PLASMA CELL MYELOMA IMMUNOPHENOTYPING

Nahla FarahatProfessor of clinical pathology

Alexandria University

Plasma cell Neoplasms

Heterogeneous group of neoplasms characterized by expansion of clonal plasma cells in the bone marrow and usually associated with a detectable monoclonal protein in serum and/or urine.

Origin of Plasma cells Plasma cells are terminally differentiated

activated B-cells, which have the ability to produce antibodies against a variety of antigens.

Once a clonal plasma cell population is established it has the potential to behave in a number of different ways.

This is translated into the clinically diverse plasma cell disorders which we encounter in everyday practice.

Monoclonal gammopathy

The monoclonal gammopathies, in the presence of a clonal plasma cell proliferation can be subdivided into the following possible entities:

1. Monoclonal gammopathy of uncertain significance (MGUS)

2. Plasma cell myeloma (PCM) 3. Isolated plasmacytoma 4. Plasma cell leukaemia (PCL) 5. Primary amyloidosis 6. Immunoglobulin heavy chain and light chain

diseases

Myeloma Pathogenesis

The diagnosis of PCM is usually suspected when monoclonal gammopathy in the serum and/or Bence–Jones protein in the urine are found.

However, several types of plasma cell dyscrasia

show no or inconspicuous monoclonal protein.

In contrast, cases of amyloidosis may show monoclonal gammopathy, yet it is not necessarily neoplastic.

Therefore, the demonstration of myeloma cells is still the most important and definitive criterion for the diagnosis of PCM.

Plasma cell Myeloma Bone marrow based multifocal Plasma cell

proliferative disorder associated with monoclonal protein in serum and/or urine and disseminated bone marrow involvement in most of cases.

In symptomatic phase, PCM is characterized by end organ affection in the form of:

Hypercalcemia Renal dysfunction Anemia Bone disease CRAB

Plasma cells can be encountered in a number of neoplastic scenarios both in bone marrow and occasionally peripheral blood.

Also polyclonal, reactive plasma cells are also encountered in a number of different infective, inflammatory and neoplastic conditions.

Reactive plasma cells

In morphological terms, it can be difficult to differentiate reactive from clonal plasma cells unless plasma cells are present in gross excess, thereby making a clonal proliferation more likely.

Reactive plasma cells can show remarkable morphological diversity; the presence of multinucleate, pleomorphic and atypical plasma cells is well recognized in reactive scenarios and cannot be relied upon to indicate neoplasia

Flow cytometry studies are very capable at differentiating reactive from neoplastic plasma cell proliferations.

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Flow cytometry and PCM In the past, MFC immunophenotyping of PC has

not been routinely applied in many laboratories for the diagnosis, classification and monitoring of patients with PC disorders.

FC is highly sensitive for the detection and characterization of plasma cells and has many advantages over morphological assessment.,

At present consensus exists about the great clinical utility of MFC in at least five different areas.

Diagnosis by confirmation of the clonal nature of the plasma cells.

Differentiation of plasma cell dyscrasias from lymphoma.

Determining risk of progression Assessing disease prognosis Monitoring disease response to treatment

MRD.

Flow cytometry and PCM

Markers of Plasma cells Normal plasma cells express CD45dim,

CD19, CD38 bright and CD138, together with restricted cytoplasmic light chain expression.

They do not express surface mmunoglobulin.

In the bone marrow, the brightest CD38+ cells are plasma cells.

CD138 is a specific marker for plasma cells

Mateo et al J.Clin Oncol 2008

Panel of MoAb (Triple combinations)FITC PE PercP

CD19 CD27 CD138

CD38 CD56 CD138

Cyt K Cytλ CD138

Gating on CD138 positive cells

Panel of MoAb (Quadruple combinations)

FITC PE PE/CY5 APCCD38 CD56 CD19 CD45CD138 CD28 CD33 CD38CD20 CD117 CD138 CD38

Gating on bright CD38 positive cells

FITC PE PerCP CY5.5

PC7 APC APCC750

1. CD27 CD56 CD19 CD38 CD138 CD452. CD81 CD117 CD19 CD38 CD138 CD45

Panel of MoAb (6 color combinations)

Leeds UK

FITC PE PerCPCY5.5

PC7 APC APCC750

V450 BV510

CD38 CD56 CD45 CD19 CD117 CD81 CD138 CD27

Panel of MoAb (8 color combinations)

PETHEMA/Euroflow Group

CD27

Expression of CD27 is uniformly seen in normal plasma cells as well as plasma cells in MGUS and its loss has been correlated with disease progression in patients with MGUS.

Progressive loss of CD27 has also been observed with disease progression in myeloma, and lack of CD27 expression at diagnosis was associated with shorter overall survival

CD28 CD28 is aberrantly expressed on

neoplastic plasma cells in a fairly consistent manner.

Its expression level appears to increase with disease progression, with one study showing CD28 expression on plasma cells from 19% of MGUS and 41% of myeloma patients, and in 100% of human myeloma cell lines.

CD117

CD117 (c-kit) is aberrantly expressed by a proportion of neoplastic plasma cells.

In one study, plasma cells from normal bone marrow samples did not show any reactivity for CD117, compared with 83 – 99% of CD117 positive plasma cells found in the marrows of one-third of myeloma patients.

Expression of CD117 has been associated with a good prognosis in myeloma

CD33

CD33 is a myeloid antigen that can be aberrantly expressed by plasma cells.

In one study, CD33 positive patients had higher beta-2 microglobulin and lactate dehydrogenase levels and a higher incidence of anemia and thrombocytopenia than did CD33 negative patients.

The overall survival was significantly shorter in the CD33 positive group,

CD20

CD20 is typically not expressed by normal plasma cells.

Several studies have suggested the presence of a small proportion of neoplastic plasma cells that express CD20 on their surface.

In one study in patients with multiple myeloma, CD20 expression was associated with small mature plasma cell morphology and with t(11;14)

CD56

Expression of CD56 is commonly used to identify abnormal plasma cells, since normal plasma cells are typically CD56 negative.

Heterogeneity exists among clonal plasma cells with respect to CD56 expression.

CD56 negative patients had a higher incidence of extramedullary disease compared to CD56 positive patients.

Plasma cell leukemia are usually CD56 negative.

CD81 is a tetraspanin widely expressed on B cells including mature PCs which is involved in the regulation of cell growth, motility, signaling and BM homing.

CD81 is strongly expressed on the surface of NPCs but MM cell lines are shown to underexpress it (negative or dim) .

CD81 positivity on myeloma cells is an independent prognostic factor for progression-free and overall survival and a marker for risk of progression in SMM

CD81

Simultaneous assessment of the expression of CD38 and CD138 represents the best combination of markers for the specific identification of PC in hematological samples and its discrimination from other populations

of leukocytes and other hematopoietic cells. CD38 and CD138 are also expressed on

neoplastic plasma cells.

Gating strategy

Positivity for these antigens is not discriminatory.

The intensity of expression does change. CD38 is dimmer and CD138 brighter in

malignant plasma cell populations compared to their normal counterpart.

They generates a well-defined gate for subsequent immunophenotypic analysis.

Gating strategy

Gating strategy Measurement of 0.5 to 1 x 105 is typically

recommended for precise identification of PC and their accurate enumeration.

In PB and in BM samples with low (<1%) PC numbers, the usage of a second data acquisition step is required for higher sensitivity.

In this second step a broad ‘‘live-gate’’ drawn to select CD38 bright or CD138 positive events consisting of a minimum of 3,000 PC/tube should be obtained and stored

In hypocellular and/or hemodiluted samples, analysis of a high number of PC is often difficult.

The European myeloma network recommends that at least 100 neoplastic plasma cell events be acquired for accurate enumeration. This will often require acquisition of a million or more total events

Gating strategy

How to differentiate between normal plasma cells and

myeloma cells The most important findings that

characterize malignant versus normal plasma cells are: absent or low expression of CD27, CD19 and/or CD45, and increased expression of CD28, CD33, CD117 and/or CD56.

Monoclonality for intracytoplasmic immunoglobulin light chains is the ultimate proof for the presence of an abnormal plasma cell population.

Classical phenotype of PCM

Phenotypically aberrant PC typically show:

(1) underexpression of CD19, CD27, CD38, and CD45

(2) overexpression of CD28, CD33, CD81 and CD56

(3) asynchronous expression of CD20, CD117, and surface immunoglobulins (sIg)

Markers on Myeloma cells

By using these markers with this gating strategy, even in cases where the expression of one of these markers is normally expressed (e.g. absence of CD56), simultaneous evaluation of antigenic aberrancies facilitates the discrimination between clonal and normal PC in virtually every PCM case.

Risk categories of PCM

There is good correlation between flow and morphology in the assessment of bone marrow plasma cells, but it is important to note that morphological assessment of bone marrow plasma cell percentages is commonly higher than that determined by flow cytometry.

Assessment of Myeloma cells

A 5- to 20-fold discrepancy in the proportion of plasma cells between the FCM sample and the aspirate smear is a common occurrence.

The finding of a plasma cell population at levels >1% as determined from the CD138/CD38 or CD45/CD38 dot plot would suggest some degree of plasmacytosis.

Why are plasma cells less in flow ? There are a number of possible reasons for

this: 1. Malignant plasma cells are fragile and

surface membrane can be disrupted or lost during flow analysis.

2. Differences in plasma cell enrichment if serial rather than single pull marrow aspirations are taken (hemodilution).

3. Plasma cells often form clusters in bone marrow which are not freed up for single cell analysis in the cytometer. (focal distribution)

Coexistence of normal and phenotypically aberrant PC is a constant finding in patients with MGUS, with most cases (>80%) displaying >5% normal PC within the overall BM PC compartment.

By contrast, only a small proportion (<15%) of all patients with symptomatic PCM display >5% N-PC/BMPC at diagnosis.

Therefore, the presence of >5% residual polyclonal PC at diagnosis has been found to be an accurate parameter for the discrimination between MGUS and PCM cases at diagnosis

How to differentiate between MGUS and PCM

How to differentiate between MGUS and PCM

How to differentiate between PCM and WM

Clonal myeloma cells Phenotype of B-NHL cells: CD19, CD20, CD45 and S

Immunoglobulins positive. CD 56 negative

Thank You