Dilip Kumar Bhattacharya

Identification of 9-O acetyl sialoglycoconjugates (9-OAcSGs) as biomarkers in childhood acute lymphoblastic leukemia using a lectin, AchatininH, as a probe.

Neoplastic transformation causes changes in cell surface architecture, most notably, aberrant sialylation. Exploiting the restricted specificity of a 9-O acetyl sialic acid (9-OAcSA) binding lectin, AchatininH (ATNH), we have identified two 9-O acetyl sialoglyconjugates (9-OAcSGs) on lymphoblasts of 87 children suffering from acute lymphoblastic leukemia (ALL). The preferential binding of ATNH to lymphoblasts induces their 11-fold increased agglutination (81 ± 7.8%) compared to peripheral blood mononuclear cells (PBMC) of normal donors (8 ± 4.3%) which corroborates with flow cytometry studies. Agglutination of MOLT-4 (87 ± 4.8%), a lymphoblastoid cell line and MDCK (91.25 ± 0.01%), a cell line expressing surface 9-OAcSA, confirms the preferential binding of ATNH to lymphoblasts through their surface 9-OAcSGs. Furthermore, fluorometric quantitation reveals a 4.6-fold increase in % of 9-OAcSA on lymphoblasts of ALL patients (42.1 ± 4.1%) compared to normal donors (9.2. ± 3.4%). Western blotting confirms that ATNH recognizes two membrane sialoglycoconjugates, of MW 120 kDa and 90 kDa, both having 9-OAcSA α2 → 6 GalNAc terminal sugar moiety as their lectinogenic epitope. We propose that these 9-OAcSGs may serve as biomarkers for detection and monitoring of lymphoblasts in ALL and accordingly merit therapeutic considerations.

A novel method for prognostic evaluation of childhood acute lymphoblastic leukemia.

Although childhood acute lymphoblastic leukemia (ALL) is highly responsive to chemotherapy, patients in remission may harbor residual leukemic blasts, the cause of disease persistence and resurgence.1 Improved assays are therefore needed to evaluate individual chemotherapeutic response and predict impending relapse. Employing a 9-O acetyl sialic acid binding lectin, ATNH, we have identified two 9-O acetylated sialoglycoconjugates (9-OAcSGs), as novel biomarkers on leukemic blasts of newly diagnosed/untreated ALL patients2 of both B and/or T lineages and assessed their differential expression at different phases of therapy.3 Presently, we describe a noninvasive, blood-based lymphoproliferation assay to evaluate the clinical status of B lineage ALL patients (HLA-DR+,CD19+, n = 16) followed up longitudinally for 3 years. ALL patients, who received treatment as per the UKALLVIII protocol, were bled at different phases of therapy. Peripheral blood mononuclear cells were isolated and cultured with ATNH (0.05–12 mg) for 96 h followed by an 18 h pulse with 3H-TdR. Radioactivity incorporated, in individual samples, was plotted against ATNH dose and the dose corresponding to maximal incorporation of radioactivity was designated as its ‘maximal lymphoproliferative dose’ (MLD). The MLD was employed to evaluate the percentage recovery of individual patients.

A colorimetric assay to evaluate the chemotherapeutic response of children with acute lymphoblastic leukemia (ALL) employing AchatininH: a 9-O-acetyl sialic acid binding lectin

Employing a 9-O-acetyl sialic acid binding lectin, Achatinin(H) (ATNH), we have reported a non-invasive, blood based lymphoproliferation assay which measures the maximal lymphoproliferative dose (MLD) of ATN(H) to assess the status of 9-O-acetylated sialoglycoconjugates (9-OAcSGs) in patients with Acute lymphoblastic leukemia (ALL) (Mandal C, Sinha D, Sharma V, Bhattacharya DK. O-acetyl sialic acid binding lectin, as a probe for detection of subtle changes on the cell surface induced during acute lymphoblastic leukemia [ALL] and its clinical application. Ind J Biochem Biophys 1997;34:82; Sinha D, Mandal C, Bhattacharya DK. Development of a simple blood based lymphoproliferation assay to assess the clinical status of patients with acute lymphoblastic leukemia. Leuk Res 1999;13:309-312; Sinha D, Mandal C, Bhattacharya DK. A novel method for prognostic evaluation of childhood acute lymphoblastic leukemia. Leukemia 1999;13[in press]). Although the expression of 9-OAcSGs clearly serves as an index of treatment outcome, the assay has limitations in that it requires radioisotopes, i.e. [3H]-TdR. Therefore a colorimetric assay was developed as an alternative approach. The pre-treatment MLD, as measured by the colorimetric assay, was 0.15 +/- 0.02 microg which progressively increased during consolidation therapy (1.40 +/- 0.39 microg), maintenance therapy (4.20 +/- 1.60 microg) and in followed-up cases (5.20 +/- 0.43 microg) but sharply declined following relapse (0.25 +/- 0.02 microg). The colorimetric assay also showed a good correlation with radiometric assay (r = + 0.93) and their mean coefficient of inter-assay precision were also comparable (15.53% versus 14.86%). We therefore propose that the colorimetric assay is a safe, non-radiometric, user-friendly alternative for assessing individual chemotherapeutic responses in childhood ALL.

O-acetylation of sialic acids is required for the survival of lymphoblasts in childhood acute lymphoblastic leukemia (ALL)

Exploiting the selective affinity of Achatinin-H towards 9-O-acetylneuraminic acid(α2-6)GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs) on hematopoietic cells of children suffering from acute lymphoblastic leukemia (ALL), indicative of defective sialylation associated with this disease. The carbohydrate epitope of Neu5,9Ac2-GPsALL was confirmed by using several synthetic sialic acid analogues. They are functionally active signaling molecules as demonstrated by their role in mediating lymphoproliferative responses and consequential increased production of IFN-γ due to specific stimulation of Neu5,9Ac2-GPs on PBMCALL with Achatinin-H. Cells devoid of 9-O-acetylations (9-O-AcSA−) revealed decreased nitric oxide production as compared to 9-O-AcSA+ cells on exposure to IFN-γ. Under this condition, a decrease in viability of 9-O-AcSA− cells as compared to 9-O-AcSA+ cells was also observed which was reflected from increased caspase 3 activity and apoptosis suggesting the protective role of this glycotope. These Neu5,9Ac2-GPs are also capable of inducing disease-specific anti-Neu5,9Ac2-GPs antibodies in ALL children. Additionally, we have observed that disease-specific anti-Neu5,9Ac2-GPs have altered glycosylation profile, and they are incapable of exerting a few Fc-glycosylation-sensitive effector functions. These observations hint toward a disbalanced homeostasis, thereby enabling the cancer cells to escape host defense. Taken together, it may be hypothesized that Neu5,9Ac2-GPs and their antibodies play a prominent role in promoting the survival of lymphoblasts in ALL.

Interferon gamma promotes survival of lymphoblasts overexpressing 9‐O‐acetylated sialoglycoconjugates in childhood acute lymphoblastic leukaemia (ALL)

An enhanced linkage‐specific 9‐O‐acetylated sialic acid (9‐O‐AcSA) on peripheral blood mononuclear cells (PBMC) of children with acute lymphoblastic leukaemia, ALL (PBMCALL, 9‐O‐AcSA+ cells) was demonstrated by using a lectin, Achatinin‐H, whose lectinogenic epitope was 9‐O‐AcSAα2‐6GalNAc. Our aim was to evaluate the in vitro contributory role of this glycotope (9‐O‐AcSAα2‐6GalNAc) towards the survival of these 9‐O‐AcSA+ cells in ALL patients. For direct comparison, 9‐O‐AcSA− cells were generated by removing O‐acetyl group of 9‐O‐AcSA present on PBMCALL using O‐acetyl esterase. An elevated level of serum interferon gamma (IFN‐γ) in affected children led us to think that PBMCALL are continuously exposed specifically to this cytokine. Accordingly, 9‐O‐AcSA+ and 9‐O‐AcSA− cells were exposed in vitro to IFN‐γ. A twofold increased NO release along with inducible NO synthase (iNOS) mRNA expression by the 9‐O‐AcSA+ cells was observed as compared to the 9‐O‐AcSA− cells. The decreased viability of IFN‐γ exposed 9‐O‐AcSA− cells as compared to 9‐O‐AcSA+ cells were reflected from a 5.0‐fold increased caspase‐3‐like activity and a 10.0‐fold increased apoptosis in the 9‐O‐AcSA− cells when production of NO was lowered by adding competitive inhibitor of iNOS in reaction mixture. Therefore, it may be envisaged that a link exists between induction of this glycotope and their role in regulating viability of PBMCALL. Taken together, it is reasonable to hypothesise that O‐acetylation of sialic acids on PBMCALL may be an additional mechanism that promotes the survival of lymphoblasts by avoiding apoptosis via IFN‐γ‐induced NO production.

Increased interferon gamma production by peripheral blood mononuclear cells in response to stimulation of overexpressed disease-specific 9-O-acetylated sialoglycoconjugates in children suffering from acute lymphoblastic leukaemia

Disease‐specific over‐expression of 9‐O‐acetylated sialoglycoconjugates (9‐O‐AcSGs) on peripheral blood mononuclear cells (PBMC) of children with acute lymphoblastic leukaemia (ALL, PBMCALL) has been demonstrated using a lectin, Achatinin‐H, with specificity towards 9‐O‐AcSAα2‐6GalNAc. This study investigated the contributory role of 9‐O‐AcSGs induced on PBMCALL. Stimulation of PBMCALL with Achatinin‐H through 9‐O‐AcSGs led to a lymphoproliferative response with a significantly increased interferon‐γ (IFN‐γ) production when compared with unstimulated cells as demonstrated by enzyme‐linked immunosorbent assay and mRNA expression. Under identical conditions, PBMCALL ablated of O‐acetylations did not respond to such stimulation. In summary, it may be concluded that stimulation of over‐expressed 9‐O‐AcSGs regulate signalling for proliferation, leading to the release of IFN‐γ. Controlled expression of these molecules may be exploited as potential targets for therapy, promising beneficial effects to children with ALL.

O-acetyl sialic acid specific IgM in childhood acute lymphoblastic leukaemia

Initial studies have revealed an enhanced surface expression of O-acetylated sialoglycoconjugates (O-AcSGs) on lymphoblasts concomitant with high titres of IgG in childhood Acute Lymphoblastic Leukaemia (ALL) (Mandal C, Chatterjee M, Sinha D, Br J Haematol 110, 801–12, 2000). In our efforts to identify disease specific markers for ALL, we have affinity-purified IgM directed against O-AcSGs that reacts with three disease specific O-AcSGs present on membrane proteins derived from peripheral blood mononuclear cells (PBMC) of ALL patients. Antibody specificity towards O-AcSGs was confirmed by selective binding to erythrocytes bearing surface O-AcSGs, decreased binding with de-O-acetylated BSM and following pretreatment with O-acetyl esterase. Competitive inhibition ELISA demonstrated a higher avidity of IgM for O-AcSG than IgG. Flow cytometry demonstrated the diagnostic potential of purified O-AcSA IgM as binding was specific with ALL patients and minimal with other haematological disorders and normal individuals. It therefore may be adopted as a non-invasive approach for detection of childhood ALL. Taken together, the data indicates that carbohydrate epitopes having terminal O-AcSA α2 → 6 GalNAc determinants induce disease specific IgG and IgM, potentially useful molecular markers for childhood ALL.

Development of a simple, blood based lymphoproliferation assay to assess the clinical status of patients with acute lymphoblastic leukemia

Although childhood acute lymphoblastic leukemia (ALL) is highly responsive to chemotherapy, reliable techniques are needed to determine treatment outcome and predict relapse. Employing a 9-O-acetyl sialic acid binding lectin, ATN(H), we have identified two 9-O-acetylated sialogycoconjugates (9-OAcSGs) as novel biomarkers expressed selectively on leukemic blasts of ALL patients. Presently, we report a non-invasive, blood based lymphoproliferation assay, which employs the maximal lymphoproliferative dose of ATN(H) (MLD) to assess the status of 9-OAcSGs with progressive therapy. A low MLD (0.18 +/- 0.01 microg) in untreated patients reflects increased expression of 9-OAcSGs which decline following therapy (MLD = 2.10 +/- 0.60 microg), persist during maintenance therapy (MLD = 4.50 +/- 1.60 microg)/follow-up (MLD = 5.50 +/- 0.85 microg) and are re-induced with relapse (MLD = 0.25 +/- 0.01 microg). Since the assay detects lymphoblasts with a sensitivity of 10(-4), shows no cross-reactivity with other hematological disorders (n = 48) and has been tested in 212 patients, it meets clinical consideration.