E2A-ZNF384 and NOL1-E2A fusion created by a cryptic t(12;19)(p13.3; p13.3) in acute leukemia.

A 5-year-old boy who initially presented with ALL and relapsed 4 months later with AML was found to have an add(19) in the leukemia cells. FISH revealed that the add(19) was really a cryptic t(l2;l9)(p13.3;p13.3) interrupting E2A (TCF3). Nucleotide sequences of cloned genomic fragments with the E2A rearrangements revealed that the der(12) contained E2A joined to an intron of the NOLI (p120) gene. Reverse transcriptase (RT)-PCR of patient lymphoblast RNA showed expression of in-frame fusion cDNAs consisting of most of NOL1 fused to the 3' portion of E2A that encoded part of the second transcriptional activation domain and the DNA binding and protein dimerization motifs. The reciprocal der(19) E2A genomic rearrangements included 5' regions of E2A joined to an intron of the ZNF384 (NMP4, CIZ) gene, located approximately 450 kb centromeric to NOL1 on chromosome 12. RT-PCR showed expression of in-frame E2A-ZNF384 fusion cDNAs. To our knowledge, this is the second report of a chromosome translocation in leukemia resulting in two different gene fusions. This is the first report of expression of E2A fusion protein that includes the DNA binding and protein dimerization domains due to a more proximal break in E2A compared to those described previously.

Lymphoblastic leukemia/lymphoma in mice overexpressing the Mer (MerTK) receptor tyrosine kinase.

Mer (MerTK) is a receptor tyrosine kinase important in platelet aggregation, as well as macrophage cytokine secretion and clearance of apoptotic cells. Mer is not normally expressed in thymocytes or lymphocytes; however, ectopic Mer RNA transcript and protein expression is found in a subset of acute lymphoblastic leukemia cell lines and patient samples, suggesting a role in leukemogenesis. To investigate the oncogenic potential of Mer in vivo, we created a transgenic mouse line (Mer(Tg)) that expresses Mer in the hematopoietic lineage under control of the Vav promoter. Ectopic expression and activation of the transgenic Mer protein was demonstrated in lymphocytes and thymocytes of the Mer(Tg) mice. At 12-24 months of age, greater than 55% of the Mer(Tg) mice, compared to 12% of the wild type, developed adenopathy, hepatosplenomegaly, and circulating lymphoblasts. Histopathological analysis and flow cytometry were consistent with T-cell lymphoblastic leukemia/lymphoma. Mer may contribute to leukemogenesis by activation of Akt and ERK1/2 anti-apoptotic signals, which were upregulated in Mer(Tg) mice. Additionally, a significant survival advantage was noted in Mer(Tg) lymphocytes compared to wild-type lymphocytes after dexamethasone treatment. These data suggest that Mer plays a cooperative role in leukemogenesis and may be an effective target for biologically based leukemia/lymphoma therapy.

Monosomy 7 associated with pediatric acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS): successful management by allogeneic hematopoietic stem cell transplant (HSCT).

Pediatric acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) with monosomy 7 is associated with poor disease-free survival when treated by conventional chemotherapy, immunosuppression or supportive measures. Hematopoietic stem cell transplant (HSCT) may improve outcomes; however, data to support this are limited. To better understand the curative potential of HSCT in these patients, all cases of AML and MDS with monosomy 7 treated by two transplant programs (1992 to present) were reviewed. A total of 16 patients were treated, all by allogeneic HSCT. Primary diagnoses were MDS (N = 5), therapy-related MDS (N = 3), AML (N = 5) and therapy-related AML (N = 3). In all, 11 patients (69%) survive event-free at 2 years with median follow-up of 986 days (range 330-2011 days). Toxicity caused deaths of the five nonsurviving patients, four of whom were transplanted with active leukemia. Allogeneic HSCT is effective therapy for childhood AML and MDS associated with monosomy 7, particularly for patients with AML in complete remission and MDS.

Genetic and clinical characterization of patients with an interstitial duplication 15q11-q13, emphasizing behavioral phenotype and response to treatment.

The clinical significance of an interstitial duplication of (15)(q11-q13) remains unclear and controversial. The reported phenotypes vary widely and appear to be influenced by the parent of origin of the duplication. Aside from cases of dup(15) reported with autism, the behavioral phenotype of individuals with dup(15) has not been described. We present three families, two with intrachromosomal duplication (15)(q11-q13) ascertained because of developmental delay in a relative. Two families show clear evidence of multigenerational maternal inheritance. The individuals discussed in this paper have minor anomalies and developmental delays. In addition, we describe a behavioral phenotype which often includes attention deficit hyperactivity disorder (ADHD) and autistic spectrum disorder. Responses to medications used to manage these behaviors are also described, including a positive response to methylphenidate and a poor response to fluoxetine. The duplication in each presenting individual, and available family members, was investigated utilizing cytogenetic and molecular techniques including high resolution cytogenetics, fluorescence in situ hybridization (FISH), DNA methylation studies, and quantitative fluorescence PCR. High resolution cytogenetic techniques alone missed some cases, demonstrating the need to confirm results with other methods.

Minimal residual disease (MRD) in remission t(8;21) AML and in vivo differentiation detected by FISH and CD34+ cell sorting.

Many patients with t(8;21) AML have residual positive cells during remission. We previously developed D-FISH probes that detect both derivative chromosomes and the normal alleles. In negative controls, only 2/44,000 (0.0045%) positive signals were observed. To investigate MRD, we examined specimens from 29 patients who had initially obtained CR. In remission patients, 61% had 1-4/2000 positive cells (0.05-0.19%). Higher frequencies were found in two patients in early relapse and in one patient in early remission. However, a negative test did not exclude relapse. Since false positives were negligible and because most t(8;21) AMLs express CD34, we asked whether cell sorting combined with FISH would increase the sensitivity. In one patient, we observed that 80% of CD34+ cells were t(8;21)+ at 2 months from initial clinical and cytogenetic remission. However, by 5 months the pre- and post-sorted populations contained 0.15% and 0.06% t(8;21) cells, respectively. Whereas essentially all t(8;21) cells in the initial specimen expressed CD34, only 0.6% were subsequently CD34+. These results are consistent with in vitro assays showing that residual t(8;21) cells undergo differentiation. Thus, FISH can identify MRD in a majority of t(8;21) patients and, combined with CD34+ selection, may provide an indirect assessment of the differentiation state of residual t(8;21) cells.

Unusual childhood extramedullary hematologic malignancy with natural killer cell properties that contains tropomyosin 4--anaplastic lymphoma kinase gene fusion.

This report describes an unusual extramedullary hematologic malignancy in an 18-month-old child who presented with a capillary leak syndrome that evolved into hyperleukocytosis with malignant cells. The circulating tumor cells did not express an antigen profile typical of any subtype of leukemia commonly observed in children. Tumor cells were CD3(-)/CD56(+); had germline TCR genes; and strongly expressed CD30, epithelial membrane antigen, and anaplastic lymphoma kinase (ALK) consistent with a null cell anaplastic large cell lymphoma (ALCL). The malignant cells contained a t(2;19)(p23;p13.1) that interrupted ALK and translocated it to the der(19). Reverse transcriptase-polymerase chain reaction and nucleotide sequence analysis revealed fusion of ALK to tropomyosin 4, an ALK fusion partner not described previously in hematologic malignancies. The clinical presentation and phenotypic features of this malignancy were not typical for ALCL because tumor cells expressed both myeloid (CD13, CD33, HLA-DR) and natural killer (NK) cell antigens. The neoplastic cells most resembled NK cells because in addition to being CD3(-)/CD56(+) with germline TCR genes, these cells were CD25(+)/CD122(+)/granzyme B(+) and possessed the functional properties of immature NK cells. The unusual clinical presentation, immunophenotype, and functional properties of these neoplastic cells suggest that this malignancy may be derived from the putative myeloid-NK precursor cell. Furthermore co-expression of NK and ALCL features supports the concept that a minority of null-ALCL may be derived from NK cells and expands the spectrum of phenotypes that can be seen in tumors produced by ALK fusion proteins. (Blood. 2001;98:1209-1216)

Detection of E2A translocations in leukemias via fluorescence in situ hybridization.

Three rearrangements in ALL disrupt E2A and create E2A fusion proteins: the t(1;19)(q23;p13) and E2A-PBX1, t(17;19)(q22;p13) and E2A-HLF and a cryptic inv(19)(p13;q13) and E2A-FB1. While E2A is fused to PBX1 in most ALLs with a t(1;19), 5-10% of cases have translocations that appear identical, but do not affect E2A or PBX1. Because more intensive therapy improves the outcome of patients with E2A-PBX1positive (1;19) translocations, it is critical to identify this subset of patients so that appropriate therapy can be administered. In addition, there are balanced and unbalanced variants of the t(1;19) and controversy exists regarding the clinical significance of this distinction. We have developed a two-color fluorescence in situ hybridization assay that accurately detects E2A translocations in metaphase and interphase cells, distinguishes between balanced and unbalanced variants and identifies patients with a t(1;19) who lack E2A-PBX1 fusion. We found that clonal microheterogeneity is common in patients with E2A translocations and most patients have mixtures of cells with balanced and unbalanced translocations, suggesting that this distinction represents two ends of a continuum rather than distinct biological entities. These reagents should have widespread clinical utility and be useful for translational and basic research studies involving E2A translocations and this region of chromosome 19p13.

Choroid plexus carcinomas and rhabdoid tumors: phenotypic and genotypic overlap.

Five of six poorly differentiated choroid plexus carcinomas identified at our institution contained cells displaying a rhabdoid phenotype. Immunoperoxidase stains showed focal positivity for cytokeratin, epithelial membrane antigen, glial fibrillary acidic protein, S100, and vimentin. The MIB-1 proliferative index ranged from 7.0% to 27.1%. All six tumors were p53 positive. Only the one child with Li-Fraumeni syndrome had a p53 germline mutation. Electron microscopy verified choroid plexus differentiation and the co-existence of rhabdoid cells. Of the five studied, four had deletions of chromosome 22 [three with monosomy 22 and one with del(22)(q12)]. Thus, there was a phenotypic and genotypic overlap between choroid plexus carcinomas and rhabdoid tumors.

Analysis of TP53 mutations in relapsed childhood acute lymphoblastic leukemia.

TP53 is the most commonly mutated gene in human cancer, but TP53 mutations are present in less than 5% of children with acute lymphoblastic leukemia (ALL) at initial presentation. Mutations are detected more frequently in children with relapsed T-cell ALL, but the potential role of TP53 mutations in relapsed B-lineage childhood ALL is not understood as well. The authors determined the nucleotide sequence of amplified DNA from exons 5 to 8 of the TP53 gene in leukemic cells obtained from 17 children with ALL at the time of first bone marrow relapse. All 17 contained only germline TP53 sequences. Review of the published literature disclosed that TP53 mutations have been found in 22% of cases of relapsed ALL. To understand the role of p53 abnormalities in this clinical setting, it will be important for future studies to analyze cases of relapsed ALL with assays capable of interrogating the functional integrity of the p53 pathway.

Rhabdoid glioblastoma.

Rhabdoid phenotypic change has been described in a number of different neoplasms from diverse organ sites. These tumors share common light and electron-microscopic features, display a polyphenotypic immunohistochemical profile and often show cytogenetic abnormalities of chromosome 22. In the central nervous system (CNS), most rhabdoid tumors occur in the posterior fossa of very young children and are associated with a primitive neuroectodermal tumor (PNET) component and are designated atypical teratoid/rhabdoid tumors. Infrequently, other rhabdoid tumors of the CNS have been described, including rhabdoid meningiomas and malignant rhabdoid tumors of uncertain histogenesis. Several examples of conventional gliomas displaying significant areas with rhabdoid morphology were also presented in an abstract by Kepes and Moral [1991], although never published in final manuscript form. We now detail the case of an 18-year-old male with an aggressive, supratentorial CNS rhabdoid tumor that was associated with an epithelioid glioblastoma and apparently arose from areas of low-grade glioma. The rhabdoid tumor component was present in the original tumor but became more predominant with each of 3 successive resections. No areas of PNET were identified. Electron microscopy and immunohistochemistry showed features classic for rhabdoid tumors and cytogenetic studies demonstrated multiple tumor clones with monosomy 22. This case documents progressive rhabdoid transformation of a glioma, expands the spectrum of CNS tumor types that can display a rhabdoid phenotype and highlights the diagnostic and therapeutic challenges with this type of tumor.

Hepatocyte transplantation in acute liver failure.

The majority of patients with acute liver failure (ALF) die waiting for orthotopic liver transplantation (OLT). No other treatment modality is shown to improve survival. This study was conducted to assess the safety and feasibility of hepatocyte transplantation (HT) and subsequent engraftment and function of donor cells. Functional and structural integrity of cryopreserved and thawed human hepatocytes were assessed by their morphological characteristics, induction of P-4501A1 transcription, and survival in vivo by xenotransplantation into rats. Five patients with severe ALF underwent intrasplenic (4 patients) and/or intrahepatic (2 patients) HT through angiography under cyclosporine immunosuppression. All patients had grade III to IV encephalopathy and factor V levels less than 0.5 U/mL, were ventilator and dialysis dependent, and were not OLT candidates. Three of the 5 patients who survived 48 hours after HT had substantial improvement in encephalopathy scores, arterial ammonia levels, and prothrombin times. Clinical improvement was paralleled by an increase in aminopyrine and caffeine clearances. All 3 patients lived substantially longer than expected based on clinical experience after HT (12, 28, and 52 days) but eventually died. Postmortem examination showed the presence of transplanted hepatocytes in liver and spleen by light microscopy and fluorescent in situ hybridization (FISH). Cryopreserved and thawed human hepatocytes can be transplanted into recipients with ALF with some acceptable but definite complications. Engraftment of donor hepatocytes was proven by histological examination and FISH by both transjugular biopsy and at autopsy. Improvement in brain edema, encephalopathy grade, and clearance of antipyrine and caffeine suggested function, albeit with a 24- to 72-hour delay posttransplantation.

Protean clinical manifestations in children with leukemias containing MLL-AF10 fusion.

Translocations involving the MLL gene on chromosome 11q23 occur in 5-10% of human leukemias, and involve fusion with more than 30 different partner genes. The MLL-AF10 fusion produced by the t(10;11)(p12;q23) or ins(10;11)(p12;q23q13) occurs in a small percentage of acute leukemias, most commonly acute myelogenous leukemia (AML) of the M5 FAB subtype. We report two cases of AML (M5a and M0) and one case of acute lymphoblastic leukemia containing MLL-AF10 fusion. Each case had varied clinical characteristics, despite expressing similar MLL-AF10 fusion transcripts. Including the three cases described in this report, we identified a total of 38 cases of leukemia with MLL-AF10 fusion. Approximately one-third of these are not M5 AML. Taken together, these findings emphasize that while the sentinel molecular event may be identical in a disease, the clinical presentation and outcome can vary widely.

Astroblastoma: ultrastructural observations on a case of high-grade type.

An astroblastoma of high-grade type arising in the brain of a 3-year-old child is reported. The first description of the ultrastructural, immunohistochemical, and cytogenetic findings in this rare tumor variant are presented.

TEL-AML1 fusion identifies a subset of children with standard risk acute lymphoblastic leukemia who have an excellent prognosis when treated with therapy that includes a single delayed intensification.

The Children's Cancer Group (CCG) found that children with moderate risk acute lymphoblastic leukemia (ALL) had an improved 5-year event-free survival (EFS) rate when treated with therapy that included a doubled delayed intensification (DDI) vs a single DI (SDI) phase. Because of increased toxicity with DDI, it is important to determine whether subgroups of children with ALL can be identified who have excellent outcomes with SDI therapy. TEL-AML1 fusion and hyperdiploid DNA content are present in the leukemic blasts of significant proportions of children with ALL and have been associated with an excellent prognosis. In this study, we retrospectively examined the impact of TEL-AML1 status and ploidy on treatment outcome in a cohort of 75 children with standard risk ALL treated at our institution between 1983 and 1993 with SDI therapy. TEL-AML1 fusion was present in 19/43 (44%) evaluable cases. Fifteen of 56 (27%) evaluable cases were classified as hyperdiploid based on a modal chromosome number of >/=51 and/or a DNA index of >/=1.16. The 7-year EFS was 81% for the 19 TEL-AML1-positive patients vs 54% for the 24 TEL-AML1-negative patients (P = 0.0264). In multivariate analyses, TEL-AML1-positive status was associated with a superior EFS (P = 0.02) even when the intial white blood count was included in the model. Overall survival (OS) at 7 years for TEL-AML1-positive patients was 100% vs 83% for TEL-AML1-negative patients (P = 0.0677). There were no differences in 7-year EFS or OS based on ploidy comparisons. These results underscore the need to examine closely the effects of treatment intensification on specific biologically defined subgroups of children with ALL.

Acquisition of p16(INK4A) and p15(INK4B) gene abnormalities between initial diagnosis and relapse in children with acute lymphoblastic leukemia.

Although numerous somatic mutations that contribute to the pathogenesis of childhood acute lymphoblastic leukemia (ALL) have been identified, no specific cytogenetic or molecular abnormalities are known to be consistently associated with relapse. The p16(INK4A) (p16), which encodes for both p16(INK4A) and p19(ARF) proteins, and p15(INK4B) (p15) genes are inactivated by homozygous deletion and/or p15 promoter hypermethylation in a significant proportion of cases of childhood ALL at the time of initial diagnosis. To determine whether alterations in these genes play a role in disease progression, we analyzed a panel of 18 matched specimen pairs collected from children with ALL at the time of initial diagnosis and first bone marrow relapse for homozygous p16 and/or p15 deletions or p15 promoter hypermethylation. Four sample pairs contained homozygous p16 and p15 deletions at both diagnosis and relapse. Among the 14 pairs that were p16/p15 germline at diagnosis, three ALLs developed homozygous deletions of both p16 and p15, and two developed homozygous p16 deletions and retained p15 germline status at relapse. In two patients, p15 promoter hypermethylation developed in the interval between initial diagnosis and relapse. In total, homozygous p16 deletions were present in nine of 18 cases, homozygous p15 deletions in seven of 18 cases, and p15 promoter hypermethylation in two of eight cases at relapse. These findings indicate that loss of function of proteins encoded by p16 and/or p15 plays an important role in the biology of relapsed childhood ALL, and is associated with disease progression in a subset of cases.

Oncogenesis in utero: fetal death due to acute myelogenous leukaemia with an MLL translocation.

The incidence of translocations involving the 11q23 gene MLL is markedly increased in leukaemias that occur in infants <1 year of age. Epidemiological and molecular data have demonstrated that at least some of these translocations occur in utero. In this report we describe a case of fetal death at 36 weeks of gestation. At autopsy the fetus was found to have widely disseminated acute myelogenous leukaemia (AML), FAB subtype M5. Molecular cytogenetic studies of nuclei recovered from paraffin-embedded tissue sections demonstrated that the leukaemic cells contained an MLL translocation. This is the first detailed report, to our knowledge, of fetal death due to acute leukaemia, and directly demonstrates oncogenesis in utero.

Different patterns of homozygous p16INK4A and p15INK4B deletions in childhood acute lymphoblastic leukemias containing distinct E2A translocations.

The p16INK4A (p16) and p15INK4B (p15) tumor suppressor genes are inactivated by homozygous gene deletion and p15 promoter hypermethylation in a significant proportion of childhood acute lymphoblastic leukemias (ALLs). However, little is known about the potential association between p16/p15 gene alterations and specific genetic abnormalities implicated in leukemogenesis. The t(1;19)(q23;p13) and t(17;19)(q21-22;p13) are non-random translocations observed in childhood ALL that create distinct E2A fusion proteins: E2A-PBX1 and E2A-HLF, respectively. Previously, a negative association was found between the t(1;19) and homozygous p16/p15 deletions. In this study we determined p16 and p15 gene status in additional t(1;19)+ ALLs and compared this incidence to that observed in t(17;19)+ ALLs. No homozygous p16 or p15 deletions were observed among 13 t(1;19)+ ALLs analyzed. In contrast, homozygous deletions of both p16 and p15 were present in two of four t(17;19)+ ALLs. None of 10 t(1;19)+ ALLs contained p15 promoter hypermethylation. In contrast, one of the two t(17;19)+ ALLs that lacked p15/p16 homozygous deletions showed probable hemizygous p15 hypermethylation. We conclude that homozygous p16 and/or p15 deletions and p15 hypermethylation rarely accompany E2A-PBX1 fusion, but occur in concert with E2A-HLF fusion in a subset of t(17;19)+ ALLs. These findings suggest that there may be different modes of cooperative leukemogenesis in ALLs associated with different E2A fusion proteins.