**4. Identification and immunophenotype of PCs**

usually equipped with 2-3 lasers allowing excitation of 6 or more standard fluorochromes and the term multiparametric and/or polychromatic flow cytometry is used for this approach [7]. The classical immunophenotypisation identifies cells based on their size and granularity/ complexity as well as by the "visualization" of antigen-antibody binding. More than 360 antigens are currently known and commercial monoclonal antibodies conjugated with

Flow cytometry has been used in diagnostics of MM since 90th years of the 20th century. Mostly ploidy and proliferative characteristics were analysed, but also the combination of DNA analysis with cytoplasmic immunoglobulin detection was done [8-10]. Discovery of new monoclonal antibodies (MoAb) against PCs helped in the development of immunophenotyp‐

It is well known that MFC underestimate the number of PCs when compared to routine morphological evaluation. However, the sensitivity of MFC is similar to light microscopy, results obtained using both approaches correlate and the percentage of PCs provided by MFC is also an independent prognostic factor affecting the overall survival of patients [13]. MFC is precise in detecting even a small number of PCs and together with analysis of expression of selected markers, normal and abnormal PCs could be easily discriminated [4]. So MFC is

Development of flow cytometry, including powerful instruments with the possibility to analyze many fluorochromes, availability of new dyes and antibodies, together with accessible specific software for complex phenotype analysis, require reviewing of current settings in MG analyses. The shift towards polychromatic analyses should be associated with standardisation and validation of this method as it is necessary to be consistent in providing analyses and reporting results. Recently, the European Myeloma Network (EMN) started to use the Euroflow settings which led to the development of a uniform protocol for the analysis of

B cells and PCs as their terminally differentiated stage play an essential role in humoral immune response. The antigen-dependent phase of B cell differentiation has been extensively

the circulation into lymph nodes. Recognition of antigen presented on a follicular dendritic cell together with a costimulatory signal from a specific T lymphocyte causes B cell activation [15,16]. The activated B cell either migrates to extrafolicullar areas where it differentiates into a short term plasma cell or moves into a lymphoid follicle to establish a germinal centre (GC) [17,18]. Massive proliferation of B cell, somatic hypermutation of variable region of Ig chains, isotype switch and subsequent affinity maturation occur in GC [19,20,18,16]. The aim of these processes is to generate B cells able to bind the appropriate antigen with a high affinity. Part

CD38+/-CD20+

CD27-

CD38++CD20- CD138-

CD138+

IgM+ IgD+

CD27+

) pass from

) migrating

) producing high-

**3. Development and differentiation of B cells as PC precursors**

different fluorochromes are widely available.

94 Multiple Myeloma - A Quick Reflection on the Fast Progress

helpful method for clinical analyses of MGs.

biological material of MG cases [14].

studied for many years. Mature naive B cell (CD19+

of these cells then differentiate into plasmablasts (CD19+

into the bone marrow where they mature into long-lived PCs (CD38+

isation in MGs [11,12].

Syndecan-1 (CD138) is a specific marker of PCs expressed on the surface of both, normal and malignant PCs from their early stages [29]. Expression of CD138 is usually missing and/or is not very intensive on circulating PCs and/or plasmablasts in peripheral blood as well as on immature PCs and/or lymphoplasmacytic cells in bone marrow. Another important marker is CD38, a non-specific marker, whose bright expression (brighter on normal than on abnormal PCs) was used to identify PCs for a long time period. Together with CD138 helps in precise identification of PCs. An important marker for pathological PCs identification is also CD45 which is usually missing on PCs. These surface antigens are still used in analyses, but adding of other antigens is necessary [30,31].

Mostly terminally differentiated clonal CD38++CD138+ CD45- PCs are available in MM bone marrow. Relative number of PCs (determined by morphology and/or flow cytometry) corresponds to type of MG, although results could be distorted by dilution of aspirated bone marrow with peripheral blood. Lower amount of PCs is characteristic for MGUS, aMM and/or amyloidosis, on the other hand higher PC infiltration occurs in MM. There is no possibility to determine PC "abnormality" in low-infiltrated cases without detailed phenotype study (Fig 2). There are also circulating pathological PCs in peripheral blood of some myeloma patients, which have usually the same phenotype as bone marrow PCs (mostly CD56- ) [32].

2) [38]. Detailed information about diagnostic and prognostic value of some interesting

markers is mentioned in Table 3. Also other markers should be more and/or less expressed by

**Abnormal expression**

CD19 Positive (>70%) negative 95% necessary

CD56 Negative (<15%) strongly positive 75% necessary

CD117 Negative (0%) positive 30% recommended

CD20 Negative (0%) positive 30% recommended

CD28 Weak Positivity (<15%) strongly positive 15-45% recommended

CD27 Strong Positivity (100%) weak/negative 40-50% recommended

**FITC PE**

1 CD45 CD138 CD38 CD28 CD27 CD19 CD117 CD81

2 CD45 CD138 CD38 CD28 CD56 β2m cIgκ cIgλ

**Table 2. EuroFlow PCD classification panel.** Tube No.1 is useful for phenotype characterization of PCs and evaluation markers with potential prognostic significance, tube No.2 is used for detection and discrimination of

**PerCP-**

**Cy5.5 PE-Cy7 APC APC-H7**

**Table 1.** List of surface antigens useful for detection of normal and abnormal CD38+CD138+ PCs in MGs [4].

**Patients with abnormal expression (%)**

Immunophenotyping in Multiple Myeloma and Others Monoclonal Gammopathies

**Requirement for diagnostics and monitoring**

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97

PCs, mostly without clinical relevance.

**Antigen Normal expression**

**Tube/ fluorochrom** **Pacific Blue**

normal PCs from aberrant and clonal PCs [38].

**Pacific Orange**

Increased absolute (>2x109 /l) and/or relative (>20% of leukocytes) count of peripheral PCs serve as diagnostic criterion of plasma cell leukaemia (PCL). Primary PCL originates *de novo*, but secondary PCL occurs in patients with relapsed/refractory myeloma [33]. Primary PCL is a distinct clinic-pathological entity with different cytogenetic and molecular findings. The clinical course is aggressive with short remissions and survival duration [34].

Mixture of lymphoplasmacytic cell (LPC) subpopulations with different maturity status (from B cells CD19+ CD20+ CD38- CD138 to PCs CD19+ CD20+/-CD38+++CD138+ ) is characteristic for Waldenström macroglobulinemia (WM), where abnormal LPCs multiply out of control and produce large amounts of IgM protein [35]. It is supposed that every MM is precede mostly by non-IgM MGUS, however Waldeström macroglobulinemia and/or B-CLL proba‐ bly arise from IgM MGUS or monoclonal B cell lymphocytosis (MBL) [36,37].

**Figure 2. Mixture of polyclonal CD19+ (blue dots) and clonal CD56+/- PCs (violet dots).** Heterogeneous expression of CD56 and nestin, positivity for CD45, negativity for CD27 and CD81 was found in clonal CD38+CD138+ PCs.

Clinically important and necessary antigens allowing discrimination of abnormal from normal PCs are known and listed in Table 1 [4]. Similar antigens were used in Euroflow settings (Table 2) [38]. Detailed information about diagnostic and prognostic value of some interesting markers is mentioned in Table 3. Also other markers should be more and/or less expressed by PCs, mostly without clinical relevance.

possibility to determine PC "abnormality" in low-infiltrated cases without detailed phenotype study (Fig 2). There are also circulating pathological PCs in peripheral blood of some myeloma

serve as diagnostic criterion of plasma cell leukaemia (PCL). Primary PCL originates *de novo*, but secondary PCL occurs in patients with relapsed/refractory myeloma [33]. Primary PCL is a distinct clinic-pathological entity with different cytogenetic and molecular findings. The

Mixture of lymphoplasmacytic cell (LPC) subpopulations with different maturity status

to PCs CD19+

for Waldenström macroglobulinemia (WM), where abnormal LPCs multiply out of control and produce large amounts of IgM protein [35]. It is supposed that every MM is precede mostly by non-IgM MGUS, however Waldeström macroglobulinemia and/or B-CLL proba‐

**Figure 2. Mixture of polyclonal CD19+ (blue dots) and clonal CD56+/- PCs (violet dots).** Heterogeneous expression of CD56 and nestin, positivity for CD45, negativity for CD27 and CD81 was found in clonal CD38+CD138+ PCs.

Clinically important and necessary antigens allowing discrimination of abnormal from normal PCs are known and listed in Table 1 [4]. Similar antigens were used in Euroflow settings (Table

/l) and/or relative (>20% of leukocytes) count of peripheral PCs

CD20+/-CD38+++CD138+

) [32].

) is characteristic

patients, which have usually the same phenotype as bone marrow PCs (mostly CD56-

clinical course is aggressive with short remissions and survival duration [34].

bly arise from IgM MGUS or monoclonal B cell lymphocytosis (MBL) [36,37].

CD138-

Increased absolute (>2x109

(from B cells CD19+

CD20+

96 Multiple Myeloma - A Quick Reflection on the Fast Progress

CD38-


**Table 1.** List of surface antigens useful for detection of normal and abnormal CD38+CD138+ PCs in MGs [4].


**Table 2. EuroFlow PCD classification panel.** Tube No.1 is useful for phenotype characterization of PCs and evaluation markers with potential prognostic significance, tube No.2 is used for detection and discrimination of normal PCs from aberrant and clonal PCs [38].


**Cluster Designation** **Normal distribution and functions**

common antigen and aids in activation and signaling processes of B

CD56 NK and NKT cells One of the most valuable

and T cells

CD45 CD45 is a leukocyte

CD81 Expressed on B cells

CD117 Progenitors of myeloid, erythroid and megakaryocytic lineage;

mast cells

including PCs and regulates CD19 expression

**Expression in plasma cells of pre-malignant (MGUS) and malignant stage of**

MGUS - heterogeneous distribution of CD45+ normal and CD45- abnormal PCs in bone marrow MM - mostly CD45 negative

CD45 expression demonstrates proliferating compartment of normal, reactive and malignant PCs; immature PCs should be CD45+ as well

markers to define the abnormal phenotype of PCs in PC proliferative disorders including myeloma. Loss of CD56 expression always associated with aggressive phenotype of myeloma cells. Lack of CD56 expression can be frequently found in patients with circulating PCs and extramedullary myeloma.

Less than 50% of MM cases express CD81 on PCs and expression is heterogeneous in most of the cases (ranging

MGUS- 50% of cases express

MM- only one third of myeloma cases express

from 5%-92%)

CD117

CD117

**Diagnostic or prognostic**

Immunophenotyping in Multiple Myeloma and Others Monoclonal Gammopathies

Patients with CD45 positive expression had better OS than patients with CD45 negative

Possess substantial diagnostic value in PC disorders when combined with CD19 marker. Patients with CD56 negative expression on PCs found to have reduced OS compared to patients with CD56 positive expression. Also, CD56 negative myeloma cases strongly associated with adverse biological parameters.

Patients with CD81 expression on myeloma cells had inferior prognostic outcome (PFS and OS) compared to patients with CD81 negative expression

CD117 expression on PCs predicted better outcome in MM patients. Combination of CD117 and CD28 markers delineated MM patients with different risks; CD117 expression is associated with an altered maturation of the myeloid and lymphoid hematopoietic cell compartments and favorable

disease features

**References**

http://dx.doi.org/10.5772/55938

99

[48-50]

[30,51-53]

[54]

[46,55-57]

**significance**

expression

**myeloma**


**Cluster Designation** **Normal distribution and functions**

98 Multiple Myeloma - A Quick Reflection on the Fast Progress

of B cells ranging from pro-B cells to PCs

CD19 Expressed in all stages

CD20 Expressed during

on PCs

CD27 Helps in differentiation

CD33 Myeloid and monocytic cells

maturation process of B cells and mostly absent

of B cells into PCs

CD28 T cell activation MGUS– only very few cases

**Expression in plasma cells of pre-malignant (MGUS) and malignant stage of**

MGUS – normal PCs express CD19 whereas malignant

MM – only negative or dim CD19 expression on PCs

Only few patients express CD20 on their PCs (< one third of patients)

MGUS - consistent expression on PCs MM- expression is heterogeneous and intensity of expression is lower compared to MGUS

express CD28

MM– CD28 expressing PC represents aggressive phenotype and associates always with tumour expansion

A very few MM cases express CD33 on the surface

of PCs

**Diagnostic or prognostic**

Facilitate as an identification marker of malignant and physiological PCs in combination with CD56. Patients with "/>5% of normal PCs (CD19+CD56-) had better PFS and OS compared to patients with ≤ 5% of normal PCs. Similarly, presence of "/>5% normal PCs or <95% of malignant PCs in MGUS and asymptomatic MM (AMM/ SMM) predicted better PFS compared to patients with ≤ 5% normal PCs or ≥ 95% of

**References**

[39-41]

[41-43]

[44,45]

[46]

[47]

**significance**

malignant PCS.

prognosis

and OS

Associated with poor

Lack of CD27 expression associated with shorter PFS

Combination of CD28 and CD117 markers identified three groups of patients with different risk. Patients with CD28-CD117+ PCs (good risk group) had better PFS and OS compared to patients with CD28+CD117- PCs (poor risk) and patients with CD28- CD117- or CD28+CD117+ PCs

(intermediate risk)

CD33 expression associated with poor OS and higher mortality rate

**myeloma**

PCs do not


Thus polychromatic FC (minimum of 6 markers, but usually 8 markers) is required for sufficient PC analysis and combination of surface and intracellular antigens is necessary for identification and clonality assessment of PCs [66-68]. Only a limited number of cases requires more than 8 markers to detect a small clonal subpopulation of PCs on the prevailing back‐ ground of polyclonal PCs. Use of marker with a known aberrant expression on analysed PCs (CD28, CD117 etc.) could help in precise identification of clonal PCs. Marker CD27 should be useful as loss of this antigen should reveal clonal PCs (Fig 3). Together with analysis of a sufficient number of PCs, the sensitivity of polychromatic FC should reach the sensitivity of

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101

FC should be used not only for assessment of PCs in peripheral blood (PB) and/or bone marrow (BM), but in simultaneous analysis of 8 markers on a single cell could identify the type of PCs

Identification and enumeration of PCs is as important as discrimination between normal polyclonal PCs in reactive plasmocytosis and clonal PCs in plasma cell disorders (MGUS, MM, PCL, extramedullary plasmocytoma) [4]. It was found that BM of MGUS cases contained a mixture of polyclonal PCs with normal phenotype and clonal PCs with aberrant phenotype, on the other hand there is a majority of clonal PCs in MM [63,65]. Presence of more than 5% normal PCs in BM should be used as a cut-off value for differentiation between MGUS and MM [40]. Surprisingly there were found symptomatic MM patients with more than 5% normal PCs in BM, these should be signed as "MGUS-like MM" and have a low incidence of high-risk cytogenetic abnormalities with a longer progression-free survival and longer overall survival as well [39]. There are clonal non-myelomatous PCs present in Waldenström macroglobuli‐ nemia (WM) so careful PC analysis should be done in these patients especially when they have low number of PCs [35]. Discrimination of myelomatous from non-myelomatous PCs then

Conventional parameters related to the higher risk of progression of MGUS into MM are monoclonal Ig level (MIG) > 15 g/l and non-IgG isotype of MIG. Even so, a new parameter is serum free light chain (FLC) ratio. These parameters were used for risk stratification model [69]. Simultaneously evolving and non-evolving theory of MGUS type, based on evolutionary pattern of MIG (increasing vs. stable) was published [70]. Mentioned parameters are important in patient monitoring for decades, but FC approach based on pathological PCs enumeration is quicker with a better predictive value [40]. Finding ≥95 % pathological PCs (from all PCs) is an independent parameter with a predictive value, in term of risk of progression MGUS and/ or a MM into symptomatic form. When compared FC results with a parameter describing

the PCR approach [5,67].

that has clinical and predictive value.

**6.1. Differential diagnostics**

**6. Clinical application of flow cytometry in MGs**

should help in determination of other lymphoproliferations [28].

**6.2. Determination of the progression risk in MGUS and MM**

**Table 3.** Myeloma cell specific antigens and their diagnostic and prognostic values. Abbreviations: PFS - progression free survival, OS - overall survival

#### **5. Abnormality vs. clonality of PCs**

The most useful antigens allowing basic orientation in context of PC normality are CD19 and CD56 which can allow relatively easy discrimination of immunophenotypically normal (CD19+ CD56- ) from immunophenotypically aberrant (CD19- CD56+ ) PCs [63-65]. As was verified by cytoplasmic analysis of immunoglobulin light chains kappa and lambda, this discrimination should be used just for orientation and does not have to correspond to a real number of polyclonal and clonal PCs, especially in unusual cases and/or time after treatment. Thus polychromatic FC (minimum of 6 markers, but usually 8 markers) is required for sufficient PC analysis and combination of surface and intracellular antigens is necessary for identification and clonality assessment of PCs [66-68]. Only a limited number of cases requires more than 8 markers to detect a small clonal subpopulation of PCs on the prevailing back‐ ground of polyclonal PCs. Use of marker with a known aberrant expression on analysed PCs (CD28, CD117 etc.) could help in precise identification of clonal PCs. Marker CD27 should be useful as loss of this antigen should reveal clonal PCs (Fig 3). Together with analysis of a sufficient number of PCs, the sensitivity of polychromatic FC should reach the sensitivity of the PCR approach [5,67].
