Detection of minimal residual disease in acute lymphoblastic leukemia: the St Jude experience.
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Flow Cytometry↗
Biomedical subjects
D Campana
Publications and source records attributed to D Campana.
Epidemiology, clinical features and diagnosis of gastroenteropancreatic endocrine tumours.
Gastroenteropancreatic (GEP) neoplasms originate from any of the various cell types belonging to the neuroendocrine system. A general characteristic of GEP endocrine tumours is that the vast majority produce and secrete a multitude of peptide hormones and amines. Many patients with malignant metastasising tumours present clinical symptoms related to hormone hyperproduction. These include the so-called carcinoid syndrome, characterised by flushing, diarrhoea, wheezing and right heart disease, which is predominantly associated with the serotonin- and tachykinins-producing carcinoids of the midgut. Several types of syndrome associated with GEP endocrine tumors are caused by overproduction of a specific hormone. For instance, the well-known Zollinger-Ellison syndrome is gastrin-mediated. The so-called 'insulinoma syndrome' depends on excessive production of insulin and proinsulin, resulting in hypoglycemia. The 'glucagonoma syndrome' is characterised by necrolytic migratory erythema, diabetes and diarrhoea. The Verner-Morrison syndrome, which is brought about by high circulating levels of vasointestinal peptide (VIP). produces severe secretory diarrhoea. Finally the 'somatostatinoma syndrome' involves gallbladder dysfunction and gallstones, diarrhoea with or without steatorrhea, and impaired glucose tolerance. The biochemical diagnosis of endocrine digestive tumors is based on general and specific markers. The best general markers are chromogranin A (CgA) and pancreatic polypeptide (PP). Specific markers for endocrine tumors include insulin, gastrin, glucagon, vaso intestinal polypeptide (VIP), somatostatin and the primary cathabolic product of serotonin, 5-hydroxyndoleacetic acid (5-HIAA). Localisation procedures commonly applied, in the diagnosis of endocrine tumours include ultrasound (US), computed tomography (CT) and somatostatin receptor scintigraphy (SRS).
Biomarkers, Tumor↗
Clinical importance of minimal residual disease in childhood acute lymphoblastic leukemia.
By using rapid flow cytometric techniques capable of detecting one leukemic cell in 10(4) normal cells, we prospectively studied minimal residual disease (MRD) in 195 children with newly diagnosed acute lymphoblastic leukemia (ALL) in clinical remission. Bone marrow aspirates (n = 629) were collected at the end of remission induction therapy and at 3 intervals thereafter. Detectable MRD (ie, > or = 0.01% leukemic mononuclear cells) at each time point was associated with a higher relapse rate (P < .001); patients with high levels of MRD at the end of the induction phase (> or = 1%) or at week 14 of continuation therapy (> or = 0.1%) had a particularly poor outcome. The predictive strength of MRD remained significant even after adjusting for adverse presenting features, excluding patients at very high or very low risk of relapse from the analysis, and considering levels of peripheral blood lymphoblasts at day 7 and day 10 of induction therapy. The incidence of relapse among patients with MRD at the end of the induction phase was 68% +/- 16% (SE) if they remained with MRD through week 14 of continuation therapy, compared with 7% +/- 7% if MRD became undetectable (P = .035). The persistence of MRD until week 32 was highly predictive of relapse (all 4 MRD(+) patients relapsed vs 2 of the 8 who converted to undetectable MRD status; P = .021). Sequential monitoring of MRD by the method described here provides highly significant, independent prognostic information in children with ALL. Recent improvements in this flow cytometric assay have made it applicable to more than 90% of all new patients. (Blood. 2000;96:2691-2696)
Adolescent↗
Immunophenotyping of leukemia.
Modern immunophenotyping of hematological malignancies by flow cytometry is assisted by a wide array of easily accessible monoclonal antibodies, by antibodies conjugated to diverse fluorochromes, and by reliable techniques for cell membrane permeabilization. Simultaneous assessment of multiple surface and intracellular markers at diagnosis reduces the number of cells required, helps the identification of the malignant cells and determines the degree of immunophenotypic heterogeneity of the malignant cell population. A few critical markers are sufficient to establish the lineage association in the majority of cases of acute and chronic leukemias and lymphomas. More extensive immunophenotyping can provide information about the cells' stage of differentiation, assess the expression of prognostically important features, and determine clonality. The identification of leukemia-associated immunophenotypes can be used for monitoring minimal residual disease during therapy. The presence of cells expressing these phenotypes in patients who are in clinical remission is associated with an increased risk of relapse.
Acute Disease↗
CD38 ligation inhibits normal and leukemic myelopoiesis.
CD38 is a transmembrane molecule whose expression varies during hematopoietic cell differentiation. We used stroma-supported cultures of human myeloid cells to assess the effects of CD38 ligation on myeloid differentiation. In 8 experiments with CD34(+ )cells purified from normal bone marrow or cord blood, flow cytometry used with antibodies to CD34 and myeloperoxidase (MPO) identified 4 cell populations after 7 days of culture. Addition of anti-CD38 (T16) to the cultures induced a profound reduction of the most mature (CD34(-)MPO(++)) cell population, which includes promyelocytes, myelocytes and metamyelocytes; mean (+/- SD) cell recovery was 12.8% +/- 9.8% of that in parallel cultures with an isotype-matched control antibody. The suppressive effect of CD38 ligation on phenotypically more immature normal cells was inconsistent but generally less pronounced. Recovery of CD34(++)MPO(-) cells was 63.3% +/- 24.4%, recovery of CD34([+/-] )MPO(- )cells was 95.3% +/- 35.1%, and recovery of CD34(-)MPO(+) cells was 42.0% +/- 18.7% of that in control cultures. However, anti-CD38 suppressed recovery of cells obtained from 6 patients with CD38(+) acute myeloid leukemia; after 7-day cultures, cell recovery was 25.2% +/- 21.7% of that in control cultures. Cell recovery was also reduced by F(ab')(2) or Fab fragments of anti-CD38. CD38 ligation dramatically suppressed recovery of murine 32D myeloid cells transfected with human CD38 and cocultured with stroma (3.8% +/- 7.3%; n = 7). CD38 ligation of CD38( + )32D cells also induced cell aggregation, tyrosine kinase activity, and Ca(++) influx. We conclude that CD38 mediates signals that culminate in suppression of myeloid cell growth and survival. (Blood. 2000;95:535-542)
ADP-ribosyl Cyclase↗
Simple methods for the rapid exchange of flow cytometric data between remote centers.
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Cancer Care Facilities↗
New definition of remission in childhood acute lymphoblastic leukemia.
The extent of clearance of leukemic cells from the blood or bone marrow during the early phase of therapy is an independent prognostic factor in acute lymphoblastic leukemia (ALL). Several methods are available to measure the minimal residual disease (MRD) remaining after initial intensive chemotherapy. The most promising are flow cytometric detection of aberrant immunophenotypes and polymerase chain reaction analysis of clonal antigen-receptor gene rearrangements. When applied together, these techniques enable one to monitor MRD in virtually all cases of ALL. Patients who achieve an 'immunologic' or 'molecular' remission (ie leukemic involvement of <0.01% of nucleated bone marrow cells at the end of remission induction therapy) are predicted to have a better clinical outcome than patients whose remission is defined solely by morphologic criteria. In studies to date, patients with MRD at a level of 10(-2) or more at the end of induction have fared almost as poorly as those with > or =5% blast cells in the bone marrow (ie induction failures). Sequential monitoring of MRD can improve the clinical utility of risk assessment still further. Additional studies are needed to determine the critical levels of MRD at various times of treatment and whether therapeutic intervention based on MRD findings can improve clinical outcome.
Child↗
Long-term results of Total Therapy studies 11, 12 and 13A for childhood acute lymphoblastic leukemia at St Jude Children's Research Hospital.
We present the long-term results of three consecutive clinical trials (Total Therapy studies 11, 12 and 13A) conducted for children with newly diagnosed acute lymphoblastic leukemia (ALL) between 1984 and 1994. In study 11 (1984-1988), the overall event-free survival rates (+/-1 s.e.) were 71.8 +/- 2.4% and 69.3 +/- 2.4%, and the cumulative risks of isolated central nervous system (CNS) relapse 5.6 +/- 1.2% and 5.9 +/- 1.3%, at 5 and 10 years, respectively. In study 12 (1988-1991), event-free survival rates were 67.6 +/- 3.4% and 61.5+/- 9.0%, and isolated CNS relapse rates were 10.4 +/- 2.3% and 10.4 +/- 2.3%, respectively. Early intensive intrathecal therapy in study 13A (1991-1994) has yielded a very low 5-year isolated CNS relapse rate of 1.2 +/- 0.9%, boosting the 5-year event-free survival rate to 76.9 +/- 3.3%. Factors consistently associated with an adverse prognosis included male sex, infant or adolescent age group, leukocyte count >100 x 10(9)/l, nonhyperdiploidy karyotype and poor early response to treatment. Risk classification based on age and leukocyte count had prognostic significance in B-lineage but not T-lineage ALL. Early therapeutic interventions or modifications for patients with specific genetic abnormalities or persistent minimal residual leukemia may further improve long-term results.
Central Nervous System Neoplasms↗
CD38 in hematopoiesis.
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ADP-ribosyl Cyclase↗
An essential role for BLNK in human B cell development.
The signal transduction events that control the progenitor B cell (pro-B cell) to precursor B cell (pre-B cell) transition have not been well delineated. In evaluating patients with absent B cells, a male with a homozygous splice defect in the cytoplasmic adapter protein BLNK (B cell linker protein) was identified. Although this patient had normal numbers of pro-B cells, he had no pre-B cells or mature B cells, indicating that BLNK plays a critical role in orchestrating the pro-B cell to pre-B cell transition. The immune system and overall growth and development were otherwise normal in this patient, suggesting that BLNK function is highly specific.
Adaptor Proteins, Signal Transducing↗
Detection of minimal residual disease in acute leukemia by flow cytometry.
Patients with acute leukemia in clinical remission may still have up to 10(10) residual malignant cells (the upper limit of detection by standard morphologic techniques). Sensitive techniques to detect minimal residual disease (MRD) may allow better estimates of the leukemia burden and help the selection of appropriate therapeutic strategies. Flow cytometry and polymerase chain reaction have emerged as the most promising methods for detecting submicrospopic levels of leukemia. Flowcytometric detection of MRD is based on the identification of immunophenotypic combinations expressed on leukemic cells but not on normal hematopoietic cells. It affords the detection of one leukemic cell among 10,000 normal bone marrow cells, and can be currently applied to at least two thirds of all patients with acute leukemia. Prospective studies in large series of patients have demonstrated a strong correlation between MRD levels during clinical remission and treatment outcome. Therefore, MRD assays can be reliably used to assess early response to treatment and predict relapse. In this review, we discuss methodologic aspects and clinical results of flowcytometric detection of MRD in patients with acute leukemia.
Adult↗
CD38-mediated signaling events in murine pro-B cells expressing human CD38 with or without its cytoplasmic domain.
To elucidate the signaling mechanism of CD38 (a transmembrane molecule highly expressed in immature hemopoietic cells), we transfected Ba/F3 murine pro-B cells with a cDNA encoding human CD38. CD38 ligation with anti-CD38 Abs caused rapid, transient, dose-dependent tyrosine phosphorylation of several proteins, including the tyrosine kinase TEC and the adaptor molecule CBL, and association of tyrosine-phosphorylated proteins with phosphatidylinositol 3-kinase p85. Exposure to anti-CD38 Abs or their F(ab')2 and Fab also induced tight aggregation of CD38-transfected Ba/F3 cells, which appeared to be Ca2+ and Mg2+ independent and did not involved LFA-1. Aggregation was abrogated by addition of the tyrosine kinase inhibitor herbimycin A and was delayed by the phosphatidylinositol 3-kinase inhibitor wortmannin, suggesting a link between biochemical events and cellular effects induced by CD38. Cell aggregation was accompanied by a decrease in cell recovery. After 3 days of culture on bone marrow-derived stroma, the mean (+/-SD) cell recovery in the presence of anti-CD38 (T16) was 10.5 +/- 9.2% (n = 7) of that in parallel cultures with an isotype-matched nonreactive Ab. Finally, CD38 ligation in Ba/F3 cells expressing a mutant human CD38 lacking the cytoplasmic domain induced tyrosine phosphorylation with intensity and kinetics similar to those seen with the entire protein. It also induced cell aggregation and decreased cell recovery. We conclude that CD38 triggers remarkably similar signaling pathways in human and murine immature B cells. This signaling is independent of the CD38 cytoplasmic domain, suggesting the existence of accessory transmembrane molecules associated with CD38.
ADP-ribosyl Cyclase↗
Hyperdiploid acute lymphoblastic leukemia with 51 to 65 chromosomes: a distinct biological entity with a marked propensity to undergo apoptosis.
To determine the cellular basis for the excellent clinical outcome of hyperdiploid acute lymphoblastic leukemia (ALL), defined by a modal chromosome number of 51 to 65, we assessed the growth potential of leukemic cells from 129 children with newly diagnosed ALL. Flow cytometric analysis was used to compare leukemic cell recoveries at the beginning and at the end of 7-day cultures on allogeneic bone marrow-derived stromal layers. The median percentage of cell recovery after culture was 91% (range, <1% to 550%). Among the 25 hyperdiploid cases, only two had cell recoveries above the median value, compared with 63 of 104 cases with different ploidies (P <.001); 21 had recoveries within the first quartile, in contrast to only 12 of the 104 other cases. Cell recoveries in the 16 cases with duplications of chromosomes 4 and 10, a feature previously associated with a superior outcome, were all within the first quartile. Flow cytometric studies indicated that rapid induction of apoptosis was the underlying cause of low cell recoveries in cases with hyperdiploidy. The demise of hyperdiploid cells on stroma was not due to failure to adhere with stromal elements (as shown by electron microscopy) or to deficiencies of interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-6, IL-7, IL-11, stem-cell factor, interferon- (IFN-), tumor necrosis factor- (TNF-), or to combinations of these cytokines. Inactivation of IL-4, IFN- and TNF-, which if secreted by stromal layers could be toxic to ALL cells, failed to improve the survival of hyperdiploid blasts. We conclude that leukemic cells bearing 51 to 65 chromosomes have a marked propensity to undergo apoptosis. The stringent survival requirements of these cells, together with their potentially higher sensitivity to antileukemic drugs, may well account for the high cure rates achieved in patients with this form of ALL.
Adolescent↗
Tandem application of flow cytometry and polymerase chain reaction for comprehensive detection of minimal residual disease in childhood acute lymphoblastic leukemia.
Children with acute lymphoblastic leukemia (ALL) with > or = 0.01% leukemic cells in the bone marrow after remission induction are at a greater risk of relapse. The most promising methods of detecting minimal residual disease (MRD) are flow cytometric identification of leukemia-associated immunophenotypes and polymerase chain reaction (PCR) amplification of antigen-receptor genes. However, neither assay can be applied to all patients. Moreover, both assays carry the risk of false-negative findings due to clonal evolution. The simultaneous use of both assays might resolve these problems, but the correlation between the methods is unknown. We studied serial dilutions of normal and leukemic cells by flow cytometry and PCR amplification of IgH genes and found the two methods highly sensitive (one leukemic cell among 10(4) or more normal cells), accurate (r2 was 0.999 for flow cytometry and 0.960 for PCR by regression analysis) and concordant (r2 = 0.962). We then examined 62 bone marrow samples collected from children with ALL in clinical remission. In 12 samples, both techniques detected MRD levels > or = 1 in 10(4). The percentages of leukemic cells measured by the two methods correlated well (r2 = 0.978). Of the remaining 50 samples, 48 had MRD levels < 1 in 10(4). In only two samples results were discordant: 2 in 10(4) and 5 in 10(4) leukemic cells by PCR but < 1 in 10(4) by flow cytometry. We conclude that immunologic and molecular techniques can be used in tandem for universal monitoring of MRD in childhood ALL.
Child↗
Mutations in Igalpha (CD79a) result in a complete block in B-cell development.
Mutations in Btk, mu heavy chain, or the surrogate light chain account for 85-90% of patients with early onset hypogammaglobulinemia and absent B cells. The nature of the defect in the remaining patients is unknown. We screened 25 such patients for mutations in genes encoding components of the pre-B-cell receptor (pre-BCR) complex. A 2-year-old girl was found to have a homozygous splice defect in Igalpha, a transmembrane protein that forms part of the Igalpha/Igbeta signal-transduction module of the pre-BCR. Studies in mice suggest that the Igbeta component of the pre-BCR influences V-DJ rearrangement before cell-surface expression of mu heavy chain. To determine whether Igalpha plays a similar role, we compared B-cell development in an Igalpha-deficient patient with that seen in a mu heavy chain-deficient patient. By immunofluorescence, both patients had a complete block in B-cell development at the pro-B to pre-B transition; both patients also had an equivalent number and diversity of rearranged V-DJ sequences. These results indicate that mutations in Igalpha can be a cause of agammaglobulinemia. Furthermore, they suggest that Igalpha does not play a critical role in B-cell development until it is expressed, along with mu heavy chain, as part of the pre-BCR.
Agammaglobulinemia↗
IL-2 adenovector-transduced autologous tumor cells induce antitumor immune responses in patients with neuroblastoma.
In many different murine models, the immunogenicity of tumor cells can be increased by transduction with a range of immunostimulatory genes, inducing an immune response that causes regression of pre-existing unmodified tumor cells. To investigate the relevance of these animal models to pediatric malignancy, we used autologous unirradiated tumor cells transduced with an adenovirus-IL-2 to immunize 10 children with advanced neuroblastoma. In a dose-escalation study, we found that this tumor immunogen induced a moderate local inflammatory response consisting predominantly of CD4(+) T lymphocytes, and a systemic response, with a rise in circulating CD25(+) and DR+ CD3(+) T cells. Patients also made a specific antitumor response, manifest by an IgG antitumor antibody and increased cytotoxic T-cell killing of autologous tumor cells. Clinically, five patients had tumor responses after the tumor immunogen alone (one complete tumor response, one partial response, and three with stable disease). Four of these five patients were shown to have coexisting antitumor cytotoxic activity, as opposed to only one of the patients with nonresponsive disease. These results show a promising correlation between preclinical observations and clinical outcome in this disease, and support further exploration of the approach for malignant diseases of children.
Adenoviridae↗
Interleukin-2 gene-modified allogeneic tumor cells for treatment of relapsed neuroblastoma.
Tumor cells that have been genetically modified to express immunostimulatory genes will induce effective antitumor responses in a range of syngeneic animal models. For human applications, transduced autologous tumor cell lines are often difficult or impossible to prepare, so that there are strong incentives for substituting a standardized allogeneic tumor cell line. However, such lines may be inferior immunogens if they differ from host tumors in the antigens they express. We have evaluated the safety, immunostimulatory, and antitumor activity of an interleukin-2-secreting allogeneic neuroblastoma cell line in 12 children with relapsed stage IV neuroblastoma. They received two to four subcutaneous injections of cells in a dose-escalating schedule, up to a maximum of 10(8) cells per injection. There was induration and pruritus at the injection site, and skin biopsies revealed mild panniculitis with CD3+ cells surrounding scanty residual tumor cells. There was a limited but significant peripheral monocytosis. No patient showed any increase in direct cytotoxic effector function against the immunizing cell line, but 3 patients had a rise in the frequency of neuroblastoma-reactive cytotoxic T lymphocyte precursor cells. One child had > 90% tumor response (PR), 7 had stable disease, and 4 had progressive disease in response to vaccine alone. Although these results offer some encouragement for the continued pursuit of allogeneic vaccine strategies in human cancer, the antitumor immune responses we observed are inferior to those obtained in an earlier immunization study using autologous neuroblastoma cells. Hence, we suggest that this earlier approach remains preferable, its difficulties notwithstanding.
Cancer Vaccines↗
Immunological detection of minimal residual disease in children with acute lymphoblastic leukaemia.
BACKGROUND: The clinical significance of submicroscopic levels of leukaemic cells in bone-marrow aspirates from children with acute lymphoblastic leukaemia (ALL) remains controversial. We prospectively determined the frequency and prognostic importance of minimal residual disease detected by a rapid immunological assay in bone-marrow aspirates of children with ALL. METHODS: 158 children with newly diagnosed ALL received 6 weeks of remission-induction chemotherapy. Once complete clinical remission was attained the patients received 2 weeks of consolidation therapy followed by continuation therapy. Bone-marrow aspirates were collected after induction therapy and during weeks 14, 32, and 56 of continuation therapy, and then at 120 weeks (end of therapy). Cells with leukaemia-associated immunophenotypes were investigated by multiparameter flowcytometry capable of detecting one leukaemic cell among 10,000 normal cells. FINDINGS: The proportion of patients with detectable leukaemic cells was 23% at remission induction and 17% at week 14 of continuation therapy, decreasing to 5% and 4% at weeks 32 and 56. None of the 65 samples examined at completion of therapy (week 120) showed evidence of disease. Detectable residual disease at the end of remission induction correlated with adverse genetic abnormalities--the Philadelphia chromosome and MLL gene rearrangements--but not with other presenting features. Detectable leukaemia was associated with subsequent relapse regardless of the time at which bone-marrow samples were examined (p < 0.002 for all comparisons). For example, 3-year cumulative incidence (SE) of relapse in patients with and without detectable leukaemia at remission induction was 32.5% (10.6) and 7.5% (4.0), respectively (p < 0.001); for tests done at week 14, it was 42.1% (14.6) and 6.6% (3.5), p < 0.001. These correlations remained significant after adjusting for adverse presenting features. A higher degree of marrow infiltration by leukaemic cells (> or = 0.1%) in week 14 samples identified a subset of patients with an especially poor prognosis. INTERPRETATION: Immunological detection of residual leukaemic cells at any point in the treatment course is a powerful predictor of relapse in children with ALL. Alternative treatment should be considered for cases with persistent disease beyond the first 3 months of continuation therapy.
Bone Marrow↗