Molecular cloning, expression, and chromosomal localization of the gene encoding a human myeloid membrane antigen (gp150).

DNA from a tertiary mouse cell transformant containing amplified human sequences encoding a human myeloid membrane glycoprotein, gp150, was used to construct a bacteriophage lambda library. A single recombinant phage containing 12 kilobases (kb) of human DNA was isolated, and molecular subclones were then used to isolate the complete gp150 gene from a human placental genomic DNA library. The intact gp150 gene, assembled from three recombinant phages, proved to be biologically active when transfected into NIH 3T3 cells. Molecular probes from the gp150 locus annealed with a 4.0-kb polyadenylated RNA transcript derived from human myeloid cell lines and from tertiary mouse cell transformants. The gp150 gene was assigned to human chromosome 15, and was subchromosomally localized to bands q25-26 by in situ hybridization. The chromosomal location of the gp150 gene coincides cytogenetically with the region assigned to the c-fes proto-oncogene, another human gene specifically expressed by myeloid cells.

The first intron in the human c-abl gene is at least 200 kilobases long and is a target for translocations in chronic myelogenous leukemia.

The c-abl protooncogene is unusual in two respects; it has multiple, widely space N-terminal coding exons transcribed by different promoters, and it is the target of the translocations that form the Philadelphia chromosome found in cells of chronic myelogenous leukemia patients. To understand the organization of the gene in normal and chronic myelogenous leukemia patient DNA we have mapped c-abl by pulsed field gradient gel electrophoresis. We find that one of the alternative 5' exons of the gene lies at least 200 kilobases upstream of the remaining c-abl exons, posing formidable transcription and splicing problems. The 5'-most c-abl exon includes an unusually long 1,276-base-pair segment that contains 15 ATG codons and multiple short open reading frames, upstream of the abl initiator codon. Its peculiar structure suggests that c-abl may be decapitated in most chronic myelogenous leukemia patients, and we demonstrate that this is the case in the chronic myelogenous leukemia cell line K562.

Screen detected sclerosing lymphocytic lobulitis and amyloid of the breast in the same patient--a possible causal link.

Sclerosing lymphocytic lobulitis (SLL) and amyloidosis of the breast are both rare. We report the case of a 59 year old woman who presented with suspicious microcalcifications on routine screening mammography. Wire-guided excision biopsy showed features typical of SLL but also localised amyloid deposits within the specimen. Amyloidosis and SLL may have similar immunological causes. This patient represents the first documented association of these two disorders.

K562 cells implicate increased chromatin accessibility in Alu transcriptional activation.

Alu repeats in K562 cells are unusually hypomethylated and far more actively transcribed than those in other human cell lines and somatic tissues. Also, the level of Alu RNA in K562 cells is relatively insensitive to cell stresses, namely heat shock, adenovirus infection and treatment with cycloheximide, which increase the abundance of Alu RNA in HeLa and 293 cells. Recent advances in understanding the interactions between DNA methylation, transcriptional activation and chromatin conformation reveal reasons for the constitutively high level of Alu expression in K562 cells. Methylation represses transcription of transiently transfected Alu templates in all cell lines tested but cell stresses do not relieve this repression suggesting that they activate Alu transcription through another pathway. A relatively large fraction of the Alus within K562 chromatin is accessible to restriction enzyme cleavage and cell stresses increase the chromatin accessibility of Alus in HeLa and 293 cells. Cell stress evidently activates Alu transcription by rapidly remodeling chromatin to recruit additional templates.

Impact of chromosomal translocations on prognosis in childhood acute lymphoblastic leukemia.

The presence of a chromosomal translocation in the leukemic cells at diagnosis of acute lymphoblastic leukemia (ALL) in children is associated with a high risk for treatment failure. We have reexamined the relationship between translocations and prognosis in 146 children with ALL who received risk-based therapy such that high-risk patients were treated with intensive drug schedules. In univariate analysis, multiple factors were associated with a relatively poor event-free survival (EFS) including age less than 2 years or greater than 10 years (combined group), WBC count greater than 10 x 10(9)/L, French-American-British (FAB) morphologic classification L2, absence of common ALL antigen (CALLA, CD10) expression, absence of hyperdiploidy with a chromosome number of 50 to 60, and presence of the specific translocations t(4; 11)(q21;q23) or t(9;22)(q34;q11) (combined group). However, there was no disadvantage with respect to EFS in patients with translocations compared with those who lacked translocations (73% at 4 years in both groups). Furthermore, when patients with specific cytogenetic abnormalities for which the prognostic significance has been well established (hyperdiploid 50 to 60, t(4;11), and t(9;22] were removed from the analysis, the remaining group with other translocations had a better EFS than the remaining group lacking translocations, although this was not statistically significant (81% v 65% at 4 years, P = .24). In a multivariate analysis, a model including WBC count and FAB classification was the strongest predictor of EFS. The presence or absence of translocations was not an independent predictor of EFS and did not contribute to the ability of any model to predict EFS. In conclusion, when effective intensive therapy is used to treat childhood ALL with high-risk clinical features, categorization of patients on the basis of chromosomal translocations without attention to the specific abnormality is not useful as a prognostic factor.

Intraocular retinoblastoma group V: an analysis of prognostic factors.

A retrospective analysis of the University of Minnesota (Minneapolis) experience with retinoblastoma is presented. Seventy-five patients were diagnosed with retinoblastoma between 1958 and 1983, of which 53 (71%) had at least one Reese-Ellsworth group V eye. Nineteen group V patients and one group II patient developed extraocular disease recurrence. The cumulative actuarial rate of recurrence at 12 years was 36% for patients with group V disease. The median time from diagnosis to recurrence for unilateral patients was seven months and for bilateral patients 28 months (P = .001). Patients developing extraocular disease had a 10-year actuarial survival rate postrecurrence of 34%. The four long-term survivors of extraocular recurrences had had isolated orbital or local soft tissue recurrences only. Features of group V patients associated with extraocular recurrences were identified by univariate life table analyses. Clinical poor-risk factors included the nongenetic form of the disease (P = .03) and male sex (P = .02). Pathologic poor risk factors included rubeosis (P = .01), undifferentiated histology (P = .03), large tumor size (P = .05), and intraocular extension to the anterior segment (P = .02), retinal pigment epithelium (P = .03), choroid (P less than .001), and optic nerve beyond the lamina cribrosa (P = .02). Treatment-associated poor-risk factors included an optic nerve length of less than 5 mm removed at enucleation (P = .003). Multivariate life table analyses demonstrated the following parameters to be independent poor-prognostic factors: optic nerve length of less than 5 mm removed at enucleation (P = .001), optic nerve involvement (P = .004), and large tumor size (P = .01). These results will help to identify patients with retinoblastoma who are at greatest risk for extraocular recurrence.

Dispersion and insertion polymorphism in two small subfamilies of recently amplified human Alu repeats.

Newly isolated members of two recently propagated (young) Alu subfamilies were examined for sequence diversity and insertion polymorphism in primate genomes. The smaller subfamily (termed HS-2) is comprised of approximately 5 to 25 members, while the larger (termed Sb2) includes approximately 125 to 600 members. Individual members of these Alu subfamilies share distinguishing sets of diagnostic mutations, are well-conserved relative to each other, and have expanded in the human lineage. At least one member from each subfamily is known to be polymorphic in humans. Three newly characterized HS-2 Alu family members as well as three Sb2 Alu repeats are monomorphic (fixed) in humans. The existence of a number of Alu subfamilies that have amplified in parallel within the human genome provides compelling evidence for the simultaneous activity of multiple dispersed Alu source genes.

Basophilic differentiation in acute promyelocytic leukemia.

A rare variant of acute promyelocytic leukemia (APL) is associated with basophilic differentiation. Such a patient presented with basophilia, headaches, and diffuse engorgement of superficial blood vessels, attributable to hyperhistaminemia. Karyotype analysis showed a clonal rearrangement of chromosome 12p13 in addition to the t(15;17). During treatment with all-trans-retinoic acid (TRA), the absolute basophil count rose steadily during the first week, then declined. By one month, the basophilia resolved, an abrupt rise occurred in both the platelet and absolute neutrophil count, and the bone core biopsy showed complete maturation of all cell lines. Abnormalities of chromosome 12p13 in acute myelogenous leukemia have been associated with basophilia. Since every cell in our patient with t(12p13;?) also had the t(15;17), we speculate that the basophilia was due to clonal evolution with acquisition of the t(12p13;?). In two out of five other reported cases, abnormalities of chromosomes known to be associated with basophilia were present in addition to t(15;17). It is possible that the basophilia in this variant is reactive; however, since TRA induces differentiation of leukemic promyelocytes into mature neutrophils, we speculate that the leukemic promyelocytes in our patient differentiated into basophils. Future studies employing either fluorescent in situ hybridization or polymerase chain reaction using a probe to the breakpoint on t(15;17) may establish whether or not the basophils derive from the leukemic clone.

Lineage involvement by BCR/ABL in Ph+ lymphoblastic leukemias: chronic myelogenous leukemia presenting in lymphoid blast vs Ph+ acute lymphoblastic leukemia.

Chronic myelogenous leukemia (CML) can sometimes present in lymphoid blast phase (L-BP), and can be difficult to distinguish from Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). Some have suggested that the determination of cell lineages involved by the Ph chromosome may be used for distinguishing CML presenting in L-BP (presumably multilineage disease) from Ph+ ALL (presumably lymphoid-restricted), although others have suggested the term 'stem cell ALL' for the multilineage process. Because it has been difficult to perform lineage studies of the Ph chromosome, we investigated the use of fluorescence in situ hybridization (FISH) with probes for BCR (on chromosome 22) and ABL (on chromosome 9) to study lineage involvement in Ph+ lymphoblastic malignancies. We analyzed routine blood and marrow specimens from eight patients who presented with Ph+ lymphoblastic leukemia and found that FISH recognized the 9;22 translocation, distinguished between the two common molecular variants, and readily identified multilineage vs lymphoblast-restricted disease. In our series, four patients had multilineage and four had lymphoblast-restricted disease. Multilineage disease was associated with morphologic features of CML at diagnosis and/or reversion to chronic phase CML after treatment leading us to consider it as CML presenting in L-BP. Patients with lymphoid-restricted disease lacked such findings. The survival of three of our four patients with multilineage disease was prolonged, at 25, 28+, and 126+ months, and when data from our entire series are added to those of 18 previously reported cases that were studied for lineage involvement (reviewed in Leukemia 1993; 7: 147), the difference in overall survival between patients with multilineage and lymphoblast-restricted disease is significant (median overall survival of 47 months vs 8 months, respectively; P=0.013, log rank). Our findings illustrate that FISH analysis can be used to recognize lineage involvement in patients presenting with Ph+ lymphoblastic malignancies, and they provide further support to the notion that multilineage and lymphoblast-restricted disease are distinct clinically as well as biologically.

The t(2;5)(p23;q35): a recurring chromosomal abnormality in Ki-1-positive anaplastic large cell lymphoma.

In lymphoid neoplasms, nonrandom cytogenetic abnormalities correlate with clinical, morphologic and immunophenotypic features. A subtype of non-Hodgkin's lymphoma, which expresses the Ki-1 antigen (CD30) and has distinct morphologic and clinical features, has recently been described. We now report the association of a reciprocal translocation involving the short arm of chromosome 2 (band p23) and the long arm of chromosome 5 (band q35), t(2;5)(p23;q35), with Ki-1 positive anaplastic large cell lymphoma. Rearrangement of the genes that are located at the breakpoints on chromosomes 2 and 5 may be a critical step in the pathogenesis of this lymphoma.

Clinical and prognostic significance of chromosomal abnormalities in childhood acute myeloid leukemia de novo.

We report on the chromosomal pattern of 120 patients with childhood AML de novo. One hundred and fifteen patients (96%) had adequate samples for analysis; 98 (85%) of these showed clonal karyotypic abnormalities. They were classified into cytogenetic subgroups which were closely correlated with FAB subtypes: t(8;21) and M2 (n = 9); t(15;17) and M3 (n = 12); inv(16) and M4Eo (n = 9); t(9;11) and M5a (n = 10); t(11q23) other than t(9;11) and M4-M5 (n = 11); and t(1;22) and M7 (n = 4). In patients with -7/del(7q) (n = 6), leukemia was preceded by MDS in half of the cases, although they had diverse FAB subtypes. Thirty-seven patients had miscellaneous abnormalities. Despite a high CR rate, patients with t(8;21) had a very poor survival: only one child was event-free at 3 years from diagnosis. One third of patients with t(15;17) died during induction. Those eight who achieved CR fared well: only two relapsed, and six were event-free survivors. Patients with inv(16) had a high remission rate and a long survival: five children were in CR 20 to 136 months. Both groups with t(9;11) and t(11q23) had a high remission rate: however, outcome was superior for the t(9;11) group when compared to either the t(11q23) group (EFS at 3 years +/- SE, 56 +/- 17% vs. 11 +/- 10%, p = 0.07) or to the remaining patients (p = 0.06). Both -7/del(7q) and t(1;22) groups had low CR rates (50%) and poor survival. Cytogenetic analysis identifies clinically distinct subsets of childhood AML and is useful in tailoring treatment for these patients. Favorable cytogenetic groups (t(15;17), inv(16), and t(9;11)) may do well with current therapy protocols, whereas unfavorable groups (t(11q23), t(8;21), -7/del(7q), and t(1;22)) require more effective therapies.

Genome-wide chromatin remodeling modulates the Alu heat shock response.

During heat shock recovery in Hela cells, the level of Alu RNA transiently increases with kinetics that approximately parallel the transient expression of heat shock protein mRNAs. Coincidentally, there is a transient increase in the accessibility of Alu chromatin to restriction enzyme cleavage suggesting that an opening and re-closing of chromatin regulates the Alu stress response. Similar changes occur in alpha satellite and LINE1 chromatin showing that heat shock induces a genome-wide remodeling of chromatin structure which is independent of transcription. The increased accessibility of restriction sites within these repetitive sequences is inconsistent with a simple lengthening of the nucleosome linker region but instead suggests a scrambling of nucleosome positions. Chromatin structure and its dynamics account for many of the principal features of SINE transcriptional regulation potentially providing a functional rationale for the dispersion and high copy number of SINEs.

Physiological stresses increase mouse short interspersed element (SINE) RNA expression in vivo.

The possible functionality of short interspersed elements (SINEs) is investigated by assaying the effects of physiological stress on their RNA polymerase-III-directed transcriptional expression in vivo. B2 RNA is expressed at moderately high levels in all mouse tissues investigated, namely liver, spleen, kidney and testis. B1 RNA is expressed in testis but is nearly undetectable in the other tissues. Following hyperthermic shock, the amounts of B1 and B2 SINE RNAs transiently increase in all tissues by as much as 40-fold in certain cases. The kinetics of these increases resemble those of heat shock protein mRNAs. An acute dose of ethanol also transiently increases the abundance of B1 and B2 RNA in liver, showing that other physiological stresses increase SINE RNA expression. The constitutive expression of B2 RNA in all tissues and tissue-specific differences in expression of B1 RNA imply that these transcripts serve a normal physiological function(s). Moreover, increased SINE RNA expression is a vital response to stress and by the criterion of their inducibility, mammalian SINEs behave like regulated cell stress genes.

Morphology in Ki-1(CD30)-positive non-Hodgkin's lymphoma is correlated with clinical features and the presence of a unique chromosomal abnormality, t(2;5)(p23;q35).

Ten patients with strongly Ki-1(CD30)-positive non-Hodgkin's lymphoma (NHL) were identified at our institution during the past 5 years. Based on morphology, the lymphomas of five of these patients were classified as anaplastic large-cell lymphoma (ALCL); the lymphomas of four patients lacked the morphologic features of ALCL (non-ALCL); and the lymphoma of one patient was unclassifiable. Significant clinical and cytogenetic differences were observed between patients with ALCL and those with non-ALCL. The patients with ALCL tended to be young at the time of diagnosis. They presented with peripheral lymphadenopathy, and two of the five patients had skin involvement. An identical reciprocal translocation involving chromosomes 2 and 5 [t(2;5)(p23;q35)] was observed in lymph nodes from each of the two ALCL patients whose chromosomes were studied. Four of the five patients with ALCL are alive and in complete remission 10-27 months after receiving systemic chemotherapy. In contrast, the patients with non-ALCL were heterogeneous with respect to clinical findings. All of the non-ALCLs were histologically aggressive; however, their morphology varied. The t(2;5) was absent in the lymphoma specimens from each of three non-ALCL patients studied. Three of the four patients died within 17 months after receiving systemic chemotherapy. Thus, differences in morphology are correlated with differences in the clinical findings, karyotype, and outcome in Ki-1-positive NHL.

Complete lymphoid chimerism and chronic graft-versus-host disease in an infant recipient of a hepatic allograft from an HLA-homozygous parental living donor.

Living-related donor liver transplantation (LDLT) is an accepted approach to pediatric liver transplantation. Parental donation imposes a significant risk of chimerism with graft-versus-host disease (GVHD) because donors homozygous at all HLA loci (1.6% of the population) present no mismatched HLA antigens to be recognized by their offspring's immune system. The case of a 9-month-old who underwent LDLT with her 23-year-old HLA-homozygous mother as a donor demonstrates the consequences of this occurrence. The patient developed GVHD with aplastic anemia; the patient's nucleated peripheral blood elements were shown to be entirely derived from the donor. Later, after some marrow recovery, the patient's circulating lymphocytes had a donor origin, while the marrow-derived neutrophils had a recipient origin. The patient suffers from chronic GVHD and debilitating skin disease several years posttransplant. Our current protocol calls for HLA typing to eliminate parents who are homozygous at all HLA loci as donors of hepatic allografts to their children.

Posttransplant lymphoproliferative disease in pediatric liver transplantation. Interplay between primary Epstein-Barr virus infection and immunosuppression.

The incidence, risk factors, and outcome of posttransplant lymphoproliferative disease (PTLD) were examined for 298 children undergoing liver transplantation. The overall incidence of PTLD was 8.4% (25 of 298). Intensity of immunosuppression was found to be a major risk factor for the development of PTLD. Cyclosporine and tacrolimus when used as primary immunosuppression were associated with the development of PTLD in 4.3% and 6.6% of cases (P=NS). OKT3 and tacrolimus, when used as rescue therapy for steroid-resistant rejection, were associated with a comparable increase in the risk of developing PTLD (10.9% and 11.1%, P=NS). Patients requiring both OKT3 and tacrolimus to treat refractory rejection were at significantly increased risk for PTLD (28.1% vs. 4.3% or 6.6%, P<0.0001). PTLD was more common in patients who received transplants for Langerhans cell histiocytosis relative to other indications for transplantation (66% vs. 8.4%, P=0.0005). The data also support an association between primary Epstein-Barr virus (EBV) infections following transplantation and the development of PTLD. While only three patients were EBV seropositive before transplantation (14%), 19 patients were EBV seropositive at the time of diagnosis of PTLD (90%), confirming a high incidence of primary EBV infections in patients with PTLD (21 patients had both pre- and posttransplant EBV serologies). In this series, PTLD was associated with a mortality rate of 60%, and 12 of the 15 patients who died had persistent tumor at the time of death. Five of the 13 patients rendered disease-free developed ductopenic rejection. Of the four with severe liver dysfunction, two have undergone successful retransplantation and are alive without evidence of PTLD. In conclusion, intense immunosuppression using OKT3 and tacrolimus as rescue agents was associated with a significant increase in the incidence of PTLD. Primary EBV infection after transplantation further accentuated this risk. Independent of these risk factors, patients with Langerhans cell histiocytosis were at significantly increased risk for PTLD. The identification of high-risk patients should allow the development of protocols to screen patients for primary EBV infections and early indications of PTLD, as well as the institution of preemptive antiviral and antitumor therapies.

Bone marrow transplantation for children with acute leukemia and Down syndrome.

Four children with acute leukemia and Down syndrome received high-dose cyclophosphamide therapy and total body irradiation in preparation for bone marrow transplantation. Skin and mucous membrane toxicity was pronounced. Furthermore, three children died during the immediate posttransplantation period of infectious and hemorrhagic pulmonary complications. One patient had hematologic recovery and is surviving disease-free 1 year following transplantation. These preliminary observations are in agreement with previous data suggesting that children with Down syndrome are at higher risk for toxicity, pneumonitis, and, possibly, death following administration of intensive therapy for leukemia in comparison with children without Down syndrome. Improvements in the management of these children in the future will depend upon a better understanding of the biologic and pathophysiologic aspects of Down syndrome and additional clinical experience.

Bone marrow transplantation for the treatment of haematological disorders in Down's syndrome: toxicity and outcome.

We report 18 patients with Down's syndrome who underwent bone marrow transplantation, and review nine previously published patients. The indications for transplant in the combined group of 27 patients were acute lymphoblastic leukaemia in 14 cases (52%), acute myeloid leukaemia in 11 cases (41%) and aplastic anemia in two cases (7%). Transplants were autologous in five cases (19%) and allogeneic in 22 cases (81%); of the 22 allogeneic transplants, 16 donors were HLA-matched siblings. In all patients the conditioning regimen included total body irradiation of 7.5 Gy or more, and/or contained cyclophosphamide of 120 mg/kg or more. Seven patients (26%) had fatal pulmonary disease including pneumonitis and pulmonary hemorrhage. Five patients (19%) had significant airway problems including three with severe mucositis who required intubation for airway protection, one with severe mucositis with partial airway obstruction that required observation in the intensive care unit but did not require intubation, and one with Candida albicans laryngitis with development of a glottic web. Nineteen patients (70%) survived beyond 100 days post-transplant. There was no clear association between 100-day survival and the use of any particular agent or regimen used for conditioning or graft-versus-host disease prophylaxis, and the majority of patients tolerated high-dose cyclophosphamide, high-dose cytosine arabinoside, high-dose busulfan, total body irradiation, cyclosporin A, and methotrexate. There appeared to be more early deaths in patients who received the combination of cyclophosphamide and total body irradiation, compared with those receiving the combination of busulfan and cyclophosphamide or those receiving the combination of cytosine arabinoside and total body irradiation. Also, the use of methotrexate was associated with a greater number of early deaths, compared with cyclosporin A. At 3 years, life table estimates of freedom from relapse, relapse-free survival and survival were 75%, 44% and 48%, respectively. The estimated cumulative risk of death due to a non-leukaemic cause at 3 years was 39%. The data show that Down syndrome patients can tolerate the commonly used transplant conditioning regimens with acceptable toxicity; however, there is a strong suggestion in the data that the rates of life-threatening and fatal toxicity are higher than would be expected to occur in patients without Down's syndrome. Patients with Down's syndrome may have a predisposition to fatal pulmonary complications and reversible airway problems during the immediate post-transplant period.

Comparison of low-osmolar contrast media in paediatric cardiac angiography.

Sixty-two children investigated by cardiac angiography for a wide spectrum of congenital heart disease were randomly assigned to one of two groups, one for iohexol, 350 mg I/ml (Omnipaque, Nycomed UK Ltd) and one for iopamidol, 370 mg I/ml (Niopam, E. Merck Ltd). Changes in systolic pressure, end-diastolic pressure when the injection was made into a ventricle, heart rate and rhythm and plasma osmolality were assessed at 1 min and 3 min after the injection of contrast medium. The angiograms were subjectively assessed for angiographic quality. No significant differences were detected between the two groups.