Co-existence of clonal expanded autologous and transplacental-acquired maternal T cells in recombination activating gene-deficient severe combined immunodeficiency.

It is commonly accepted that the presence of high amounts of maternal T cells excludes Omenn syndrome (OS) in severe combined immunodeficiency (SCID). We report a SCID patient with a novel mutation in the recombination activating gene (RAG)1 gene (4-BP DEL.1406 TTGC) who presented with immunodeficiency and OS. Several assays, including representatives of specific T cell receptors (TCR), Vß families and TCR-γ rearrangements, were performed in order to understand more clearly the nature and origin of the patient's T cells. The patient had oligoclonal T cells which, based on the patient-mother human leucocyte antigen (HLA)-B50 mismatch, were either autologous or of maternal origin. These cell populations were different in their numbers of regulatory T cells (T(reg)) and the diversity of TCR repertoires. This is the first description of the co-existence of large amounts of clonal expanded autologous and transplacental-acquired maternal T cells in RAG1-deficient SCID.

P53 in blind subterranean mole rats--loss-of-function versus gain-of-function activities on newly cloned Spalax target genes.

A tumor suppressor gene, p53, controls cellular responses to a variety of stress conditions, including DNA damage and hypoxia, leading to growth arrest and/or apoptosis. Recently, we demonstrated that in blind subterranean mole rats, Spalax, a model organism for hypoxia tolerance, the p53 DNA-binding domain contains a specific Arg174Lys amino acid substitution. This substitution reduces the p53 effect on the transcription of apoptosis genes (apaf1, puma, pten and noxa) and enhances it on human cell cycle arrest and p53 stabilization/homeostasis genes (mdm2, pten, p21 and cycG). In the current study, we cloned Spalax apaf1 promoter and mdm2 intronic regions containing consensus p53-responsive elements. We compared the Spalax-responsive elements to those of human, mouse and rat and investigated the transcriptional activity of Spalax and human Arg174Lys-mutated p53 on target genes of both species. Spalax and human-mutated p53 lost induction of apaf1 transcription, and increased induction of mdm2 transcription. We conclude that Spalax evolved hypoxia-adaptive mechanisms, analogous to the alterations acquired by cancer cells during tumor development, with a bias against apoptosis while favoring cell arrest and DNA repair.

Gene expression profiles of AML derived stem cells; similarity to hematopoietic stem cells.

Tumors contain a fraction of cancer stem cells that maintain the propagation of the disease. The CD34(+)CD38(-) cells, isolated from acute myeloid leukemia (AML), were shown to be enriched leukemic stem cells (LSC). We isolated the CD34(+)CD38(-) cell fraction from AML and compared their gene expression profiles to the CD34(+)CD38(+) cell fraction, using microarrays. We found 409 genes that were at least twofold over- or underexpressed between the two cell populations. These include underexpression of DNA repair, signal transduction and cell cycle genes, consistent with the relative quiescence of stem cells, and chromosomal aberrations and mutations of leukemic cells. Comparison of the LSC expression data to that of normal hematopoietic stem cells (HSC) revealed that 34% of the modulated genes are shared by both LSC and HSC, supporting the suggestion that the LSC originated within the HSC progenitors. We focused on the Notch pathway since Jagged-2, a Notch ligand was found to be overexpressed in the LSC samples. We show that DAPT, an inhibitor of gamma-secretase, a protease that is involved in Jagged and Notch signaling, inhibits LSC growth in colony formation assays. Identification of additional genes that regulate LSC self-renewal may provide new targets for therapy.

Cellular and molecular characteristics of an immortalized ataxia-telangiectasia (group AB) cell line.

Ataxia-telangiectasia (A-T) is a multisystem hereditary disease featuring neurodegeneration, immunodeficiency, extreme cancer proneness, chromosomal instability, and radiosensitivity. A-T is found in many ethnic groups, and is genetically heterogeneous: four complementation groups have been identified in A-T so far. Attempts to isolate the A-T gene are based in part on gene transfer experiments, using permanent A-T fibroblast lines, obtained by transformation with SV40. "Immortalization" of A-T primary diploid fibroblasts using SV40 is difficult, possibly because of the chromosomal instability of these cells. The number of currently available permanent A-T fibroblast lines is small, and not all of them have been assigned to specific complementation groups. Using the assay of X-ray induced inhibition of DNA synthesis, we have assigned the A-T strain AT22IJE to complementation group AB. Origin-defective SV40 was used to transfect these cells, and one transformant (AT22IJE-T), which survived crisis, was found to have the typical characteristics of permanent cell lines obtained in this way. "In-gel renaturation" analysis did not show any DNA amplification of high degree in AT22IJE-T. Cytogenetic analysis showed considerable chromosomal instability in the new cell line, and medium conditioned by these cells contained the clastogenic activity which is characteristic of the parental strain as well. Other parameters of the "cellular A-T phenotype" have also been retained in the immortalized cells: hypersensitivity to the lethal effects of X-rays and neocarzinostatin, as well as "radioresistant" DNA synthesis. However, the sensitivity of AT22IJE-T to both DNA-damaging agents is less pronounced than that of the parental cells. The capacity of the cells for uptake of foreign DNA was tested by introducing into them the plasmid pRSVneo, using three different transfection methods. Satisfactory frequency of G418-resistant transfectants (0.66%) was achieved using a protocol recently published by Chen and Okayama (Mol. Cell Biol., 7: 2745-2752, 1987), which was found to be superior to the traditional calcium phosphate transfection method and to the polybrene-based method.

Deletion of chromosome 2 is an early event in the development of radiation-induced myeloid leukemia in SJL/J mice.

In this study we have analyzed the chromosomal changes in the preleukemic phase in SJL/J mice treated with radiation and acute myeloid leukemias (AMLs) induced by radiation alone or with additional corticosteroid treatment. SJL/J mice exposed to 300 rad whole body irradiation developed a low incidence of AML (20-25%) that could be markedly increased (to 50-70%) by additional coleukemogenic treatment with corticosteroids. Partial deletion in one chromosome 2 was found in 100% of bone marrow and spleen cells of leukemic animals in both treatment modalities, whereas the age-matched controls exhibited a normal karyotype. Five types of deletion were observed according to site and size, but region D through G was the common missing part in all five types of chromosome 2 deletion. The occurrence of chromosome 2 deletion was also tested among bone marrow cells removed from 17 mice, 4 months after exposure to 300 rad whole body irradiation, long before the time when AML development is expected. About 80% of the mice tested had different levels of deleted chromosome 2 among their bone marrow population. Cytological and histological examination of bone marrow and spleen of most tested animals showed a normal hematologic picture. These results suggest that the marker chromosome is related to the process of radiation-induced initiation of AML in SJL/J mice.

Chromosomal mapping of the murine c-abl proto-oncogene by in situ hybridization.

Deletion and rearrangement of chromosome 2 were shown to be major cytogenetic characteristics of radiation-induced murine myeloid leukemias. Analysis of the localization of the murine protooncogene c-abl, previously assigned by Goff et al. to chromosome 2, was done using the in situ hybridization method. The c-abl was located close to the centromere, within bands 2A-2B. This site does not correspond to the common characteristic deleted segments (2C-2D) predominantly observed in radiation induced murine myeloid leukemias.

Absence of negative growth regulation in three new murine radiation-induced myeloid leukemia cell lines with deletion of chromosome 2.

Murine radiation-induced acute myeloid leukemia (RI-AML) may be considered as the experimental counterpart of human secondary leukemia. Three new myelomonocytic cell lines derived from RI-AML and carrying a partially deleted chromosome 2 are described. The RI-AML cells responded with increased proliferation after being incubated with the hemopoietic growth factors rG-CSF, rGM-CSF and IL-3. Increased proliferation of the same extent without any effect in differentiation, was also demonstrated in the RI-AML cells after incubation with IL-6 and with mouse lung conditioned medium (CM) and Krebs ascites tumor cells CM which induce differentiation in normal and most leukemic myeloid cells. Down-regulation of the c-myc gene and induction of (2'-5') oligo-adenylate synthetase (reflecting autocrine interferon secretion), two essential mechanisms operating during arrest of growth and concomitant differentiation, were demonstrated to be absent in RI-AML cells. In contrast, the M1 cells responded to the above differentiating factors with growth arrest and differentiation and with appropriate c-myc down-regulation and synthetase induction. The genetic basis for the distinct RI-AML cells' behavior may be connected with the loss or structural and/or functional abnormalities of DNA sequences located in the deleted part of chromosome 2 or in the respective allele. The presently described new RI-AML cell lines may be used for studies concerning myeloid leukemogenesis in general and secondary leukemia in particular.

Chimerism testing and detection of minimal residual disease after allogeneic hematopoietic transplantation using the bioView (Duet) combined morphological and cytogenetical analysis.

Recurrent disease remains a major obstacle to cure after allogeneic transplantation. Various methods have been developed to detect minimal residual disease (MRD) after transplantation to identify patients at risk for relapse. Chimerism tests differentiate recipient and donor cells and are used to identify MRD when there are no other disease-specific markers. The detection of MRD does not always correlate with relapse risk. Chimerism testing may also identify normal hematopoietic cells or other cells not contributing to relapse. In this study we report our initial experience with a novel system that provides combined morphological and cytogenetical analysis on the same cells. This system allows rapid automatic scanning of a large number of cells, thus increasing the sensitivity of detection of small recipient population. The clinical significance of MRD detection is improved by identifying the morphology of recipient cells. Identification of recipient characteristics within blasts predicts overt relapse in leukemia patients and precedes it by a few weeks to months. Identification within mature hematopoietic cells may not be closely associated with relapse. The system also allows chimerism testing after sex-mismatched transplants, within cellular subsets, with no need for sorting of cells. The system merits further study in larger scale trials.

Tetraploid myeloid cells in donors of peripheral blood stem cells treated with rhG-CSF.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is frequently used to mobilize CD34+ cells in healthy donors and patient with malignant diseases prior to peripheral blood stem cell (PBSC) harvest. To analyze the effects of rhG-CSF on morphology and genotype of white blood cells, a novel multiparametric cell scanning system that combines morphologic, immune and genotypic analyses of the same cells was used. We report here that tetraploid myeloid cells are present in the peripheral blood of donors treated with rhG-CSF. The tetraploidy was detected in up to 0.6% of differentiated myeloid cells and all observed CD34+ cells were diploid. Thus, short treatment with rhG-CSF of PBSC donors induces numerfical chromosomal alterations in a small subset of mature myeloid cells.

Multilineage hematopoietic engraftment after allogeneic peripheral blood stem cell transplantation without conditioning in SCID patients.

Successful stem cell transplantation for patients with severe combined immunodeficiency (SCID) from matched family donors without conditioning results in engraftment of T lymphocytes. B lymphocytes engraft in only 50% of the cases, while myelopoiesis and erythropoiesis remain of host origin. Full hematopoietic engraftment was reported in one case after bone marrow transplantation without conditioning for a SCID patient. We studied three SCID patients who were transplanted with unmodified mobilized peripheral blood from HLA-identical family sex-mismatched members. They received megadoses of stem cells (18-23 x 10(6)CD34/kg). In contrast to the expected mixed chimerism that usually occurs in the absence of conditioning, we found in our patients 100% donor cell engraftment based on fluorescence in situ hybridization (FISH) and microsatellite techniques. Subset analysis of the engrafted cells using a multiparametric system enabling a combined analysis of morphology, immunophenotyping and FISH showed that both T and B lymphocytes and myeloid cells were of donor origin in two patients, while T lymphocytes and myeloid cells were of donor origin in the third. In the two cases with ABO incompatibility, erythroid engraftment was evidenced by blood group conversion from recipient to donor type. Multilineage donor engraftment is possible in SCID patients even without conditioning.

Subgroup of patients with Philadelphia-positive chronic myelogenous leukemia characterized by a deletion of 9q proximal to ABL gene: expression profiling, resistance to interferon therapy, and poor prognosis.

A major deletion of the region proximal to the rearranged ABL gene on 9q was found in 14/94 (15%) of chronic myelogenous leukemia Philadelphia-positive patients by interphase fluorescent in situ hybridization with the BCR/ABL extra signal dual-color probe. Preliminary results indicated that the prognosis of the deletion 9q patients is probably worse than that of the non-deletion 9q patients. Twelve of the 14 deletion 9q patients were treated with alpha-interferon and none had a major cytogenetic response. The median duration of the chronic phase in patients not undergoing BMT was significantly shorter for the deletion 9q patients as compared to the non-deletion 9q patients (p =.0144). DNA microarray technology was performed in order to compare the gene expression patterns between the two groups of patients. A number of genes exhibiting differential expression, especially involving cell adhesion and migration, were identified. This finding may identify a sub-group of CML patients with different cell properties and a relatively poor prognosis.

Standardization criteria for the detection of BCR/ABL fusion in interphase nuclei of chronic myelogenous leukemia patients by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH), as a new clinical test, is not presently standardized. For practical reasons, each laboratory must build its own criteria. In this work, we present our standardization criteria for clinical practice, which include not only the methods for cell fixation, specimen preparation, and hybridization conditions, but mainly the definition of false-positive range and the scoring criteria of microscopic analysis. These include signal assessment, difference between individual microscopists, evaluation of specimen homogeneity, and the minimum number of scored nuclei required for a clinically reliable result. For this purpose, we analyzed by FISH 24 healthy volunteer donors, 31 patients affected by non-chronic myelogenous leukemia (CML) hematological malignancies, 47 CML patients at diagnosis, and 82 CML patients during treatment for the BCR/ABL fusion. In this article, we present several quality control and assurance methods that can be useful in providing standardization of the FISH technique.

Hexasomy of chromosome 8 and trisomy of chromosome 11 characterize two karyotypically independent clones in a case of acute non-lymphocytic leukemia. Conventional cytogenetic and FISH investigation.

A case of ANLL following a myelodysplastic syndrome, probably resulting from occupational exposure to ionizing irradiation, with two cytogenetically unrelated clones, hexasomy 8 and trisomy 11, was investigated by conventional cytogenetics and FISH. Significant quantitative differences between data obtained by metaphase and interphase analysis of the hexasomy 8 clone were observed. A difference in the sensitivity to chemotherapy of the two clones was found: while the hexasomy 8 clone markedly decreased in response to treatment, the trisomy 11 clone remained unchanged.

In vitro proliferative advantage of bone marrow cells with tetrasomy 8 in Ewing sarcoma.

We describe a case of a 14.5-year-old boy with a clinically aggressive pelvic Ewing sarcoma. The tumor cells showed the presence of a typical t(11;22)(q24;q12) aberration and gains of chromosomes 8, 10, 14, and 21. To determine the size of the trisomy and tetrasomy 8 clones an interphase analysis by fluorescence in situ hybridization with a centromere-specific chromosome 8 probe was performed. Significant quantitative differences between metaphase and interphase data were obtained. It was shown that culturing of bone marrow cells leads to enrichment of tetrasomy 8 population that may be explained by the proliferative advantage of the tetrasomy 8 cells.

Coexistence of several unbalanced translocations in a case of neuroblastoma: the contribution of multicolor spectral karyotyping.

Spectral karyotyping (SKY) is based on the simultaneous hybridization of a set of 24 chromosome-specific DNA painting probes, each labeled with a different fluor combination. Automatic classification, based on the measurement of the spectrum for each chromosome, was applied to metaphases obtained from the affected bone marrow of a neuroblastoma case. Spectral karyotyping allowed the identification of chromosomal aberrations that could not be identified by the use of the G-banding technique, and revealed a number of gains and unbalanced translocations.

Chromosomal numerical aberrations in oral lichen planus.

The malignant potential of oral lichen planus (OLP) has been a matter of serious controversy. We aimed to detect chromosomal numerical aberrations in cells of brush samples collected from affected mucosa. The samples were simultaneously analyzed for morphology and fluorescent in situ hybridization (FISH) with chromosomes 2 and 8 centromeric probes. We analyzed 57 persons with OLP and 33 control individuals. A cut-off value of aneuploid cells was determined as 1.1%. Aneuploid cells were found in 16 persons with OLP (28.1%); in 10 individuals (17.5%), over 5% of the cells were aneuploid. Aneuploid cells were also detected in normal-looking mucosa of seven persons with OLP. One person with OLP developed squamous cell carcinoma; 10% of the cells examined were aneuploid. OLP carries an increased risk for chromosomal instability. Identifying aneuploid cells in a brush sample and the combined morphological and FISH analysis can increase the specificity in predicting the malignant potential of OLP.

New insights to the MLL recombinome of acute leukemias.

Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements.

Evolutionary regulation of the blind subterranean mole rat, Spalax, revealed by genome-wide gene expression.

We applied genome-wide gene expression analysis to the evolutionary processes of adaptive speciation of the Israeli blind subterranean mole rats of the Spalax ehrenbergi superspecies. The four Israeli allospecies climatically and adaptively radiated into the cooler, mesic northern domain (N) and warmer, xeric southern domain (S). The kidney and brain mRNAs of two N and two S animals were examined through cross-species hybridizations with two types of Affymetrix arrays (mouse and rat) and muscle mRNA of six N and six S animals with spotted cDNA mouse arrays. The initial microarray analysis was hypothesis-free, i.e., conducted without reference to the origin of animals. Principal component analysis revealed that 20-30% of the expression signal variability could be explained by the differentiation of N-S species. Similar N-S effects were obtained for all tissues and types of arrays: two Affymetrix microarrays using probe oligomer signals and the spotted array. Likewise, ANOVA and t test statistics demonstrated significant N-S ecogeographic divergence and region-tissue specificity in gene expression. Analysis of differential gene expression between species corroborates previous results deduced by allozymes and DNA molecular polymorphisms. Functional categories show significant N-S ecologic putative adaptive divergent up-regulation of genes highlighting a higher metabolism in N, and potential adaptive brain activity and kidney urine cycle pathways in S. The present results confirm ecologic-genomic separation of blind mole rats into N and S. Gene expression regulation appears to be central to the evolution of blind mole rats.