Plasma Cell Myeloma and Related Disorders

Plasma Cell Myeloma and Related Disorders GENERAL CONSIDERATIONS The monoclonal proliferation of plasma cells, also known as “monoclonal gammopathies,” “plasma cell dyscrasias,” or “paraproteinemias,” consists of several clinicopathologic entities. It is characterized by a single class of immunoglobulin (Ig) product, referred to as “M-component ” (monoclonal component), present in serum and/or urine protein electrophoresis (Figure 16.1) [1–3]. In addition to protein electrophoresis, other more sensitive and specific techniques, such as immunoelectrophoresis and immunofixation, have been used to detect the specific class or subtype of monoclonal Ig in these disorders (Figures 16.2 and 16.3). The following categories are discussed in this chapter. Waldenstrom macroglobulinemia (lymphoplasmacytic lymphoma) has already been discussed in Chapter 15. Monoclonal gammopathy of undetermined significance (MGUS) Plasma cell myeloma (PCM) and variants Plasmacytoma and variants Monoclonal Ig deposit diseases Heavy chain diseases.

Etiology and Pathogenesis The etiology of these related disorders is not known. Several environmental and genetic factors have been associated with the increased risk of PCM. Increased incidence of PCM has been observed in the Japanese survivors of atomic bomb and in radiologists exposed to long-term low doses of radiation [4–6]. Occupations in

agriculture and metal industries and exposure to benzene and hair dyes have been reported to be in association with increased risk of PCM [7–9]. There are also reports linking obesity and diet to the increased risk of PCM [10, 11]. There are several reports of familial clusters of PCM affecting two or more first-degree relatives suggesting that hereditary factors may be involved in the development of monoclonal gammopathies in certain conditions [12–14]. Viral infections, such as hepatitis C, HIV, and HHV-8 (Kaposi-sarcoma-associated herpes virus), have been associated with increased risk of PCM [15–17]. Dysregulation in immune function may also play a role in the pathogenesis of monoclonal plasma cell disorders [18–20]. Molecular genetic studies have shed some light on the pathogenesis of these disorders (Figure 16.4) [21, 22]. A broad spectrum of abnormalities have been identified in the signaling pathways, cell cycle, apoptotic processes, and bone marrow microenvironment. For example, the monoclonal plasma cells are capable of producing IL-6 and IL-6 receptor, leading to autocrine stimulation [23–25]. IL-6 activates two pathways: the JAK2-STAT3 and the RAS-MAP. The first pathway upregulates antiapoptotic proteins Mcl-1 and Bcl-X, and the second pathway leads to the upregulation of transcription factors, such as ELK-1 and AP-1 [25–28]. Gene expression profiling studies have identified three genes which may play important roles in pathogenesis and clinical outcome of PCM: RAN, CHC1L, and ZHX-2 [29]. The RAN gene maps to 12q24, is a member of the Ras family, and has a role in regulating chromosome condensation, spindle formation, and cell-cycle progression. The CHC1L gene maps

16 CHAPTER

Faramarz Naeim, P. Nagesh Rao and Wayne W. Grody

373

Hematopathology: Morphology, Immunophenotype, Cytogenetics and Molecular Approaches Gamma Albumin

Albumin

Alpha1

Alpha2

Beta

Beta Gamma

Alpha1

Alpha2

(b)

(a)

FIGURE 16.1 Schematics of serum protein electrophoresis: (a) demonstrates a normal profile and (b) shows a spike in the gamma region. ()

()

1 A

anti-human

B

anti-IgG

C

anti-IgA

D

anti-IgM

E

anti-kappa

F

anti-lambda

2 3 4 5 6

ELP

G

A

M

K

L

7 FIGURE 16.3 Immunofixation analysis of a serum demonstrating IgA-kappa monoclonal gammopathy. Courtesy of Eugene Dinovo, Ph.D., Department of Veterans Affairs, Greater Los Angeles Healthcare System.

FIGURE 16.2 Serum immunoglobulin electrophoresis with a panel of IgG, IgA, IgM, kappa, and lambda antibodies demonstrating an IgG/kappa spike. Wells 1–7 contain control serum (odd number) and patient serum (even number). From Bossuyt X, Bogaerts A, Schiettekatte G, Blanckaert N. (1998). Serum protein electrophoresis and immunofixation by a semiautomated electrophoresis system. Clin Chem 44, 944–9.

B-cell

MGUS

SM

PCM

EM

Karyotypic abnormalities 10 Ig TLC

20 Ig TLC del(13q)

Hyper/hypodiploidy N-ras, K-ras, FGFR3 mutations p18 deletion c-MYC dysregulation p53 mutation Bone destruction Angiogenesis Increased DNA labeling index

374

FIGURE 16.4 Schematic stages of molecular, cytogenetic, and clinical events in plasma cell disorders from monoclonal gammopathy with undetermined significance (MGUS) to smoldering myeloma (SM), plasma cell myeloma (PCM), and extramedullary myeloma (EM). Adapted from Ref. [21].

Plasma Cell Myeloma and Related Disorders to 13q14.3 and appears to harbor a tumor suppressor gene for PCM. The ZHX-2 gene maps to 8q24, near the MYC oncogene, and is a negative regulator of the NF-Y transcription factor. This factor is a master transcriptional regulator of numerous genes involved in cell-cycle control and proliferation. The levels of cyclin D1, cyclin D2, or cyclin D3 mRNA in the tumor cells of almost all patients with MGUS and PCM are relatively high. Increased expression of one of the cyclin D proteins facilitates activation of CDK4, leading to inactivation of the RB1 (retinoblastoma 1) gene, and consequently cell-cycle progression [21].

16

Pathology Morphology The morphologic hallmark of PCM and related disorders is an increase in bone marrow plasma cells. The neoplastic plasma cells have a tendency to appear in clusters or sheets in the biopsy sections and, unlike reactive plasmacytosis, are not located around the vascular structures (Figure 16.5). The bone marrow biopsy sections are usually hypercellular, but occasionally they may appear hypocellular (Figure 16.6). Prominent osteoclastic activity may be evident, adjacent to the bony trabeculae.

FIGURE 16.5 Plasma cell myeloma. Bone marrow biopsy section (a) and bone marrow smear (b) demonstrating numerous plasma cells.

(a)

(b)

375

Hematopathology: Morphology, Immunophenotype, Cytogenetics and Molecular Approaches FIGURE 16.6 Hypocellular plasma cell myeloma with interstitial infiltration of plasma cells around fatty tissue: (a) low power and (b) high power views.

(a)

(b)

The cytologic features of plasma cells in the bone marrow smears may vary from normal-appearing mature plasma cells to immature and anaplastic forms (Figures 16.7–16.9). Plasmablasts show a high nuclear:cytoplasmic ratio, deep blue cytoplasm, with or without perinuclear hof, round or irregular nucleus, fine chromatin, and one or several prominent nucleoli (Figure 16.9). Multinucleated or multilobated plasma cells may be present. Cells with cherry-red, round cytoplasmic (Russell bodies) or nuclear (Dutcher bodies) inclusions, as well as cytoplasmic crystals may be present (Figures 16.10 and 16.11). Some plasma cells may appear like grapes and demonstrate numerous, round, Ig-containing cytoplasmic structures (Mott and Morula cells) (Figure 16.12). The blood 376

smears may show rouleaux formation of the red blood cells or the presence of plasma cells in various proportions (Figure 16.9c). Solitary neoplasms of plasma cells (plasmacytoma) may involve bone or other extramedullary sites.

Immunophenotype Plasma cells in myeloma and related disorders, similar to normal plasma cells, lack surface Ig and express cytoplasmic Ig, CD38, CD138, and CD79a. CD138 is more specific than CD38 in the detection of plasma cells (Figures 16.13 and 16.14) [30–34]. However, the CD138 molecules disintegrate and disappear fast and therefore may not be detected

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Therefore, if the percentage of plasma cells in the bone marrow smears is not high, immunohistochemistry on the bone marrow biopsy sections is recommended. Plasma cells in PCM and related disorders are monoclonal and express only one type of Ig light chain. The class of Ig is usually IgG, less frequently IgA, and occasionally IgD, IgE, or IgM. The neoplastic plasma cells often lack CD19 and CD20 expressions but may aberrantly express CD10, CD56, or, less frequently, myelomonocytic or T-cell-associated markers [30–34]. Both normal and neoplastic plasma cells may express CD117.

(a)

(b)

(c)

FIGURE 16.7 Plasma cell myeloma. Bone marrow biopsy section demonstrating a paratrabecular aggregate of plasma cells: (a) low power and (b) high power views. Bone marrow smear shows clusters of plasma cells (c).

by flow cytometry if the specimen is not prepared immediately. Also, most flow cytometry laboratories have experienced a significant loss in the number of plasma cells during the cell preparation for the flow cytometry procedures.

Cytogenetic and Molecular Studies PCM is a genetically unstable malignancy of post-germinal center B-lineage cells. Chromosome analysis in PCM is hampered by the low proliferative fraction in most cases. Recent studies using cytokine-stimulated bone marrow cultures and FISH and microarray techniques have increased the proportion of informative cases. Active myeloma is often preceded by an indolent phase of MGUS, where the plasma cells are already abnormal with an aneuploid DNA content. In fact, almost all myeloma tumors and most cases of MGUS are aneuploid as demonstrated by DNA content measurements using flow cytometry, conventional cytogenetics, or molecular cytogenetics (FISH) [35, 36]. By classical cytogenetics, only one-third of PCM patients have a complex abnormal karyotype. The remaining two-thirds have normal karyotypes [37, 38]. However, the observed normal karyotypes are often derived from the other non-neoplastic hematopoietic cells, rather than from abnormal plasma cells, because the plasma cells fail to grow. Plasma cells may fail to grow in culture for three major reasons. First, samples from PCM patients may fail to grow because of the low proliferative capacity of the myeloma cells [39]. Myeloma cells (especially in early myeloma) are stroma dependent, so removing the cells from their supportive microenvironment results in apoptosis and lack of growth. However, if myeloma cells have become stroma independent (i.e. in advanced stages of the disease), removal of the myeloma cells from their microenvironment can result in proliferation and an abnormal karyotype [39]. The second explanation for the laboratory’s inability to obtain abnormal metaphases lies in the quality of the bone marrow aspirates received for cytogenetic studies. Aspirates frequently contain drastically fewer plasma cells than the corresponding smears used for morphological assessment since the number of tumor cells in a given specimen largely depends on the level of local bone marrow infiltration and the degree of sample dilution by blood [38]. For this reason, it is essential that the first few milliliters of the bone marrow drawn be sent for cytogenetic analysis. Also, the needle should be repositioned during aspiration, rather than simply continuing to withdraw marrow from the initial puncture site, to ensure that adequate numbers of abnormal cells are submitted to the laboratory. Finally, aspirates should be processed as soon as possible if FISH is requested. Several techniques have been created to selectively culture plasma cells [40], but these methods may not be helpful if the sample provided to the laboratory is of poor quality. 377

378 (b)

FIGURE 16.8 Plasma cell myeloma. Bone marrow biopsy section (a) and bone marrow smear (b) demonstrate numerous, large, atypical plasma cells with abundant cytoplasm, irregular nuclear borders, and prominent nucleoli.

(a)

(b)

FIGURE 16.9 Plasmablastic myeloma. Bone marrow smear demonstrating cells with variable amounts of blue cytoplasm, round nuclei with finely dispersed chromatin, and a single prominent nucleolus: (a) low power and (b) high power views.

Hematopathology: Morphology, Immunophenotype, Cytogenetics and Molecular Approaches

(a)

FIGURE 16.10 Plasma cell myeloma. Bone marrow biopsy section demonstrating plasma cells with nuclear inclusions (Dutcher bodies, black arrows) and cytoplasmic inclusions (Russell bodies, green arrows): (a) low power and (b) high power views. Blood smear showing formations (c).

16

(a)

(b)

(c)

379

Hematopathology: Morphology, Immunophenotype, Cytogenetics and Molecular Approaches FIGURE 16.11 Plasma cell myeloma. Bone marrow smear demonstrating plasma cells with cytoplasmic needle-like immunoglobulin crystals: (a) low power and (b) high power views.

(a)

(b)

Several recurring aberrations are observed in karyotypically abnormal PCM. The majority of PCM cases demonstrate chromosomal aneuploidy. Four categories of aneuploidy can be defined by karyotyping: 1. Hypodiploidy (46 chromosomes) (Figure 16.15). 2. Pseudodiploidy (46–47 chromosomes but with structural or numerical abnormalities). 3. Hyperdiploidy (50 chromosomes, HRD) (Figure 16.16). 4. Near-tetraploidy (75 chromosomes). Multiple non-random trisomies are associated with hyperdiploid tumors [41], especially trisomies of 380

odd-numbered chromosomes [40]. It was initially believed that trisomies were more common than monosomies in PCM, but the opposite is true. The most common trisomies are of chromosomes 3, 5, 7, 9, 11, 15, 19, and 21 [42]. The most common monosomies are of chromosomes 13, 14, 16, and 22. No specific numerical chromosomal abnormality is constant or predictive of disease progression. The prevalence of aneuploidy is independent of stages. Karyotypes are typically complex and exhibit 10 abnormalities in almost half of the patients and even 20 aberrations in about 10% of the cases. HRD, present in nearly 60% of PCM, is most often associated with trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19, and 21 and represents one of the central genetic

Plasma Cell Myeloma and Related Disorders

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(b)

(a)

(c)

CD56 CD38

SSC

CD38

FSC

CD45

FIGURE 16.13 Flow cytometry of plasma cell myeloma demonstrating a population of cells (red) which are negative for CD45 and express CD38, CD138, CD56, and cytoplasmic kappa light chain.

CD38 CD38

SSC

CD138

FIGURE 16.12 Plasma cell myeloma with cytoplasmic vacuoles. (a) Bone marrow biopsy section, (b) immunohistochemical stain for Ig kappa light chain, and (c) bone marrow smear. From Naeim F. (1997). Pathology of Bone Marrow, 2nd ed. Williams & Wilkins, Baltimore, by permission.

cyKappa

cyLambda

381

Hematopathology: Morphology, Immunophenotype, Cytogenetics and Molecular Approaches

(a)

(b)

(c)

FIGURE 16.14 Plasma cell myeloma. Bone marrow biopsy section demonstrating a large population of plasma cells (a). Immunohistochemical stains show expression of CD138 (b) and kappa light chain (c) by the plasma cells.

pathways in the development of PCM, and this type of disease has been previously shown to have distinct gene expression signature [21]. Hyperdiploidy is seen in about 40% of PCM patients, with an incidence of 25% of the hypo- and 382

pseudodiploid karyotypes. Triploidy and tetraploidy are very rare, usually not observed over 10%. Although the reasons are still unclear, the frequency of HRD has been shown to be more common in elderly patients [43]. The vast majority of PCM involves the translocation of the IGH gene on 14q32 to one of several non-random partners (40 gene regions) and this is considered to be an initial event in the genesis of PCM (seen in about 40% of patients) [40]. Standard cytogenetic analyses identify abnormalities of 14q32 in up to 40% of cases (Figures 16.17–16.19), while this detection rate nearly doubles with interphase FISH studies. Illegitimate recombination of the IGH gene also occurs in MGUS but at a slightly lower frequency (50%). Similar rearrangements of the IGK and IGL genes have been found to occur in a small subset of PCM and MGUS [44]. The rearrangements involving the IGH gene are commonly simple reciprocal translocations, but more complex recombination events, such as insertions and duplications, are also observed (Tables 16.1 and 16.2). Cyclin D1 (most common), D2, and D3 genes (CCND1, CCND2, and CCND3) on chromosomes 11q13, 12p13, and 6p21, respectively, MAF family member genes (c-MAF, MAFA, and MAFB) on chromosomes 16q23, 8q24, and 20q11, respectively, and the fibroblast growth factor receptor 3 gene (FGFR3) on chromosome 4p16 are commonly observed as IGH translocation partners [1]. These translocations are markers for distinct subtypes of myeloma with important prognostic implications. IGH gene translocations are found more frequently in nonHRD tumors (70%) than in HRD tumors (20%). The t(11;14) (Figure 16.16) and t(4;14) are the most common IGH translocations followed by t(14;16) (Figure 16.19), and t(14;20) is the least common [38, 40]. The t(14;16)(q32;q23) and t(14;20)(q32;q11.2) result in the activation of c-MAF and MAFB proto-oncogenes, respectively, and are together seen in approximately 6% of cases. The reciprocal t(4;14)(p16;q32) translocation results in the hyperactivation of both the FGFR3 and MMSET genes. Two cyclin D family members are activated by translocations in PCM: cyclin D1 by the t(11;14)(q13;q32) in 17% and CCND3 by t(6;14)(p21;q32) (Figure 16.18) in 2%. The overall rate of 14q32 translocations, however, significantly increases with disease progression and reaches up to 90% in advanced tumors and human myeloma cell lines. IGH translocation and HRD act similarly through the upregulation of one of the cyclins (D1, D2, or D3) [39, 41]. Translocations between the Ig heavy chain locus and CCND1, CCND3, c-MAF, MAFB, FGFR3, and MMSET represent recurrent genetic lesions in approximately 40% of PCM [45, 46]. Although numerical and gross structural changes can be diagnosed by karyotyping without difficulty, small interstitial deletions, partial genomic gains, and cryptic translocations (e.g. IGH translocations) can be easily overlooked due to the karyotype’s limited spatial resolution. Modern molecular-based techniques, such as CGH and FISH, allow the detection of genetic abnormalities independent of proliferating cells. With these methods, chromosomal aberrations are found in 90% of patients with PCM and most (if not all) patients with MGUS. Considering that analyses with interphase FISH and molecular genetic techniques reveal

Plasma Cell Myeloma and Related Disorders

1

2

6

3

7

13

8

9

14

19

6

21

13

3

8

14

19

22

2

7

17

18

X

Y

11

17

22

chromosomal abnormalities in close to 100% of PCM cases and in the majority of MGUS cases, the normal karyotypes are most likely not representative of the neoplastic clone. FISH permits reliable identification of both translocations and small deletions or gains in PCM [4]. Most clinical laboratories currently test for 13q14 (RB1) and 17p13.1 (TP53) deletions as well as the primary translocations t(4;14)(p16.3;q32) and t(11;14)(q13;q32). Ploidy should be determined in all tumors; for example, disomy of chromosome band 13q14 in a near-tetraploid karyotype is functionally a deletion of the region. Polyploidy can be reliably excluded by the use of control probes mapped to genomic regions that rarely display aneuploidy (e.g. chromosomes 2, 10, and 12) [35, 37]. Finally, some laboratories test for t(6;14)(p21;q32), t(14;16)(q32;q23), and t(14;20), and

FIGURE 16.16 Bone marrow karyotype in a patient with plasma cell myeloma demonstrating hyperdiploidy with 66,XX,X,6,del(11)(q23), del(11)(q23), 12,13,der(14)t(11;14)(q13;q32)2, 16, 19, 21,22.

5

10

16

21

12

4

9

15

20

11

16

FIGURE 16.15 Bone marrow karyotype in a patient with plasma cell myeloma demonstrating hypodiploidy with 30,X,X,3,4,5,6,7,10 ,11,12,13,14,15,17,del(17)(p13),18,2 0,21,22.

5

10

15

20

1

4

16

12

18

X

Y

detection of the most frequent chromosomal abnormalities (e.g. 1q, 9q, 11q) [36]. Cytogenetics is helpful in determining myeloma patients’ clinical outcome [36]. Normal metaphases and normal FISH, HRD tumors, and CCND1 gene activation are associated with a better prognosis [1]. However, patients with c-MAF, MAFB, or FGFR3 activation, del(13q), del(17p), hypodiploidy, 1q abnormalities, or 9q trisomies are associated with a worse prognosis [35, 39]. A strong association between chromosome 1q abnormalities and the etiology of PCM disease has been suggested. Tandem duplications and jumping translocations of 1q21 occur frequently in this malignancy [46]. The gains of 1q is one of the most common abnormalities in PCM [42]. In addition, hyperdiploidy with 1q was found to have a less 383

Hematopathology: Morphology, Immunophenotype, Cytogenetics and Molecular Approaches FIGURE 16.17 Bone marrow karyotype in a patient with plasma cell myeloma demonstrating 46,XY,t(13;14)(q14;q32).

1

6

2

7

3

8

4

9

5

10

11

12

13

14

15

16

17

18

19

20

21

22

X

Y

FIGURE 16.18 Bone marrow karyotype in a patient with plasma cell myeloma demonstrating 45,X,Y,t(6;14) (p21;q32),iso(9)(q10),t(11;12)(q21;p13). 1

6

2

7

3

8

13

14

19

20

4

9

15

21

10

16

22

favorable clinical outcome than hyperdiploidy lacking this feature [47]. Additional copies of 1q21 have been reported to accompany the progression of smoldering to overt PCM by molecular and FISH studies such as array-CGH (aCGH) and interphase FISH [48]. Also it has been noted that patients with gains or amplifications of 1q21 were linked to inferior survival, and thus these events may be linked to PCM pathogenesis and progression. Among losses, monosomy or partial deletion of chromosome 13 (13q14) is the most common finding, occurring 384

5

11

17

X

12

18

Y

in 15–40% of the newly diagnosed cases (Figure 16.20). Patients with a 13q14 deletion have been reported to have significant reductions in the rate of response to conventional dose chemotherapy (41% versus 79%) and overall survival (24 versus 60 months) compared to patients without this deletion. When present, it was the most important independent variable associated with unfavorable outcome [49]. PCM and related disorders show clonal Ig gene rearrangements and high frequency of Ig VH gene somatic mutation. Approximately 5% of the cases of PCM may show more than one rearranged Ig bands best seen by Southern

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FIGURE 16.19 Bone marrow karyotype in a patient with plasma cell myeloma demonstrating 45,XX,t(2;11)(p11.2;p11.2),10, t(14;16)(q32;q23). 1

6

2

7

3

8

4

9

5

10

11

12

13

14

15

16

17

18

19

20

21

22

X

Y

TABLE 16.1 Chromosomal regions/genes as translocation partners with 14q32(IGH) in PCM/MGUS with known frequencies.

TABLE 16.2 Frequency of IGH translocation and clinical outcome.

Abnormality

PCM and smoldering PCM (%)

PCM (%)

Prognosis

IRF4

IGL translocations

20

20

Unknown

6p21

CCND3 (15–20%)

IGH translocations t(4;14)

35–50

50–70

Poor

8q24

c-MYC

t(11;14)

2–10

15

Poor

11q13

CCND1

t(14;16)

15–30

15

Good

t(6;14)

2–5

5

Poor

4p16

FGFR3,WHSC1 (15%)

4p13

ARHH

6p23-25

16q23

MAF (5–10%)

18q21

BCL2

20q11-q13

MAFB FIGURE 16.20 Bone marrow karyotype in a patient with plasma cell myeloma demonstrating 46,XX,del(13)(q13q22).

1

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2

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9

5

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13

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18

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Y

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Hematopathology: Morphology, Immunophenotype, Cytogenetics and Molecular Approaches TABLE 16.3 Clinicopathological features of MGUS according to the International Myeloma Working Group.* 1. Serum M-protein level  3 g dL 2. Bone marrow monoclonal plasma cells 10% 3. No evidence of other B-cell lymphoproliferative disorders 4. No evidence of organ or tissue impairment or bone lesions *From Ref. [51].

blot [1]. Approximately 50% of the tumors carry the translocation of Ig heavy chain (IGH) locus (14q32) with various oncogenes and suppressor genes, such as cyclin D1, cyclin D3, PGFR3, CHC1L, ZHX-2, MAF, and MAFB at 11q13, 6p21, 4p16, 13q14.3, 8q24.3, 16q23, and 20q12, respectively [50]. Because the diagnosis of myeloma is straightforward, such gene rearrangement studies are usually not required. However, detection of clonality by PCR may be helpful for monitoring minimal residual disease. The importance of cytogenetic and molecular features as determinants of outcome is being increasingly recognized. The present data support that PCM is characterized by marked inter- and intratumor cytogenetic heterogeneity that may account for the diverse clinical behavior of this neoplasm. Multivariate analysis of all cytogenetic and clinicopathologic features, including patient survival, is needed to identify the cytogenetic variables of independent prognostic significance and also to define the predictive role of the proposed cytogenetic classification for response to treatment and survival of patients with PCM.

protein concentration on a routine blood test, followed by demonstration of a monoclonal spike by serum protein electrophoresis. The presence of light chain in the urine (BenceJones protein) is generally suggestive of PCM. The nature of serum M-component in MGUS is IgG in about 75%, IgM in 15%, and IgA in 10% of the cases. Plasma cells in bone marrow biopsy sections and smears appear mature and account for 10% of the total bone marrow nucleated cells (Figure 16.21). Immunophenotypic studies reveal a monoclonal population expressing Ig molecules corresponding to the patient’s serum M-component and often an abnormal population of CD19– and CD56 plasma cells. The elevated levels of monoclonal protein, presence of IgA or IgM class or an abnormal free light chain ratio, and a high percentage of plasma cells are predictors of MGUS progressing to a more aggressive B-cell lymphoproliferative disorder [55].

PLASMA CELL MYELOMA Plasma cell myeloma (PCM) (multiple myeloma, myelomatosis, Kahler’s disease) is a multifocal bone-marrow-based plasma cell neoplasm with the production of monoclonal Ig, often associated with bone destruction and osteolytic lesions, hypercalcemia, and anemia [1, 51]. PCM has been divided into several clinicopathologic entities: Asymptomatic myeloma (smoldering myeloma) Indolent myeloma Symptomatic myeloma (or symptomatic plasma cell myeloma)

MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE The term “monoclonal gammopathy of undetermined significance” (MGUS) stands for a clinical condition defined by the presence of monoclonal Ig production without evidence of PCM, amyloidosis, Waldenstrom macroglobulinemia, or other related plasma cell or lymphoproliferative disorders (Table 16.3) [51]. Several other terms have been used for this condition, such as idiopathic, asymptomatic, non-myelomatous, cryptogenic, and benign monoclonal gammopathy. However, the term “benign monoclonal gammopathy” is inappropriate, because a significant proportion of patients with MGUS eventually develop PCM or other lymphoproliferative disorders. The cumulative probability of progression of MGUS to PCM or other lymphoproliferative disorders in one large study was 12% at 10 years, 25% at 20 years, and 30% at 25 years [52, 53]. Clearly, MGUS and PCM represent different time points along the same disease spectrum, and so far, no molecular or cytogenetic test can reliably distinguish them. The incidence of MGUS increases by age. In a recent study, the prevalence of MGUS was 3.21% in individuals older than 50 and 5.3% in those older than 70 years [54]. It affects more African Americans than Caucasians. MGUS is usually an incidental finding detected by elevated total 386

Non-secretory myeloma Plasma cell leukemia.

Asymptomatic (Smoldering) Myeloma Smoldering myeloma represents the point of transition from MGUS to PCM without anemia, skeletal lesions, hypercalcemia, or renal insufficiency. The serum M-protein level is 3 g/dL and the bone marrow plasma cells are

10% but 30% (Table 16.4). These patients do not need treatment but should be followed up closely, because many of them eventually become symptomatic.

Indolent Myeloma This category is described by the WHO but not included in the report of criteria for the classification of plasma cell disorders by the International Myeloma Working Group (IMWG) [51]. According to the WHO and Alexanian [1, 56], indolent myeloma is similar to smoldering myeloma in that there is no evidence of anemia, hypercalcemia, or renal insufficiency, but unlike smoldering myeloma there are up to three lytic bone lesions and the serum M-component is at intermediate levels (IgG 3 and 7 g /dL).

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TABLE 16.4 Criteria for asymptomatic myeloma (smoldering myeloma).* Serum M-protein

3 g dL

Bone marrow clonal plasma cells

10%

No related tissue or organ impairment or symptoms *From Ref. [51].

TABLE 16.5 Myeloma-related organ or tissue impairment.* (a)

Elevated serum calcium levels

0.25 mmol L above the upper limit of normal or 2.75 mmol L

Renal insufficiency

Creatinine 173 mmol/L

Anemia

Hemoglobin 2 g/dL below the lower limit of normal or 10 g dL

Bone lesions

Lytic lesions or osteoporosis with compression fractures

Others

Symptomatic hyperviscosity, amyloidosis, recurrent bacterial infections

*From Ref. [51].

(b)

TABLE 16.6 Criteria for the diagnosis of plasma cell myeloma set by the International Myeloma Working Group.* M-protein in serum and or urine Bone marrow clonal plasma cells or plasmacytoma** Related organ or tissue impairment (end-organ damage, including bone lesions)*** *From Ref. [51]. **Immunophenotypic studies demonstrate monoclonal population of abnormal plasma cells. ***Some patients may have no symptoms but show evidence of organ or tissue impairment (see Table 15.3).

(c)

FIGURE 16.21 Monoclonal gammopathy of undetermined significance. Bone marrow smear (a) and biopsy section (b) demonstrate modest plasmacytosis. Plasma cells show kappa light chain restriction (brown) by dual immunohistochemical stains (c).

Symptomatic PCM Symptomatic PCM, or myelomatosis, is characterized by monoclonal proliferation of plasma cells in the bone marrow and an M-protein production (see Figures 16.5–16.12).

This neoplastic proliferation leads to bone destruction and pathological fractures, particularly in the spine and ribs [51]. Anemia, hypercalcemia, and renal insufficiency are other common features (Table 16.5). The criteria for the diagnosis of PCM proposed by the IMWG are shown in Table 16.6. According to the IMWG, the most critical criterion for symptomatic myeloma is the evidence of organ or tissue impairment manifested by anemia, hypercalcemia, lytic bone lesions (Figure 16.22), renal insufficiency, hyperviscosity, amyloidosis, or recurrent infections [51]. Unlike the WHO criteria for the diagnosis of PCM (Table 16.7), no level of serum or urine M-protein and no minimal percentage of clonal bone marrow plasma cells are 387

Hematopathology: Morphology, Immunophenotype, Cytogenetics and Molecular Approaches criteria, they should carry the diagnosis of PCM [51] and be treated appropriately. Approximately 50% of the patients with PCM show IgG, 25% IgA M-proteins, and about 20% demonstrate only monoclonal light chain production [29, 30, 57]. IgD and IgM PCM are rare [58, 59]. Bence–Jones protein is detected in the urine of about 75% of patients. Over 95% of the patients with PCM show an M-protein in the serum or urine at the time of diagnosis [51]. Approximately 20% of patients show hypercalcemia. Conventional radiologic studies reveal lytic bone lesions, osteoporosis, or fractures in about 80% of the patients at diagnosis. The bone marrow biopsies more often show focal lesions than diffuse, and therefore repeated biopsies may be required to establish the diagnosis. The morphologic features of the neoplastic plasma cells may vary from normalappearing mature forms to blastic or anaplastic forms. As mentioned earlier, the neoplastic plasma cells, unlike normal plasma cells, lack the expression of CD19 and may show aberrant expression of CD56, CD10, and a number of other myeloid or T-cell-associated CD molecules [30–34]. An international staging system has been proposed based on the serum beta-2 microglobulin (β2 M) and serum albumin levels [30, 60]:

(a)

Stage I: Serum β2 M 3.5 mg/L and serum albumin

3.5 g/dL Stage II: Neither stage I nor stage III

(b)

Stage III: Serum β2 M 5.5 mg/L

FIGURE 16.22 (a) Skull X-ray of a patient with plasma cell myeloma demonstrating punched out lytic lesions. (b) Gross specimen of a vertebrate showing several bone marrow lesions of plasma cell myeloma (arrows).

TABLE 16.7 WHO diagnostic criteria for plasma cell myeloma.* Major criteria A. Marrow plasmacytosis (30%) B. Plasmacytoma on biopsy C. M-component: i. Serum: IgG  3 g/dL, IgA 2 g dL ii. Urine 1 g 24 h of Bence-Jones protein Minor criteria A. B. C. D.

Marrow plasmacytosis (10–30%) M-component present but less than above Lytic bone lesions Reduced normal Ig (50% normal levels)

The diagnosis of plasma cell myeloma requires a minimum of one major and one minor criteria or three minor criteria which must include (A) and (B). *From Ref. [1].

required in the proposed diagnostic criteria by the IMWG. Approximately 5% of patients with evidence of tissue or organ impairment may demonstrate 10% plasma cells in their bone marrow, and therefore based on the IMWG 388

The median survival times for patients with stages I, II, and III are 62, 44, and 29 months, respectively [30]. Hyperdiploidy and t(11;14)(q13;q32) are reported in association with favorable prognosis (median survival

50 months), and t(4;14)(p16;q32.3), t(14;16)(q32.3;q23), del(17p13) (locus for p53), and hypodiploidy are considered indicators of poor prognosis (median survival 25 months). The del(13q14) is associated with an intermediate prognosis with median survival of 42 months. In one flow cytometric study, patients with 10 circulating plasma cells (CD38, CD45) per 50,000 mononuclear cells had a significantly lower median survival than those with 10 circulating plasma cells [61]. Table 16.8 provides a list of the adverse prognostic factors. Conventional chemotherapy, autologous and allogeneic stem cell transplantation, and more recently targeted therapies have been used to increase the survival rate [29].

Non-secretory Myeloma Non-secretory myeloma accounts for 1–5% of all myelomas and is characterized by the absence of detectable M-protein in the serum and urine [62, 63]. However, utilization of more sensitive techniques such as serum-free light chain assay may significantly reduce the number of these cases [64, 65]. The reports on non-secretory myelomas suggest a lower incidence of renal failure and hypogammaglobulinemia, lower median percentage of bone marrow plasma cells, higher incidence of neurological presentation, and longer survival than the secretory myelomas [66]. The therapeutic

Plasma Cell Myeloma and Related Disorders

16

TABLE 16.8 Adverse prognostic factors in plasma cell myeloma.* Age

70 years

Serum albumin

3 g/dL

Serum creatinine

2 mg/dL

Beta-2 microglobulin

4 mg/L

Plasma cell labeling index

1%

Circulating plasma cells

10 per 50,000 mononuclear cells

Serum calcium

11 mg/dL

Platelet count

15,000/μL

Hemoglobin

10 g/dL

Cytogenetics

t(4;14)(p16;q32.3)

(a)

t(14;16)(q32.3;q23) del(17p13) (p53 locus) hypodiploidy * Adapted from Kyle RA, Gertz MA, Witzig TE, Lust JA, Lacy MQ, Dispenzieri A, Fonseca R, Rajkumar SV, Offord JR, Larson DR, Plevak ME, Therneau TM, Greipp PR. (2003). Review of 1027 patients with newly diagnosed multiple myeloma. Mayo Clin Proc 78, 21–33, and Ref. [30].

approaches for non-secretory myeloma are similar to those for secretory PCM.

Plasma Cell Leukemia Plasma cell leukemia is a rare event characterized by the presence of 2,000/μL of circulating plasma cells accounting for 20% of the white cell differential count (Figure 16.23) [1, 67–69]. Plasma cell leukemia is divided into two categories: primary and secondary. Primary plasma cell leukemia constitutes about 60% of the cases and is manifested de novo without evidence of previous history of PCM. Secondary plasma cell leukemia accounts for the remaining 40% and represents leukemic transformation in patients with a history of PCM. Patients with primary plasma cell leukemia are younger, have fewer lytic bone lesions and smaller amounts of serum M-protein, demonstrate higher incidence of hepatosplenomegaly, and show a longer survival than patients with secondary plasma cell leukemia [51]. The immunophenotype of primary plasma cell leukemia is frequently IgD, IgE, or light chain only [1], but clonal gene rearrangement studies targeting the IgM region will be sufficient to establish a monoclonal plasma cell disorder if needed.

PLASMACYTOMA Plasmacytoma is a solitary neoplasm of plasma cells involving bone or extramedullary sites. Plasmacytoma demonstrates identical morphologic and immunophenotypic features of PCM [1].

(b)

(c)

FIGURE 16.23 Blood smears of patients with plasma cell leukemia demonstrating circulating plasma cells with various morphologic features (a, b and c). © From Naeim F. (1997). Pathology of Bone Marrow, 2nd ed. Williams & Wilkins, Baltimore, by permission.

Solitary Plasmacytoma of Bone Solitary plasmacytoma of bone is a rare condition and accounts for about 3–5% of the plasmacytic neoplasms. The 389

Hematopathology: Morphology, Immunophenotype, Cytogenetics and Molecular Approaches TABLE 16.9 Characteristics of plasmacytoma of bone and extramedullary sites.* No M-protein in serum or urine** Solitary bone or extramedullary tumor of monoclonal plasma cells Bone marrow not consistent with plasma cell myeloma Normal skeletal survey No related organ or tissue impairment *From Ref. [51].

cell disorder (M), and skin changes (S) [75, 76]. Other frequent features include sclerotic bone lesions, Castleman’s disease, papilledema, serous effusions, and thrombocytosis. The biopsy sections of sclerotic lesions show a monoclonal population of plasma cells. Plasmacytosis is usually modest (median 5%), but the bone marrow is often hypercellular with myeloid preponderance, increased megakaryocytes, and thick bone trabeculae. The M-protein is typically small and sometimes undetectable by routine serum protein electrophoresis [76]. The median age and survival reported in a Mayo Clinic study of 99 patients were 51 years and 165 months, respectively [76].

**A small amount of M-component may be present in some cases.

median age is around 55 years and the male:female ratio is about 2:1. It often involves the axial skeleton, particularly thoracic vertebrae and ribs. Involvement of distal bones, particularly below the knees or elbows, is extremely rare [51, 70]. Bone pain at the site of the lesion is one of the most common presenting symptoms. Infiltration of the tumor cells into the surrounding soft tissue may result in a palpable mass. Radiologic studies show no evidence of additional lesions. Morphologic and immunophenotypic features are identical to those of PCM, but no serum or urine M-protein is detected, and there is no evidence of anemia, hypercalcemia, renal insufficiency, or other organ or tissue impairment (Table 16.9). Multiple solitary plasmacytomas without evidence of PCM occur in up to 5% of the cases [51]. Approximately 50% of patients with solitary plasmacytoma of bone eventually develop PCM [51]. Plasmacytomas of 5 cm in diameter have a greater chance of conversion to PCM. Radiotherapy is the treatment of choice.

Extramedullary Plasmacytoma Extramedullary plasmacytoma is a monoclonal plasma cell neoplasm that arises outside the bone marrow. The most frequent site of involvement is the upper respiratory tract, including the nasal cavity and sinuses, nasopharynx, and larynx [51, 70–74], but any organ or tissue may be involved, such as gastrointestinal tract and urinary tracts, thyroid, male and female reproductive systems, parotid gland, lymph nodes, and central nervous system (Figure 16.24). The diagnosis is made based on the monoclonality of the plasma cell tumor and lack of evidence for PCM and serum or urine M-protein (Table 16.9). IgA is the most frequent immunophenotype, but again it is IgM that is typically assessed at the molecular level for monoclonality (Figure 16.25). Approximately 15% of the patients may eventually develop symptomatic PCM [74]. Surgery and/ or radiation are the treatment of choice.

OSTEOSCLEROTIC MYELOMA Osteosclerotic myeloma, or POEMS syndrome, is characterized by a combination of peripheral neuropathy (P), organomegaly (O), endocrinopathy (E), monoclonal plasma 390

MONOCLONAL IMMUNOGLOBULIN DEPOSITION DISEASES Monoclonal immunoglobulin deposition diseases are monoclonal gammopathies characterized by the deposition of Ig-derived proteins in the organs and tissues causing impairment of their function. These disorders are divided into two major groups: (1) disorders with the deposition of fibrillary proteins (primary amyloidosis) and (2) disorders with the deposition of an amorphous, non-fibrillary protein, known as monoclonal light and heavy chain deposition diseases.

Primary Amyloidosis “Amyloidosis” is a general term referring to a heterogeneous group of disorders characterized by the extracellular deposition of fibrillar proteins with antiparallel beta-pleated sheet configuration on X-ray diff raction [77–85]. These fibrillar structures are identified on biopsy sections by an intense yellow-green fluorescence by thioflavine T and by binding to Congo red stain, leading to apple green birefringence under polarized light [77, 78]. So far, 23 different proteins have been identified that form fibrillar extracellular amyloid deposits in tissues that bind Congo red [79]. The major categories of amyloidosis include (1) primary amyloidosis, (2) secondary amyloidosis, (3) dialysis-related amyloidosis, (4) heritable amyloidosis, and (5) senile amyloidosis (Table 16.10). Primary amyloidosis (AL) is referred to a specific type of amyloidosis in which the fibrillar protein is derived from monoclonal Ig light chains. Primary amyloidosis is considered a variant of monoclonal plasma cell proliferative disorder, and in about 10% of the cases it is associated with symptomatic PCM. In approximately 75% of the cases, the fibrillary protein is derived from the variable region of lambda light chain. The kappa light chain is involved in the remaining 25%. There appears to be a correlation between the site of involvement and the involved variable region of the light chain. For example, the amyloid deposit in patients with dominant renal involvement is often derived from V lambda IV, whereas in patients with dominant cardiac involvement the amyloid deposit is derived from V lambda II or III [77–85]. A serum or urinary monoclonal protein could be

Plasma Cell Myeloma and Related Disorders

(a)

(c)

(b)

(d)

16

FIGURE 16.24 Solitary plasmacytoma of conjunctiva. Biopsy section demonstrating a large aggregate of plasma cells: (a) low power and (b) intermediate power. Immunohistochemical stains for CD138 (c) and lambda light chain (d, pink) demonstrate a monoclonal population of plasma cells. Courtesy of G. Pezeshkpour, M.D., Department of Pathology, VA Greater Los Angeles Healthcare System.

FIGURE 16.25 IgH PCR study of an extramedullary plasmacytoma showing a prominent clonal peak (framework 1 primer set) amid a weaker polyclonal background.

detected in over 85% of the patients using immunofixation techniques. Also, a protein known as serum amyloid P component (SAP) is detected by scintiography in patients with primary amyloidosis. The diagnosis of amyloidosis is based on the biopsies obtained from the affected organs. Liver and kidney biopsies are positive in over 90% of the cases, followed by

abdominal fat pad aspirate and biopsies of rectum, bone marrow (Figure 16.26), and skin [78]. Monosomy of chromosome 18 is the most common abnormality in primary amyloidosis followed by t(11;14)(q13;q32) and del(13q14) [85, 86]. The most frequent clinical symptoms in primary amyloidosis are (1) nephrotic syndrome with or without renal insufficiency, (2) cardiomyopathy, (3) peripheral neuropathy, (4) hepatomegaly, and (5) macroglossia [78]. Elevated serum β-2 microglobulin and bone marrow plasma cells 10%, dominant cardiac involvement, and circulating plasma cells 1% correlate with poor prognosis [78–83]. The actuarial survival for 810 patients studied at the Mayo Clinic was 51% at 1 year, 16% at 5 years, and 4.7% at 10 years [85]. Progression to PCM is rare and in one large study it was reported in only 0.4% of the patients between 10 and 81 months [86]. Therapeutic approaches include chemotherapy, such as melphalan with or without prednisone or dexamethasone and stem cell transplantation. The most common form of heritable amyloidosis is familial Mediterranean fever, an autosomal recessive autoinflammatory disorder characterized by periodic 391

Hematopathology: Morphology, Immunophenotype, Cytogenetics and Molecular Approaches TABLE 16.10 Major categories of amyloidosis.* Amyloid protein

Precursor

Clinical status

AL

Ig light chain

Primary amyloidosis, local or systemic, associated with monoclonal plasma cell disorders

AA

Apolipoprotein AA

Secondary amyloidosis, systemic, associated with chronic infections

Aβ2M

Beta-2 microglobulin

Hemodialysis, systemic

AApoAI, AApoAII, AGel, ALys, ACys, and others

Apolipoprotein AI and AII, gelsolin lysozyme, crystatin C, and others

Familial, systemic

Ab, APro, ATTR, AMed

Ab protein precursor, prolactin, transthretin, lactadherin

Senile, local, or systemic

*Adapted from Ref. [79].

(a)

fevers, abdominal pain (peritonitis), pleuritis, arthritis, pericarditis, and skin rash. It is most prevalent in individuals of Armenian descent, in whom the carrier frequency approaches 1 in 7 but is also found in Arabs, Jews, Persians, Italians, and Greeks [1, 87–90]. In addition to the periodic attacks of pain and fever, the life-threatening complication of the disease is amyloid deposition, particularly in the kidneys. However, this form of amyloidosis is to be distinguished from the others discussed here since it is neither related to plasma cell proliferation nor is it composed predominantly of Ig protein components. Differential diagnosis is made by the clinical history and genetic testing. The causative gene, MEFV, will often, but not always, demonstrate mutations in the homozygous or compound heterozygous state. Since over 60 different mutations have been reported, practical testing is usually limited to a subset of the more common ones found in Mediterranean populations [91]. Technical approaches include DNA sequencing (Figure 16.27a) and allele-specific DNA probe hybridization (Figure 16.27b).

Monoclonal Light and Heavy Chain Diseases The light chain deposition disease (LCDD) and heavy chain deposition disease (HCDD) are clinical variants of monoclonal plasma cell disorders characterized by the deposition of abnormal light chain, heavy chain, or both in the tissues or organs [75, 92, 93]. The deposits, unlike primary amyloidosis, are not fibrillar, do not bind Congo red, and do not contain SAP. LCDD is more common than primary amyloidosis and often consists of kappa light chain (Table 16.11). The primary defect in LCDD appears to be mutations in the Ig light chain variable region, with predominant involvement of VkIV of kappa light chain [1, 78, 29]. Deletion of the CH1 constant domain and point mutation of variable regions of the heavy chain are the primary events in HCDD [1, 78, 93]. These events lead to premature secretion of heavy chain binding protein and increased tendency for tissue deposition. HCDD of IgG1 and IgG3 isotypes are associated with reduced complement activities [1, 94]. Many organs may be involved, including kidneys, liver, heart, nerves, and blood vessels. Approximately 85% of the cases show a detectable serum M-component.

Heavy Chain Diseases

(b)

FIGURE 16.26 Bone marrow biopsy section demonstrating primary amyloidosis: (a) low power and (b) high power views.

392

The heavy chain disease is a monoclonal lymphoplasmacytic disorder characterized by the production of incomplete Ig molecules because of the lack of gamma chain binding sites for light chains. There are three major categories of heavy chain diseases: α, γ, and μ [1, 95, 96]. The α heavy chain disease (αHCD, Mediterranean lymphoma) is considered a variant of MALT-type lymphoma [1]. It occurs in older children and young adults and is associated with gastrointestinal symptoms, such as malabsorption, intestinal obstruction, and diarrhea [1, 97–99].

Plasma Cell Myeloma and Related Disorders A

C

16

The γ heavy chain disease (γHCD, Franklin disease) is a rare condition presenting with lymphadenopathy, splenomegaly, and hepatomegaly with lymphoplasmacytic infiltration similar to lymphoplasmacytic lymphoma [1, 100]. Immunofixation studies may show the presence of serum IgG without light chain. Some patients may demonstrate autoimmune disorders or chronic lymphocytic leukemia [100]. The μ heavy chain disease is a rare lymphoproliferative disorder with clonal IgM molecules with defective variable region [101–103]. Clinically, it resembles chronic lymphocytic leukemia and is often associated with hepatosplenomegaly [1]. The bone marrow is infiltrated by mature small lymphocytes admixed with vacuolated plasma cells [1]. Lymphadenopathy is unusual.

G T

References

(a)

3 4 (b) FIGURE 16.27 (a) DNA sequencing gel showing heterozygosity for the V726A mutation in the 2MEFV gene of a patient with familial Mediterranean fever (the second mutation was M694V). The red dots mark the nucleotide position showing both T (normal) and C (mutant). (b) Reverse hybridization strips using allele-specific oligonucleotide probes directed against the most prevalent mutations in the MEFV gene associated with familial Mediterranean fever. Patient #3 is homozygous for mutation K695R (single arrow), and patient #4 is compound heterozygous for mutations P369S and E148Q (double arrows).

TABLE 16.11 Comparison between primary amyloidosis and light chain deposition disease.

Type

Ig

Deposition

Congo red

Primary amyloidosis

Light chain, often λ

Fibrillary

Positive

Light chain deposition

Light chain, often κ

Non-fibrillary

Negative

αHCD is the most frequent heavy chain disease with the majority of the reported cases being from Middle East, North and South Africa, and the Far East. The pathologic features are more or less similar to those described in MALT lymphoma depicted by a mucosal infiltrate of centrocyte-like lymphocytes and plasma cells. An abnormal heavy chain protein is detected in the serum of 20–90% of the patients [1, 97]. Antibacterial therapy may completely resolve the disease in early stages. Some cases may eventually transform to large B-cell lymphoma. Despite the similar ethnic appellation, this disorder has nothing to do with familial Mediterranean fever, which is not a B-cell disorder.

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