ALBERT

Description: Purpose: To determine the safety and efficacy of high dose cyclophosphamide, BCNU and etoposide (CBV) chemotherapy followed by autologous non-purged PBSCT in children with Hodgkin’s (HD) and non-Hodgkin’s lymphoma (NHL) who have either failed primary induction therapy or have experienced a first relapse. Methods: At study entry, patients received 2–4 courses of reinduction therapy as determined by the local institution. When a CR or PR was achieved, G-CSF mobilized PBSCs were harvested and patients underwent autologous PBSCT after a preparative therapy of cyclophosphamide 6000 mg/m2, BCNU 450–300 mg/m2, and etoposide 2400 mg/m2. Patients were to receive a set dose of 5x106 CD34 cells/kg. Patients were followed for disease status and toxicities. 69 patients (38 HD, 31 NHL) were entered onto study between April 1998 and June 2002. Survival statistics were calculated as Kaplan Meier estimates. Results: 41/69 (27/38 HD, 14/31 NHL) achieved a CR/PR at the conclusion of reinduction; 14 failed due to PD; 7 had SD; 7 were inevaluable. 38 patients (27 HD, 11 NHL) proceeded to autologous PBSCT. Overall 28/38 (20 HD, 8 NHL) of the transplanted patients survive and 21/28 (15 HD, 6 NHL) are progression free. The survival rates at 12 months and 24 months were 88%(±6) (HD 92%(±6), NHL 78%(±14)) and 73%(±10) (HD 76%(±11), NHL 65%(±22)), respectively. The rates of PFS at 12 months and 24 months were 65%(±8)(HD 72%(±9); NHL 46%(±17)) and 53%(±11) (HD 56%(±12); NHL 46%(±19)), respectively. 10 patients died after PBSCT. The causes of death were PD (N=5), infection (N=4), and toxicity (N=1). The first 17 patients received BCNU 450 mg/m2 and 8 of these experienced grade 3 or 4 pulmonary or renal toxicity. The dose was thus dropped to 300 mg/m2 and an additional 21 patients underwent PBSCT and 3 of these patients developed grade 3 or 4 pulmonary or renal toxicity. The incidence of grade 3 or 4 pulmonary of renal toxicity at 300mg/m2 was significantly lower than that at 450 mg/m2 (p=0.04, Fisher’s exact test). Conclusions: For children with primary resistant or first relapsed lymphoma who achieve a CR or PR after reinduction, CBV preparative therapy followed by PBSCT for children with primarily resistant or relapsed lymphoma results in an acceptable survival but a lower PFS due to a high relapse rate following transplant. A BCNU dose of 300 mg/m2, but not a dose of 450 mg/m2 is well tolerated by these heavily treated patients.

Description: Abstract 3202 Poster Board III-139 Background Diamond Blackfan Anemia (DBA) is a rare disorder characterized by red cell aplasia, congenital anomalies and a predisposition to cancer. Corticosteroids and red cell transfusions are the mainstays of therapy. Hematopoietic stem cell transplantation (HSCT) is curative in DBA, however outcomes have been variable. Nonetheless, preliminary data suggest improvement in HSCT outcomes since the year 2000. Purpose To compare the degree of HLA compatibility, pre-transplantation iron burden, and survival before and since the year 2000 in sibling and alternative donor HSCT recipients, in order to evaluate if enhanced HLA matching techniques along with improved control of pre-transplant iron burden contribute to improved outcomes. Methods The DBA Registry (DBAR) is a comprehensive database of patients with DBA who are enrolled after informed consent is obtained. The patients, their families, and their physicians complete a detailed questionnaire. A review of medical records and telephone interviews are performed to complete and clarify the information provided. Presence of iron burden prior to transplant was determined based upon fulfillment of any of the following criteria: 1) serum ferritin level 〉 2000 ng/mL within 2 months prior to HSCT, 2) liver iron concentration greater than 8 micrograms per gram, dry weight within 6 months prior to HSCT (measured by MR or biopsy), 3) liver histology from biopsy or autopsy within 6 months of HSCT demonstrating 3+ or greater hemosiderosis or any level of fibrosis. Results As of August 11, 2009, 583 patients have been enrolled in the DBAR, with 55 having undergone an allogeneic sibling or alternative donor HSCT. The median age at HSCT for all patients was 7 years, 8 months; 7 years, 2 months versus 9 years, 8 months for the 32 sibling and 23 alternative donor HSCT, respectively. There were 8 sibling and 13 alternative donor HSCT done prior to the year 2000 compared to 24 and 10 HSCT performed since the year 2000, respectively. Prior to 2000, 8 of 8 sibling HSCT performed were HLA matched, whereas since the year 2000, there were 19 complete and 1 partial HLA matched sibling HSCT done. HLA reports on 4 sibling HSCT were not available for review. There were 5 full and 7 partial matches amongst the alternative donor HSCT done prior to 2000 and 6 full and 2 partial matches since the year 2000. Prior to the year 2000 HLA typing was done by serologic typing alone or by serologic methods for Class I and molecular methods for Class II. HLA typing was done by molecular techniques after the year 2000. Overall survival at 5 years for all patients is 56.6 ± 8.7%; 77.3 ± 8.3% for sibling HSCT and 31.5 ± 12.7% for alternative donor HSCT (p=0.012). For sibling HSCT done prior to 9 years of age survival is 90.0 ± 9.5% and for those older than 9 years of age is 70.0 ± 11.6% (p=0.007). Survival for all patients prior to 2000 and since 2000 is 47.1 ± 11.0% and 72.4 ± 9.3%, respectively (p=0.184). Survival for alternative donor HSCT is 23.1 ± 11.7% prior to 2000 and 85.7 ± 13.2% since 2000 (p=0.047). From data which are available, there are no reported pre-2000 cases of sibling HSCT where absence of iron is documented. Two cases of sibling HSCT pre-2000 had known significant iron overload. One of these patients is the only fatality amongst sibling donor HSCT pre-2000. Post-2000, there were 3 cases of sibling HSCT with significant iron overload and 7 cases of proven absence of iron burden (data on remaining patients are pending). In the alternative donor HSCT pool prior to 2000, 5 of 13 had proven pre-HSCT iron overload and 1 without (data on remaining 7 patients are pending). Since the year 2000, of the alternative donor HSCT, there were 2 proven cases with iron overload and 2 with proven absence of iron burden (data on 6 remaining patients are pending). Overall survival at 5 years for patients without evidence of iron overload is 53.3 ± 18.6% and for those with iron overload pre-transplant is 38.5 ± 23.3 (p=0.97). Conclusions The best outcomes for stem cell transplantation in DBA patients occur when using HLA-matched sibling donors in younger patients. There has been a significant improvement in survival with alternative donor SCT since the year 2000 which may be a result of improved HLA matching techniques. There is a trend to improved survival with lower pre-transplant iron burden. Disclosures No relevant conflicts of interest to declare.

Description: Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia with marked clinical heterogeneity, an increased risk of malignancy and mutations in ribosomal protein (RP) S19 in 25% of probands. To identify other gene(s) mutated in DBA and investigate their expression and function, we performed a genome-wide screen using a 10,000 single nucleotide polymorphism mapping set (Affymetrix) on a large family comprising 10 informative meioses. We found linkage of the DBA phenotype to regions on chromosome 8q, 10 and 6. The RP gene RPS24, is located in the linked region on chromosome 10; we sequenced exons, intron-exon boundaries and the promoter regions in this family, and found a nonsense mutation (316C〉T) in exon 4 of RPS24 in five affected individuals, and a wild type sequence in five unaffected family members. This mutation causes the change of Gln106STOP and is predicted to result in formation of a truncated RPS24 protein. Subsequently, we sequenced DNA from 215 unrelated DBA probands, 30 with RPS19 mutations and 185 without. We found another nonsense mutation in exon 2 in a sporadic case, and a splice site deletion resulting in skipped exon 2 in another proband and in his father; over 200 control individuals did not have any of the above sequence changes, indicating that they are pathogenic mutations. To explore the normal role of RPS24 and consider how its dysfunction might result in DBA we performed real time RT-PCR (rt-PCR) and western blotting experiments on 20 normal human tissues and on lymphoblastoid cell lines from diseased and control individuals. Interestingly, rt-PCR of total human RPS24 and RPS19 mRNA revealed a tissue-specific variation in expression level. We found co-ordinate expression of both genes in the majority of studied tissues. Lymphoblastoid cell lines from both probands with nonsense mutations showed a reduced level of RPS24 mRNA, suggesting degradation of mutated transcripts due to nonsense mediated decay, while the RPS19 mRNA level in these patients was normal or elevated. Western blot experiments revealed a reduction of RPS24 protein in lymphoblastoid cell lines from all three mutated probands compared to control samples. Interestingly, co-ordinate expression of RPS24 and RPS19 protein was found in these patients as well as in other patients with RPS19 mutations or without any mutations, suggesting co-regulation of RP expression. To determine whether recruitment of mRNA to polysomes was impaired in DBA patients, we separated lymphoblast cell line lysates from nine diseased and four control individuals on sucrose gradients. We did not detect any significant difference in the RNA ratio of polysome-bound/free ribosomal subunits between diseased and control samples (p

Description: Diamond Blackfan anemia (DBA) is a rare, congenital bone marrow failure syndrome characterized by severe macrocytic anemia, usually without perturbation of other hematopoietic lineages. DBA patients are generally diagnosed during infancy or early childhood, have a high frequency of congenital anomalies, and a predisposition to cancer. Approximately 65% of DBA patients have heterozygous mutations or deletions in ribosomal protein genes. Additionally, mutations in the GATA1 gene, which encodes the GATA1 erythroid transcription factor, have been demonstrated in two DBA patients (Sankaran VG et al. J Clin Invest. 122: 2439-43, 2012). The genetic cause of DBA in the remaining 35% of patients is unknown. Despite our knowledge of the genotypes, the mechanism underlying the erythroid failure in DBA is not completely understood. This is largely due to the inability to study primary erythroid cells from DBA patients. To begin to delineate the mechanisms regulating erythroid differentiation in DBA, we developed an in vitro culture system starting with CD34+ progenitor cells isolated from approximately 20 ml of DBA patient peripheral blood collected prior to transfusion at the DBA Registry of North America. Using this system, we have characterized DBA patients with mutations in large (RPL5) and small (RPS17) subunit ribosomal protein genes, one individual with a mutation in the GATA1 gene, and several patients with unknown mutations. At the end of the culture, we routinely obtained 6x107 CD235+ erythroid cells from an initial population of 5x104 control CD34+ progenitor cells. In contrast to control cells, cells from the DBA patients exhibited a significantly reduced growth rate and generated approximately 100-fold fewer CD235+ erythroid cells, with a two day delay in the acquisition of the CD235 marker. Using flow cytometry, we isolated populations of CD41-, CD44+, CD235+ erythroid cells from both control and patient cell cultures from which we extracted RNA. This allowed the first time comparison of mRNA expression in DBA erythroid cells. Protein coding and long non-coding RNA transcripts were compared using Affymetrix GeneChip Human Gene ST Arrays and RNASeq. Compared to controls, CD235+ cells from the patient with the RPL5 mutation showed decreased levels of the GAS5 (growth arrest) and NOP56 (large ribosomal subunit assembly) mRNAs, as well as small nucleolar RNAs. Ingenuity Pathway Analysis (IPA) identified the tRNA charging and RNA Polymerase II assembly pathways as significantly perturbed in this patient. The DBA-associated splice donor mutation in GATA1 results in the exclusive expression of the short form of the GATA1 protein (GATA1s), which lacks the transactivation domain. Western blot analysis of CD235+ control cells showed expression of both full length GATA1 and GATA1s, with the full length protein predominating. In the patient cells with the GATA1 mutation, only the GATA1s protein was expressed and exceeded the combined level of both GATA1 isoforms in the controls. Northern blot analysis demonstrated that the GATA1 mutation did not affect ribosomal RNA processing. The microarray and RNASeq expression profiles of the patient with the GATA1 mutation differed significantly from controls and from that of the patient with the RPL5 mutation. Many known GATA1 target genes including SLC4A1, AHSP, and TRIM10 were down regulated in the CD235+ erythroid cells of the patient with the GATA1mutation compared to control cells, clearly indicating that these genes depend on full length GATA1 for activation. IPA identified the heme biosynthesis pathway as significantly perturbed in this patient and GATA1 as the top regulator. In summary, we have shown that DBA patient cells show decreased proliferative and erythroid differentiation capabilities in vitro. RNA transcript analysis in DBA patient cells has revealed significant differences between DBA patients with a ribosomal protein gene mutation and a mutation in the GATA1 gene. These data delineate multiple signaling pathways or mechanisms involved in DBA and erythroid differentiation. Finally, we have demonstrated that many GATA1 target genes depend on full length GATA1 for activation. Disclosures No relevant conflicts of interest to declare.

Description: Background: Diamond Blackfan anemia (DBA) is a congenital anemia characterized by failure of adequate erythrocyte expansion from hematopoietic precursors. The genetic basis of DBA is largely established, with mutation or deletion of at least 19 structural ribosomal protein (RP) genes, a RP chaperone (TSR2), or a pivotal erythroid transcription factor (GATA1) identifiable in most DBA cases. However, the marked clinical variability in DBA-including varying ages of presentation, severity of anemia, responsiveness to corticosteroids, and sporadic hematologic remissions-remains unexplained by genotype and may be modulated by epigenetic factors. Further understanding of this variability is of potential therapeutic relevance for biomarkers of steroid response and remission as well as in application to novel treatment approaches. Aim: We characterized genome-wide methylation and chromatin accessibility of erythroid progenitors from normal controls and DBA patients during erythroid commitment in order to identify the epigenetic features associated with erythroid failure in DBA, steroid response, and remission. Methods: We expanded and sorted CD71+/CD235- (transferrin receptor/glycophorin A) and CD71+/CD235+ erythroid cell fractions from DBA patients and controls after isolation of primary circulating CD34+ cells from peripheral blood (O'Brien et al, Blood 129(23):3111, 2017). We performed DNA methylation analysis using Illumina Epic arrays in 9 control and 22 DBA subjects (11 transfusion-dependent, 6 steroid responsive, 5 remission), characterizing differentially methylated probes and regions among groups. To define broad chromatin domains, we identified A/B chromatin compartments (representing open/closed chromatin) using long-range correlations in methylation data as previously described (Fortin et al. Genome Biol 16:180, 2015). To identify discrete local changes in chromatin accessibility, we performed ATAC-sequencing in 9 controls and 17 DBA patients (10 transfusion, 6 steroid, 1 remission). Results: Global DNA methylation falls during erythroid commitment, with 258,618 differentially hypermethylated CpG sites in normal control GlyA- cells compared to their more differentiated GlyA+ counterparts. This pattern is exaggerated in DBA samples, with 297,926 sites hypermethylated in GlyA- cells. We identified 17,392 CpC sites that distinguish GlyA- DBA progenitors from normal progenitors (16,953 hyper- and 439 hypomethylated). We identified 1,749 differentially methylated sites in comparison of transfusion dependent and remission DBA, as well as 79 sites different between transfusion dependent and steroid responsive DBA. Using genome-wide methylation data, we evaluated A/B compartment organization among these groups to identify large regions of open and closed chromatin during normal and DBA early erythroid differentiation. We observe significant shifts in A/B compartments in normal cells concurrent with the acquisition of GlyA surface expression. At genome scale, transfusion dependent and steroid resistant DBA samples are generally similar to each other, with thousands of regions where A/B identity are closely matched in DBA, but diametrically opposed to the configuration in stage-matched normal controls. Intriguingly, remission samples generally matched A/B compartments of other DBA samples in GlyA- fractions but GlyA+ compartments more closely resemble those of the controls, indicating that normalization of chromatin structural maturation accompanies hematologic remission. We generated a uniform set of 8,877 enriched ATAC-seq peaks on autosomes for differential chromatin accessibility analysis. As with methylation data, a large proportion (25%; 1085 up and 1114 down, B-H adj. P 〈 0.1) showed differential accessibility in normal control GlyA- vs GlyA+ cells. Steroid-responsive cases showed additional regions of differential accessibility during early maturation, with 31% of regions (1515 up and 1248 down) differentially accessible. Among transfusion dependent DBA patients, this count was much higher, with over half of peak regions (52%, 2400 up and 2216 down, B-H adj. P 〈 0.1) showing differential accessibility. Conclusion: Epigenetic maturation is broadly altered in DBA erythroid progenitors compared to stage matched normal controls, with specific changes identifiable in patients responding to steroids and in remission. Disclosures No relevant conflicts of interest to declare.

Description: Background: RPL35A, a gene encoding a large ribosomal subunit protein located at the telomeric end of chromosome 3q (3q29-qter) is essential for rRNA processing, ribosomal biogenesis, cell proliferation, and apoptosis, and accounts for a subset of patients with Diamond Blackfan anemia (DBA). Reported pathogenic RPL35A mutations include single-nucleotide variants (SNVs), small insertion/deletions (indels), and large contiguous gene deletions associated with 3q29 microdeletion syndrome. 3q29 deletion syndrome is an overlapping syndrome that consists of developmental and intellectual disability with or without dysmorphic features, and other congenital anomalies but no anemia or cytopenia. The clinical phenotype and disease severity of patients with RPL35A-related DBA may be influenced by other genes deleted within 3q29 and be different in patients with large deletions compared to those with SNVs or small indels. Objectives: To determine whether DBA patients with large deletions of the 3q29 region have a more severe disease phenotype than those with SNVs or small indels in RPL35A, and whether other genes deleted within the 3q29 region might contribute to some of the features. Methods and Results: We identified 40 patients in a multi-institutional, international collaborative study of patients with DBA with RPL35A haploinsufficiency: 21 had deletion of RPL35A as part of 3q29 contiguous gene deletion, ranging in size from 0.012 Mb to 11 Mb in 16 patients; the extent of the deletion beyond RPL35A in either direction was unknown in 5 patients. Nineteen patients had SNVs or small indels (7 missense, 1 nonsense, 3 splice site, 6 indels and 2 unclear pathogenicity). Thirty-nine of 40 patients had severe anemia, 32 had neutropenia at some time and 3 had thrombocytopenia. Compared to the patients with SNVs or small indels, a significantly higher proportion of patients with RPL35A haploinsufficiency due to 3q29 deletion had steroid-resistant anemia (17 vs 7; p=0.009), severe chronic or intermittent neutropenia requiring treatment with G-CSF (7 vs 0; p=0.009), and humoral and/or cellular immunodeficiency (7 vs 1; p=0.046) diagnosed in some patients due to recurrent infections requiring hospitalizations (10 vs 2; p=0.03). Learning difficulties (12 vs 2; p=0.003), craniofacial abnormalities (11 vs 3; p=0.02), skeletal and limb defects (9 vs 2; p=0.03) or multiple physical anomalies (≥3) were also more frequent in patients with large deletions than in those with SNVs or indels (11 vs 3; p=0.02). Microcephaly (28%), short stature (33%), cardiac defects (28%) and/or urogenital abnormalities (23%) were equally distributed. The potential genes of interest near RPL35A that may be associated with immune dysregulation and/or neutropenia are RNF168, TFRC, PAK2, PIGZ, DLG1 and LMLN. One or more of these genes were deleted in at least 6 of 7 patients with neutropenia or immunodeficiency. Eight of 9 patients with malformations involving extremities, skeleton and ribs had deletions of TCTEX1D2 which is associated with rib/thoracic dysplasia and polydactyly. The genes of interest deleted in patients with developmental delay and learning disabilities included PAK2 and DLG1 in 9 patients, as well as RNF168, PPP1R2, TNK2 and q29 KIAA0226 in 8 of 12 patients in whom the extent of the deletion was known. Summary and Conclusion: Patients with DBA due to RPL35A contiguous gene deletions are clearly different from those with SNVs or small indels and have increased frequency of steroid-resistant transfusion-dependent anemia, severe neutropenia, immunodeficiency, learning/developmental delay, and craniofacial/skeletal/limb anomalies. Distinction of this subtype of DBA with RPL35A haploinsufficiency due to 3q29 contiguous gene deletion is relevant to their management, and evaluations should include a work-up for immunodeficiency. Further studies are needed to determine whether the complex phenotypes and severe disease manifestations in these patients are solely due to RPL35A haploinsufficiency or to the potential effect of other genes deleted in the 3q29 region. Genotype-phenotype characterization and comparison of DBA patients with 3q29 deletion with those due to deletions in other ribosomal protein genes may determine similarities or differences in disease phenotypes related to large gene deletions versus the influence of multigenic contiguous deletions. Supported by: AZV 16-32105A Disclosures Kattamis: Novartis: Consultancy, Honoraria; CELGENE: Consultancy, Honoraria; ApoPharma: Honoraria; Vifor Pharma: Consultancy. Niemeyer:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Description: Diamond Blackfan anemia (DBA) is a rare, inherited bone marrow failure syndrome characterized by anemia, congenital anomalies and a predisposition to cancer. The patients usually present during infancy or early childhood, but can also present in adulthood. In the majority of cases DBA is due to a mutation in a small or large ribosomal protein (RP) subunit leading to RP haploinsufficiency. The only treatments for the anemia of DBA are red cell transfusions (accompanied by iron chelation), oral corticosteroid therapy or stem cell transplantation. Pospisilova et al. (Haematologica 2007; 92(5):e66-67) reported one complete and two partial erythroid responses after the use of the branched chain amino acid L-leucine in 6 select patients. In skeletal muscle, leucine supplementation can upregulate components of the protein synthetic machinery, including ribosomal proteins, promoting protein translation. Mouse and fish models of DBA respond with amelioration of anemia to L-leucine. We therefore proposed to study the effect of L-leucine on transfusion dependence and growth in subjects with DBA. Methods: The primary objectives were to determine the feasibility of administering L-leucine in subjects with DBA who are red cell transfusion-dependent and to determine the efficacy of L-leucine to produce a hematologic and growth response. The secondary objective was to determine the safety profile of L-leucine. Twelve study sites were involved in this multi-center, Phase I/II study with an anticipated accrual of 50 subjects. A dose of 700 mg/M2 orally three times per day for 9 months was used. Inclusion criteria included age 〉 2 years, the diagnosis of DBA and transfusion dependence with adequate kidney and liver function. Response was evaluated at 9 months with Complete Response (CR) defined as no further transfusions required and Hb 〉9; Partial Response (PR): Hb 〈 9 gm/dL with an increase in reticulocyte count and transfusion interval; and No Response (NR): no change in transfusion needs, Hb or reticulocyte count . Growth percentiles were evaluated at baseline and at completion of 9 months of treatment and the growth velocity change was calculated. Results: The study opened July 2014 and closed February 2017; 55 subjects were consented; 12 were screen failures; 43 subjects were evaluable. There were 21 males; the median age was 9 years 1 month (2 years 5 months - 46 years 1 month). There were no untoward side effects experienced by any subject that were attributable to the L-leucine. Two subjects had an erythroid CR and 1 subject had a PR. The CRs occurred at 1 month and 3 months after start of L-leucine. The subject with PR had an elevated reticulocyte count but was not able to maintain a Hb 〉9 gm/dL without a transfusion and thus was not transfusion independent. Of the 30 subjects with growth potential who received L-leucine 10 experienced a positive growth velocity change at 9 months of therapy compared to baseline. At a median age of 7.5 years, the mean pre-leucine height percentile was 27 +/- 17.9 and the post-leucine height percentile was 35 +/- 19.9 (p