Gene expression profiling is a powerful tool to classify and predict subtypes of pediatric acute lymphoblastic leukemia (ALL). In addition to accurately classifying known subtypes of ALL, microarray analysis of 327 cases of ALL at our institution using Affymetrix U95 arrays identified a previously uncharacterized novel subgroup of 14 cases of B-ALL with a gene expression signature distinct from other leukemias, suggesting a unique pathogenesis. These cases lacked a recurring cytogenetic anomaly, showed a high frequency of aberrant expression of CD2, lacked molecular markers of other subtypes of ALL, and had a favorable prognosis. We have used several complementary candidate and genome-wide approaches to identify the molecular basis of this novel subtype. Gene expression profiling using Affymetrix U133A and B arrays was used to refine the signature, and when applied to an additional 36 cases of cytogenetically normal B-ALL, 5 cases were identified that share the novel gene expression profile. Intriguingly, the tyrosine kinase PDGFRA was overexpressed in the majority of the novel cases, with 6 novel cases showing exceedingly high levels of PDGFRA expression, which was confirmed by western blotting and flow cytometry. Unexpectedly, 3 of these cases harbored the FIP1L1-PDGFRA fusion characteristic of idiopathic hypereosinophilic syndrome. However, FISH examination demonstrated that this fusion was present in a minor subclone only. RT-PCR and FISH approaches failed to identify alternative PDGFRA fusions, and PDGFRA sequencing failed to identify evidence of activating mutations. Furthermore, in unstimulated cultures or following PDGF-AA stimulation, PDGFRA-overexpressing ALL blasts were not sensitive to imatinib (Gleevec). Other genes in the gene expression profile with potential roles in leukemogenesis, include include the B-cell receptor signaling mediator BRDG1 and the tyrosine phosphatase PTPRM. Using FISH and genomic sequencing of multiple candidates, no potentially leukemogenic abnormality has been identified. To determine if gene amplification, deletion, or copy-neutral loss of heterozygosity is characteristic of this subtype of ALL, genome-wide array-based comparative genomic hybridization and Affymetrix 100K single nucleotide polymorphism arrays were performed on all cases. Deletion of the CDKN2A/2B locus, and deletion of a region at 8p22–23.1 flanking the SPAG11 locus were each found in multiple cases, however no single unifying abnormality was identified. In summary, we have identified and characterized a novel subtype of B-ALL with unique gene expression signature, high incidence of CD2 and PDGFRA overexpression, and favorable outcome. Although no uniform causal molecular lesion has been identified, FIP1L1-PDGFRA fusion may represent one mechanism of PDGFRA overexpression. Further studies and analysis of additional cases may prove informative.

The extent and rapidity with which T cells are regenerated from graft-derived precursor cells directly influences the incidence of infection and the T-cell-based graft-versus-tumor effect. Measurement of T-cell receptor excision circles (TRECs) in peripheral blood is a means of quantifying recent thymic T-cell production and has been used after transplantation in many studies to estimate thymus-dependent T-cell reconstitution. We hypothesized that the quality of thymic function before transplantation affects thymus-dependent T-cell reconstitution after transplantation. We used real-time polymerase chain reaction (PCR) to quantify signal-joint TRECs (sjTRECs) before and after transplantation. T-cell reconstitution was evaluated by T-cell receptor beta (TCRbeta) CDR3 size spectratyping. We tested 77 healthy sibling donors and 244 samples from 26 pediatric recipients of allogeneic hematopoietic stem cell transplantation (AHSCT). Blood from the healthy donors contained 1200 to 155,000 sjTREC copies/mL blood. Patients who had greater than 1200 copies/mL blood before transplantation showed early recovery of sjTREC numbers and TCRbeta repertoire diversity. In contrast, patients who had fewer than 1200 copies/mL blood before transplantation demonstrated significantly slower restoration of thymus-dependent T cells. We conclude that the rate of reconstitution of thymus-dependent T cells is dependent on the competence of thymic function in the recipients before transplantation. Therefore, pretransplantation measurement of sjTREC may provide an important tool for predicting thymus-dependent T-cell reconstitution after transplantation.

Contemporary treatment of pediatric acute myeloid leukemia (AML) requires the assignment of patients to specific risk groups. To explore whether expression profiling of leukemic blasts could accurately distinguish between the known risk groups of AML, we analyzed 130 pediatric and 20 adult AML diagnostic bone marrow or peripheral blood samples using the Affymetrix U133A microarray. Class discriminating genes were identified for each of the major prognostic subtypes of pediatric AML, including t(15;17)[PML-RARalpha], t(8;21)[AML1-ETO], inv(16) [CBFbeta-MYH11], MLL chimeric fusion genes, and cases classified as FAB-M7. When subsets of these genes were used in supervised learning algorithms, an overall classification accuracy of more than 93% was achieved. Moreover, we were able to use the expression signatures generated from the pediatric samples to accurately classify adult de novo AMLs with the same genetic lesions. The class discriminating genes also provided novel insights into the molecular pathobiology of these leukemias. Finally, using a combined pediatric data set of 130 AMLs and 137 acute lymphoblastic leukemias, we identified an expression signature for cases with MLL chimeric fusion genes irrespective of lineage. Surprisingly, AMLs containing partial tandem duplications of MLL failed to cluster with MLL chimeric fusion gene cases, suggesting a significant difference in their underlying mechanism of transformation.

BACKGROUND. The purpose of the current study was to evaluate the cytogenetic features of the hypodiploid leukemic cells of pediatric patients with this rare subgroup of acute lymphoblastic leukemia (ALL). In addition, the authors determined whether subdivision of the hypodiploid category served a prognostic purpose for these patients.METHODS. The authors evaluated the cytogenetic records of 979 patients with ALL admitted to St. Jude Children's Research Hospital (Memphis, TN) between 1984 and 1999.RESULTS. Of 67 patients (6.8%) whose leukemic cells contained a modal number (MN) of chromosomes less than or equal to 45 (i.e., hypodiploid leukemic cells), 57 had an MN of 45 and 10 had an MN of less than 45. In 19 patients, cells with an MN of 45 had a whole chromosome missing (42%), which was a sex chromosome in 12 patients (63%). Leukemic cells with an MN of 45 contained dicentric chromosomes (n = 33) formed from chromosome 9p (55%), 12p (18%), or both (21%). The ETV6- CBFA2 fusion was present in 39% of 28 evaluable B-lineage cases with an MN of 45. The event-free survival rate (EFS) for patients with hypodiploid leukemic cells of MN less than 45 (5-year EFS 20.0% +/- 10.3%) was significantly (P < 0.001) lower than that for patients with leukemic cells of MN greater than or equal to 45 (5-year EFS = 74.9% +/- 1.6%).CONCLUSIONS. Low hypodiploidy (MN < 45) should be recognized as a high-risk feature in pediatric ALL. Only two hypodiploid groups (MN < 45 and MN = 45) may be necessary in prognostic assessments. (C) 2003 American Cancer Society.

Summary. In children with acute myeloid leukaemia (AML), morphological and karyotypic studies cannot precisely assess response to treatment, and less than one‐third of patients have genetic markers for molecular studies of residual disease. We determined the usefulness of a four‐colour flow cytometric strategy developed in our laboratory to study residual disease. We first compared the immunophenotypes of AML cells obtained from 54 children at diagnosis with those of cells from 59 normal or regenerating bone marrow samples. Forty‐six of the 54 AML cases (85·2%) had immunophenotypes that allowed detection of 0·1–0·01% residual leukaemic cells. Of 230 bone marrow samples obtained from those 46 patients during and off treatment, 61 (26·5%) had ≥ 0·1% AML cells by flow cytometry. We found that core binding factor‐associated AML had a significantly better early treatment response. Mean (± standard error) 2‐year survival estimate was 33·1 ± 19·1% for patients with ≥ 0·1% AML cells by flow cytometry after induction therapy, but 72·1 ± 11·5% for those with < 0·1% AML cells (P = 0·022); overt recurrence of AML within the subsequent 6 months was significantly more likely in the former group. The assay described here holds promise for guiding the choice of post‐remission treatment options in children with AML.

While most anaplastic lymphoma kinase (ALK)-positive non-Hodgkin lymphomas (NHLs) are of T-cell lineage, a small number of B-lineage tumors with plasmablastic morphology and expression of the full-length ALK protein have been described in the literature. All of these reported tumors lacked the NPM-ALK fusion transcript. There is controversy regarding the existence of ALK fusion-positive B-cell NHL, with many investigators contending that ALK fusions are expressed uniquely in T- or null-cell lymphomas. Here we describe 2 well-characterized cases of ALK-positive B-cell lymphoma expressing the NPM-ALK fusion. Both tumors occurred in pediatric patients and showed poor response to chemotherapy. Each had plasmablastic morphology, showed immunoglobulin A restriction, and was ALK positive and CD30- by immunohistochemistry. One tumor showed the t(2;5)(p23;q35) chromosomal translocation by conventional cytogenetics. Both were positive for NPM-ALK by reverse transcriptase-polymerase chain reaction. Thus, ALK-positive plasmablastic B-cell lymphomas are more heterogeneous at the molecular level than previously recognized.

PURPOSE:To determine the impact of MLL rearrangements on the outcome of children with acute myeloid leukemia (AML).PATIENTS AND METHODS:We analyzed the clinical and biologic features of 298 infants and children with primary AML treated on four consecutive institutional clinical trials. The Kaplan-Meier method was used in survival analysis and the Cox proportional-hazards model was used to analyze the effect of potential prognostic factors on event-free survival (+/- 1 SE).RESULTS:Molecular studies of 152 cases detected 42 with MLL rearrangements. The karyotypes of these 42 revealed the t(9;11) (15 cases), abnormalities of chromosomes 10 and 11 (nine cases), the t(11;19) (four cases), other abnormalities of 11q23 (seven cases), and miscellaneous rearrangements (seven cases). Among these 42 patients, the 15 whose leukemic cells carried the t(9;11) had a better outcome (66% +/- 15%) than the other 27 (25.9% +/- 11.2%; P =.004). Cases with the t(9;11) were also characterized by M5 AML morphology (21 of 23 cases). Of the 63 patients with M5 AML, the 21 whose leukemic cells demonstrated the t(9;11) had a better outcome (71.1% +/- 11%) than the other 42 (25.8% +/- 7.9%; P =.0004). The only independent factors indicating a favorable prognosis were presenting leukocyte count less than 50 x 10(9)/L (relative risk of relapse, 0.73; 95% confidence interval, 0.55 to 0.97; P =.03) and the t(9;11) (relative risk of relapse, 0.32; 95% confidence interval, 0.16 to 0.64; P =.002).CONCLUSION:We conclude that the t(9;11) is the most favorable genetic factor for patients with AML treated at our institution.

Treatment of pediatric acute lymphoblastic leukemia (ALL) is based on the concept of tailoring the intensity of therapy to a patient's risk of relapse. To determine whether gene expression profiling could enhance risk assignment, we used oligonucleotide microarrays to analyze the pattern of genes expressed in leukemic blasts from 360 pediatric ALL patients. Distinct expression profiles identified each of the prognostically important leukemia subtypes, including T-ALL, E2A-PBX1, BCR-ABL, TEL-AML1, MLL rearrangement, and hyperdiploid >50 chromosomes. In addition, another ALL subgroup was identified based on its unique expression profile. Examination of the genes comprising the expression signatures provided important insights into the biology of these leukemia subgroups. Further, within some genetic subgroups, expression profiles identified those patients that would eventually fail therapy. Thus, the single platform of expression profiling should enhance the accurate risk stratification of pediatric ALL patients.

In childhood B-lineage acute lymphoblastic leukemia (ALL), the most common genetic change, the ETV6-CBFA2 (TEL-AML1) fusion resulting from the cryptic t(12;21)(p13;q22) is associated with a favorable outcome. Therefore, it is important to identify patients with this translocation so that they can receive appropriate treatment. To identify new partner breakpoints for ETV6 and CBFA2, we selected 30 patients with childhood ALL in whose leukemic cells a t(12;21) had been detected by RT-PCR. Conventional cytogenetics revealed that 12p abnormalities were present in 10 patients and that other random abnormalities were present in another 15, including 9 with a numerical or structural abnormality of chromosome 21. Normal karyotypes were observed in the leukemic blasts of five patients. Interphase fluorescence in situ hybridization (FISH) confirmed the RT-PCR finding of the t(12;21) in each patient and detected the loss of the wild-type ETV6 allele in 14 (47%) patients. Metaphase cells from only 20 patients were available for additional FISH analysis. In 13 patients, the expected fusion signal of t(12;21) was observed on der(21)t(12;21), and the reciprocal CBFA2 signal was observed on der(12)t(12;21). However, in six patients with the ETV6-CBFA2 fusion on chromosome 21, the reciprocal CBFA2 signal was observed not on 12p13 but on 4q21, 4q27, 8q24, 11q24, 14q11.2, or 16p13.1. In four of these six patients, we found interstitial insertions of part of CBFA2. In another patient, the ETV6-CBFA2 fusion was observed on 4q21 rather than on 21q. Thus, seven (35%) of the 20 patients with a t(12;21) revealed complex rearrangements. Our findings also indicate the importance of analyzing metaphase chromosomes in identifying cryptic and complex rearrangements involving ETV6 and CBFA2.

Purpose To compare the use of reverse transcriptase polymerase chain reaction (RT-PCR) with that of morphology-based methods for diagnosis, staging, and detection of metastatic disease in pediatric alveolar rhabdomyosarcoma (ARMS), Ewing sarcoma family of tumors (ESFT), and desmoplastic small round cell tumors (DSRCT). Materials and Methods RT-PCR assays for the EWS-FLI1, EWS-ERG, PAX3-FKHR, PAX7-FKHR, and EWS-WT1 fusion transcripts were performed on RNA extracted from the primary tumor tissue, bone marrow, and body fluids obtained at initial presentation and relapse. Molecular findings were compared with original histologic diagnoses and results of staging procedures. Results Eighty-eight samples from 47 patients with ARMS (n = 13), ESFT (n = 31), or DSRCT (n = 3) were analyzed. The detection rate of metastatic disease was significantly higher with RT-PCR (95%) as compared with the morphologic methods (70%) for the three pediatric sarcomas studied. In primary tumors with characteristic fusion transcript, RT-PCR was positive in all cases with morphologic evidence of metastatic disease. Moreover, in six patients (3 with ARMS, 2 with DSRCT, and 1 with ESFT) with metastatic disease, micrometastases in bone marrow (4) and other sites (2) were detected by RT-PCR alone. Importantly, none of the patients with localized disease diagnosed had micrometastases detected by RT-PCR in bone marrow. Conclusions The high sensitivity and specificity of RT-PCR for the characteristic fusion transcripts of pediatric sarcomas make it an ideal method to aid in the routine staging of these patients. In addition, the 100% sensitivity of RT-PCR in detection of micrometastasis makes it useful for follow-up and detection of minimal residual disease. However, the clinical significance of molecularly-detectable disease remains unknown. Further studies should aim to elucidate the therapeutic and prognostic implications of micrometastases detected by RT-PCR alone.

To determine whether the BCL10 mutation plays a role in the oncogenesis of plasma cell dyscrasias, we used polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing analysis and examined the genomic BCL10 mutations in 57 patients with multiple myeloma or plasma cell leukemia and 52 normal bone marrow samples. We found three polymorphic sequence variants, either alone or in combination, at codons 5 and 8, and in intron 1 at base 58 of the BCL10 gene in 37 patients with plasma cell dyscrasia. Identical aberrant band shifts were also observed in 34 normal marrow samples. No polymorphic variants were identified in exon 2 or 3 in either patient or control samples, and no pathogenic mutations were detected. Patients with plasma cell dyscrasias in Taiwan appeared to have a higher frequency of polymorphisms at codon 5 and intron 1 at base 58, and a lower frequency at codons 8 and 213. Our results suggest that BCL10 is not involved in the oncogenesis of plasma cell dyscrasias.

The t(12;21)(p13;q22) is a cryptic abnormality observed in 25% of children with B-lineage acute lymphoblastic leukemia (ALL), associated with a favorable prognosis. To determine whether specific cytogenetic abnormalities accompany the t(12;21), we analyzed the cytogenetic profiles of blast cells from 169 ALL cases positive for the t(12;21), previously identified by molecular methods. Only 13.6% of samples had normal karyotypes. Structural changes were detected in 89.7% of abnormal karyotypes, and numerical abnormalities in 47%. Rearrangements of 12p were the most frequent structural aberration (57 out of 146 patients with chromosomal abnormalities). Nonspecific deletions of chromosomes 6 and 9 were also found. The most frequent numerical abnormalities was trisomy for chromosomes 21. Blast cells were pseudodiploid (45.6%), hyperdiploid with 47 to 51 chromosomes (24.3%), hypodiploid with 44 to 45 chromosomes (10%), near-triploid (0.6%), or near-tetraploid (5.9%). Our results show that the t(12;21) is not associated with hyperdiploidy of 52 to 68 chromosomes or with the prognostic t(1;19), t(4;11) or t(9;22). Only children with B-lineage ALL who lack these abnormalities detected by conventional cytogenetics will probably benefit from additional testing by molecular methods to detect the t(12;21).

Modern therapy for pediatric acute lymphoblastic leukemia (ALL) is based on the principle of risk stratification. One of the most important laboratory features used to accurately risk stratify patients is the presence of specific chromosomal translocation within the leukemic blasts. In this paper, we describe a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the accurate, sensitive, and rapid identification of chimeric transcripts encoded by the major risk-stratifying translocations of pediatric ALL. This assay will identify both the CML- and ALL-type BCR-ABL transcripts encoded by the t(9;22), all described variants of the E2A-PBX1 transcripts encoded by the t(1;19), the MLL -AF4 transcripts encoded by the t(4;11), and all variants of TEL-AML1 encoded by the t(12;21). In addition, we have developed a reverse dot-blot detection system as an alternative to traditional post-PCR Southern blot analysis. Application of this combined assay to the analysis of 70 leukemic samples and five cell lines resulted in a complete concordance between this multiplex assay and individual PCR reactions. The characteristics of the multiplex assay suggest that its application to routine clinical screening will significantly improve the ability of clinical laboratories to accurate risk stratify pediatric ALL patients.

PURPOSE:Recently recognized as a distinct clinicopathologic entity, desmoplastic small round-cell tumors typically affect young men. These aggressive tumors usually arise in the abdomen; other sites of primary disease have been described only rarely. We report the case of an extraabdominal primary tumor with widespread dissemination, including the subcutaneous tissue, a previously unrecognized metastatic site. PATIENT AND METHODS:We describe the case of a 16-year-old boy with a primary extraabdominal metastatic desmoplastic small round-cell tumor. RESULTS:Our patient had a primary intrathoracic desmoplastic small round-cell tumor and widespread dissemination involving the subcutaneous tissue, kidney, liver, bone, and lymph nodes. Histopathologic analysis found intense desmoplasia and polyphenotypic expression of neural, muscle, and epithelial markers. Reverse transcriptase-polymerase chain reaction analysis of fresh tumor tissue confirmed the characteristic EWS-WT1 transcript. CONCLUSIONS:Broader than originally anticipated, the clinical spectrum of desmoplastic small round-cell tumors continues to evolve. Primary intrathoracic tumors with soft-tissue dissemination and polyphenotypic expression should prompt suspicion of this malignancy. Molecular analysis of fresh tumor tissue is an important adjunct to diagnosing this rare neoplasm.