**4. Conclusions and discussion**

Successful treatment of patients with acute leukemia is associated with escalation of chemotherapy doses which requires effective monitoring of response. Assessment of response in key points of chemotherapy protocols helps both to stratify patients by risk groups more accurately and to avoid serious long-term side effects or patient overtreatment.

Search for adequate approaches to assessment of response was started since the 1970s. For a long time, response to therapy was assessed basing on the number of blasts in BM specimens as determined by morphological study, and as a result so called M-types were defined as follows: M1: <5.0% of blasts, M2: 5–25% of blasts, and M3: ≥25% of blasts [22].

However, the progress in molecular methods such as polymerase chain reaction (PCR) and flow cytometry (FC) allowed quantification of minimal number of residual blasts at submicroscopic level (<0.01%).

In recent years, quantification of residual tumor cells in BM, that is, minimal residual disease (MRD) was a main criterion of response to chemotherapy. Opposite to myeloid leukemias when molecular genetic methods play the leading role in MRD detection, it is immunological approaches involving FC alone that can detect MRD cells in more than 95.0% of cases [23].

Both PCR and FC have their advantages and disadvantages. Implementation of multiparameter FC able to assess up to 12 parameters of a single cell and development of computer software that allows tens of millions of cells to be examined makes FC close to PCR by sensitivity [24]. FC is currently the most rapid and less costly approach to MRD monitoring.

In adults, problems of the most important detection points are a matter of discussion [2]. The MRD significance is most vivid in pediatrics. MRD is found to play a role both in ALL prognosis and recurrence prediction [1]. Key points of MRD monitoring and their clinical significance have been determined as well as MRD levels that allow detailed risk stratification of patients.

It is in childhood oncology that the MRD effects of the patient faith have been studied [1, 25]. The current protocols defined three mandatory time points such as the middle and end of remission induction chemotherapy (days 15 and 33) and the end of consolidation induction (day 78).

MRD monitoring plays different roles at different chemotherapy stages. The most important point in terms of the patient faith is the middle of induction chemotherapy when primary tumor response and prognosis are determined.

For a long time, there were multiple attempts to make the earliest possible assessment of response. One of the approaches is count of blasts in peripheral blood on day 8 of treatment. Patients with blast reduction below 1000 cells/mcl are a group of good response and good prognosis. However, the blast clearance on day 8 is found to reflect response to steroids only [5], while MRD level on day 15 of induction chemotherapy is the most accurate reflection of leukemic cytoreduction, that is, primary response to treatment [5, 14].

The key moment is the selection of good response patients (MRD < 0.01%) in whom reduction of anthracycline dose may be considered [11, 26]. In our study, FC discovered MRD-negativity in 11.5% of patients in whom reduction of anthracycline dose could be considered. These patients did not develop recurrence.

The situation in BM by day 15 of therapy is unique in terms of immunology. Since BCP are highly sensitive to glucocorticoids [17] that are the basis for treatment at the given stage, these cells are fully eliminated from BM [16]. This observation was a basis for a FC protocol developed by the St Jude Children's Research Hospital (Memphis, USA) study group for detection of MRD cells on day 15. This protocol

**121**

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

involves MRD monitoring within BM mononuclear fraction, while the BFM proto-

We compared the two approaches to conclude that recount for NC (the St. Jude Hospital protocol) is the most robust criterion for risk stratification of patients. Moreover, analysis of MRD cell immunophenotype with respect to CD58 and CD38

CD58++/CD38low/− BCP in 36.4% of cases only. While the number of CD58++CD38low

BCP. We conclude therefore that quantification of MRD should be based on detec-

MRD cells on day 15 are BCP. We used a variety of monoclonal antibodies for their monitoring. In pre-pre-B and pre-B ALL immunosubtypes, it is just enough to

Important properties of BM on day 15 are hypocellularity and a marked proportion of debris, that is, destroyed cells under the effect of systemic chemotherapy [29, 30]. This requires modification of FC protocols for MRD monitoring, namely use of special dies able to identify viable, not destroyed cells in specimens. Our study showed reasonable to use Syto nucleotropic dies that can clearly select NC and as a consequence help to avoid debris dilution that may result in a considerable

By the end of induction chemotherapy, appearance (regeneration) of normal

1.Different levels of Ag expressions (overexpression of some molecules [CD58,

2.Expression of Ag of uncommon differentiation lineages (e.g., co-expression of

3.Asynchronous expression of lineage-specific Ag (expression of Ag of mature

A combination of CD58 and CD38 is the most frequently used Ag combination for assessment of B-lymphoblast immunophenotype aberrance in the AIEOP-BFM

Normal BCP are characterized by CD58+/low/CD38+/++ immunophenotype, while

As shown by the I-BFM-ALL-MRD Study group, CD58 overexpression is present on overwhelming majority of tumor B-lymphoblasts and is found in 93.5% of cases

CD38 molecule with receptor and enzymatic activities that is involved in B- and T-cell ontogenesis is brightly expressed on normal BM progenitor cells. Low CD38 expression in BCP is considered aberrant [19, 41] and is found in 61.2% of B-ALL

In our study, CD58 was overexpressed in 71.7% of B-ALL cases, that is, less frequently that by literature data. An explanation may be that the boundary between

aberrant immunophenotypes demonstrate CD58 overexpression and no or low

B-lineage progenitors in BM makes MRD detection more difficult. Clear-cut distinguishing between MRD cells and regenerating BCP is possible basing on LAIP

CD10, CD9], low or no expression of Ag such as CD38, CD81)

myeloid Ag such as CD13, CD33, CD66c, CD123 on tumor blasts)

BCP)

CD34<sup>−</sup>), MRD detection should involve a combination of nuclear TdT with cytoplasmatic CD22 or CD79α that show stable expression as demonstrated already in

underestimation of MRD count and interfere with risk stratification.

B-cells was similar to that of

B-cells in the remaining specimens of aberrant

BCP. In case of pro-B immunosubtype (CD10<sup>−</sup> and/or

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

col uses recount for NC in the specimen.

BCP was less as compared to CD10+

count CD34+

tion of BCP rather than of aberrant blasts.

and/or CD10<sup>+</sup>

early studies of B-cell ontogenesis [27, 28].

or aberrant immunophenotype.

B-cells, e.g., CD20 on CD34<sup>+</sup>

and COG FC protocols.

according to Min Xia et al.

CD38 expression.

[31, 32, 19].

Aberrance is currently defined as follows:

expression demonstrated that the number of CD10+

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

involves MRD monitoring within BM mononuclear fraction, while the BFM protocol uses recount for NC in the specimen.

We compared the two approaches to conclude that recount for NC (the St. Jude Hospital protocol) is the most robust criterion for risk stratification of patients. Moreover, analysis of MRD cell immunophenotype with respect to CD58 and CD38 expression demonstrated that the number of CD10+ B-cells was similar to that of CD58++/CD38low/− BCP in 36.4% of cases only. While the number of CD58++CD38low BCP was less as compared to CD10+ B-cells in the remaining specimens of aberrant BCP. We conclude therefore that quantification of MRD should be based on detection of BCP rather than of aberrant blasts.

MRD cells on day 15 are BCP. We used a variety of monoclonal antibodies for their monitoring. In pre-pre-B and pre-B ALL immunosubtypes, it is just enough to count CD34+ and/or CD10<sup>+</sup> BCP. In case of pro-B immunosubtype (CD10<sup>−</sup> and/or CD34<sup>−</sup>), MRD detection should involve a combination of nuclear TdT with cytoplasmatic CD22 or CD79α that show stable expression as demonstrated already in early studies of B-cell ontogenesis [27, 28].

Important properties of BM on day 15 are hypocellularity and a marked proportion of debris, that is, destroyed cells under the effect of systemic chemotherapy [29, 30]. This requires modification of FC protocols for MRD monitoring, namely use of special dies able to identify viable, not destroyed cells in specimens. Our study showed reasonable to use Syto nucleotropic dies that can clearly select NC and as a consequence help to avoid debris dilution that may result in a considerable underestimation of MRD count and interfere with risk stratification.

By the end of induction chemotherapy, appearance (regeneration) of normal B-lineage progenitors in BM makes MRD detection more difficult. Clear-cut distinguishing between MRD cells and regenerating BCP is possible basing on LAIP or aberrant immunophenotype.

Aberrance is currently defined as follows:


A combination of CD58 and CD38 is the most frequently used Ag combination for assessment of B-lymphoblast immunophenotype aberrance in the AIEOP-BFM and COG FC protocols.

Normal BCP are characterized by CD58+/low/CD38+/++ immunophenotype, while aberrant immunophenotypes demonstrate CD58 overexpression and no or low CD38 expression.

As shown by the I-BFM-ALL-MRD Study group, CD58 overexpression is present on overwhelming majority of tumor B-lymphoblasts and is found in 93.5% of cases [31, 32, 19].

CD38 molecule with receptor and enzymatic activities that is involved in B- and T-cell ontogenesis is brightly expressed on normal BM progenitor cells. Low CD38 expression in BCP is considered aberrant [19, 41] and is found in 61.2% of B-ALL according to Min Xia et al.

In our study, CD58 was overexpressed in 71.7% of B-ALL cases, that is, less frequently that by literature data. An explanation may be that the boundary between

*Normal and Malignant B-Cell*

**4. Conclusions and discussion**

effects or patient overtreatment.

and M3: ≥25% of blasts [22].

residual blasts at submicroscopic level (<0.01%).

levels that allow detailed risk stratification of patients.

cells in more than 95.0% of cases [23].

costly approach to MRD monitoring.

end of consolidation induction (day 78).

is, primary response to treatment [5, 14].

Successful treatment of patients with acute leukemia is associated with escala-

Search for adequate approaches to assessment of response was started since the 1970s. For a long time, response to therapy was assessed basing on the number of blasts in BM specimens as determined by morphological study, and as a result so called M-types were defined as follows: M1: <5.0% of blasts, M2: 5–25% of blasts,

However, the progress in molecular methods such as polymerase chain reaction (PCR) and flow cytometry (FC) allowed quantification of minimal number of

In recent years, quantification of residual tumor cells in BM, that is, minimal residual disease (MRD) was a main criterion of response to chemotherapy. Opposite to myeloid leukemias when molecular genetic methods play the leading role in MRD detection, it is immunological approaches involving FC alone that can detect MRD

Both PCR and FC have their advantages and disadvantages. Implementation of multiparameter FC able to assess up to 12 parameters of a single cell and development of computer software that allows tens of millions of cells to be examined makes FC close to PCR by sensitivity [24]. FC is currently the most rapid and less

In adults, problems of the most important detection points are a matter of discussion [2]. The MRD significance is most vivid in pediatrics. MRD is found to play a role both in ALL prognosis and recurrence prediction [1]. Key points of MRD monitoring and their clinical significance have been determined as well as MRD

It is in childhood oncology that the MRD effects of the patient faith have been studied [1, 25]. The current protocols defined three mandatory time points such as the middle and end of remission induction chemotherapy (days 15 and 33) and the

MRD monitoring plays different roles at different chemotherapy stages. The most important point in terms of the patient faith is the middle of induction chemo-

For a long time, there were multiple attempts to make the earliest possible assessment of response. One of the approaches is count of blasts in peripheral blood on day 8 of treatment. Patients with blast reduction below 1000 cells/mcl are a group of good response and good prognosis. However, the blast clearance on day 8 is found to reflect response to steroids only [5], while MRD level on day 15 of induction chemotherapy is the most accurate reflection of leukemic cytoreduction, that

The key moment is the selection of good response patients (MRD < 0.01%) in whom reduction of anthracycline dose may be considered [11, 26]. In our study, FC discovered MRD-negativity in 11.5% of patients in whom reduction of anthracycline dose could be considered. These patients did not develop recurrence.

The situation in BM by day 15 of therapy is unique in terms of immunology. Since BCP are highly sensitive to glucocorticoids [17] that are the basis for treatment at the given stage, these cells are fully eliminated from BM [16]. This observation was a basis for a FC protocol developed by the St Jude Children's Research Hospital (Memphis, USA) study group for detection of MRD cells on day 15. This protocol

therapy when primary tumor response and prognosis are determined.

tion of chemotherapy doses which requires effective monitoring of response. Assessment of response in key points of chemotherapy protocols helps both to stratify patients by risk groups more accurately and to avoid serious long-term side

**120**

Ag normal and overexpression may be unclear. To make a more accurate evaluation of aberrant immunophenotype in primary diagnosis, Ag tumor expression should be compared with the number of residual normal BCP that is not always large. To make visualization more determinant, one has to select a considerable amount of cells (≥1 million), which was not possible in early studies.

There were only 58.6% of ALL with aberrant (low) CD38 expression which was in accord with foreign data.

If one of two Ag is not aberrant, MRD may be assessed basing on alternative combinations such as CD58/CD10, CD58/CD34, CD38/CD10, and CD38/CD34.

Of note, in our study, about one fourth of all cases (24.0%) had tumor B-lymphoblasts with no aberrance by this Ag combination. Therefore, additional criteria to identify aberrant immunophenotype are needed.

This requires MRD criteria to be used at time of diagnosis and most informative MRD markers to be selected in every specific case at the stage of ALL primary diagnosis.

Multicolor FC that simultaneously evaluates up to 12 various cell characteristics makes the most complete characterization of LAIP. This approach may be implemented by use of the EuroFlow consortium 8-color panels with optimal combination of stable markers and fluorochromes [33–35].

The panel for more accurate B-ALL diagnosis includes 3 basic (repeated in all specimens) Ag and 20 specific Ag. This helps to make multiple aberrance evaluation by expression of nonlineage-specific CD45, CD58, CD38, CD9, CD123, and CD81; by expression of noncommon differentiation lineages such as pan-myeloid CD13, CD33, CD66c, CD15, CD65; and by asynchronous expression of Ag of different stages of B-cell differentiation CD10, CD34, CD, CD22, CD20, CD21, CD24. It is of much importance that expression of all above-mentioned markers may be assessed within a single cell population, that is, CD19<sup>+</sup> CD34+ CD45low BCP.

The protocol makes possible indirect prediction of clinically significant abnormalities basing on a proper immunophenotype [36]. For instance, immunophenotypes of cells with mutations BCR-ABL, MLL, Tel-AML-1, and E2APBX1 are described.

When analyzing capacities of EuroFlow 8-color protocols with respect to MRD monitoring, we paid attention to CD81 [35]. The CD81 molecule belongs to the tetraspanin family and is directly associated with CD19, thus forming a signal complex CD19-CD21-CD81 that realizes its functional activity in normal B-cell ontogenesis. By the literature [37], aberrant (weak) CD81 expression is found in 82% of B-ALL cases. Our evaluation of CD81 expression in primary diagnosis coincided with international study data. For instance, aberrant expression was found in 85.7% of primarily diagnosed ALL.

Another molecule from the tetraspanin family is of interest as a criterion for MRD detection, that is, CD9 [38–40]. Its expression is evaluated both in 4- and 6-color FC protocols of the COG study group (M. Borowitz). CD9 is brightly expressed on early BCP, disappears at the pre-B stage, and appears again on mature B-cells. Its monomorphic overexpression on BCP is considered aberrant. In our study, 87.5% of B-ALL cases demonstrated CD9 aberrant expression on tumor B-lymphoblasts.

In case of monomorphous co-expression of myeloid Ag such as CD13, CD33 and CD66c [31, 41] and also CD123 [42], they may be used as LAIP of tumor B-lymphoblasts. Prognostic significance of myeloid Ag co-expression on tumor cells is disputable, though CD66c (KORSA) is shown to be associated with BCR/ ABL1 reconstructions [43, 44]. N. Guillaume et al. analyzed immunophenotype of ALL from BCP to discover CD66c co-expression to be the most frequent (40.0%), while pan-myeloid Ag CD13 and CD33 were detected in 15.0% of cases only.

**123**

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

In our evaluation of primary immunophenotype of tumor B-lymphoblasts, CD66c co-expression was found in 75.0% of cases, while CD13 and CD33 were pres-

study, only 14.7% of ALL patients demonstrated CD20 co-expression on tumor

Remission completeness is clinically assessed on day 33 of treatment. In our

Day 78 of treatment (end of consolidation induction) is the third time point with undoubted prognostic value for MRD detection. Basing on this evaluation, a group of so called slow response may be identified basing on MRD status. These patients are MRD-positive on days 15 and 33 and reach MRD negativity by day 78

In our study, 64.3% of patients were MRD negative, with 7 of 14 patients

Tumor immunophenotyping in disease recurrence is of special interest. Changes in Ag expression during treatment are studied and described rather well. According to international study data, changes as compared to disease onset may occur also in

We assessed blast immunophenotype both in disease onset and relapse in five patients. Tumor blast immunophenotype was the same by the main diagnostic Ag in disease onset and recurrence in four of the five patients. Of interest is that change in blast phenotype as concerns intensity of CD58/CD38 expression versus the primary

Clinical value of immunophenotype change as concern aberrance markers needs

further clarification and may become an additional criterion in the protocol. Novel targeted therapies that influence disease at molecular level are implemented in clinical practice every year and many are at various stages of clinical trial. A new stage of progress in targeted therapy is associated with development of biospecific antibodies that are a new class of monoclonal antibodies (MAb) binding with a surface antigen target, on the one hand, and with T-cell receptor, on the other hand, thus recruiting effector T-cells and enhancing tumor response. Blinatumomab (Blinzito) is the first therapy in this class that is approved by FDA for the treatment of Ph-negative refractory/relapse ALL from B-LP [38]. This approval was based on results of phase II clinical trials with 43% of 189 adults achieving complete response and 82% achieving MRD-negativity [46].

followed-up in all the three points belonging to the slow response group.

Most patients (65.0%) receiving treatment by ALL IC BFM 2009 schedule were

So in terms of FC approaches to evaluation of small populations which may also include MRD determination of LAIP is most effective using a combination of two Ag with aberrance manifesting itself as overexpression or a combination of molecules with over- and low/no expression. We propose the following Ag combina-

As concerns interleukin-3 receptor α-chain (CD123), all B-ALL were aberrant (i.e., the Ag was expressed on tumor cells), which is in accord with L. Munoz et al. [45]. Asynchronous expression of Ag of noncharacteristic differentiation lineages is an additional criterion for tumor B-lymphocyte LAIP. CD20 is such a marker in CD20-positive B-ALL whose expression is characteristic of mature B-cells only. However, CD20 co-expression on tumor B-lymphoblasts is found in 6.4–15.3% of B-ALL [29, 30]. CD20 is included in most FC panels for MRD diagnosis though it is appropriate rather for more clear-cut identification of CD20<sup>−</sup> BCP than for evaluation of its aberrant expression. This finding may be explained by a small cohort of B-ALL patients (up to 16.0% of all B-ALL patients) in whom residual

CD20<sup>−</sup> immunophenotype. In our

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

blasts may be identified on the basis of CD34<sup>+</sup>

CD81low, and CD9++CD81low.

study, MRD-negative status was detected in 600% of patients.

stratified into the intermediate risk group by MRD content.

only. This cohort is characterized by good prognosis.

tumor cell immunophenotype in disease recurrence.

diagnosis was found in two of the five patients.

B-lymphoblasts.

tions: CD123+

ent in 46.0%, which is similar to international study data.

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

In our evaluation of primary immunophenotype of tumor B-lymphoblasts, CD66c co-expression was found in 75.0% of cases, while CD13 and CD33 were present in 46.0%, which is similar to international study data.

As concerns interleukin-3 receptor α-chain (CD123), all B-ALL were aberrant (i.e., the Ag was expressed on tumor cells), which is in accord with L. Munoz et al. [45].

Asynchronous expression of Ag of noncharacteristic differentiation lineages is an additional criterion for tumor B-lymphocyte LAIP. CD20 is such a marker in CD20-positive B-ALL whose expression is characteristic of mature B-cells only. However, CD20 co-expression on tumor B-lymphoblasts is found in 6.4–15.3% of B-ALL [29, 30]. CD20 is included in most FC panels for MRD diagnosis though it is appropriate rather for more clear-cut identification of CD20<sup>−</sup> BCP than for evaluation of its aberrant expression. This finding may be explained by a small cohort of B-ALL patients (up to 16.0% of all B-ALL patients) in whom residual blasts may be identified on the basis of CD34<sup>+</sup> CD20<sup>−</sup> immunophenotype. In our study, only 14.7% of ALL patients demonstrated CD20 co-expression on tumor B-lymphoblasts.

So in terms of FC approaches to evaluation of small populations which may also include MRD determination of LAIP is most effective using a combination of two Ag with aberrance manifesting itself as overexpression or a combination of molecules with over- and low/no expression. We propose the following Ag combinations: CD123+ CD81low, and CD9++CD81low.

Remission completeness is clinically assessed on day 33 of treatment. In our study, MRD-negative status was detected in 600% of patients.

Most patients (65.0%) receiving treatment by ALL IC BFM 2009 schedule were stratified into the intermediate risk group by MRD content.

Day 78 of treatment (end of consolidation induction) is the third time point with undoubted prognostic value for MRD detection. Basing on this evaluation, a group of so called slow response may be identified basing on MRD status. These patients are MRD-positive on days 15 and 33 and reach MRD negativity by day 78 only. This cohort is characterized by good prognosis.

In our study, 64.3% of patients were MRD negative, with 7 of 14 patients followed-up in all the three points belonging to the slow response group.

Tumor immunophenotyping in disease recurrence is of special interest. Changes in Ag expression during treatment are studied and described rather well. According to international study data, changes as compared to disease onset may occur also in tumor cell immunophenotype in disease recurrence.

We assessed blast immunophenotype both in disease onset and relapse in five patients. Tumor blast immunophenotype was the same by the main diagnostic Ag in disease onset and recurrence in four of the five patients. Of interest is that change in blast phenotype as concerns intensity of CD58/CD38 expression versus the primary diagnosis was found in two of the five patients.

Clinical value of immunophenotype change as concern aberrance markers needs further clarification and may become an additional criterion in the protocol.

Novel targeted therapies that influence disease at molecular level are implemented in clinical practice every year and many are at various stages of clinical trial.

A new stage of progress in targeted therapy is associated with development of biospecific antibodies that are a new class of monoclonal antibodies (MAb) binding with a surface antigen target, on the one hand, and with T-cell receptor, on the other hand, thus recruiting effector T-cells and enhancing tumor response. Blinatumomab (Blinzito) is the first therapy in this class that is approved by FDA for the treatment of Ph-negative refractory/relapse ALL from B-LP [38]. This approval was based on results of phase II clinical trials with 43% of 189 adults achieving complete response and 82% achieving MRD-negativity [46].

*Normal and Malignant B-Cell*

in accord with foreign data.

diagnosis.

described.

primarily diagnosed ALL.

Ag normal and overexpression may be unclear. To make a more accurate evaluation of aberrant immunophenotype in primary diagnosis, Ag tumor expression should be compared with the number of residual normal BCP that is not always large. To make visualization more determinant, one has to select a considerable amount of

There were only 58.6% of ALL with aberrant (low) CD38 expression which was

If one of two Ag is not aberrant, MRD may be assessed basing on alternative combinations such as CD58/CD10, CD58/CD34, CD38/CD10, and CD38/CD34. Of note, in our study, about one fourth of all cases (24.0%) had tumor B-lymphoblasts with no aberrance by this Ag combination. Therefore, additional

This requires MRD criteria to be used at time of diagnosis and most informative MRD markers to be selected in every specific case at the stage of ALL primary

Multicolor FC that simultaneously evaluates up to 12 various cell characteristics makes the most complete characterization of LAIP. This approach may be implemented by use of the EuroFlow consortium 8-color panels with optimal combina-

The panel for more accurate B-ALL diagnosis includes 3 basic (repeated in all specimens) Ag and 20 specific Ag. This helps to make multiple aberrance evaluation by expression of nonlineage-specific CD45, CD58, CD38, CD9, CD123, and CD81; by expression of noncommon differentiation lineages such as pan-myeloid CD13, CD33, CD66c, CD15, CD65; and by asynchronous expression of Ag of different stages of B-cell differentiation CD10, CD34, CD, CD22, CD20, CD21, CD24. It is of much importance that expression of all above-mentioned markers may be assessed

The protocol makes possible indirect prediction of clinically significant abnormalities basing on a proper immunophenotype [36]. For instance, immunophenotypes of cells with mutations BCR-ABL, MLL, Tel-AML-1, and E2APBX1 are

When analyzing capacities of EuroFlow 8-color protocols with respect to MRD monitoring, we paid attention to CD81 [35]. The CD81 molecule belongs to the tetraspanin family and is directly associated with CD19, thus forming a signal complex CD19-CD21-CD81 that realizes its functional activity in normal B-cell ontogenesis. By the literature [37], aberrant (weak) CD81 expression is found in 82% of B-ALL cases. Our evaluation of CD81 expression in primary diagnosis coincided with international study data. For instance, aberrant expression was found in 85.7% of

Another molecule from the tetraspanin family is of interest as a criterion for MRD detection, that is, CD9 [38–40]. Its expression is evaluated both in 4- and 6-color FC protocols of the COG study group (M. Borowitz). CD9 is brightly expressed on early BCP, disappears at the pre-B stage, and appears again on mature B-cells. Its monomorphic overexpression on BCP is considered aberrant. In our study, 87.5% of B-ALL

In case of monomorphous co-expression of myeloid Ag such as CD13, CD33 and CD66c [31, 41] and also CD123 [42], they may be used as LAIP of tumor B-lymphoblasts. Prognostic significance of myeloid Ag co-expression on tumor cells is disputable, though CD66c (KORSA) is shown to be associated with BCR/ ABL1 reconstructions [43, 44]. N. Guillaume et al. analyzed immunophenotype of ALL from BCP to discover CD66c co-expression to be the most frequent (40.0%), while pan-myeloid Ag CD13 and CD33 were detected in 15.0% of cases

cases demonstrated CD9 aberrant expression on tumor B-lymphoblasts.

CD34+

CD45low BCP.

cells (≥1 million), which was not possible in early studies.

criteria to identify aberrant immunophenotype are needed.

tion of stable markers and fluorochromes [33–35].

within a single cell population, that is, CD19<sup>+</sup>

**122**

only.

#### *Normal and Malignant B-Cell*

Notwithstanding initial treatment results, some patients fail to respond to blinatumomab or develop progressive disease after initial response. Recurrence rate is 30%.

Characterization of blast immunophenotype in recurrence on blinatumomab therapy discovered no CD19 expression on tumor cells. This interferes with BCP identification and requires new methodological approaches to MRD assessment.

Inclusion of blinatumomab into B-ALL therapy requires an alternative MRD evaluation strategy.

There was one patient receiving blinatumomab in our study [47]. The MRD monitoring and immunophenotyping at second recurrence was difficult due to the absence of CD19+ B-cells. An algorithm for BCP detection was proposed and tested that was based on alternative B-lineage differentiation markers. Nuclear nuTdT in combination with cytoplasmatic CD22 as most stably expressed Ag were chosen.

So, given the appearance of new targeted therapies, FC algorithms for both MRD diagnosis and monitoring require certain flexibility and timely rational changes.

There is an equivocal situation with T-ALL. On the one hand, search for aberrant immunophenotype for TCP ended in failure. Expression of main Ag studied with respect to LAIP, that is, CD99 and nuTdT is variable [21]. On the other hand, taking into account normal T-cell ontogenesis, MRD assessment may be based on the absence of TCP of the cyCD3++smCD3−nuTdT+ CD7+ level in BM. One has therefore just to assess TCP number in order to quantify MRD at any therapy stage.

Correct choice of antibody CD3 clone is of much importance for TCP identification. For instance, clone UCHT1 should be used to detect CD3 cytoplasmatic expression, while CD7 is needed to detect the membrane determinant.

The AIEOP-BFM protocol suggests that MRD detection in T-ALL should be based on CD7.

We compared the two approaches to TCP identification to find optimal TCP detection with CD7<sup>+</sup> / ++smCD3+ immunophenotype within cyCD3+ population basing on T-cell ontogenesis.

All T-ALL patients in our study were stratified into the intermediate and high risk groups. According to MRD levels on day 15, most patients remained in the initial stratification group though 25.0% were transferred from the intermediate into the high risk group.

Of note that all patients demonstrated MRD-positivity on day 15, that is, none of the T-ALL patients achieved complete leukemic cytoreduction on ALL-IC-BFM 2009 therapy.

So, clinical significance of MRD in ALL arises no doubt. It is reasonable to make MRD quantification immunologically using FC assay.

Since there are no normal BCP on day 15 of induction chemotherapy, MRD quantification in case of pre-pre-B and pre-B ALL immunosubtypes should be based on detection of CD10<sup>+</sup> /CD34+ population within CD19+ B-cells. In case of pro-B (CD10<sup>−</sup> and/or CD34<sup>−</sup>), ALL immunosubtype MRD detection should be based on expression of nuclear nuTdT in combination with cyCD22.

During BM regeneration (end of remission induction therapy and long-term treatment stages), MRD quantification is based on identification of BCP with aberrant immunosubtype. Alongside with the most common CD58/CD38, the following Ag should be used in LAIP assessment: CD81, CD9, CD13, CD33, CD66c, CD123, and CD20. At the stage of diagnosis, the most informative personalized Ag combinations should be selected for further MRD monitoring to make a more accurate risk stratification of patients at different therapy stages and are an effective tool for its modification.

Tumor immunophenotype in disease recurrence should be compared with that in disease onset taking into account its possible changes.

In case of targeted therapy, for example, anti-recurrence treatment, FC protocol should be changed with respect to treatment features. For instance, BCP evaluation

**125**

**Author details**

provided the original work is properly cited.

and Nikolay Nikolayevich Tupitsyn

TCP T-cell precursors

(N.N. Blokhin NMRCO), Moscow, Russia

\*Address all correspondence to: dr.chernysheva@mail.ru

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Olga Chernysheva\*, Lyudmila Yuryevna Grivtsova, Alexander Popa

Federal State Budgetary Institution, "N.N. Blokhin National Medical Research Center of Oncology" оf the Ministry of Health of the Russian Federation

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

on blinatumomab therapy should be based on expression of nuTdt in combination

ALL-IC-BFM intercontinental acute lymphoblastic leukemia protocol of Berlin-

Frankfurt-Munster study group

BFM Berlin-Frankfurt-Munster study group

cyCD cytoplasmic cluster of differentiation

LAIP leukemia-associated immunophenotype

smCD membrane cluster of differentiation

In T-ALL, MRD assessment at any therapy stage may be limited to quantification

population.

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

of CD7+/++smCD3<sup>−</sup> cells within cyCD3<sup>+</sup>

**Appendixes and nomenclature**

The authors declare no conflict of interests.

ALOT acute leukemia orientation tube ALL acute lymphoblastic leukemia

B-ALL ALL from B-cell precursors

CD cluster of differentiation

COG children oncology group

MAb monoclonal antibodies MRD minimal residual disease MNC mononuclear cells NC nucleated cells

T-ALL ALL from T-cell precursors

BCP B-cell precursors

BM bone marrow

FC flow cytometry

with cyCD22.

**Conflict of interest**

Ab antibody Ag antigene

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

on blinatumomab therapy should be based on expression of nuTdt in combination with cyCD22.

In T-ALL, MRD assessment at any therapy stage may be limited to quantification of CD7+/++smCD3<sup>−</sup> cells within cyCD3<sup>+</sup> population.
