Identification of unique genomic signatures in patients with fibromyalgia and chronic pain.

Fibromyalgia (FM) is a chronic pain syndrome characterized by widespread pain. The pathophysiology of fibromyalgia is not clearly understood and there are no specific biomarkers available for accurate diagnosis. Here we define genomic signatures using high throughput RNA sequencing on 96 fibromyalgia and 93 control cases. Our findings revealed three major fibromyalgia-associated expression signatures. The first group included 43 patients with a signature enriched for gene expression associated with extracellular matrix and downregulation of RhoGDI signaling pathway. The second group included 30 patients and showed a profound reduction in the expression of inflammatory mediators with an increased expression of genes involved in the CLEAR signaling pathway. These results suggest defective tissue homeostasis associated with the extra-cellular matrix and cellular program that regulates lysosomal biogenesis and participates in macromolecule clearance in fibromyalgia. The third group of 17 FM patients showed overexpression of pathways that control acute inflammation and dysfunction of the global transcriptional process. The result of this study indicates that FM is a heterogeneous and complex disease. Further elucidation of these pathways will lead to the development of accurate diagnostic markers, and effective therapeutic options for fibromyalgia.

In-Depth Comparison of Genetic Variants Demonstrates a Close Relationship Between Invasive and Intraductal Components of Prostate Cancer.

Intraductal carcinoma (IDC) of the prostate is often associated with concurrent high-grade invasive prostate cancer (PCa) and poor clinical outcomes. In this context, IDC is thought to represent the retrograde spread of invasive prostatic adenocarcinoma into the acini and ducts. Prior studies have demonstrated a concordance of PTEN loss and genomic instability between the IDC and high-grade invasive components of PCa, but larger genomic association studies to solidify our understanding of the relationship between these 2 lesions are lacking. Here, we evaluate the genomic relationship between duct-confined (high-grade prostatic intraepithelial neoplasia and IDC) and invasive components of high-grade PCa using genetic variants generated by whole exome sequencing. High-grade prostatic intraepithelial neoplasia and IDC were laser-microdissected, and PCa and nonneoplastic tissue was manually dissected from 12 radical prostatectomies. A targeted next-generation sequencing panel was used to identify disease-relevant variants. Additionally, the degree of overlap between adjacent lesions was determined by comparing exome-wide variants detected using whole exome sequencing data. Our results demonstrate that IDC and invasive high-grade PCa components show common genetic variants and copy number alterations. Hierarchical clustering of genome-wide variants suggests that in these tumors, IDC is more closely related to the high-grade invasive components of the tumor compared with high-grade prostatic intraepithelial neoplasia. In conclusion, this study reinforces the concept that, in the context of high-grade PCa, IDC likely represents a late event associated with tumor progression.

The role of next-generation sequencing in the differential diagnosis of composite neoplasms.

Composite neoplasms (CNs) are rare and diagnostically challenging lesions that require differentiating between mixed clonal tumors with divergent phenotypes (MT), collision of 2 independent tumors adjacent to each other (CT), and tumor-to-tumor metastasis (TTM). To that end, pathologists have traditionally used immunohistochemistry and limited molecular studies, such as Sanger sequencing. Herein we evaluate the potential application of NGS in the differential diagnosis of these rare neoplasms. Four CNs were included in the study. Two were diagnosed as MT (mixed adenoneuroendocrine carcinoma of the gallbladder and metastatic papillary thyroid carcinoma with squamous dedifferentiation) and 2 were interpreted as TTM (esophageal adenocarcinoma to lung adenocarcinoma and small cell carcinoma of the lung to meningeal melanoma). Diagnoses were made using clinical, histologic, and immunophenotypic information, with the aid of limited molecular studies in 2 cases. Formalin-fixed, paraffin-embedded tissue was dissected for DNA and RNA extraction, and NGS was performed using the Oncomine Comprehensive Panel. The 2 tumors initially interpreted as MT showed shared genetic aberrations in the different neoplastic components, supporting the pathologic diagnosis. NGS results for the lesion diagnosed as esophageal adenocarcinoma metastatic to lung adenocarcinoma did not support the histopathologic interpretation and were deemed inconclusive. However, the identification of an identical CDKN2A mutation in all components and in the adjacent benign lung parenchyma suggests a possible germline aberration. Sequencing results in the last case were clearly supportive of TTM. This study illustrates the role of NGS in the diagnostic workup of CNs, as an adjunct to light microscopy and immunohistochemistry.

Unique immunologic patterns in fibromyalgia.

BACKGROUND: Fibromyalgia (FM) is a clinical syndrome characterized by chronic pain and allodynia. The diagnosis of FM has been one of exclusion as a test to confirm the diagnosis is lacking. Recent data highlight the role of the immune system in FM. Aberrant expressions of immune mediators, such as cytokines, have been linked to the pathogenesis and traits of FM. We therefore determined whether cytokine production by immune cells is altered in FM patients by comparing the cellular responses to mitogenic activators of stimulated blood mononuclear cells of a large number of patients with FM to those of healthy matched individuals. METHODS: Plasma and peripheral blood mononuclear cells (PBMC) were collected from 110 patients with the clinical diagnosis of FM and 91 healthy donors. Parallel samples of PBMC were cultured overnight in medium alone or in the presence of mitogenic activators; PHA or PMA in combination with ionomycin. The cytokine concentrations of IFN-γ, IL-5, IL-6, IL-8, IL-10, MIP-1ß , MCP-1, and MIP1-α in plasma as well as in cultured supernatants were determined using a multiplex immunoassay using bead array technology. RESULTS: Cytokine levels of stimulated PBMC cultures of healthy control subjects were significantly increased as compared to matched non-stimulated PBMC cultures. In contrast, the concentrations of most cytokines were lower in stimulated samples from patients with FM compared to controls. The decreases of cytokine concentrations in patients samples ranged from 1.5-fold for MIP-1ß to 10.2-fold for IL-6 in PHA challenges. In PMA challenges, we observed 1.8 to 4-fold decreases in the concentrations of cytokines in patient samples. CONCLUSION: The cytokine responses to mitogenic activators of PBMC isolated from patients with FM were significantly lower than those of healthy individuals, implying that cell-mediated immunity is impaired in FM patients. This novel cytokine assay reveals unique and valuable immunologic traits, which, when combined with clinical patterns, can offer a diagnostic methodology in FM.

Early T-cell precursor leukaemia: a subtype of very high-risk acute lymphoblastic leukaemia.

BACKGROUND: About a fifth of children with acute T-lymphoblastic leukaemia (T-ALL) succumb to the disease, suggesting an unrecognised biological heterogeneity that might contribute to drug resistance. We postulated that T-ALL originating from early T-cell precursors (ETPs), a recently defined subset of thymocytes that retain stem-cell-like features, would respond poorly to lymphoid-cell-directed therapy. We studied leukaemic cells, collected at diagnosis, to identify cases with ETP features and determine their clinical outcome. METHODS: Leukaemic cells from 239 patients with T-ALL enrolled at St Jude Children's Research Hospital (n=139) and in the Italian national study Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) ALL-2000 (n=100) were assessed by gene-expression profiling, flow cytometry, and single nucleotide polymorphism array analysis. Probabilities of survival and treatment failure were calculated for subgroups considered to have ETP-ALL or typical T-ALL. FINDINGS: 30 patients (12.6%) had leukaemic lymphoblasts with an ETP-related gene-expression signature or its associated distinctive immunophenotype (CD1a(-), CD8(-), CD5(weak) with stem-cell or myeloid markers). Cases of ETP-ALL showed increased genomic instability, in terms of number and size of gene lesions, compared with those with typical T-ALL. Patients with this form of leukaemia had high risk of remission failure or haematological relapse (72% [95% CI 40-100] at 10 years vs 10% [4-16] at 10 years for patients with typical T-ALL treated at St Jude Children's Research Hospital; and 57% [25-89] at 2 years vs 14% [6-22] at 2 years for patients treated in the AIEOP trial). INTERPRETATION: ETP-ALL is a distinct, previously unrecognised, pathobiological entity that confers a poor prognosis with use of standard intensive chemotherapy. Its early recognition, by use of the gene expression and immunophenotypic criteria outlined here, is essential for the development of an effective clinical management strategy. FUNDING: US National Cancer Institute, Cariplo Foundation, Citta della Speranza Foundation, Italian Association for Cancer Research (AIRC), Italian Ministry for University and Research, and American Lebanese Syrian Associated Charities (ALSAC).

Acute mixed lineage leukemia in children: the experience of St Jude Children's Research Hospital.

To characterize the biology and optimal therapy of acute mixed-lineage leukemia in children, we reviewed the pathologic and clinical features, including response to therapy, of 35 patients with mixed-lineage leukemia. The majority of cases (91%) had blasts cells that simultaneously expressed either T-lineage plus myeloid markers (T/myeloid, n = 20) or B-lineage plus myeloid markers (B/myeloid, n = 12). Overall survival rates for the B/myeloid and T/myeloid subgroups were not significantly different from each other or from the rate for acute myeloid leukemia (AML) but were inferior to the outcome in children with acute lymphoblastic leukemia (ALL). Patients who failed to achieve complete remission with AML-directed therapy could often be induced with a regimen of prednisone, vincristine, and L-asparaginase. Analysis of gene-expression patterns identified a subset of biphenotypic leukemias that did not cluster with T-cell ALL, B-progenitor ALL, or AML. We propose that treatment for biphenotypic leukemia begin with one course of AML-type induction therapy, with a provision for a switch to lymphoid-type induction therapy with a glucocorticoid, vincristine, and L-asparaginase if the patient responds poorly. We also suggest that hematopoietic stem cell transplantation is often not required for cure of these patients.

Rapid immune reconstitution after a reduced-intensity conditioning regimen and a CD3-depleted haploidentical stem cell graft for paediatric refractory haematological malignancies.

The main obstacles to successful haploidentical haematopoietic stem cell transplantation from a mismatched family member donor are delayed immune reconstitution, vulnerability to infections and severe graft-versus-host disease (GvHD). We designed a reduced-intensity conditioning regimen that excluded total body irradiation and anti-thymocyte globulin in order to expedite immune reconstitution after a CD3-depleted haploidentical stem cell transplant. This protocol was used to treat 22 paediatric patients with refractory haematological malignancies. After transplantation, 91% of the patients achieved full donor chimaerism. They also showed rapid recovery of CD3(+) T-cells, T-cell receptor (TCR) excision circle counts, TCRbeta repertoire diversity and natural killer (NK)-cells during the first 4 months post-transplantation, compared with those results from a group of patients treated with a myeloablative conditioning regimen. The incidence and extent of viremia were limited and no lethal infection was seen. Only 9% of patients had grade 3 acute GvHD, while 27% patients had grade 1 and another 27% had grade 2 acute GvHD. This well-tolerated regimen appears to accelerate immune recovery and shorten the duration of early post-transplant immunodeficiency, thereby reducing susceptibility to viral infections. Rapid T-cell reconstitution, retention of NK-cells in the graft and induction of low grade GvHD may also enhance the potential anti-cancer immune effect.

Near-triploidy and near-tetraploidy in childhood acute lymphoblastic leukemia: association with B-lineage blast cells carrying the ETV6-RUNX1 fusion, T-lineage immunophenotype, and favorable outcome.

The prognostic significance of near-triploidy (68-80 chromosomes) and near-tetraploidy (>80 chromosomes) in childhood acute lymphoblastic leukemia (ALL) is unclear. Therefore, we retrospectively evaluated the incidence of and outcome associated with these subtypes of ALL. In 620 children with ALL diagnosed between 1988 and 1999, the leukemic cells were near-triploid (DNA index, 1.50-1.73) in 4 and near-tetraploid (DNA index, 1.79-2.28) in 14. Of 15 patients with B-lineage ALL, 11 (73.3%) had an ETV6-RUNX1 (previously TEL-AML1 and then ETV6-CBFA2) fusion. No differences in age (P = 0.99), leukocyte count (P = 0.99), or immunophenotype (P = 0.99) were observed between patients with near-triploidy and those with near-tetraploidy. Patients with near-triploidy or near-tetraploidy were more likely than those with high-hyperdiploidy (51-67 chromosomes) (n = 159) to be female (P = 0.05) and have T-lineage ALL (P = 0.02), L2 morphology (P < 0.0001), or the ETV6-RUNX1 fusion (P < 0.0001). The median follow-up period was 10.4 years. The 5-year event-free survival estimates (+/- SE) were 75% +/- 19% for patients with near-triploidy, 93% +/- 7% for those with near-tetraploidy, and 84% +/- 3% for those with high-hyperdiploidy. Although near-triploidy and near-tetraploidy are biologically different from high-hyperdiploidy, the favorable outcomes of patients with any one of these abnormalities suggest that patients with B-lineage ALL and a DNA index >or= 1.16 can be included in the low-risk arm of treatment protocols. We cannot make similar recommendations for patients with T-lineage ALL because of the small number of cases (n = 3) in this study.

Comparison of killer Ig-like receptor genotyping and phenotyping for selection of allogeneic blood stem cell donors.

The repertoire of killer Ig-like receptors (KIRs) can be determined at the level of DNA, RNA, or surface protein expression for selection of blood stem cell donors. We compared genotyping and phenotyping of the four inhibitory KIRs that are important in transplantation for leukemia in 73 unrelated persons. In 5 (7%) of the 68 individuals in whom the KIR2DL1 gene was present and in 10 (15%) of the 67 in whom KIR3DL1 was present, the corresponding receptor was not expressed by NK cells, as determined by flow cytometry analysis. In contrast, one or both allelic forms of KIR2DL2/KIR2DL3 were expressed by a high proportion of NK cells in all 73 individuals. However if both KIR2DL2 and KIR2DL3 genes were present, KIR2DL3 was preferentially expressed, as transcripts of KIR2DL2 was not detectable by RT-PCR in 42% of these individuals. In total, repertoire assessment for the four KIRs by genotyping vs phenotyping was not in complete agreement in 18 (25%) of the 73 individuals. Furthermore, among the samples that tested positive for the expression of a certain KIR gene, the levels of transcripts and surface expression varied considerably as measured by both real-time quantitative PCR and flow cytometry analysis. Extension of this comparative analysis to include all 12 KIR family members showed that KIR2DL3 and KIR3DL2 were the only genes whose transcripts were consistently detectable. These results caution the use of genotyping alone for donor selection or leukemia-relapse prognostication because some KIRs may be expressed at a very low level.

Prediction of T-cell reconstitution by assessment of T-cell receptor excision circle before allogeneic hematopoietic stem cell transplantation in pediatric patients.

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.

Improved outcome for children with acute lymphoblastic leukemia: results of Total Therapy Study XIIIB at St Jude Children's Research Hospital.

St Jude Total Therapy Study XIIIB for childhood acute lymphoblastic leukemia (ALL) incorporated more stringent risk classification, early intensification of intrathecal chemotherapy, reinduction treatment, and the addition of dexamethasone to postremission therapy to increase the proportion of event-free survivors without jeopardizing their quality of life. Cranial irradiation was reserved for the 12% of patients who had T-cell ALL and a presenting leukocyte count of 100 x 10(9)/L or more, or CNS-3 (5 or more leukocytes/microL with identifiable blast cells in an atraumatic sample or the presence of cranial nerve palsy) status. Among the 247 consecutive patients enrolled in the study, 117 were classified as having lower-risk leukemia and received mainly antimetabolite-based continuation therapy; the 130 cases with higher-risk leukemia received more intensive continuation chemotherapy with multiple drug pairs administered in weekly rotation. The 5-year event-free survival estimate was 80.8% +/- 2.6% (SE); the 8-year rate was 78.6% +/- 5.8%. The 5-year cumulative risk of an isolated central nervous system (CNS) relapse was 1.7% +/- 0.8%, and that of isolated plus combined CNS relapse was 3.0% +/- 1.1%. The 5-year cumulative risks of etoposide-related myeloid malignancies were 1.8% +/- 1.3% in the lower-risk patients who received a cumulative dose of 1.2 g/m(2) and 5.0% +/- 2.0% in the higher-risk patients who received a cumulative dose of up to 14.4 g/m(2) (P = .18). Independent adverse prognostic features included the presence of MLL-AF4 or BCR-ABL fusion gene and minimal residual leukemia of 0.01% or more at the end of the 6-week remission induction phase. Our results suggest the efficacy of early intensification of intrathecal chemotherapy and provide the basis for studies omitting cranial irradiation altogether.

Reassessment of the prognostic significance of hypodiploidy in pediatric patients with acute lymphoblastic leukemia.

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.

ALK-positive anaplastic large cell lymphoma with leukemic peripheral blood involvement is a clinicopathologic entity with an unfavorable prognosis. Report of three cases and review of the literature.

Leukemic peripheral blood involvement in anaplastic large cell lymphoma (ALCL) is uncommon. We describe 3 children with such manifestations and review the features of 9 pediatric and adult patients previously described in the literature. Leukemic involvement in ALCL may occur at the time of initial diagnosis or develop during the course of disease. It most often is associated with the small cell histologic features and the t(2;5)(p23;q35). Clinical features commonly include significant respiratory distress, diffuse lung infiltrates or pleural effusions, and hepatosplenomegaly. Most cases have an aberrant T-cell immunophenotype with frequent expression of myeloid antigens, most often CD11b or CD13. Ten of the 12 cases reviewed had a poor response to therapy or early relapse. Thus, while anaplastic lymphoma kinase-positive ALCL and young patient age generally are associated with a favorable prognosis, leukemic involvement seems to identify a high-risk malignant neoplasm that requires more aggressive therapy, including hematopoietic stem cell transplantation.

Clinical significance of residual disease during treatment in childhood acute myeloid leukaemia.

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.

Results of therapy for acute lymphoblastic leukemia in black and white children.

CONTEXT: Treatment results for acute lymphoblastic leukemia (ALL) clearly have improved over the past decade, but black children have not fared as well as white children in large national trials. OBJECTIVE: To compare the clinical outcomes of therapy for black and white children with ALL treated at a single institution. DESIGN, SETTING, AND PATIENTS: A retrospective analysis of 412 children and adolescents (68 black, 338 white, and 6 other race) with newly diagnosed ALL who were treated consecutively at a pediatric cancer center in Memphis, Tenn. Patients were enrolled from December 1991 to July 1998 in successive Total Therapy studies regardless of race, ethnicity, or ability to pay and received risk-directed therapy according to stringent criteria. INTERVENTIONS: All patients received the same intensive, remission-induction therapy followed by 120 weeks of risk-assigned postremission therapy that included reinduction treatment, pulses of high-dose methotrexate, and early intensification of intrathecal chemotherapy. MAIN OUTCOME MEASURES: Event-free and overall survival rates for black and white children were estimated by the method of Kaplan and Meier and compared with the Mantel-Haenszel test and by Cox proportional hazards regression analysis, adjusting for known prognostic factors. RESULTS: The 68 black children were significantly more likely than the 338 white children to have higher-risk prognostic features, including an initial leukocyte count greater than 100 x 10(3)/ microL, a T-cell immunophenotype, and the t(1;19) chromosomal translocation with E2A-PBX1 fusion, and were less likely to have hyperdiploid blast cells, a favorable prognostic factor in childhood ALL. However, the clinical outcomes for these 2 cohorts were not significantly different: 5-year event-free and overall survival rates were 80.7% (95% confidence interval [CI], 70.3%-91.1%) and 86.2% (95% CI, 77.2%-95.2%) for black children vs 79.4% (95% CI, 74.7%-84.1%) and 85.0% (95% CI, 80.9%-89.1%) for white children. Ten-year results also were comparable, but the CIs were wide because of the small numbers of patients who had been followed up for 10 years or more. The lack of a racial effect on the long-term outcome of therapy was still apparent in a multivariate Cox regression analysis, adjusting for sex, age, presenting leukocyte count, leukemic cell DNA index, immunophenotype, and central nervous system status. CONCLUSION: With equal access to effective antileukemic therapy, black and white children with ALL can expect the same high rate of cure.

ALK-positive plasmablastic B-cell lymphoma with expression of the NPM-ALK fusion transcript: report of 2 cases.

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.

Characterization of the MLL partner gene AF15q14 involved in t(11;15)(q23;q14).

Translocations interrupting the mixed lineage leukemia gene (MLL) occur in 7-10% of acute lymphoblastic leukemia (ALL) and 5-6% of acute myeloid leukemia (AML) cases. One of these translocations, t(11;15)(q23;q14), occurs rarely in both ALL and AML. The gene on chromosome 15, AF15q14, was cloned recently in a patient with AML-M4. We have identified the same gene in a de novo T-ALL patient. However, both the MLL and AF15q14 breakpoints in these patients differed: in the previously reported AML-M4, both gene breaks were within exons, while in our ALL case the MLL break is intronic and the AF15q14 break is exonic. The MLL-AF15q14 fusion described previously shares no AF15q14 residues in common with the chimera reported here. The fusion proteins also differ with respect to MLL--the previously described fusion contains 55 extra amino acids as its MLL break is in exon 11, while the chimera we report breaks in intron 9. Contrary to the originally described normal AF15q14 (5925-bp cDNA encoding a 1833-aa protein), we identify a 7542-bp cDNA and a 2342-aa AF15q14 protein. AF15q14 appears identical to an mRNA previously found to be expressed in melanoma rendered nontumorigenic by microcell-mediated introduction of normal chromosome 6, suggesting the gene may function normally to suppress cell growth and/or enhance maturation.

Granulocyte colony-stimulating factor and the risk of secondary myeloid malignancy after etoposide treatment.

Event-free survival for children with acute lymphoblastic leukemia (ALL) now exceeds 80% in the most effective trials. Failures are due to relapse, toxicity, and second cancers such as therapy-related myeloid leukemia or myelodysplasia (t-ML). Topoisomerase II inhibitors and alkylators can induce t-ML; additional risk factors for t-ML remain poorly defined. The occurrence of t-ML among children who had received granulocyte colony-stimulating factor (G-CSF) following ALL remission induction therapy prompted us to examine this and other putative risk factors for t-ML in 412 children treated on 2 consecutive ALL protocols from 1991 to 1998. All children received etoposide and anthracyclines, 99 of whom received G-CSF; 284 also received cyclophosphamide, 58 of whom also received cranial irradiation. There were 20 children who developed t-ML at a median of 2.3 years (range, 1.0-6.0 years), including 16 cases of acute myeloid leukemia, 3 myelodysplasia, and 1 chronic myeloid leukemia. Stratifying by protocol, the cumulative incidence functions differed (P =.017) according to the use of G-CSF and irradiation: 6-year cumulative incidence (standard error) of t-ML of 12.3% (5.3%) among the 44 children who received irradiation without G-CSF, 11.0% (3.5%) among the 85 children who received G-CSF but no irradiation, 7.1% (7.2%) among the 14 children who received irradiation plus G-CSF, and 2.7% (1.3%) among the 269 children who received neither irradiation nor G-CSF. Even when children receiving irradiation were excluded, the incidence was still higher in those receiving G-CSF (P =.019). In the setting of intensive antileukemic therapy, short-term use of G-CSF may increase the risk of t-ML.

Genetic polymorphisms in CYP3A5, CYP3A4 and NQO1 in children who developed therapy-related myeloid malignancies.

Therapy-related acute myeloid leukemia and myelodysplastic syndrome (t-ML) are serious complications that affect some patients after acute lymphoblastic leukemia (ALL) treatment. Genetic polymorphisms in the promoter of CYP3A4 (CYP3A4*1B) and in NAD(P)H:quinone oxidoreductase (NQO1609C-->T substitution) have been associated with the risk of t-ML. A polymorphism in CYP3A5 (CYP3A5*3) affects CYP3A activity and the wild-type allele (CYP3A5*1) is in partial linkage with the CYP3A4*1B allele. We compared the genotype frequencies for the CYP3A5*3, the CYP3A4*1B and the NQO1609C-->T substitution in 224 children with ALL who did not develop t-ML (controls) and in 53 children with ALL who did develop the complication. The allele frequencies differed significantly among whites, blacks and Hispanics (P < 0.001 for CYP3A5*3, P < 0.001 for CYP3A4*1B and P = 0.004 for NQO1609), thus we performed the comparisons between ALL controls and t-ML patients after accounting for race. We found no differences in the CYP3A4*1B allele distribution between ALL controls and t-ML patients in whites (P = 0.339, 6.6% vs. 9.8%), blacks (P = 0.498, 93.8% vs. 87.5%) or Hispanics (P = 0.523, 39.1% vs. 25.0%). The frequencies for the NQO1609C-->T allele did not differ between control and t-ML groups in whites (P = 0.191, 35.0% vs. 44.9%), blacks (P = 0.664, 37.5% vs. 37.5%) or Hispanics (P = 0.447, 65.2% vs. 50.0%). We found no differences between the control and t-ML group in the incidence of homozygous CYP3A5*3 genotypes: 82.0% vs. 85.4% in whites (P = 0.403), 6.5% vs. 12.5% in blacks (P = 0.508), and 69.6% vs. 75.0% in Hispanics (P= 0.663). Our data do not support an association between common CYP3A4, NQO1 or CYP3A5 polymorphisms and the risk of t-ML in children treated for ALL.