AIMS—To examine the clinical and
biological features of acute lymphoblastic leukaemia in children with
Down's syndrome (DS), to compare their survival with other children,
and to determine if entry to trials and survival has improved.
METHODS—Examination of presenting
features and response to treatment in patients treated in two
consecutive national trials, MRC UKALL X and XI.
RESULTS—The proportion of children
with DS was significantly higher in UKALL XI (1.9%) than UKALL X
(0.9%). Children with DS tended to be under 10 years and to have the
common ALL subtype. Cytogenetic analysis showed that favourable
features, such as high hyperdiploidy and t(12;21) were less frequent
but also that there was a lack of translocations associated with a poor
prognosis. Children with DS showed no increase in risk of relapse at
any site but their survival and event free survival were inferior to
other children. These results were caused by an increased number of
infective deaths during remission (11% compared to 2%). At five years
overall survival was 73% in DS children compared with 82% in other
children; event free survival was 53% compared to 63% in non-DS children.
CONCLUSIONS—Entry of children with
DS to national trials has increased and survival has improved. However
they remain at risk of relapse and also of treatment related mortality.
These findings emphasise the need for both intensive chemotherapy and
optimal supportive care.
The benefits of achieving a long term event free survival of 60-70% by using increasingly intense treatment regimens must be weighed against the increased risk of treatment toxicity. From 1985 to 1990, 1612 children with childhood acute lymphoblastic leukaemia (ALL) in the UK were treated on MRC UKALL X with intensive induction therapy, central nervous system directed therapy (cranial irradiation and intrathecal methotrexate), and continuing treatment for two years. There was a randomisation to receive blocks of additional intensification treatment at five weeks, 20 weeks, not at all, or both. The five year disease free survival was 71% for children randomised to two blocks of intensification, a 14% improvement on children randomised to no intensification treatment. Treatment related mortality in this national multicentre study has been analysed for induction and first remission (including those after intensification treatment). There were 38 induction deaths, 2.3% and 53 deaths in first remission, 3.3% (including those from a second malignancy). Thirty one (84%) of the induction deaths followed an infection: bacterial in 22 and fungal in nine. Thirty seven infective remission deaths occurred: bacterial in 11, viral in 16, fungal in seven, and three caused by Pneumocystis carinii pneumonia. Ten of these deaths followed a block of intensification treatment. The majority of noninfective remission deaths followed the development of a second tumour. Risk analysis for an induction death showed girls and children with Down's syndrome to be at greater risk. For deaths in first remission analysis showed an increased risk for bone marrow transplant (BMT) patients and children with Down's syndrome. There was no effect of age and leucocyte count for either group. Most significantly when BMT patients were excluded from the analysis, intensification treatment did not increase the risk of remission death.
Between 1980 and 2001, the United Kingdom Medical Research Council Childhood Leukemia Working Party has conducted 4 clinical trial in acute lymphoblastic leukemia, which have recruited a total of 6516 patients. UKALL VIII examined the role of daunorubicin in induction chemotherapy, and UKALL X examined the role of post-induction intensification. Both resulted in major improvement in the outcomes. UKALL XI examined the efficacy of different methods of CNS-directed therapy and the effects of an additional intensification. ALL97, which was initially based on the UKALL X D template (two intensification phases), examined the role of different steroids in induction and different thiopurines through continuing chemotherapy. A reappraisal of results from UKALL XI compared to other cooperative group results led to a redesign in 1999, which subsequently resulted in a major improvement in outcomes. Additionally, ALL97 and 97/99 demonstrated a significant advantage for the use of dexamethasone rather than prednisolone; although the use of 6-thioguanine resulted in fewer relapses, this advantage was offset by an increased incidence of deaths in remission. Over the era encompassed by these four trials there has been a major improvement in both event-free and overall survival for children in the UK with ALL.
acute leukemia; therapy; clinical trial
As part of the Medical Research Council Leukaemia Trial UKALL VIII, 738 unselected children with acute lymphoblastic leukaemia (ALL) had the morphology of their marrow blast cells reviewed by a panel of three haematologists. Ninety four (13%) showed appearances classifiable as type L2 by the French American and British (FAB) cooperative group's criteria, five (0.7%) were typed L3, and the remaining 639 (86%) as L1. Disregarding the patients classified as L3, those with the L2 variant showed an inferior disease free survival to that of the remainder (p less than 0.01), and more of them failed to remit after receiving "standard" remission induction treatment (p less than 0.01). They included an excess of older children (p less than 0.01) with less profound marrow failure at diagnosis, and fewer of them expressed the common ALL antigen (p = 0.05). There was no association between L2 morphology and the diagnostic white cell count, sex, or the presence of a mediastinal mass. These findings confirm earlier reports that FAB L2 ALL is associated with a poor prognosis and that it occurs more commonly in older children. The high remission failure rate is a recent observation and indicates that alternative early treatment may be appropriate for such patients.
Childhood B-cell precursor (BCP) ALL is thought to be caused by a delayed immune response to an unidentified postnatal infection. An association between BCP ALL and HLA class II (DR, DQ, DP) alleles could provide further clues to the identity of the infection, since HLA molecules exhibit allotype-restricted binding of infection-derived antigenic peptides. We clustered >30 HLA-DPB1 alleles into six predicted peptide-binding supertypes (DP1, 2, 3, 4, 6, and 8), based on amino acid di-morphisms at positions 11 (G/L), 69 (E/K), and 84 (G/D) of the DPβ1 domain. We found that the DPβ11-69-84 supertype GEG (DP2), was 70% more frequent in BCP ALL (n=687; P<10−4), and 98% more frequent in cases diagnosed between 3 and 6 years (P<10−4), but not <3 or >6 years, than in controls. Only one of 21 possible DPB1 supergenotypes, GEG/GKG (DP2/DP4) was significantly more frequent in BCP ALL (P=0.00004) than controls. These results suggest that susceptibility to BCP ALL is associated with the DP2 supertype, which is predicted to bind peptides with positively charged, nonpolar aromatic residues at the P4 position, and hydrophobic residues at the P1 and P6 positions. Studies of peptide binding by DP2 alleles could help to identify infection(s) carrying these peptides.
HLA-DPB1; supertypes; BCP ALL; case–control comparison; allele frequency; peptide-binding pockets
The degree of drug-induced neutropenia resulting from a controlled trial (UKALL I) of treatment in acute lymphoblastic leukaemia was analysed. The main agent associated with severe neutropenia was methotrexate, and methotrexate-induced neutropenia was significantly greater in patients who had received craniospinal irradiation. The synergistic toxic effect of irradiation followed by methotrexate treatment seems to have contributed to three of the five deaths which occurred in complete remission in this trial; all deaths in remission occurred in patients who had received central nervous system prophylaxis. Analysis of patients who subsequently relapsed compared with those still in remission after 18 months of treatment indicated that the former, on average, had slightly lower neutrophil counts. This suggests that the children who relapsed did not receive any less aggressive treatment than those who remained in remission.
Forty-two adults and children with previously untreated acute lymphoblastic leukaemia (ALL) were entered into a programme of chemotherapy in which three combinations, each of four drugs were administered in a predetermined cyclical rotation together with cranial irradiation and intrathecal injections of methotrexate. Forty-one patients (98%) entered remission and no patient developed neuroleukaemia. Relapse of ALL occurred in 10 patients, and three patients died during remission, while eight patients stopped treatment after two and a half years and have remained in remission for two to 26 months. Comparison of remission and survival experience in this mixed group of children and adults with the experience of children treated at Memphis and in the Medical Research Council's UKALL-I trial showed no significant differences. On the other hand, analysis by prognostic factors showed that neither age nor blast cell count at presentation had any adverse effect in patients treated in this study. No relapses occurred in nine patients with blast cell counts greater than 20 x 109/1 at presentation. This regimen is effective treatment for ALL and may be of special value in patients with poor prognoses. The regiment has not as yet proved superior for the treatment of children with ALL who do not have adverse prognostic features.
Two groups of four-year survivors with acute lymphoblastic leukaemia (ALL) are compared: an 'old' series consisting of 83 patients diagnosed before 1968, and a 'new' series of 366 patients included in Medial Research Council trials (UKALL I-III) and diagnosed in 1970-4. Both series differed significantly from a group of ALL patients who survived less than four years in having lower total leucocyte and blast-cell counts at diagnosis, but the new series did not show the significant differences in organ involvement and platelet count seen in the old series. In both series, girls were more likely than boys to survive for four years and less likely to have relapsed meanwhile; in the new series, relapse rates were also lower for girls than for boys after four years, and subsequent survival was significantly better. There was no difference between the two series in survival rates of those patients who had relapsed before reaching four years. A much higher proportion of the new series, however, had reached four years without prior relapse, and these had a more favourable subsequent survival than the corresponding group of the old series. About 90% of patients achieving four years' continuous complete remission on these UKALL regimes seem likely to survive for 10 years.
Three hundred and twenty consecutive children with lymphoblastic leukaemia (ALL), treated on the Medical Research Council UKALL VIII schedule, had their Romanowsky stained diagnostic marrows reviewed for the presence of azurophil granules in blast cell cytoplasm. Twenty patients (7%) had greater than 5% blasts showing this feature; 19 had the cell phenotype of "common ALL." Male children and those with French-American-British (FAB) L2 morphology predominantly showed this feature. There was also a strong correlation between granularity and non-diffuse acid phosphate positivity, but no obvious difference between the 20 patients in their response to treatment emerged during a minimum follow up of 15 months. The "granular" variant occurs in around 7% of children with ALL, but has no clear prognostic importance. Morphologists should be aware of its existence and incidence to avoid confusion with acute myeloid leukaemia.
Fifteen children with mediastinal non-Hodgkin's lymphoma were treated with MRC UKALL X, the current national protocol for acute lymphoblastic leukaemia. The treatment was well tolerated, and in a minimum follow up period of 46 months the event free survival of 93% was significantly better than that in a group of historical controls treated with intermittent chemotherapy regimens whose survival was only 57%. We conclude that intensive induction and consolidation treatment, with continued oral drugs, provides an effective approach to the management of mediastinal non-Hodgkin's lymphoma.
Eighty two eligible children with 'standard risk' lymphoblastic leukaemia were entered into the Medical Research Council UKALL VII trial. Three failed to remit. With a minimum follow up time of four years, actuarial relapse free survival for the remainder was 65%; a significant improvement over the two preceding 'standard risk' trials at the same stage. Only one of five treatment variables possibly affected relapse free survival; this being whether methotrexate was given orally or parenterally during remission maintenance treatment. Twenty seven of 36 patients (75%) who were given intramuscular methotrexate remain alive and in their first remission compared with 23 of 41 (56%) given the drug orally. Although statistically significant differences in small trials should be interpreted with caution, this finding raises the possibility that orally administered methotrexate is not completely absorbed.
AIMS--To assess the prevalence and degree of periodic acid Schiff (PAS) positivity in blast cells from children with lymphoblastic leukaemia (ALL); its association with other disease characteristics; and its clinical importance in predicting the outcome of treatment. METHODS--Marrow slides from entrants to a large United Kingdom multicentre ALL trial (UKALL X) were batch processed and assessed blind for PAS positivity by one morphologist. Patients were classified into groups A, B, and C, corresponding to less than 1% PAS positive cells, 1-10%, and over 10%, respectively. Their PAS pattern was then compared with other clinical and pathological features of ALL and with treatment outcome. RESULTS--Slides from 921 children were examined of which 371 (40%) were categorised as group A, 324 (35%) as group B, and 226 (25%) as group C. There was a clear association between the presence of blast cell vacuoles on Romanowsky staining and PAS positivity. Group A (PAS negative) patients included a disproportionate excess of those with L2 morphology, those under 2 or over 6 years of age, those with an initial white cell count over 50 x 10(9)/l, those with a T or null cell immunophenotype, and those with chromosomal abnormalities other than "high hyperdiploidy". Four years from diagnosis, group C patients had an 8% disease free survival advantage over those in group A (2p = 0.01). This was irrespective of initial white cell count, but not of immunophenotype or the presence of vacuoles. CONCLUSIONS--Strong PAS positivity is a feature of "common" ALL and is particularly associated with blast cell vacuoles. It does occasionally occur in other disease subtypes with or without vacuoles. It predicts a better response to current treatment, but not independently of other cell characteristics.
As most children with acute lymphoblastic leukaemia (ALL) achieve long-term survival, minimising late effects of treatment is a priority. Acute lymphoblastic leukaemia survivors treated historically with protocols including cranial irradiation demonstrate increased weight gain.
We retrospectively studied all 134 patients treated on the MRC/UKALL97 protocol (without cranial irradiation as standard therapy) at a single centre, with 77 inclusions. Height-, weight- and body mass index (BMI) standard-deviation scores (SDS) were recorded at diagnosis and annually until 3 years out (YO) from end of treatment (EoT); changes across time were explored using a univariate model (significance P⩽0.001 to account for multiple comparisons).
Whole-group height SDS was lower from 1 year into treatment until 2 YO, whereas weight- and BMI-SDS remained higher until 3 YO. In females, height-SDS was lower until EoT, but higher weight- and BMI-SDS persisted until 3 YO. In males, height-SDS was lower at EoT and at 2 YO; differences in BMI-SDS had resolved by 2 YO. By WHO criteria, more patients were overweight or obese at 3 YO than at diagnosis (P=0.01).
Survivors of childhood ALL, particularly females, exhibit adverse changes in height-, weight- and BMI-SDS, which arise during treatment and persist into follow-up. Patients should be supported with appropriate dietary and lifestyle advice during ALL treatment and follow-up, which may minimise these changes and reduce associated long-term morbidity.
acute lymphoblastic leukaemia; auxological; body mass index; childhood; late effects; weight
Anthracyclines are used to treat childhood acute lymphoblastic leukaemia (ALL) but non-randomised studies suggest that cardiotoxicity may be a problem.
Individual patient data from trials in childhood ALL which randomized anthracyclines or methods of reducing cardiotoxicity were analysed by standard meta-analysis methods. Results were grouped and combined according to: addition of an anthracycline to standard therapy, type of anthracycline, mode of administration, and the use of a cardioprotectant.
Data from 958 patients in 4 trials, recruiting between 1972 and 1984, showed that addition of an anthracycline reduced bone marrow relapse and, non-significantly, non-bone marrow relapse, resulting in increased relapse free interval. However there was a non-significant increase in induction failures, and in deaths in first remission. Event free survival at 5 years was 56.7% with anthracycline versus 52.8% without (OR=0.91; 95% CI=0.76-1.10; p=0.3). There were no significant differences found in other treatment comparisons.
The limited data from trials did not demonstrate differences in clinically evident cardiotoxicity. Anthracyclines are effective against bone marrow relapse but have not been shown to significantly increase event free survival in childhood ALL. The evidence on type of anthracycline, method of administration or use of cardioprotectant is insufficient to be able to rule out important differences.
Anthracycline; leukaemia; childhood ALL; meta-analysis; randomized
The MRC UKALLXI trial tested the efficacy of different central nervous system (CNS) directed therapies in childhood acute lymphoblastic leukaemia (ALL). To evaluate morbidity 555/1826 randomised children underwent prospective psychological evaluations. Full Scale, verbal and performance IQs were measured at 5 months, 3 years and 5 years. Scores were compared in; (1) all patients (n = 555) versus related controls (n = 311), (2) low-risk children (presenting white cell count (WCC) < 50 × 109/l) randomised to intrathecal methotrexate (n = 197) versus intrathecal and high-dose intravenous methotrexate (HDM) (n = 202), and (3) high-risk children (WCC ≥ 50 × 109/l, age ≥ 2 years) randomised to HDM (n = 79) versus cranial irradiation (n = 77).
There were no significant differences in IQ scores between the treatment arms in either low- or high-risk groups. Despite similar scores at baseline, results at 3 and 5 years showed a significant reduction of between 3.6 and 7.3 points in all three IQ scores in all patient groups compared to controls (P < 0.002) with a higher proportion of children with IQs < 80 in the patient groups (13% vs. 5% at 3 years p = 0.003).
Children with ALL are at risk of CNS morbidity, regardless of the mode of CNS-directed therapy. Further work needs to identify individuals at high-risk of adverse CNS outcomes.
acute lymphoblastic leukaemia; IQ; central nervous system; morbidity; cranial radiotherapy; methotrexate; neuropsychometric; paediatric
The frequency and pattern of obesity in survivors of acute lymphoblastic leukaemia (ALL) was examined in a retrospective analysis of height and weight at zero, two, and four years from diagnosis in 40 children (19 boys and 21 girls). The children had been treated according to the Medical Research Council protocols UKALL VIII and X, both of which included cranial radiotherapy at a dose of 1800 cGy. Body mass index (BMI), determined as weight/height, was used as a measure of fatness. The BMI Z scores were calculated for each patient from standard tables. The ALL group was compared with a control group of 18 age matched children who had received chemotherapy but no radiotherapy. Changes in BMI between diagnosis and two and four years later were analysed by paired t tests. Mean BMI Z scores at diagnosis were similar between ALL boys, ALL girls, and the control group. Two years after diagnosis the ALL group, particularly the girls, showed a significant increase in BMI. By four years BMI had decreased slightly in the ALL boys, but had increased still further in the ALL girls with 57% having BMI Z scores greater than 2. In the control group BMI increased, but not significantly, at two and four years. It is concluded that the obesity seen in patients treated for ALL is more pronounced in girls than boys, and that cranial irradiation is an important factor.
Sequential methotrexate (Mtx) absorption studies were undertaken in 127 children undergoing treatment for childhood non-T acute lymphoblastic leukaemia (ALL) to determine whether serum drug concentration, clearance and dosage affect event free survival (EFS). Higher serum concentration and area under the plasma concentration curve (AUC) were not associated with an improved EFS. Methotrexate clearance was not found to be of prognostic significance. Patients who tolerated only low 6-mercaptopurine (6-MP) doses because of neutropaenia and those who randomly were prescribed higher doses of Mtx had a lower rate of leukaemia relapse after the completion of therapy. This suggests that the use of maintenance therapy in maximally tolerated doses may be associated with an increased survival in childhood ALL.
Mercaptopurine has been used in continuing treatment for childhood acute lymphoblastic leukaemia since the mid 1950s. Recent advances in the understanding of thiopurine pharmacology indicated thioguanine might be more effective than mercaptopurine. The US and UK cooperative groups began randomized thiopurine trials and agreed prospectively to a meta-analysis. All randomized trials of thioguanine versus mercaptopurine were sought and data on individual patients were analysed by standard methods. Combining three trials (from US, UK and Germany), the overall event free survival (EFS) was not significantly improved with thioguanine (Odds ratio (OR) = 0.89; 95% confidence interval 0·78–1·03). Apparent differences in results between trials may be partly explained by the different types of patients studied. The larger treatment effect reported in males in the US trial was confirmed in the other trials. There was heterogeneity between sex/age subgroups (p=0·001), with significant EFS benefit of thioguanine only seen for males aged under 10 years old (OR=0·70; 0·58–0·84), although this did not result in a significant difference in overall survival (OR=0·83; 0·62–1·10). Additional toxicity occurs with thioguanine. Mercaptopurine remains the standard thiopurine of choice, but further study of thioguanine may be warranted to determine whether it may benefit particular subgroups.
Thiopurine; mercaptopurine; thioguanine; leukaemia; childhood; systematic review
Children from the UKALL V trial were studied to assess the clinical importance of myelosuppression during uninterrupted 'maintenance' treatment of 'standard risk' lymphoblastic leukaemia. Those receiving daily 6-mercaptopurine and weekly methotrexate who were in first remission 20 months from diagnosis were divided into two groups on the basis of whether or not they had ever had an absolute neutrophil count of less than 0.5 x 10(9)/l recorded during maintenance treatment up to that time. Of 105 evaluable children, 45 (43%) became neutropenic at least once, and 60 (57%) did not. Seven (16%) of the neutropenic group subsequently relapsed compared with 27 (45%) of the remainder. This difference was still significant if the analysis was stratified by total treatment time (two or three years), age, sex, or diagnostic white cell count. Seven (16%) neutropenic children died in remission, compared with one (2%) of the non-neutropenic children. Therapeutic myelosuppression during standard maintenance treatment of 'standard risk' lymphoblastic leukaemia is associated with increased toxicity but a reduced risk of relapse. The unexplained improvement in long term survival in the United Kingdom in recent years may in large part be due to this.
Epidemiological studies suggest that childhood common acute lymphoblastic leukaemia (c-ALL) may be the rare outcome of early post-natal infection with a common infectious agent. One of the factors that may determine whether a child succumbs to c-ALL is how it responds to the candidate infection. Since immune responses to infection are under the partial control of (human leucocyte antigen) HLA genes, an association between an HLA allele and c-ALL could provide support for an infectious aetiology. To define the limit of c-ALL susceptibility within the HLA region, we have compared HLA-DQB1 allele frequencies in a cohort of 62 children with c-ALL with 76 newborn controls, using group-specific polymerase chain reaction (PCR) amplification, and single-strand conformation polymorphism (SSCP) analysis. We find that a significant excess of children with c-ALL type for DQB1*05 [relative risk (RR): 2.54, uncorrected P=0.038], and a marginal excess with DQB1*0501 (RR: 2.18; P=0.095). Only 3 of the 62 children with c-ALL have the other susceptibility allele, DPB1*0201 as well as DQB1*0501, whereas 15 had one or the other allele. This suggests that HLA-associated susceptibility may be determined independently by at least two loci, and is not due to linkage disequilibrium. The combined relative risk of the two groups of children with DPB1*0201 and/or DQB1*0501 is 2.76 (P=0.0076). Analysis of amino acids encoded by exon 2 of DQB1 reveal additional complexity, with significant (P<0.05) or borderline-significant increases in Gly26, His30, Val57, Glu66-Val67 encoding motifs in c-ALL compared with controls. Since these amino acids are not restricted to DQB1*0501, our results suggest that, as with DPB1, the increased risk of c-ALL associated with DQB1 is determined by specific amino acid encoding motifs rather than by an individual allele. These results also suggest that HLA-associated susceptibility to c-ALL may not be restricted to the region bounded by DPB1 and DQB1.
A child with acute lymphoblastic leukaemia, being treated in the UKALL II Trial, had while in remission an attack of measles and made a normal recovery. Four months later she developed an acute encephalopathy and died within two weeks. The brain showed mild inflammatory features and widespread inclusion bodies in neurones and glial cells. Immunofluorescence proved an infection with measles virus. Similar cases have been called SSPE; reasons are given for preferring the term "measles inclusion-body encephalitis".
Treatment of the central nervous system (CNS) is an essential therapy component for childhood acute lymphoblastic leukaemia (ALL). Individual patient data from 47 trials addressing 16 CNS treatment comparisons were analyzed. Event-free survival (EFS) was similar for radiotherapy versus IT, and radiotherapy plus IT versus IVMTX plus IT. Triple intrathecal therapy (TIT) gave similar EFS but poorer survival than ITMTX, but additional IVMTX improved both outcomes. One trial resulted in similar EFS and survival with IVMTX plus ITMTX versus TIT alone. Radiotherapy can generally be replaced by IT therapy. TIT should be used with effective systemic therapy such as IVMTX.
Leukaemia; Meta-analysis; childhood leukaemia; CNS; triple intrathecal therapy
Survival rates were studied among 1258 children with acute non-lymphocytic leukaemia diagnosed in 1971-88 and included in the population based National Registry of Childhood Tumours. Of the total, 147 (12%) died without receiving treatment. Among the remaining treated children, actuarial five year survival rates were 6% in 1971-4, 15% in 1975-9, 23% in 1980-3, and 40% in 1984-8. Infants aged less than 1 year had a significantly worse prognosis and there was a significant trend towards lower survival rates with increasing white cell count. No independent significant effects on survival were found with sex, French-American-British (FAB) subtype, or the presence or absence of Down's syndrome. Children entered in national trials had a higher survival rate than those who were not entered, and children treated at teaching hospitals had a higher survival rate than those who were treated elsewhere. Among the 535 (43%) children who survived at least one year from diagnosis no factor studied had a significant effect on survival, emphasising the importance of achieving first remission as a determinant of long term survival.
Serial measurements of enolase in the cerebrospinal fluid were made in 19 children with lymphoblastic leukaemia undergoing their first 6 weeks of antineoplastic treatment. The neurone-specific gamma enolase value rose appreciably in nearly all patients during the first two weeks of treatment, which comprised chemotherapy only, but the mean values for this isoenzyme failed to show any further rise during the subsequent cranial irradiation. In contrast the alpha enolase value, which is derived predominantly from glial tissue, rose progressively to attain its highest value during radiotherapy. A consideration of the likely rate of clearance of gamma enolase from the cerebrospinal fluid and the time sequence of administration of the several chemotherapeutic agents in UKALL VIII suggests that asparaginase may be the main causative agent in the rise of this marker of neuronal damage.
A positive selection method, D-aspartic acid beta-hydroxamate resistance, was used to isolate Saccharomyces cerevisiae strains lacking the ability to synthesize asparaginase II. Of 100 such mutant strains, 93 exhibited mutations which were allelic with asp3, a previously characterized mutation. The other seven strains carried a new mutation, asp6. The asp6 mutation segregated 2:2 in asp6 X wild-type crosses and assorted from the asp3 mutation in asp6 X asp3 crosses. All seven asp6 mutant isolates reverted at a relatively high frequency, whereas the asp3 mutant isolates did not revert under the same conditions. Various independent asp3 isolates were mated to give heteroallelic diploids, which when sporulated and spread on D-asparagine medium yielded no recombinant strains.