New markers for minimal residual disease detection in acute lymphoblastic leukemia

To identify new markers for minimal residual disease (MRD) detection in acute lymphoblastic leukemia (ALL), we compared genome-wide gene expression of lymphoblasts from 270 patients with newly diagnosed childhood ALL to that of normal CD19?CD10? B-cell progenitors (n = 4). Expression of 30 genes differentially expressed by ≥ 3-fold in at least 25% of cases of ALL (or 40% of ALL subtypes) was tested by flow cytometry in 200 B-lineage ALL and 61 nonleukemic BM samples, including samples containing hematogones. Of the 30 markers, 22 (CD44, BCL2, HSPB1, CD73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, PBX1, CTNNA1, ITGB7, CD69, CD49f) were differentially expressed in up to 81.4% of ALL cases; expression of some markers was associated with the presence of genetic abnormalities. Results of MRD detection by flow cytometry with these markers correlated well with those of molecular testing (52 follow-up samples from 18 patients); sequential studies during treatment and diagnosis-relapse comparisons documented their stability. When incorporated in 6-marker combinations, the new markers afforded the detection of 1 leukemic cell among 10(5) BM cells. These new markers should allow MRD studies in all B-lineage ALL patients, and substantially improve their sensitivity.     

Autologous bone marrow transplantation in high-risk remission B-lineage acute lymphoblastic leukemia using a cocktail of three monoclonal antibodies (BA-1/CD24, BA-2/CD9, and BA-3/CD10) plus complement and 4-hydroperoxycyclophosphamide for ex vivo bone marrow purging.

Fourteen patients with high-risk B-lineage acute lymphoblastic leukemia (ALL) in complete remission underwent autologous bone marrow transplantation (BMT) using a combined immunochemopurging protocol. A monoclonal antibody (MoAb) cocktail of BA-1, BA-2, and BA-3 plus rabbit complement (C') plus 4-hydroperoxycyclophosphamide (4-HC) was used to eliminate residual occult leukemia cells from autografts. All patients were conditioned with single-dose total body irradiation (TBI) followed by high-dose Ara-C. All 14 patients engrafted at a median of 24 days (range, 12 to 36 days). Three patients are alive and disease free at 3.5 years, 3.9 years, and 4.1 years post-BMT. The Kaplan-Meiser estimate and standard error of the probability of sustained remission was 23% +/- 12% at 3.5 years post-BMT with a mean relapse-free interval of 1.4 +/- 0.4 years. The disease-free survival (DFS) at 3.5 years was 21% +/- 11%, with a mean DFS time of 1.3 +/- 0.4 years. A novel and quantitative minimal residual disease (MRD) detection assay, which combines fluorescence-activated multiparameter flow cytometry and cell sorting with leukemic progenitor cell (LPC) colony assays, was used to analyze remission BM samples from B-lineage ALL patients for residual LPC, and to evaluate the efficacy of ex vivo BM purging. Notably, the minimal residual leukemia burden before BMT, as measured by the percentage of B-lineage LPC in the pre-BMT remission BM samples, indicated the outcome of the BMT. The median value for the minimal residual leukemia burden before BMT was 0.0035% (35 LPC/10(6) mononuclear cells). The Kaplan-Meier estimates and standard errors of the probability of remaining in remission after BMT were 43% +/- 19% for patients whose BM samples contained less than or equal to 0.0035% LPC and 0% +/- 0% for patients whose BM samples contained greater than 0.0035% B-lineage LPC (P less than .05). In contrast to the minimal residual leukemia burden measured by the described MRD assay system, the percentage of blasts or TdT+ cells in the remission BM samples did not correlate with the probability of relapse. The applied purging protocol showed variable success in destroying target B-lineage LPC populations contaminating the autografts. While in some cases purging was highly effective, eliminating up to greater than or equal to 4 logs of residual B-lineage LPC, in other cases only 0.1 to 0.2 logs of B-lineage LPC were purged.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of novel markers for monitoring minimal residual disease in acute lymphoblastic leukemia

To identify new markers of minimal residual disease (MRD) in B-lineage acute lymphoblastic leukemia (ALL), gene expression of leukemic cells obtained from 4 patients with newly diagnosed ALL was compared with that of normal CD19(+)CD10(+) B-cell progenitors obtained from 2 healthy donors. By cDNA array analysis, 334 of 4132 genes studied were expressed 1.5- to 5.8-fold higher in leukemic cells relative to both normal samples; 238 of these genes were also overexpressed in the leukemic cell line RS4;11. Nine genes were selected among the 274 overexpressed in at least 2 leukemic samples, and expression of the encoded proteins was measured by flow cytometry. Two proteins (caldesmon and myeloid nuclear differentiation antigen) were only weakly expressed in leukemic cells despite strong hybridization signals in the array. By contrast, 7 proteins (CD58, creatine kinase B, ninjurin1, Ref1, calpastatin, HDJ-2, and annexin VI) were expressed in B-lineage ALL cells at higher levels than in normal CD19(+)CD10(+) B-cell progenitors (P <.05 in all comparisons). CD58 was chosen for further analysis because of its abundant and prevalent overexpression. An anti-CD58 antibody identified residual leukemic cells (0.01% to 1.13%; median, 0.03%) in 9 of 104 bone marrow samples from children with ALL in clinical remission. MRD estimates by CD58 staining correlated well with those of polymerase chain reaction amplification of immunoglobulin genes. These results indicate that studies of gene expression with cDNA arrays can aid the discovery of leukemia markers. (Blood. 2001;97:2115-2120)     

Minimal residual disease monitoring in adult T-cell acute lymphoblastic leukemia: a molecular based approach using T-cell receptor G and D gene rearrangements

BACKGROUND AND OBJECTIVES: Minimal residual disease (MRD) is important in the measurement of response to treatment in childhood B- and T-cell acute lymphoblastic leukemia (ALL) and in adult B-cell ALL. Little is known about MRD evaluation in adult T-cell ALL. This study aimed to determine the prognostic significance of MRD measurements in adult T-cell ALL. DESIGN AND METHODS: T-cell receptor (TCR) gamma (G) and TCR delta (D) gene analyses were carried out at presentation in 49 patients with de novo T-ALL using a polymerase chain reaction (PCR) approach. In 26 of the patients bone marrow (BM) samples were collected at sequential time points (0-2, 3-5, 6-9, 10-24 months) after diagnosis for MRD investigation. The relationship between MRD status and clinical outcome was investigated and correlated with age, gender and white blood cell count at presentation. RESULTS: TCRG clonal gene rearrangements were found in 40 patients (82%). Eleven patients showed TCRD rearrangements (22%), in one of them as the sole molecular marker. V(gamma)I family rearrangements predominated (45 of 65 alleles) together with V(delta)1-J(delta)1/2 (9 of 13 alleles). Continuous clinical remission (CCR) occurred in 17 patients while nine patients relapsed. MRD analysis showed that negative tests during the first 6 months post-induction, and persisting negative MRD after induction were the best predictors of CCR. A positive test after 5 months was better at predicting relapse. In only four of seven patients was relapse preceded by a positive test the 5 months preceding relapse. INTERPRETATION AND CONCLUSIONS: Overall the ability of positive and negative tests to predict relapse or CCR was weaker in this cohort of adult T-ALL patients than in T- and B-lineage childhood ALL and B-lineage adult ALL. TCRG and TCRD gene analysis provides a clonal marker in the majority of adult T-ALL. These results suggest that caution should be taken in using MRD data based on TCR gene rearrangements to predict prognosis in adult T-ALL. Biological reasons may underlie differences between the performance of MRD tests in B- and T-lineage ALL. Further studies in a larger cohort of patients are needed to determine the exact role that MRD determination has in the management of T-ALL in adults.     

Detection of abnormalities in B-cell differentiation pattern is a useful tool to predict relapse in precursor-B-ALL

Immunophenotypic investigation of minimal residual disease (MRD) has traditionally been based on the investigation of phenotypic aberrants at diagnosis to be used later as a target for MRD detection. This approach has several shortcomings (it is only applicable to patients with aberrant phenotypes, requires a diagnostic sample, and is patient-specific) and therefore a search for simpler alternatives is warranted. The present study is based on the hypothesis that in precursor-B-ALL patients the persistence of residual leukaemic cells may induce abnormalities in the precursor-B-cell compartment in bone marrow (BM) and these could be used as a criteria to predict relapse. These abnormalities may include: (1) the presence of an increase in the frequencies of immature B cells (CD34+/CD19+ or CD20-/CD19+) or (2) the existence of an altered B-cell differentiation pathway due to a blockade or to the presence of B cells outside the normal pathway. A total of 180 BM samples from 45 consecutive precursor-B-ALL patients who achieved morphological complete remission (CR) were analysed by multiparametric flow cytometry. Our results show that a significant increase in immature B-cell subsets or an altered B-cell differentiation predicts a high relapse rate (P<0.01) and a shorter disease-free survival (P<0.01). Moreover, abnormalities in either of these two criteria detected at specific time points during follow-up (end of induction, maintenance, or after treatment) were associated with a significantly shorter disease-free survival (P<0.01). In summary, the investigation of abnormalities in B-cell differentiation is a relatively simple and cheap approach for predicting relapse in precursor-B-ALL patients.     

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.     

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.     

A simplified flow cytometric assay identifies children with acute lymphoblastic leukemia who have a superior clinical outcome

Bone marrow normal lymphoid progenitors (CD19+, CD10+, and/or CD34+) are exquisitely sensitive to corticosteroids and other antileukemic drugs. We hypothesized that, in patients with B-lineage acute lymphoblastic leukemia (ALL), cells with this phenotype detected early in treatment should be leukemic rather than normal. We therefore developed a simple and inexpensive flow cytometric assay for such cells and prospectively applied it to bone marrow samples collected on day 19 from 380 children with B-lineage ALL. In 211 patients (55.5%), these cells represented 0.01% or more of the mononuclear cells; results correlated remarkably well with those of more complex flow cytometric and molecular minimal residual disease (MRD) evaluations. Among 84 uniformly treated children, the 10-year incidence of relapse or remission failure was 28.8% +/- 7.1% (SE) for the 42 patients with 0.01% or more leukemic cells on day 19 detected by the simplified assay versus 4.8% +/- 3.3% for the 42 patients with lower levels (P = .003). These assay results were the strongest predictor of outcome, even after adjustment for competing clinicobiologic variables. Thus, this new assay would enable most treatment centers to identify a high proportion of children with ALL who have an excellent early treatment response and a high likelihood of cure.     

Autologous bone marrow transplantation in high-risk remission T-lineage acute lymphoblastic leukemia using immunotoxins plus 4-hydroperoxycyclophosphamide for marrow purging

Fourteen patients with high-risk T-lineage acute lymphoblastic leukemia (ALL) in complete remission underwent autologous bone marrow transplantation (BMT) in an attempt to eradicate their residual disease burden. A combined immunochemotherapy protocol using a cocktail of two immunotoxins directed against CD5/Tp67 and CD7/Tp41 T-lineage differentiation antigens in combination with the in vitro active cyclophosphamide congener 4-hydroperoxy-cyclophosphamide (4-HC) was used to purge autografts. Despite high dose pretransplant radiochemotherapy and effective purging of autografts, 9 of 14 patients relapsed at a median of 2.5 months (range, 1.2 to 16.8 months) post BMT. Two patients remain alive and disease free at 26 and 28 months post BMT. We used a novel quantitative minimal residual disease (MRD) detection assay, which combines fluorescence activated multiparameter flow cytometry and cell sorting with leukemic progenitor cell (LPC) assays, to analyze remission bone marrow (BM) samples from T-lineage ALL patients for the presence of residual LPCs. Notably, high numbers of residual LPC detected in remission BM before BMT constituted a poor prognostic indicator, providing the first evidence for the biologic significance and clinical value of in vitro T-lineage ALL LPC assays. The median value for the residual leukemia burden before BMT, was approximately 8.6 x 10(3) LPC/10(8) mononuclear cells (MNC) (approximately 0.0086% LPC). Patients with a residual leukemia burden less than this median value appeared to have a better outlook for remaining free of relapse after autologous BMT than patients with a greater leukemia burden (53 +/- 25% v 14 +/- 13%, P = .006, Mantel-Cox). By comparison, the log kill efficacy of purging, the remaining numbers of LPC in purged autografts, or the estimated numbers of reinfused LPC, did not correlate with the probability of disease-free survival (DFS). These results indicate that the primary reason for the recurrence of leukemia was inefficient pretransplant radiochemotherapy rather than inefficient purging of autografts.     

Developmental hierarchy during early human B-cell ontogeny after autologous bone marrow transplantation using autografts depleted of CD19+ B-cell precursors by an anti-CD19 pan-B-cell immunotoxin containing pokeweed antiviral protein.

Sequential immunophenotypes of bone marrow (BM) and peripheral blood (PBL) lymphoid cells from 15 B-lineage acute lymphoblastic leukemia (ALL) patients who underwent autologous bone marrow transplantation (BMT) during complete remission were determined by dual-color immunofluorescence and multiparameter flow cytometry. Autografts were depleted of CD19+ B-cell precursors by an immunochemopurging protocol that combines B43-PAP, a potent anti-CD19 immunotoxin, and the cyclophosphamide congener 4-hydroperoxycyclophosphamide (4-HC). A marked interpatient variation was observed in the appearance and expansion of B-cell precursors repopulating the posttransplant marrow. The expression of CD10 and CD19 antigens during early B-cell ontogeny post-BMT preceded the expression of CD20, CD21, CD22, CD40, and sIgM. The surface antigen profiles of the emerging B-cell precursors were similar to those of fetal liver or fetal bone marrow B-cell precursors. Our comparisons of BM and PBL samples from patients in the early post-BMT period demonstrated that (1) PBL initially contains fewer B-lineage cells than does BM, and (2) circulating B-lineage lymphoid cells have a more mature immunophenotype than do BM B-lineage lymphoid cells. Comparison of the surface antigen profiles of day 30 versus day 100 or year 1 BM or PBL lymphoid cells showed an increase in the percentages of CD10+CD22- undifferentiated lymphocyte precursors, as well as CD19+sIgM- B-cell precursors (pre-pre-B), consistent with a time-dependent expansion of these B-cell precursor populations post-BMT. Importantly, the percentages of CD10+CD22+ and CD19+sIgM+ B-cell precursor (pre-B) populations also increased between 30 days and 1 year post-BMT, confirming the ability of emerging immature B-cell precursors to differentiate along the B-precursor pathway. The acquisition and expression of B-lineage differentiation antigens at different stages of the post-BMT B-cell ontogeny support the notion that the expression of these antigens is developmentally programmed. Similar to patients in previous autologous BMT studies, recipients of B-cell precursor-depleted autografts had normal or nearly normal serum immunoglobulin levels, suggesting that the maturing B-cell/plasma cell populations can produce and secrete immunoglobulins. The development of a functional CD19+ B-lineage lymphoid compartment in recipients of autografts which were depleted of CD19+ B-cell precursors corroborates the previously postulated existence of CD19- B-lineage lymphoid progenitor cells.     

Flow cytometry of peripheral blood and bone marrow cells from patients with hairy cell leukemia: phenotype of hairy cells, lymphocyte subsets and detection of minimal residual disease after treatment

By flow cytometry (FC) and an extensive panel of markers we characterized leukemia cells from the peripheral blood (PB) and bone marrow (BM) of 13 symptomatic patients with hairy cell leukemia (HCL). Hairy cells (HCs) identified in the large cell gate always expressed B-cell markers - CD19, CD20, CD22, HLA-DR, and 'HCL-restricted' markers - CD22+CD11c, CD25 and CD103. Other markers, not followed regularly, were occasionally expressed, such as CD34, CD38, CD71, CD15, CD10 and kappa/lambda light chains. Furthermore, in one patient with suspect but not proved HCL in PB or BM, neither morphologically nor immunologically, we confirmed the diagnosis of HCL. Only the immunophenotyping of splenic cells after splenectomy confirmed HCL diagnosis. Flow cytometry was repeated at 3-5 month intervals, after treatment with 2-Chlorodeoxyadenosine (CdA) or less frequently alpha-interferon (IFN). We investigated serially lymphocyte subsets after treatment and we found profound and persistent CD4+ lymphopenia in majority of studied patients after CdA treatment. Simultaneously we investigated the value of FC to detect minimal residual disease (MRD) and to establish, whether MRD+ could predict relapse. Detection of MRD in our series predicted hematological relapse only in one case with persistent MRD+, in majority of cases with occasionally found MRD+ phenotype, did not. Using quantitative immunophenotyping we observed significantly higher values of molecule numbers of hairy cell B-cell markers, comparing to B-cells in nonleukemic gate of the same sample. Our study showed 1) the diagnostic value of FC in management of HCL patients, 2) long-lasting response in the majority of patients after CdA, 3) a profound and persistent CD4+ lymphopenia in CdA treated patients, 4) some correlation between persistent MRD staining and hematological relapse, and 5) further, till now not described activated feature of HCs, given by the increased values of molecular numbers (molecules of equivalent soluble fluoresceine - MESF) in B-cell antigens of HCL.     

The significance of minimal residual disease in hairy cell leukaemia treated with deoxycoformycin: a long-term follow-up study

We investigated the clinical significance and long-term follow-up of detecting minimal residual disease (MRD) in hairy cell leukaemia (HCL) in complete remission (CR) after treatment with deoxycoformycin (DCF). MRD was assessed in 23 patients by immunophenotyping peripheral blood and bone marrow frozen sections using a panel of antibodies, CD11c, CD25, CD103 and HC2, which detect hairy cells. 31 cases with active HCL were used as controls. 10/23 patients (43%) had MRD in bone marrow (seven), blood (one) or both sites (two) which was not detected on haematoxylin–eosin bone marrow sections nor by immunohistochemistry on paraffin sections in six cases tested. Sequential studies in four cases did not show changes in the amount of MRD. At a median follow-up of 72 months (range 15–108), 5/23 (22%) patients have relapsed with a median time of 59 months (range 15–105). MRD was detected in three of the five patients who relapsed. In the two patients with negative MRD, one relapsed with an abdominal mass and the other was a late relapse at 84 months. MRD was also documented in 7/18 patients who continued in clinical CR for a median of 80 months (range 63–98). There were no statistical differences in disease-free survival between MRD⁺ and MRD⁻ patients ( P   =  0.8). Our findings indicate that relapse after long-term remission achieved with DCF cannot be predicted when MRD is detected by sensitive methods. A search for other parameters such as proliferative rate of the residual cells or chest and abdominal CT scan might identify patients with a higher probability of disease recurrence.     

Methodological approach to minimal residual disease detection by flow cytometry in adult B-lineage acute lymphoblastic leukemia

A flow cytometric approach to minimal residual disease (MRD) monitoring useful in childhood B-lineage acute lymphoblastic leukemia (ALL) is discussed here in the context of ALL in adults. Of 64 leukemia samples analyzed, 95.3% had at least one abnormal phenotype (57.3% had two or more) as compared to physiologic B-cell precursors in adult bone marrow. The method was sensitive enough to detect one leukemic cell among 10,000 normal cells in 16/19 experiments (84.2%). Blast phenotypes were stable in culture and at relapse, and were useful for MRD monitoring in patients. Marker combinations for childhood ALL are also applicable to adult cases.     

Quantitative analysis of minimal residual disease predicts relapse in children with B-lineage acute lymphoblastic leukemia in DFCI ALL Consortium Protocol 95-01

In a prospective trial in 284 children with B-lineage acute lymphoblastic leukemia (ALL), we assessed the clinical utility of real-time quantitative polymerase chain reaction analysis of antigen receptor gene rearrangements for detection of minimal residual disease (MRD) to identify children at high risk of relapse. At the end of induction therapy, the 5-year risk of relapse was 5% in 176 children with no detectable MRD and 44% in 108 children with detectable MRD (P < .001), with a linear association of the level of MRD and subsequent relapse. Recursive partitioning and clinical characteristics identified that the optimal cutoff level of MRD to predict outcome was 10(-3). The 5-year risk of relapse was 12% for children with MRD less than one leukemia cell per 10(3) normal cells (low MRD) but 72% for children with MRD levels greater than this level (high MRD) (P < .001) and children with high MRD had a 10.5-fold greater risk of relapse. Based upon these results we have altered our treatment regimen for children with B-lineage ALL and children with MRD levels greater than or equal to 10(-3) at the end of 4 weeks of multiagent induction chemotherapy now receive intensified treatment to attempt to decrease their risk of subsequent relapse.     

Status of minimal residual disease after induction predicts outcome in both standard and high-risk Ph-negative adult acute lymphoblastic leukaemia. The Polish Adult Leukemia Group ALL 4-2002 MRD Study

The treatment of adults with Philadelphia-negative acute lymphoblastic leukaemia (ALL) depends on the presence of risk factors including age, white blood cell count, immunophenotype and time to complete remission. In recent years, status of minimal residual disease (MRD) has been postulated as an additional risk criterion. This study prospectively evaluated the significance of MRD. Patients were treated with a uniform Polish Adult Leukemia Group (PALG) 4-2002 protocol. MRD status was assessed after induction and consolidation by multiparametric flow cytometry. Out of 132 patients included (age, 17–60 years), 116 patients were suitable for analysis. MRD level ≥0·1% of bone marrow cells after induction was found to be a strong and independent predictor for relapse in the whole study population ( P  < 0·0001), as well as in the standard risk (SR, P  = 0·0003) and high-risk ( P  = 0·008) groups. The impact of MRD after consolidation on outcome was not significant. The combination of MRD status with conventional risk stratification system identified a subgroup of patients allocated to the SR group with MRD <0·1% after induction who had a very low risk of relapse of 9% at 3 years as opposed to 71% in the remaining subjects ( P  = 0·001). We conclude that MRD evaluation after induction should be considered with conventional risk criteria for treatment decisions in adult ALL.     

Advances in the immunological monitoring of childhood acute lymphoblastic leukaemia

In children with acute lymphoblastic leukaemia (ALL), measurements of minimal residual disease (MRD) during therapy provide crucial information about the response to treatment and the risk of relapse. Flow cytometry is a practical and widely applicable tool for monitoring MRD in patients with ALL. This approach is based on the identification of immunophenotypes expressed by leukaemic cells but not by normal lympho-haematopoietic cells in bone marrow and peripheral blood. These phenotypes can identify one leukaemic cell among 10 000 normal cells and are currently applicable to at least 90% of patients with ALL. A strong correlation between flow cytometric measurements of MRD during clinical remission and treatment outcome has been demonstrated, suggesting that these assays should be incorporated into treatment protocols.     

Changing antigen receptor gene rearrangements in a case of early pre-B cell leukemia: evidence for a tumor progenitor cell with stem cell features and implications for monitoring residual disease.

A case of acute lymphoblastic leukemia (ALL) was encountered in which the two clonal gamma T-cell receptor gene (TCR gamma) rearrangements found in bone marrow (BM) samples at relapse both differed from the single clonal TCR gamma rearrangement present in BM obtained at diagnosis 5 years previously. In contrast, two clonal Ig heavy chain gene (IgH) rearrangements present at relapse were identical to those present at diagnosis. Comparison of the DNA sequences of the relapse TCR gamma rearrangements with that of the diagnostic TCR gamma rearrangement indicated that they must have been generated de novo from TCR gamma loci in germline configuration. By polymerase chain reaction using clonotypic N-region oligonucleotide primers (N-PCR), cells bearing the diagnosis or relapse TCR gamma rearrangements were undetectable in the sample from the opposite time point. Two BM samples obtained at different times in clinical remission were both devoid of detectable residual tumor when analyzed by N-PCR, indicating a depth of remission of less than 1 tumor cell per 4 x 10(5) BM mononuclear cells. The tumor cells showed a primitive phenotype: T-cell antigen-negative, CALLA/CD10-negative, CD20-negative, CD19-positive, and positive for the myeloid marker My9. This case, which appears to represent a tumor arising from a progenitor cell with both early B-lineage and certain stem cell features, has implications for monitoring residual ALL and possibly also for treatment of the disease.