Thirty‐eight HLA matched and mismatched patients given combined living donor kidney and enriched CD34+ hematopoietic cell transplants were enrolled in tolerance protocols using posttransplant conditioning with total lymphoid irradiation and anti‐thymocyte globulin. Persistent chimerism for at least 6 months was associated with successful complete withdrawal of immunosuppressive drugs in 16 of 22 matched patients without rejection episodes or kidney disease recurrence with up to 5 years follow up thereafter. One patient is in the midst of withdrawal and five are on maintenance drugs. Persistent mixed chimerism was achieved in some haplotype matched patients for at least 12 months by increasing the dose of T cells and CD34+ cells infused as compared to matched recipients in a dose escalation study. Success of drug withdrawal in chimeric mismatched patients remains to be determined. None of the 38 patients had kidney graft loss or graft versus host disease with up to 14 years of observation. In conclusion, complete immunosuppressive drug withdrawal could be achieved thus far with the tolerance induction regimen in HLA matched patients with uniform long‐term graft survival in all patients.

A hematopoietic cell transplantation regimen was adapted from a preclinical model that used reduced-intensity conditioning (RIC) and protected against graft-versushost disease (GVHD) by skewing residual host T-cell subsets to favor regulatory natural killer T cells. One hundred eleven patients with lymphoid (64) and myeloid (47) malignancies received RIC using total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) followed by the infusion of granulocyte colony-stimulating factor-mobilized grafts. Included were 34 patients at least 60 years of age, 32 patients at high risk of lymphoma relapse after disease recurrence following prior autologous transplantation, and 51 patients at high risk of developing GVHD due to lack of a fully human leukocyte antigen (HLA)-matched related donor. Durable chimerism was achieved in 97% of patients. Cumulative probabilities of acute GVHD (grades II-IV) were 2 and 10% of patients receiving related and unrelated donor grafts. Nonrelapse mortality (NRM) at 1 year was less than 4%. Cumulative incidence of chronic GVHD was 27%. The 36-month probability of overall and eventfree survival was 60% and 40%, respectively. Disease status at start of conditioning and the level of chimerism achieved after transplantation significantly impacted clinical outcome. The high incidence of sustained remission among patients with active disease at time of transplantation suggests retained graftversus-tumor reactions. Active trial registration currently at clinicaltrials.gov un-

Purpose To evaluate the prognostic value of metabolic tumor volume measured on 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) imaging and other clinical factors in patients treated for locally advanced head and neck cancer at a single institution. Materials/Methods From March 2003 to August 2007, 85 patients received PET/CT-guided chemoradiotherapy for HNC. Metabolically active tumor regions were delineated on pretreatment PET scans semi-automatically using custom software. We evaluated the relationship of FDG-PET maximum standardized uptake value (SUV) and total metabolic tumor volume (MTV) with disease-free survival (DFS) and overall survival (OS). Results Mean follow-up for surviving patients was 20.4 months. The estimated 2-year locoregional control, DFS, and OS for the group were 88.0%, 69.5% and 78.4%, respectively. The median time to first failure was 9.8 months among the 16 patients with relapse. An increase in MTV of 17.4 mL (difference between the 75th and 25th percentiles) was significantly associated with an increased hazard of first event (recurrence or death) (1.9-fold, p<0.001), even after controlling for Karnofsky performance status (KPS) (1.8-fold, p=0.001), and of death (2.1-fold, p<0.001). We did not find a significant relationship of maximum SUV, stage, or other clinical factors with DFS or OS. Conclusion Metabolic tumor volume is an adverse prognostic factor for disease recurrence and death in HNC. MTV retained significance after controlling for KPS, the only other significant adverse prognostic factor found in this cohort. MTV is a direct measure of tumor burden and is a potentially valuable tool for risk stratification and guiding treatment in future studies.

During mouse development, primordial germ cells (PGCs) that give rise to the entire germ line are first identified within the proximal epiblast. However, long-term tracing of the fate of the cells has not been done wherein all cells in and around the germ-cell lineage are identified. Also, quantitative estimates of the number of founder PGCs using different models have come up with various numbers. Here, we use tetrachimeric mice to show that the progenitor numbers for the entire germ line in adult testis, and for the initiating embryonic PGCs, are both 4 cells. Although they proliferate to form polyclonal germ-cell populations in fetal and neonatal testes, germ cells that actually contribute to adult spermatogenesis originate from a small number of secondary founder cells that originate in the fetal period. The rest of the ''deciduous'' germ cells are lost, most likely by apoptosis, before the reproductive period. The second ''actual'' founder germ cells generally form small numbers of large monoclonal areas in testes by the reproductive period. Our results also demonstrate that there is no contribution of somatic cells to the male germ cell pool during development or in adulthood. These results suggest a model of 2-step oligoclonal development of male germ cells in mice, the second step distinguishing the heritable germ line from cells selected not to participate in forming the next generation.stem cells ͉ testis ͉ premordial germ cells ͉ apoptosis chimeras T he germ line is made up of a highly protected and strictly regulated group of cells that transmit genetic information accurately to the next generation. It was initially proposed (1), and later shown that they usually originate as a very small founding population that is segregated from somatic cells early in development, at least in organisms where the overall body plan is established early (2, 3). However, retroviral marking of mouse early embryos at the 4-to 16-cell stage suggests that at least 3 cells contribute to the germ line and are set aside before somatic tissue allocation (4), indicating that the origin of germ cells is not generally monoclonal. In species such as Drosophila and Caenorhabditis elegans, germ plasm, a unique cluster of mRNAs and organelles, is a good marker to follow the origin and development of early germ cells (3). However, there is no visible germ plasm in mammals, making it difficult to follow early populations of germ cells (5). It has been shown that inner cell mass (ICM) or epiblast cells earlier than embryonic day (E)5.5 can contribute to any lineage of somatic and germ cells (6-8).Tissue nonspecific alkaline phosphatase (TNAP) has long been used as a marker for candidate primordial germ cells (PGCs) (9, 10). Also, many markers for PGCs have been identified, including SSEA-1 (11), Oct3/4 (12), Stella/PGC-7 (13, 14), Fragilis (14), and Blimp-1 (15). By using these markers, earlier candidate PGCs that cannot be identified with TNAP could be detected. It has been shown that they are stella positive and fragilis highly posit...

IntroductionThe DNA-hypomethylating drugs 5-azacytidine (5AC) and 5-aza-2Ј-deoxycytidine (DAC) are in clinical use for the treatment of myelodysplastic syndrome (MDS) 1,2 and have been shown to improve overall response rates and increase the time to progression to acute myelogenous leukemia compared with best supportive care. 3,4 Several studies have documented an increase in genomewide promoter methylation in mononuclear cells and in CD34 ϩ hematopoietic progenitor cells (HPCs) derived from the BM of MDS patients. 5,6 However, drug-induced differences in genomewide promoter methylation signatures and in gene-expression profiles have not been found to be correlated with the clinical response to hypomethylating agents. 7 In addition, a correlation between changes in promoter methylation profiles and acquired resistance to 5AC or DAC has never been demonstrated 8,9 Therefore, the role of DNA hypermethylation in the pathogenesis of MDS remains to be determined, as does the question of whether the therapeutic responses to 5AC and DAC result from the induction of gene-specific DNA hypomethylation as opposed to other sequelae such as the induction of a DNA-damage response, the activation of an immune response, or the induction of senescence. 7 The nucleolus contains clusters of genes that encode the 45S pre-rRNA precursor of rRNA that is subsequently processed to generate the 18S, 5.8S, and 28S rRNA components. 10,11 In human cells, there are approximately 400 copies of rDNA genes arranged as tandem repeats within nucleolar organizer regions located on chromosomes 13-15, 21, and 22, although not all of the genes are actively transcribed. Transcription of the rRNA genes depends on the chromatin structure of the promoter region. 12-14 Promoters of active rRNA genes are devoid of CpG methylation and are associated with acetylated histones, whereas the reverse is found for silenced genes. 15 DNA methyltransferase 1 (DNMT1) controls rDNA gene transcription 16 and also regulates the nucleolar architecture by maintaining the heterochromatin state within intergenic repetitive sequences. 17,18 Several recent observations have suggested that ribosomal protein synthesis is an essential component of normal hematopoietic stem cell (HSC) function. Haploinsufficiency of the ribosomal gene RPS14 in 5qϪ syndrome, 19,20 the presence of RPS19 mutations in Diamond-Blackfan anemia, 21 and the aberrant expression of multiple ribosomal proteins in MDS 22-24 all suggest that altered ribosome biogenesis could play a major role in the pathogenesis of BM failure syndromes. To determine whether rRNA synthesis might be altered in HPCs from a broader spectrum of MDS patients, in the present study, we compared the level of rRNA expression and the extent of rDNA promoter methylation in CD34 ϩ cells derived from BM of MDS patients with those in normal CD34 ϩ cells. Our data demonstrate that rRNA expression is decreased in HPCs from MDS patients, whereas methylation within the rDNA upstream regulatory region is increased. We also show that rRNA expres...