**1. Introduction**

Control of response to chemotherapy is an intrinsic part of current treatment protocols [1–5]. Assessment of response in key points of chemotherapy programs helps both to stratify patients by risk groups more accurately and to avoid serious chronic side effects and patient overtreatment [6–12]. In case of acute leukemia, the number of tumor blasts undetectable in bone marrow (BM) morphologically at different therapy stages or minimal residual disease (MRD) is a criterion for such assessment.

In adult patients, problems of most significant points of immunological detection and the role of MRD levels are a matter of discussion [2]. The MRD significance is demonstrated in full in pediatric oncology. The MRD is of importance both in prognosis of acute lymphoblastic leukemia (ALL) and in prediction of recurrence [13]. Key points for MRD assessment are determined as well as their clinical significance and MRD levels that help in detailed risk stratification of patients [1].

Immunological quantification of MRD cells solves different problems depending on chemotherapy stages. In the middle of induction therapy (day 15), it evaluates primary response [14]. While at the end of induction therapy (day 33), the purpose is final risk stratification of patients with respect to clinical and immunological prognostic factors [1, 14, 15]. MRD assessment at the end of induction consolidation (day 78) identifies a patient group with so called slow response. These patients remain MRD-positive by days 15 and 33 and reach MRD-negativity by day 78 only. This group is characterized by good prognosis [3].

Many international research groups have developed flow cytometry (FC) protocols for MRD diagnosis; however, there is not a common approach yet. The St Jude Children's Research Hospital (Memphis, USA) has proposed a simplified 3-color FC assay to detect MRD cells on day 15 of induction therapy [16] that is based on elimination of normal B-cell precursors (BCP) under the effect of corticosteroids [17] that are a basis for therapy at the given stage. The BFM international study group has developed an MRD monitoring protocol basing on the detection of B-LP with aberrant (leukemia-associated) immunophenotype (LAIP). Antigens CD58 and CD38 are most common markers to characterize the aberrant immunophenotype [18, 19]. However, tumor lymphoblasts show no aberrance by these antigens [20]. There are no clearly identified alternative combinations of aberrant markers.

In ALL from T-cell precursors (T-ALL), there are no clearly cut criteria for MRD assessment [21] and research in this field is ongoing.

The aim of this chapter is to compare immunophenotyping features of normal B-cell precursors and B-lymphoblasts in acute leukemia and to show possibilities of use of a leukemia-associated immunophenotype in monitoring of the MRD.

#### **2. Materials and methods**

The study involved 191 ALL cases (160 B-ALL [142—primary diagnosis, 18 diagnosis in relapse] and 31 T-ALL). The diagnosis was made basing on a combination of morphocytochemical and immunophenotyping assays of BM puncture biopsies.

In most cases (88.8% of B-ALL and 87.1% of T-ALL), the immunophenotyping at diagnosis was performed by 3-color FC (at least 20 markers was analyzed). The immunophenotyping using EuroFlow 8-color standardized panels was made in 11.2% of B-ALL and 12.9% of T-ALL cases. Lineage of blasts was determined using ALOT (acute leukemia orientation tube) (**Table 1**).

A more accurate B-ALL (**Table 2**) or T-ALL (**Table 3**) 8-color standardized panel was used depending upon the identified blast lineage. The BCP aberrance was assessed basing on expression of the following antigens: CD58, CD38, CD81, CD9, CD123, CD66c, CD13, CD33, CD20, CD21, and CD24; T-cell precursors (TCP) were characterized with respect to CD99, CD56 expression.

MRD quantification was made in 397 BM specimens from ALL patients at different therapy stages (days 15, 33, and 78, **Table 4**).


**109**

*B-Cell Precursors: Immunophenotypic Features in the Detection of Minimal Residual Disease…*

 CD58 CD66c CD34 CD19 CD10 CD38 CD20 CD45 cyIgM CD33 CD34 CD19 CD117 + sIgM sIg-λ sIg-κ CD45 nuTdT CD13 CD34 CD19 CD22 CD24 CD9 CD45 CD15 + CD65 NG2 CD34 CD19 CD123 CD81 CD21 CD45

 nuTdT CD99 CD5 CD10 CD1a smCD3 cyCD3 CD45 CD2 CD117 CD4 CD8 CD7 smCD3 cyCD3 CD45 TCRγδ TCRαβ CD33 CD56 cyTCRαβ smCD3 cyCD3 CD45 CD44 CD13 HLA-DR CD45RA CD123 smCD3 cyCD3 CD45

**Markers**

**FITC PE PerCP-cy5.5 PE-cy7 APC APC-117 V450 V500**

**FITC PE PerCP-cy5.5 PE-cy7 APC APC-117 V450 V500**

**MRD monitoring points** Day 15 Day 33 Day 78 **3 col. 4–6 col. 8 col. 3 col. 4–6 col. 8 col. 3 col. 4–6 col. 8 col.**

At diagnosis, every BM specimen was characterized both morphologically and immunologically. Myelogram count was made by two morphologists (250 cells each) on Giemsa stained BM smears. The following M-types were identified basing on standard morphological criteria for the number of blasts: M1—specimens with ≤5% of blasts, M2—specimens with 5.0–25.0% of blasts, and M3—specimens with ≥25% of blasts. After that, a more detailed analysis was made, and the morphological criteria were compared with immunological findings. Detailed analysis of MRD levels was made in M1, M2, and M3 specimens. Subgroups of specimens within each group were identified with respect to the number of MRD cells. The subgroups were identified according to the BFM protocol for MRD assessment on induction chemotherapy as follows: standard risk <0.1% (including negative cases), medium risk 0.1–10.0%, and high risk ≥10%/the level of 0.01% of tumor cells in a specimen

В-ALL Total 139 148 54

Т-ALL Total 30 26 —

No. 59 68 12 51 79 18 3 38 13 Percentage 42.4 49.0 8.6 34.4 53.4 12.2 5.6 70.4 24.0

No. 9 19 2 6 18 2 — — — Percentage 30.0 63.3 6.7 23.1 69.2 7.7 — — —

was taken as a standard threshold for MRD negativity.

*The number of analyses at each MRD monitoring point.*

*DOI: http://dx.doi.org/10.5772/intechopen.84223*

*EuroFlow consortium 8-color standardized panel for B-ALL diagnosis.*

**No./fluorochrome Markers**

*EuroFlow consortium 8-color standardized panel for T-ALL diagnosis.*

**No./ fluorochrome**

**Table 2.**

**Table 3.**

**Table 4.**

**ALL immuno-subtype FC color number**

**Table 1.**

*Acute leukemia orientation tube.*

*B-Cell Precursors: Immunophenotypic Features in the Detection of Minimal Residual Disease… DOI: http://dx.doi.org/10.5772/intechopen.84223*


**Table 2.**

*Normal and Malignant B-Cell*

This group is characterized by good prognosis [3].

assessment [21] and research in this field is ongoing.

ALOT (acute leukemia orientation tube) (**Table 1**).

characterized with respect to CD99, CD56 expression.

ent therapy stages (days 15, 33, and 78, **Table 4**).

cy CD79a

**2. Materials and methods**

biopsies.

Immunological quantification of MRD cells solves different problems depending on chemotherapy stages. In the middle of induction therapy (day 15), it evaluates primary response [14]. While at the end of induction therapy (day 33), the purpose is final risk stratification of patients with respect to clinical and immunological prognostic factors [1, 14, 15]. MRD assessment at the end of induction consolidation (day 78) identifies a patient group with so called slow response. These patients remain MRD-positive by days 15 and 33 and reach MRD-negativity by day 78 only.

Many international research groups have developed flow cytometry (FC) protocols for MRD diagnosis; however, there is not a common approach yet. The St Jude Children's Research Hospital (Memphis, USA) has proposed a simplified 3-color FC assay to detect MRD cells on day 15 of induction therapy [16] that is based on elimination of normal B-cell precursors (BCP) under the effect of corticosteroids [17] that are a basis for therapy at the given stage. The BFM international study group has developed an MRD monitoring protocol basing on the detection of B-LP with aberrant (leukemia-associated) immunophenotype (LAIP). Antigens CD58 and CD38 are most common markers to characterize the aberrant immunophenotype [18, 19]. However, tumor lymphoblasts show no aberrance by these antigens [20]. There are no clearly identified alternative combinations of aberrant markers.

In ALL from T-cell precursors (T-ALL), there are no clearly cut criteria for MRD

The aim of this chapter is to compare immunophenotyping features of normal B-cell precursors and B-lymphoblasts in acute leukemia and to show possibilities of

The study involved 191 ALL cases (160 B-ALL [142—primary diagnosis, 18 diagnosis in relapse] and 31 T-ALL). The diagnosis was made basing on a combination of morphocytochemical and immunophenotyping assays of BM puncture

In most cases (88.8% of B-ALL and 87.1% of T-ALL), the immunophenotyping at diagnosis was performed by 3-color FC (at least 20 markers was analyzed). The immunophenotyping using EuroFlow 8-color standardized panels was made in 11.2% of B-ALL and 12.9% of T-ALL cases. Lineage of blasts was determined using

A more accurate B-ALL (**Table 2**) or T-ALL (**Table 3**) 8-color standardized panel was used depending upon the identified blast lineage. The BCP aberrance was assessed basing on expression of the following antigens: CD58, CD38, CD81, CD9, CD123, CD66c, CD13, CD33, CD20, CD21, and CD24; T-cell precursors (TCP) were

MRD quantification was made in 397 BM specimens from ALL patients at differ-

**Markers FITC PE PerCP-cy5.5 PE-cy7 APC APC-117 V450 V500**

CD34 CD19 CD7 sm

CD3

cy CD3 CD45

use of a leukemia-associated immunophenotype in monitoring of the MRD.

**108**

**No./ fluorochrome**

**Table 1.**

1 cy

*Acute leukemia orientation tube.*

MPO

*EuroFlow consortium 8-color standardized panel for B-ALL diagnosis.*


#### **Table 3.**

*EuroFlow consortium 8-color standardized panel for T-ALL diagnosis.*


#### **Table 4.**

*The number of analyses at each MRD monitoring point.*

At diagnosis, every BM specimen was characterized both morphologically and immunologically. Myelogram count was made by two morphologists (250 cells each) on Giemsa stained BM smears. The following M-types were identified basing on standard morphological criteria for the number of blasts: M1—specimens with ≤5% of blasts, M2—specimens with 5.0–25.0% of blasts, and M3—specimens with ≥25% of blasts. After that, a more detailed analysis was made, and the morphological criteria were compared with immunological findings. Detailed analysis of MRD levels was made in M1, M2, and M3 specimens. Subgroups of specimens within each group were identified with respect to the number of MRD cells. The subgroups were identified according to the BFM protocol for MRD assessment on induction chemotherapy as follows: standard risk <0.1% (including negative cases), medium risk 0.1–10.0%, and high risk ≥10%/the level of 0.01% of tumor cells in a specimen was taken as a standard threshold for MRD negativity.

Statistical analysis was made using IBM-SPSS Statistics v.17 software. Parametric data were analyzed by comparison of means using Student's t-test. Comparison of nonparametric data was made by Pearson's χ2-test.
