Detection of minimal residual disease in B-lineage acute lymphoblastic leukaemia by quantitative flow cytometry

The clinical significance of detecting minimal residual disease (MRD) in B-lineage acute lymphoblastic leukaemia (ALL) was evaluated by quantitative flow cytometry using a combination of TdT with CD10 and CD19. 53 patients with B-cell precursor ALL were followed during and after completion of treatment (median follow-up 23 months). Nine patients relapsed and MRD had been detected in six of them, 5–15 weeks before relapse despite morphological complete remission. 43 patients remain in clinical remission and in none of these was MRD detected. Disease-free survival based on the detection of MRD by flow cytometry showed a statistically significant difference between both groups ( P  < 0.0001). The absence of MRD correlates with a low relapse rate, whereas the presence of MRD predicted early relapse. This study has shown that flow cytometry can improve the morphologic assessment of bone marrow (BM) remission status in B-lineage ALL. The finding of < 5% blasts in BM aspirates did not correlate with 'true' remission in a proportion of cases as residual leukaemic blasts were detected by flow cytometry in nine samples from six patients. On the other hand, the presence of > 5% blasts assessed by morphology was not necessarily a feature of relapse in five patients as these cells were shown to have a phenotype identical to normal TdT-negative B-cell precursors. Quantitative flow cytometry was more informative than conventional morphology to assess remission status and showed a strong correlation with clinical outcome. This methodology is useful to define MRD in the majority of patients with B-lineage ALL and should be tested in prospective clinical trials.     

CD22ΔE12 as a molecular target for RNAi therapy

B‐precursor acute lymphoblastic leukaemia (BPL) is the most common form of cancer in children and adolescents. Our recent studies have demonstrated that CD22ΔE12 is a characteristic genetic defect of therapy‐refractory clones in paediatric BPL and implicated the CD22ΔE12 genetic defect in the aggressive biology of relapsed or therapy‐refractory paediatric BPL. The purpose of the present study is to evaluate the biological significance of the CD22ΔE12 molecular lesion in BPL and determine if it could serve as a molecular target for RNA interference (RNAi) therapy. Here we report a previously unrecognized causal link between CD22ΔE12 and aggressive biology of human BPL cells by demonstrating that siRNA‐mediated knockdown of CD22ΔE12 in primary leukaemic B‐cell precursors is associated with a marked inhibition of their clonogenicity. Additionally, we report a nanoscale liposomal formulation of CD22ΔE12‐specific siRNA with potent in vitro and in vivo anti‐leukaemic activity against primary human BPL cells as a first‐in‐class RNAi therapeutic candidate targeting CD22ΔE12.     

Quantitative flow cytometry can distinguish between normal and leukaemic B-cell precursors

Summary. The immunological detection of minimal residual disease in B-lineage acute lymphoblastic leukaemia (ALL) has been hampered by the fact that the leukaemic cells represent the malignant counterparts of normal haemo-poietic precursors expressing terminal deoxynucleotidyl transferase (TdT), CD10 and CD19. We have used quantitative double-labelling flow cytometry with standard fluorescent beads to convert the mean fluorescence to the number of antigen molecules per cell. The number of TdT, CD10 and CD19 molecules per cell was determined in normal B-cell precursors from 22 healthy donors and eight regenerating marrows from patients with various malignancies and in 20 cases of B-lineage ALL. In normal bone marrow we characterized two different B-cell populations: TdT⁺/CD10⁺/ CD19⁺ and TdT/CD10⁺/CD19⁺. We demonstrated a major difference in the level of expression of TdT, CD 10 and CD 19 between normal bone marrow and B-lineage ALL blasts. Normal TdT⁺ precursors have significantly higher number of TdT (>100×l0³) and lower number of CD10 (<50×l0³) and CD19 (<10×10³) molecules per cell than B-lineage ALL blasts (<100, >50, >10×l0³ molecules per cell respectively); these differences were statistically highly significant. Furthermore, regenerating marrows had a significantly higher percentage of B-cell precursors than healthy donors. This increase was at the expense of the TdT^?/ CD10⁺/CD19⁺ population which, in the context of B-lineage ALL, could be wrongly interpreted as evidence of relapse if TdT is not included in the analysis. Therefore the quantitative analysis of TdT combined with CD10 and CD19 may allow a clear distinction between normal precursors and minimal residual leukaemia in B-lineage ALL and avoid the pitfall of misinterpreting regenerating B-cells as evidence of relapse.     

Leukaemia-associated immunophenotypes (LAIP) are observed in 90% of adult and childhood acute lymphoblastic leukaemia: detection in remission marrow predicts outcome

Analysis of differentiation antigens on leukaemic blasts is routinely done for diagnostic purposes, i.e. determination of stage of differentiation and lineage assignment. Acute lymphoblastic leukaemias are also frequently characterized by a leukaemia-associated immunophenotype (LAIP), either the coexpression of differentiation antigens physiologically restricted to other stages of differentiation (asynchronous LAIP) or cell lineages (aberrant LAIP). We defined LAIP in 241 consecutive unselected B-lineage (n = 193) and T-lineage (n = 48) ALL by three-colour flow cytometry using directly conjugated monoclonal antibodies. The incidence of LAIP was found to be 91.7%. In 63% of patients two to six leukaemia-associated expression patterns were detected. In order to study the specificity of LAIP in a therapy-relevant setting, remission bone marrow samples from patients with B-lineage ALL were analysed for the expression of T-lineage-associated phenotypes on the normal bone marrow cells and vice versa. The frequency of all T-lineage LAIP+ cells and all aberrant B-lineage LAIP+ cells was <1% in regenerating bone marrow samples at different timepoints. The incidence and clinical significance of LAIP+ cells was studied in 196 remission marrows of 70 ALL patients (55 remaining in CCR, 14 with bone marrow relapse, one with isolated CNS relapse). The presence of >1% LAIP+ at two consecutive timepoints predicted 5/8 bone marrow relapses in B-lineage ALL. The occurrence of LAIP+ cells >1% in T-lineage ALL after induction therapy predicted relapse in 7/7 cases. In conclusion, flow cytometric detection of LAIP+ cells appears to be a powerful tool for the prediction of outcome in ALL.     

Growth factor receptor profile of CD34+ cells in AML and B-lineage ALL and in their normal bone marrow counterparts

Leukaemic cells show a low clonogenic activity and a heterogeneous proliferative response to growth factors. We investigated whether this could be due to an altered expression of growth factor receptors on the leukaemic precursors. Receptors for G-CSF, stem cell factor (SCF), IL-3, IL-6 and IL-7 were detected on CD34+ cells in AML and B-lineage ALL with monoclonal antibodies and flow cytometry. The expression was compared with that on myeloid and B-lymphoid CD34+ cells in normal bone marrow. Leukaemic CD34+ cells expressed the same receptors as their normal counterparts. AML and B-lineage ALL could be distinguished by the growth factor receptor profile of their CD34+ cells. SCFR, G-CSFR and IL-6Ralpha were found in AML, IL-7R in B-lineage ALL and IL-3Ralpha in both. IL-3Ralpha was upregulated in AML and B-lineage ALL CD34+ cells, while samples with low or high expression were present for the other receptors. This variable expression could correlate with the heterogeneous response of leukaemic cells to growth factors. Functional studies on isolated CD34+ cells are needed to investigate this further.     

High expression of connective tissue growth factor in pre-B acute lymphoblastic leukaemia

In recent years microarrays have been used extensively to characterize gene expression in acute lymphoblastic leukaemia (ALL). Few studies, however, have analysed normal haematopoietic cell populations to identify altered gene expression in ALL. We used oligonucleotide microarrays to compare the gene expression profile of paediatric precursor-B (pre-B) ALL specimens with two control cell populations, normal CD34(+) and CD19(+)IgM(-) cells, to focus on genes linked to leukemogenesis. A set of eight genes was identified with a ninefold higher average expression in ALL specimens compared with control cells. All of these genes were significantly deregulated in an independent cohort of 101 ALL specimens. One gene, connective tissue growth factor (CTGF, also known as CCN2), had exceptionally high expression, which was confirmed in three independent leukaemia studies. Further analysis of CTGF expression in ALL revealed exclusive expression in B-lineage, not T-lineage, ALL. Within B-lineage ALL approximately 75% of specimens were consistently positive for CTGF expression, however, specimens containing the E2A-PBX1 translocation showed low or no expression. Protein studies using Western blot analysis demonstrated the presence of CTGF in ALL cell-conditioned media. These findings indicate that CTGF is secreted by pre-B ALL cells and may play a role in the pathophysiology of this disease.     

In vitro and in vivo radiation resistance associated with CD3 surface antigen expression in T-lineage acute lymphoblastic leukemia.

The radiobiologic features of primary clonogenic blasts (referred to also as T-lineage leukemic progenitor cells) from newly diagnosed and relapsed T-lineage acute lymphoblastic leukemia (ALL) patients were analyzed. Intrinsic radiation sensitivity differed substantially among primary clonogenic blasts from 34 newly diagnosed patients. The mean D0 (37% dose slope), SF2 (surviving fraction at 200 cGy), and alpha values (initial slope of the survival curve) were 141 +/- 15 cGy, 0.31 +/- 0.04, and 0.630 +/- 0.093 Gy-1, respectively. Among newly diagnosed cases, nine had SF2 values of greater than or equal to 0.50 and alpha values of less than or equal to 0.2 Gy-1, consistent with a marked intrinsic radiation resistance at the level of clonogenic blasts using the multitarget and linear quadratic models of cell survival. Of these nine radiation resistant cases, seven were CD3+. Furthermore, the mean D0 (162 +/- 20.8 cGy) and SF2 (0.377 +/- 0.057) values for the 20 CD3+ cases were significantly higher than the D0 (108.6 +/- 18.2 cGy) and SF2 (0.204 +/- 0.051) values for the 14 CD3- cases (P less than or equal to .05). Thus, clonogenic blasts from CD3+ newly diagnosed T-lineage ALL patients were more resistant to radiation than clonogenic blasts from CD3- newly diagnosed T-lineage ALL patients. Nineteen T-lineage ALL patients received autologous bone marrow transplants during complete remission. Pretransplant conditioning consisted of total body irradiation (TBI) combined with high-dose chemotherapy. Primary clonogenic blasts from patients who relapsed after bone marrow transplantation (BMT) displayed a particularly high degree of intrinsic radiation resistance with a mean D0 value of 333 cGy and an alpha value of 0.112 Gy-1. The expression of CD3 antigen appeared to predict the outcome of relapsed T-lineage ALL patients undergoing autologous BMT after TBI plus high-dose chemotherapy. The Kaplan-Meier estimates and standard errors of the probability of remaining in remission after BMT were 60% +/- 22% (mean relapse - free interval = 1.6 +/- 0.7 years) for CD3- patients and 0% +/- 0% (mean relapse - free interval = 0.2 +/- 0.0 years) for CD3+ patients (P = .002). Furthermore, the mean percentage of CD3-positive leukemic marrow blasts at presentation or relapse before BMT was significantly lower than the mean percentage of CD3-positive leukemic marrow blasts at relapse after BMT. Notably, in cultured leukemic bone marrow specimens from newly diagnosed as well as relapsed patients, colony blasts surviving in vitro radiation expressed CD3 more vividly than did colony blasts in unirradiated cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bortezomib reinduction chemotherapy in high-risk ALL in first relapse: a report from the Children's Oncology Group

While survival in paediatric acute lymphoblastic leukaemia (ALL) is excellent, survival following relapse is poor. Previous studies suggest proteasome inhibition with chemotherapy improves relapse ALL response rates. This phase 2 Children's Oncology Group study tested the hypothesis that adding the proteasome inhibitor bortezomib to chemotherapy increases complete response rates (CR2). Evaluable patients (n = 135, 103 B-ALL, 22 T-ALL, 10 T-lymphoblastic lymphoma) were treated with reinduction chemotherapy plus bortezomib. Overall CR2 rates were 68 ± 5% for precursor B-ALL patients (<21 years of age), 63 ± 7% for very early relapse (<18 months from diagnosis) and 72 ± 6% for early relapse (18–36 months from diagnosis). Relapsed T-ALL patients had an encouraging CR2 rate of 68 ± 10%. End of induction minimal residual disease (MRD) significantly predicted survival. MRD negative (MRDneg; MRD <0·01%) rates increased from 29% (post-cycle 1) to 64% following cycle 3. Very early relapse, end-of-induction MRDneg precursor B-ALL patients had 70 ± 14% 3-year event-free (EFS) and overall survival (OS) rates, vs. 3-year EFS/OS of 0–3% (P = 0·0001) for MRDpos (MRD ≥0·01) patients. Early relapse patients had similar outcomes (MRDneg 3-year EFS/OS 58–65% vs. MRDpos 10–19%, EFS P = 0·0014). These data suggest that adding bortezomib to chemotherapy in certain ALL subgroups, such as T-cell ALL, is worthy of further investigation. This study is registered at http://www.clinical.trials.gov as NCT00873093.     

Postrelapse survival in childhood acute lymphoblastic leukemia is independent of initial treatment intensity: a report from the Children's Oncology Group

While intensification of therapy has improved event-free survival (EFS) and survival in newly diagnosed children with acute lymphoblastic leukemia (ALL), postrelapse outcomes remain poor. It might be expected that patients relapsing after inferior initial therapy would have a higher retrieval rate than after superior therapy. In the Children's Oncology Group Study CCG-1961, significantly superior EFS and survival were achieved with an augmented (stronger) versus standard intensity regimen of postinduction intensification (PII) for children with newly diagnosed high-risk ALL and rapid day 7 marrow response (EFS/survival 81.2%/88.7% vs 71.7%/83.4%, respectively). This provided an opportunity to evaluate postrelapse survival (PRS) in 272 relapsed patients who had received randomly allocated initial treatment with augmented or standard intensity PII. As expected, PRS was worse for early versus late relapse, marrow versus extramedullary site, adolescent versus younger age and T versus B lineage. However, no difference in 3-year PRS was detected for having received augmented versus standard intensity PII (36.4% ± 5.7% vs 39.2% ± 4.1%; log rank P = .72). Similar findings were noted within subanalyses by timing and site of relapse, age, and immunophenotype. These findings provide insight into mechanisms of relapse in ALL, and are consistent with emergence of a resistant subclone that has acquired spontaneous mutations largely independent of initial therapy. This study is registered at www.clinicaltrials.gov as NCT00002812.     

Long-term outcome of initially homogenously treated and relapsed childhood acute lymphoblastic leukaemia in Austria – A population-based report of the Austrian Berlin-Frankfurt-Münster (BFM) Study Group

Relapsed acute lymphoblastic leukaemia (ALL) is the most common cause for a fatal outcome in paediatric oncology. Although initial ALL cure rates have improved up to 80%, the prognosis of recurrent ALL remains dismal with event-free-survival (EFS) rates about 35%. In order to analyse a population-based cohort with uniform treatment of initial disease, we examined the outcome of children suffering from relapsed ALL in Austria for the past 20 years and the validity of the currently used prognostic factors (e.g. time to and site of relapse, immunophenotype). Furthermore, we compared survival rates after chemotherapy alone with those after allogeneic stem cell transplantation (SCT). All 896 patients who suffered from ALL in Austria between 1981 and 1999 were registered in a prospectively designed database and treated according to trials ALL-Berlin-Frankfurt-Münster (BFM)-Austria (A) 81, ALL-A 84 and ALL-BFM-A 86, 90 and 95. Of these, 203 (23%) suffered from recurrent disease. One-hundred-and-seventy-two patients (85%) achieved second complete remission. The probability of 10-year EFS for the total group was 34 ± 3%. Clinical prognostic markers that independently influenced survival were time to relapse, site of relapse and the immunophenotype. Additionally, a Cox regression model demonstrated that allogeneic SCT after first relapse was associated with a superior EFS compared with chemo/radiotherapy only (hazard ratio = 0·254; P  = 0·0017).     

Use of peripheral blood instead of bone marrow to monitor residual disease in children with acute lymphoblastic leukemia

In children with acute lymphoblastic leukemia (ALL), response to treatment is assessed by bone marrow aspiration. We investigated whether minimal residual disease (MRD) can be effectively monitored in peripheral blood. We used flow cytometric techniques capable of detecting 1 leukemic cell among 10 000 or more normal cells to compare MRD measurements in 718 pairs of bone marrow and peripheral blood samples collected from 226 children during treatment for newly diagnosed ALL. MRD was detected in marrow and blood in 72 pairs and in marrow but not in blood in 67 pairs; it was undetectable in the remaining 579 pairs. Remarkably, findings in marrow and blood were completely concordant in the 150 paired samples from patients with T-lineage ALL: for each of the 35 positive marrow samples, the corresponding blood sample was positive. In B-lineage ALL, however, only 37 of 104 positive marrow samples had a corresponding positive blood sample. Notably, peripheral blood MRD in these patients was associated with a very high risk for disease recurrence. The 4-year cumulative incidence of relapse in patients with B-lineage ALL was 80.0% +/- 24.9% for those who had peripheral blood MRD at the end of remission induction therapy but only 13.3% +/- 9.1% for those with MRD confined to the marrow (P =.007). These results indicate that peripheral blood may be used to monitor MRD in patients with T-lineage ALL and that peripheral blood MRD may provide strong prognostic information in patients with B-lineage ALL.     

The UK experience in treating relapsed childhood acute lymphoblastic leukaemia: a report on the medical research council UKALLR1 study

We have examined the toxicity and overall outcome of the Medical Research Council UKALL R1 protocol for 256 patients with relapsed childhood acute lymphoblastic leukaemia (ALL). Second remission was achieved in over 95% of patients. Two patients died during induction and seven patients died of resistant disease. The overall actuarial event-free survival (EFS) at 5 years for all patients experiencing a first relapse was 46% (95% CI 40-52). Duration of first remission, site of relapse, age at diagnosis and sex emerged as factors of prognostic significance. Five-year EFS was only 7% for children relapsing in the bone marrow within 2 years of diagnosis, but was 77% for those relapsing without bone marrow involvement > 2.5 years from diagnosis. All analyses in this report are by treatment received. For those receiving chemotherapy alone, the 5-year EFS was 48%; for autologous bone marrow transplantation (BMT), the 5-year EFS was 47%; for unrelated donor BMT, it was 52%; and for related donor BMT, the 5-year EFS was 45%. The groups, however, were not comparable with respect to risk factor profile, and therefore direct comparison of EFS is misleading. Adjustment for time to transplant and prognostic factors was used to reduce the effects of biases between treatment groups, but did not suggest benefit for any particular treatment. There was failure of our planned randomization scheme in this trial with only 9% of those eligible being randomized, which highlights the difficulties in running randomized trials especially in patients who have relapsed from a previous trial. The optimal treatment for relapsed ALL therefore remains uncertain. Alternative approaches are clearly needed for those with early bone marrow relapse if outcome is to improve.     

ALL R-87 protocol in the treatment of children with acute lymphoblastic leukaemia in early bone marrow relapse

Seventy-three children with acute lymphoblastic leukaemia (ALL) in first bone marrow (BM) relapse, occurring within 30 months from complete remission (CR), were enrolled in an Italian cooperative study (ALL R-87 protocol). This treatment programme consisted of an induction phase with intermediate-dose cytarabine (IDARA-C) plus idarubicin (IDA) and prednisone (PDN), followed by a multidrug consolidation therapy and bone marrow transplant (BMT). 55/73 children achieved CR (75.3%); 15 (20.5%) failed to respond and three (4.2%) died during induction. The response rate was significantly higher for children with a first CR duration 12 months (P = 0.0005) and for those with a white blood cell (WBC) count at relapse <20 × 10⁹/l (P=0.004). The estimated disease-free survival (DFS ± SE) at 82 months was 0.18 ± 0.05 for all responders, and 0.70 ± 0.14 for allotransplanted patients versus 0.05 ± 0.05 for those autografted (P = 0.001). The estimated probabilities of survival ± SE and event-free survival (EFS ± SE) at 83 months were 0.16 ± 0.07 and 0.13 ± 0.04, respectively, for all enrolled children. Univariate analysis showed that age <10 years at initial diagnosis and B-lineage immunophenotype favourably influenced both DFS (P = 0.001) and EFS probabilities (P = 0.0014 and P = 0.012, respectively), whereas a first CR duration 12 months and a WBC count at relapse <20 × 10⁹/l were associated only with a better EFS rate (P = 0.026 and P = 0.004, respectively). Our results show the efficacy of the IDA plus IDARA-C schedule used in the ALL R-87 protocol in high-risk relapsed ALL children. Allogeneic BMT proved effective for patients with an HLA sibling donor. In a multivariate analysis, age 10 years at initial diagnosis (P = 0.016) and WBC count at relapse 20 × 10⁹/l (P = 0.048) were independently associated with a worse disease outcome.     

Risk-directed treatment of infant acute lymphoblastic leukaemia based on early assessment of MLL gene status: results of the Japan Infant Leukaemia Study (MLL96)

We studied the effectiveness of risk-directed therapy for infants younger than 13 months of age with acute lymphoblastic leukaemia (ALL). Fifty-five infants were assigned to different treatment programs (from December 1995 to December 1998) on the basis of their MLL gene status at diagnosis. Forty-two cases (76.3%) had a rearranged MLL gene (MLL+) and were treated with remission induction therapy followed by sequential intensive chemotherapy, including multiple genotoxic agents (MLL9601 protocol). Haematopoietic stem cell transplantation (HSCT) was attempted if suitable donors were available. Thirteen infants (23.7%) were classified as MLL- and treated for 2.5 years with intensive chemotherapy for high-risk B-ALL (MLL9602 protocol). Complete remission was induced in 38 of the 42 infants (90.5%) with MLL+ ALL and in all 13 patients (100%) with MLL- disease. In the MLL+ subgroup, the estimated event-free survival (EFS) rate at 3 years post diagnosis was 34.0% +/- 7.5%, compared with 92.3% +/- 7.4% in the MLL- subgroup (overall comparison, P = 0.001 by log-rank analysis). Both age less than 6 months (hazard ratio = 6.87, 95% CI = 0.91-52.3; P = 0.013) and central nervous system (CNS) involvement at diagnosis (hazard ratio = 2.92 95% CI = 1.29-6.63; P = 0.015) were significant independent predictors of an inferior outcome. These findings indicate a strategic advantage in classifying infant ALL as either MLL+ or MLL- early in the clinical course and selecting therapy accordingly. Standard chemotherapy for high-risk B-lineage ALL appeared adequate for MLL- cases. Novel therapeutic initiatives are warranted for infants with MLL+ disease, particularly those with initial CNS leukaemic involvement or age less than 6 months, or both.     

Functional response of leukaemic blasts to stromal cell-derived factor-1 correlates with preferential expression of the chemokine receptor CXCR4 in acute myelomonocytic and lymphoblastic leukaemia

The chemokine stromal cell-derived factor-1 (SDF-1) that is released by bone marrow (BM) stromal cells and contributes to stem cell homing may also play a role in the trafficking of leukaemic cells. We analysed SDF-1-induced intracellular calcium fluxes in leukaemic blasts from the peripheral blood of patients with newly diagnosed acute myeloid leukaemia (AML) and lymphoblastic leukaemia (B-lineage ALL), determined the effect of BM stromal cell-conditioned medium on in vitro transendothelial migration (TM) and measured expression of the SDF-1 receptor, CXCR4, by flow cytometry. AML FAB M1/2 blasts did not show calcium fluxes and TM was not stimulated. In myelomonocytic AML (M4/5), however, SDF-1 induced significant calcium fluxes and TM was increased twofold by the conditioned medium. M3 and M4 blasts with eosinophilia (M4eo) showed intermediate activity and M6 blasts showed no functional activity. In ALL, strong calcium fluxes and increased TM (2.5-fold) were observed. Accordingly, expression of CXCR4 was low in undifferentiated (M0) AML, myeloid (M1/2) AML and erythroid (M6) AML, but high [mean fluorescence (MF) > 50] in promyelocytic (M3) AML, myelomonocytic (M4/5) AML and B-lineage ALL. We conclude that, in AML, SDF-1 is preferentially active in myelomonocytic blasts as a result of differentiation-related expression of CXCR4. Functional activity of SDF-1 and high expression of CXCR4 in B-lineage ALL is in accordance with the previously described activity of SDF-1 in early B cells. SDF-1 may contribute to leukaemic marrow infiltration, as suggested by increased CXCR4 expression and migratory response in BM-derived blasts compared with circulating cells.     

Soluble l-selectin (sCD62L) in relapsed childhood acute lymphoblastic leukaemia

Soluble l-selectin (sCD62L) plasma concentrations at diagnosis and outcome were investigated in 193 children at first relapse of acute lymphoblastic leukaemia (ALL) after treatment according to the Berlin-Frankfurt-Münster relapsed ALL multicentre trials, ALL-REZ BFM 95 and 96. sCD62L was low (< fifth paediatric reference percentile) in 63 (33%) and high (> 95th percentile) in 36 (19%) children, and was independent of remission duration, sex, BCR-ABL fusion or extramedullary disease. High sCD62L was associated with circulating blasts and T-cell phenotype. More initial adverse events occurred in children with high and low levels of sCD62L (23 out of 99) than in those with normal levels (9 out of 94, P = 0.018). Among 75 worst-prognosis patients (risk groups S3/S4, isolated bone marrow relapse occurring less than 6 months after elective cessation of front-line therapy, or T-cell phenotype with bone marrow involvement), 27 had low sCD62L and decreased event-free survival (EFS) probability (PEFS5 = 0.09 at 5 years) and duration (219 d) compared with normal sCD62L (29 out of 75, PEFS5 = 0.24, 640 d, P = 0.01). Low (44 out of 118), normal (72 out of 118), and high (19 out of 118) sCD62L non-S3/S4 patients fared similarly (average PEFS5 = 0.45, 1369 d; P = 0.5). Low sCD62L may be a marker of malignant blasts replacing normal sCD62L-producing haematopoietic cells. In children with first relapse of ALL and worst prognosis, plasma sCD62L may be useful for risk-adapted stratification.     

Flow cytometric analysis of de novo acute lymphoblastic leukemia in childhood: report from the Japanese Pediatric Leukemia/Lymphoma Study Group

Although the antigen expression patterns of childhood acute lymphoblastic leukemia (ALL) are well known, little attention has been given to standardizing the diagnostic and classification criteria. We retrospectively analyzed the flow cytometric data from a large study of antigen expression in 1,774 children with newly diagnosed ALL in JPLSG. T- and B-lineage ALL accounted for 13 and 87% of childhood ALL cases, respectively. Cytoplasmic CD3 and CD7 antigens were positive in all T-ALL cases. More than 80% of T-ALL cases expressed CD2, CD5 and TdT. In B-lineage ALL, the frequencies of early pre-B, pre-B, transitional pre-B and B-ALL were 81, 15.5, 0.6 and 2.9%, respectively. More than 90% of early pre-B ALL cases expressed CD19, CD79a, CD22, CD10 and TdT. CD34 was expressed in three-fourths of early pre-B ALL cases. The frequencies of TdT and CD34 expression were lower in pre-B ALL than in early pre-B ALL. B-ALL showed less frequent expression of CD22, CD10, CD34 and TdT than other B-lineage ALL cases. Expression of CD13 and CD33, aberrant myeloid antigens, was significantly more frequently associated with B-lineage ALL than with T-ALL. Based on this retrospective study of antigen expression in 1,774 de novo childhood ALL cases in JPLSG, we propose standardized clinical guidelines for the immunophenotypic criteria for diagnosis and classification of pediatric ALL.     

Flow cytometric assessment of CD15+CD117+ cells for the detection of minimal residual disease in adult acute myeloid leukaemia

There is little information available regarding immunophenotypic monitoring of minimal residual disease (MRD) in acute myeloid leukaemia (AML). We investigated leukaemic cells co‐expressing CD15 and CD117 (CD15+CD117+) in 72 adult AML cases at diagnosis. In 22 cases (31%) with various AML subtypes, more than 5% of leukaemic cells showed the CD15+CD117+ phenotype (range 5.22–55.48%). These 22 cases were younger and had a higher complete remission (CR) rate than the other AML cases, but the CD15+CD117+ cell percentage at diagnosis showed no correlation with the CR duration among the 72 cases. The CD15+CD117+ cell percentage showed a range of 0.00–0.08% in bone marrow cells from 10 haematologically normal subjects. We also investigated CD15+CD117+ cells in sequential bone marrow samples from 17 AML patients who achieved CR and who had had more than 5% CD15+CD117+ leukaemic cells at diagnosis. Because the CD15+CD117+ cell percentage varied among these AML cases, we calculated the percentage of MRD {MRD% = [CD15+CD117+ cells (%) in each sequential marrow sample] ÷ [CD15+CD117+ cells (%) at diagnosis of the corresponding case] × 100}. A high MRD% after 10 months of CR was significantly associated with a short CR duration (P = 0.0004), whereas continuation of a well‐reduced MRD% was associated with a long CR duration. The leukaemic cells conserved the CD15+CD117+ phenotype in all of the eight cases who relapsed. Flow cytometric monitoring of CD15+CD117+ cells is simple and can be applied to a substantial fraction of AML cases. This monitoring may be useful for predicting relapse of adult AML.