Bernard Lemieux

A population-based study of the usefulness of screening for neuroblastoma

BACKGROUND Neuroblastoma has many characteristics which suggest that preclinical detection might improve outcome. The Quebec Neuroblastoma Screening Project was initiated to determine whether mass screening could reduce mortality in a large cohort of infants. As an early endpoint, we report whether screening could reduce the incidence of poor-prognosis neuroblastoma in children with advanced-stage disease over 1 year of age. METHODS All 476,603 children born in the province of Quebec during the 5-year period of May 1, 1989, to April 30, 1994, were eligible for urinary assay of homovanillic acid and vanillylmandelic acid at 3 weeks and 6 months of age. Children with a positive screen were referred to one of four paediatric cancer centres in the province for uniform evaluation and treatment if necessary. Standardised incidence ratios (SIRs) were calculated for neuroblastoma in the province and two similar population-based controls, the state of Minnesota and the province of Ontario, during the same period of time and with similar ascertainment procedures. FINDINGS Compliance with screening in Quebec province was 91% at 3 weeks (n = 425,816) and 74% at 6 months (n = 349,706). Through July 31, 1995, with a follow-up of the birth cohort of 15-75 months, 118 cases of neuroblastoma were diagnosed, 43 detected preclinically by screening, 20 detected clinically before screening at 3 weeks of age, and 55 detected clinically after 3 weeks of age having normal screens (52) or never screened (3). Retrospective analysis of stored samples confirmed that 49 of 52 patients missed by screening had levels of catecholamine metabolites that were too low to be detected at 6 months or earlier. Based on US Surveillance, Epidemiology and End Results data, 54.5 cases of neuroblastoma would have been expected in Quebec province during the study period, for an SIR of 2.17 (95% CI 1.79-2.57, p < 0.0001). For the two control groups, 43 and 80 cases of neuroblastoma were detected, respectively, compared with 37.9 and 85.4 expected, overall SIR 1.00 (not significant). SIRs for Quebec province by age at diagnosis in yearly intervals show a marked increased incidence under 1 year of age (SIR 2.85, 2.26-3.50), with no reduction in incidence in subsequent years. Limiting analysis to only patients diagnosed over 1 year of age with advanced-stage disease, 22 cases were detected in Quebec province versus 14.4 expected (SIR 1.52, 0.95-2.23). Data in the two control groups show no significant increase or decrease in any-stage disease in children under or over the age of 1 year, except for an increase in early-stage disease in Minnesota children over 1 year: 10 versus 3.8 expected (SIR 2.67, 1.27-4.58). INTERPRETATION Screening for neuroblastoma increases the incidence in infants without decreasing the incidence of unfavourable advanced-stage disease in older children. It is unlikely that screening for neuroblastoma in infants will reduce mortality for this disease.

Outcome of individuals with low-moderate methylmalonic aciduria detected through a neonatal screening program

BACKGROUND The clinical spectrum of methylmalonic aciduria (MMAuria) ranges from severe, neonatal acidosis to benign asymptomatic organic aciduria. In 1975, screening for MMAuria was established in the province of Quebec. Although newborn screening programs facilitate presymptomatic detection and treatment and also detect asymptomatic variants, uncertainties about potential long-term hazards of mild to moderate elevations of MMA create concern. The objective of this study was to examine the outcome of individuals excreting low to intermediate quantities of MMA, ascertained by a newborn screening program. RESULTS AND STUDY DESIGN One hundred and thirty-six individuals with elevations of urinary MMA were initially identified by the screening program; 122 individuals were noted to have excretion of urinary MMA <1400 micromol/mmol creatinine. At follow-up assessment at 1 year of age, in 65 of these 122 individuals, the MMA excretion had resolved. Of the remaining individuals, 9 were lost to follow-up, 13 had symptoms, and the remaining 35 were free of symptoms. Among the 35 individuals with asymptomatic persistent MMAuria, MMA excretion has resolved in 13 over 1 year; 22 individuals exhibit persistent low-moderate MMAuria (range, 210 to 1133 micromol/mmol creatinine). CONCLUSION Follow-up examination of individuals in the latter asymptomatic cohort with persistent low-moderate MMAuria indicates normal somatic and cognitive outcomes.

Biological aspects of neuroblastomas identified by mass screening in quebec

BACKGROUND Neuroblastoma has several characteristics that suggest that preclinical diagnosis might improve outcome. Therefore, the Quebec Neuroblastoma Screening Project was undertaken from 1989 to 1994 to examine infants at 3 weeks and 6 months by measuring urinary catecholamine metabolites. PROCEDURE Over the 5-yr period, 45 tumors were detected by screening, 20 were identified clinically prior to the third week, and 64 were identified clinically at a later time. We analyzed available tumors for Shimada histopathology, tumor ploidy, MYCN copy number and serum ferritin. RESULTS Of the tumors detected by screening, only 2 of 45 tested had unfavorable histology, 2 of 45 had diploid or tetraploid DNA content, 0 of 43 had MYCN amplification, and 4 of 44 had elevated serum ferritin. All of these patients are alive and well. The 20 patients detected prior to the 3-week screen had similar biological characteristics. In contrast, of the patients detected clinically after 3 weeks of age, 19 of 51 testedhad unfavorable histology, 25 of 66 had diploid or tetraploid tumors, 12 of 56 had MYCN amplification, and 14 of 54 had elevated ferritin. CONCLUSIONS The difference between the screened and clinically detected cases was highly significant for each biological variable. Preliminary data on other biological variables, such as neurotrophin expression and allelic loss on 1 p in these patients are consistent with the above findings. These data suggest that mass screening for neuroblastoma at or before 6 months of age detects almost exclusively tumors that have favorable biological characteristics, many of which might have regressed spontaneously. Thus, continued mass screening for neuroblastoma at 6 months is unlikely to accomplish its intended goal, and should probably be discontinued.

Screening Newborns for Multiple Organic Acidurias in Dried Filter Paper Urine Samples: Method Development

Screening urine for inherited and acquired organic acidurias in newborns has the potential of preventing severe disease, mental retardation, and death. A method for screening dried urine filter paper samples for acidic markers of at least 20 different metabolic conditions has been developed. These conditions include, among others, maple syrup urine disease; methylmalonic, propionic, isovaleric, glutaric, and hydroxymethylglutaric acidurias; methylcrotonylglycinuria; medium-chain acyl-CoA dehydrogenase deficiency; inherited vitamin responsive disorders (B12, biotin, B2), and acquired deficiencies of these vitamins. The preparation of the urine extract is identical to the method we use to screen infants for neuroblastoma. Screening is based on a highly sensitive and specific determination of eight organic acid markers by an automated computerized gas chromatography mass spectrometry system using selected ion monitoring. The markers used for screening are methylmalonic acid, 2-hydroxyisocaproic acid, glutaric acid, propionylglycine, isovalerylglycine, 3-methylcrotonylglycine, hexanoylglycine, and 3-phenylpropionylglycine. The extraction efficiencies of these acids from dried filter paper were similar to extraction from water, ranging from about 40% to 80%, except for propionylglycine which showed a low extraction efficiency of 11–13%. The stability of these acids on filter paper exposed to room air and temperature over a period of 15 d was adequate for the use of this collection method for organic aciduria screening. Normal levels, adjusted to urinary creatinine, were established for these acids in 519 urine filter paper samples obtained from 3-wk-old newborns. This screening method was tested on samples obtained from 12 patients with known organic acidurias including stored urine filter paper collected at 3-wk of age from two infants later found to have organic acidurias.

Screening for neuroblastoma is ineffective in reducing the incidence of unfavourable advanced stage disease in older children

Neuroblastoma exhibits many characteristics which would suggest that preclinical detection may improve outcome. The Quebec Neuroblastoma Screening Project was initiated to determine whether mass screening could reduce mortality in a large cohort of infants. All 476,603 children born in the province of Quebec during a 5-year period of time (1 May 1989 to 30 April 1994) were eligible for determinations of urinary catecholamine metabolites at 3 weeks and 6 months of age. Children with positive screening were referred to one of four paediatric cancer centres in Quebec for uniform evaluation and treatment. Standardised incidence ratios (SIRs) were calculated for neuroblastoma in Quebec and two comparable population-based controls during the same period of time using similar ascertainment procedures. Compliance with screening in Quebec was 91% at 3 weeks (n = 425,816) and 74% at 6 months (n = 349,706). Up to 31 July 1995 with a follow-up of the birth cohort of 15-75 months, 118 cases of neuroblastoma were diagnosed, 43 detected preclinically by screening, 20 detected clinically prior to screening at 3 weeks of age and 55 detected clinically after 3 weeks of age having normal screens (n = 52) or never screened (n = 3). Based on data from concurrent control populations, 54.5 cases of neuroblastoma would have been expected in Quebec during the study period for an SIR of 2.17 (95% CI 1.79-2.57, P < 0.0001). For the two control groups, the overall SIR was 1.00 (NS). SIRs for Quebec by age at diagnosis in yearly intervals show a marked increased incidence under 1 year of age (SIR = 2.85, 95% CI 2.26-3.50), with no reduction in incidence in subsequent years. We conclude that screening for neuroblastoma markedly increases the incidence in infants without decreasing the incidence of unfavourable advanced stage disease in older children. It is unlikely that screening for neuroblastoma in infants will reduce the mortality of this disease.

Screening for neuroblastoma in north america. Preliminary results of a pathology review from the quebec project

Background. The Quebec Neuroblastoma Screening Project was initiated to assess clinical and biologic aspects of neuroblastomas detected by screening infants born in the province of Quebec from May 1, 1989, to April 30, 1994.

Enzymologic and Metabolic Studies in Two Families Affected by Argininosuccinic Aciduria

Summary: Both the affected families studied provide another example of the autosomal recessive inheritance of argininosuccinic aciduria. The fasting plasma levels of argininosuccinic acid in the two propositi did not correlate with the levels of argininosuccinic acid lyase (ASAL) in erythrocytes. There was 210 μM argininosuccinic acid with indications of anhydride B content in the family I propositus, having an enzyme activity of 13%; while the family II propositus gave an argininosuccinic acidemia reading of 64.6 μM with no activity of RBC ASAL. There was a reduced enzyme activity in all the members of affected families due to a significantly reduced Vmax value as compared to control. Within family I, the differences were solely determined by the Vmax of the enzymes. In family II, the two siblings had a significantly reduced Km value as compared to the parents (0.09 and 0.10 against 0.16 and 0.18 mM).Plasma citrulline and glutamine were generally elevated in the two affected families, particularly in the propositi, whereas arginine was normal. A dietary protein-loading trial conducted on members of family II showed more urinary ammonia and α-amino nitrogen excretion as compared to a control family, while urea excretion was comparable. Only the propositus showed a urinary excretion of argininosuccinic acid.Speculation: Research work on the structural and kinetic characterization of human ASAL from various organs and tissues may throw light on the importance of RBC enzyme in the pathogenesis and diagnosis of argininosuccinic aciduria. Such studies will also help in clarifying the regulatory gene hypothesis of ASAL activity in various organs.

Single column high pressure liquid chromatographic determination of drugs in blood.

1. Analysis of anticonvulsants (phenobarbital, diphenylhydantoin and carbamazepine), theophylline and an antiarrhythmic agent (disopyramide) in blood using a simple high pressure liquid chromatography apparatus equipped with a reversed -- phase column is described. A simple extraction of plasma or serum with organic solvent is used to isolate the anticonvulsants and theophylline. Disopyramide is extracted with ether and is further purified by a back extraction into acid. 2. Hexanesulfonic acid -- methanol solutions are used for chromatography of the anticonvulsants and disopyramide while the mobile phase for theophylline is a NH4H2PO4 -- methanol mixture. Chromatographic analysis time for the drugs is approximately 15 minutes. The drugs are monitored by a UV detector at 254 nm except for theophylline which is measured at 280 nm. Quantitation is accomplished by comparison of peak heights with those of internal standards. Quantities of serum or plasma routinely used for analysis are: 200 ul for the anticonvulsants, 100 ul for theophylline and 0.5 ml for disopyramide. Detection limits are less than 1 ug/ml for these quantities.

541 NEONATAL SCREENING FOR AMINOACIDURIA IN THE PROVINCE OF QUEBEC, 1971–1977

Between August 1971 and 1973, 113,680 5-day-old newborns were screened for aminoaciduria by one-dimensional TLC using dry urine samples. Only 5 abnormal patterns were observed: Histidinuria (emia)(2), tyrosinuria (emia)(2), cystinuria (5) and dicarboxylic aminoaciduria (1). The frequency was 2.64 per 100,000 for “overflow” and 6.16 per 100,000 for “renal” aminoacidurias. From September 1973 to June 1977, 276,000 14-day-old newborns were studied. 5 additional pathological patterns were found: Argininosuccinic aciduria (emia)(3), phenylketonuria (9), dibasic aminoaciduria (1), generalized (3) and neutral (Hartnup) aminoaciduria (5). The frequency of all 10 abnormal patterns increased to 15.92 per 100,000 for “overflow” and 26.76 per 100,000 for “renal” aminoacidurias. Non-pathological aminoacidurias were found in 10.49 per 100,000 newborns. Our results agree with others regarding the frequency of pathological aminoacidurias except for tyrosinuria (emia) and dicarboxylic aminoaciduria, both found with a higher frequency in the Quebec population. We conclude that neonatal screening for aminoacidurias at 14 days of age offers a valuable back-up system for blood screening programs and permits the detection of new inborn errors of metabolism in a given population.

KINETIC ABNORMALITIES OF CARBAMYL PHOSPHATE SYNTHETASE CPS-I) IN A CASE OF CONGENITAL HYPERaMMONEMIA

Late onset hyperammonemia caused by partial CPS-I deficiency is a rare disorder. We report a 2½ month old girl who was symptom-free till 9 weeks of age. She was hospitalized for vomiting and metabolic acidosis. Plasma aminogram showed increased alanine, with a higher excretion of alanine, proline, serine and threonine. Urinary orotate was normal. Plasma NH3 on the third day was 96 μmol/l. The child died due to complications of an intestinal hemorrhage. Brain pathology was indicative of delayed my-elinization and proliferation of astrocytes. A post-mortem liver sample showed normal ornithine transcarbamylase, argininosuccinate lyase, and arginase. CPS-I measured by a direct colorimetric method (Piersen and Brien, J. Biol Chem 255:7891,1980) showed a partial deficiency (10-25 % of normal controls). Mancini radial immunodiffusion with rabbit antiserum against human CPS-I showed 10 % of normal cross-reacting material. A study of pH dependence using a triethanolamine buffer gave a flat curve over the pH range 7.0-9.0. Two normal controls had a pH optimum of 7.8, with 70 % activity at pH 9.0. Apparent Km HCO4- (5.6 mM) was normal (controls: 6.0-9.5). Km NH4+ (0.73 mM) was abnormally increased (normal range: 0.24-0.51).Kinetic and immunochemical characterization of CPS-I mutants is important to understand variations in the clinical expression of congenital hyperammonemia in children. The above work is a preliminary step in defining the pathology of this rare syndrome.