Improved detection of p53 point mutations by dideoxyfingerprinting (ddF).

Two screening techniques for identifying point mutations (single-strand conformational polymorphism (SSCP) and dideoxyfingerprinting (ddF)) were compared to sequencing to determine their efficiency in detecting mutations in exons 5-8 of the p53 tumor suppressor gene. Twelve human glioblastoma cell lines were studied by each of the three methods. Ten mutations were identified by sequencing; of these, 10/10 were detected by ddF, while SSCP detected 6/10 true mutations and falsely identified two presumed mutations not confirmed by sequencing. We examined the impact of parameters which influence DNA conformation (gel temperature, gel composition, and PCR product size) on the ability of SSCP and ddF to detect mutations. The sensitivity of SSCP varied with both gel temperature and the size of the PCR product; in contrast, ddF was not influenced by either gel temperature or product length (up to 460 nucleotides). We conclude that the increased sensitivity of ddF, together with its greater ease of application due to the lack of need for optimization, provides significant advantages over SSCP in screening DNA sequences for the presence of point mutations. Our results also suggest that the incidence of p53 mutations may be underestimated in studies of human cancers which utilize SSCP as the method of mutational screening.

Detection of mutations by automated fluorescence/RNA-based dideoxy fingerprinting (ARddF)

Dideoxy fingerprinting (ddF) is a hybrid technique which combines aspects of single strand conformational polymorphism (SSCP) and dideoxy sequencing to detect the presence of single base changes in a defined fragment of nucleic acid. ddF is no more technically demanding than SSCP, yet it is more sensitive in detecting point mutations. We describe here the adaptation of conventional ddF to an automated sequencing system using fluorescent Cy5 labeled primers. We show that automated RNA-based ddF (ARddF) has several advantages over conventional radioisotope-based ddF, including: (1) analysis of larger nucleic acid fragments (up to 10(3) bp), due to the ability to continuously analyse and compile sequencing information; (2) greater reliability for distinguishing mutant sequences from wild type sequences (particularly when the mutation leads to gain or loss of a dideoxy termination segment); (3) the use of fluorescent labeled primers, making ARddF less hazardous than methods requiring radionucleotides. The use of ARddF in conjunction with new methods for isolating RNA from a [corrected] small number of cells facilitates mutational analysis of small tissue biopsies and other limited samples, and will allow more widespread application of mutational screening in the setting of clinical diagnostic laboratories.

t(5;14)(q33-34;q11), a new recurring cytogenetic abnormality in childhood acute leukemia.

A novel translocation, t(5;14)(q33-34;q11), was identified in the leukemic cells of four children presenting with acute lymphoblastic leukemia (ALL). The patients were 14 months, 2 years, 10 years, and 12 years old; each presented with one or more features of bulky disease, including lymphadenopathy, organomegaly or a mediastinal mass. The leukemic blasts were B lineage in two cases and T lineage in two cases. The patients with B-lineage ALL remain in continuous remission at 22 and 19 months following diagnosis. One patient with T-lineage ALL relapsed 6 months after diagnosis. The other patient with T-lineage ALL developed acute myelocytic leukemia (AML) 17 months after diagnosis; t(5;14)(q33-34;q11) was present in both the lymphoblasts at diagnosis and the myeloblasts at relapse, consistent with a lineage switch from ALL to AML. Rearrangement of the T-cell receptor delta chain (TCRD) gene at 14q11 was demonstrated in the three cases studied, suggesting its involvement in the pathogenesis of these leukemias by alteration of the structure or expression of an unidentified gene(s) on the long arm of chromosome 5.

High-dose cytosine arabinoside and etoposide: an effective regimen without anthracyclines for refractory childhood acute non-lymphocytic leukemia.

The purpose of this report is to describe the tolerability and activity of the combination of high-dose cytosine arabinoside (Ara-C) given at the maximum tolerated dose of 36 g/m2, together with high doses of etoposide in relapsed and refractory childhood acute leukemias. Eighteen children with relapsed or refractory acute leukemia were treated with Ara-C 3 g/m2 every 12 h on days 1-6, followed by etoposide 400 mg/m2 on days 7-9 (HDAC/VP-16). Eight children with refractory disease received HDAC/VP-16 as salvage induction therapy after failing conventional induction regimens; four of five refractory ANLL patients (80%) had a complete response (CR) after HDAC/VP-16 therapy. Ten patients received HDAC/VP-16 as post-remission intensification therapy; five patients (four ANLL, one relapsed ALL) remain in second CR at 56, 26, 9, 5 and 2 months. Toxicities were primarily hematologic and dermatologic. Seven patients (39%) developed bacterial or fungal infections; four patients developed grade 3 or 4 acral erythema. No patient died of therapy-related toxicity. The combination of 36 g/m2 cytosine arabinoside and 1200 mg/m2 etoposide is an effective regimen for children with relapsed or refractory acute nonlymphocytic leukemia, with tolerable toxicities; the absence of anthracyclines makes this regimen suitable for patients who have previously received maximal doses of anthracyclines or who have evidence of cardiac dysfunction. Further evaluation of this regimen in acute nonlymphocytic leukemia is presently being investigated.

Disruption of the RanBP17/Hox11L2 region by recombination with the TCRdelta locus in acute lymphoblastic leukemias with t(5;14)(q34;q11).

The t(5;14)(q33-34;q11) translocation constitutes a recurrent rearrangement in acute lymphoblastic leukemia involving the T cell receptor (TCR) delta locus on chromosome 14. Breakpoint sequences of the derivative chromosome 5 were isolated by application of a ligation-mediated PCR technique using TCR delta-specific primers to amplify genomic DNA from the leukemic cells of a patient with t(5;14). Through exon trap analysis, we identified various putative exons of the chromosome 5 target gene of the translocation; compilation of sequence information of trapped exons and available expressed sequence tags (ESTs) from the GenBank database allowed us to assemble 1.2 kb of the cDNA. Full-length cDNAs were isolated from a human testis cDNA library and sequence analysis predicted a putative Ran binding protein, a novel member of the importin-beta superfamily of nuclear transport receptors, called RanBP17. The t(5;14) breakpoint maps to the 3' coding region of the gene. The breakpoint of a second t(5;14) positive patient was mapped about 8 kb downstream of the most 3' RanBP17 exon and 2 kb upstream of the first exon of the orphan homeobox gene, Hox11L2. In both cases TCR delta enhancer sequences are juxtaposed downstream of the truncated or intact RanBP17 gene, respectively on the derivative chromosome.

Phase I study of topotecan administered as a 21-day continous infusion in children with recurrent solid tumors: a report from the Children's Cancer Group.

The purpose of this study was to determine the toxicity, maximum tolerated dose, and pharmacokinetics of a 21-day continuous infusion of topotecan in children with relapsed solid tumors. Fifteen patients received 40 courses of continuous ambulatory infusions of topotecan every 28 days or when there was resolution of hematological toxicity and any grade 2 or greater nonhematological toxicity. The starting dose was 0.4 mg/m2/day. Total topotecan levels were measured on days 1, 7, 14, and 21. Three of four patients who received a starting dose of 0.4 mg/m2/day experienced dose-limiting myelosuppression. At the reduced dose of 0.3 mg/ m2/day, only two of the seven patients experienced dose-limiting myelosuppression. Subsequently, four patients with more limited prior therapy were treated with 0.4 mg/m2/ day; three had dose-limiting myelosuppression. Two patients with a dose-limiting toxicity at 0.4 mg/m2/day tolerated additional courses at 0.3 mg/m2/day. An equal number of patients had grade 4 neutropenia or thrombocytopenia. Other adverse events were rare. Two patients with ependymoma, one with rhabdomyosarcoma, and one with retinoblastoma metastatic to the brain had objective responses. The steady state plasma concentration and clearance of topotecan (Css) was achieved by day 1. Css in six patients with complete data were 1.44 +/- 0.50 and 2.13 +/- 0.83 ng/ml at 0.3 and 0.4 mg/m2/day, respectively. Thus, a 21-day topotecan infusion was well-tolerated at 0.3 mg/m2/day. Myelo-suppression was the dose-limiting toxicity at 0.4 mg/m2/day. The steady state and clearance of topotecan in this study are similar to those reported in adult patients.

A PrP gene codon 178 base substitution and a 24-bp interstitial deletion in familial Creutzfeldt-Jakob disease.

Several mutations in the prion protein (PrP) gene are associated with familial Creutzfeldt-Jakob disease (FCJD). We describe a family in which five members in three generations have had FCJD. The proband and some descendants of the affected members carried an abnormal PrP gene allele. This allele contained a 24-bp deletion from the tandem repeat region of the open reading frame and a codon 178 missense substitution. Observations suggest that the codon 178 mutation is involved in the pathogenesis of FCJD in the family described here. The 24-bp deletion may be an uncommon polymorphism.

A small-cell-predominant variant of primary Ki-1 (CD30)+ T-cell lymphoma.

We describe nine patients with a primary Ki-1 (CD30)+ T-cell lymphoma containing numerous, often CD30-negative, small lymphocytes with irregular nuclei and a minor population of large CD30+ tumor cells. All previously described primary Ki-1+ lymphomas have been large-cell neoplasms. In this small-cell variant, the diagnosis of lymphoma was difficult to make because there was a predominance of small lymphocytes and, in some cases, clinical features suggested an inflammatory process. Patients were young (age range 0.3-40 years, median 14 years), and frequently had B symptoms (56%); sites of involvement were predominantly skin (78%) and lymph node (67%). The actuarial 2-year disease-free survival was 14%, and the overall survival was 51%. Two patients had a rapidly fatal course. In all cases histologic sections showed a predominance of small lymphocytes with marked nuclear irregularity and often a perivascular/intravascular distribution of CD30+ large cells. All cases had a T-cell phenotype. In four cases the large and small cells could be compared and had a similar aberrant T-cell phenotype. Large cells were CD30+, but only rare small cells expressed CD30. Cytogenetic studies revealed a t(2;5)(p23;q35) in four of four cases studied. Four patients had numerous large cells on repeat biopsies; two of these developed sheets of large CD30+ cells typical of anaplastic large-cell lymphoma (ALCL). These cases provide further evidence that primary Ki-1+ lymphoma has a morphologic spectrum that includes a small-cell variant. Although very different morphologically from previously described Ki-1+ ALCL, this small-cell variant is clearly part of the disease spectrum on the basis of clinical features, the presence of the t(2;5)(p23;q35), the aberrant T-cell phenotype in the small and large cells, as well as histologic progression seen in several patients.

Relation of silent myocardial ischemia after coronary artery bypass grafting to angiographic completeness of revascularization and long-term prognosis.

The prevalence and characteristics of silent myocardial ischemia as detected by 24-hour ambulatory electrocardiography ST-segment depression were prospectively assessed in 94 patients examined early (1 to 3 months) and 184 patients examined late (12 months) after coronary artery bypass grafting (CABG), and followed for a mean of 48 +/- 11 (range 4 to 62) months. The relation of ambulatory electrocardiographic silent ischemia to evidence of completeness of revascularization as defined by cardiac angiography performed 1 and 12 months after CABG, and to prognosis by follow-up of adverse clinical events was analyzed. Silent ischemia was detected early in 20% (19 of 94) and late in 27% (50 of 184) of patients, and showed a mean frequency of episodes ranging from 6 to 10 episodes/24 hours with a mean duration ranging from 15 to 23 minutes. The circadian distribution of episodes disclosed a significant peak of ischemic activity during the period of 6 A.M. to noon and a secondary peak between 6 P.M. and midnight (p less than 0.01 and p less than 0.001, respectively). Silent ischemia was not found by univariate analysis to be associated with graft or anastomotic site occlusions, low graft flow rates, grafted arteries with significant distal residual stenoses or ungrafted stenotic native coronary arteries. Kaplan-Meier analysis of time to cardiac event showed that silent ischemia was not predictive of an adverse clinical event in the early years after CABG. Cox regression analysis of 30 covariates only disclosed age (relative risk 1.06 [95% confidence interval, 1.01 to 2.94]) as having an effect on time to adverse clinical event.(ABSTRACT TRUNCATED AT 250 WORDS)

DiGeorge anomaly and chromosome 10p deletions: one or two loci?

We report on a patient with DiGeorge syndrome (DGS) phenotype or anomaly and an unbalanced translocation [45,XY,-10,-22,+der(10),t(10;22)(p13;q11)] resulting in monosomy of 10p13-pter and 22q11-pter. Because both regions involved in this rearrangement have been implicated in DGS, we performed a molecular cytogenetic analysis of both loci in this patient. Results indicate that the chromosome 22 DGS locus is intact but that the terminal deletion of the short arm of chromosome 10 is adjacent to or partially overlapping with the recently defined consensus deleted region observed in DGS patients with 10p deletions. We conclude that the DGS anomaly in our patient is likely to be due to haploinsufficiency of genes located on chromosome 10p. Most, if not all, of the region included in the previously described 10p smallest region of deletion overlap is not deleted in our patient. Therefore, this deletion breakpoint either narrows the previously proposed 10p region or defines a second region within 10p critical for the DGS anomaly.

Use of the BCR probe to demonstrate extramedullary recurrence of CGL with a T cell lymphoid phenotype following bone marrow transplantation.

A case of Philadelphia chromosome (Ph1) positive chronic granulocytic leukemia (CGL) is described in which the patient underwent successful treatment with supralethal chemoradiotherapy and allogeneic bone marrow transplantation (BMT) after transformation to blast crisis. Supraclavicular adenopathy developed 5 months after BMT and biopsy revealed a hematopoietic lymphoid neoplasm with an early T cell phenotype. A concurrent bone marrow was microscopically and cytogenetically normal. A metaphase chromosome preparation could not be obtained from nodal tissue. Lymph node DNA, however, was easily extracted and a rearrangement of BCR identical to that in the bone marrow prior to BMT was demonstrated indicating recurrent CGL rather than a de novo lymphoproliferative process. Appropriate therapy for lymphoid blast crisis resulted in a marked regression of measurable disease. The BCR probe may prove to be a useful tool for the diagnosis of CGL when standard cytogenetic techniques cannot be applied.

Childhood acute lymphocytic leukemia with a preleukemic phase: report of an associated translocation and review of the literature.

A fourteen month old boy presented with hepatosplenomegaly and pancytopenia. An extensive evaluation, including bone marrow aspiration and biopsies of both liver and lymph nodes, revealed a polyclonal B cell proliferation consistent with a reactive process, with no evidence of leukemia. After receiving transfusions of red cells and platelets, his blood counts recovered. Five weeks later, he returned with an elevated white blood count and bone marrow findings diagnostic of acute lymphocytic leukemia. The leukemic blasts contained a novel chromosomal translocation, t(5;14) (q34;q12). We describe the clinical, immunophenotypic and cytogenetic features of this case, review the literature of acute lymphocytic leukemia associated with a preleukemic phase, and discuss the relationship of this clinical entity to the 5q-abnormality associated with myelodysplasia.

Unusual thalamic inclusions in an infant with progressive neurologic dysfunction and blindness.

A 25-month-old boy had a progressive neurologic disorder which started at two months of age and was characterized by seizures, blindness, partial deafness and severe hypotonic quadriparesis. Neuropathologic examination demonstrated the presence of intracytoplasmic neuronal inclusions which were entirely restricted to the thalamus. The inclusions had the histochemical staining characteristics of a glycolipid, were autofluorescent, and ultrastructurally consisted of concentric and parallel arrays of double membranes and curvilinear profiles. These features suggest that the infant may have had a disorder related to neuronal ceroid lipofuscinosis. Review of the literature failed to disclose a similar case.

Management of pleural effusions in children with malignant lymphoma.

PURPOSE: The aim of this study was to determine potential problems in the diagnosis and management of children with pleural effusions and malignant lymphoma as well as the efficacy of thoracentesis. METHODS: The case histories of six children with malignant lymphoma who presented with pleural effusions were reviewed. Thoracentesis was performed using the Seldinger technique. RESULTS: Four of the children presented with symptoms and chest radiograph findings similar to pneumonia. A large mediastinal mass was present in two children. Pleural fluid analysis resulted in a definitive diagnosis of lymphoma in five of the six children. Two of the children had symptoms of reexpansion pulmonary edema after removal of pleural fluid. An empyema developed in one child after thoracotomy and chest tube placement. Reaccumulation of pleural fluid was common before initiating chemotherapy. CONCLUSIONS: Malignant pleural effusions frequently are present in children with non-Hodgkin's lymphoma. They may present with respiratory distress because of the size of the effusion, the mediastinal mass, or both. Management of these pleural effusions is associated with potential complications, some of which are life threatening. Thoracentesis is the initial diagnostic and therapeutic procedure of choice. The use of a Seldinger technique for thoracentesis has proved useful and safe. In patients with large effusions, aggressive removal of the pleural fluid may be followed by reexpansion pulmonary edema.

Functional outcome of low-level traumatically brain-injured admitted to an acute rehabilitation programme.

A retrospective analysis of functional status at discharge, disposition, Glasgow Outcome Scale (GOS) score at 6 months and 1 year was undertaken for 23 consecutive 'low-level' traumatic brain injury victims admitted in a state of complete dependency to an acute rehabilitation programme. All patients met criteria for extremely severe traumatic brain injury, with an average Glasgow Coma Scale score of 8.7 at admission to the rehabilitation facility (an average of 44 days post-injury). All but three patients made significant functional gains during the acute rehabilitation stay. Almost half of the study patients (48%) were discharged at home while the rest went to sub-acute rehabilitation programmes. At 6 months post-injury eight (35%) met GOS criteria for 'good' outcome or 'moderate disability'. A review of clinical features, categories of functional progress, and relationships of outcome and disposition to variables known to have predictive value in such a patient population is described. Analysis of variables related to admission selection criteria that have been used in this rehabilitation programme is presented and the basis and implications of selection criteria discussed.