins. In one case, immunofluorescence of bone marrow smears with double labels demonstrated three different plasma cell populations: those producing either monoclonal immunoglobulins M ( IgM) or A (IgA) and those synthesizing simultaneously IgM and IgA. Dual immunoglobulin production, although known to occur in myelomas with paraproteinemia, has not been previously documented in the nonsecretory variety. The presence of a homogeneous immunoglobulin in serum and/or urine is a cardinal point in the diagnosis of multiple myeloma. However, in an estimated 1 to 5 per cent of cases [l-4], a monoclonal spike cannot be identified. The term nonsecretory, coined for this variety of myeloma, implies that a block in release rather than synthesis accounts for the absence of paraproteinemia. This is probably true for the cases in which abnormal cytoplasmic immunoglobulins are demonstrated by immunofluorescence methods [5-81. However, the absence of intracellular immunoglobulins in some cases [9-l l] suggests an actual lack of production. These concepts are supported by electromicroscopic and immunohistologic correlations we have made in two cases of nonsecretory multiple myeloma. One case was of particular interest in that immunohistologic studies documented the production of two abnormal immunoglobulins by the myeloma cells. A description of our findings and a review of the pertinent literature are the bases of this report. CASES REPORTS Patient 1. In January 1975, a 62 year old white man was admitted to the hospital with a five week history of anorexia, weight loss and pain aggravated by movement in the anterior and lateral areas of the chest and the left hip. Physical examination revealed only bilateral chest and lumbar tenderness to percussion. A roentgenographic bone survey showecl destruction and
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collapse of the fourth, sixth and seventh thoracic vertebrae, and multiple lytic lesions in the mandible and intertrochanteric area. Laboratory data included a hemoglobin level of 11.2 g/l00 ml, a hematocrit of 34 mm/100 ml, a white blood cell count of 2,25O/ml with a normal differential and a platelet count of 420,00O/ml. Blood urea nitrogen, serum creatinine, uric acid, calcium, phosphorus, alkaline phosphatase and urinalysis were within normal limits. Cellulose acetate serum electrophoresis was normal showing 4.02 g/ 100 ml albumin, 2.88 g/ml globulins (alpha, globulin 0.20 g, alphas globulin 0.73 g. beta globulin 1.10 g and gamma globulin 0.84 g). Urine electrophoresis was also within normal limits. Immunoelectrophoresis of serum and concentrated urine showed no monoclonal immunoglobulins. Quantification of serum immunoglobulins showed normal levels of IgG (804 mg/lOO ml) and IgA (121 mg/lOO ml), and slightly low levels of IgM (49 mg/lOO ml). A bone marrow biopsy specimen and aspirate were diagnostic of multiple myeloma showing 32 per cent plasma cells in different stages of maturation (Figure 1). The patient was given 2,150 rads to the lumbar spine with control of the pain. Treatment with melphalan and prednisone was also started with good subjective response. On his last admission (December 1975) a marked increase in the number of osteolytic lesions was noticed. Case 2. In 1965, a 46 year old man was admitted with signs and symptoms of spinal cord compression. An x-ray examination showed destruction of the fourth, fifth and sixth thoracic vertebrae. A laminectomy was performed, and a diagnosis of plasmacytoma was made (Figure 2). Radiation was given to the tumor area and the patient did well until 1969, when he was readmitted with a pathologic fracture of the left femur. A needle biopsy specimen from this area was diagnosed as plasmacytoma. The lesion was pinned, and a new
course of local radiotherapy was started. One year later the patient was readmitted because of pain in the chest and extremities. A roentgenographic bone survey disclosed multiple lytic lesions in the spine, radii, ribs and hips. A diagnosis of multiple myeloma was established, and treatment with melphalan and prednisone was started with good response. In July 1974, the patient was readmitted with pain in the ankles, left forearm and knee. Roentgenographic bone survey showed an increased number of lytic lesions. In a surgical biopsy specimen of one of the lytic lesions, the diagnosis of multiple myeloma was confirmed. Three months later he was admitted for the last time. On this admission the skeletal destructive process was more marked; lytic lesions were now present in the skull, humeri, radii, femurs and left ulna. The ulnar lesion was biopsied primarily to obtain tissue for immunofluorescence and electronmicroscopic studies. Laboratory examination disclosed a hemoglobin level, hematocrit value and white blood cell counts within normal limits. Other laboratory studies, including urinalysis, blood urea nitrogen, serum creatinine, calcium and phosphorus, have also yielded normal results. Since 1969 several serum and urine electrophoreses have failed to disclose a monoclonal spike. Examinations for Bence Jones protein have also been negative. On the patient’s last admission, serum and urine immunoelectrophoresis showed no abnormal immunoglobulins. Quantification of serum immunoglobulins revealed immunoglobulin G (IgG) and M IgM levels within the lower normal limits (727 and 63 mg/lOO ml, respectively) and also normal IgA levels (94 mg/lOO ml). MATERIAL
AND METHODS
Electronmicroscopy. Small blocks of biopsy specimens and bone marrow aspirates were fixed for 1 hour in phosphate buffered 2.5 per cent glutaraldehyde solution, pH 7.2, and
Figure 2. Case 2. Immature plasma cells with abundant granular cytoplasm and round nuclei. A trinucleated cell is seen at the top. Hematoxylin and eosin stain; magnification X600, reduced by 10 per cent.
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FlguIre 3. Two myeloma cells of patient (Case 1) showing immature nuclei and abundant r(,ugh end ?plasmic reticulum (RER). The cell at the right shows dilated RER sacs filled with gr: 3r)u/ar and osmiophilic material. ( X 12,000). Original magnification X 72,000, reduced by 14 per cent
Figure 4. Case 1. Myeloma cell with very atypical nucleus. In contrast the cytoplasm is highly developed showing prominent Golgi apparatus, abundant rough endoplasmic reticulum and numerous round electrodense bodies. (X 75,500). Some dense bodies are surrounded by double membranes and others are probably located within rough endoplasmic reticulum cysternae. Original magnification X 15,500 (insert X 56,500); reduced by 4 per cent.
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Case 2. Two plasmacytoid ceils showing poor RER development Original magnification X 7,000; reduced by 14 per cent.
and no Golgi.
Figure 6. Case 2. This myeloma cell shows more RER, although the cysternae are small, flattened and contain no electrodense materials. A single, small dense body is seen near the Golgi area. Original magnification X 76,000; reduced by 14 per cent.
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Figure 7. Case 1. A, bone marrow smear stained with fluorescent antiserum to lambda light chains. Many cells contain bright, intracytoplasmic fluorescent granules. B, myeloma cell showing bright cyfoplasmic fluorescence with antiserum to IgA. An intranuclear fluorescent inclusion body is seen. postfixed in phosphate-buffered 1 per cent osmium tetroxide solution. Blocks were dehydrated in graded concentrations of ethanol and embedded in epoxy resin. Ultrathin sections were double stained with uranyl acetate and lead citrate, and examined with a Phillips EM-300 or 201C electron microscope. lmmunohistopathology. Cryostat sections of rapidly frozen tissue and frozen marrow smears were rinsed in phosphate buffer solution, pH 7.2, briefly fixed in cold acetone or absolute alcohol and incubated for 30 minutes with commercially purchased heavy chain specific fluorescein-labelled goat antiserums to human IgG, IgM, IgA, IgE, IgD, and kappa and lambda light chains (Hyland Laboratories, Costa Mesa, Calif.; Meloy Laboratories, Springfield, Va.). In addition, some smears from one patient (Case 1) were double stained in succession or with mixtures of fluorescein-labelled antiserum to IgM and rhodamine-labelled antiserum to IgA (Cappel Laboratories, Downington, Pa.). The monospecificity of the antiserums was previously assessed by immunoelectrophoresis. Inhibition controls were carried out by overlying slides with unlabelled antiserum and then with the corresponding labelled antiserum. Fluorescent microscopy was performed with a Leitz Ortholux microscope equipped with a vertical illuminator. The light source was an Osram HBO mercury lamp. A filter system as described by Bouvet et al. 1121was used. PATHOLOGIC OBSERVATIONS
Electronmicroscopy. In Case 1 all examined cells asynshowed varying degrees of nucleus-cytoplasm chrony, with primitive nuclei. The nucleus to cytoplasm ratio was increased in most cells, and infoldings and other irregularities of nuclear contour were common. The chromatin pattern varied from cell to cell, from fine granules randomly distributed throughout the nucleus to scattered coarser granules peripherally located. The margination of the chromatin never reached the proportions seen in normal plasma cells. Some nuclei
displayed 1 or 2 prominent nucleoli. Nuclear inclusion bodies were not seen. In contrast to the nuclear immaturity, the cytoplasm was highly differentiated showing very prominent Golgi apparatus and large amounts of ribosome-studded endoplasmic reticulum (RER) (Figure 3). In some cells, the cysternae were dilated and filled with finely granular, osmiophilic material (Figure 3). In many cells, moderately sized, round dense bodies were seen within the RER sacs (Russell bodies) (Figure 4). Extracysternal dense bodies were also common, particularly in the Golgi regions. In Case 2 a different electronmicroscopic picture was observed. The nucleus-cytoplasm asynchrony was reversed with the nucleus appearing more mature than the cytoplasm. In many cells, the chromatin pattern was similar to that observed in normal plasma cells and nucleoli were not prominent. Most cells had a “plasmacytoid” appearance as the RER was scanty and flattened, and the Golgi apparatus was poorly developed (Figures 5 and 6). In a few cells the cysternae were moderately dilated but they did not c’ontain granular material or Russell bodies. Few, small, dense bodies were seen in the intercysternal cytoplasm of some cells. Immunohistology. In Case 2, immunofluorescent examination of two different biopsy specimens gave consistently negative results. In Case 1, exposure of bone marrow smears to fluorescein-labelled antiserum to lambda light chains resulted in bright cytoplasmic fluorescence in approximately 30 per cent of the nucleated cells (Figure 7A). Their morphology was consistent with immature plasma cells. In some cells, intranuclear fluorescent inclusion bodies were seen (Figure 78). Smears stained with antiserum to IgM showed many fluorescent cells in slightly fewer numbers than those stained with antiserum to lambda light
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Figure 8. Case 1. Composite picture of plasma cells stained with a mixture of fluorescein-labelled anti-IgM and rhodamin-labelled anti-IgA antiserums. At the left, the cells stain green for IgM. The same cells viewed with a different filter system also stain red for IgA (right). One cell contains only IgA (arrow)
chains. Cells containing IgA were also found but in fewer numbers than those containing IgM and lambda chains. Staining with antiserums to IgG, IgD, IgE and kappa chains was negative except by an exceptional IgG- or kappa-positive cell. On marrow smears stained sequentially or with mixtures of fluorescein-labelled antiserum to IgM and rhodamine-labelled antiserum to IgA, the use of suitable filter combinations [ 121 allowed the characterization of three different cell populations. Approximately 40 per cent of the positive plasma cells displayed exclusively green fluorescence (IgM), 40 per cent had only red fluorescence (IgA) and the remaining 20 per cent of the cells displayed both green and red cytoplasmic fluorescence (Figure 8), indicating the simultaneous production of IgM and IgA. Appropriate filters revealed a weak yellow fluorescence on these cells. COMMENTS Other than the absence of a monoclonal spike, few features differentiate secretory from nonsecretory multiple myeloma. The levels of normal immunoglobulins in nonsecretory multiple myeloma may be severely depressed [ 2,131. However, in our two patients and in other cases, normal or near normal values were found [3,9,11,14]. The group of patients studied by Azar et al. [ 131 had a median survival of only 7.5 months in contrast with the 23 months median survival in patients
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with myelomas of all types which suggests that patients with nonsecretory multiple myeloma have shorter life spans [2,15]. More recent data, however, indicate that the prognosis is not necessarily dismal. Forssman and Nilsson [ 161, and Kim et al. [3] have observed patients who survived several years, and one of our patients (Case 2) is alive 10 years after diagnosis. The clinical course, however, seems to differ in that renal complications, which are frequent and severe in secretory myeloma [4], occur rarely in nonsecretory multiple myeloma [ 131. The phenomenon of “nonsecretion” in multiple myeloma remains speculative. Most electronmicrostopic studies have failed to reveal abnormalities which explain the lack of a monoclonal spike. Indeed, as in multiple myeloma with an M component, nonsecretory multiple myeloma cells possess well developed RER and Golgi regions, electrodense material within RER sacs and dense bodies in the Golgi region [ 13,17,18]. This impression, however, is not unanimous. In a case studied by Gach et al. [9] the cells had a “plasmacytoid” appearance, the RER was scanty and the Golgi apparatus inconspicuous. The functional status of nonsecretory multiple myeloma cells has been evaluated at the cellular level by immunofluorescence in 10 instances. In three cases [9-l I] neither heavy nor light chains were demonstrated, and in the remaining cases the cells contained intracy-toplasmic immunoglobulins of a monoclonal type [5-81. A combined ultrastructural and immunohistologic approach, as in the present study, has been undertaken only twice before [8,9]. In Case 1, as in that studied by Whither et al. [ 81, most cells contained an abnormal immunoglobulin; ultrastructurally, the cells appear to be actively engaged in protein synthesis as seen in multiple myeloma with a monoclonal spike [ 18,191. In contrast, the cells of our patient (Case 2) displayed a paucity of RER and Glogi regions. It is interesting that, as in Gach’s case [9], the immaturity of organelles known to participate in protein production correlated with an absence of intracellular immunoglobulins as revealed by the immunofluorescent method. From these data two possible explanations emerge for the absence of paraproteinemia in multiple myeloma. First, a lack of synthetic activity when immunohistologic methods fail to uncover intracytoplasmic immunoglobulins. The term nonproducer is tentatively applied to this type of myeloma. However, it is possible that, as seen in mouse myeloma [20], more refined technics of protein analysis could eventually demonstrate immunoglobulin synthesis in apparently nonproducing cells. A second possibility is that nonsecretory multiple myeloma cells yet capable of immunoglobulin synthesis are unable to release their product. That this is probably the most common situation is strongly suggested by the
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presence of intracellular immunoglobulins in most cases to which immunohistologic methods have been applied. These will be true nonsecretory myelomas. To explain a block in the secretory process, several possibilities are entertained. Prior to secretion the assembled immunoglobulin molecule is carried through the endoplasmic reticulum by a transport protein which recognizes light chains, the recognition site probably being located at the constant region [ 211. It is conceivable, therefore, that abnormal light chains or absence of the transport protein could result in block of this initial secretory step. Because attachment of carbohydrate has been considered a prerequisite for the release [22] of the immunoglobulin molecule, a block in this step may account for a lack of secretion in nonsecretory myelomas [ 131. However, this possibility is negated by the fact that myelomas secreting immunoglobulins devoid of carbohydrate are known to occur [ 231. An increased intracellular breakdown [6] and a faulty heavy and/or light chain synthesis prior to assembly [5] have also been considered. The latter possibility is supported by Cowan’s [20] observations documenting a defective heavy chain synthesis in nonsecreting clones of mouse myeloma. The immunohistologic findings in Case 1 are, to our knowledge, previously unreported. By double label technics we documented the presence of three functionally different plasma cell populations. Approximately 80 per cent of the positive cells produced either IgM and IgA. The possibility that some of the positive cells were residual normal plasma cells is negated by the fact that only lambda light chains were found and that in multiple blocks examined by electron microscopy virtually all cells showed nucleus-cytoplasm asynchrony indicative of a neoplastic transformation [ 181. Our immunofluorescent findings suggest that nonsecretory variants may share, at the cellular level, the
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functional diversity of secretory myelomas. Indeed, two abnormal immunoglobulins belonging to different classes may be rarely found in the serum of patients with multiple myeloma. The most common is a combination of IgG and IgA [24-321 or IgG and IgM [33-361, generally sharing the same type of light chain. To our knowledge, double IgM-IgA paraproteinemia has not been documented in patient with classic multiple myeloma. Such combination, however, has been detected in patients with lymphosarcoma [37], WaldenStrom’s macroglobulinemia [38] and other plasma cell disorders [39]. In six patients with double myelomas the dual immunoglobulin production was documented at the cellular level by immunofluorescence of bone marrow smears. In four cases it was found that each immunoglobulin was produced by different cell lines [27,28,33,34], and in two instances double label technics revealed the two myeloma Iproteins being produced simultaneously by single cells [24,25]. Thus, the first case reported herein appears to be the nonsecretory counterpart of the latter type of double myeloma. The existence of myelomas with a double M component has raised questions concerning their monoclonal origin. However, detailed immunologic studies in some cases have shown that both myeloma proteins share similar if not identical idiotypic determinations [24-26,33,35,36] thus indicating a common origin from a single ancestor cell. Since during the antibody response a clone of cells may initially produce IgM and switch over to IgG synthesis, Levin et al. [36] proposed that a malignant transformation occurring during the switch over phase may result in myelomas capable of producing IgM and IgG sharing the same antigenic determinants. A similar sequence of events has been suggested to explain the existence of IgGlgA myelomas [25]. Whether this also applies to myelomas producing other immunoglobulin combinations has yet to be documented.
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Rosen BJ, Smith TW, Block KJ: Multiple myeloma associated with two serum M components; yG type K and yA type L. N Engl J Med 227: 902. 1967. Bachmann R: Simultaneous occurrence of two immunologically different M-components in serum. Acta Med Stand 177: 593, 1965. Kistner S, Norberg R: Simultaneous occurrence of two different myeloma proteins. Stand J Clin Lab Invest 17: 32 1, 1965. Radl J. Chott L, Richova D: Ein Fall von myelom und bildung zweier in antigener hinsicht unterschiedlicher paraprotein. Klin Wochenschr 44: 1117, 1966. Wetter 0, Hertenstein C: Die optiscne rotationsdispersion isolierter paraproteine. IV. Untersuchungen bei multiplem myelom mit doppelter proteinanomalie. Klin Wochenschr 44: 1111, 1966. Wang AC, Wang IYF, McCormack JN, et al.: The identity of the light chains of monoclonal IgG and monoclonal IgM in one patient. Immunochemistry 6: 451, 1969. Curtain CC: Immuno-cytochemical localization of two abnormal serum globulins in the one bone marrow smear. Australs Ann Med 13: 136, 1964. Penn GM, Kunkel GH, Grey HM: Sharing of individual antigenic determinants between a yo and a yu protein in the myeloma serum. Proc Sot Exp Biol Med 135: 660, 1970. Levin AC, Fudenberg HH, Hooper JE, et al.: Immunofluorescence evidence for cellular control of synthesis of variable regions of light and heavy chains of immunoglobulins G and M by the same gene. Proc Natl Acad Sci USA 68: 169, 1971. Yagi Y, Pressman D: Monoclonal IgA and IgM in the serum of a single patient (SC). I. Sharing of individually specific determinants between IgA and IgM. J lmmunol 110: 335, 1973. Fateh-Moghadam A, Beil E, Borchers H, et al.: Plasmozytom. Makroglobulinamie Waldenstrcm und Morbus Paget bei einem Patienten mit IgAK+lgMK-Doppelparaproteinamie und Bence-Jones-Protein (Typ K). Blut 21: 146, 1970. lmhof JW, Ballieux RE, Mul NA, et al.: Monoclonal and biclonal gammopathies. Acta Med Stand 445 (suppl): 102, 1966.
