Number Of Residual Leukemic Cells Research Articles

Relevance of Minimal Residual Disease in the Era of Targeted Agents.

The evaluation of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) has evolved in parallel with the enormous progresses in the therapeutic armamentarium and the application of cutting-edge diagnostic techniques the CLL community witnessed in the past few years. Minimal residual disease is considered an objective measure of disease status defined by the number of residual leukemic cells detected in a sample of peripheral blood and/or bone marrow as proportion of the total white blood cells and defined undetectable if fewer than 1 CLL cell among 10,000 white blood cells (10 or 0.01%) is detected. In this review, we aim at shedding light on how to evaluate MRD, what we already know about MRD from the experience with chemoimmunotherapy, and why MRD evaluation remains still relevant in the era of targeted agents.

Analysis of Early Disease Response in Childhood ALL: Can Peripheral Blood Replace Bone Marrow Analysis?.

Abstract 2523▪▪This icon denotes a clinically relevant abstractThe detection of minimal residual disease (MRD) is highly predictive of outcome in pediatric acute lymphoblastic leukemia (ALL) and has now been integrated into the majority of treatment regimens. Early studies demonstrated the strong prognostic value of post-induction MRD measurement, using both molecular and flow cytometric techniques, allowing treatment intensity to be tailored to response. More recent studies have examined the value of MRD measurement at earlier time points and using peripheral blood (PB) in place of bone marrow (BM) in an attempt to reduce invasive procedures.Within the United Kingdom (UK), previous national trials have shown that post-induction MRD assessment, using molecular techniques, is highly predictive of relapse, allowing treatment stratification based on MRD levels. We have previously demonstrated the value of flow cytometric MRD assessment at early time points in response prediction (Motwani et al, Blood 2007a), however, as yet there has been no assessment of MRD in peripheral blood at early time points using flow cytometric methods in the context of a UK national trial.To address this we assessed MRD by flow cytometry using Day 8 (D8) and/or D15 paired PB and BM samples taken from children being treated for ALL at our unit on the UK MRC UKALL2003 trial or ALLR3 relapse trial. Samples were analyzed by 4-color flow cytometry using a large panel of monoclonal antibodies to identify all leukemia-associated immunophenotypes (LAIP). For follow up samples a minimum of 2 markers were identified for each patient and sequential gating was used for analysis. 500 000 to 106cells were acquired for each antibody combination for analysis to be sufficiently sensitive (0.01%). The number of residual leukemic cells was calculated as the percentage of blasts present within total nucleated cells counted. Sensitivity experiments were performed using dilutions of leukemic blasts in normal bone marrow or peripheral blood and demonstrated a sensitivity of 0.01%.Fifty paired BM and PB samples from 46 patients (41 newly diagnosed, 5 relapse) were included in the study (Age 2–22 years, mean 7.8 years). Of these, 22 were taken at D8 and 28 at D15. Forty-two samples were from patients with B-cell ALL, whilst 8 were from patients with T-cell ALL. The most frequent LAIPs used to identify blasts in B-ALL were CD58 (90% of cases), CD45 (75%), CD38 (86%), CD123 (77%). In T-ALL the most common LAIP combinations were CD34 (75%), CD5/CD56 (62.5%), CD5/CD2 (50%) and CD5/CD99 (50%).In keeping with previous reports, B-ALL MRD levels in PB blood were approximately 1.3-log lower than BM MRD levels (D8 mean 2.27% vs. 27.8%; D15 levels 0.63% vs. 7.99%). T-ALL levels were approximately 0.5-log lower. Importantly, the level of MRD in BM and PB in B-ALL showed a strong correlation (r=0.77).Whilst there were insufficient relapses to allow correlation with long-term outcome, PB MRD levels at D8 showed good prediction of post-induction molecular MRD, the current time point used for risk stratification in the UK trial. In patients with PB MRD >1% at D8, 100% (5/5) were in the high-risk group post-induction, significantly more than those with PB MRD <1% at D8, of which only 36.4% (4/11) were allocated to the high risk group post-induction (p=0.023). MRD levels in BM at D8 did not show similar predictive value. Importantly, this early prediction of risk could offer an early opportunity to intensify treatment in this high-risk group, potentially enhancing disease clearance during the induction phase.This study further supports that early assessment of MRD using flow cytometric techniques enhances treatment stratification in pediatric ALL. Furthermore, it shows that PB sampling may provide a markedly less invasive means of MRD assessment in these patients. Validation in a larger cohort of patients is warrented. Disclosures:No relevant conflicts of interest to declare.

Simultaneous Use of Histone Deacetylase Inhibitor Valproic Acid Potentiates Natural Killer Cell Cytotoxicity against T-Lineage Acute Lymphoblastic Leukemia

Leukemic cells could survive after aggressive therapies including mega-dose chemotherapy. Natural killer (NK) cells have great promise for treatment of such refractory disease because many kinds of hematopoietic malignancies and solid tumors are reported to be intrinsically sensitive to NK cell lysis. Indeed, NK cell allo-reactivity has been shown to reduce leukemia relapse after haploidentical stem cell transplantation (SCT) and cord blood transplantation (CBT) in patients with myeloid leukemia (Ruggeri et al. Science 2002; Willemze, et al. EBMT 2008 abstract). However, NK cell allo-reactivity against acute lymphoblastic leukemia (ALL) has been reported to be generally weak regardless of the presence of killer immunoglobulin receptor (KIR) ligand mismatch. We previously showed that genetic modification of primary NK cells with anti-CD19 chimeric receptor could overcome the refractoriness of B-lineage ALL cells to NK cell lysis (Imai et al, Blood 2005). For T-lineage ALL, however, development of antigen-specific artificial receptors against T-ALL cells has been hampered by the similarity in immunophenotype between NK cells and T cells. Novel strategy to augment anti-T-ALL activity of NK cells, such as combined use of a kind of agents which has anti-leukemic activity and does not inhibit NK cell activity, is needed. Valproic acid (VPA) has been widely used as an anticonvulsant and a mood stabilizer for a long time, and its safety has been established. Recently, a number of studies have shown that VPA could act as a histone deacetylase (HDAC) inhibitor and possess anti-cancer activity. We thus studied the effect of VPA on NK cell cytotoxicity against T-ALL cell lines and asked whether VPA could be incorporated into NK cell therapy. NK cells were activated and expanded by coculture of peripheral blood mononuclear cells with K562 cells transfected with 4-1BB ligand and membrane-bound interleukin-15. After 7 days, cells were subjected to CD3-depletion procedure to earn highly pure (>99%) NK cells, and then cultured in the presence of high dose interleukin (IL)-2 (1000 U/ml). Efficient killing of T-ALL cell lines MOLT4, Jurkat and CEM-C7 by activated NK cells were observed in 4-hour cytotoxicity assay, even if KIR ligand mismatch were not present. VPA suppressed leukemic cell proliferation and induced apoptosis in MOLT4, Jurkat and CEM-C7 at clinically achievable concentrations and in a dose-dependent fashion. Exposure of activated NK cells to VPA at 100 mg/ml for 48 hours in the presence of IL-2 resulted in mild inhibition of NK cell proliferation by approximately 20% and decreased surface expression of activating receptors such as NKG2D, 2B4, and natural cell cytotoxicity receptors p30, p44, p46 (10–40% decrease in mean fluorescence intensity). However, the addition of VPA to coculture of NK cells with MOLT-4, Jurkat and CEM-C7 did not result in interfering the anti-leukemia activity of NK cells. When anti-leukemic activity was evaluated in more prolonged culture experiments at very low effector: target (E/T) ratio, the number of residual leukemic cells was significantly lower in culture with VPA compared to those without VPA (MOLT-4: residual leukemia cells 12.2 ± 1.9% vs 46.0 ± 2.3% of control culture at E/T ratio of 0.13:1; CEM-C7: 21.0 ± 0.4% vs 40.1 ± 1.1% of control culture at E/T ratio of 0.13:1, 72-hour incubation). Similar results were also obtained when VPA was substituted by another HDAC inhibitor Suberoylanilide hydroxamic acid (SAHA). We found that upregulation of NK-activating receptor ligands MICA/B, ULBP1 and ULBP2 occurred in MOLT-4, Jurkat and CEM-C7 after 24–48 hour exposure to VPA at 100 mg/ml. On the contrary, T-ALL cells did not show increased sensitivity to NK cell lysis after pretreatment by VPA for 48 hours. Therefore, we thought that the synergistic effect of VPA observed in prolonged culture experiments could be mainly attributed to growth suppression of continuously growing leukemic cells, not to its immunomodulatory activity. These data suggest that simultaneous use of VPA would not abrogate NK cell cytotoxic activity and VPA might be used in combination with infusion of NK cells for patients bearing VPA-sensitive tumors. In patients after haploidentical SCT and CBT, NK cell allo-reactivity might be potentiated by the use of VPA.

Quantitative Detection of NPM1 Mutations as Marker of Minimal Residual Disease (MRD) in the Large Majority of AML with Normal Karyotype.

Normal cytogenetics in AML is a major drawback to detect minimal residual disease. We have identified heterozygous NPM1 mutations as the most frequent genetic lesion in AML with normal karyotype (NEJM 2005:352,254). Mutations A, B and D, all characterized by tetranucleotide insertions, account for more than 90% of all mutations. NPM1 mutations are new important prognostic markers in AML with normal cytogenetics, being predictive of successful response to induction treatment (NEJM 2005:352,254) and longer overall survival in cases without FLT3 mutations (Dohner K et al, and Schnittger S et al, Blood 2005, on line). Their clinical impact prompted us to develop a Real Time Quantitative PCR assay for the identification and monitoring of NPM1 mutants.Tests were set up using either cDNA (13 adult cases with mutation A and 1 with mutation B) or genomic DNA (gDNA) (7 pediatric and 8 adult patients).cDNA RQ-PCR. Forward primer was designed in exon 11, probe in exon 11/exon 12 junction (5′-FAM-3′-MGB) and reverse primers in exon 12. Samples were analyzed in ABI PRISM 7700 Sequence Detection System (Applied Biosystems). ABL1 gene was used as control. For the absolute quantitative assessment of NPM1 mutation A copies a standard curve with serial dilutions of a plasmid containing the target sequences was used. The highest reproducible sensitivity of this RQ-PCR assay was 10 molecules. The highest reproducible sensitivity of the relative quantification RQ-PCR assay for mutation A and B was 10−4. Thirteen AML NPM mutation A patients were monitored at diagnosis and over follow up. The number of mutated copies was high in all cases at diagnosis and significantly decreased after induction treatment in all cases with complete hematological remission. Only a slight decrease was observed in the case who did not reach remission. Patients with a complete hematological remission but MRD decrease <3log showed a higher risk of relapse.gDNA RQ-PCR. specific forward primers for six different mutations were designed using Primer Express software to anneal to the mutated region of NPM exon 12. Reverse primers were designed on NPM1 exon 12. The TaqMan core reagent kit (Applied Biosystems) was used for RQ-PCR. The albumin gene was used as control. A reproducible sensitivity of 10−4 was reached in all but one case. The mean Ct of undiluted DNA samples was 23.3 (range 22.1–24.8). The mean slope of the dilution curves was 3.6 (2.9–4.0). High correlation coefficients (0.99 in all but one) were obtained.Using both cDNA and gDNA we set up sensitive and reproducible systems to detect minimal residual disease in 60% of AML with normal karyotype and NPM gene mutations. While gDNA RQ-PCR has the advantage to be directly related to the number of residual leukemic cells, cDNA RQ-PCR can be easily applied in samples collected in the routine diagnostic testing for common translocations. Large prospective studies are necessary to clarify the clinical impact of NPM1-based MRD monitoring of AML with normal karyotype.

Detection of Translocation 8;21 on Interphase Cells from Acute Myelocytic Leukemia by Fluorescence In Situ Hybridization and Its Clinical Application

To detect a translocation (8;21)(q22;q22) in interphase cells by fluorescence in situ hybridization (FISH), we investigated three probe combinations: single-color hybridization with two cosmid probes (cY8 and cY3), single-color hybridization with four cosmid probes (cY8, cY3, cY107, and cYR4), and dual-color hybridization with two cosmid probes (cY107 and cYR4) from the AML1 gene flanking or overlapping the breakpoint region. Over 95% of nuclei gave sufficient signals in all three probe combinations, and the detection rates were not statistically different among them. Among 18 patients examined at diagnosis, 12 with t(8;21) were also monitored for the number of residual leukemic cells after chemotherapy or bone marrow transplantation (BMT). There were some discrepancies between morphology and genetic (especially FISH) results at partial, or even complete remission. As leukemic cells with t(8;21) can maturate, morphological assessment alone is insufficient to evaluate the residual leukemic cells. Interphase FISH provided more precise information about the clinical status of patients with an 8;21 translocation after treatment.

Quantitation of minimal residual disease in acute myelogenous leukemia and myelodysplastic syndromes in complete remission by molecular cytogenetics of progenitor cells.

Detection of karyotypic clonal abnormalities are prognostically useful in patients with acute myelogenous leukemia (AML) and myelodysplastic syndromes (MDS), but cytogenetic methods are not sensitive enough to detect low numbers of residual leukemic cells in patients who have achieved complete remission (CR). Fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) were used to investigate the frequency and presence of minimal residual disease (MRD) in AML and MDS patients (n = 28) with monosomy of chromosomes 7, 17 and 18 and trisomy of chromosomes 6, 8, 9 and 10 in CR. MRD was detected in all patients with monosomy 7 (n = 10) and followed by relapse in eight patients after 4.8 +/- 3.1 months. In contrast, persistent leukemic cells occurred in 11/12 patients with trisomy 8, but only three of them relapsed after 7.7 +/- 4.0 months. Cox regression analysis showed that cytogenetic class and levels of clonal cells at CR were related to time to relapse (P = 0.001). The level of MRD identified patients at high and low risk of relapse. High absolute levels of proliferating residual leukemic cells correlated with monosomy 7 and high risk of relapse.

Immunophenotyping Investigation of Minimal Residual Disease Is a Useful Approach for Predicting Relapse in Acute Myeloid Leukemia Patients

A high complete remission rate is currently achieved in patients with acute myeloid leukemia (AML). However, many patients eventually relapse due to the persistence of low numbers of residual leukemic cells that are undetectable by conventional cytomorphologic criteria (minimal residual disease [MRD]). Using immunophenotypic multiparametric flow cytometry, we have investigated in sequential studies (diagnosis and follow-up) the impact of MRD detection on the outcome of 53 AML patients that had achieved morphologic remission with standard AML protocols and displayed at diagnosis an aberrant phenotype. Patients were studied at diagnosis with a panel of 35 monoclonal antibodies in triple staining combinations for detection of aberrant or uncommon phenotypic features. According to these features, a patient's probe was custom-built at diagnosis for the identification of possible residual leukemic cells during follow-up. The level of MRD at the end of induction and intensification therapy correlated with the number of relapses and relapse-free survival (RFS). Thus, patients with more than 5 × 10−3 residual cells (5 residual cells among 1,000 normal bone marrow [BM] cells) identified as leukemic by immunophenotyping in the first remission BM showed a significant higher rate of relapse (67% v 20% for patients with less than 5 × 10−3 residual cells; P = .002) and a lower median RFS (17 months v not reached; P = .01). At the end of intensification, with a cut-off value of 2 × 10−3 leukemic cells, AML patients also separated into two distinct groups with relapse rates of 69% versus 32% (P = .02), respectively, and median RFS of 16 months versus not reached (P = .04). In addition, overall survival was also significantly related to the level of residual cells in the marrow obtained at the end of induction and particularly after intensification therapy (P = .008). Furthermore, we have explored whether residual disease was related with the functional expression of multidrug resistance (MDR-1) at diagnosis as assessed by the rhodamine-123 assay. Patients with ≥5 × 10−3 residual leukemic cells at the end of induction therapy had a significantly higher rhodamine-123 efflux (mean, 56% ± 24%) than those with less than 5 × 10−3 residual cells (mean, 32% ± 31%; P = .04). Finally, multivariate analysis showed that the number of residual cells at the end of induction or intensification therapy was the most important prognostic factor for prediction of RFS. Overall, our results show that immunophenotypical investigation of MRD strongly predicts outcome in patients with AML and that the number of residual leukemic cells correlates with multidrug resistance.

Immunophenotyping Investigation of Minimal Residual Disease Is a Useful Approach for Predicting Relapse in Acute Myeloid Leukemia Patients

AbstractA high complete remission rate is currently achieved in patients with acute myeloid leukemia (AML). However, many patients eventually relapse due to the persistence of low numbers of residual leukemic cells that are undetectable by conventional cytomorphologic criteria (minimal residual disease [MRD]). Using immunophenotypic multiparametric flow cytometry, we have investigated in sequential studies (diagnosis and follow-up) the impact of MRD detection on the outcome of 53 AML patients that had achieved morphologic remission with standard AML protocols and displayed at diagnosis an aberrant phenotype. Patients were studied at diagnosis with a panel of 35 monoclonal antibodies in triple staining combinations for detection of aberrant or uncommon phenotypic features. According to these features, a patient's probe was custom-built at diagnosis for the identification of possible residual leukemic cells during follow-up. The level of MRD at the end of induction and intensification therapy correlated with the number of relapses and relapse-free survival (RFS). Thus, patients with more than 5 × 10−3 residual cells (5 residual cells among 1,000 normal bone marrow [BM] cells) identified as leukemic by immunophenotyping in the first remission BM showed a significant higher rate of relapse (67% v 20% for patients with less than 5 × 10−3 residual cells; P = .002) and a lower median RFS (17 months v not reached; P = .01). At the end of intensification, with a cut-off value of 2 × 10−3 leukemic cells, AML patients also separated into two distinct groups with relapse rates of 69% versus 32% (P = .02), respectively, and median RFS of 16 months versus not reached (P = .04). In addition, overall survival was also significantly related to the level of residual cells in the marrow obtained at the end of induction and particularly after intensification therapy (P = .008). Furthermore, we have explored whether residual disease was related with the functional expression of multidrug resistance (MDR-1) at diagnosis as assessed by the rhodamine-123 assay. Patients with ≥5 × 10−3 residual leukemic cells at the end of induction therapy had a significantly higher rhodamine-123 efflux (mean, 56% ± 24%) than those with less than 5 × 10−3 residual cells (mean, 32% ± 31%; P = .04). Finally, multivariate analysis showed that the number of residual cells at the end of induction or intensification therapy was the most important prognostic factor for prediction of RFS. Overall, our results show that immunophenotypical investigation of MRD strongly predicts outcome in patients with AML and that the number of residual leukemic cells correlates with multidrug resistance.

Leukemic Burden in Subpopulations of CD34+ Cells Isolated From the Mobilized Peripheral Blood of α-Interferon-Resistant or -Intolerant Patients With Chronic Myeloid Leukemia

We attempted to determine the frequency of normal hematopoietic stem cells (HSC) and contaminating leukemic cells in mobilized peripheral blood (MPB) collected from chronic myeloid leukemia (CML) patients, intolerant of alpha-interferon or with interferon-resistant disease. A total of 14 MPB samples, six from patients in chronic phase (CP) and eight from patients in accelerated phase or blast crisis (AP/BC) were studied. Cytogenetic analysis of MPB collected from AP/BC patients showed that 100% of the cells were Ph+, whereas cells from four of five CP MPB were Ph-. By contrast, fluorescence in situ hybridization (FISH) analysis of CP MPB showed a mean frequency of 14.7% Ph+ cells, while AP/BC MPB contained 39.2% Ph+ cells. In an attempt to purify normal HSC, subpopulations of the MPB CD34+ cells were isolated based on expression of the Thy-1 antigen (CDw90). The mean Ph+ cell frequency as determined by FISH within the CD34+Thy-1+Lin- and CD34+Thy-1-Lin- populations from CP patients was 19.2% and 33.9%, respectively. In the AP/BC patients, levels of residual leukemic cells were significantly greater with mean Ph+ cell frequencies of 59.2% and 72.7% for the CD34+Thy-1+Lin- and CD34+Thy-1-Lin- fractions, respectively. The frequency of cobblestone area forming cells (CAFC) was used as a means of quantitating the numbers of functional HSC within these cell subpopulations. The mean CAFC frequency was 1 of 19 for the CD34+Thy-1+Lin- cells as compared with 1 of 133 for the Thy-1-fraction indicating a higher frequency of primitive progenitor cells in the Thy-1+ subpopulation. CD34+ cell subsets from two patients were also injected into SCID-hu bone assays to determine the in vivo behavior of these cell populations. After 8 weeks, multilineage donor engraftment was observed in these grafts. FISH analysis of the donor cells within the grafts showed that 55.3% and 60.0% of the cells were Ph+. We conclude that unfractionated MPB from this patient population is not leukemia-free and that the CD34+Thy-1+Lin- cell subpopulation, although predominantly enriched for normal HSC, still contains substantial numbers of residual leukemic cells.

Value of PCR analysis for long term survivors after allogeneic bone marrow transplant for chronic myelogenous leukemia: a comparative study.

At present, allogeneic bone marrow transplantation (BMT) seems to be the only curative treatment for patients with chronic myeloid leukemia (CML). The mechanisms of this therapeutic approach probably include anti-leukemic immune response or "graft-versus-leukemia" (GVL) reaction against leukemic cell clones, since even aggressive chemotherapy is not sufficient per se to cure this type of disease. In many patients, allogeneic BMT results in the disappearance of the Ph chromosome; this negativity does not exclude the presence of residual leukemic cells since sensitivity of cytogenetic analysis is low. The chimeric BCR-ABL gene in CML is a tumor-specific marker that is well suited to allowing detection of low numbers of residual leukemic cells through the extremely sensitive detection method of polymerase chain reaction (PCR). Using this method, 1 leukemic cell in 10(5) or 10(6) normal cells can be detected. Many studies have evaluated the clinical significance and predictive value of BCR-ABL gene rearrangement detected by PCR assay after BMT, most often on heterogeneous populations. Results from such studies have been conflicting. We have conducted a review of the literature, focussing on long-term survivors (> 3 years from BMT) of CML to determine the value of PCR in such patients (n = 183). With the consideration that such a population is obviously heterogenous, long-term PCR negativity strongly correlated with achieving a cure since no patient relapsed in this population (n = 117). Among the PCR positive patients (n = 66), only 5 (8%) relapsed, suggesting a poor prognostic value for a PCR+ test > 36 months post-transplant. Besides the potential clinical value of these data, they contribute to the understanding of the biology of the disease and may suggest a crucial role for the long term GVL effect of marrow transplantation.

PERSISTENCE OF bcr-abl GENE EXPRESSION FOLLOWING BONE MARROW TRANSPLANTATION FOR CHRONIC MYELOGENOUS LEUKEMIA IN CHRONIC PHASE

The bcr-abl RNA transcript is the molecular counterpart of the Philadelphia chromosome and is detectable by an extremely sensitive polymerase chain reaction assay in most patients with chronic myelogenous leukemia. To determine the effectiveness of ablative radiochemotherapy and bone marrow transplantation in eradicating molecular evidence of the malignant clone, we assayed for bcr-abl RNA expression in specimens from 19 patients with CML in chronic phase (CP) who have survived for at least one year post-BMT. We correlated these results with the patients' remission status based on cytogenetic analysis and BM morphology, and with evidence of mixed hematopoietic chimerism by analysis of RBC antigen and DNA restriction fragment length polymorphism patterns. Thirteen of the 19 patients had detectable bcr-abl RNA at some time following BMT. Twelve of these patients have remained in remission by morphologic and karyotypic criteria from 16.6 to 63.7 months following BMT. One of these 13 patients relapsed both by cytogenetic and clinical criteria at 28.1 months after BMT. Six of these 13 patients are still positive at the time of their most recent analysis. Only two patients have evidence for mixed chimerism of normal hematopoietic elements by either RBC antigen or DNA RFLP patterns. These results suggest that, in some patients transplanted for CML in CP, small numbers of residual leukemic cells may persist or reappear transiently without leading to clinical relapse. The definition of complete remission in CML may need to be revised in light of the enhanced ability to detect minimal residual disease by PCR technology.

Detection of minimal disease in hematopoietic malignancies of the B-cell lineage by using third-complementarity-determining region (CDR-III)-specific probes.

Approximately 80% of hematopoietic malignancies of the B-cell lineage carry only one or two immunoglobulin heavy chain gene rearrangements indicating their clonal origin. These rearrangements due to the recombination of various variable, diversity, and joining regions of the heavy-chain gene segments during B-cell commitment result in a region called complementarity-determining region III (CDR-III). This region, which encompasses the diversity region of the heavy-chain segment, because of extensive somatic mutations, provides a DNA-encoded signature specific for each B-cell clone. CDR-III sequences were obtained from DNA of pre-B-cell acute lymphoblastic leukemia by using suitable primers and the polymerase chain reaction. The sequences were used to generate diagnostic probes that hybridized only to the amplified CDR-III of leukemic cells from which the sequences were derived. With these probes, leukemic cells could be detected when diluted 1:10,000 with other cells. By cloning the amplified CDR-III into recombinant libraries residual leukemic cells were accurately quantitated in bone-marrow samples from repeated relapses and remissions in one case of acute lymphoblastic leukemia. During a clinical remission lasting greater than 7 mo, malignant cells were present in marrow at greater than 1 per 1000 cells. These findings indicate that custom-made diagnostic probes will be useful in accurate quantitation of malignant cells in acute lymphoblastic leukemia patients in clinical remission and will allow investigation of the biological significance of low or high numbers of residual leukemic cells in evolution of that disease.

High-dose cytosine arabinoside as the initial treatment of poor-risk patients with acute nonlymphocytic leukemia: a Leukemia Intergroup Study.

Sixty-seven patients with newly diagnosed acute nonlymphocytic leukemia (ANLL) who were considered to be poor candidates for treatment with cytosine arabinoside (ara-C)/anthracycline antibiotic therapy were treated with high-dose ara-C (HDara-C) remission induction therapy. Thirty-four of the 67 patients had a hematologic disorder before developing acute leukemia or had a history of exposure to marrow toxins, 23 patients were greater than 70 years old, and 10 patients had medical problems that were felt to be a contraindication to therapy with an anthracycline antibiotic. Forty-two percent of patients entered complete remission (CR), whereas 22% failed to enter remission because of persistent leukemia. Treatment was associated with substantial toxicity varying from nausea and vomiting to irreversible cerebellar toxicity. Thirty-four percent of patients died during therapy. Poor performance status, a low serum albumin, and a low platelet count were associated with death during remission induction therapy, whereas a high pretherapy leukemic cell mass and a large number of residual leukemic cells in the marrow after six days of therapy were associated with treatment failure due to persistent leukemia.

Detection of residual acute lymphoblastic leukemia cells in cultures of bone marrow obtained during remission.

We used a semisolid culture assay to quantitate leukemia cells in the bone marrow of patients with childhood acute lymphoblastic leukemia (ALL). In bone marrow cultures from 40 patients with newly diagnosed disease, the colonies that developed in vitro consisted of lymphoblasts with the same surface markers and abnormal karyotype as the original diagnostic marrow specimens. We also studied marrow cultures from 13 patients in chemotherapy-induced remission; 6 of these, including 1 obtained from a patient during successful engraftment after marrow transplantation, also yielded lymphoblast colonies in culture, with the same immunologic phenotype or abnormal karyotype as the original leukemic marrow. Four of these patients, including the one who underwent marrow transplantation, relapsed within 2 to 30 months of the abnormal cultures; the other two are still in remission, one of them 30 months after diagnosis. Bone marrow cultures from eight normal controls and from the other seven patients in remission did not yield lymphoblast colonies; all seven of the latter are still in remission. This assay appears to allow detection of small numbers of residual leukemic cells. We conclude that the technique will be valuable in monitoring the efficacy of chemotherapy and allogeneic bone marrow transplantation in acute lymphoblastic leukemia, as well as in evaluating the quality of purged marrow for autologous marrow transplantation.

Total body irradiation of chronic lymphocytic leukemia: incidence and duration of remission.

The results of total body irradiation for 17 consecutive patients with symptomatic (active) chronic lymphocytic leukemia who had received no prior therapy are reviewed. All patients were given an induction course of treatment and subsequently observed without maintenance therapy to determine the duration of remission. Using a described classification to measure therapeutic response, 16 of the 17 patients were evaluated to have had favorable clinical responses. Eight of these responders had complete disappearance of palpable disease and symptoms, correction of anemia when present, and restoration of the white blood count and differential count to normal following completion of therapy (Type I response). The remaining 8 responders had less complete but definite improvement both objectively and subjectively (Type II response). The duration of unmaintained remission was measured from completion of induction therapy to relapse, the latter being defined as any evidence of recurring disease, even though asymptomatic. The median duration of remission was significantly longer for the Type I (19 months) as compared to the Type II (4 months) responders. This parallels the experience with both experimental L1210 and human acute leukemia wherein the duration of unmaintained remission is inversely related to the number of residual leukemic cells surviving treatment. The ability to achieve these high quality remissions offers some prospect of materially improving both the quality of life and survival time of chronic lymphocytic leukemia patients in the future.