**3. Results**

## **3.1 General patient characteristics**

A total of 186 patients with ALL (five patients were treated in debut and in relapse of the disease) managed at the N.N. Blokhin Cancer Research Center during 2006 through 2017 were entered in the study.

The B-ALL group included 155 patients 1–18 years of age: 78 boys (48.7%) and 82 girls (51.3%) (**Table 5**). In 142 cases (88.8%), the diagnosis was made at the disease onset. In 18 cases (11.2%), the diagnosis was made in recurrence stage. Five patients were followed up both in the debut and recurrences stages.

The T-ALL group consisted of 31 patients aged 2–17 years including 26 boys (83.9%) and 5 girls (16.1%) (**Table 6**).

Treatment was given according to the ALL IC BFM 2002 and 2009 protocols. Data on risk stratification are shown for 79 patients with B-ALL receiving treatment by the ALL IC BFM 2009 protocol (data for 63 patients with B-ALL onset are not available). The majority of patients (67.1%) were classified into an intermediate risk group. Only 5.1% of patients were a high risk group. And 27.8% of patients were assigned to a standard risk group.

71.0% of T-ALL patients received treatment by ALL IC BFM 2009 protocol. The majority of patients (72.7%) were classified into an intermediate risk group. Other patients (27.3%) were assigned to a high risk group.


#### **Table 5.**

*Patient characteristics in the B-ALL group.*


**111**

**Table 7.**

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

*3.2.1 Immunological characterization of blasts at primary diagnosis*

CD38 (immunophenotype CD58++CD38low/<sup>−</sup>, **Figure 1**).

CD123 and CD81 in B-lymphoblasts at primary diagnosis.

*3.2.2 Immunophenotypic characterization of blasts at relapse*

**Antigen No. of cases** 

MRD monitoring was made on days 15, 33, and 78 of chemotherapy according to the treatment protocols used at the Research Institute of Pediatric Oncology and Hematology of the FBI N.N. Blokhin NMRCO and international recommendations.

**Table 7** shows B-lymphoblast immunophenotype characteristics at primary

CD58 and CD38 are currently most commonly used antigens as main criteria of tumor B-lymphoblast aberrance. Blasts are considered aberrant if they are characterized by overexpression of CD58 in combination with low or no expression of

Analysis for this antigen combination demonstrated that blasts do not always have the CD58++CD38low/<sup>−</sup> immunophenotype, that is, aberrance by both antigens is found in 54.3% of cases only (**Table 8**). **Figure 2** shows an example of no aberrance

The following markers were studied as additional criteria of aberrance: CD123 (**Figure 3**), CD66c, CD81 (**Figure 4**), CD9, CD21, CD24 with their expression presented in **Table 9**. **Figures 3** and **4** show an example of aberrant expression of

A total of 18 patients were examined at relapse of the disease. Five patients were examined at both disease onset and relapse. Blast characterization was made using a

**analyzed**

CD10 142 93.0 CD34 139 74.1 nuTdT 24 100.0 smCD22 137 50.3 CD20 129 14.7 Pan-myeloid (CD13, CD33) 137 46.0 CD58++CD38low/<sup>−</sup> 50 54.0 CD38+/++ 93 54.8

CD123 8 100.0 CD66c 8 75.0 CD81 7 14.3 CD9 8 87.5 CD21 7 0.0 CD24 7 100.0

*Tumor blast immunophenotype characteristics at primary diagnosis at disease onset.*

**Frequency of Ag-positive cases (%)**

diagnosis. These immunological characteristics were used further in MRD

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

**3.2 ALL from B-cell precursors**

by CD58 and CD38 antigens.

Additional criteria of aberrance

monitoring.

#### **Table 6.**

*Patient characteristics in the T-ALL group.*

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

MRD monitoring was made on days 15, 33, and 78 of chemotherapy according to the treatment protocols used at the Research Institute of Pediatric Oncology and Hematology of the FBI N.N. Blokhin NMRCO and international recommendations.

#### **3.2 ALL from B-cell precursors**

*Normal and Malignant B-Cell*

**3. Results**

nonparametric data was made by Pearson's χ2-test.

**3.1 General patient characteristics**

2006 through 2017 were entered in the study.

(83.9%) and 5 girls (16.1%) (**Table 6**).

assigned to a standard risk group.

patients (27.3%) were assigned to a high risk group.

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

A total of 186 patients with ALL (five patients were treated in debut and in relapse of the disease) managed at the N.N. Blokhin Cancer Research Center during

patients were followed up both in the debut and recurrences stages.

The B-ALL group included 155 patients 1–18 years of age: 78 boys (48.7%) and 82 girls (51.3%) (**Table 5**). In 142 cases (88.8%), the diagnosis was made at the disease onset. In 18 cases (11.2%), the diagnosis was made in recurrence stage. Five

The T-ALL group consisted of 31 patients aged 2–17 years including 26 boys

Treatment was given according to the ALL IC BFM 2002 and 2009 protocols. Data on risk stratification are shown for 79 patients with B-ALL receiving treatment by the ALL IC BFM 2009 protocol (data for 63 patients with B-ALL onset are not available). The majority of patients (67.1%) were classified into an intermediate risk group. Only 5.1% of patients were a high risk group. And 27.8% of patients were

71.0% of T-ALL patients received treatment by ALL IC BFM 2009 protocol. The majority of patients (72.7%) were classified into an intermediate risk group. Other

**Characteristic Number Percentage** Gender M 78 48.4

Time of diagnosis Debut 142 88.8

Immunosubtype Pre-pre-В 150 93.8

**Characteristic Number Percentage** Gender М 29 83.9

Time of diagnosis Debut 31 100

Age 1–17 years (mean 7.3 ± 4.7 years)

Age 1–18 years (mean 5.9 ± 0.3 years)

F 7 16.1

F 83 51.6

Recurrence 18 11.2

Pro-В 10 6.2

Pre-T-cell 12 38.7 Cortico-thymocytic 19 61.3

**110**

**Table 6.**

**Table 5.**

*Patient characteristics in the T-ALL group.*

*Patient characteristics in the B-ALL group.*

#### *3.2.1 Immunological characterization of blasts at primary diagnosis*

**Table 7** shows B-lymphoblast immunophenotype characteristics at primary diagnosis. These immunological characteristics were used further in MRD monitoring.

CD58 and CD38 are currently most commonly used antigens as main criteria of tumor B-lymphoblast aberrance. Blasts are considered aberrant if they are characterized by overexpression of CD58 in combination with low or no expression of CD38 (immunophenotype CD58++CD38low/<sup>−</sup>, **Figure 1**).

Analysis for this antigen combination demonstrated that blasts do not always have the CD58++CD38low/<sup>−</sup> immunophenotype, that is, aberrance by both antigens is found in 54.3% of cases only (**Table 8**). **Figure 2** shows an example of no aberrance by CD58 and CD38 antigens.

The following markers were studied as additional criteria of aberrance: CD123 (**Figure 3**), CD66c, CD81 (**Figure 4**), CD9, CD21, CD24 with their expression presented in **Table 9**. **Figures 3** and **4** show an example of aberrant expression of CD123 and CD81 in B-lymphoblasts at primary diagnosis.

#### *3.2.2 Immunophenotypic characterization of blasts at relapse*

A total of 18 patients were examined at relapse of the disease. Five patients were examined at both disease onset and relapse. Blast characterization was made using a


**Table 7.**

*Tumor blast immunophenotype characteristics at primary diagnosis at disease onset.*

#### **Figure 1.**

*Blasts with aberrant immunophenotype CD58++/CD38low. Cytogram A: blasts (gate 2, green) demonstrate bright CD19 expression (X-axis) against side scattering parameters (SSC, Y-axis). Cytogram B: B-lymphoblasts demonstrate bright CD58 expression (Y-axis) in combination with low CD38 expression (X-axis) as compared to normal BCP (CD58<sup>+</sup> CD38+ , gate 4).*


#### **Table 8.**

*Blast immunophenotypes with respect to CD58 and CD38 expression at primary diagnosis.*

#### **Figure 2.**

*Tumor B-lymphoblasts with immunophenotype CD58<sup>+</sup> /CD38+ . Cytogram A: tumor B-lymphoblasts are identified basing on CD19 expression (X-axis) and low SSC characteristics (Y-axis). Blasts (CD45lowCD19+ ) are characterized by bright CD38 expression (X-axis) in combination with normal CD58 expression (Y-axis) (cytogram B).*

**113**

**Figure 4.**

**Figure 3.**

*CD123 expression (X-axis).*

assessed in 13 patients.

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

*Tumor B-lymphoblast aberrance by CD123 at primary diagnosis. Cytogram A: blasts (gate 1, red) are detected by low CD45 expression (X-axis) and low SSC characteristics (Y-axis). Cytogram B: tumor lymphoblasts demonstrate bright expression of pan-B-cell antigen CD19 (Y-axis) in combination with bright (aberrant)* 

3-color FC protocol in 66.7% (12/18) patients. In six patients (33.3%), immunophe-

*Normal (A) and aberrant (B) expression of CD81 on B-LP. Cytogram A: normal bright CD81 expression on tumor B-lymphoblasts (green) in X-axis (CD81) against Y-axis (CD45) coordinates. Cytogram B: tumor B-lymphoblasts (green) with aberrant (weak) CD81 expression in X-axis (CD81) against Y-axis (CD45).*

Expression of main CD58/CD38 aberrance markers at disease recurrence was

Blast immunophenotype with respect to CD58 and CD38 expression intensity as compared with the primary diagnosis changed in 2 of 5 cases. In the first case, blasts at primary diagnosis were immunophenotypically close to normal BCP as

opposite, blasts lost aberrant CD38 sign and became close to normal non-tumor

CD38++), while acquiring aberrant

) at relapse. In the second case, on the

notyping used the EuroFlow consortium 8-color protocol.

concerns CD58 and CD38 expression (CD58<sup>+</sup>

CD58 and CD38 expression (CD58++CD38+

B-lymphoblasts by this antigen expression.

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

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

#### **Figure 3.**

*Normal and Malignant B-Cell*

**Figure 1.**

*to normal BCP (CD58<sup>+</sup>*

CD58+

CD58+

**Table 8.**

*CD38+*

*, gate 4).*

*Blasts with aberrant immunophenotype CD58++/CD38low. Cytogram A: blasts (gate 2, green) demonstrate bright CD19 expression (X-axis) against side scattering parameters (SSC, Y-axis). Cytogram B: B-lymphoblasts demonstrate bright CD58 expression (Y-axis) in combination with low CD38 expression (X-axis) as compared* 

**CD58/CD38 expression Number Percentage** CD58++/CD38low/<sup>−</sup> 25 54.3

/CD38low/<sup>−</sup> 2 4.3 CD58++/CD38+ 8 17.4

/CD38+ 11 24.0 Total 46 100.0

*/CD38+*

*identified basing on CD19 expression (X-axis) and low SSC characteristics (Y-axis). Blasts (CD45lowCD19+*

*are characterized by bright CD38 expression (X-axis) in combination with normal CD58 expression (Y-axis)* 

*. Cytogram A: tumor B-lymphoblasts are* 

*)* 

*Blast immunophenotypes with respect to CD58 and CD38 expression at primary diagnosis.*

**112**

**Figure 2.**

*(cytogram B).*

*Tumor B-lymphoblasts with immunophenotype CD58<sup>+</sup>*

*Tumor B-lymphoblast aberrance by CD123 at primary diagnosis. Cytogram A: blasts (gate 1, red) are detected by low CD45 expression (X-axis) and low SSC characteristics (Y-axis). Cytogram B: tumor lymphoblasts demonstrate bright expression of pan-B-cell antigen CD19 (Y-axis) in combination with bright (aberrant) CD123 expression (X-axis).*

#### **Figure 4.**

*Normal (A) and aberrant (B) expression of CD81 on B-LP. Cytogram A: normal bright CD81 expression on tumor B-lymphoblasts (green) in X-axis (CD81) against Y-axis (CD45) coordinates. Cytogram B: tumor B-lymphoblasts (green) with aberrant (weak) CD81 expression in X-axis (CD81) against Y-axis (CD45).*

3-color FC protocol in 66.7% (12/18) patients. In six patients (33.3%), immunophenotyping used the EuroFlow consortium 8-color protocol.

Expression of main CD58/CD38 aberrance markers at disease recurrence was assessed in 13 patients.

Blast immunophenotype with respect to CD58 and CD38 expression intensity as compared with the primary diagnosis changed in 2 of 5 cases. In the first case, blasts at primary diagnosis were immunophenotypically close to normal BCP as concerns CD58 and CD38 expression (CD58<sup>+</sup> CD38++), while acquiring aberrant CD58 and CD38 expression (CD58++CD38+ ) at relapse. In the second case, on the opposite, blasts lost aberrant CD38 sign and became close to normal non-tumor B-lymphoblasts by this antigen expression.


#### **Table 9.**

*Detection frequency of antigens associated with various TCP differentiation stages in T-ALL.*

#### *3.2.3 MRD assessment on day 15 of therapy*

We compared morphological and immunological findings from BM analysis on day 15 of therapy. In group M1, morphological and FC data were fully similar (no blasts by morphology and complete MRD negativity [MRD cells < 0.01%] by FC) in 11.5% of cases.

Specimens from the M1 group were characterized by a marked percentage of lymphocytes (mean > 60.0%). The presence of MRD-positive specimens (FC) in the M1 group (good morphological response) may be explained by blast mimicry, that is, similarity with lymphocytes. This makes difficult accurate morphological verification of residual leukemic blasts.

In group M1, there was no contradiction between morphological and FC data. Most M3 specimens (85.7%) contained ≥10.0% of MRD-positive cells by FC. The fraction of MRD-positive cells was 5.5% in one case only; that is, in terms of FC, this patient might be referred to the intermediate risk group and did not require therapy intensification.

Comparison of two FC protocols for MRD monitoring on day 15 (3-color St. Jude [28] based on identification of CD10+ /CD34+ BCP among BM mononuclear fraction, and BFM protocol based on identification of BCP with aberrant immunophenotype among nucleated cells [NC]) demonstrated that the mononuclear approach was a more strict criterion for risk stratification of patients.

MRD on day 15 of induction therapy in B-ALL is always represented by BCP. Their identification is based on detection of CD10 and CD34 expression or no expression of pan-leukocyte antigen CD45 and combination of the above mentioned markers in B-cells. Quantification of MRD cells on the basis of each of the progenitor markers demonstrated that CD10 was the most reliable criterion for evaluation of BCP cells on day 15 of chemotherapy in children with pre-pre-B ALL. While in pro-B B-ALL, the count of BCLP should be made basing on nuTdT-positive cyCD22-expressing B-cells.

CD58/CD38 expression was assessed on day 15 of induction chemotherapy in 28 patients. All BCP, that is, MRD cells demonstrated aberrant immunophenotype CD58++/CD38low only in 15 of 28 patients (54%) (**Figure 5**).

CD58++/CD38low/<sup>−</sup> BCP content was analyzed in patients with no aberrance by this antigen combination at primary diagnosis (n = 11). The proportion of CD10+ B-cells was similar to that of CD58++/CD38low/<sup>−</sup> BCP in four specimens only. This example is illustrated in **Figure 5**. In the remaining cases, the number of CD58++/ CD38low BCP was less than the number of CD10+ B-cells.

The analysis demonstrated a significant predominance of MRD on day 15 of therapy in pro-B ALL as compared to pre-B (p = 0.016).

Day 15 of induction chemotherapy is characterized by BM hypocellularity and a marked fraction of debris that can interfere with results (**Figure 6**). To avoid this,

**115**

detection panel.

*recount for NC CD19+*

**Figure 6.**

**Figure 5.**

*CD38 [X-axis] against CD58 [Y-axis]).*

rence in this subgroup.

*3.2.4 MRD assessment on day 33 of therapy*

*negativity. Recount with respect to Syto16 results in CD34+*

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

*BCP immunophenotype features on day 15 of induction therapy. Cytogram A: 16.6% CD19+*

*3, green) in CD19 (X-axis) against CD45 (Y-axis) coordinates. The cells demonstrate bright CD10 (cytogram B), CD19 (X-axis) expression against CD10 (Y-axis) and marked CD34 expression (cytogram C), CD19 (X-axis) against CD34 (Y-axis). MRD cells show aberrant immunophenotype CD58++CD38low (cytogram D,* 

*CD45low BCP (gate* 

 *BCP are 0.0021% without NC, that is, MRD-*

 *BCP being 0.06%, that is, MRD-positivity.*

a nucleotropic Syto die (Sito16 and Sito16 in our study) was included in the MRD

*Day 15 of induction therapy. The specimen has much debris. Cytogram A: NC detection basing on expression of nucleotropic die Syto16 against SSC. The specimen has 3.65% of NC (gate 1, red). Cytogram B: evaluation of B-cell fraction (gate 2, blue): CD19 (X-axis) against SSC. The B-cell fraction without NC is 0.57%, with* 

In the group, as a whole MRD-negative status was found on day 15 of induction chemotherapy in 11.5% of patients, which allowed reduction in anthracycline dose to be considered with respect to clinical prognostic factors without the risk of recur-

MRD quantification was based on count of CD58++CD38low/<sup>−</sup> BCP. If specimens demonstrated no aberrance by this antigen combination at primary diagnosis, MRD

According to the morphological risk stratification criteria, all specimens contained less than 5.0% of blasts (1.2% ± 0.1, n = 80) and were included in the M1 group. There were no blasts in 6.25% of specimens (5/80). MRD-positivity by immunology was identified in 40.0% of cases (32/80). In two patients (2.5%), MRD cells were >1.0%. There were no high risk specimens (MRD ≥ 10.0%) in our study. CD58 and CD38 primary tumor aberrance on day 33 of induction chemotherapy

was assessed in 50.0% of patients. CD58 aberrance was found in 28 of 40 cases (70%), and CD38 aberrance was detected in 60.0% of cases (24/40). 55.0% of cases (22/40) were CD58/CD38 aberrant and 45.0% (18/40) demonstrated no aberrance.

was assessed by CD58++CD10++ or CD38lowCD10++ combinations.

 *cells are 15.6% in this specimen. CD34+*

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

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

#### **Figure 5.**

*Normal and Malignant B-Cell*

*3.2.3 MRD assessment on day 15 of therapy*

verification of residual leukemic blasts.

[28] based on identification of CD10+

was a more strict criterion for risk stratification of patients.

CD58++/CD38low only in 15 of 28 patients (54%) (**Figure 5**).

CD38low BCP was less than the number of CD10+

therapy in pro-B ALL as compared to pre-B (p = 0.016).

11.5% of cases.

CD4+

**Table 9.**

We compared morphological and immunological findings from BM analysis on day 15 of therapy. In group M1, morphological and FC data were fully similar (no blasts by morphology and complete MRD negativity [MRD cells < 0.01%] by FC) in

**Antigen No. of cases studied Frequency of Ag-positive cases (%)**

nuTdT 31 80.6 CD34 31 19.4 CD10 31 38.7

CD8+ 31 35.5 CD1a 31 54.8 CD5 31 90.3 CD7 31 96.8

*Detection frequency of antigens associated with various TCP differentiation stages in T-ALL.*

Specimens from the M1 group were characterized by a marked percentage of lymphocytes (mean > 60.0%). The presence of MRD-positive specimens (FC) in the M1 group (good morphological response) may be explained by blast mimicry, that is, similarity with lymphocytes. This makes difficult accurate morphological

In group M1, there was no contradiction between morphological and FC data. Most M3 specimens (85.7%) contained ≥10.0% of MRD-positive cells by FC. The fraction of MRD-positive cells was 5.5% in one case only; that is, in terms of FC, this patient might be referred to the intermediate risk group and did not require therapy intensification. Comparison of two FC protocols for MRD monitoring on day 15 (3-color St. Jude

/CD34+

tion, and BFM protocol based on identification of BCP with aberrant immunophenotype among nucleated cells [NC]) demonstrated that the mononuclear approach

MRD on day 15 of induction therapy in B-ALL is always represented by BCP. Their identification is based on detection of CD10 and CD34 expression or no expression of pan-leukocyte antigen CD45 and combination of the above mentioned markers in B-cells. Quantification of MRD cells on the basis of each of the progenitor markers demonstrated that CD10 was the most reliable criterion for evaluation of BCP cells on day 15 of chemotherapy in children with pre-pre-B ALL. While in pro-B B-ALL, the count of BCLP should be made basing on nuTdT-positive cyCD22-expressing B-cells. CD58/CD38 expression was assessed on day 15 of induction chemotherapy in 28 patients. All BCP, that is, MRD cells demonstrated aberrant immunophenotype

CD58++/CD38low/<sup>−</sup> BCP content was analyzed in patients with no aberrance by this antigen combination at primary diagnosis (n = 11). The proportion of CD10+ B-cells was similar to that of CD58++/CD38low/<sup>−</sup> BCP in four specimens only. This example is illustrated in **Figure 5**. In the remaining cases, the number of CD58++/

The analysis demonstrated a significant predominance of MRD on day 15 of

Day 15 of induction chemotherapy is characterized by BM hypocellularity and a marked fraction of debris that can interfere with results (**Figure 6**). To avoid this,

B-cells.

BCP among BM mononuclear frac-

**114**

*BCP immunophenotype features on day 15 of induction therapy. Cytogram A: 16.6% CD19+ CD45low BCP (gate 3, green) in CD19 (X-axis) against CD45 (Y-axis) coordinates. The cells demonstrate bright CD10 (cytogram B), CD19 (X-axis) expression against CD10 (Y-axis) and marked CD34 expression (cytogram C), CD19 (X-axis) against CD34 (Y-axis). MRD cells show aberrant immunophenotype CD58++CD38low (cytogram D, CD38 [X-axis] against CD58 [Y-axis]).*

#### **Figure 6.**

*Day 15 of induction therapy. The specimen has much debris. Cytogram A: NC detection basing on expression of nucleotropic die Syto16 against SSC. The specimen has 3.65% of NC (gate 1, red). Cytogram B: evaluation of B-cell fraction (gate 2, blue): CD19 (X-axis) against SSC. The B-cell fraction without NC is 0.57%, with recount for NC CD19<sup>+</sup> cells are 15.6% in this specimen. CD34+ BCP are 0.0021% without NC, that is, MRDnegativity. Recount with respect to Syto16 results in CD34+ BCP being 0.06%, that is, MRD-positivity.*

a nucleotropic Syto die (Sito16 and Sito16 in our study) was included in the MRD detection panel.

In the group, as a whole MRD-negative status was found on day 15 of induction chemotherapy in 11.5% of patients, which allowed reduction in anthracycline dose to be considered with respect to clinical prognostic factors without the risk of recurrence in this subgroup.

#### *3.2.4 MRD assessment on day 33 of therapy*

MRD quantification was based on count of CD58++CD38low/<sup>−</sup> BCP. If specimens demonstrated no aberrance by this antigen combination at primary diagnosis, MRD was assessed by CD58++CD10++ or CD38lowCD10++ combinations.

According to the morphological risk stratification criteria, all specimens contained less than 5.0% of blasts (1.2% ± 0.1, n = 80) and were included in the M1 group. There were no blasts in 6.25% of specimens (5/80). MRD-positivity by immunology was identified in 40.0% of cases (32/80). In two patients (2.5%), MRD cells were >1.0%. There were no high risk specimens (MRD ≥ 10.0%) in our study.

CD58 and CD38 primary tumor aberrance on day 33 of induction chemotherapy was assessed in 50.0% of patients. CD58 aberrance was found in 28 of 40 cases (70%), and CD38 aberrance was detected in 60.0% of cases (24/40). 55.0% of cases (22/40) were CD58/CD38 aberrant and 45.0% (18/40) demonstrated no aberrance.

Cases without CD58/CD38 aberrance at the primary diagnosis were examined for markers identifying cells of residual tumor by the literature.

We analyzed CD81, CD123, CD9, CD21, CD24 expression in 21 BM specimens. As a result, most informative marker combinations for detection of aberrant B-lymphoblasts were identified: combinations of CD58 or CD38 with CD10 (aberrant immunophenotypes CD58++CD10+ or CD38+ CD10++) were the most informative in terms of FC. In case of CD20-positive ALL MRD could be detected by asynchronous CD20 and CD34 expression (aberrance characterized by CD34+ CD20+ phenotype, **Figure 7**). In most cases, residual tumor may be detected by marked expression of CD123 in combination with weak or no expression of CD81 (aberrant immunophenotype CD123++CD81low, **Figure 8**). In some cases, CD9 monomorphic tumor expression especially in combination with one of clear-cut

#### **Figure 7.**

*MRD quantification basing on asynchronous expression of stage-specific Ag CD34/CD20 in a patient with CD20+ pre-pre-B B-ALL immunosubtype. Cytogram A: tumor B-lymphoblasts (CD45lowCD19+ ) at primary diagnosis (X-axis CD20 against Y-axis CD10), of which 60.0% express CD20. Cytogram B: MRD detection basing on simultaneous CD20 (X-axis) and CD34 (Y-axis) expression. MRD cells are 0.027% of NC specimen, that is, MRD-positivity.*

#### **Figure 8.**

*MRD quantification basing on aberrance by CD58/CD38 and CD123/CD81. Cytogram A: BCP detection basing on bright CD10 (Y-axis) expression in combination with low CD45 expression (X-axis), gate 4 (0.03% of NC) within CD19+ B-cells. Cytogram B: BCP are characterized CD58 overexpression (Y-axis) in combination with no CD38 expression (X-axis) and demonstrate CD123++CD81low immunophenotype (cytogram C: CD123 [Y-axis] against CD81 [X-axis]). Therefore, B-lymphoblasts are aberrant by all four Ag analyzed, and there are no normal BCP in this specimen. Aberrant BCP are 0.027% of NC specimen, that is, MRD-positivity.*

**117**

study.

and CD19+

**Figure 9.**

*(CD45lowCD19+*

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

aberrance criteria (CD58++, CD123++, CD38low, CD81low, **Figure 9**) are a sign of

*MRD quantification basing on aberrance by CD9 and CD81. Cytogram A: tumor B-lymphoblasts* 

In cases with monomorphic co-expression of myeloid antigens (CD66c, CD13 or

*) at primary diagnosis with bright nuTdT expression (Y-axis) in combination with monomorphous CD9 overexpression (X-axis). Cytogram B: tumor cells are aberrant by CD81 (X-axis CD81 against Y-axis CD45). As compared to residual normal BCP (gate 4, pink) with bright CD81 expression, tumor B-lymphoblasts demonstrate low (aberrant) Ag expression. Cytogram C: MRD detection basing on bright monomorphous CD9 overexpression (Y-axis) in combination with low CD81 expression (X-axis) (gate* 

Basing on FC quantification of MRD, most patients (65.0%) receiving treatment

MRD assessment on day 78 of therapy was made in 42 patients and was based on

As recommended by BFM protocols, BM specimens were divided into MRDpositive and MRD-negative subgroups. Most BM specimens were MRD-negative

In 14 patients, MRD status was assessed at three points of treatment protocol (days 15, 33 and 78). Seven of them achieved MRD-negativity by day 78 and met the

After 1 block of anti-recurrence therapy, MRD was detectable in 15 BM specimens. By FC, 40.0% of patients (0.004 ± 0.0008%, n = 6) were MRD-negative and 60.0% of patients (7.3 ± 4.8%, n = 9) were MRD-positive with 77.0% of the MRDpositive specimens (0.9 ± 0.6%, n = 7) containing <5.0% of blasts by morphological

In our study, one patient with early recurrence of ALL (pre-pre-B immunosubtype) received anti-recurrence targeted agent blinatumomab (biospecific

by ALL IC BFM 2009 protocol were included in the intermediate risk group.

detection of BCP with aberrant immunophenotype (CD58++CD38low/<sup>−</sup>).

(n = 27), and 15 of 42 specimens (35.7%) were MRD-positive.

*3.2.6 MRD assessment after 1 block of anti-relapse therapy (day 35)*

Of the whole patients, cohort (80 patients) MRD-negative status was determined by FC in 60.0% (48 patients), and 40.0% (32 patients) were found MRDpositive. Comparison of basic morphological and immunological characteristics in these groups identified significant differences in the number of blasts (p = 0.012)

tumor aberrance and criterion for MRD assessment.

*4, dark blue). They are 0.015% of NC specimen: MRD-positivity.*

B-cells (p = 0.044).

*3.2.5 MRD assessment on day 78 of therapy*

criterion of slow response to therapy.

*3.2.7 MRD monitoring on targeted therapy*

CD33), MRD assessment may be based on these markers.

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

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

#### **Figure 9.**

*Normal and Malignant B-Cell*

CD34+

**Figure 7.**

*that is, MRD-positivity.*

*CD20+*

CD20+

Cases without CD58/CD38 aberrance at the primary diagnosis were examined

We analyzed CD81, CD123, CD9, CD21, CD24 expression in 21 BM specimens.

informative in terms of FC. In case of CD20-positive ALL MRD could be detected

by marked expression of CD123 in combination with weak or no expression of CD81 (aberrant immunophenotype CD123++CD81low, **Figure 8**). In some cases, CD9 monomorphic tumor expression especially in combination with one of clear-cut

*MRD quantification basing on asynchronous expression of stage-specific Ag CD34/CD20 in a patient with* 

*diagnosis (X-axis CD20 against Y-axis CD10), of which 60.0% express CD20. Cytogram B: MRD detection basing on simultaneous CD20 (X-axis) and CD34 (Y-axis) expression. MRD cells are 0.027% of NC specimen,* 

*MRD quantification basing on aberrance by CD58/CD38 and CD123/CD81. Cytogram A: BCP detection basing on bright CD10 (Y-axis) expression in combination with low CD45 expression (X-axis), gate 4* 

*in combination with no CD38 expression (X-axis) and demonstrate CD123++CD81low immunophenotype (cytogram C: CD123 [Y-axis] against CD81 [X-axis]). Therefore, B-lymphoblasts are aberrant by all four Ag analyzed, and there are no normal BCP in this specimen. Aberrant BCP are 0.027% of NC specimen, that is,* 

 *B-cells. Cytogram B: BCP are characterized CD58 overexpression (Y-axis)* 

 *pre-pre-B B-ALL immunosubtype. Cytogram A: tumor B-lymphoblasts (CD45lowCD19+*

or CD38+

phenotype, **Figure 7**). In most cases, residual tumor may be detected

CD10++) were the most

*) at primary* 

As a result, most informative marker combinations for detection of aberrant B-lymphoblasts were identified: combinations of CD58 or CD38 with CD10

by asynchronous CD20 and CD34 expression (aberrance characterized by

for markers identifying cells of residual tumor by the literature.

(aberrant immunophenotypes CD58++CD10+

**116**

**Figure 8.**

*MRD-positivity.*

*(0.03% of NC) within CD19+*

*MRD quantification basing on aberrance by CD9 and CD81. Cytogram A: tumor B-lymphoblasts (CD45lowCD19+ ) at primary diagnosis with bright nuTdT expression (Y-axis) in combination with monomorphous CD9 overexpression (X-axis). Cytogram B: tumor cells are aberrant by CD81 (X-axis CD81 against Y-axis CD45). As compared to residual normal BCP (gate 4, pink) with bright CD81 expression, tumor B-lymphoblasts demonstrate low (aberrant) Ag expression. Cytogram C: MRD detection basing on bright monomorphous CD9 overexpression (Y-axis) in combination with low CD81 expression (X-axis) (gate 4, dark blue). They are 0.015% of NC specimen: MRD-positivity.*

aberrance criteria (CD58++, CD123++, CD38low, CD81low, **Figure 9**) are a sign of tumor aberrance and criterion for MRD assessment.

In cases with monomorphic co-expression of myeloid antigens (CD66c, CD13 or CD33), MRD assessment may be based on these markers.

Of the whole patients, cohort (80 patients) MRD-negative status was determined by FC in 60.0% (48 patients), and 40.0% (32 patients) were found MRDpositive. Comparison of basic morphological and immunological characteristics in these groups identified significant differences in the number of blasts (p = 0.012) and CD19+ B-cells (p = 0.044).

Basing on FC quantification of MRD, most patients (65.0%) receiving treatment by ALL IC BFM 2009 protocol were included in the intermediate risk group.

#### *3.2.5 MRD assessment on day 78 of therapy*

MRD assessment on day 78 of therapy was made in 42 patients and was based on detection of BCP with aberrant immunophenotype (CD58++CD38low/<sup>−</sup>).

As recommended by BFM protocols, BM specimens were divided into MRDpositive and MRD-negative subgroups. Most BM specimens were MRD-negative (n = 27), and 15 of 42 specimens (35.7%) were MRD-positive.

In 14 patients, MRD status was assessed at three points of treatment protocol (days 15, 33 and 78). Seven of them achieved MRD-negativity by day 78 and met the criterion of slow response to therapy.

#### *3.2.6 MRD assessment after 1 block of anti-relapse therapy (day 35)*

After 1 block of anti-recurrence therapy, MRD was detectable in 15 BM specimens. By FC, 40.0% of patients (0.004 ± 0.0008%, n = 6) were MRD-negative and 60.0% of patients (7.3 ± 4.8%, n = 9) were MRD-positive with 77.0% of the MRDpositive specimens (0.9 ± 0.6%, n = 7) containing <5.0% of blasts by morphological study.

#### *3.2.7 MRD monitoring on targeted therapy*

In our study, one patient with early recurrence of ALL (pre-pre-B immunosubtype) received anti-recurrence targeted agent blinatumomab (biospecific

anti-CD19 monoclonal antibody). Blinatumomab mechanism of action involves specific binding with CD19<sup>+</sup> cells and recruitment of effector T-cells to enhance response. According to the literature data, CD19 is not expressed on cells during blinatumomab therapy which makes difficult BCP identification. We chose an alternative BCP identification procedure to monitor MRD in this patient category: BCP identification and characterization on blinatumomab (anti-CD19) therapy should be based on expression of cyCD22 and nuTdT due to lost of CD19 expression.

#### **3.3 ALL from T-lineage precursors**

A total of 31 patients with T-ALL were included in our study. About 61.3% of patients (n = 19) presented with cortico-thymocytic immunosubtype (CD1a expression and/or the presence of CD4<sup>+</sup> CD8+ population) as determined by primary immunophenotyping. **Table 9** shows characteristics of blast immunophenotype at the stage of primary diagnosis.

Since T-cell ontogenesis is characterized by the absence of T-cell progenitors in normal BM, MRD monitoring by FC at all therapy stages involves detection and count of TCP.

According to BFM protocol, MRD detection in T-ALL is based on identification of T-cells with aberrant immunophenotype within CD7-positive fraction. However, CD7 demonstrates a broad range of expression and is present not only on TCP (NK-cells and myeloid progenitor cells); therefore, a cytometric protocol for TCP detection in BM (MRD cells) was developed that was based on expression of pan-T-cell antigen CD3 in cytoplasm (cyCD3) as the most stable lineage-specific pan-T-cell antigen. Choice of appropriate antigen clone is an important requirement for detection of cytoplasmic CD3 expression. For instance, SK7 clone is used to detect membrane determinant, while UCHT-1 binds to cytoplasmatic determinant. So, only UCHT-1 monoclonal antibody should be used in analysis of cyCD3.

In our study, quantification of TCP in BM was based on detection of subpopulations smCD3<sup>−</sup>CD7+ , smCD3<sup>−</sup>TdT+ , smCD3<sup>−</sup>CD1a+ , and CD4<sup>+</sup> CD8+ within cyCD3 positive BM T-cells at all time points of MRD monitoring.

To simplify recount and exclusion of debris, the quantification was made within NC of specimens as identified by Syto dies. The highest values were used as recommended by MRD detection protocols.

#### *3.3.1 MRD assessment on day 15 of therapy*

Quantification of residual blasts on day 15 of induction chemotherapy was made in 30 BM specimens.

The morphological and immunological findings were as a whole similar. The group M2 was heterogeneous demonstrating variability in blast numbers and MRD levels; however, these specimens belonged to intermediate risk group both by M-subtypes and MRD levels and therefore did not interfere in patient stratification.

BM characteristics on day 15 of treatment were analyzed with respect to T-ALL immunosubtype. The pre-T immunosubtype was characterized by significantly higher MRD level on day 15 as compared to cortico-thymocytic one (p = 0.044).

About 76.2% of patients composed the intermediate risk group (according to ALL-IC-BFM 2009 protocol), and 23.8% were stratified into the high risk group. There were no patients meeting criteria for standard risk in our study

Basing on MRD level on day 15 of induction chemotherapy, 25.0% were transferred from the intermediate to the high risk group.

**119**

within cyCD3+

smCD3<sup>−</sup> populations is more accurate.

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

It is important to note that MRD negativity was not reported on day 15 of induc-

 *T-cells within NC (gate 2, blue) with low SSC parameters (cyCD3, X-axis* 

Quantification of residual blasts on day 33 of induction chemotherapy was made

Most BM specimens (25/26) were included into group M1, while there was only

We compared morphological and immunological findings. Specimens from the standard and intermediate risk groups were analyzed in detail with respect to MRD

All BM specimens from group M1 were MRD-positive. Basing on the immunological criteria, 16.0% of BM specimens (n = 4) were included in the standard risk group (MRD < 0.1%). The intermediate risk group consisted of 20 of 25 BM specimens (80.0%). One BM specimen from this group contained no blast cells by

morphology, though FC discovered 2.69% of MRD cells (**Figure 10**).

referred to the standard risk group (MRD cells 0.04%).

One specimen was included in the high risk group by MRD assessment

Note that specimens from this sample demonstrated rather high lymphocytosis

By morphological criteria, only one BM specimen was included in the M2 group (blasts 10.8%), while by immunological characteristics, this specimen might be

CD7 in combination with CD3 are used to assess MRD in T-ALL in the BFM protocol. However, CD7 is commonly found on myelokaryocytes including both lymphoid and myeloid progenitors. Basing on ontogenetic characteristics of expression of T-lineage antigens, we propose to detect TCP (that is MRD cells) by expres-

Comparison of these two approaches to MRD monitoring (the standard BFM protocol basing on MRD cell count within CD7-positive cells and MRD count

to make the conclusion that MRD quantification in T-ALL by detection of cyCD3<sup>+</sup>

cells), both on days 15 and 33 of induction chemotherapy allowed us

sion of cytoplasmatic CD3 as the earliest lineage-specific T-cell marker.

one specimen meeting the M2 criteria. None of the specimens was included in

tion chemotherapy in any of the patients, that is, none of the patients achieved complete leukemic cytoreduction in the middle of remission induction therapy.

*An example of MRD-positivity with no blasts in the BM specimen by morphology on day 33 of therapy. Cytogram A: shows NC of the specimen (gate 1, red) basing on reactivity with Syto16 (X-axis) against* 

 *smCD3<sup>−</sup> immunophenotype that are 2.69% of myelokaryocytes: MRD-positivity.*

*against SSC Y-axis). Cytogram C: CD7, X-axis against smCD3, Y-axis (gate 2) shows TCP (MRD cells) with* 

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

*3.3.2 MRD assessment on day 33 of therapy*

group M3 basing on morphological characteristics.

in 26 patients.

**Figure 10.**

*CD3+ CD7+*

*SSC. Cytogram B: cyCD3+*

content.

(MRD ≥ 10.0%).

(more than 28.0%).

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

#### **Figure 10.**

*Normal and Malignant B-Cell*

specific binding with CD19<sup>+</sup>

**3.3 ALL from T-lineage precursors**

sion and/or the presence of CD4<sup>+</sup>

the stage of primary diagnosis.

expression.

count of TCP.

tions smCD3<sup>−</sup>CD7+

in 30 BM specimens.

stratification.

anti-CD19 monoclonal antibody). Blinatumomab mechanism of action involves

A total of 31 patients with T-ALL were included in our study. About 61.3% of patients (n = 19) presented with cortico-thymocytic immunosubtype (CD1a expres-

immunophenotyping. **Table 9** shows characteristics of blast immunophenotype at

According to BFM protocol, MRD detection in T-ALL is based on identification of T-cells with aberrant immunophenotype within CD7-positive fraction. However, CD7 demonstrates a broad range of expression and is present not only on TCP (NK-cells and myeloid progenitor cells); therefore, a cytometric protocol for TCP detection in BM (MRD cells) was developed that was based on expression of pan-T-cell antigen CD3 in cytoplasm (cyCD3) as the most stable lineage-specific pan-T-cell antigen. Choice of appropriate antigen clone is an important requirement for detection of cytoplasmic CD3 expression. For instance, SK7 clone is used to detect membrane determinant, while UCHT-1 binds to cytoplasmatic determinant.

So, only UCHT-1 monoclonal antibody should be used in analysis of cyCD3.

, smCD3<sup>−</sup>TdT+

mended by MRD detection protocols.

*3.3.1 MRD assessment on day 15 of therapy*

positive BM T-cells at all time points of MRD monitoring.

In our study, quantification of TCP in BM was based on detection of subpopula-

To simplify recount and exclusion of debris, the quantification was made within NC of specimens as identified by Syto dies. The highest values were used as recom-

Quantification of residual blasts on day 15 of induction chemotherapy was made

BM characteristics on day 15 of treatment were analyzed with respect to T-ALL immunosubtype. The pre-T immunosubtype was characterized by significantly higher MRD level on day 15 as compared to cortico-thymocytic one (p = 0.044). About 76.2% of patients composed the intermediate risk group (according to ALL-IC-BFM 2009 protocol), and 23.8% were stratified into the high risk group.

Basing on MRD level on day 15 of induction chemotherapy, 25.0% were trans-

The morphological and immunological findings were as a whole similar. The group M2 was heterogeneous demonstrating variability in blast numbers and MRD levels; however, these specimens belonged to intermediate risk group both by M-subtypes and MRD levels and therefore did not interfere in patient

There were no patients meeting criteria for standard risk in our study

ferred from the intermediate to the high risk group.

, smCD3<sup>−</sup>CD1a+

Since T-cell ontogenesis is characterized by the absence of T-cell progenitors in normal BM, MRD monitoring by FC at all therapy stages involves detection and

CD8+

response. According to the literature data, CD19 is not expressed on cells during blinatumomab therapy which makes difficult BCP identification. We chose an alternative BCP identification procedure to monitor MRD in this patient category: BCP identification and characterization on blinatumomab (anti-CD19) therapy should be based on expression of cyCD22 and nuTdT due to lost of CD19

cells and recruitment of effector T-cells to enhance

population) as determined by primary

, and CD4<sup>+</sup>

CD8+

within cyCD3-

**118**

*An example of MRD-positivity with no blasts in the BM specimen by morphology on day 33 of therapy. Cytogram A: shows NC of the specimen (gate 1, red) basing on reactivity with Syto16 (X-axis) against SSC. Cytogram B: cyCD3+ T-cells within NC (gate 2, blue) with low SSC parameters (cyCD3, X-axis against SSC Y-axis). Cytogram C: CD7, X-axis against smCD3, Y-axis (gate 2) shows TCP (MRD cells) with CD3+ CD7+ smCD3<sup>−</sup> immunophenotype that are 2.69% of myelokaryocytes: MRD-positivity.*

It is important to note that MRD negativity was not reported on day 15 of induction chemotherapy in any of the patients, that is, none of the patients achieved complete leukemic cytoreduction in the middle of remission induction therapy.

#### *3.3.2 MRD assessment on day 33 of therapy*

Quantification of residual blasts on day 33 of induction chemotherapy was made in 26 patients.

Most BM specimens (25/26) were included into group M1, while there was only one specimen meeting the M2 criteria. None of the specimens was included in group M3 basing on morphological characteristics.

We compared morphological and immunological findings. Specimens from the standard and intermediate risk groups were analyzed in detail with respect to MRD content.

All BM specimens from group M1 were MRD-positive. Basing on the immunological criteria, 16.0% of BM specimens (n = 4) were included in the standard risk group (MRD < 0.1%). The intermediate risk group consisted of 20 of 25 BM specimens (80.0%). One BM specimen from this group contained no blast cells by morphology, though FC discovered 2.69% of MRD cells (**Figure 10**).

One specimen was included in the high risk group by MRD assessment (MRD ≥ 10.0%).

Note that specimens from this sample demonstrated rather high lymphocytosis (more than 28.0%).

By morphological criteria, only one BM specimen was included in the M2 group (blasts 10.8%), while by immunological characteristics, this specimen might be referred to the standard risk group (MRD cells 0.04%).

CD7 in combination with CD3 are used to assess MRD in T-ALL in the BFM protocol. However, CD7 is commonly found on myelokaryocytes including both lymphoid and myeloid progenitors. Basing on ontogenetic characteristics of expression of T-lineage antigens, we propose to detect TCP (that is MRD cells) by expression of cytoplasmatic CD3 as the earliest lineage-specific T-cell marker.

Comparison of these two approaches to MRD monitoring (the standard BFM protocol basing on MRD cell count within CD7-positive cells and MRD count within cyCD3+ cells), both on days 15 and 33 of induction chemotherapy allowed us to make the conclusion that MRD quantification in T-ALL by detection of cyCD3<sup>+</sup> smCD3<sup>−</sup> populations is more accurate.
