Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages.

The transcription factor PU.1 is a hematopoietic-specific member of the ets family. Mice carrying a mutation in the PU.1 locus were generated by gene targeting. Homozygous mutant embryos died at a late gestational stage. Mutant embryos produced normal numbers of megakaryocytes and erythroid progenitors, but some showed an impairment of erythroblast maturation. An invariant consequence of the mutation was a multilineage defect in the generation of progenitors for B and T lymphocytes, monocytes, and granulocytes. Thus, the developmental programs of lymphoid and myeloid lineages require a common genetic function likely acting at the level of a multipotential progenitor.

Vitamin E. An inhibitor of the platelet release reaction.

Formation of lipid peroxides rises sharply when platelets undergo the release reaction. In this study the in vitro effect of vitamin E on platelet aggregation was investigated. alpha-Tocopherol, an anitoxidant of known inhibitory action on lipid peroxidation, was added to platelet suspensions in concentrations up to 1.5 mM. A dose-dependent reduction in platelet aggregation was observed, with complete inhibition of the secondary wave of aggregation at greater than or equal to 0.9 mM alpha-tocopherol. The inhibitory effect of alpha-tocopherol on the platelet release reaction was further documented by the decrease in aggregation-induced release of [14C]5-hydroxytryptamine from prelabeled platelets and by the reduction of N-acetylglucosaminidase activity released into the medium. The sharp rise in lipid peroxides normally associated with platelet aggregation was markedly reduced by alpha-tocopherol and also by acetylsalicylic acid, a known inhibitor of the platelet release reaction. In vivo studies examined the effect of oral vitamin E administration (1,200-2,400 IU daily) on plasma and platelet levels of alpha-tocopherol. Up to 1,800 IU daily, increasing dosages of vitamin E resulted in increasing concentrations of alpha-tocopherol in plasma and platelets, but intake of vitamin E in excess of this dosage failed to show any further increase in plasma or platelet levels.

Establishment of a leukemia cell line with i(12p) from a patient with a mediastinal germ cell tumor and acute lymphoblastic leukemia.

We report the establishment of a leukemia cell line (UoC-B10) from a patient who developed leukemia several months after the diagnosis of a mediastinal yolk sac tumor. The patient's yolk sac tumor responded to combination chemotherapy, and a mature teratoma with focal areas of hematopoiesis was subsequently resected. However, 5 months after the initial diagnosis, the patient developed an acute lymphoblastic leukemia with a precursor B-cell phenotype. Cytogenetic analysis showed an i(12p) abnormality in the patient's leukemia cells and in the UoC-B10 cell line. The i(12p) was also identified retrospectively in the mediastinal tumor cells by fluorescent in situ hybridization analysis. The UoC-B10 cell line, which has been growing continuously for > 24 months in culture, was Epstein-Barr virus negative and was generally concordant with the patient's leukemia cells by analysis of immunophenotype, karyotype, and genotype. The UoC-B10 cell line possesses receptors for granulocyte-colony-stimulating factor, a cytokine which the patient received as part of his treatment protocol. This cell line may be useful in studying the relationship between i(12p) and hematological differentiation of human mediastinal germ cell tumors.

Cytogenetic characterization of B-cell lymphomas from severe combined immunodeficiency disease mice given injections of lymphocytes from Epstein-Barr virus-positive donors.

We analyzed the karyotype of 27 B-cell lymphomas of human origin that developed in mice with severe combined immunodeficiency disease following the injection of peripheral blood leukocytes from Epstein-Barr virus-seropositive donors. Three tumors had clonal abnormalities detected with conventional techniques, 2 had trisomy 11, and 1 had a del(6)(q21q25). One other tumor had trisomy 11 detected with fluorescence in situ hybridization. Twelve tumors had a normal karyotype, 11 tumors had nonclonal abnormalities (which included trisomy 9 or 12 in 3 or 2 tumors, respectively), and one tumor had a karyotype of 92,XXXX(75%)/46,XX(25%) by conventional cytogenetic analysis. Trisomy for chromosomes, 9, 11, and 12 are recurring abnormalities that have been observed in lymphomas associated with an immunocompromised state. Clonal or nonclonal abnormalities were observed in 8 of 11 tumors derived from 3 donors whose peripheral lymphocytes induced a high incidence of tumors in mice with severe combined immunodeficiency disease compared with a clonal abnormality and 2 nonclonal abnormal cells in 2 of 5 tumors derived from 3 donors whose lymphocytes induced an intermediate to low incidence. These observations suggest an association between a higher incidence of karyotypically abnormal cells in lymphomas and the increased tumorigenic potential of the lymphocytes that induced these tumors.

Lymphadenopathy, splenomegaly, and altered immunoglobulin production in BCL3 transgenic mice.

The candidate proto-oncogene BCL3 was isolated through its involvement in the t(14;19) found in chronic lymphocytic leukemia and other B-cell neoplasms. As a member of the I kappaB family, BCL3 plays a role in the immune response by interactions with the NF-kappaB family of transcription factors. In order to study the role of BCL3 overexpression in lymphoid malignancies, we generated five lines of E mu-BCL3 transgenic mice. Transgenic animals develop normally but show splenomegaly and an accumulation of mature B cells in lymph nodes, bone marrow and peritoneal cavity. A hyperresponsive immune system is suggested by the follicular hyperplasia and plasmacytosis in lymph nodes of unimmunized animals, increased incidence of antibodies to self-antigens, and a heightened response to cross-linking of surface IgM. Statistically significant decreases in serum IgM and IgG3, but an increase in IgG1 and IgA were also observed. No lymphoid neoplasms have been identified in transgenic animals. The expansion of B cells in vivo is consistent with the overexpression of BCL3 as being one step in the multi-step process of leukemogenesis. The phenotype also suggests that BCL3 plays a part in B cell proliferation and isotype switching.

High-dose cladribine therapy for chronic myelogenous leukemia in the accelerated or blast phase.

PURPOSE: A phase II clinical trial was performed to evaluate the effectiveness of high-dose cladribine (2CDA) for treatment of chronic myelogenous leukemia (CML) in the accelerated or blast phase. PATIENTS AND METHODS: Nineteen patients were treated. The median age was 55 years (range, 30 to 73). Six were older than 60 years. Eight had progressed after intensive combination chemotherapy and three after allogeneic or autologous transplantation. For the first course, 16 patients received 2CDA at 15 mg/m2/d intravenously (i.v.) over 1 hour for 5 days. Two received 18 mg/m2 and one received 21.5 mg/m2 daily. The second course was escalated to 20 mg/m2/d in five patients. RESULTS: Rapid cytoreduction of leukemia occurred in the blood, with the nadir at 10 to 12 days. The median WBC count decreased from 36,900/microL before treatment to 500/microL at the nadir and recovered to 5,200/microL at day 30. The median platelet count changed from 113,000/microL to 24,000/microL at the nadir and 71,000/microL at day 30. The complete remission (CR) plus partial remission (PR) rate was 47% (95% confidence interval [CI], 23% to 72%). One 64-year-old man with lymphoid blast phase of CML had a morphologic and cytogenetic CR that lasted 9 months. The median survival for all patients was 34 weeks, and the median survival for the eight responders was 56 weeks (range, 11 to 167). The median number of days spent in hospital over the entire treatment period was 19 (range, 4 to 60). CONCLUSION: High-dose 2CDA therapy provides effective palliation for CML in accelerated or blast phases, even for heavily pretreated patients.

Pseudo-Gaucher histiocytes identified up to 1 year after transplantation for CML are BCR/ABL-positive.

The results of polymerase chain reaction (PCR) analysis after transplantation for chronic myelogenous leukemia (CML) are difficult to interpret clinically. Positive findings for BCR/ABL can be seen not only in patients who go on to relapse but also in patients who, after years of follow-up, remain in complete remission. The cause for the lack of concordance between PCR findings and relapse is not clear. We identified two patients with CML who had rare pseudo-Gaucher cells in their bone marrow aspirate specimens prior to, and at 1, and 6 or 12 months following syngeneic or allogeneic hematopoietic transplantation. After the transplant, the patients obtained clinical remission and were shown to be cytogenetically normal and to have germline MBCR in blood or bone marrow by Southern analysis. One patient was PCR-positive for BCR/ABL in the marrow at 12 months. In order to determine whether the pseudo-Gaucher histiocytes were BCR/ABL-positive, we used fluorescence in situ hybridization and probes for MBCR and ABL and analyzed Wright-stained smears to correlate molecular cytogenetic findings with cell type. On three aspirate smears from each patient (at 6 or 12 months post-transplant), all of the pseudo-Gaucher cells studied (10/10 in one patient and 12/12 in the other) showed the fusion for BCR/ABL. Other cells analyzed randomly (erythroid precursors, granulocytes and rare monocytes, lymphocytes and plasma cells) did not. Our cases provide the first proof that pseudo-Gaucher cells carry the BCR/ABL fusion. Furthermore, they illustrate that these cells can be found in the marrow for up to 12 months following transplantation. Our results permit speculation that pseudo-Gaucher cells or other long-lived histocytes may be one cause of persistent PCR positivity after transplantation that is not predictive of disease relapse.

Cytogenetic abnormalities in a secondary lymphoma complicating cardiac transplantation.

We report a case of secondary lymphoma in the brain in a cardiac transplant recipient. Cytogenetic analysis revealed the presence of clonal cytogenetic abnormalities. Two clones with unrelated chromosomal abnormalities were noted; in both abnormal clones, trisomy for the long arm of chromosome 11 was observed. Our observations, combined with single cases previously reported, suggest that a gain of the long arm of chromosome 11 may represent a characteristic cytogenetic abnormality that is associated with secondary lymphoma.

The relationship between secondary chromosomal abnormalities and blast transformation in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a stem cell disorder which progresses from a chronic phase (CP) to an accelerated phase (AP), and/or a blast phase (BP) of myeloid (M) or lymphoid (L) phenotype. This progression is frequently preceded or accompanied by recurring secondary chromosomal abnormalities which are believed to play a role in the transformation. In order to investigate the relationship between the secondary change and the development of BP, we undertook a study using fluorescence in situ hybridization to determine in which cells the secondary abnormalities were present. We observed that in one case of L-BP, the secondary change (trisomy 8) appeared to be in a subclone that was different from the blast cells, as it was absent from the lymphoblasts but present in differentiating erythroid, monocytic and granulocytic cells. In two cases, the secondary change (trisomy 8, extra Ph) probably occurred prior to an acute transforming event as it was present in CP or AP predominantly in differentiated granulocytic or monocytic cells. In one case of M-BP, the secondary change (trisomy 8) probably occurred after the acute transformation, as it appeared in only a subset of the blasts. Lastly, in four cases of L-BP, the secondary change (monosomy 7, extra Ph or hyperdiploidy) was closely associated with the BP as it was present in all of the blasts. The findings indicate that some secondary abnormalities may be directly related to the development of BP and may provide clues to the identity of genes responsible for the acute phase transition. Other abnormalities occurring before, or after the acute transformation or in a different subclone from the acute phase blasts, may be more important for denoting genomic instability than for helping to understand the mechanism of blast transformation.

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.

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.

Myelodysplasia and acute leukemia following high-dose chemotherapy and autologous bone marrow or peripheral blood stem cell transplantation.

Therapy-related myelodysplastic syndrome (t-MDS)/acute myeloid leukemia (t-AML) has been reported after autologous bone marrow or peripheral blood stem cell transplantation (ABMT/PBSCT) for various malignancies. We retrospectively reviewed all adult ABMT/PBSCT cases performed at the University of Chicago Medical Center from 1985 to 1997 in order to determine the incidence of therapy-related leukemia. Among 649 patients, seven (1.1%) developed therapy-related acute lymphoblastic leukemia (one patient) or t-MDS/t-AML (six patients). Of these seven, primary malignancies included one case of breast carcinoma, five cases of Hodgkin's disease (HD) and one case of non-Hodgkin's lymphoma (NHL). Disease-specific incidences for therapy-related leukemia occurring after ABMT/PBSCT were one in 354 (0.3%) for breast carcinoma, five in 79 (6.3%) for HD and one in 103 (1%) for NHL. The median latency periods for the development of therapy-related leukemia from the time of initial diagnosis and of ABMT/PBSCT were 5.5 and 1.5 years, respectively, for the combined HD and NHL group of patients and 4.4 and 2.8 years, respectively, for the one breast carcinoma patient. All seven patients had clonal cytogenetic abnormalities, and five had recurring abnormalities typical of myeloid disorders. Given the similar latency period observed in patients treated with conventional chemotherapy alone, our findings support the hypothesis that therapy-related leukemia after ABMT/PBSCT likely results from pre-transplant therapy. Early detection of therapy-related leukemia is therefore critical to exclude these patients from undergoing ABMT/PBSCT.

Interphase cytogenetic analysis in the diagnosis and study of neoplastic disorders.

Cytogenetic information usually is obtained through the direct analysis of chromosomes from cells arrested in metaphase. Recently, advances in molecular genetics have made it possible to acquire cytogenetic information through the study of interphase and terminally differentiated cells. By using chromosome-specific DNA probes or probes that are specific for certain chromosomal regions, and by employing techniques of in situ hybridization along with nonradioactive detection methods, it is now possible to detect numerical and structural chromosomal abnormalities from nonmetaphase cells. When used as an adjunct to conventional cytogenetic analysis or when used together with knowledge of established cytogenetic findings for a particular malignancy, this new technology can serve to broaden the scope and utility of cytogenetic analysis beyond the limits of the present metaphase-based technology. Interphase cytogenetic analysis has application to the diagnosis and study of neoplastic disorders and, thus, has particular importance in pathology.

Hodgkin's disease associated with chronic lymphocytic leukemia. Eight additional cases, including two of the nodular lymphocyte predominant type.

Several reports of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and of coexisting or subsequent Hodgkin's disease (HD) have raised the question how these two disorders are related. The authors have identified eight new cases of B-cell low-grade lymphoproliferative disorders (LGLPD) and HD. Six of these cases were similar to those previously reported on by others in that the HD were mixed cellularity, nodular sclerosing, and lymphocyte depleted subtypes. The morphology in these cases was typical of HD, as was the immunohistochemical profile. However, the two remaining cases were notable in that the HD was of the nodular lymphocyte predominant type (NLPHD). To our knowledge, this association has not been well documented previously. In the two cases in this study, CLL and NLPHD were found simultaneously when each patient presented with lymphadenopathy and a lymphocytosis that was comprised of small monoclonal B lymphocytes coexpressing CD5. Lymph node biopsies in each case revealed typical NLPHD, with large, indistinct nodules containing scattered lymphocytic-histiocytic (L&H) cells. Focal, but distinct areas of CLL/SLL were also present. Immunostaining of the lymph node biopsy specimens showed the L&H cells to be CD20- and CD45 positive, and to lack CD15 or evidence of light chain restriction. In one of these patients, a NLPHD-associated large cell lymphoma developed 8 months later. The large cells were CD20- and CD45 positive, with lambda light chain restriction. In contrast, the original CLL cells in this patient expressed kappa light chains. This report indicates that LGLPD can be associated with all subtypes of HD, including the NLP type. The discordant light chain restriction between the CLL and the NLPHD-associated large cell lymphoma in one of these cases indicates that the CLL and HD were probably not derived from the same clone.

HIV and diarrhea in the era of HAART: 1998 New York State hospitalizations.

BACKGROUND: This study reflects an attempt to identify the causes of diarrheal illness in hospitalized HIV patients in light of therapeutic advancements in HIV management. METHODS: The study identifies the various etiologies associated with diarrhea among HIV patients hospitalized in New York State in 1998. Data for this study were extracted from the New York State Department of Health Statewide Planning and Research Cooperative System. Pathogens recognized to cause diarrhea in persons with HIV and general codes identifying diarrhea were examined by using the principal and all secondary diagnoses based on the International Classification of Diseases 9th Revision Clinical Modification codes. RESULTS: Based on the Statewide Planning and Research Cooperative System data set, more than 15,000 patients with HIV were hospitalized in 1998. Among the HIV patients hospitalized, 2.8% were admitted with a diarrheal diagnosis. The following diagnoses occurred the most frequently among HIV patients hospitalized with a diarrheal illness: Clostridium difficile (51.3%), other protozoal diseases (18.1%), and other organisms, not elsewhere specified (11.7%). CONCLUSIONS: In the era of highly active antiretroviral therapy, diarrhea is still an occurring symptom in HIV patients. Despite the relatively small percentage of hospitalizations attributed to diarrhea, clinicians must remember that even "mild" to "moderate" diarrhea can have a debilitating impact among persons with the symptom.

A foregut carcinoid tumor causing Zollinger-Ellison syndrome.

A patient had severe peptic ulcer disease complicated by gastric outlet obstruction and choledochoduodenal fistula. Serum gastrin levels were elevated preoperatively to 340 ng/L. A 1.5-cm histologically benign carcinoid tumor of the antrum of the stomach was found at surgery, and surgical resection of the tumor resulted in normalization of serum gastrin levels and amelioration of the peptic acid diathesis. The patient remains asymptomatic at one year. Immunohistochemical staining demonstrated that the carcinoid indeed contained gastrin along with chromogranin, cholecystokinin, and neuron-specific enolase. This is a case of Zollinger-Ellison syndrome caused by a benign foregut carcinoid (gastric carcinoid-gastrinoma).

Improved oxygen delivery to the myocardium during hypothermia by perfusion with 2,3 DPG-enriched red blood cells.

The oxygen affinity of red cells increases stepwise with temperature reductions below 37 degrees C. In vitro studies demonstrated that biochemically modified red cells with increased 2,3 diphosphoglycerate (2,3 DPG) (150% and 250% of normal) exhibited significantly less oxygen affinity at 24 degrees C than did unmodified cells. At 15 degrees C, significant attenuation of affinity was observed with 250%, but not 150%, of normal 2,3 DPG cells. Measurements made of isolated fibrillating dog hearts during perfusion at 24 degrees C alternately with unmodified (80% of normal 2,3 DPG) and modified (300% of normal 2,3 DPG) red cells demonstrated significantly greater oxygen consumption, higher coronary sinus partial pressures of oxygen and carbon dioxide, higher in vitro P50 values, and lower arterial and coronary sinus lactate levels during perfusion with modified as compared with unmodified cells. This evidence, indicating improved oxygen delivery to hypothermic dog hearts by red cells with 300% of normal 2,3 DPG activity, suggests that high 2,3 DPG cells might protect myocardial tissue in patients undergoing hypothermic cardiac operation.

A recurring chromosome rearrangement, dic(16;22), in acute nonlymphocytic leukemia.

A dicentric chromosome, dic(16;22), resulting in the loss of 16q and 22p was seen in bone marrow cells from two patients with acute myelomonocytic leukemia and one patient with a therapy-related myelodysplastic syndrome that evolved to leukemia. Review of the clinical findings and of the bone marrow morphology failed to reveal any distinctive features in common among these patients. The dic(16;22) may be a new, rare, recurring abnormality associated with malignant myeloid disorders.

Issues in the pathology of the myelodysplastic syndromes.

The diagnosis and subclassification of myelodysplastic syndromes (MDS) are usually straightforward if the criteria established by the French-American-British Cooperative Group are carefully followed. Some cases can be diagnostically challenging, however. This article discusses some of the special diagnostic problems such as hypocellular MDS, MDS with fibrosis, and cases that show morphologic overlap with myeloproliferative disorders and acute leukemia. Additional problems sometimes encountered in the diagnosis of MDS such as extramedullary disease, associated lymphoproliferative disease, and the 5q-syndrome are also reviewed.

The histopathologic diagnosis and subclassification of Hodgkin's disease.

Although Hodgkin's disease is considered a distinct clinical entity, it exhibits a wide range of histologic and cytologic features. It is important to recognize histologic subtypes and their variants for diagnostic reasons, as well as for their clinical significance. An appreciation of the histologic diversity of Hodgkin's disease may also be critical for the interpretation of data regarding the origin of the RS cell.