Related Articles

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.

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.

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.

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.

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.

Images

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.

Background:

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.

Methods:

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).

Results:

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).

Conclusion:

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.

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.

Images

Background

High accrual to clinical trials enables new treatment strategies to be tested rapidly, accurately and with generalisability. Ethical standards also must be high so that participation is voluntary and informed. However, this can be difficult to achieve in trials with complex designs and in those which are closely embedded in clinical practice. Optimal recruitment requires a balance of both ethical and accrual considerations. In the context of a trial of stratified treatments for children with acute lymphoblastic leukaemia (UKALL2003) we examined how recruitment looked to an observer and how it felt to the parents, to identify how doctors' communication could promote or inhibit optimal recruitment.

Methods

We audio-recorded, transcribed and analysed routine doctor-patient consultations (n = 20) and interviews between researchers and parents (n = 30 parents) across six UK treatment centres. Analysis was informed by the constant comparative method. For consultation transcripts, analysis focussed on how doctors presented the trial. We compared this with analysis of the interview transcripts which focussed on parents' perceptions and understanding of the trial.

Results

Parents and doctors discussed the trial in most consultations, even those that did not involve a decision about randomisation. Doctors used language allying them both with the trial and with the parent, indicating that they were both an 'investigator' and a 'clinician'. They presented the trial both as an empirical study with a scientific imperative and also as offering personalisation of treatment for the child. Parents appeared to understand that trial involvement was voluntary, that it was different from routine care and that they could withdraw from the trial at any time. Some were confused about the significance of the MRD test and the personalisation of treatment.

Conclusions

Doctors communicated in ways that generally promoted optimal recruitment, indicating that trials can be embedded into clinical practice. However, parents were unclear about some details of the trial's rationale, suggesting that recruitment to trials with complicated designs, such as those involving stratified treatments, might need enhanced explanation.

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.

Background

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).

Results

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).

Conclusion

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.

Trial registration

ISRCTN: ISRCTN16757172

SUMMARY

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.

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.

The serotonin (5HT3) antagonist ondansetron was compared in a randomised study with metoclopramide and dexamethasone for the prevention of chemotherapy induced emesis. Thirty children aged 1-15 years with acute lymphoblastic leukaemia received 'intensification modules' according to the MRC United Kingdom acute lymphoblastic leukaemia regimen UKALL XI. This contains the moderately emetogenic drugs daunorubicin, etoposide, and cytarabine. Fifteen children received an intravenous loading dose of ondansetron followed by intravenous or oral doses 12 hourly for five days. Fifteen children received intravenous metoclopramide every six hours for three days with a loading dose of dexamethasone, repeated every eight hours for three days intravenously or orally. Efficacy was assessed by a diary card documenting the incidence of nausea, retching, or vomiting. In the 24 hour period after starting chemotherapy, ondansetron was more effective, with a complete or major response rate of 93%, compared with 33% using metoclopramide/dexamethasone.

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.

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.

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".

Images

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.

The true survival rates for the various forms of childhood cancer are best determined from a population-based study rather than from the results of clinical trials. Population-based survival rates have been calculated for four periods between 1956 and 1980 in Queensland. There was a significant improvement in survival for children who developed cancer after 1973 compared with those diagnosed before this date. There has however been no significant improvement in the survival rate for childhood cancer overall, or for acute lymphoblastic leukaemia since 1973. Over the 25 year period significant trends in survival rates were seen in acute lymphoblastic leukaemia, non-Hodgkin's lymphoma, Hodgkin's disease, Wilms' tumour, medulloblastoma, and retinoblastoma. No such trend was seen for acute non-lymphoblastic leukaemia, neuroblastoma, rhabdomyosarcoma, juvenile or anaplastic astrocytoma, brain stem glioma, histiocytosis X, or bone tumours. There is a need for continuing research into better methods of treatment of childhood cancer.

D-Aspartate (D-Asp) can substitute for L-Glutamate (L-Glu) at excitatory Glu receptors, and occurs as free D-Asp in the mammalian brain. D-Asp electrophysiological responses were studied as a potential correlate of aging in the California sea hare, Aplysia californica. Whole cell voltage- and current clamp measurements were made from primary neuron cultures of the pleural ganglion (PVC) and buccal ganglion S cluster (BSC) in 3 egg cohorts at sexual maturity and senescence. D-Asp activated an inward current at the hyperpolarized voltage of −70 mV, where molluscan NMDA receptors open free of constitutive block by Mg2+. Half of the cells responded to both D-Asp and L-Glu while the remainder responded only to D-Asp or L-Glu, suggesting that D-Asp activated non-Glu channels in a subpopulation of these cells. The frequency of D-Asp-induced currents and their density were significantly decreased in senescent PVC cells but not in senescent BSC cells. These changes in sensory neurons of the tail predict functional deficits that may contribute to an overall decline in reflexive movement in aged Aplysia.