Prognostic significance of detection of monoclonality in remission marrow in acute lymphoblastic leukemia in childhood. Australian and New Zealand Children's Cancer Study Group.

Techniques based on the polymerase chain reaction (PCR) to detect rearrangement of the immunoglobulin or T-cell receptor genes can detect residual disease in leukemia and hence have the potential to improve prognosis and treatment. Such techniques may involve either detection of monoclonality, which is simple and quick but has limited sensitivity, or specific detection of the leukaemic clone, which is complex and time-consuming but has high sensitivity. The PCR was used to detect monoclonal rearrangements of the immunoglobulin heavy chain and/or T-cell receptor gamma chain genes in archival marrow specimens from 185 children with acute lymphoblastic leukemia who achieved remission during two consecutive Australasian trials of treatment. A monoclonal rearrangement was detected at diagnosis in 152 (84%) patients and in these patients detection of the same rearrangement in the remission marrow at the end of induction therapy was highly significantly correlated with outcome. There were nine patients in whom polymerase chain reaction showed only the monoclonal rearrangement and eight (89%) relapsed; there were 26 patients in whom PCR showed the leukemic monoclonal rearrangement as well as polyclonal rearrangements from normal lymphocytes and 12 (46%) relapsed; and there were 117 patients in whom only polyclonal rearrangements could be detected and only 29 (25%) relapsed. In patients who relapsed, remissions were shorter in those patients in whom the leukemic rearrangements had been detected in the remission marrow. Treatment in the later trial was more intensive than in the earlier trial, the results were better and the PCR detected the leukemic rearrangement in the remission marrow in significantly fewer patients. We conclude that detection by PCR of the monoclonal gene rearrangement of the leukemic clone in remission marrow indicates that numerous leukemic cells have survived induction therapy and is a good predictor of relapse. However, due to limited sensitivity of the test, failure to detect the leukemic clone by PCR is not a sufficiently good predictor of ultimate cure.

Characterization of clonal immunoglobulin heavy chain and I cell receptor gamma gene rearrangements during progression of childhood acute lymphoblastic leukemia.

Instability of antigen receptor gene rearrangements during progression of acute lymphoblastic leukemia (ALL) has important implications for polymerase chain reaction (PCR)-based techniques using these genes for the detection of minimal residual disease (MRD). Antigen receptor gene instability may lead to false negative results in bone marrow samples taken during remission. Utilizing the PCR and consensus primers for rearranged immunoglobulin heavy chain (IgH) and T cell receptor gamma (TCR gamma) gene sequences, we analyzed the bone marrow samples at diagnosis and first relapse for 37 children with ALL. The incidence of clonal evolution at the IgH locus was 9/33 (27%) and at the TCR gamma locus 1/15 (7%). In four of the nine patients with clonal evolution at the IgH locus, the sequence at relapse retained the diversity and joining region (D-N-J) sequences from diagnosis. Patients with clonal evolution were characterized by a higher incidence of more than one IgH PCR band at diagnosis and by late relapse (> 18 months from diagnosis). These results suggest that, where possible, patients with more than one IgH PCR rearrangement at diagnosis should be monitored using another antigen receptor gene, such as TCR gamma, since evolution for this gene was found to be a rare event. By combining this approach with a strategy directed at the more stable D-N-J region of the IgH gene, MRD false negativity would have occurred in less than 10% of patients in the present study.

Effect of the Philadelphia chromosome on minimal residual disease in acute lymphoblastic leukemia.

The Philadelphia translocation is associated with a poor prognosis in adults and children with acute lymphoblastic leukemia, even though the majority of patients achieve remission. To test the hypothesis that the translocation leads to drug resistance in vivo, we studied 61 children and 20 adults with acute lymphoblastic leukemia and used the level of minimal residual disease at the end of induction as the measure of drug resistance in vivo. In children the presence of the translocation was associated with a significant increase in residual disease, indicating higher drug resistance in vivo; five of seven Philadelphia-positive children but only five of 54 Philadelphia-negative children had a minimal residual disease level >10(-3), a level which is associated with a high risk of relapse in childhood acute lymphoblastic leukemia of standard risk. By contrast, in adults, residual disease and hence drug resistance was already higher than in children, and the presence of the Philadelphia translocation in seven patients had no obvious additional effect. We conclude that the Philadelphia chromosome may increase resistance to drugs in vivo in children, but not detectably in adults.

The quantitation of haemoglobin A2 and haemoglobin F: a report of the techniques review group of the Thalassaemia Society of Victoria.

A techniques review group was established in 1975 to investigate the reproducibility of tests for HbA2 and HbF for the diagnosis of beta thalassaemia. Over a 3-year period, 22 samples were distributed for analysis by up to 14 laboratories. Of 250 estimations of HbA2, 26 (10.4%) were at variance with the majority result. The largest number of variant results were obtained in samples with a HbA2 close to the upper limit of normal. Of 238 HbF determinations, 29 (12%) were equivocal or at variance with the majority result. It is concluded that the techniques of HbA2 and HbF estimation as currently practised by several laboratories are not sufficiently accurate, and that, therefore, a number of cases of beta thalassaemia are not being diagnosed. It is the opinion of those participating in the study that the survey is of value in providing a continuing review of techniques and a forum for discussion.

The role of filtration in the provision of leukocyte poor red cells to multitransfused patients.

Five techniques for the preparation of leukocyte poor red cells were investigated to compare their efficiency in preventing febrile transfusion reactions with their cost of production, in order to establish a cost-effective transfusion programme for chronically anemic patients. Leukocyte depletion and preparation costs per unit for the products were:--microaggregate filtered red cells (MF): 25% leucocyte removal at a cost of $A2; buffy coat poor red cells (BCP): 70% at $A2; washed buffy coat poor red cells (WBCP): 88% at $A28; cotton wool filtered red cells (CWF): 93% at $A23; reconstituted frozen red cells (FC): 97% at $A80. The 5 products were transfused into 103 thalassemia patients with a documented history of febrile transfusion reactions. Reaction rates, expressed as a percentage of units transfused over a 12 mth period (MF 7%, BCP 0.3%, WBCP 0.1%, CWF 0.1%, FC 0.2%) correlated well with the degree of leukocyte depletion. The cost of CWF was reduced by a further 10% using the filter in line during the transfusion. Serum from these patients was screened for leukocyte and platelet antibodies using microlymphocytotoxic, granulocyte immunofluorescence and platelet immunofluorescence assays. There was no correlation between the antibodies present and the type of leukocyte poor product required by a patient to prevent a febrile reaction. A progressive regimen of transfusion (BCP to CWF to FC as the patient reacts) has now been adopted, with considerable cost saving.

Long-term follow up of patients with transcobalamin II deficiency.

Five cases of transcobalamin II deficiency presenting to our institution were reviewed. A delay in diagnosis often led to acute deterioration. Two patients have long term neurological sequelae. Minimal treatment in these patients may be dangerous. While haematological normality may be maintained, the adequate therapeutic dose of vitamin B-12 to allow normal neurological development and function is not easily determined and damage sustained early in life may be irreversible.

Acute lymphocytic leukemia in children presenting with bone marrow necrosis.

Bone marrow necrosis has been regarded as an indicator of very poor prognosis in malignant disease. The cause and incidence are unknown, and reports of treatment response are few. We describe four children with marrow necrosis at presentation with acute lymphocytic leukemia (ALL), all of whom entered remission with standard treatment showing complete marrow healing. The bleak outlook for patients with marrow necrosis based on early experience in adults with disseminated malignancy does not appear to apply to children with ALL. The incidence of marrow necrosis at diagnosis of childhood ALL is 1%.

Infantile megaloblastosis secondary to maternal vitamin B12 deficiency.

We reviewed six cases of infantile megaloblastosis secondary to maternal vitamin B12 deficiency, the most common cause of infantile megaloblastosis in our institution. Two patients had long-term neurological sequelae, with a further patient remaining abnormal but at short follow-up. In 50% of cases the mother was asymptomatic, with subtle or no peripheral blood abnormalities, having early pernicious anaemia. Any infant which fails to thrive, with progressive neurological deterioration and haematological cytopenias should have their vitamin B12 and folate status rapidly assessed. This is one of the few potentially reversible causes of failure to thrive and neurological deterioration. Early diagnosis and treatment may prevent significant long-term sequelae.

Fetal brain disruption sequence in sisters.

We report two female siblings with the fetal brain disruption sequence. Extensive investigation of both children failed to define a definitive aetiology but clinical and laboratory findings are consistent with a hitherto unknown storage disease. We postulate that the accumulation of a neurotoxic metabolite may be responsible for the disease phenotype observed. This is the first report of recurrence of the fetal brain disruption sequence and supports the existence of a genetic form of this condition. Previous reports have emphasized possible environmental aetiologies. Infants with fetal brain disruption sequence should be investigated exhaustively and, in the absence of definitive evidence of an environmental cause, the possibility of a genetic aetiology should be considered. In some families the recurrence risk may be as high as one in four.

Hemophagocytic reticulosis. A case report with investigations of immune and white cell function.

A 5-month-old child with hemophagocytic reticulosis is described. Investigations revealed a grossly defective PHA response of the patient's lymphocytes which improved with chemotherapy. Defective glucose oxidation by phagocytosing cells and low IgA levels were demonstrated at diagnosis and have persisted despite chemotherapy. HL-A typing and chromosome studies did not reveal maternal lymphocytes in the child's circulation. The patient was treated with vinblastine and prednisolone and remains well after 11 months of treatment.

Normalization of vitamin B12 absorption after ileal resection in children.

Impaired Vitamin B12 absorption after significant ileal resection has been reported to be permanent, although partial recovery after ileal bypass can occur. Three children are presented in whom Vitamin B12 malabsorption returned to normal 6-8 years after ileal resection. This was due probably to adaptation of the remaining small bowel, although spontaneous resolution of bacterial overgrowth is a possible explanation. An abnormal Schilling test after ileal resection does not automatically imply the need for life-long Vitamin B12 injections.

Bronchoalveolar lavage cellularity in healthy children.

The role of bronchoalveolar lavage (BAL) in children remains to be defined, and to date there has been no standardization of acquisition and processing of the BAL fluid. The aim of the present study was to evaluate a standardized protocol for obtaining, processing, and analyzing BAL fluid and to establish reference values for healthy children. Eighteen children 3 mo to 10 yr of age were lavaged with three 1-ml/kg aliquots of normal saline, using a flexible bronchoscope into the right middle lobe. The first aliquot was processed separately; the second and the third were pooled. There was a higher percentage of neutrophils and epithelial cells in the first aliquot. The number of total cells per milliliter (median, Q1 to Q3) in the first aliquot was 60 (45 to 90) x 10(3)/ml; in the pooled sample it was 155 (75 to 257) x 10(3)/ml. Percentages (median, Q1 to Q3) of different cell types in the pooled sample were: macrophages, 91% (84 to 94); lymphocytes, 7.5% (4.7 to 12.8); neutrophils, 1.7% (0.6 to 3.5); eosinophils, 0.15% (0.0 to 0.3). The ratio of helper/cytotoxic T-cells was 0.58 (0.4 to 1). The results of this study demonstrate that BAL, using a standardized protocol adjusting instilled fluid volume by weight, appears to be appropriate in children and that cellular and protein values from healthy children are, apart from differences in lymphocyte subsets, similar to those found in healthy adults.

Isolation of malignant cells from human bone marrow using a discontinuous Percoll gradient.

A technique for examining relatively large volumes of bone marrow for involvement by malignancy is described. The use of discontinuous Percoll gradients offers no advantage over conventional methods in the diagnosis of hematological malignancy. Its usefulness in detecting infiltration by solid tumor is uncertain. Complete exclusion of malignancy from the fraction containing hematologic stem cells in three patients raises the possibility that this technique is a useful adjunct to other methods of marrow purging before autologous marrow rescue in malignant disease.

Deficiency of immunological and phagocytic function in aboriginal children with protein-calorie malnutrition.

Infection, associated with protein-calorie malnutrition (PCM), is a widespread and important health problem in young Aboriginal children. Clinical obervations have suggested these children to have impaired immune resistance to infection. Children were fivided by anthropometric criteria into three groups: moderately malnourished; showing effects of previous PCM; normally nourished. Numbers and function of T and B lymphocytes and neutrophils were measured in these groups to give an assessment of systemic immune resistance. Primary antigen recognition and blastogenic response of T-lymphocytes were significantly impaired in the malnourished groups. Normal or increased numbers of B and T lymphocytes, and normal secondary antibody response to tetanus toxoid inoculations were found in all groups. Serum opsonin levels, C3 concentrations, immunoglobulin levels, neutrophil numbers and phagocytic activity were normal or increased in all groups. The malnourished children showed relative impairment of neutrophil chemotaxis, metabolic response to phagocytosis and intraphagocytic bactericidal activity. The findings suggested that children with moderate or lasting effects of PCM had multiple dificiencies in the funnction of their immune defence mechanism which may profoundly influence the prevalence, chronicity and mortality of infections diseases in Aboriginal communities.

Autologous bone marrow rescue in the treatment of advanced tumors of childhood.

High-dose multiagent chemotherapy followed by autologous marrow rescue was used in the treatment of 13 patients with Stage III or IV childhood tumors. Encouraging results are being obtained in abdominal lymphoma (1/3 complete remissions (CR); rhabdomyosarcoma (2/4 CR); and retinoblastoma (1/2 CR). In neuroblastoma, the results are disappointing, with only one of four patients in CR; this patient developed a lymphoma associated with Epstein-Barr virus infection. Marrow reconstitution was obtained in 11 patients, with recovery of neutrophils to greater than 0.5 x 10(9)/liter between six and 30 days and platelet recovery to greater than 50 x 10(9)/liter between seven and 38 days. Investigations on the numbers of cells or committed granulocyte precursors ()CFU-c's) infused and parameters of hematologic recovery show poor correlation and suggest that a more accurate and reliable assay for the predictability of cryopreserved marrow to reconstitute marrow function within a reasonable time is necessary. Nonhematologic toxicities of high-dose multiagent chemotherapy are the principal dose-limiting factors.

Results of intensive therapy in childhood acute myeloid leukemia, incorporating high-dose melphalan and autologous bone marrow transplantation in first complete remission.

Childhood acute myeloid leukemia (AML) has a poor prognosis with standard chemotherapy. Allogeneic bone marrow transplantation (BMT) in remission improves the outlook only for the one third of patients with sibling donors. Autologous BMT with a lower morbidity and mortality is available to all. In this study, maximum cytoreduction was achieved by intensive early chemotherapy. Final intensification, with autologous BMT was offered to all those remaining in first complete remission (CR). Patients received two induction and two consolidation courses of intensively scheduled chemotherapy. Cytoreduction was assessed on day 14 and remission was assessed after courses 2 and 4. Bone marrow was harvested after recovery from the second consolidation course or after the first maintenance course and separated on a discontinuous percoll gradient before cryopreservation. Twenty-eight of 31 consecutively enrolled patients achieved CR. Three relapsed early and, of the 25 eligible, 24 underwent autologous BMT. Twenty-three patients received high-dose melphalan and 1 received busulphan and cyclophosphamide before autologous BMT at a median of 113 days (range, 86 to 301) after initial CR. Trilineage engraftment occurred in all. Neutrophil recovery to greater than 0.5 x 10(9)/L occurred at a median of 46 days (range, 13 to 92) after autologous BMT. Platelet recovery was delayed, with a median time to achieve greater than 20 x 10(9)/L of 42 days (range, 18 to 215). With a minimum follow up of 25 months following autologous BMT only 3 children have relapsed. The 5-year event-free survival rate (EFS) from diagnosis is 68% (95% confidence interval, 46% to 90%). Five-year EFS following autologous BMT is 87% (95% confidence interval, 67% to 100%). Autologous BMT with high-dose melphalan administration after intensive chemotherapy has produced EFS equivalent to allogeneic BMT and is associated with a strikingly low relapse rate. High-dose melphalan appears to be a valuable agent for conditioning therapy in AML.

Left shift in the peripheral blood count at diagnosis in acute lymphocytic leukemia is significantly correlated with duration of complete remission.

The prognostic significance of a left shift in the peripheral blood at the time of diagnosis of acute lymphocytic leukemia was investigated by a retrospective analysis of 109 patients treated on the same protocol in a single institution. Left shift was defined as the presence of 1% or more of metamyelocytes, myelocytes, or promyelocytes. All peripheral blood films were checked at the time of diagnosis by one of the authors. It was found that the duration of complete remission at 92 mo was 74% in patients with left shift and 42% in those without left shift (p less than 0.05, log-rank test). By Cox regression analysis, only the total white cell count (p less than 0.001) and the presence or absence of left shift (p less than 0.01) were independently significant in determining the proportion of patients in complete remission. Patients with a left shift had a significantly higher granulocyte count at diagnosis (p less than 0.05). We postulate that left shift in the peripheral blood count at the time of diagnosis may be an indirect measure of the total leukemia cell load. It is a new prognostic factor of significance in determining the likely outcome of the disease.

Prospective study of childhood acute lymphocytic leukemia: hematologic, immunologic, and cytogenetic correlations.

We studied the karyotype in 81 consecutively diagnosed children with acute lymphocytic leukemia (ALL) treated at one institution on a randomized treatment protocol. In 75 patients (93%), a morphological cytogenetic result was obtained, and 57 (65%) were successfully G-banded. Of the 75 patients, 46 (61%) showed abnormal chromosomes, mainly hyperdiploidy and pseudodiploidy, and 29 had no detectable abnormality. Our findings confirmed that the karyotype has prognostic significance. Duration of complete remission was 93% at 42 months for patients with high hyperdiploidy (greater than 50). For patients with an apparently normal karyotype, it was 58%; and for patients with structural abnormalities it was 15%. The significance of these findings was confirmed by multivariate analysis, which showed age and karyotype to be the most important determinants of duration of remission.

Outcome prediction in childhood acute lymphoblastic leukaemia by molecular quantification of residual disease at the end of induction.

Methods to detect and quantify minimal residual disease (MRD) after chemotherapy for acute lymphoblastic leukaemia (ALL) could improve treatment by identifying patients who need more or less intensive therapy. We have used a clone-specific polymerase chain reaction to detect rearranged immunoglobulin heavy-chain gene from the leukaemic clone, and quantified the clone by limiting dilution analysis. MRD was successfully quantified, by extracting DNA from marrow slides, from 88 of 181 children with ALL, who had total leucocyte counts below 100 x 10(9)/L at presentation and were enrolled in two clinical trials, in 1980-84 and 1985-89. Leukaemia was detected in the first remission marrow of 38 patients, in amounts between 6.7 x 10(-2) and 9.9 x 10(-7) cells; 26 of these patients relapsed. Of 50 patients with no MRD detected, despite study of 522-496,000 genomes, only 6 relapsed. The association between MRD detection and outcome was significant for patients in each trial. In the first trial, patients relapsed at all levels of detected MRD, whereas in the later trial, in which treatment was more intensive and results were better, the extent of MRD was closely related to the probability of relapse (5 of 5 patients with > 10(-3) MRD, 4 of 10 with 10(-3) to 2 x 10(-5), 0 of 3 with levels below 2 x 10(-5), and 2 of 26 with no MRD detected). Early quantification of leukaemic cells after chemotherapy may be a successful strategy for predicting outcome and hence individualizing treatment in childhood ALL, because the results indicate both in-vivo drug sensitivity of the leukaemia and the number of leukaemic cells that remain to be killed by post-induction therapy.