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Flow-cytometric monitoring of disease-associated expression of 9-O-acetylated sialoglycoproteins in combination with known CD antigens, as an index for MRD in children with acute lymphoblastic leukaemia: a two-year longitudinal follow-up study.

Chowdhury S, Bandyopadhyay S, Mandal C, Chandra S, Mandal C - BMC Cancer (2008)

Bottom Line: Over expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs, abbreviated as OAcSGP) has been demonstrated as a disease-associated antigen on the lymphoblasts of childhood acute lymphoblastic leukaemia (ALL).Hence, MRD study beyond two-years follow-up is necessary to investigate further reduction in MRD, thereby ensuring their disease-free survival.Therefore, we suggest use of these templates for MRD detection, during and post-chemotherapy for proper patient management strategies, thereby helping in personalizing the treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Immunobiology Division, Indian Institute of Chemical Biology, 4, Raja S, C, Mullick Road, Kolkata 700032, Kothari Medical Centre 8/3, Alipore Road, Kolkata 700027, India. suchandra_chowdhury@rediffmail.com

ABSTRACT

Background: Over expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs, abbreviated as OAcSGP) has been demonstrated as a disease-associated antigen on the lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). Achatinin-H, a lectin, has selective affinity towards terminal 9-O-acetylated sialic acids-alpha2-6-Nacetylated galactosamine. Exploring this affinity, enhanced expression of OAcSGP was observed, at the onset of disease, followed by its decrease with chemotherapy and reappearance with relapse. In spite of treatment, patients retain the diseased cells referred to as minimal residual disease (MRD) responsible for relapse. Our aim was to select a suitable template by using the differential expression of OAcSGP along with other known CD antigens to monitor MRD in peripheral blood (PB) and bone marrow (BM) of Indian patients with B- or T-ALL during treatment and correlate it with the disease status.

Methods: A two-year longitudinal follow-up study was done with 109 patients from the onset of the disease till the end of chemotherapy, treated under MCP841protocol. Paired samples of PB (n = 1667) and BM (n = 999) were monitored by flow cytometry. Three templates selected for this investigation were OAcSGP+CD10+CD19+ or OAcSGP+CD34+CD19+ for B-ALL and OAcSGP+CD7+CD3+ for T-ALL.

Results: Using each template the level of MRD detection reached 0.01% for a patient in clinical remission (CR). 81.65% of the patients were in CR during these two years while the remaining relapsed. Failure in early clearance of lymphoblasts, as indicated by higher MRD, implied an elevated risk of relapse. Soaring MRD during the chemotherapeutic regimen predicted clinical relapse, at least a month before medical manifestation. Irrespective of B- or T-lineage ALL, the MRD in PB and BM correlated well.

Conclusion: A range of MRD values can be predicted for the patients in CR, irrespective of their lineage, being 0.03 +/- 0.01% (PB) and 0.05 +/- 0.015% (BM). These patients may not be stated as normal with respect to the presence of MRD. Hence, MRD study beyond two-years follow-up is necessary to investigate further reduction in MRD, thereby ensuring their disease-free survival. Therefore, we suggest use of these templates for MRD detection, during and post-chemotherapy for proper patient management strategies, thereby helping in personalizing the treatment.

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Quantification of leukemic blasts by flow cytometry using admix assay. a. Dilutions of diagnostic samples admixed to PB or BM samples, from various time-points of follow-up during chemotherapy for lineage-unrelated leukaemia (e.g. B-lineage blasts into T-ALL follow-up BM and vice-versa) and analysed for MRD in R3 region using the same template as described in Materials and Methods. Correlation of the data was examined by regression analysis; line of identity is shown by (-). Diagnostic PB from B-ALL mixed with T-ALL follow-up (52 weeks of treatment, black rhombus); diagnostic BM from B-ALL mixed with T-ALL follow-up (52 weeks of treatment, black triangle); diagnostic BM from T-ALL mixed with B-ALL follow-up (90 weeks of treatment, O); diagnostic BM from T-ALL mixed with B-ALL follow-up (104 weeks of treatment, black square) b. Dilutions of the REH (B-ALL, white triangle) and MOLT-4 (T-ALL, white rhombus) cell lines were made with normal PBMC and analysed as above.
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Figure 7: Quantification of leukemic blasts by flow cytometry using admix assay. a. Dilutions of diagnostic samples admixed to PB or BM samples, from various time-points of follow-up during chemotherapy for lineage-unrelated leukaemia (e.g. B-lineage blasts into T-ALL follow-up BM and vice-versa) and analysed for MRD in R3 region using the same template as described in Materials and Methods. Correlation of the data was examined by regression analysis; line of identity is shown by (-). Diagnostic PB from B-ALL mixed with T-ALL follow-up (52 weeks of treatment, black rhombus); diagnostic BM from B-ALL mixed with T-ALL follow-up (52 weeks of treatment, black triangle); diagnostic BM from T-ALL mixed with B-ALL follow-up (90 weeks of treatment, O); diagnostic BM from T-ALL mixed with B-ALL follow-up (104 weeks of treatment, black square) b. Dilutions of the REH (B-ALL, white triangle) and MOLT-4 (T-ALL, white rhombus) cell lines were made with normal PBMC and analysed as above.

Mentions: Initially to simulate the in vivo scenario, MRD was assessed using leukemic blasts from diagnostic samples admixed to PB or BM samples, as the case may be, from various time-points of follow-up during chemotherapy for lineage-unrelated leukaemia (Figure 7a). One OAcSGP+CD10+CD19+, OAcSGP+CD34+CD19+ or OAcSGP+CD7+CD3+ cell could be detected in a total population of 10,000 lymphocytes. The estimate of leukemic cell content in each mixture was extremely accurate (r2 = 0.96). Similarly, the assay was repeated using a heterogeneous cell population of PBMCN with ALL cell lines. In this mixed population, similar detection of MRD was possible for OAcSGP+CD10+CD19+ or OAcSGP+CD7+CD3+ population (r2 = 0.99, Figure 7b).


Flow-cytometric monitoring of disease-associated expression of 9-O-acetylated sialoglycoproteins in combination with known CD antigens, as an index for MRD in children with acute lymphoblastic leukaemia: a two-year longitudinal follow-up study.

Chowdhury S, Bandyopadhyay S, Mandal C, Chandra S, Mandal C - BMC Cancer (2008)

Quantification of leukemic blasts by flow cytometry using admix assay. a. Dilutions of diagnostic samples admixed to PB or BM samples, from various time-points of follow-up during chemotherapy for lineage-unrelated leukaemia (e.g. B-lineage blasts into T-ALL follow-up BM and vice-versa) and analysed for MRD in R3 region using the same template as described in Materials and Methods. Correlation of the data was examined by regression analysis; line of identity is shown by (-). Diagnostic PB from B-ALL mixed with T-ALL follow-up (52 weeks of treatment, black rhombus); diagnostic BM from B-ALL mixed with T-ALL follow-up (52 weeks of treatment, black triangle); diagnostic BM from T-ALL mixed with B-ALL follow-up (90 weeks of treatment, O); diagnostic BM from T-ALL mixed with B-ALL follow-up (104 weeks of treatment, black square) b. Dilutions of the REH (B-ALL, white triangle) and MOLT-4 (T-ALL, white rhombus) cell lines were made with normal PBMC and analysed as above.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2268943&req=5

Figure 7: Quantification of leukemic blasts by flow cytometry using admix assay. a. Dilutions of diagnostic samples admixed to PB or BM samples, from various time-points of follow-up during chemotherapy for lineage-unrelated leukaemia (e.g. B-lineage blasts into T-ALL follow-up BM and vice-versa) and analysed for MRD in R3 region using the same template as described in Materials and Methods. Correlation of the data was examined by regression analysis; line of identity is shown by (-). Diagnostic PB from B-ALL mixed with T-ALL follow-up (52 weeks of treatment, black rhombus); diagnostic BM from B-ALL mixed with T-ALL follow-up (52 weeks of treatment, black triangle); diagnostic BM from T-ALL mixed with B-ALL follow-up (90 weeks of treatment, O); diagnostic BM from T-ALL mixed with B-ALL follow-up (104 weeks of treatment, black square) b. Dilutions of the REH (B-ALL, white triangle) and MOLT-4 (T-ALL, white rhombus) cell lines were made with normal PBMC and analysed as above.
Mentions: Initially to simulate the in vivo scenario, MRD was assessed using leukemic blasts from diagnostic samples admixed to PB or BM samples, as the case may be, from various time-points of follow-up during chemotherapy for lineage-unrelated leukaemia (Figure 7a). One OAcSGP+CD10+CD19+, OAcSGP+CD34+CD19+ or OAcSGP+CD7+CD3+ cell could be detected in a total population of 10,000 lymphocytes. The estimate of leukemic cell content in each mixture was extremely accurate (r2 = 0.96). Similarly, the assay was repeated using a heterogeneous cell population of PBMCN with ALL cell lines. In this mixed population, similar detection of MRD was possible for OAcSGP+CD10+CD19+ or OAcSGP+CD7+CD3+ population (r2 = 0.99, Figure 7b).

Bottom Line: Over expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs, abbreviated as OAcSGP) has been demonstrated as a disease-associated antigen on the lymphoblasts of childhood acute lymphoblastic leukaemia (ALL).Hence, MRD study beyond two-years follow-up is necessary to investigate further reduction in MRD, thereby ensuring their disease-free survival.Therefore, we suggest use of these templates for MRD detection, during and post-chemotherapy for proper patient management strategies, thereby helping in personalizing the treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Immunobiology Division, Indian Institute of Chemical Biology, 4, Raja S, C, Mullick Road, Kolkata 700032, Kothari Medical Centre 8/3, Alipore Road, Kolkata 700027, India. suchandra_chowdhury@rediffmail.com

ABSTRACT

Background: Over expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs, abbreviated as OAcSGP) has been demonstrated as a disease-associated antigen on the lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). Achatinin-H, a lectin, has selective affinity towards terminal 9-O-acetylated sialic acids-alpha2-6-Nacetylated galactosamine. Exploring this affinity, enhanced expression of OAcSGP was observed, at the onset of disease, followed by its decrease with chemotherapy and reappearance with relapse. In spite of treatment, patients retain the diseased cells referred to as minimal residual disease (MRD) responsible for relapse. Our aim was to select a suitable template by using the differential expression of OAcSGP along with other known CD antigens to monitor MRD in peripheral blood (PB) and bone marrow (BM) of Indian patients with B- or T-ALL during treatment and correlate it with the disease status.

Methods: A two-year longitudinal follow-up study was done with 109 patients from the onset of the disease till the end of chemotherapy, treated under MCP841protocol. Paired samples of PB (n = 1667) and BM (n = 999) were monitored by flow cytometry. Three templates selected for this investigation were OAcSGP+CD10+CD19+ or OAcSGP+CD34+CD19+ for B-ALL and OAcSGP+CD7+CD3+ for T-ALL.

Results: Using each template the level of MRD detection reached 0.01% for a patient in clinical remission (CR). 81.65% of the patients were in CR during these two years while the remaining relapsed. Failure in early clearance of lymphoblasts, as indicated by higher MRD, implied an elevated risk of relapse. Soaring MRD during the chemotherapeutic regimen predicted clinical relapse, at least a month before medical manifestation. Irrespective of B- or T-lineage ALL, the MRD in PB and BM correlated well.

Conclusion: A range of MRD values can be predicted for the patients in CR, irrespective of their lineage, being 0.03 +/- 0.01% (PB) and 0.05 +/- 0.015% (BM). These patients may not be stated as normal with respect to the presence of MRD. Hence, MRD study beyond two-years follow-up is necessary to investigate further reduction in MRD, thereby ensuring their disease-free survival. Therefore, we suggest use of these templates for MRD detection, during and post-chemotherapy for proper patient management strategies, thereby helping in personalizing the treatment.

Show MeSH
Related in: MedlinePlus