—  SPECIALTY CONFERENCE  —

Hematopathology

Case 2 - CD20(+), Cyclin D1(+)Plasma Cell Myeloma

Robert W. McKenna
University of Minnesota
Minneapolis, MN





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Clinical History
The patient is an 81 year-old female who presented with pancytopenia with the following blood counts:
  • WBC—0.8X109/L

  • Hematocrit—27.2%

  • MCV—92fL

  • RDW—14.5

  • Platelet count—16X109/L
A blood smear (Figures 1 and 2) and bone marrow specimen (Figures 3 to 5) were obtained. Immunohistochemical stains were performed on marrow particle sections (Figures 6 to 10).

Flow cytometry was performed on the bone marrow specimen. The predominant cells had the following immunophenotype: CD20(+), CD19(-), CD5(-), CD10(-), (s)kappa(-), (s)lambda(-), CD56(-).


Case 2 - Slide 1
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Case 2 - Figure 1
Blood smear showing marked pancytopenia.
Case 2 - Figure 2
Higher magnification of the blood smear showing a small plasma cell or plasmacytoid lymphocyte.
Case 2 - Figure 3
Bone marrow aspirate smear showing small, relatively mature appearing plasma cells.

Case 2 - Figure 4
Normocellular bone marrow particle section for the patient's age with decreased hematopoietic cells replaced by a monomorphic cell infiltrate.
Case 2 - Figure 5
A higher magnification of figure 4 showing a predominance of plasmacytic cells
Case 2 - Figure 6
CD138 immunohistochemical stain of the marrow section. The majority of the cells are CD138 positive.

Case 2 - Figure 7
Immunohistochemical stain for kappa light chain. Nearly all of the plasmacytic cells express kappa light chain.
Case 2 - Figure 8
Immunohistochemical stain for lambda light chain. The plasmacytic cells are negative.

Case 2 - Figure 9
CD20 immunohistochemical stain. Nearly all of the plasmacytic cells express surface CD20.
Case 2 - Figure 10
Cyclin D1 stain--There is nuclear cyclin D1 expression in most of the plasmacytic cells.


Differential Diagnosis
  • Plasma cell myeloma (mature morphologic type)

  • Lymphoplasmacytic lymphoma

  • B cell lymphoma with extreme plasma cell differentiation

Diagnosis
CD20(+), Cyclin D1(+)Plasma Cell Myeloma


Case Discussion
In the past decade several distinctive genetic subsets of plasma cell myeloma with characteristic clinical and biological features have been identified. [1] The present case appears to be an example of a recently described possibly distinct subgroup of myeloma associated with small mature (lymphoplasmacytoid) plasma cells that expresses CD20, strong cyclin D1 and a t(11;14)(q13;q32), have a favorable prognosis, and potential for anti-CD20 directed therapy. [2, 3, 4, 5, 6, 7, 8]

The diagnosis in this case is somewhat difficult due to the lymphoplasmacytoid morphology of the plasma cells. The differential diagnosis of CD20 positive myelomas would include lymphomas with extreme plasma cell differentiation. The lymphomas that most commonly exhibit marked plasma cell differentiation and may potentially be confused with a myeloma or plasmacytoma are lymphoplasmacytic lymphoma, marginal zone lymphoma with extreme plasma cell differentiation or large cell lymphoma with immunoblastic or plasmablastic features. Any of these may show morphologic similarities to myeloma and be associated with an M-protein but their clinical presentation is usually more consistent with a lymphoma, and some of the diagnostic criteria for myeloma are nearly always lacking.

As with the present case, careful morphologic study of both bone marrow sections and smears will usually distinguish these lymphomas from plasma cell myeloma. In problematic cases, immunophenotyping will often help. A clonally related B lymphocyte population may be identified by flow cytometry in lymphomas with plasma cell differentiation. The lack of expression of pan-B-cell antigens (CD19, CD20) in myeloma and the aberrant expression of CD56 in most cases is also helpful in distinguishing myeloma from B cell lymphomas with plasma cell differentiation. [9] Immunophenotyping was less definitive in the present case, which expressed CD20 and lacked expression of CD56 although like most myelomas the plasma cells were CD19 negative. Cases of plasma cell leukemia in which the plasma cells are small with features of plasmacytoid lymphocytes must be distinguished from peripheralized lymphoplasmacytic lymphoma. The clinical presentation, bone marrow findings, M-protein type, and genetics help in the differential diagnosis.

Plasma Cell Myeloma—Immunophenotype, Genetics and Prognosis
The discussion that follows is a brief update on plasma cell myeloma that focuses on some of the important features of immunophenotyping, genetic studies, and prognostic indicators.

Immunophenotype and Immunohistochemistry
The plasma cells in myeloma typically express CD79a, VS38c, CD138, bright CD38 and monotypic cytoplasmic Ig and lack surface Ig. Unlike normal plasma cells they are nearly always CD19 negative and CD56 is aberrantly expressed in about 75% of cases. [10, 11, 12] Other aberrantly expressed antigens include CD117, CD20, CD52 and CD10, in decreasing order of frequency; occasionally other myeloid and monocytic antigens are found. [10, 13] In contrast to most myeloma, about 80% of plasma cell leukemias are CD56 negative. [11, 12]

Immunohistochemical stains on marrow biopsy sections are often valuable to essential in the diagnosis of plasma cell neoplasms. They may be used to:
  • Assess the quantity of plasma cells in marrow biopsies

  • Identify monoclonal plasma cell proliferations

  • Distinguish plasma cell myeloma from other neoplasms
CD138 (syndecan-1) is expressed on normal plasma cells and in 60% to 100% of myelomas. Immunohistochemical stains for CD138 are useful in characterizing and quantifying plasma cells in marrow biopsy sections. Generally from 70% to 100% of the neoplastic cells are positive in myeloma. CD138 appears to be plasma cell specific among normal hematopoietic cells in the marrow, however; other neoplastic B-cell disorders such as chronic lymphocytic leukemia and primary effusion lymphomas react with some anti-CD138 antibodies. CD20 is negative in most myelomas but CD79a and CD38 although not plasma cell specific, are usually expressed.

Immunohistochemical stains and in situ hybridization for kappa and lambda light chains are useful for characterizing clonal plasma cell proliferations. Normal and myeloma plasma cells are rich in cytoplasmic immunoglobulin and generally react strongly with antibodies to kappa or lambda light chains. In cases of myeloma, the plasma cells exhibit a monoclonal pattern of reactivity. [14, 15, 16]

In normal marrow and in reactive plasma cell proliferations there is a mixture of kappa and lambda reacting plasma cells. The kappa:lambda staining ratio in marrow biopsies has been proposed as a useful tool for distinguishing myeloma from MGUS. [15] Kappa and lambda stains are often particularly useful in cases with a relatively low percentage of marrow plasma cells, such as may be encountered in post-treatment biopsies for assessment of residual disease. Immunohistochemical staining for plasma cell associated antigens, e.g., CD138 and kappa/lambda, and a panel of appropriate antigens associated with other neoplasms is useful in differentiating myeloma from other hematopoietic neoplasms or a metastatic tumor in morphologically equivocal cases.

Genetics
Until recently, the relationship of chromosome abnormalities to tumor biology and survival in plasma cell myeloma was controversial. As more cases have been studied by improved conventional cytogenetics, FISH and spectral karyotyping (SKY) it has become clear that there are significant biologic and prognostic relationships to cytogenetic abnormalities.

Conventional cytogenetics studies reveal abnormalities in about one third of myelomas. By FISH analysis the proportion with chromosomal aberrancies increases to more than 90%. [17, 18, 19] Both structural and numerical changes are found and include translocations, trisomies, and whole or partial chromosome deletions; complex cytogenetic abnormalities are frequent. Abnormalities of nearly every chromosome have been reported. Translocations involving the heavy chain locus (IgH) on chromosome 14q32 are the most frequent. They are found in 55-70% of myeloma and most frequently involve 5 recurrent major oncogenes: CYCLIN D1 (11q13) (15-18%); C-MAF (16q23) (5%); FGF-R3/MMSET (4p16.3) (15%); CYCLIN D3 (6p21) (3%); and MAFB (20q11) (2%). [17, 18, 20, 21, 22, 23] Together these 5 translocations are found in about 40% of cases of myeloma; most of which are non-hyperdiploid (<48 and/or >75 chromosomes). The remaining myelomas are mostly hyperdiploid, usually with gains in the odd numbered chromosomes, 3, 5, 7, 9, 11, 15, 19, and 21. [18, 19, 24, 25] Both hyperdiploidy and IgH translocations seem to be early, and possibly initiating, events in the genesis of plasma cell neoplasms. They are unified by associated up-regulation of one of the CYCLIN D genes (D1, D2, D3). [21, 25, 26] Over-expression of oncogenes dysregulated by the five recurrent IgH translocations and expression levels of CYCLIN D1, D2, and D3 can be determined by gene expression profiling.

Plasma cell myelomas can be classified into 8 groups that are based mostly on initiating or early pathogenic events using patterns of translocations and cyclin D expression (TC groups). [22] These groups may represent distinct entities that require different therapies. Another molecular classification identifies 7 groups that are similar to the TC groups but not identical. This classification is based on unsupervised clustering of tumors by gene expression profiles. [27] One of these 7 molecular groups is defined by progression events that lead to increased proliferation. Another early event in pathogenesis of plasma cell neoplasia is monosomy or partial deletion of chromosome 13 (13q14), which is found in nearly half of myelomas by FISH. [17, 18] The precise timing of these various early events is poorly understood.

Recurrent genetic changes associated with disease progression in myeloma include secondary IgH or IgL translocations, deletion or mutation of p53 (17p13), translocations involving c-MYC or N-MYC, losses of chromosome 1p and gains of 1q, mutations of FGFR3 in myeloma with t(4;14), mutations of genes resulting in activation of the NF-kappa B pathway, and inactivation of p18INK4c or RB1. [17, 20, 22, 25, 28, 29, 30, 31, 32] Activating mutations of K- or N-RAS are present in about 30-40% of tumors, and are thought to represent an early event in progression. [18, 33]

Prognosis
The range of survival for patients with plasma cell myeloma varies from less than 6 months to greater than 10 years with a median survival of 3 to 4 years. For most patients the disease is incurable. [34] Length of survival is closely related to clinical stage of disease at diagnosis. The Durie and Salmon (DS) staging system, shown in Table 1, is based on a combination of presenting laboratory and radiographic studies. [35]

TABLE 1 Myeloma Staging System

Stage I:
  • Low M-protein levels: IgG <5g/dl, IgA <3g/dl; Urine Bence-Jones <4g/24hr

  • Absent or solitary bone lesions.

  • Normal hemoglobin, serum calcium, Ig levels (non-M protein).
Stage III: Any one or more of the following:
  • High M-protein: IgG >7g/dl, IgA>5g/dl; Urine light chain 12g/24hr

  • Advanced, multiple lytic bone lesions.

  • Hemoglobin <8.5g/dl, serum calcium >12mg/dl.
Stage II: Overall values between I and III
Subclassification: Based on renal function
  • A = serum creatinine <2mg/dl

  • B = serum creatinine >2mg/dl

[Modified from: Durie, B.G. and Salmon, S.E. Cancer 1975; 36:842-854] [36]

There is a significant difference in survival between each of the three tumor burden stages. Renal insufficiency further defines higher risk patients in each stage. [34]

Morphologic features that are associated with aggressive disease include plasmablastic and anaplastic cytologic features and extensive replacement of marrow in trephine biopsy sections. Very low polyclonal serum immunoglobulins and IgA and light chain only types of myeloma have been associated with a poorer response to therapy and shorter survival. [1]

Elevated levels of serum b 2 -microglobulin reflect increased plasma cell tumor mass and/or deteriorating renal function. The serum b 2 -microglobulin alone or in combination with the serum albumin is a reliable predictor of prognosis in myeloma and is the basis of an International Staging System (ISS) that provides highly significant prognostic correlations (Table 2). [34]

TABLE 2 International Staging System for Plasma Cell Myeloma

Stage Criteria Median Survival (mo.)
I Serum b2-microglobulin <3.5 mg/L 62
Serum albumin >3.5g
II Not stage I or III* 44
III Serum b2-microglobulin >5.5mg/L 29

*There are two categories for stage II: serum b 2 -microglobulin <3.5 mg/L but serum albumin <3.5g/dL; or serum b 2 -microglobulin 3.5 to 5.5 mg/L irrespective of the serum albumin level.

[From Greipp PR, et al International staging system for multiple myeloma. J Clin Oncol 2005;23:3412-3420] [3, 4]

There are several other laboratory findings that are indicators of higher risk patients. These include high C-reactive protein, elevated lactate dehydrogenase, increased plasma cell proliferative activity, elevated serum soluble receptor for interleukin 6 (sIL-6R), and cytogenetic abnormalities. [35, 37, 38] Patients with chromosome karyotypic abnormalities by conventional cytogenetics banding studies have a significantly shorter median survival than those without. [34] Prognostic correlations with specific genetics abnormalities are listed in Table 3. [17, 25, 28, 39, 40, 41, 42, 43, 44]

TABLE 3 Prognostic Correlations with Genetic Findings in Plasma Cell Myeloma

Unfavorable:
  • Deletion 13 or aneuploidy by conventional karyotype analysis

  • t(4;14) or t(14;16) or t(14;20) by FISH

  • Deletion 17p13 by FISH

  • Hypodiploidy
Favorable:
  • Absence of unfavorable genetics and presence of:

  • Hyperdiploidy

  • t(11;14) or t(6;14) by FISH

[Modified from: Stewart AK, et al. Leukemia 2007;21:529-534] [25]

The most robust negative prognostic indicators are: high levels of serum b2 M, deletion of 17p/p53 sequences, t(4;14) and the MAF translocations t(14;16) and t(14;20). A high-risk molecular profile based on the expression of either 17 or 70 genes may provide the best independent prognostic indicator. [45]

Take Home Points
1. Immunophenotyping by immunohistochemistry and flow cytometry is important in the characterization and differential diagnosis of mature plasma cell neoplasms.

2. Genetics provides essential biologic and prognostic information in plasma cell neoplasms.

3. Plasma cell neoplasms may be classified according to genetic translocations and type of cyclin overexpression (D1, D2, D3), both of which appear to be early and possibly initiating events in the pathobiology of plasma cell neoplasms.

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