Favorable impact of pre-transplant ATG on outcomes of reduced-intensity hematopoietic cell transplants from partially mismatched unrelated donors.

Reduced-intensity conditioning (RIC) permits allogeneic hematopoietic progenitor cell transplantation in patients who would not be considered candidates for transplantation using a myeloablative preparative regimen because of age, comorbidities or prior therapy. In the setting of myeloablative transplantation, use of antithymocyte globulin (ATG) can reduce the risk of GVHD without negatively affecting transplant outcomes; however, limited data exist on the impact of ATG in the setting of RIC, particularly when there is HLA-mismatch. We performed a retrospective analysis of 85 patients who received unrelated donor transplants at our institution for hematologic malignancies following conditioning with fludarabine and melphalan (FluMel), with or without rabbit ATG (6 mg/kg). ATG was targeted to patients receiving HLA-mismatched grafts. With a median follow-up of 36 months, those receiving ATG and a mismatched graft had similar rates of acute and chronic GVHD, relapse, and similar OS compared with those receiving HLA-matched grafts without ATG. In a multivariate analysis, HLA-mismatched donor was not associated with a decrement in OS. We conclude that this intermediate dose of ATG is effective in preventing severe GVHD in the setting of HLA-mismatch, without undue compromise of the graft versus tumor effects on which RIC transplants depend.

Eosinophilic pulmonary syndrome as a manifestation of GVHD following hematopoietic stem cell transplantation in three patients.

Eosinophilic pulmonary syndrome is an uncommon problem in SCT recipients that can mimic an infectious process. We report the occurrence of eosinophilic pulmonary syndrome in three patients following allogeneic hematopoietic stem cell transplantation (HSCT), and postulate that this entity is part of the clinicopathologic spectrum of pulmonary GVHD. In all three cases, active chronic GVHD of the skin preceded or coincided with the development of pulmonary involvement. Other common features included peripheral blood eosinophilia, diffuse bilateral pulmonary infiltrates and lung biopsies showing pronounced infiltrates of eosinophils involving the small bronchioles. All patients responded promptly to systemic steroid therapy, with improvement of their pulmonary symptoms and the resolution of peripheral blood eosinophilia. Clinicians should be aware that eosinophilic pulmonary syndrome can occur following HSCT, may be associated with other manifestations of chronic GVHD, and generally responds well to corticosteroid therapy.

Posaconazole as salvage treatment of invasive fungal infections in patients with underlying renal impairment.

OBJECTIVES: The aim of this study is to determine the efficacy and safety of posaconazole in patients with underlying renal impairment. Patients and methods We analysed the efficacy and safety of posaconazole in patients with renal impairment in a post hoc subanalysis of a Phase 3, multicentre, open-label trial in patients with invasive fungal infections (IFIs). In the Phase 3 study, 330 patients intolerant of or with IFIs refractory to standard antifungal therapy received posaconazole 800 mg daily in divided doses. In our subanalysis, 238 patients with proven/probable IFIs, including 65 patients with renal impairment (creatinine clearance < 50 mL/min or serum creatinine (sCR) level >2 mg/dL at baseline) and 173 patients with greater renal function [creatinine clearance >/= 50 mL/min (acceptable renal function)], formed the modified intent-to-treat population. Success was defined as complete or partial response, and non-success was defined as stable disease or treatment failure. RESULTS: Overall response rates were similar in the renal impairment group (49%) and in the acceptable renal function (50%) group. Seventeen of the 41 patients with renal impairment and aspergillosis responded. Adverse events occurred in 32/65 (49%) patients with renal impairment and in 72/173 (42%) patients with acceptable renal function. The most common adverse events in both groups were nausea (14% patients with renal impairment versus 8% with acceptable renal function), altered/elevated levels of other medications (8% versus 2%), increased sCR levels (6% versus 0%), vomiting (6% versus 4%), abdominal pain (5% versus 5%) and dizziness (5% versus 1%). CONCLUSIONS: These results suggest that posaconazole is effective and well tolerated in patients with refractory IFIs regardless of renal impairment.

Long-term progression-free survival after early autologous transplantation for mantle-cell lymphoma.

Autologous hematopoietic progenitor SCT (HPCT) has been studied both as a consolidative and salvage maneuver in mantle-cell lymphoma (MCL), and may improve failure-free survival rates as well as overall survival. We describe 21 patients with MCL who received autologous HPCT at Emory University Hospital as part of the primary treatment strategy. Sixteen patients were in CR1 and five in PR1 at the time of HPCT. The most commonly used induction chemotherapy was the hyper-CVAD (cyclophosphamide, vincristine, doxorubicin and dexamethasone) regimen with or without rituximab. At the last follow-up, 17 patients were in continuous CR, and there were four relapses. There were no transplant-related deaths. With a median follow-up of 54 months from HPCT, 5-year progression-free survival and overall survival are 73% and 76%, respectively. Our retrospective analysis provides the longest follow-up to date for patients with MCL who received an autologous HPCT as part of primary treatment. This lengthy follow-up helps define the natural course of MCL after autologous transplantation.

Rapid hematopoietic engraftment following fractionated TBI conditioning and transplantation with CD34(+) enriched hematopoietic progenitor cells from partially mismatched related donors.

Nineteen adult patients with poor-risk hematologic malignancy received T cell-depleted (TCD) hematopoietic progenitor cell (HPC) transplant from partially mismatched related donors (PMRD). The preparative regimen (FITFA) included fractionated TBI, thiotepa, fludarabine, and horse (n = 3) or rabbit (n = 16) anti-thymocyte anti-sera (ATG). GVHD prophylaxis consisted of TCD by positive/negative selection using the Isolex 300i system and pre-transplant ATG with no post-transplant immunosuppression. The mean number (+/-s.d.) of transplanted CD34(+) and CD3(+) cells were 8.9 x 10(6)/kg +/-4.3 (range 2.6-19.3) and 1.4 x 10(4)/kg +/-1.2 (range 0.3-4.6) respectively. Seventeen patients evaluable for neutrophil engraftment achieved an ANC >0.5 x 10(9)/l at a median of 12 days (range 9-27), with evidence of full donor chimerism. Thirteen patients died of the following causes: relapse (n = 6), infections (n = 5), interstitial pneumonia (n = 1), and unknown causes (n = 1) None of the recipients of rabbit ATG required therapy for acute or chronic GVHD. Five patients are alive and disease-free at a median time of 303 days post transplant (range 100-660). The FITFA preparative regimen using fractionated TBI is well tolerated and is sufficiently immunosuppressive to allow rapid and stable donor origin hematopoietic engraftment without 'mega' doses of CD34(+) cells. Combination of stringent ex vivo TCD and pre-transplant ATG is effective GVHD prophylaxis.

A novel retinal degeneration locus identified by linkage and comparative mapping of canine early retinal degeneration.

Early retinal degeneration (erd) is an early onset progressive retinal atrophy, a hereditary canine retinal disease phenotypically similar to human retinitis pigmentosa (RP). In previous efforts to identify the erd locus, canine homologs of genes causally associated with RP in humans, such as opsin (RHO), the beta-subunit gene for cyclic GMP phosphodiesterase (PDE6B), and RDS/peripherin, were excluded. A genome-wide screen was undertaken on canine families segregating the erd disease. Analysis of over 150 canine-specific markers has localized erd to a single linkage group comprising two previously identified canine linkage groups, 20 and 26, corresponding to canine radiation hybrid groups RH.34-a and RH.40-a. Multipoint analysis places erd in the interval between marker FH2289 (distance 23.6 cM) and FH2407 (5.9 cM) with a lod score of 12.23. Although the erd linkage group has not been assigned to an identified canine chromosome, conserved synteny of this linkage group with human 12p13-q13 suggests several candidates for erd and identifies a novel retinal degeneration locus. The rapid progress now occurring in canine genetics will expedite identification of the genes and molecular mechanisms underlying the inherited traits and diseases that make the dog a unique asset for study of mammalian traits.

Evaluation of the APOH gene as a positional candidate for prcd in dogs.

PURPOSE: Progressive rod-cone degeneration (prcd) is an autosomal recessive retinal degeneration of dogs characterized by abnormalities in lipid metabolism. It has recently been mapped to the centromeric region of canine chromosome 9, homologous to human 17q, which contains the apolipoprotein H (apoH, protein; APOH, gene) gene involved in lipid metabolism and regulation of triglycerides. The present study was undertaken to evaluate APOH as a positional candidate for prcd. METHODS: Expression of APOH in the retina was examined by reverse transcription-polymerase chain reaction (RT-PCR) and by immunocytochemistry in normal and prcd-affected dogs. The level of apoH in the plasma was determined by western blot analysis. Intragenic polymorphic markers were identified and typed in the prcd pedigree. Canine-rodent hybrid cell lines were analyzed to detect canine APOH. RESULTS: ApoH has been localized to the photoreceptor outer segment layer by immunocytochemistry. Its expression in the retina of normal and prcd-affected dogs was confirmed by RT-PCR. The levels of antihuman apoH cross-reacting material in plasma were similar in all dogs, regardless of disease status. Finally, linkage analysis of the APOH gene with the disease locus in the prcd pedigree detected 3 recombinants among 70 informative offsprings (lod score 15.09 at 0 = 4.3 centimorgan [cM]). CONCLUSIONS: APOH is expressed in the retina and tightly linked to the prcd locus. However, despite its potential role in phenotypes of abnormal lipid metabolism associated with prcd, the gene has been excluded as a primary candidate for prcd by linkage analysis.

Toward a framework linkage map of the canine genome.

Selective breeding to maintain specific physical and behavioral traits has made the modern dog one of the most physically diverse species on earth. One unfortunate consequence of the common breeding practices used to develop lines of dogs with the desired traits is amplification and propagation of genetic diseases within distinct breeds. To map disease loci we have constructed a first-generation framework map of the canine genome. We developed large numbers of highly polymorphic markers, constructed a panel of canine-rodent hybrid cell lines, and assigned those markers to chromosome groups using the hybrid cell lines. Finally, we determined the order and spacing of markers on individual canine chromosomes by linkage analysis using a reference panel of 17 outbred pedigrees. This article describes approaches and strategies to accomplish these goals.

Acquired amegakaryocytic thrombocytopenia treated with allogeneic BMT: a case report and review of the literature.

Despite recent advances in understanding the biology of thrombopoiesis, autoimmune thrombocytopenia caused by inhibition of megakaryocytic precursors, remains a treatment dilemma. We report a case of a 43-year-old female who developed amegakaryocytic thrombocyto- penia refractory to intravenous immunoglobulin (IVIG), prednisone, cytoxan and vincristine. She was subsequently treated with myeloablative chemotherapy (busulfan and cyclophosphamide) followed by allogeneic bone marrow transplant from a 6/6 HLA-matched sibling. The patient is currently more than 1 year after transplant with complete donor chimerism and restoration of normal thrombopoiesis. A review of the literature shows that the clinical syndrome known as amegakaryocytic thrombocytopenia represents a heterogeneous group of disorders, and clinical experience with immunosuppression varies. Appropriate initial treatment for these patients requires immunosuppressive agents, including antithymocyte globulin (ATG) for steroid refractory disease. However, in the case of symptomatic patients who have an appropriate sibling donor, early hematopoietic progenitor cell transplant, even before administration of ATG, may be necessary. Further studies are needed to better define the pathogenesis and mechanism of this heterogeneous disorder before more definitive treatment algorithms can be established.

Identification of a RAPD marker linked to progressive rod-cone degeneration in dogs.

Random amplified polymorphic DNA (RAPD) analysis has been used widely in plant and fungi for identification of markers linked to genetic traits and mapping, but its use is limited to identification of intra- and inter-species difference in domestic mammals. We report here identification of a RAPD-derived marker linked to progressive rod-cone degeneration (prcd), an inherited autosomal recessive retinal disease of dogs. A total of 400 standard 10-mer primers were used for amplification by use of DNA samples from normal (+/+) and affected (prcd/prcd) dogs. A single primer was identified which amplified a 1.5-kb DNA fragment only from normal dogs. PCR with longer primers designed from the sequence-characterized amplified region of the 1.5-kb DNA fragment identified a co-dominant multi-allelic polymorphism in the prcd-informative pedigree. Three recombinants were identified among 34 informative offsprings, yielding a LOD score of 5.568 at theta = 0.091. This marker was mapped to two canine-rodent hybrid cell lines in which two genes (canine homologues of human breast cancer 1 susceptibility gene, and cGMP phosphodiesterase gamma-subunit gene), and three anonymous microsatellites have been identified. This is the first reported identification of a RAPD-derived marker with multiple alleles linked to a mammalian disease locus.

Linkage analysis and comparative mapping of canine progressive rod-cone degeneration (prcd) establishes potential locus homology with retinitis pigmentosa (RP17) in humans.

Progressive rod-cone degeneration (prcd) is the most widespread hereditary retinal disease leading to blindness in dogs and phenotypically is the canine counterpart of retinitis pigmentosa (RP) in humans. In previous efforts to identify the genetic locus for prcd, the canine homologs for many of the genes causally associated with RP in humans, such as RHO, PDE6B, and RDS/peripherin, have been excluded. In parallel with a recent undertaking to establish a framework map of the canine genome, multiple prcd-informative pedigrees have been typed with a panel of more than 100 anchor loci and microsatellite-based markers. Identification of a linkage group flanking prcd ([TK1, GALK1, prcd]-[MYL4, C09.173, C09.2263]-RARA-C09.250-C09.474-NF1) localizes prcd close to the centromeric end of canine chromosome 9 (CFA9), and excludes RARA as a candidate gene. The conserved synteny of this region of CFA9 and distal human chromosome 17q establishes the potential locus homology of prcd in the dog with RP17, a human retinitis pigmentosa locus for which no gene has yet been identified. Assignment of the prcd disease locus to an identified canine autosome represents a powerful application of the developing canine linkage map in medical genetics. The usefulness of this approach is further demonstrated by identification of the correspondence of the prcd interval to homologous human and mouse chromosomal regions. The rapid progress that is now occurring in the field of canine genetics will expedite the identification of the genes underlying many of the inherited traits and diseases that make the dog a unique asset for the study of mammalian traits.

Hereditary ovarian cancer.

Ovarian cancer has been described in association with three autosomal dominant syndromes: familial site-specific ovarian cancer, familial breast and ovarian cancer, and the hereditary nonpolyposis colon cancer syndrome. It appears that most breast-ovarian and site-specific ovarian cancer families are explained by mutations in the BRCA1 tumor suppressor gene. Other genes associated with inherited susceptibility to ovarian cancer include BRCA2, p53, and the DNA mismatch repair genes.

Construction of a panel of canine-rodent hybrid cell lines for use in partitioning of the canine genome.

We have constructed a collection of canine-rodent microcell hybrid cell lines by fusion of canine fibroblast microcell donors with immortalized rodent recipient cells. Characterization of the hybrid cell lines using a combination of fluorescence in situ hybridization and PCR analysis of canine microsatellite repeat sequences allowed selection of a panel of hybrids in which most canine chromosomes are represented. Approximately 90% of genetic markers and genes that were tested could be assigned to 1 of 31 anonymous canine chromosome groups, based on common patterns of retention in the hybrid set. Many of these putative chromosome groups have now been validated by linkage analysis. This panel of cell lines provides a tool for development of genetic, physical, and comparative maps of the canine genome.

A linkage map of the canine genome.

A genetic linkage map of the canine genome has been developed by typing 150 microsatellite markers using 17 three-generation pedigrees, composed of 163 F2 individuals. One hundred and thirty-nine markers were linked to at least one other marker with a lod score > or = 3.0, identifying 30 linkage groups. The largest chromosome had 9 markers spanning 106.1 cM. The average distance between markers was 14.03 cM, and the map covers an estimated 2073 cM. Eleven markers were informative on the mapping panel, but were unlinked to any other marker. These likely represent single markers located on small, distinct canine chromosomes. This map will be the initial resource for mapping canine traits of interest and serve as a foundation for development of a comprehensive canine genetic map.

A class of highly polymorphic tetranucleotide repeats for canine genetic mapping.

We have identified and characterized a new class of polymorphic markers for the canine genome from a simple tetranucleotide repeat sequence, (GAAA)n. Genetic markers derived from this repeat are highly polymorphic compared with other canine microsatellites, yet are stable enough to be useful for following Mendelian inheritance in multigeneration pedigrees. We show further that (GAAA)n repeats are distributed throughout the canine genome and occur with sufficient frequency to be useful in the development of a framework map of the canine genome.

Allogeneic marrow transplantation for aplastic anaemia associated with dyskeratosis congenita.

Eight patients with aplastic anaemia associated with dyskeratosis congenita received allogeneic marrow grafts from either HLA-identical siblings (six patients) or HLA-matched unrelated donors (two patients). Patients who received marrow from HLA-identical siblings were conditioned with cyclophosphamide (140-200 mg/kg), with or without antithymocyte globulin. Patients who received unrelated donor marrow were conditioned with cyclophosphamide (120 mg/kg) and total body irradiation (1200 cGy). The six patients who survived for >2 weeks following transplant all had haematological evidence of engraftment, and all three patients who survived for at least a year following transplant recovered normal haematological function. Three patients died with respiratory failure and pulmonary fibrosis at 70 d. 8 years and 20 years posttransplant; three patients died during the neutropenic period of invasive fungal infections; one patient died on day 44 of refractory acute graft-versus-host disease; and one patient remains alive 463 d following transplant. The surviving patient recently underwent surgical resection of a Dukes' stage C rectal carcinoma diagnosed 14 months posttransplant. The aplastic anaemia associated with dyskeratosis congenita can be successfully treated by allogeneic bone marrow transplantation; however, this approach does not reverse the other systemic manifestations of the syndrome. The pathogenesis of the intestinal lung disease observed in dyskeratosis congenita patients following marrow transplantation is not understood.

BRCA1 mutations in a population-based sample of young women with breast cancer.

BACKGROUND: Inherited mutations in the BRCA1 gene are associated with a high risk of breast and ovarian cancer in some families. However, little is known about the contribution of BRCA1 mutations to breast cancer in the general population. We analyzed DNA samples from women enrolled in a population-based study of early-onset breast cancer to assess the spectrum and frequency of germ-line BRCA1 mutations in young women with breast cancer. METHODS: We studied 80 women in whom breast cancer was diagnosed before the age of 35, and who were not selected on the basis of family history. Genomic DNA was studied for BRCA1 mutations by analysis involving single-strand conformation polymorphisms and with allele-specific assays. Alterations were defined by DNA sequencing. RESULTS: Germ-line BRCA1 mutations were identified in 6 of the 80 women. Four additional rare sequence variants of unknown functional importance were also identified. Two of the mutations and three of the rare sequence variants were found among the 39 women who reported no family history of breast or ovarian cancer. None of the mutations and only one of the rare variants was identified in a reference population of 73 unrelated subjects. CONCLUSIONS: Alterations in BRCA1 were identified in approximately 10 percent of this cohort of young women with breast cancer. The risk of harboring a mutation was not limited to women with family histories of breast or ovarian cancer. These results represent a minimal estimate of the frequency of BRCA1 mutations in this population. Comprehensive methods of identifying BRCA1 mutations and understanding their importance will be needed before testing of women in the general population can be undertaken.

Tissue-specific extinguisher loci in the human genome: a screening study based on random marking and transfer of human chromosomes.

Expression of many liver-specific genes is extinguished when cultured hepatoma cells are fused with fibroblasts, but liver genes can be reexpressed in hybrid segregants that have lost fibroblast chromosomes. To map extinguisher loci involved in this process, hepatoma microcell hybrids retaining single fibroblast chromosomes have been employed. Two different, transdominant loci that affect liver gene expression have been defined in this way. To determine whether other monochromosomal extinction phenotypes could be observed, we inserted a selectable marker into many human chromosomal sites and transferred the marked human chromosomes into rat hepatoma recipient cells by microcell fusion. Nearly 200 microcell hybrid clones were isolated and screened for expression of liver-specific mRNAs. Most liver transcripts continued to be expressed. However, PEPCK mRNA was extinguished in 12 hybrid clones. Some of these hybrids contained human TSE1, the previously characterized extinguisher locus on chromosome 17, but others contained a novel extinguishing function that mapped to human chromosome 14. The implications of these findings are discussed.