Ewing Sarcoma tumor cells express CD34: implications for autologous stem cell transplantation.

The significance of tumor cell contamination in marrow and peripheral blood stem cell (PBSC) collections of patients with solid tumors remains controversial. Various methods have been developed to purge tumor cells from autologous stem cell products, including CD34+ selection. PBSC harvests from patients with Ewing family of tumors (EFT) were analyzed for contaminating tumor cells prior and after CD34+ selection using reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry (FC) analyzes. The expression of CD34 was studied by RT-PCR and FC in 14 primary tumors and 13 PBSC harvests, respectively. Tumor cells were identified in the harvests by both methods. In two patients, contaminating tumor cells were evident by RT-PCR only after positive selection. FC analysis confirmed a higher level of tumor cells in the CD34+ fraction. In an attempt to explore this finding, expression of CD34 was detected in 93% of primary tumors and 67% of contaminated harvests. As CD34 is expressed on EFT cells, these cells may be enriched following CD34+ selection of harvests, although the total number of tumor cells is reduced. Other methods of purging, rather than CD34+ selection, should be explored in patients with EFT undergoing autologous stem cell transplantation.

A novel germ line p53 mutation in intron 6 in diverse childhood malignancies.

Screening for p53 mutations in exons 5 to 8 in 124 pediatric malignancies identified 18 abnormal shifts using single strand conformation polymorphism: 12 were missense mutations and in 6, no mutation was detected in the exon or in the splice donor acceptor sequences. Sequencing was then performed in the adjacent introns, revealing a G to A base substitution at 39 base pairs upstream to exon 7. This mutation was identified in the germ line of five of the patients, and also in the father of one, whose parents were available. For comparison, of the 184 normal controls similarly screened, only one had this mutation (P=0.036). Positive staining of p53 protein was observed in three of the paraffin embedded tissues that were available: brain tumor, rhabdomyosarcoma, and lymphocytes from a normal lymph node from the rhabdomyosarcoma patient. All tumors with the identified intron mutation were Li-Fraumeni syndrome tumors. Sequencing of all exons including splice sites was performed and revealed no mutation. We suggest that this mutation in intron 6 of the p53 gene stabilizes the wild type p53 protein, resulting in its abnormal accumulation. Mutations in the noncoding region of p53 should be further studied.

Human growth hormone and insulin-like growth factor-1 enhance the proliferation of human leukemic blasts.

As the number of long-term survivors of childhood leukemia increases, growth retardation has emerged as a significant complication. Treatment of these children with growth hormone (GH) has been suggested and sporadically implemented. We, therefore, studied the effect of human GH (hGH) and its by-product insulin-like growth factor-1 (IGF-1) on the growth of leukemic cells in vitro. Under serum-free conditions hGH and IGF-1 induced a significant dose-dependent proliferative effect on promyelocytic leukemia (HL60) and Burkitt's lymphoma (Daudi) cell lines. Anti-hGH antibodies negated the stimulatory effect of hGH and anti-IGF-1 serum abrogated the growth-promoting effect enhanced by IGF-1. Similar statistically significant stimulatory properties were found when freshly obtained marrow cells from four of five acute lymphoblastic leukemia (ALL) of childhood and four acute myelogenous leukemia (AML) patients were studied in ALL and AML blast-cell clonogenic assays. ALL colonies increased numerically by 72% (P less than .025) and AML colonies by 92% (P less than .01) in the presence of hGH at concentrations of 2.5 x 10(2) and 3.0 x 10(2) ng/mL, respectively. IGF-1 stimulated ALL and AML blast-colony growth at concentrations ranging from 0.05 to 0.5 ng/mL by up to 105% (P less than .025) and 65% (P less than .03), respectively. Our in vitro data suggest that circulating hGH and IGF-1 may promote leukemic blast cell replication in vivo, and the supplemental administration of hGH to leukemia patients in remission must be carefully monitored for early relapse.

Association between telomerase activity and outcome in patients with nonmetastatic Ewing family of tumors.

PURPOSE: Telomerase is considered a molecular marker for malignancy. The aim of this study was to determine telomerase activity (TA) as a prognostic factor at diagnosis and as a marker for minimal residual disease during therapy and follow-up in nonmetastatic Ewing family of tumors (EFT). PATIENTS AND METHODS: Primary tumor specimens and 97 peripheral blood (PBL) samples from 31 EFT patients were analyzed for TA by the Telomeric Repeat Amplification Protocol (TRAP assay). The telomerase catalytic subunit (human telomerase reverse transcriptase [hTERT]) gene expression was evaluated by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and telomere length was determined by Southern blotting. The presence of the EFT chimeric transcripts was analyzed by RT-PCR. Correlations with progression-free survival were evaluated. RESULTS: At diagnosis, TA in primary tumors did not correlate with outcome. During therapy and follow-up, highly significant correlation was observed between high TA in PBL samples and adverse prognosis (P <.0001). None of the patients harboring low TA progressed, with a long follow-up (median, 60 months) and a progression-free survival (PFS) of 100%. In nine patients, high TA actually could predict relapse, long before overt clinical relapse. The group of patients with high TA and positive RT-PCR had the most adverse outcome; PFS of 20% (P =.0025). TA was found to be a better prognostic factor than RT-PCR and histopathologic response at surgery. CONCLUSION: The results suggest that TA is a significant prognostic variable, superior to the established clinical prognostic parameters during therapy and tumor surveillance. It could be used in combination with RT-PCR for a new risk classification.

HLA-A11 is associated with poor prognosis in childhood acute lymphoblastic leukemia (ALL).

A possible association between HLA antigens, susceptibility or resistance to leukemia, and responsiveness to treatment has been studied in 144 patients with childhood acute lymphoblastic leukemia (ALL) and compared to other prognostic factors, i.e. white blood cell (WBC) counts, age at onset, sex, ethnic origin, and cell surface markers. All sequentially newly diagnosed children (97) comprised the group for the prospective study (PSG) and were followed for 6 years. The group included 37 patients classified as T-ALL, 41 as CALLA+, 27 as NULL, 12 as B and pre-B, and 27 unclassified patients, who were diagnosed before 1980. During the follow-up period, 45 patients of the PSG died. Forty-seven patients designated long-term survivors (LTS) have been followed 6-20 years after diagnosis, having completed a 3-5 year course of anti-leukemia therapy, and having remained disease free thereafter. High WBC counts at diagnosis and T-cell-surface markers were associated with poor prognosis, as were enthnic origin and specific HLA antigens. Thus, there was one (1) a significant increase in HLA-A30 and a decrease in HLA B-14 in the PSG Jewish patients; and (2) a complete absence of HLA-ALL in LTS while, in the PSG, 8 of 9 HLA-All-positive patients died during the follow-up period. This suggests that HLA-All is associated with poor prognosis in childhood ALL.

More than two immunoglobulin heavy chain J region genes in the majority of infant leukemia.

Congenital and infant leukemia are rare conditions associated with a very poor prognosis due to the high frequency of adverse clinical and laboratory parameters. As the occurrence of multiple immunoglobulin heavy chain hybridization band in childhood leukemia has been associated with poor prognosis, we studied whether it was present in this type of leukemia as well. Seven cases were examined, 4 of them less than 7 months of age. The immunophenotype was lymphoid in 5 and hybrid in 2. Most had abnormal karyotypes. In 5 of the 7, including all with congenital leukemia, an immunoglobulin heavy chain J region multiband pattern was found by Southern blot. The multiband pattern, whether primary or due to clonal evolution, seems to be associated with poor prognosis.

Effect of intrauterine growth on erythropoiesis.

Erythropoietic activity, as reflected in the reticulocyte count and the incorporation of 59Fe into red cells, was studied in relation to intrauterine growth of rabbit fetuses during the last third of the period of gestation. Radioiron (10 microci) was administered by i.v. injection into the mothers and placental transfer to the fetuses. The fetuses were extracted after 48 hours. The amount of radioiron received by each individual fetus was determined by whole body counting. Radioactivity in red cells was determined and calculated as a percentage of the dose received. From day 21 to day 31 of gestation, the hemoglobin concentration increased by a factor of 1.5, from 8 to 12.2 g/100 ml, while the body mass increased by a factor of 7, from 6.6 to 47.5 g. During the same period the rate of growth declined from 45 to 20% in 24 hours. This was paralleled by a drop in 59Fe incorporation from 35 to 12% and in the reticulocyte count from 85 to 14.5%. It is concluded that the rate of erythropoiesis in fetal life is largely controlled by the rate of growth and the corresponding increase in hemoglobin mass.

Thymic humoral factor-gamma 2, an immunoregulatory peptide, enhances human hematopoietic progenitor cell growth.

Thymus humoral factor-gamma 2 (THF gamma 2), an octapeptide important for T-lymphocyte regulation, was assessed for its effect on the in vitro growth of human hematopoietic progenitor cells. This was achieved using a recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF)-stimulated myeloid cell colony formation (granulocyte-macrophage colony-forming cells, GM-CFC) assay as well as a recombinant erythropoietin (rEpo)-stimulated erythroid burst formation (erythroid burst-forming units, BFU-E) assay. Cells were obtained from bone marrow (BM) and peripheral blood (PB) of normal healthy donors and from patients with suppressed bone marrows. The latter group included aplastic anemia, leukemia, and lymphoma patients and patients with solid tumors who responded to intensive chemotherapy with significant pancytopenia. THF gamma 2 significantly enhanced normal BM and PB GM-CFC and PB BFU-E by 2- to 2.5-fold. This effect was totally dependent on the presence of the respective growth factors, that is, rGM-CSF or rEpo, and was specifically reversed by an anti-THF gamma 2 antiserum. Furthermore, although THF gamma 2-induced enhancement of GM-CFC colony formation was not affected by lymphocyte or monocyte depletion, the augmenting effect of the peptide on BFU-E was completely abrogated in the absence of lymphocytes. THF gamma 2-induced augmented growth of progenitor cells derived from severely suppressed marrows was minimal. However, cells from moderately neutropenic patients with leukemia in remission or with lymphoma under chemotherapy responded to the peptide similarly to cells from normal donors. These results suggest a stimulatory role for THF gamma 2 on human myeloid and erythroid hematopoietic progenitor cells. They also suggest the lymphocyte dependence of BFU-E enhancement and lymphocyte independence of GM-CFC stimulation by THF gamma 2. In the former case the thymus-derived peptide may act through the induction of certain erythroid-enhancing lymphokines.

Long-term proliferation of human leukemia cells induced by mouse stroma.

Acute lymphocytic leukemias (ALL) of infants and children were found to preferentially survive in coculture with a cloned cell line of endothelial adipose cells (14F1.1) from mouse bone marrow. One of these ALLs expressed a phenotype compatible with an early stage of differentiation (HLA-DR+, CD19+, and CD34+) and exhibited extensive growth in the presence of the mouse stromal cells during a period greater than 25 weeks following seeding. These ALL cells were strictly dependent upon the mouse stromal clone 14F1.1 and failed to proliferate in the absence of the endothelial adipocytes or with a variety of "feeder cells." Throughout the culture period the cells died if removed from the stroma. No similarly proliferative cell population with strict dependence upon stromal cells was found among a variety of other leukemias including hairy cell, acute myeloid, and chronic lymphocytic leukemia. The 14F1.1 clone has been previously found to promote the renewal of mouse and human stem cells. It is therefore possible that leukemias with a stem cell-like phenotype depend upon stromal cell factors similar to those affecting the growth of normal stem cells. These factors appear to operate across genetic barriers.

Metastatic brain involvement in Ewing family of tumors in children.

OBJECTIVE: To evaluate the incidence and clinical characteristics of CNS involvement in Ewing family of tumors (EF) in children. METHODS: Chart reviews of children with EF treated in our center from 1972 to 1997. Clinical and imaging data regarding possible CNS involvement were collected. RESULTS: During this 25-year period, 80 children with EF were treated. Intracranial involvement was found in eight (10%) children: the brain was involved in seven children (8.8%) and a retro-orbital metastasis without parenchymal brain involvement was noted in one child. Metastases were localized intrahemispherically, or in the cerebellum or the basal ganglia. Intracranial spread was hematogenous in five children and by contiguous spread from the skull in three children. Intracranial involvement was diagnosed 1.3 to 11 years from initial presentation. Seizures and hemiparesis were the main neurologic complications. CONCLUSIONS: The rate of parenchymal brain involvement in our patients with EF was 8.8%. Spread was mainly hematogenous. Substantial morbidity was associated with CNS disease, which appeared in most patients late in the course of disease.

Prognostic relevance of genetic alterations in the p32 region of chromosome 1 in neuroblastoma.

Thirty-six neuroblastomas were analysed for chromosome 1p alterations and their prognostic relevance. In 72% (26/36) of the patients, 1p alterations were identified in the tumours using 24 polymorphic loci ranging 1p22-1p36.3. LOH was identified in 25 children, and in 10 additional allelic imbalance was identified. In 1 child allelic imbalance was the sole alteration. Imbalance was termed as gain in intensity of one allele with or without reduction of the second allele (< 50%). The imbalance was identified in adjacent regions to the LOH. Two distinct regions of LOH were identified: 1p36.1-p36.3 and 1p31-p32. The common imbalance regions overlapped the common LOH regions. The children with LOH and imbalance had improved survival (100%) compared to the children with LOH only (26%) after 48 months of follow-up. The imbalance had an advantageous effect that is reflected by the improved outcome in children with other unfavourable clinical features.

Aminohydroxy propylidene bisphosphonate (APD) treatment improves the clinical skeletal manifestations of Gaucher's disease.

OBJECTIVE: To evaluate the long-term effects and safety of aminohydroxy propylidene bisphosphonate (APD) treatment on the frequency and severity of the clinical skeletal manifestations of Gaucher's disease. METHODOLOGY: Five adolescents who suffered from recurrent bone crisis episodes and atraumatic bone fractures due to Gaucher's disease were treated with APD for 14 to 83 months. RESULTS: During the 6 years before treatment, the patients suffered from 6 to 17 bone crisis episodes, or 1 to 2.8 episodes per patient per year. Three patients were free from bone crisis episodes during 14 to 32 months of ADP treatment, while two patients had two such episodes during 60 and 83 months of APD treatment (these represent a decrease in bone crisis episodes from 1 and 2.8 per year to 0.4 and 0.3 per year, respectively). Although four patients suffered from 1 to 3 atraumatic bone fractures during the 6 years preceding treatment (a total of 10 fractures), only one patient sustained a fracture on APD treatment (total of 219 months of treatment). Using APD was not associated with clinical side effects, biochemical aberrations, significant changes in liver and kidney function, or changes in serum levels of the hormones regulating mineral metabolism. In all patients, a band-like metaphyseal sclerosis appeared on radiography of the long bone. However, APD did not interfere with bone growth. CONCLUSIONS: The marked clinical improvement in the clinical skeletal manifestations of Gaucher's disease and the absence of toxic side effects in adolescent patients treated with APD support previous findings in three adult patients on the efficacy of APD and indicate possibilities for its use in inducing prolonged remissions in affected patients.

Ala244Val is a common, probably ancient mutation causing factor VII deficiency in Moroccan and Iranian Jews.

We investigated the molecular basis for factor VII (FVII) deficiency in Israel and found that 13 patients were homozygous and 10 heterozygous for a C to T substitution at nucleotide 10648 of the FVII gene. This predicted an Ala244Val change and was associated with decreased FVII activity and antigen level. Of the 36 Ala244Val positive alleles, 20 were observed in patients of Moroccan origin, 10 in Iranian-Jewish patients and 6 in patients of other origins. A computer model of the serine protease domain of FVII suggested that the Ala244Val substitution may cause distortion of the entire protein structure. Intragenic polymorphic sites analyses disclosed a founder effect for the Moroccan and Iranian-Jewish patients. A survey of the Ala244Val mutation revealed an allele frequency of 1:42.5 in Moroccan Jews and 1:40 in Iranian Jews. As Moroccan Jews have been separated from Iranian Jews for more than two millennia, the data suggest that the Ala244Val mutation occurred in ancient times.

Phenotypic characterization of acute leukemia with monoclonal antibodies using the microlymphocytotoxicity assay.

Surface antigens of lymphoblasts from 56 pediatric ALL patients were studied with a set of complement fixing monoclonal antibodies. This group of lymphoblasts was comprised of 22 T-cell ALL, 22 CALLA+ Ia+ ALL and 12 non-T-non-B, CALLA- Ia+ ALL. For comparison, two adult T-cell CLL and six B-cell CLL were also studied. It was found that by using the microlymphocytotoxicity technique, the lymphoblasts can be assigned their immunophenotype and thus be classified into their respective lineage and stage of differentiation. In the samples tested, concordant reactivity was observed when FACS fluorescence profile was compared with that of microlymphocytotoxicity suggesting that the latter can be used especially when qualitative estimates are required.

Expression of HLA-DR alloantigens on acute lymphoblastic leukemia lymphoblasts.

We have described the expression of HLA-DR alloantigens on the surface of lymphoblasts from patients with acute lymphoblastic leukemia (ALL). The patient groups included 49 acute lymphoblastic leukemia (ALL) patients (15 common ALL [CALLA +]; 11 "Null" ALL [CALLA-]; 19 T-Cell ALL; 4 Pre-B ALL, and one patient with hairy cell leukemia (HCL). Thirty one of these patients, who exhibited Ia-like antigens demonstrable by monoclonal antibodies, expressed HLA-DR utilizing alloantisera to Class II histocompatibility alloantigens (25/26 non-T-ALL; 2/4 Pre-B ALL; 3/19 T-ALL, and one HCL). The expression of HLA-DR on lymphoblasts was confirmed by family studies of five patients, indicating that ALL lymphoblasts can be used to perform HLA-DR phenotyping or genotyping of such patients. Another important finding was the coexpression on T-cell ALL lymphoblasts of markers for T-helper, T-supressor/cytotoxic, and thymic differentiation marker T6, together with Ia-like and HLA-DR, in one patient, and markers for T-helper, T6, and CALLA in another patient.

Expression of extra class I major histocompatibility antigens on T-cell acute lymphoblastic leukemia (ALL) lymphoblasts.

The presence of extra HLA antigens has been demonstrated, serologically and biochemically, on the surface of lymphoblasts from a patient with acute lymphoblastic leukemia of the T-cell subtype (T-ALL). Family analysis of this patient revealed the presence of the expected antigens, plus an additional HLA antigen (A24) which could be demonstrated by cytotoxicity on the lymphoblasts. Absorption studies revealed that the lymphoblasts had the ability to remove cytotoxic antibodies from alloantisera; similarly, absorption of these alloantisera with normal cells removed the reaction against the extra antigen from the lymphoblasts. The extra HLA molecules were also demonstrated by one-dimensional IEF. Two heavy chain-like molecules, together with the beta 2m subunit, were obtained after removal of appropriate antigens from externally labeled leukemia cells by the use of monoclonal antibody W6/32, which detects a class I specific determinant. The pI of the one heavy chain was shown to be very similar to that of the serologically detected A24. Our data thus suggest that the extra antigens detected by serological reagents may have been due to activation of silent class I MHC gene(s) at the protein level.

Mutations of the adenomatous polyposis coli and p53 genes in a child with Turcot's syndrome.

Turcot's syndrome is a rare heritable complex that is characterized by an association between a primary neuroepithelial tumor of the central nervous system and multiple colonic polyps. The aim of this study was to analyze genetic alterations in a case of Turcot's syndrome in a 10.5-year-old boy in whom a colorectal tumor developed 3.5 years following astrocytoma. An APC germline non-sense mutation at codon 1284 leading to a truncated protein was identified, as was a somatic p53 mutation in the colorectal carcinoma in exon 7, codon 244. The latter was not identified in the primary astrocytoma. However, immunohistochemistry revealed high p53 protein expression in both tumors, suggesting an additional p53 mutation in the primary astrocytic tumor. The diverse p53 mutations observed in this unique syndrome in two different sites and stages of the disease may shed light on the multistep progression of the malignant events.

Antibodies to HIV in Israeli hemophiliacs: relationship between serological profile and disease development.

We studied 66 Israeli hemophiliacs for antibodies to HIV in blood samples collected between 1978 and 1985. By May 1985, 2 had AIDS, 2 had ARC, 4 had lymphadenopathy with some immunologic dysfunction, and 58 were asymptomatic. Antibodies to HIV were detected in 40 (60.6%) patients, including all 8 with disease. Presence of HIV antibodies was significantly associated with receipt of non-heat-treated commercial factor VIII concentrates (NHT fac VIII) between 1980 and 1983. Thirty-eight of 45 (84.44%) patients treated with NHT fac VIII developed antibodies to HIV, compared to 1 of 16 (6.25%) treated with cryoprecipitates and fresh plasma only. Of 40 seropositive patients, 1 (2.5%) had antibodies by 1980, 4 (10%) by 1982, 14 (35%) by 1983, 10 (25.0%) by 1984, and 11 (27.5%) by May 1985. The decline in the rate of seroconversion can be attributed to the replacement of NHT fac VIII concentrate with heat-inactivated factor VIII (HT fac VIII) concentrate by November 1983. As of January 1984 only HT fac VIII was administered. Twenty-nine multitransfused thalassemia patients as well as 20 healthy Israeli blood donors were seronegative to HIV. All 40 (100%) seropositive hemophiliacs had antibodies to viral env gene encoded gp120/gp160 antigens. Twenty-four (60.05%) also had antibodies to viral gag gene encoded p24 and/or p55 antigens. While antibodies to gp120/160 persisted during the follow-up time, a loss of antibodies to p24/55 was observed in 5 of 16 (31.25%) seropositive patients from whom multiple samples were available. gp120/160 positive, p24/55 negative hemophiliacs had significantly lower absolute T-helper cell counts and reversed Th/Ts ratios when compared to gp120/160 p24/55 seropositive patients. Four of the 16 (25.0%) asymptomatic gp120/160 positive, p24/55 negative patients developed overt disease within 15 months of the last blood collection. The data suggest that exposure to HIV antigens is widespread among hemophiliacs in Israel, and can be attributed to receipt of NHT fac VIII concentrates prior to 1984. Antibodies to gp120/160 are of the most important diagnostic value while loss of antibodies to p24/p55 may be of prognostic value.

The epidemiology of childhood acute lymphoblastic leukemia and non-Hodgkin's lymphoma in Israel between 1976 and 1981.

n epidemiologic survey of childhood acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL) occurring in Israel, Judea, Samaria and the Gaza Strip between the years 1976 and 1981, revealed 205 cases of ALL and 69 of NHL. The mean annual incidence of lymphatic malignancies was 3.1/10(5) in the Israeli Jews, 2.3/10(5) in the Israeli Arabs and 2.5/10(5) in the Gaza Strip. In the Jewish population there was a peak in the incidence of lymphatic malignancies at the 2-5 years age group, while in the Israeli Arabs this was less prominent. There were no significant differences in the incidence or type of lymphatic malignancies in the various Jewish or Arab groups but there was a trend for a high leukemia to lymphoma ratio (LLR) in the patients from the Gaza Strip. A relatively higher LLR was observed in families of a high socioeconomic status, but it did not reach statistical significance. T-cell ALL comprised about a third of the typed ALL cases. A high proportion of the patients with ALL belonged to the high-risk category: 46% of the Jewish children and 76% of the Gaza Strip children. White blood cell count above 100,000/mm3 were found at presentation in 36.7% of the Gaza Strip patients but only in 9.4% of the Jewish patients. In spite of that, the survival at 4 years of the Jewish and Arab patients was similar. However, the patients with T-cell ALL had a significantly worse survival than the standard risk or the non-T high-risk group: 43.3 +/- 9.7, 66.6 +/- 7.1 and 63.6 +/- 10.4%, respectively. Compared to a previous study conducted in this country in the sixties it appears that the epidemiologic differences that were observed at that time between the various Jewish ethnic groups have practically disappeared.

Exon 5 mutations in the p53 gene in relapsed childhood acute lymphoblastic leukemia.

Thirty seven children with relapsed acute lymphoblastic leukemia (ALL), 25 B-lineage and 12 T-lineage, were analyzed for p53 alterations at different stages of the disease. Loss of heterozygosity (LOH) was detected in the relapse phase in three patients. p53 mutations were identified by single strand conformation polymorphism (SSCP) and sequencing analyzes in seven of the 37 ALL patients (19%); three B-lineage (12%) and four T-lineage (33%). Most of the mutations were identified in the relapse phase. In two exceptional cases, one of the mutations was indicated as a germ line and the other was already present at diagnosis. No p53 mutation was identified in any of the other 20 available bone marrow samples obtained at diagnosis. No correlation between the p53 status and clinical outcome could be determined. The majority of the mutations (four out of seven, 57%) were clustered at exon 5. Our data implicate that p53 exon 5 is a frequent site of mutations in relapsed childhood ALL.