ALBERT

Description: Varicella-zoster virus (VZV) infection remains a common complication after hematopoietic stem cell transplantation (HSCT). The introduction of long-term prophylaxis with low-dose acyclovir against VZV reactivation has been investigated, because VZV-related complications including post-herpetic neuralgia and secondary infection significantly affect the patient’s quality of life. We started long-term oral acyclovir at 200 mg/day in July 2001. Acyclovir was continued until the end of immunosuppressive therapy and at least one year after transplantation. To evaluate the efficacy of this long-term prophylaxis with ultra low-dose acyclovir against VZV reactivation, we analyzed the records of 242 Japanese adult patients who underwent allogeneic HSCT for the first time from June, 1995 to November, 2006 at University of Tokyo Hospital. Sixty-six patients developed VZV reactivation at a median of 248 days after HSCT, with a cumulative incidence of 34.7%. There was no VZV-related death. Only one breakthrough reactivation occurred during long-term acyclovir, responding well to the therapeutic dose of valacyclovir. The use of long-term acyclovir was the only independent determinant that significantly decreased the overall incidence of VZV reactivation (20.4% vs 50.5%, P

Description: Myelodysplastic syndrome(MDS)is a clonal disorder of hematopoietic stem cells characterized by ineffective hematopoiesis and propensity to acute myeloid leukemias. The conversion of a normal stem cell into a preleukemic and ultimately leukemic state is thought to be a multistep process requiring accumulation of a number of genetic changes. Conventional cytogenetic analysis has disclosed a number of chromosome abnormalities common to MDS and provided valuable clues to characterize these genetic lesions, rarity of balanced translocations and relative predominance of unbalanced abnormalities in MDS, including gene deletions and amplifications. However conventional analytical methods provide only limited resolutions of analysis for identification of genetic gains and losses and prevent further molecular delineation of relevant genes to the pathogenesis of MDS. Array-based comparative genomic hybridization (CGH) is a robust technique to enable rapid and comprehensive genome-wide analysis of genetic aberrations in cancers, in which differentially labeled DNAs from both tumor and normal samples are comparatively hybridized to a large number of genomic DNAs. In this study, we constructed a high-quality array-based CGH system for genome-wide analysis of chromosomal abnormalities to identify candidate target genes of MDS. Our whole genome arrays consisted of 3,300 BAC/PAC clones, thus having an average resolution of 1.0 Mb over the whole human genome. Each clone was amplified with degenerated oligonucleotide primed-PCR (DOP-PCR) and the amplified products were spotted in duplicate grids onto aminosilan-coated glass slides. For more high-resolution analysis, we employed the GeneChip Mapping 100k arrays (Affymetrix), originally developed for large-scale SNP typing, as a tool for detection of copy number changes in selected MDS cases. It contains 116,204 different SNPs on two separate arrays, covering the whole human genome with an average resolution of 21 kb. With this arrays DNA copy number’s changes could be estimated by comparing intensity of SNP signals of tumor cells with that of normal cells from the same patients. In addition, using paired samples from tumor cells and normal cells, large-scale LOH analysis became also possible. In total, 54 MDS samples were analyzed using our array CGH system. In addition to large chromosomal changes, including loss of 5q, 7q, 13q, and 20q, and gain of the whole chromosome 8, a number of small, cryptic chromosomal abnormalities were identified that would escape from conventional cytogenetic detection. Many of these abnormalities were represented only by a single PAC/BAC clone. In several chromosome regions, including 3q13, 5p15, 13p33, and 20q12, there existed commonly deleted regions, which could be confirmed by FISH analysis. Similarly gains of genetic materials were found on 8p23 and 17p13. Several genes were identified within these regions that may be candidates for relevant genes to these genetic alterations. In conclusion, genome-profiling using array CGH techniques were highly useful tools for delineating the pathogenesis of MDS.

Description: Defects in genes involved in DNA mismatch repair have been detected in both hereditary and sporadic tumors of colon, endometrium, and ovary and suggested to be associated with tumorigenesis. To investigate disruptions of the mismatch repair system in hematological malignancies, we examined alterations of the human mutL homologue 1 (hMLH1) gene, a member of the mismatch repair gene family, in a total of 43 human leukemia and lymphoma cell lines, by polymerase chain reaction–single-strand conformation polymorphism (PCR-SSCP) and sequencing analyses. Mutations of the hMLH1 gene were detected in three cell lines established from lymphoid leukemias. Moreover, Northern and Western blot analyses showed that expression of hMLH1 transcript or protein was abrogated in these three leukemia cell lines. Further studies for microsatellite loci showed that these cell lines without hMLH1 expression showed microsatellite instability. This is the first report that describes mutations and inactivation of the hMLH1 gene in human leukemia cells, suggesting that disruption of DNA mismatch repair system may play an important role in the development of human lymphoid leukemias.

Description: Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic progenitors characterized by impaired blood cell production due to ineffective hematopoiesis and high propensity to acute myeloid leukemias. One of the prominent features of MDS is the high frequency of unbalanced chromosomal abnormalities that result in genetic imbalances and copy number alterations. Although the chromosomal segments involved in these abnormalities are thought to contain relevant genes to the pathogenesis of MDS, conventional analyses including FISH have failed to identify critical regions small enough to pinpoint their target genes. Affymetrix® GeneChip® 100K/500K mapping arrays were originally developed for large-scale genotyping of more than 100,000/500,000 SNPs in two separate arrays, but the quantitative nature of the preparative whole-genome amplification and array hybridization thereafter also allows for accurate copy number estimate of the genome using these platforms at the resolutions of 21.3 kb and 5.4 kb with 116,204 and 520,000 oligonucleotide probes, respectively. Here we developed robust algorithms (CNAG) for copy number detection using 100K and/or 500K arrays and analyzed 88 MDS samples on these platforms in order to identify relevant genes for development of MDS. With these huge numbers of uniformly distributed SNP probes, numerous copy number alterations were sensitively detected in cases with MDS with more numbers of abnormalities found in advanced diseases (RAEB and RAEB-t). In addition to large-scale alterations of various chromosomal segments previously reported in these syndromes, a number of small cryptic chromosomal abnormalities were identified that would escape conventional cytogenetic analysis or array CGH analysis. Minimum overlapping deletions in 5q, 7q, 12p, 13q, and 20q were precisely defined, although no pinpoint homozygous deletions were detected within these regions. A common 20q deletion spans a 400 kb segment harboring five transcriptomes and the common 12p deletion defines a 1.3 Mb region that contains the ETV6 gene. Other common overlapping abnormalities include deletions in 21q22, 17q13, and gains of 11q25. Genome-wide analysis of copy number changes using high-density oligonucleotide arrays provides valuable information about genetic abnormalities in MDS.

Description: Background: Although the onset of invasive pulmonary aspergillosis (IPA) after allogeneic hematopoietic stem cell transplantation (HSCT) was bimodal, IPA early after HSCT has become less frequent, partly due to the shortening of neutropenic periods by the use of peripheral blood stem cells or colony-stimulating factors. Therefore, the validity of empiric anti-Aspergillus treatments based on sustained febrile neutropenia according to the IDSA guideline should be re-evaluated. Patients and Methods: We retrospectively reviewed the clinical records of 114 adult patients who underwent allogeneic HSCT between September 2002 and December 2005 in HEPA-filtered clean rooms at the University of Tokyo Hospital. Fluconazole at 200 mg/day was given as anti-Candida prophylaxis. In general, anti-Aspergillus agents were not started empirically, but presumptively started after the detection of positive Aspergillus antigen, positive beta-D-glucan, halo-sign on chest X-ray or CT-scan, and so on, associated with sustained febrile neutropenia. For the definition of early IPA, we employed proven or probable IPA according to the EORTC/MSG criteria that developed between the day of HSCT and seven days after engraftment. Results: All but two who experienced early death showed neutrophil engraftment at a median of 16.5 days after HSCT. Although 15 patients developed IPA after HSCT, early IPA was observed only in 2 (1.8 %) patients. Among 73 patients who experienced sustained febrile neutropenia for more than 7 days before engraftment, we empirically started anti-Aspergillus agents in 13 patients a median of 9 days after the development of febrile neutropenia, whereas fluconazole was continued in 60 patients who had febrile neutropenia for 15 days in median. Four of the 60 patients received presumptive anti-Aspergillus therapy, one of whom developed probable IPA after the initiation of treatment. There was another patient who received anti-Aspergillus treatment only after the development of probable IPA because he showed no prior signs for presumptive therapy. There was no difference in the incidence of early IPA between patients who received empiric anti-Aspergillus therapy and those who did not (0% vs. 3.3%, P〉0.99). The two patients who developed early probable IPA were successfully treated with anti-Aspergillus agents. Conclusions: These findings throw doubt on the validity of empiric anti-Aspergillus treatments for allogeneic HSCT recipients with sustained febrile neutropenia, provided that they are treated in clean units with anti-Candida prophylaxis. A randomized controlled trial is warranted to compare empiric and presumptive anti-Aspergillus treatments for allogeneic HSCT recipients with sustained febrile neutropenia.

Description: Notch signaling is involved in cell fate decisions in many systems including hematopoiesis. It has been shown that expression of an activated form of Notch1 (aNotch1) in 32D mouse myeloid progenitor cells inhibits the granulocytic differentiation induced by granulocyte colony-stimulating factor (G-CSF). Results of the current study show that aNotch1, when expressed in F5-5 mouse erythroleukemia cells, also inhibits erythroid differentiation. Comparison of the expression levels of several transcription factors after stimulation for myeloid and erythroid differentiation, in the presence or absence of aNotch1, revealed that aNotch1 did not change its regulation pattern with any of the transcription factors examined, except for GATA-2, despite its inhibitory effect on differentiation. GATA-2 was down-regulated when the parental 32D and F5-5 were induced to differentiate into granulocytic and erythroid lineages, respectively. In these induction procedures, however, the level of GATA-2 expression was sustained when aNotch1 was expressed. To ascertain whether maintenance of GATA-2 is required for the Notch-induced inhibition of differentiation, the dominant-negative form of GATA-3 (DN-GATA), which acted also against GATA-2, or transcription factor PU.1, which was recently shown to be the repressor of GATA-2, was introduced into aNotch1-expressing 32D (32D/aNotch1) cells that do not express GATA family proteins other than GATA2. Both DN-GATA and PU.1 reversed the phenotype of 32D/aNotch1 inducing its differentiation when G-CSF was added. Furthermore, enforced expression of HES-1, which is involved in Notch signaling, delayed differentiation of 32D, and again this phenotype was neutralized by DN-GATA. These results indicate that GATA-2 activity is necessary for the Notch signaling in hematopoietic cells.

Description: Defects in genes involved in DNA mismatch repair have been detected in both hereditary and sporadic tumors of colon, endometrium, and ovary and suggested to be associated with tumorigenesis. To investigate disruptions of the mismatch repair system in hematological malignancies, we examined alterations of the human mutL homologue 1 (hMLH1) gene, a member of the mismatch repair gene family, in a total of 43 human leukemia and lymphoma cell lines, by polymerase chain reaction–single-strand conformation polymorphism (PCR-SSCP) and sequencing analyses. Mutations of the hMLH1 gene were detected in three cell lines established from lymphoid leukemias. Moreover, Northern and Western blot analyses showed that expression of hMLH1 transcript or protein was abrogated in these three leukemia cell lines. Further studies for microsatellite loci showed that these cell lines without hMLH1 expression showed microsatellite instability. This is the first report that describes mutations and inactivation of the hMLH1 gene in human leukemia cells, suggesting that disruption of DNA mismatch repair system may play an important role in the development of human lymphoid leukemias.

Description: Non-Hodgkin lymphomas (NHL) are hematopoietic malignancies originated from diversity of peripheral lymphoid organs. During the past two decades, there have been significant advances in the pathogenesis of NHL including identification of a number of genes associated with the disease-specific translocations and other genetic alterations. In view of cytogenetics, however, NHL frequently shows complex chromosomal abnormalities involving copy number alterations as well as other unbalanced translocations, many of which have not been unveiled at the molecular levels. Affymetrix® 100K/500K mapping arrays were originally developed for large-scale SNP typing required for genome-wide association studies, but the quantitative nature of the whole-genome amplification and hybridization used in these platforms also makes them powerful tools for genome-wide analysis of cancer genomes with use of uniformly distributed 116,204/520,000 SNP-specific probes. Moreover the use of SNP specific probes enables allele-specific copy number analysis that is totally impossible with other platforms. Here we developed the robust algorithms (Copy number analyzer for Affymetrix® GeneChip®; CNAG) for high-quality processing of 100K/500K data and analyzed a total of 72 NHL samples (61 primary samples including 34 diffuse large B-cell lymphoma, 18 follicular lymphoma and 11 cell lines including 3 adult T cell leukemia/ lymphoma) for genome-wide copy number alterations, LOH, and allelic imbalances at the resolutions of 23.6/5.4 kb. In 100K analysis, 34 homozygous deletions and 42 high-grade amplifications and other numerous copy number alternations and/or LOH, were identified together with possible gene targets as for some regions. 500K analysis disclosed even more subtle changes. Common overlapping alternations included deletions in 1p31.1 and 9p21.3, and 19p13.32 and high-grade amplifications in 3p14.2–p14.1,7q21.13–q21.3, and 20q11.21. Of particular importance is, however, the finding of otherwise undetected copy number neutral LOHs, which are revealed only by allele-specific copy-number analysis. In fact the copy number neutral LOHs represented a novel type of genetic abnormality in NHL because they were very frequent and found in more than 87% (20/23) of NHL cases examined with allele-specific copy number analysis, making a stark contrast to ALL, in which these abnormalities were rare. They typically involved chromosomal ends, indicating somatic recombinations are the potential mechanism of generating these abnormalities. Notably, there was a clear predisposition of the copy number neutral LOH to specific chromosomal loci including 1p, 1q, 6p, 9p, 17q, and 19p suggesting existence of relevant genes to NHL pathogenesis within these common regions. In conclusion, Affymetrix® SNP-genotyping microarrays and our CNAG algorithms provide a powerful platform of dissecting NHL genomes and could facilitate identification of the novel molecular mechanisms for lymphomagenesis.