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

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 that has clinical and predictive value.

#### **6.1. Differential diagnostics**

**Cluster Designation**

CD200 Member of

CD221 (insulin like growth factor-1 receptor)

**Normal distribution and functions**

100 Multiple Myeloma - A Quick Reflection on the Fast Progress

immunoglobulin superfamily and expressed on endothelial cells, neurons, B cells and a subset of T cells

Tyrosine kinase receptor family, expressed widely on all types of cells

activation molecules (SLAM) family member

intermediate filaments, marker of multipotent proliferative precursors found in some embryonic and fetal

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

CD229 Signaling lymphocytic

nestin Protein of class VI

tissues

free survival, OS - overall survival

(CD19+

CD56-

CD138 Plasma cells Both normal and malignant

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

PCs from MGUS and MM cases express CD138 but the expression of CD38 marker is lower in malignant PCs

MM - more than 70% of cases do express CD200

MM - more than 70% and 85% of medullary and extramedullary cases express CD221 on the surface of PCs, respectively

MM- consistent expression

MGUS - less than 30% express nestin; MM - more than 45% and 80% of medullary and extramedullary myeloma cases express nestin in the cytoplasma of PCs, respectively

on PCs

**Diagnostic or prognostic**

Universal marker of PCs and provides basis to quantify or to assess disease burden in PC proliferative disorders

MM - more than 70% of cases

do express CD200

Patients with CD221 expression had worse prognosis and CD221+ PCs were associated with adverse cytogenetic abnormalities

Might represent an attractive diagnostic and therapeutic

Patients with nestin expression should have higher risk to develop extramedullary type of

CD56+

target for MM

MM

**Table 3.** Myeloma cell specific antigens and their diagnostic and prognostic values. Abbreviations: PFS - progression

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

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.

) from immunophenotypically aberrant (CD19-

**References**

Absence of CD200 expression on myeloma cells associated with better PFS

[55,60]

[61]

[62]

) PCs [63-65]. As was

[58, 59]

[30]

**significance**

**myeloma**

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 should help in determination of other lymphoproliferations [28].

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

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 evolution of monoclonal component, the risk of progression was better described by immu‐ nophenotypisation [71]. Multiparametric FC is thus capable to distinguish patients which need more frequent monitoring and which need to start treatment earlier than usual. There is still not any marker allowing discrimination between benign MGUS and its malignant form at this moment.

positive immunofixation (IF) after autologous transplantation (regarding to time to progression and overall survival), so FC is sufficiently sensitive method and should be

Immunophenotyping in Multiple Myeloma and Others Monoclonal Gammopathies

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

103

Using new treatment protocols led The International Myeloma Working Group (IMWG) to review treatment response criteria. There was included also FC analysis in the assessment of stringent complete response (sCR), more precisely the absence of phenotypically aberrant PCs in 3000 PC analysed by multiparametric FC (≥ 4 colours) [79]. Criteria of MRD level assessment are changing nowadays as newer more efficient treatment protocols are available and FC has technically developed. When used flow cytometry for confirmation of (s)CR, the new term "immunophenotype remission (iCR)" - a state without presence of any clonal PCs should be used [79, 80]. The evidence suggests that the 4-colour FC is not sufficiently sensitive for confirmation of iCR and standardized polychromatic flow cytometry is the best approach (Fig

Flow cytometry analysis was performed only in a limited number of subjects with monoclonal gammopathies in the late 1990's and early 2000's. During the past decade and present, many analyses showed importance of MFC in differential diagnostics and monitoring (management) of plasma cell diseases. The MFC has developed significantly and with better understanding of PC pathophysiology is the mandatory diagnostic tool which should be included as a routine

,

Lucie Rihova1,2, Karthick Raja Muthu Raja2,3, Luiz Arthur Calheiros Leite4

4 Department of Biochemistry, Federal University of Pernambuco, Brazil

1 Department of Clinical Hematology, University Hospital Brno, Brno, Czech Republic

2 Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Ma‐

3 Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech

used for routine MRD analysis [78].

3).

**7. Conclusion**

**Author details**

Republic

*6.4.1 Better definition of complete remission*

assay in monoclonal gammopathy patients.

Pavla Vsianska1,2,3 and Roman Hajek1,2,5

saryk University, Brno, Czech Republic

#### **6.3. Prognostic markers in MGs**

Determination of immunophenotype should be used not only for discrimination of normal and pathological PCs, but it has also prognostic value. The loss of CD56 (neural cell adhesion molecule, NCAM) should be joined with extramedullary spread [72]. An association between the phenotype profile and cytogenetic abnormalities was found. Expression of CD19 and CD28 and/or absence of the CD117 on pathological PCs are joined with significantly shorter time without progression and overall survival in transplanted patients [46]. Expression of CD28 correlated with t(14;16) and del(17p), on the other hand no presence of CD117 was joined with t(4;14) and del(13q). The analysis combining both CD28 and CD117 was able to divide patients into 3 risk groups with different time without progression and overall survival. The correlation of CD117 expression with hyperdiploidy was found as well [73]. The expression of CD117 on PCs is associated with changes in production of haematopoietic stem cells from BM, lead to a decreasing number of neutrophils in PB and the presence of normal PCs in BM [57]. Recently, a rare MM case was described with PCs phenotype: CD19+ CD56- CD20+ CD22+ CD28+ CD33+ CD117+ HLA-DR+ . Moreover, the cytogenetic analysis of this case revealed a hyperdiploid karyotype and no rearrangement of the IgH gene or deletion of 13q14 [74]. The very important genetic change in MM is loss of the gene for CD27 which is linked with clinically aggressive disease, but in about 50% of MM is expression of CD27 preserved and these patients have better prognosis [44]. Probably the best prognostic information until now serves a combination of two independent parameters: the presence of high-risk cytoge‐ netics by FISH and persistent minimal residual disease evaluated by multiparameter flow cytometry at day +100 after autologous transplantation. These two parameters were able to identify patients in complete remission at risk of early progression [75]. The important thing is that these two methods are available in most hospitals taking care of patients with haema‐ tological malignancies.

#### **6.4. Minimal residual disease analysis**

It is known that conventional parameters (% PCs, MIG level) are not sensitive enough for analysis of treatment response in MM patients. As FC is applicable up to 80-90% of patients, this method is able to reach the sensitivity of allelic-specific oligonucleotide (ASO)-PCR (sensitivity 10-4 for FC vs. 10-5 for PCR) and is less time and monetary consuming as well. Hence FC looks as the optimal method for minimal residual disease (MRD) assessment after any treatment [76,77]. The advantage of FC in MRD analysis is the versatility of used markers allowing assessment of normal and abnormal PCs (CD19/CD56), removing the need to know the original phenotype of PCs before treatment. MRD negativity proved by FC (detection <10-4 myeloma PC within all nucleated cells) was more informative then positive immunofixation (IF) after autologous transplantation (regarding to time to progression and overall survival), so FC is sufficiently sensitive method and should be used for routine MRD analysis [78].

#### *6.4.1 Better definition of complete remission*

Using new treatment protocols led The International Myeloma Working Group (IMWG) to review treatment response criteria. There was included also FC analysis in the assessment of stringent complete response (sCR), more precisely the absence of phenotypically aberrant PCs in 3000 PC analysed by multiparametric FC (≥ 4 colours) [79]. Criteria of MRD level assessment are changing nowadays as newer more efficient treatment protocols are available and FC has technically developed. When used flow cytometry for confirmation of (s)CR, the new term "immunophenotype remission (iCR)" - a state without presence of any clonal PCs should be used [79, 80]. The evidence suggests that the 4-colour FC is not sufficiently sensitive for confirmation of iCR and standardized polychromatic flow cytometry is the best approach (Fig 3).
