A cost-effective, high sensitivity 10-color single tube flow-cytometry (FC) based B-cell precursor acute lymphoblastic leukemia (BCPALL) minimal residual disease (MRD) assay

Abstract Flow cytometry for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) has been widely used in pediatric patients to quantify therapeutic response and to assess the risk of relapse. Flow cytometry for MRD provides roughly the same level of sensitivity (0.01%) as molecular methods but at lower cost and with faster turnaround time. MRD assessment in ALL currently requires an evaluation of 20 or more parameters divided among multiple tubes. In part due to the assessment complexity, the use of flow cytometry for MRD detection in adult ALL patients has been relatively limited. We developed a 6-color, single-tube, flow cytometry assay to detect MRD in bone marrow (BM) aspirate specimens from adult ALL patients. The 73 patients included 52 patients with B-ALL (71%), 19 patients with T-ALL (26%) and 2 patients with T/myeloid leukemia (3%) and were treated with one of several standard chemotherapeutic regimens or targeted therapies. Patients were tested for MRD by flow cytometry after induction or re-induction therapy and serially thereafter. The 6-marker MRD panel was customized for each patient based on the 18-20-marker diagnostic immunophenotype. Sixty-three percent of B-ALL patients (n=33) had lymphoblasts with an aberrant immunophenotype; expression of a myeloid marker (e.g., CD13, CD15 or CD33) was the most common aberrancy. The remaining 37% of B-ALL patients (n=19) had disease with a hematogone immunophenotype, which comprised surface expression of CD10, CD19, CD20, CD34, CD38 and CD45; in the majority of these cases, leukemic cells were distinguishable from normal hematogones based on the intensity of surface marker expression. Forty-seven percent of T-ALL patients (n=9) had an aberrant immunophenotype, most often characterized by CD33 expression. One-hundred forty-six consecutive specimens analyzed for MRD by flow cytometry were classified as positive (23%), negative (72%) or uncertain (5%). Of the 34 samples classified as positive, 14 (41%) showed morphologic (i.e., BM aspirate or biopsy) evidence of disease; nineteen (65%) samples did not show morphologic evidence of disease and 1 sample did not have a concurrent morphologic assessment. Of the 105 samples classified as negative by flow cytometry, 103 (98%) were also negative by morphology and 1 sample did not have a concurrent morphologic assessment. One sample that was negative by flow cytometry had morphologic evidence of disease in the biopsy (10-20% blasts) but not the aspirate, suggesting that aspirate sampling artifact was responsible for the discrepancy. None of the 7 samples classified as uncertain by flow cytometry had morphologic evidence of disease; five out of 7 uncertain classifications were in B-ALL patients with hematogone immunophenotypes. Overall, MRD flow cytometry showed 86% concordance with the results of morphologic assessment. We evaluated outcomes in all patients with negative morphologic results and any positive MRD flow cytometry result(s). Of the 73 patients in this study, 61 had morphology-negative results that were either MRD-negative (n=45) or MRD-positive (n=16). Patients in this group were at various points of treatment post-induction or re-induction. Four out of 45 patients (9%) with MRD-negative results relapsed during a median follow-up period of 22 months, and 8 out of 16 patients (50%) with an MRD-positive result relapsed during a median follow-up period of 15 months (odds ratio for relapse 10.3, 95% confidence interval 2.5-42.4, P=0.001). In addition, relapse-related and overall mortality (Figure 1) were higher in patients with MRD-positive results (P=0.0023 and P=0.0016, respectively, by the log-rank test). In summary, we present a simplified, single-tube, flow cytometry assay that can be used to detect MRD in adult ALL at relatively low cost with rapid turnaround time; our approach was applicable to cases with either hematogone or aberrant immunophenotype, yielding a definitive result in 95% of cases. Notably, the presence of MRD was associated with relapse and mortality, suggesting that our method of MRD assessment could be used to guide treatment of adult ALL. Further analysis of the correlations between MRD results, clinical management and patient outcomes is ongoing. Disclosures: No relevant conflicts of interest to declare.

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Heterogeneity Of CXCR4 Expression In Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v122.21.4952.4952 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4952-4952

Author(s):

Tomasz Szczepanski ◽

Urszula Malek ◽

Lukasz Sedek ◽

Alicja Sonsala ◽

Joanna Zawitkowska ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Acute Lymphoblastic Leukemia ◽

Residual Disease ◽

Risk Group ◽

Lymphoblastic Leukemia ◽

Cxcr4 Expression ◽

Study Group ◽

Cell Precursor ◽

Minimal Residual

Abstract Background CXCR4 (CD184) is a receptor specific to the Stromal Derived Factor 1 (SDF-1), a ligand also known as CXCL12. The ligand-receptor interaction has a pleiotropic effect on hematopoietic cell proliferation, migration and activation through several signaling pathways. CXCR4 expression on neoplastic cells might be responsible for their dissemination to particular organs with cells expressing CXCL12 (e.g. lymph nodes, bones, and within bone marrow). In B-cell precursor acute lymphoblastic leukemia (BCP-ALL), expression of CXCR4 was associated with higher capacity of leukemic blasts to seed into bone marrow niches. Aim of the study The study aimed at thorough analysis of CXCR4 expression on BCP-ALL blasts and correlation of CXCR4 expression with the expression of other antigens such as CD66c, CD34, CD10, CD38, CD20 and CD45 as well as with the levels of minimal residual disease on day 15. Patients and Methods The study group consisted of 198 consecutive children aged 0-18 years (median 4.4 years) treated for BCP-ALL in the centers of the Polish Pediatric Leukemia/Lymphoma Study Group. Bone marrow samples obtained at initial diagnosis were stained with monoclonal antibodies (CD58, CD66c, CD34, CD19, CD10, CD38, CD20, CD45, CXCR4) in two 8-color tubes and analyzed with multiparameter flow cytometry (BD FACSCanto II, Becton Dickinson, San Jose, CA, USA) according to the EuroFlow standard protocols. The expression of particular antigens on BCP-ALL blasts was defined by median fluorescence. In 177 patients the samples from day 15 were available and analyzed for the presence of minimal residual disease (MRD) with multicolor flow cytometry. Infinicyt software (Cytognos, Salamanca, Spain) was used for more detailed analyses of the flow cytometric data. Results The expression of CXCR4 in BCP-ALL was highly variable with median fluorescence ranging from 252 to 24 388 (median 4011). There was no obvious correlation of CXCR4 expression with immunophenotype and with the expression of other analyzed markers (CD66, CD34, CD10, CD38 i CD45). The only borderline significant correlation found was between CXCR4 and CD20 expression. On day 15, 70 children (39%) demonstrated MRD levels below 0.1%, which is consistent with MRD-based low-risk group. Among these patients, 41 children had undetectable MRD already at this time point. In contrary, MRD levels > 10% were recorded in 21 patients (12%), who were stratified to high-risk group, accordingly. Maximal MRD levels recorded at day 15 were 85.6%. In remaining 86 children (49%), MRD levels at day 15 were in-between 0.1% and 10%, which reflects intermediate response to the treatment. There was no correlation between CXCR4 expression and MRD levels at day 15. Conclusion CXCR4 expression on BCP-ALL blasts is highly heterogeneous and is not associated with particular leukemia immunophenotype. Further analyses should characterize clinical features of leukemia and treatment response with regard to CXCR4 expression. The study was supported by Polish National Center of Science grant N N407 687040. Disclosures: No relevant conflicts of interest to declare.

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Heterogeneity Of CXCR4 Expression In Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v122.21.4652.4652 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4652-4652

Author(s):

Tomasz Szczepanski ◽

Urszula Malek ◽

Lukasz Sedek ◽

Alicja Sonsala ◽

Joanna Zawitkowska ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Acute Lymphoblastic Leukemia ◽

Residual Disease ◽

Risk Group ◽

Lymphoblastic Leukemia ◽

Cxcr4 Expression ◽

Study Group ◽

Cell Precursor ◽

Minimal Residual

Background CXCR4 (CD184) is a receptor specific to the Stromal Derived Factor 1 (SDF-1), a ligand also known as CXCL12. The ligand-receptor interaction has a pleiotropic effect on hematopoietic cell proliferation, migration and activation through several signaling pathways. CXCR4 expression on neoplastic cells might be responsible for their dissemination to particular organs with cells expressing CXCL12 (e.g. lymph nodes, bones, and within bone marrow). In B-cell precursor acute lymphoblastic leukemia (BCP-ALL), expression of CXCR4 was associated with higher capacity of leukemic blasts to seed into bone marrow niches. Aim of the study The study aimed at thorough analysis of CXCR4 expression on BCP-ALL blasts and correlation of CXCR4 expression with the expression of other antigens such as CD66c, CD34, CD10, CD38, CD20 and CD45 as well as with the levels of minimal residual disease on day 15. Patients and methods The study group consisted of 198 consecutive children aged 0-18 years (median 4.4 years) treated for BCP-ALL in the centers of the Polish Pediatric Leukemia/Lymphoma Study Group. Bone marrow samples obtained at initial diagnosis were stained with monoclonal antibodies (CD58, CD66c, CD34, CD19, CD10, CD38, CD20, CD45, CXCR4) in two 8-color tubes and analyzed with multiparameter flow cytometry (BD FACS Canto II, Becton Dickinson, San Jose, CA, USA) according to the EuroFlow standard protocols. The expression of particular antigens on BCP-ALL blasts was defined by median fluorescence. In 177 patients the samples from day 15 were available and analyzed for the presence of minimal residual disease (MRD) with multicolor flow cytometry. Infinicyt software (Cytognos, Salamanca, Spain) was used for more detailed analyses of the flow cytometric data. Results The expression of CXCR4 in BCP-ALL was highly variable with median fluorescence ranging from 252 to 24 388 (median 4011). There was no obvious correlation of CXCR4 expression with immunophenotype and with the expression of other analyzed markers (CD66, CD34, CD10, CD38 i CD45). The only borderline significant correlation found was between CXCR4 and CD20 expression. On day 15, 70 children (39%) demonstrated MRD levels below 0.1%, which is consistent with MRD-based low-risk group. Among these patients, 41 children had undetectable MRD already at this time point. In contrary, MRD levels > 10% were recorded in 21 patients (12%), who were stratified to high-risk group, accordingly. Maximal MRD levels recorded at day 15 were 85.6%. In remaining 86 children (49%), MRD levels at day 15 were in-between 0.1% and 10%, which reflects intermediate response to the treatment. There was no correlation between CXCR4 expression and MRD levels at day 15. Conclusion CXCR4 expression on BCP-ALL blasts is highly heterogeneous and is not associated with particular leukemia immunophenotype. Further analyses should characterize clinical features of leukemia and treatment response with regard to CXCR4 expression. The study was supported by Polish National Center of Science grant N N407 687040. Disclosures: No relevant conflicts of interest to declare.

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