MULTIPLE MYELOMA AND RELATED DISORDERS

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1 SEVEN MULTIPLE MYELOMA AND RELATED DISORDERS Multiple myeloma Multiple myeloma or myelomatosis is a disease resulting from the proliferation in the bone marrow of a clone of neoplastic cells that are closely related, both morphologically and functionally, to plasma cells. In the great majority of cases, the neoplastic cells secrete a protein that is either a complete immunoglobulin (Ig) or an immunoglobulin light chain. Clinical features result either directly from the effects of the neoplastic proliferation or indirectly from effects of the protein, often designated a paraprotein, which the myeloma cells produce. The nature of this disease, in which cells of the abnormal clone are widely, randomly and diffusely distributed in the red marrow, is better reflected by the term myelomatosis than by the more commonly used multiple myeloma. It might be preferable if the latter term was restricted to those rare cases, more closely related to solitary myeloma (plasmacytoma), in which there are several isolated lesions but intervening bone marrow is normal. However, use of the term multiple myeloma for the disease we are describing is so common that we shall retain it. Multiple myeloma is a disease predominantly of the middle-aged and elderly. It has an incidence of 2 4/ /year. The disease is more common in Blacks than in Caucasians and somewhat more common in men than in women. Characteristic clinical features are anaemia, bone pain, pathological fractures, hypercalcaemia and renal failure. A minority of patients have hepatomegaly or lymphadenopathy. Splenomegaly is occasionally present. Patients with symptomatic bone lesions usually have either generalized osteoporosis or discrete osteolytic lesions but occasional patients have osteosclerosis. The paraprotein secreted is IgG in about 60% of cases and IgA in about 20%; some patients secrete excess monoclonal light chain (Bence-Jones protein) in addition to complete Ig and, in about 15 20% of patients, only light chain is produced (Bence-Jones myeloma). A minority of patients produce an IgM, IgD or IgE paraprotein. Occasional patients have two distinct paraproteins. A small minority of patients have no paraprotein in serum or urine (non-secretory myeloma). Any paraprotein secreted will, since it arises from a single clone of cells, contain only a single light chain type, either κ or λ. Monoclonal Ig, being of high molecular weight, are usually detected only or mainly in the serum whereas, unless there is co-existing renal failure, the low molecular weight Bence-Jones protein is detected only in the urine. Multiple myeloma can be diagnosed with reasonable certainty if two of the following three criteria are met: 1 more than 10% plasma cells in a bone marrow aspirate; 2 a paraprotein in serum or urine; and 3 osteolytic bone lesions or osteoporosis [1]. The probability of myeloma is high if an IgG paraprotein exceeds 30 g/l or if an IgA paraprotein exceeds 10 g/l. It is useful to distinguish smouldering multiple myeloma from other cases. Smouldering multiple myeloma is an appropriate diagnosis if there are no symptoms, normal serum calcium concentration and renal function (creatinine <170 µmol/l), a relatively low paraprotein concentration (IgG paraprotein less than 70 g/l or IgA paraprotein less than 50 g/l), no bone lesions and no infections. The median survival in multiple myeloma is

2 MULTIPLE MYELOMA 333 Fig. 7.1 BM aspirate, multiple myeloma, showing a range of cells from a plasmablast to mature plasma cells. MGG 940. years. The condition usually terminates in refractory disease, sometimes with a leukaemic phase. In some cases there is transformation to immunoblastic lymphoma. A significant minority of patients (about 10% of those who survive 5 10 years after treatment) succumb to a secondary myelodysplastic syndrome or to acute myeloid leukaemia (AML); it is likely that these complications result from the administration of alkylating agents. Peripheral blood The great majority of patients have anaemia which is either normocytic, normochromic or, less often, macrocytic. In most patients there is increased rouleaux formation and increased background basophilic staining due to the presence of the paraprotein in the blood. These features do not occur in the minority of patients whose cells secrete only Bence-Jones protein or are non-secretory. The blood film is occasionally leuco-erythroblastic and it is often possible to find a small number of plasma cells or plasmacytoid lymphocytes. In patients with more advanced disease, there may be thrombocytopenia and neutropenia. Occasionally, there are large numbers of circulating neoplastic cells, showing mild to marked atypia, either at presentation or during the terminal phase of the disease. The designation plasma cell leukaemia is then used (see page 345). Occasional patients have a neutrophilic leukaemoid reaction. Bone marrow cytology The bone marrow features are very variable. Plasma cells are usually increased, often constituting between 30 and 90% of bone marrow nucleated cells. The myeloma cells may be morphologically fairly normal, showing the eccentric nucleus, clumped chromatin and Golgi zone typical of a normal plasma cell, or may be moderately or severely dysplastic. A common cytological abnormality is nucleocytoplasmic asynchrony; the cytoplasm is mature but the nucleus either has a diffuse chromatin pattern or contains a prominent nucleolus (Figs 7.1 and 7.2). Other cytological abnormalities include: (i) marked pleomorphism; (ii) increased size of cells; (iii) a high nucleocytoplasmic ratio; (iv) multinuclearity (Fig. 7.3); (v) nuclear lobulation (Fig. 7.4); (vi) phagocytosis by myeloma cells (Fig. 7.3); and (vii) the presence of mitotic figures. Occasionally, multinuclearity is extreme with up to 40 nuclei per cell [2]. Sometimes there are giant dysplastic plasma cells. Cytoplasmic abnormalities include uniform basophilia without a distinct Golgi zone, cytoplasmic eosinophilia and flaming eosinophilic margins (Fig. 7.3). The cytoplasm may be voluminous or may contain crystals (Fig. 7.5), vacuoles or dilated sacs of endoplasmic reticulum (Fig. 7.6). Myeloma cells may have multiple small vacuoles containing weakly basophilic material (Fig. 7.7), the term Mott cell then being used. They may have giant spherical inclusions, referred to as

3 334 CHAPTER SEVEN Fig. 7.2 BM aspirate, multiple myeloma, showing plasmablasts, one of which is trinucleated. MGG 940. Fig. 7.3 BM aspirate, multiple myeloma, showing bi- and trinucleated myeloma cells and myeloma cells with flaming cytoplasm. MGG 940. Fig. 7.4 BM aspirate, multiple myeloma, showing one myeloma cell with a bilobed nucleus and another which has ingested an erythrocyte. MGG 940.

4 MULTIPLE MYELOMA 335 Fig. 7.5 BM aspirate, multiple myeloma, showing a myeloma cell containing crystals of immunoglobulin. MGG 940. Fig. 7.6 BM aspirate, multiple myeloma, showing myeloma cells, one of which has gross distension of the endoplasmic reticulum by immunoglobulin. MGG 940. Fig. 7.7 BM aspirate, multiple myeloma, showing a Mott cell. MGG 970.

5 336 CHAPTER SEVEN Fig. 7.8 BM aspirate, multiple myeloma, showing numerous Russell bodies. MGG 940. Fig. 7.9 BM aspirate, multiple myeloma, showing formation of a Dutcher body by invagination from the cytoplasm. There are also flame cells. MGG 940. Russell bodies (Fig. 7.8). Nuclear inclusions may be present (Fig. 7.9). Rarely the bone marrow aspirate contains amyloid (see Fig. 7.29). It is not always possible to make a diagnosis of multiple myeloma on the basis of bone marrow morphology alone. Multiple myeloma characteristically affects the bone marrow in a patchy manner and an aspirate will not necessarily contain a large number of plasma cells; nor will the morphology of the myeloma cells necessarily be very abnormal. Non-diagnostic aspirates are obtained in about 5% of patients. There is no particular percentage of plasma cells which reliably separates multiple myeloma from reactive plasmacytosis or from monoclonal gammopathy of undetermined significance (see below). It is necessary, in doubtful cases, to assess not only the bone marrow cytology and histology but also the clinical, radiological and biochemical features. The bone marrow aspirate is of value not only in making a diagnosis of multiple myeloma but also in determining the prognosis. Both the percentage of plasma cells in the aspirate [3 5] and their degree of dysplasia [4,6 8] correlate with prognosis.

6 MULTIPLE MYELOMA 337 Fig BM trephine biopsy section, multiple myeloma, showing interstitial infiltration of the marrow by plasma cells. Paraffin-embedded, H&E 940. Flow cytometry and immunocytochemistry Myeloma cells give negative reactions with most B-cell markers but positive reactions are often obtained with CD79a. In addition, both normal plasma cells and myeloma cells express CD38 and CD138 and do not express CD45. Normal plasma cells usually express CD19 and not CD56, whereas myeloma cells often over-express CD56 and fail to express CD19 [9,10]. Other antigens which are not expressed in normal plasma cells but may show aberrant expression in myeloma cells include CD20, CD28 and CD117. SmIg may be expressed whereas normal plasma cells express only cytoplasmic Ig [9]. Bone marrow histology Bone marrow biopsy can be of use both in the diagnosis of multiple myeloma and in assessing the prognosis. Non-diagnostic biopsies are obtained in 5 10% of cases, either because of early disease or because the pattern of infiltration is nodular rather than diffuse and the biopsy has included only noninfiltrated marrow [11]. In addition, increased reticulin may mean that myeloma cells are not readily aspirated. Because a larger volume of tissue is sampled than in an aspirate and since the pattern of infiltration can be ascertained, a biopsy may confirm a diagnosis of multiple myeloma when the aspirate has not done so. However, on occasions, more diagnostic information is obtained from the aspirate than from the trephine biopsy, so the two investigations should be regarded as complementary. Three major patterns of infiltration are seen: (i) interstitial, with or without paratrabecular seams of plasma cells; (ii) nodules or broad bands; and (iii) a packed marrow [11]. When infiltration is interstitial myeloma cells are dispersed among haemopoietic and fat cells (Fig. 7.10), whereas in a packed marrow (Fig. 7.11) the normal architecture of the marrow is obliterated. In reactive plasmacytosis, infiltration is interstitial and large accumulations of plasma cells are not seen. Some histopathologists have considered that plasma cells sited around capillaries are indicative of reactive plasmacytosis rather than multiple myeloma [11], but others have observed this feature also in multiple myeloma [12]. A paratrabecular pattern of infiltration (Fig. 7.12) may occur but is uncommon. Myeloma cells show varying degrees of dysplasia. In some cases myeloma cells are morphologically very similar to normal plasma cells. In others, they are very large, have nucleoli, are pleomorphic (Fig. 7.13) or are frankly blastic with a diffuse chromatin pattern and a prominent nucleolus (Fig. 7.14). Nucleocytoplasmic asynchrony is common. Dutcher bodies (Fig. 7.13), Russell bodies (Fig. 7.15), cytoplasmic crystals (Fig. 7.16) and Mott cells (Fig. 7.17) may be apparent. Additional changes which may be associated with multiple myeloma include reduced haemopoiesis, even in the absence of heavy infiltration, increased

7 338 CHAPTER SEVEN Fig BM trephine biopsy section, multiple myeloma, showing diffuse infiltration by plasma cells (packed marrow). Paraffin-embedded, H&E 195. Fig BM trephine biopsy section, multiple myeloma, showing a paratrabecular myelomatous infiltrate. Paraffinembedded, H&E 188. Fig BM trephine biopsy section, multiple myeloma, showing plasma cells with moderate nuclear pleomorphism and numerous eosinophilic intranuclear (Dutcher) and intracytoplasmic (Russell) inclusion bodies. Paraffinembedded, H&E 970.

8 MULTIPLE MYELOMA 339 Fig BM trephine biopsy section, multiple myeloma (plasmablastic), showing plasmablasts with marked variation in nuclear size and shape and prominent central nucleoli. Plastic-embedded, H&E 940. Fig BM trephine biopsy section, multiple myeloma, showing abundant Russell bodies. Paraffin-embedded, H&E 390. reticulin deposition, lymphoid infiltrates and, occasionally, the presence of granulomas (see Fig. 3.29). Bone marrow vascularity is increased and is indicative of a worse prognosis [13,14]. Patients with a neutrophilic leukaemoid reaction show striking granulocytic hyperplasia. Occasionally, reticulin deposition is markedly increased and there is also collagen deposition; this appears to be particularly associated with IgD myeloma [15,16]. Collagen deposition is preferentially type II rather than type I [17], as occurs also in idiopathic myelofibrosis. If systematic evaluation is carried out, amyloid deposition is demonstrated in the bone marrow in up to 10% of patients [18]. The bone changes usually associated with multiple myeloma are diffuse osteoporosis, with thinning of all trabeculae, and osteolytic lesions with resorption of bone by osteoclasts. In multiple myeloma there is no relationship between osteoclastic and osteoblastic activity [19]. Diffuse osteoporosis has been found to be associated with a packed marrow pattern of infiltration, while osteolytic lesions are found particularly in those with nodular infiltration [11]. Osteoclastic activity correlates with the degree

9 340 CHAPTER SEVEN Fig BM trephine biopsy section, multiple myeloma, showing a striking deposition of crystals of immunoglobulin. Paraffin-embedded, H&E 940. (By courtesy of Dr P Hayes, Chatham.) Fig BM trephine biopsy section, multiple myeloma, showing myeloma cells with endoplasmic reticulum distended by immunoglobulin (Mott cells). Paraffin-embedded, H&E 940. of plasma cell infiltration. Occasional patients with multiple myeloma have osteosclerosis (Fig. 7.18). The prognosis in myeloma can be related to: (i) the extent of plasma cell infiltration (histological stage); (ii) the pattern of infiltration; and (iii) the cytological features of the cells (histological grade) [11]. Bartl et al. [11] found that a nodular pattern of infiltration correlated with more aggressive disease and with a worse prognosis than was seen when the pattern of infiltration was interstitial, with or without paratrabecular seams of plasma cells; a packed marrow indicated a worse prognosis than either of the other patterns. This has been confirmed by Pich et al. [20]. These two groups and a number of other investigators have been able to relate prognosis also to the degree of dysplasia of the myeloma cells [19 22]. Bartl and Frisch [19] have suggested a classification that divides myeloma into three groups: (i) low grade, in which the plasma cells are mature with minimal dysplasia; (ii) intermediate grade, in which the plasma cells are dysplastic but not frankly blastic; and (iii) high grade, comprising plasmablasts. The three grades have median survivals of 60, 32 and 10 months, respectively [7,11].

10 MULTIPLE MYELOMA 341 Fig BM trephine biopsy section in osteosclerotic multiple myeloma. Paraffin-embedded, H&E 188. The best prognosis in multiple myeloma is found in those with smouldering disease. Histologically, these patients have minimal interstitial infiltration by mainly mature plasma cells [19]. The minority of cases of multiple myeloma which show a high count of mitotic figures (more than one per high power field) have a shorter survival [23]. The presence of amyloid deposits in the bone marrow or elsewhere has been found not to influence prognosis in patients who are treated with intensive chemotherapy followed by autologous stem cell transplantation [18]. Response to treatment is associated with a reduction of plasma cell burden and reduced osteoclastic activity. Corticosteroid-treated patients may have aggravation of osteoporosis. Patients treated with biphosphonates may show new bone formation with extensive osteoid seams [19]. Immunohistochemistry Neoplastic cells in the great majority of cases of multiple myeloma express monotypic immunoglobulin, i.e. there is expression of κ or λ light chains but not both. There is usually expression of CD38 and CD138 and reactions with VS38c are positive [24 26]. Cells from cases with plasmacytic morphology, but rarely those with plasmablastic features, express the CD38 ligand CD31 [27]. Expression of CD79a is usual but, in a substantial proportion of cases, cells fail to express this antigen. Cells in a proportion of cases express CD45 or CD20 but most do not. A variety of other antigens, including CD22, CD45RO, CD30 and non-haemopoietic antigens, such as epithelial membrane antigen (EMA) and vimentin, are sometimes expressed [28]. However, in contrast to normal plasma cells, the expression of CD30 and EMA is not at all common in multiple myeloma. Nuclear expression of cyclin D1 is observed in the majority of patients in whom t(11;14)(q13;q32) is detected and in a minority of other patients [29]. Expression of CD45RO correlates with a worse prognosis [30]. Cytogenetics and molecular genetics Because of the low proliferation rate of myeloma cells, cytogenetic analysis demonstrates a clonal abnormality in only 30 50% of cases of multiple myeloma, whereas fluorescence in situ hybridization (FISH) techniques demonstrate an abnormality in % of cases. No specific cytogenetic or molecular genetic abnormalities have been demonstrated but aneuploidy and chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus at 14q32 are both common. Translocations with a 14q32 breakpoint are demonstrable by FISH in about 60% of patients [31]. They include t(4;14)(p16;q32), t(6;14)(p25;q32), t(8;14)(q24;q32), t(9;14)(p13;q32), t(11;14)(q13;q32) (the

11 342 CHAPTER SEVEN Fig BM trephine biopsy section, showing heavy reactive plasma cell infiltration in HIV infection. Paraffin-embedded, H&E 940. commonest translocation demonstrated) and t(14;16)(q32;q22 23). Other translocations demonstrated have sometimes had a 22q11 breakpoint, e.g. t(8;22)(q24;q11) and t(16;22)(q23;q11). Frequently demonstrated aneuploidies include monosomy 13 and trisomies 3, 5, 6, 7, 9, 11, 15, 17 and 19 [31 34]. Common deletions are 13q14, 17p13 and 11q [35]. Hyperdiploidy is seen in more than 50% of patients with the remainder showing hypodiploidy, pseudodiploidy and near tetraploidy [34]. Deletions of 11q, 13q14 and 17p13 have been found to have a poor prognostic significance [35]. Problems and pitfalls Problems can occur in distinguishing multiple myeloma from reactive plasmacytosis and from other lymphoproliferative disorders with plasmacytic differentiation. The diagnosis must therefore be based on correlation of clinical, biochemical, radiological, cytological and histological features. Increased numbers of plasma cells can be seen as a reactive phenomenon in a wide range of conditions (page 116) and they can comprise more than 10% of bone marrow cells (Fig. 7.19). In assessing bone marrow aspirates, there is no cut-off point that reliably distinguishes multiple myeloma from reactive conditions. Rarely, as many as 30 50% of plasma cells are present as a reactive change. Cytological features must be assessed as well as plasma cell number. The presence of plasmablasts and marked plasma cell dysplasia, e.g. giant forms, striking nuclear lobulation and prominent nucleoli, are strongly suggestive of multiple myeloma. Three trephine biopsy features have been reported to be specific for multiple myeloma: (i) homogeneous nodules of plasma cells occupying at least half a high power field; (ii) monotypic plasma cell aggregates occupying the space between fat cells; and (iii) marked diffuse plasmacytosis with monotypic light chain expression [23]. Increased numbers of plasma cells are seen in monoclonal gammopathy of undetermined significance (see below) and this condition therefore needs to be distinguished from multiple myeloma [36,37]. In some cases of multiple myeloma, the neoplastic cells have the features of lymphoplasmacytoid lymphocytes rather than of plasma cells; such cases need to be distinguished from lymphoplasmacytic lymphoma. In the rare cases of multiple myeloma which show a very marked fibroblastic response, it may be difficult to recognize distorted plasma cells (Fig. 7.20); immunohistochemistry is useful to confirm their presence. In some patients with multiple myeloma, the neoplastic cells are so immature or atypical that they are cytologically indistinguishable from large cell lymphoma or other anaplastic tumours, including carcinoma, melanoma and AML. Plasmablastic

12 MULTIPLE MYELOMA 343 Fig BM trephine biopsy section, multiple myeloma, showing a fibroblastic reaction to myelomatous infiltration. Immunohistochemistry is useful for identifying the distorted neoplastic cells in the fibrous tissue. Paraffin-embedded, H&E 376. (By courtesy of Dr J Jip, Bolton.) Fig BM aspirate, multiple myeloma, showing a giant myeloma cell which could easily be confused with a megakaryocyte; the presence of an intranuclear inclusion (a Dutcher body) is a clue to the correct diagnosis. MGG 940. myeloma and large cell lymphoma with cells having the features of immunoblasts are particularly likely to be confused. Myeloma cells sometimes also resemble carcinoma cells in forming cohesive masses [38]. Giant, pleomorphic plasma cells may be confused with megakaryocytes (Figs 7.21 and 7.22). A Giemsa stain can be very useful in highlighting plasma cell differentiation in an apparently anaplastic tumour but immunohistochemistry, using an appropriate panel of antibodies, is often crucial in making a correct diagnosis. Large cell lymphoma can be distinguished from multiple myeloma immunohistochemically since lymphoma cells are usually positive for CD45, CD79a and CD20 and do not express cytoplasmic immunoglobulin. Myeloma cells are usually negative for CD45 and CD20, vary in their expression of CD79a and show strong cytoplasmic expression of monotypic immunoglobulin. AIDS-related large cell lymphomas without any apparent plasmacytic differentiation may fail to express CD20 but usually express CD45. Anaplastic large cell lymphoma (see page 306) is among the large cell lymphomas which can be confused with anaplastic multiple myeloma; both conditions are usually negative for B-cell markers and CD45 but positive for CD30 and both may express EMA.

13 344 CHAPTER SEVEN (a) (b) (c) Fig BM trephine biopsy sections (same case as in Fig. 7.21), showing giant myeloma cells which were confused with megakaryocytes. However, the giant cells were negative for CD61, positive for a plasma cell marker and showed light chain restriction. Paraffin-embedded. (a) H&E 188. (b) Positive reaction with VS38c. Immunoperoxidase 376. (c) Positive reaction with antikappa. Immunoperoxidase 376.

14 MULTIPLE MYELOMA 345 Fig (d) Negative reaction with anti-lambda. Immunoperoxidase 376. (d) Staining for ALK1 can be useful since many cases of anaplastic large cell lymphoma are positive (see page 309). CD45RO, often used as a T-cell marker, is expressed in some cases of multiple myeloma [30]. The demonstration of light chain restriction using labelled oligonucleotide or peptide nucleic acid probes complementary to κ and λ light chain mrna (see page 80) can be useful in distinguishing myeloma from large cell lymphoma and from other tumours. Anaplastic carcinoma cells do not express CD45 or CD20 and usually express EMA and high or low molecular weight cytokeratins. However, in this context, it should be noted that some myeloma cells also express EMA and, likewise, CD138 may be expressed by non-lymphoid tumours including some anaplastic carcinomas. It is usual practice to apply a broad panel of monoclonal antibodies in an attempt to determine the nature of anaplastic tumours. If reactions are all negative it is important to consider anaplastic myeloma and carry out immunostaining for κ and λ light chains. Immunostaining for CD138 and the antigen reactive with VS38c can also be useful in this context [25,26]. Plasma cell leukaemia The term plasma cell leukaemia may be used to designate a de novo leukaemia of neoplastic plasma cells or the terminal phase of multiple myeloma when myeloma cells are present in the peripheral blood in large numbers. Plasma cell leukaemia has been defined by the presence in the circulating blood of at least /l plasma cells which also constitute at least 20% of circulating cells [39]. Patients with de novo or primary plasma cell leukaemia show clinical features that are common in multiple myeloma such as bone pain, lytic lesions, hypercalcaemia and renal failure, but they have a higher incidence of extramedullary lesions and, in addition, often have hepatomegaly and splenomegaly. The disease is more aggressive than multiple myeloma with a median survival of less than a year. Patients with established multiple myeloma who develop a secondary plasma cell leukaemia have advanced disease which is usually refractory to treatment. Their prognosis is likewise poor. Peripheral blood The blood film shows large numbers of circulating neoplastic plasma cells (Fig. 7.23), the morphology of which varies between patients from cells resembling normal plasma cells to primitive, blastic cells showing only minimal evidence of plasma cell differentiation. Anaemia is almost invariable and neutropenia and thrombocytopenia are common. Increased rouleaux formation and increased background staining are usual; in patients with plasma cell leukaemia as the terminal phase of multiple

15 346 CHAPTER SEVEN Fig PB film in plasma cell leukaemia, showing marked rouleaux formation and two dysplastic plasma cells, one with an intranuclear inclusion (Dutcher body). MGG 940. myeloma these abnormalities are often striking since the paraprotein level is commonly high. Bone marrow cytology The bone marrow is heavily infiltrated with neoplastic cells showing the same morphological features as those in the peripheral blood. Normal haemopoietic elements are reduced. Bone marrow histology There is a diffuse infiltrate of plasma cells which make up the majority of cells in the marrow [40,41]. In the majority of cases, the cytological features are similar to those of multiple myeloma; in a minority of cases the cells are very immature with little morphological evidence of plasma cell differentiation. Cytogenetics and molecular genetics Clonal cytogenetic abnormalities are common in plasma cell leukaemia. Although no consistent association is recognized, monosomy 13 is common [32,33] and there are often chromosomal rearrangements, such as t(11;14)(q13;q32), with a 14q32 breakpoint. Hypodiploidy is common whereas, in contrast to multiple myeloma, hyperdiploidy is not [32 34]. Deletion of the cancer-suppressing RB gene is common [32,33]. Monoclonal gammopathy of undetermined significancedbenign monoclonal gammopathy Monoclonal gammopathy of undetermined significance (MGUS) denotes a condition in which there is proliferation of a clone of plasma cells with production of a paraprotein but without the signs of disease which are characteristic of multiple myeloma and related plasma cell disorders. The paraprotein is usually an immunoglobulin (either IgG, IgA or IgM) but is occasionally a Bence-Jones protein. The paraprotein concentration is relatively low (in the case of an IgG paraprotein less than 30 g/l, in the case of an IgA or IgM paraprotein less than 20 g/l) and is usually stable. Immune paresis is not a feature. Since there are no features of disease, the diagnosis is necessarily made incidentally. Monoclonal gammopathy of uncertain significance is common. Paraproteins can be detected in about 1% of adults under the age of 70 and in about 3% of adults over this age. It is not always possible to make a distinction between multiple myeloma and MGUS on the basis of any single feature. It is necessary to assess clinical and haematological features, bone marrow cytology and bone marrow histology in order to make the distinction. A period of observation may also be necessary to establish that the concentration of the paraprotein is stable and no disease features are appearing. It should be noted that although this

16 MULTIPLE MYELOMA 347 condition is clinically benign, it does represent a neoplastic proliferation. If patients are followed for a prolonged period, a significant proportion eventually develop overt evidence of multiple myeloma or a related condition. In one series of patients, over a 20-year period of observation, the actuarial risk of developing multiple myeloma, Waldenström s macroglobulinaemia or primary (AL) amyloidosis was found to be approximately 30% [42]. In another small series of patients, followed for up to 31 years, seven of 11 patients developed a plasma cell neoplasm or non-hodgkin s lymphoma (NHL) [43]. Since progression may occur, but the likelihood of this cannot be predicted currently for individual patients, the designation monoclonal gammopathy of undetermined significance is preferred to benign monoclonal gammopathy. Peripheral blood The peripheral blood film may show increased rouleaux formation but this is generally less marked than in multiple myeloma; anaemia does not occur and circulating plasma cells are not present. Bone marrow cytology The bone marrow may appear completely normal or there may be an increase of plasma cells. Plasma cells do not usually exceed 5% of nucleated cells but may comprise up to 10%. The plasma cells are usually morphologically fairly normal but minor dysplastic features may be noted. Bone marrow histology Plasma cell infiltration is usually minimal with focal accumulation around capillaries which is often indistinguishable from a reactive plasmacytosis [11]. Immunohistochemistry Immunohistochemical staining with anti-κ and anti-λ antisera shows κ:λ or λ:κ ratios of greater than four in approximately 65% of cases, the majority having ratios between four and 16. In comparison, almost all cases of overt myeloma have κ:λ or λ:κ ratios of greater than 16, the majority being greater than 100 [44]. Cytogenetics and molecular genetics Because the neoplastic cells make up a low proportion of bone marrow cells and have a low proliferative rate, it is uncommon for cytogenetic analysis to demonstrate any clonal abnormality in patients with MGUS. Nevertheless, FISH techniques show that translocations with a 14q32 breakpoint, such as t(4;14)(p16;q32) and t(11;14)(q13;q32), occur with a similar frequency to that seen in multiple myeloma [31]. Various aneuploidies have also been demonstrated. Monosomy 13, however, has been found to be much less common than in multiple myeloma and, when present, is likely to be predictive of disease progression [31]. Problems and pitfalls It can be difficult to estimate κ:λ ratios when plasma cells constitute less than 5% of total cells [45]. However, it should be noted that this is not an essential investigation if it is known that the patient has a paraprotein. The major purpose of the trephine biopsy in this context is to distinguish between MGUS and multiple myeloma. Waldenström s macroglobulinaemia The condition described by Waldenström as essential hyperglobulinaemia is a disease characterized by a lymphoplasmacytic neoplasm with secretion of large amounts of an IgM paraprotein. It is a variant of lymphoplasmacytic lymphoma (see page 258) and is distinguished from other cases of this lymphoma on the basis of clinical and biochemical features rather than by cytological or histological criteria. Predominant signs and symptoms are either characteristic of a lymphoma or are caused by the hyperviscosity of blood consequent on the high concentration of IgM. Hepatomegaly, splenomegaly and lymphadenopathy are common. Clinical features resulting from the high concentration of the paraprotein include anaemia (due to a greatly increased plasma volume), impaired vision, cerebral effects, cardiac failure and a bleeding tendency. In some patients the paraprotein has characteristics of a cold agglutinin or a cryoglobulin and in others it is amyloidogenic. Peripheral neuropathy is common and the paraprotein can sometimes be shown

17 348 CHAPTER SEVEN Fig BM aspirate, Waldenström s macroglobulinaemia, showing a mast cell and mature small lymphocytes. MGG 940. Fig BM aspirate, Waldenström s macroglobulinaemia (same case as in Fig. 7.24), showing mature small lymphocytes and one plasmacytoid lymphocyte clustered around a macrophage (reticulum cell). MGG 940. to have antibody activity against neural antigens. The incidence of Waldenström s macroglobulinaemia is about a tenth that of multiple myeloma. Peripheral blood There is usually a normocytic normochromic anaemia, increased rouleaux formation and increased background staining. Some patients have thrombocytopenia. When the paraprotein has the characteristics of a cold agglutinin or of a cryoglobulin, either red cell agglutinates or a cryoglobulin precipitate may be detected in the blood film. The lymphocyte count may be normal or elevated, with the lymphocytes usually being mature small lymphocytes showing some features of differentiation to plasma cells. Bone marrow cytology The bone marrow is infiltrated by neoplastic cells which most often have the morphology of small lymphocytes, with a variable proportion of cells showing some plasmacytoid features (Figs 7.24 and 7.25). Plasma cells are usually increased and, in some patients, plasmacytic differentiation is prom-

18 MULTIPLE MYELOMA 349 Fig BM trephine biopsy section, Waldenström s macroglobulinaemia, showing a diffuse infiltrate of small lymphoid cells, some of which show evidence of plasmacytoid differentiation (Dutcher bodies, clock-face chromatin pattern and/or a Golgi zone). Plastic-embedded, H&E 377. inent. Intranuclear or intracytoplasmic inclusions, which are usually periodic acid Schiff (PAS)- positive, are sometimes present. Macrophages and mast cells (Fig. 7.25) are often increased. Lymphoplasmacytoid cells may be clustered around macrophages. Bone marrow histology The bone marrow histology is not uniform [46 48]. In some patients, the infiltrating cells are predominantly lymphocytes, including plasmacytoid lymphocytes whereas, in others, there is prominent plasma cell differentiation. Some patients have an interstitial infiltrate or well-circumscribed nodules of lymphoid cells but, in the majority, there is either diffuse infiltration or a mixed nodular diffuse pattern. Paratrabecular infiltration may also occur, often accompanied by increased reticulin deposition. A variable proportion of cells show features of plasmacytic differentiation, including Russell bodies and Dutcher bodies (Fig. 7.26). Mature plasma cells are usually increased, as are macrophages, mast cells and, sometimes, eosinophils. Mast cells may be specifically associated with lymphoid infiltrates. Overall, about half of the cases show increased reticulin deposition. Trephine biopsy sections sometimes show infiltration when the bone marrow aspirate is normal. Rare observations in Waldenström s macroglobulinaemia include the deposition of paraprotein in the bone marrow interstitium and an abnormal staining pattern caused by intrasinusoidal paraprotein (Fig. 7.27). Immunohistochemistry Immunohistochemical features vary between and within cases, depending on the degree of plasmacytic differentiation shown. There is usually strong expression of CD79a by most of the cells, while any neoplastic plasma cells show monotypic cytoplasmic immunoglobulin (κ or λ) and react with VS38c. Other syndromes associated with secretion of a paraprotein There are a number of other, relatively uncommon, syndromes associated with the secretion of a paraprotein. In some of these the features of a lymphoproliferative disorder are prominent; in others the clinical and pathological features relate to the characteristics of the paraprotein and only detailed investigation reveals the presence of an abnormal clone of plasma cells, plasmacytoid lymphocytes or lymphocytes. These conditions are summarized in Table 7.1. Light chain-associated amyloidosis Light chain-associated amyloidosis (AL-type of

19 (a) (b) Fig Paraffin-embedded BM trephine biopsy sections, Waldenström s macroglobulinaemia, showing: (a) interstitial deposition of IgM paraprotein adjacent to a trabecula, PAS stain 188; and (b) intrasinusoidal paraprotein, PAS stain 94. Table 7.1 Monoclonal gammopathies (modified from [49]). Multiple myeloma (including smouldering multiple myeloma) Solitary plasmacytoma (of bone or extramedullary) Lymphoplasmacytic lymphoma including Waldenström s macroglobulinaemia and other types of non-hodgkin s lymphoma with a serum or urinary paraprotein Monoclonal gammopathy of undetermined significance including idiopathic Bence-Jones proteinuria Light chain-associated amyloidosis Light chain or other immunoglobulin deposition disease Adult acquired Fanconi s syndrome (renal tubular deposition and excretion of monoclonal light chain) Essential cryoglobulinaemia (type I and type II cryoglobulinaemia) Chronic cold haemagglutinin disease Alpha, gamma and mu (α, γ and µ) heavy chain disease The POEMS syndrome Acquired angio-oedema associated with monoclonal gammopathy Monoclonal gammopathy with peripheral neuropathy Scleromyxoedema (mucinous deposition in the skin plus serum paraprotein) Systemic capillary leak syndrome with paraproteinaemia Acquired C1 inhibitor deficiency with paraproteinaemia

20 MULTIPLE MYELOMA 351 amyloidosis) is a disease consequent on a plasma cell disorder in which there is production of an amyloidogenic light chain [50 54]. The features of the disease are usually caused by the amyloidosis rather than by other features of the plasma cell proliferation. Heart failure and renal failure are common manifestations. In a minority of patients, light chain-associated amyloidosis is associated with overt multiple myeloma, Waldenström s macroglobulinaemia or other forms of lymphoplasmacytic lymphoma (see page 258) [53]. However, in the majority of patients, the plasma cell disorder would be classified as MGUS if it were not for the amyloidogenic characteristics of the light chains. In a small minority of patients no paraprotein is detectable in serum or urine but, in this group also, the disease results from a neoplastic proliferation of plasma cells, albeit occult. The term primary amyloidosis may be used to describe cases without evidence of multiple myeloma or other overt plasma cell neoplasm. Amyloid can be formed from either κ or λ light chains but, in the majority of patients, it is derived from λ light chains. Bone marrow histology Bone marrow biopsy sections may be normal or may show increased plasma cells. Sometimes, characteristic features of multiple myeloma or lymphoplasmacytic lymphoma are present. In addition, amyloid may be detected either in the walls of small blood vessels (Figs 7.29 and 7.30) or in the interstitium (Fig. 7.31). Amyloid was detected in bone marrow sections in 56% of one large series of patients [55]. In sections stained with H&E, amyloid is homogeneous and pink (Fig. 7.29a) whereas with a Giemsa stain it is blue (Fig. 7.30). It stains with Congo red (Fig. 7.29b) and, following Congo red staining, shows apple-green birefringence on examination with polarized light (Fig. 7.29c). It stains metachromatically with crystal violet and methyl violet and fluoresces after staining with thioflavine-t [56]. Light chain-associated (AL) amyloid can be distinguished from amyloid of AA type by the abolition of Congo red staining by prior treatment with potassium permanganate in the latter type [56]. Peripheral blood The peripheral blood may be normal or may show the features usually associated with multiple myeloma or lymphoplasmacytic lymphoma. Occasionally, features of hyposplenism are present, indicating that the spleen is infiltrated by amyloid and has become hypofunctional. Thrombocytosis, observed in 9% of cases in one large series, may be indicative of hyposplenism [54]. Bone marrow cytology The bone marrow aspirate varies from normal through increased plasma cells of normal morphology to overt multiple myeloma or lymphoplasmacytic lymphoma. In a large series of patients, 40% had at least 10% bone marrow plasma cells [54]. When the bone marrow aspirate is apparently normal, κ:λ imbalance may indicate the presence of an abnormal clone of cells, even though the total number of plasma cells is not increased. Very rarely, a bone marrow aspirate from a patient with amyloidosis contains either neutrophils which have ingested amyloid [55] or extracellular amyloid deposits (Fig ). Immunohistochemistry Amyloid can be demonstrated using a monoclonal antibody that reacts with the P protein common to light chain-associated and other forms of amyloid. In the majority of cases of light chain-associated amyloid, a positive identification can be made with type-specific anti-light chain sera. Neoplastic lymphocytes and plasma cells, when present in detectable numbers, show the expected patterns of reactivity. Cytogenetics and molecular genetics Clonal chromosome abnormalities are common in primary amyloidosis but, because the neoplastic cells constitute a low percentage of bone marrow cells, demonstration of these abnormalities requires FISH techniques [57]. Numerical abnormalities, both monosomies and trisomies, are common as are complex cytogenetic abnormalities. The pattern of abnormal findings is similar to that observed in MGUS. Light chain deposition disease [56,58 60] Light chain deposition disease describes a syndrome

21 352 CHAPTER SEVEN (a) (b) Fig BM aspirate, showing; (a) a deposit of amyloid, MGG 940; and (b) amyloid within a BM fragment, Congo red 470. of organ damage consequent on the systemic deposition of free light chains. There is an associated neoplastic proliferation of plasma cells which may be occult or overt. About 70% of patients have relevant clinical features, most often of multiple myeloma but occasionally of solitary plasmacytoma, lymphoplasmacytic lymphoma or other NHL. The remaining patients either have features which would usually be interpreted as MGUS (about 15% of cases) or have no serum or urinary paraprotein detectable (also about 15% of cases) [56]. Those without a detectable paraprotein nevertheless have an occult plasma cell neoplasm, the neoplastic cells producing a light chain type which has a propensity to deposit in and damage tissues. Cases with κ light chains are over-represented relative to cases with λ light chains. In occasional patients, a similar syndrome is associated with systemic deposition of heavy chains in addition to light chains; the term monoclonal immunoglobulin deposition disease has been suggested to include these cases [60,61]. The predominant organ damage is renal, with glomerular and tubular deposition causing the nephrotic syndrome, renal failure or both. Occasional patients have presented with clinical features of hepatic, cardiac or adrenal involvement.

22 MULTIPLE MYELOMA 353 (a) (b) Fig BM trephine biopsy sections, showing amyloid in the walls of small blood vessels. Paraffin-embedded. (a) H&E 97. (b) Congo red stain 390. (c) Congo red stain viewed under polarized light 390. (c)

23 354 CHAPTER SEVEN Fig BM trephine biopsy section, showing amyloid in the walls of small blood vessels. Paraffin-embedded, Giemsa stain 376. Fig BM trephine biopsy section, light chain-associated amyloidosis (AL type), showing deposition of amorphous eosinophilic material in the interstitium. Paraffin-embedded, H&E 94. Peripheral blood and bone marrow There are no specific peripheral blood findings other than those usually associated with renal failure or with multiple myeloma or other plasma cell neoplasm. The bone marrow may appear normal or may show features usually associated with MGUS, multiple myeloma or a related condition. Some patients who initially have no evidence of multiple myeloma subsequently develop typical features of this disease. In those cases in which the bone marrow is apparently normal, it may be possible to demonstrate a monoclonal population of plasma cells by flow cytometry. Rarely, light chains are deposited in the bone marrow, either in the interstitium or in the walls of blood vessels [56,59]. Light chain deposits are morphologically similar to amyloid deposits when stained with H&E but they do not stain with Congo red, or show birefringence with polarized light, and are negative or react weakly with thioflavine-t; they are PAS-positive and stain blue with a Giemsa or Gomori s trichrome stain. The nature of light chain deposits can be confirmed by immunohistochemistry with anti-κ or anti-λ antiserum.

24 MULTIPLE MYELOMA 355 Essential and other paraprotein-associated cryoglobulinaemia A plasma cell disorder may lead to cryoglobulinaemia, either when there is secretion of a paraprotein with the characteristics of a cryoglobulin (type I cryoglobulinaemia) or when a paraprotein has antibody activity against another immunoglobulin and the immune complex formed is a cryoglobulin (type II cryoglobulinaemia); in the latter type, the paraprotein is usually IgM with antibody activity against polyclonal IgG. In about a quarter of patients, cryoglobulinaemia is a manifestation of multiple myeloma or of Waldenström s macroglobulinaemia. In the other three quarters of patients, in whom no overt neoplastic proliferation of plasma cells is present, the term essential cryoglobulinaemia is appropriate; in these cases the clone of cells secreting the paraprotein is too small to produce any pathological manifestations other than those due to the characteristics of the cryoglobulin. A significant proportion of cases of type II cryoglobulinaemia are secondary to hepatitis C infection and some of these have an overt low grade NHL; these cases are not designated essential cryoglobulinaemia. The clinical features of cryoglobulinaemia, e.g. purpura and peripheral cyanosis, may relate to precipitation of the immunoglobulin on chilling or, in the case of type II cryoglobulinaemia, to immune complex formation. Peripheral blood In the absence of overt multiple myeloma or lymphoplasmacytic lymphoma, the peripheral blood film is often normal. In a minority of patients, a cryoglobulin precipitate is present, usually as weakly basophilic globular masses, less often as crystals or a fibrillar deposit. Occasionally, cryoglobulin precipitates have been ingested by neutrophils or monocytes and are seen as globular, variably basophilic intracytoplasmic inclusions. Bone marrow cytology and histology The bone marrow findings are either normal or are those of multiple myeloma, a low grade NHL or MGUS. Chronic cold haemagglutinin disease Chronic cold haemagglutinin disease (CHAD) is a disease characterized by chronic, cold-induced haemolytic anaemia consequent on a lymphoplasmacytic neoplasm which may be either occult or overt. In patients who do not have clinical or pathological features of a lymphoma at presentation, these may evolve subsequently. Peripheral blood Unless it has been prepared from warmed blood, the blood film shows red cell agglutinates. If there has been a recent episode of haemolysis a few spherocytes may be present together with polychromatic macrocytes. Some patients have lymphocytosis, with the cells either having the morphology of normal mature lymphocytes or showing some plasmacytic features. Bone marrow cytology and histology The bone marrow appearances vary from normal to those of an overt lymphoplasmacytic lymphoma. There is usually erythroid hyperplasia. α heavy chain disease α heavy chain disease is a lymphoplasmacytic neoplasm, usually affecting predominantly the bowel, associated with the secretion of the heavy chain of IgA into the serum or into the bowel lumen. Some cases evolve into large cell lymphoma. Peripheral blood and bone marrow The peripheral blood shows no specific abnormality. The bone marrow is usually normal but may be infiltrated by plasma cells. γ heavy chain disease γ heavy chain disease is a lymphoplasmacytic neoplasm characterized by lymphadenopathy, hepatomegaly and splenomegaly and by the secretion of the heavy chain of IgG. Some patients have developed amyloidosis, indicating that light chain as well as heavy chain may be secreted.

25 356 CHAPTER SEVEN Peripheral blood Anaemia, leucopenia and thrombocytopenia are common. In about half of cases there are atypical lymphoplasmacytoid cells and some plasma cells in the peripheral blood. Some patients have eosinophilia. Bone marrow cytology The bone marrow is infiltrated by lymphocytes, lymphoplasmacytoid cells or plasma cells. µ heavy chain disease [62 65] µ heavy chain disease is a lymphoproliferative disorder characterized by the secretion of the heavy chain of IgM. Most patients described in the published literature have had the pathological features of chronic lymphocytic leukaemia (CLL) although one recent case has been reported in association with Waldenström s macroglobulinaemia [66]. Hepatomegaly and splenomegaly are usual; abdominal lymphadenopathy is more prominent than peripheral lymphadenopathy. Light chain secretion may also occur and may give rise to amyloidosis. splenomegaly and lymphadenopathy. The pathological features of the enlarged lymph nodes approximate to those of the hyaline-vascular type of Castleman s disease; there is follicular hyperplasia, vascular proliferation and an interfollicular infiltrate of lymphocytes, plasma cells and immunoblasts. The endocrinopathy may include primary gonadal failure, hypothyroidism, Addison s disease and diabetes mellitus. The monoclonal gammopathy, present in about three quarters of patients, is usually an IgGλ or IgAλ paraprotein. Skin manifestations include skin thickening resembling that of scleroderma, oedema, hypertrichosis, hyperpigmentation and Raynaud s phenomenon. Other features of the syndrome include pleural effusion, ascites, papilloedema and finger clubbing. Peripheral blood and bone marrow There are no specific peripheral blood features. The bone marrow findings range from normal to those of overt lymphoplasmacytic lymphoma or multiple myeloma. About 60% of patients have a significant bone marrow plasmacytosis. In those with multiple myeloma, osteosclerosis is usual but some patients have osteolytic lesions. Peripheral blood and bone marrow The majority of patients show features indistinguishable from CLL. The POEMS syndrome [67 70] The POEMS, or Polyneuropathy, Organomegaly, Endocrinopathy, M-protein, Skin changes, syndrome describes a curious constellation of pathological manifestations which has been associated with multiple myeloma (particularly, but not exclusively, osteosclerotic multiple myeloma), solitary plasmacytoma and lymphoplasmacytic lymphoma. In addition, POEMS has been described in patients with bone marrow appearances which, but for the many associated pathological features, would be designated MGUS. The syndrome is rare and occurs at a younger age than is usual in multiple myeloma. The polyneuropathy is both motor and sensory. The organomegaly refers to hepatomegaly, Acquired angio-oedema associated with plasma cell neoplasia [71 73] The majority of cases of acquired angio-oedema described have been associated with a neoplastic plasma cell proliferation or with other B-lineage neoplasms, with or without secretion of a paraprotein. Associated conditions have included multiple myeloma, lymphoplasmacytic lymphoma, splenic lymphoma with villous lymphocytes and other NHL, essential cryoglobulinaemia, MGUS and CLL. The mechanism of the acquired angiooedema is C1 inhibitor deficiency consequent on excessive consumption; it is likely that consumption of C1 inhibitor can be due to precipitation of a cryoglobulin, to an immunological reaction involving the paraprotein or to reaction of autoantibodies with neoplastic cells. Deficiency of C1 inhibitor may precede the development of overt lymphoproliferative disease by many years [74].