Patients Develop Bone Marrow Failure Research Articles

The Clinical and Hematologic Course of Fanconi Anemia Amongst Affected Siblings

Fanconi anemia is the most common inherited bone marrow failure syndrome characterized by impaired DNA repair pathway. Patients with Fanconi anemia often present with congenital anomalies and up to 70% of patients develop bone marrow failure (BMF) during the first decade of life, with high risk of progression to myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). While high penetrance of BMF phenotype is a key characteristic of Fanconi anemia, patients may have variable onset and severity. Despite extensive research, there are no available data whether affected siblings with Fanconi anemia have a similar or different clinical course.First, we reviewed medical records of 9 sets of probands and affected siblings with Fanconi anemia who were seen at the Memorial Sloan Kettering Cancer Center (MSKCC) in the last 30 years (Figure 1). As longitudinal and detailed clinical information was available for these sibling pairs, we collected data by retrospective chart review on the onset of BMF, MDS or AML, time to hematopoietic stem cell transplantation (HSCT), the presence of any congenital anomaly, microcephaly, renal anomaly, low birth weight, short stature, learning disability and frequent infections between older and younger siblings. After observing a signal of positive correlation in BMF onset between siblings, we moved onto reviewing additional sibling pairs in the International Fanconi Anemia Registry (IFAR), where additional 62 sibling pairs with available hematologic onset information were registered. IFAR defines hematologic onset as the onset of BMF, MDS or AML.The Spearman rank correlation coefficient was computed to assess correlation between sibling pair's clinical manifestation for those seen at MSKCC. We observed a positive correlation in the time to bone marrow failure, defined by development of any peripheral blood cytopenias, between sibling pairs, although the p value was borderline due to small sample size (correlation coefficient r=0.75, p=0.05, number of pairs = 7). We excluded two pairs of siblings in which one of the siblings was known to have blood mosaicism. The sample size was too small to compute statistics for the time to MDS/AML or time to HSCT. We also calculated the Cohen's kappa coefficient to determine whether a particular clinical event was likely to occur in both siblings or neither of siblings. We found that kidney anomaly (kappa coefficient = 0.5714; 95% CI 0.0982 - 1) and microcephaly (kappa coefficient = 1; 95% CI 1-1) showed good agreement between siblings, whereas we did not find good agreement between siblings in skeletal anomaly, cardiac anomaly, low birth weight, short stature, growth retardation, learning disability or frequent infections. To assess correlation of hematologic onset in all available sets of probands and affected siblings, the Spearman's rank correlation was calculated on total 69 sibling pairs. We found a positive correlation in the hematologic onset, defined as time to bone marrow failure, MDS or AML, whichever comes first (correlation coefficient r=0.6767, p <0.0001; Figure 2).In summary, affected siblings with Fanconi anemia tend to develop hematologic manifestations at similar ages with a median age difference of 2.18 years (range 0 - 15.36 years; 95% CI 2.61 - 4.17 years). Among other clinical manifestations, kidney anomaly and microcephaly appeared to occur concordantly between siblings. We plan to reach out to other institutions to enhance collaboration and increase number of research participants. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characterization and genotype-phenotype correlation of patients with Fanconi anemia in a multi-ethnic population.

Fanconi anemia (FA), an inherited bone marrow failure (BMF) syndrome, caused by mutations in DNA repair genes, is characterized by congenital anomalies, aplastic anemia, high risk of malignancies and extreme sensitivity to alkylating agents. We aimed to study the clinical presentation, molecular diagnosis and genotype-phenotype correlation among patients with FA from the Israeli inherited BMF registry. Overall, 111 patients of Arab (57%) and Jewish (43%) descent were followed for a median of 15 years (range: 0.1-49); 63% were offspring of consanguineous parents. One-hundred patients (90%) had at least one congenital anomaly; over 80% of the patients developed bone marrow failure; 53% underwent hematopoietic stem-cell transplantation; 33% of the patients developed cancer; no significant association was found between hematopoietic stem-cell transplant and solid tumor development. Nearly 95% of the patients tested had confirmed mutations in the Fanconi genes FANCA (67%), FANCC (13%), FANCG (14%), FANCJ (3%) and FANCD1 (2%), including twenty novel mutations. Patients with FANCA mutations developed cancer at a significantly older age compared to patients with mutations in other Fanconi genes (mean 18.5 and 5.2 years, respectively, P=0.001); however, the overall survival did not depend on the causative gene. We hereby describe a large national cohort of patients with FA, the vast majority genetically diagnosed. Our results suggest an older age for cancer development in patients with FANCA mutations and no increased incidence of solid tumors following hematopoietic stem-cell transplant. Further studies are needed to guide individual treatment and follow-up programs.

Characterization of Pathogenic Variants and Clinical Phenotypes in 117 Japanese Fanconi Anemia Patients

Objective: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome associated with multiple congenital abnormalities and predisposition to malignancies, resulting from mutations in one of the 22 known FA genes (FANCA to W). The proteins encoded by these genes participate in DNA repair pathway (the FA pathway) for endogenous aldehyde damage. Compared to the situation in the US or Europe, the number of Japanese FA patients with genetic diagnosis was relatively limited. In this study, we reveal the genetic subtyping and the characteristics of mutated FA genes in Japanese population and clarify the genotype-phenotype correlations.Results: We studied 117 Japanese FA patients from 103 families (1996 to 2018). The diagnosis of FA was confirmed on the basis of chromosomal breakage tests and clinical features. Molecular diagnosis was obtained in 107 (91.5%) of the 117 patients through direct sequencing of FANCA and FANCG, MLPA analysis for FANCA, targeted exome sequencing (targeted-seq), and whole exome sequencing (WES) analysis (Figure 1). To provide genetic subtyping for the 10 unclassified cases, we tried to apply various technologies. Array CGH revealed large deletions in two FA-B and one FA-T cases. Whole genome sequencing and RNA-sequencing analysis identified splicing site or aberrant splicing mutations among three cases (one FA-B, one FA-C, and one FA-N). Collectively, 113 (97%) of Japanese 117 FA patients were successfully subtyped and a total of 219 mutated alleles were identified. FA-A and FA-G accounted for the disease in 58% and 25% of FA patients, respectively, whereas each of the other complementation groups accounted for less than 5% of FA cases. FANCB was the third most common complementation group (n=4) and only one FA-C case was identified in Japanese FA patients.In the 68 FA-A patients, we identified 130 mutant alleles that included 55 different FANCA variants (17 nucleotide substitutions, 16 small deletions/insertions, 12 large deletions, 1 large duplication and 9 splice site mutation). FANCA c.2546delC was the most prevalent (41/130 alleles; 32%). In the 29 FA-G patients, 57 mutant alleles were identified and seven different FANCG variants were detected. FANCG c.307+1G>C and 1066C>T accounted for most of FANCG mutant alleles (49/57; 88%) in the Japanese FA-G patients. The three hotspot mutations (FANCA c.2546delC, FANCG c.307+1G>C and c.1066C>T) existed at low prevalence (0.04-0.1%) in the whole-genome reference panel of 3554 Japanese individuals (3.5KJPN, Tohoku Megabank). Consistent with the paucity of the FA-C patients as opposed to the previous report (Blood 2000), the FANCC IVS4+4A mutation was absent in the 3.5KJPN database.We were able to examine the hematological outcomes in a subset of our cases (52 FA-A and 23 FA-G). Interestingly, the FA-G patients developed bone marrow failure (BMF) at a significantly younger age than FA-A patients (median age at onset of BMF: 3.1 years vs 5 years). Furthermore, the patients with the FANCA c.2546delC mutation had an increased risk of developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), compared to FA-A patients without the mutation.In the rare complementation groups of FA, two FA-B cases with complete loss of FANCB gene and one FA-I patient with N-terminal premature termination codons revealed severe somatic abnormalities, consistent with VACTERL-H association. Two FANCD1 (BRCA2) patients and one FANCN (PALB2) patients did not experience bone marrow failure but developed early-onset malignancies (immature teratoma, T-lymphoblastic lymphoma, adenosquamous lung carcinoma, Wilms tumor).Conclusion: This is the largest series of subtyped Japanese FA patients to date and the results would be useful for future clinical management. To provide molecular diagnosis for FA in Japan, we suggest to start with PCR-direct sequencing of the three common mutations (FANCA c.2546delC, FANCG c.307+1G>C and FANCG c.1066C>T) along with MLPA assay for FANCA. These analyses would enable the identification of about 50% of the mutant alleles. For the rest of the cases, WES or targeted-seq analysis should be useful, however, large deletions and aberrant splicing need to be kept in mind. [Display omitted] DisclosuresTakaori-Kondo:Pfizer: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Janssen Pharmaceuticals: Honoraria.

Hyperactive Non-Canonical TGF-β Pathway Signaling in Fanconi Anemia Bone Marrow Stromal Cells Contributes to Growth Suppression

Introduction: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. FA patients develop bone marrow failure during the first decade of life due to attrition of hematopoietic stem cells (HSCs). FA is caused by autosomal recessive or X-linked mutations in one of nineteen FANC genes, the products of which cooperate in the FA/BRCA DNA repair pathway and regulate cellular resistance to genotoxic DNA cross-linking agents. Although its mechanism is unknown, bone marrow failure in FA may be the result, directly or indirectly, of hyperactivation of cell-autonomous or microenvironmental growth-suppressive pathways induced, in part, due to genotoxic stress. We have recently identified canonical transforming growth factor-β (TGF-β) pathway-mediated growth suppression of HSCs as a cause of bone marrow failure in FA (Zhang H et al, Cell Stem Cell, 2016). We have shown that TGF-β pathway inhibition rescues genotoxic stress, proliferation defects and engraftment defects of FA-deficient HSCs, and ameliorates bone marrow failure in FA mice. Previous studies have suggested that bone marrow stromal fibroblasts from human FA patients and FA pathway-deficient mouse models, like HSCs, are hypersensitive to genotoxic stress and have impaired growth. Here, we therefore investigated the possible suppressive function of the TGF-β pathway in bone marrow stromal cells derived from FA mice and patients with FA.Methods: We established primary stromal cell lines from bone marrow of FA-deficient mice (Fancd2-/- mice) or wild-type sibling control mice. Primary bone marrow stromal cultures were also established from FA patients or normal healthy donors. The stromal cells were characterized and evaluated for growth kinetics, mitomycin C (MMC) sensitivity, chromosome breakage, inflammatory signals and response to the TGF-β inhibitors. CRISPR/Cas-9 technology was used to knockdown specific genes in stromal cells.Results: As expected,the primary bone marrow stromal cells from Fancd2-/- mice exhibited classical FA phenotypes, including hypersensitivity to a DNA cross-linking agent, MMC, and increased MMC-induced chromosomal radials. Fancd2-/- stromal cells also demonstrated a growth defect characterized by an enrichment of cells in G1 and elevated p21 expression. Interestingly, the FA stromal cells derived from FA patients or from Fancd2-/- mice expressed constitutively elevated levels of phosphorylated (activated) ERK1/2 (pERK) compared to control cells. In order to determine whether the factor responsible for inducing ERK1/2 phosphorylation in the murine FA stromal cells was cell-intrinsic or cell-extrinsic, we examined the conditioned media from the stromal cells. Indeed the FA stromal cells secreted a high level of TGF-β cytokine responsible for increased pERK levels, and expressed a high level of secreted TGF-β mRNA. The high level of pERK indicated that the TGF-β non-canonical pathway was hyperactive in the FA stromal cells. Interestingly, CRISPR/Cas9-mediated knockdown of Tgfbr1 or inhibition of the TGF-β pathway by a treatment with a small molecule inhibitor of TGFβR1 or a neutralizing antibody against TGF-β in these cells reduced pERK levels, promoted DNA repair and rescued MMC sensitivity. In addition, a MEK inhibitor also significantly improved the clonogenic growth of Fancd2-/- stromal cells. However, CRISPR/Cas9-mediated knockdown of Smad3, a downstream target of the canonical TGF-β pathway, did not rescue the growth inhibition of FA stromal cells in MMC, further indicating that hyperactivation of the canonical pathway is less relevant to their growth defect. Collectively, these results demonstrated that the hyperactive TGF-β pathway increases phosphorylation of ERK1/2 in FA stromal cells through the non-canonical signaling pathway and impairs their growth after genotoxic stress.Conclusions: The primary FA bone marrow stromal cells exhibit hyperactive non-canonical TGF-β pathway signaling and blocking this pathway improves their growth under genotoxic stress. The TGF-β signaling pathway-mediated growth suppression in bone marrow stromal cells may account, at least in part, for defective microenvironment, impaired HSC function and bone marrow failure in FA. This work suggests that the TGF-β signaling pathway may be a potential therapeutic target for the treatment of bone marrow failure in FA. DisclosuresShimamura:TransCellular Therapeutics: Other: Husband is founder. No revenue to date.; Novartis: Other: In discussion regarding possible clinical trial for aplastic anemia; Glaxo Smith Kline: Honoraria.

TGF-β Pathway Inhibition Rescues the Function of Hematopoietic Stem and Progenitor Cells Derived from Patients with Fanconi Anemia

Introduction: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. FA patients develop bone marrow failure during the first decade of life, and frequently require an allogeneic or unrelated donor bone marrow transplant. FA patients also develop other hematologic manifestations, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) due to clonal evolution. FA is caused by biallelic mutation in one of eighteen FANC genes, the products of which cooperate in the FA/BRCA DNA repair pathway and regulate cellular resistance to DNA cross-linking agents. Bone marrow failure in FA is attributable to an impaired hematopoietic stem and progenitor cell (HSPC) pool. HSPCs in FA patients and FA mice exhibit reduced cell number and compromised stem cell function. Recent studies suggest that bone marrow failure in FA and impaired HSPC function result from the genotoxicity of endogenous cross-linking agents or from physiological stress. A greater understanding of the mechanisms of impairment of HSPC function could improve the therapeutic options for FA patients. Using a whole genome-wide shRNA screen, we have recently identified that the canonical transforming growth factor-β (TGF-β) pathway plays an important growth suppressive role in FA and targeting this pathway can reduce the genotoxic stress-induced growth inhibition of FA cells. Here, we investigated the possible suppressive function of the TGF-β pathway in HSPCs derived from patients with FA.Methods: We performed in vitro colony-forming assays using primary FA patient- derived bone marrow CD34+ cells which were either transduced with shRNA targeting SMAD3 or treated with the anti-human TGF-β neutralizing antibody GC1008. FA-like HSPCs were generated by stably knocking down FANCD2 with lentivirus encoded shRNA in primary human cord blood CD34+ cells. An in vivo engraftment assay was performed by transplanting the FA-like HSPCs into irradiated NSG mice.Results: The primary human FA bone marrow cells displayed elevated mRNA expression of multiple TGF-β pathway components. The TGF-β pathway inhibition, by knockdown of SMAD3 or anti-human TGF-β neutralizing antibody GC1008, rescued the in vitro clonogenic defects of primary CD34+ cells from bone marrow of five different FA patients. Similarly, the TGF-β pathway disruption by depletion of SMAD3 or GC1008 antibody in primary FA-like HSPCs, also rescued their clonogenic defect, and partially restored genotoxic stress-induced growth inhibition. Further, as the very low number of CD34+ cells in FA patients did not allow efficient xenograft assay to analyze in vivo clonogenicity, we performed a surrogate in vivo xenograft assay using FA-like primary CD34+ cells. Importantly, blockade of the TGF-β pathway by GC1008 antibody treatment enhanced the engraftment potential of primary FA-like CD34+ cells in vivo. Collectively, these results demonstrated that increased TGF-β pathway signaling impairs the hematopoietic function of primary human FA HSPCs.Conclusions: The TGF-β pathway signaling is increased in primary FA patient-derived hematopoietic cells and blockade of this pathway can restore the function of human FA-deficient primary HSPCs. The TGF-β signaling pathway-mediated growth suppression may account, at least in part, for bone marrow failure in FA. This work suggests that the TGF-β signaling pathway provides a novel therapeutic target for the treatment of bone marrow failure in FA. DisclosuresNo relevant conflicts of interest to declare.

Exhaustion in Myeloid Lineage and Very Early Defect in HSPC Pool: An Embryonic Origin of Fanconi Haematological Disorders

Fanconi anemia (FA) is a genetic disorder due to mutations in one of the sixteen FANC genes involved in DNA repair. Many FA patients develop bone marrow failure (BMF) during childhood, and FA strongly predisposes to myelodysplasia syndrome and/or acute myeloid leukaemia. The pathogenesis of the BMF remains uncompletely understood. Low hematopoietic progenitor cell (HPCs) counts observed early in life and preceeding the onset of blood cytopenia in patients led we, and other, to hypothesize that the hematopoietic development might be abnormal in the FA embryo. Indeed, unlike adult hematopoietic stem cells (HSCs) which are quiescent in the BM niche, during embryonic life HSCs are in active proliferation in sites of expansion such as fetal liver and placenta, where they get amplified and acquire properties of adult HSC .We hypothesized that in FA, the FA defect in response to the replicative stress could impair the expension of the HSC pool.In order to investigate this hypothesis, we carried out studies in Fancg-/- knock out mice and in human FA fetuses obtained with informed consent from medical abortion.In Fancg-/- mice, FACS analysis revealed a 1,5- to 3-fold deficiency in hematopoietic stem and progenitor cells (HSPC) very early during embryonic development (i.e 11.5 days of gestation - E11.5) in fetal liver (FL) and placenta (Pl) (p <0.001). In both organs, this defect persists during the whole period of amplification (until E14.5 for FL and E12.5 for Pl). In vitro clonogenic assays also demonstrated a 2- fold defect in granulocyte, erythrocyte and macrophage (GEM) progenitors both in Fancg-/- FL or Pl compared to WT (p <0.001), and 4 to 5- fold defect in more immature mixed GEM progenitors in FL (p <0.001). LTC-IC frequency of the HSC-enriched Lineage- Sca1+ AA4.1+ population (LSA) of E14.5 Fancg-/- FL comforted this later result, since it was 5-fold lower than for WT.In vivo long-term hematopoietic reconstitution (LTR) assays confirmed a deficit of the HSC enriched LSA population of E14.5 Fancg-/- FL. Indeed, although the percentage of mice reconstituted was as good as that obtained with the same number of WT LSA, the CD45 Ly5.2 chimerism was reduced (49±20% vs 84±4% for 1000 LSA injected, and 56±12% vs 87±2% for 5000 LSA). Interestingly, bone marrow analysis of mice reconstituted with Fancg-/- LSA 22 weeks after injection showed a level of CD45 Ly5.2 chimerism 3-fold lower than that found in blood, spleen and thymus, as well as a very low chimerism for myeloid GEM lineages, contrasting with a high chimerism for B and T lymphoid lineages. Moreover, we were able to demonstrate that this deficit is already present at E12.5, both in Fancg-/- FL and Pl. Indeed, no mice reconstituted with 3.105 total Fancg-/- fetal liver cells, while 100% injected with the same number of WT FL cells got reconstituted with a chimerism of 59,5±5%. For Pl, when 500 000 cells were injected, reconstitution was observed in only 1 out of 3 mice for Fancg-/- (29% chimerism), and in 3 out of 3 mice for WT (88±4% chimerism).In human FA FL of 14 weeks of gestation, we also observed a 4-fold defect of HSPC with a total lack of in vitro amplification compared to control, in agreement with the mice data.Taken together, these data demonstrate that a profound deficit of HSCs and progenitors cells is present since the earlier stages of embryonic development in FA. In addition, using organotypic cultures of E11 aortas, we could show that this defect of amplification is already present in HSCs emerging from Fancg-/- aorta, which showed a 2-fold lower rate of amplification compared to WT. More importantly, our results show for the first time exhaustion in myeloid lineage of FA, in agreement with what is observed in children with FA disease. Altogether, our work suggests a role of the FA pathway during the development of the hematopoietic system leading to a deficit of amplification of HSC. Comparison of FA HSC transcriptome with that of control HSC in FL and Pl is in progress. It should allow to identify the key pathways involved in the embryonic HSC amplification that are deregulated in FA, and hopefully getting more insights in the pathogenesis of the BMF and leukemogenesis in FA patients. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Failure in Fanconi Anemia from Hyperactive TGF-β Signaling

Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. FA patients develop bone marrow failure during the first decade of life due to attrition of hematopoietic stem and progenitor cells (HSPCs). FA patients also develop other hematologic manifestations, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) due to clonal evolution. FA is caused by biallelic mutants in one of sixteen FANC genes, the products of which cooperate in the FA/BRCA DNA repair pathway and regulate cellular resistance to DNA cross-linking agents. Bone marrow failure in FA may result, directly or indirectly, from hyperactivation of cell autonomous or microenvironmental growth suppressive pathways induced due to genotoxic stress. Recent studies suggest that one suppressive pathway may be the hyperactive p53 response observed in HSPCs from FA patients.In order to further identify suppressive mechanisms accounting for bone marrow failure in FA, we performed a whole genome-wide shRNA screen in FA cells. Specifically, we screened for candidate genes whose knockdown would rescue cellular growth inhibition and genotoxic stress induced by a DNA cross-linking agent mitomycin C (MMC). We transduced a FA-deficient human fibroblast line with pools of shRNAs and screened for rescue of MMC-inhibited growth. Selected shRNA inserts were identified by next generation sequencing.The top hits in the screen were shRNAs directed against multiple components of the TGF-β signaling pathway. Consistent with this, disruption of the TGF-β signaling pathway by shRNA/sgRNA-mediated knockdown of SMAD3 or TGFR1 (downstream components of the TGF- β pathway) rescued growth of multiple cell lines from several FA complementation groups in presence of genotoxic agents (e.g. MMC or acetaldehyde). Pharmacologic inhibition of the TGF- β pathway using small molecule inhibitors resulted in improved survival of FA-deficient lymphoblast cells in presence of MMC or acetaldehyde, suggesting that a hyperactive, TGF-β-mediated, suppression pathway may account, at least in part, for reduced FA cell growth. Interestingly, genes encoding TGF-β pathway signaling components were highly expressed in the bone marrow from FA patients and FA mice. Moreover, disruption of the TGF- β pathway by shRNA-mediated knockdown of SMAD3 rescued the growth defects of primary HSPCs from FA-deficient murine bone marrow. To further implicate the TGF-β pathway, we established primary stromal cell lines from the bone marrow of FA-deficient mice as well as human FA patients. We confirmed that TGF-β signaling was hyperactive in these stroma cells resulting in growth suppression and elevated phospho-ERK levels due to non-canonical signaling of the pathway. Inhibitors of TGF-β signaling partially rescued the growth defects and reduced phospho-ERK levels in these FA stroma cells.The deficiency of FA DNA repair pathway leads to cellular defects in homologous recombination (HR) repair and hyperactivation of toxic non-homologous end joining (NHEJ)-mediated repair. We therefore tested whether inhibition of the TGF-β pathway in FA cells could rescue HR defects and account for the improvement of FA cellular growth. Interestingly, disruption of the TGF-β signaling pathway caused a decrease in NHEJ activity. Disruption of the TGF-β pathway also resulted in reduced MMC-mediated DNA damage and increased HR.Taken together, our results demonstrate that primary FA hematopoietic and bone marrow stromal cells exhibit hyperactive TGF-β signaling accounting at least in part for the bone marrow failure in FA. Inhibitors of the TGF-β signaling pathway may therefore be useful in the clinical treatment of patients with bone marrow failure and Fanconi anemia. DisclosuresNo relevant conflicts of interest to declare.

Kinase Inhibitors Reduce TNF-Alpha Over-Production in Monocytes From Fanconi Anemia Group A Patients

Abstract 2409Fanconi Anemia (FA) is a chromosomal instability syndrome with hypersensitivity to alkylating agents as principal diagnostic feature. Many laboratories have demonstrated the involvement of FA proteins in DNA repair mechanisms. Recent work has also demonstrated other functions of some FA proteins suggesting that they play alternative roles in other regulatory pathways, particularly those that influence hematopoiesis.Eighty percent of FA patients develop bone marrow failure with a high incidence of evolution in myelodysplasia and/or acute leukemia. Each of these abnormalities has been related to TNF-alpha hypersensitivity in the stem and progenitor cell pools and the toll-like receptor dependent overproduction of TNF-alpha by FA macrophages. The TNF-hypersensitive phenotype involves at least two kinases, PKR and ASK1, each of which is hyperactivated in FA cells and induce apoptotic responses both in ground state and after TNF-alpha and IFN-gamma stimulation. Recent evidence showed that R848 (a TLR8 ligand) and endotoxin (LPS, a TLR4 ligand)-induced TNF-alpha gene expression in Fancc-deficient mononuclear phagocytes cells is inhibited by kinase inhibitors dasatinib and BIRB796 We sought to evaluate the activity of these agents in primary mononuclear phagocytes obtained from children with FA-A. Objectives:To determine whether primary monocytes from the peripheral blood of FANCA-deficient patients: (a) exhibit the TNF-overproduction phenotype in response to LPS and the TLR8 ligand R848, and (b) respond to dasatinib and BIRB796 by suppressing TNF-production. Methods:Six FA patients with mild to severe marrow failure on no treatment were included in this study. Healthy subjects were recruited as normal controls that were run in parallel in each case. CD14+ monocytes freshly isolated from peripheral blood were cultured for 24 hours with LPS and R848 with or without dasatinib or BIRB796. Supernatant media were collected and frozen at −80 degrees. After thawing the samples, TNF-alpha content was quantified by ELISA. Results:Baseline TNF-alpha concentration (without any TLR stimulation) was higher in FA patients than control. After LPS or R848 stimulation FA-A monocytes produced substantially more TNF-alpha than did the control samples. Both dasatinib and BIRB796 suppressed TLR-induced (both LPS and R848) TNF-alpha production. Specifically, with R848 as the agonist, BIRB 769 suppressed TNF-alpha production by 60% and dasatinib by 42%. Both inhibitors were even more potent in suppressing LPS induced TNF-alpha expression as both reduced TNF-alpha by 75%. In the absence of TLR stimulation, the presence of BIRB or dasatinib in culture reduced TNF-alpha by >50% compared to baseline in patient samples. The inhibitory effect of kinase inhibitors was observed also in the normal control. Conclusions:These findings: (a) demonstrate for the first time that TLR-induced TNF-alpha gene expression in primary FANCA deficient mononuclear phagocytes is aberrantly regulated and (b) that in FANCA-deficient macrophages the TNF-alpha overproduction phenotype can be controlled by therapeutically achievable doses of BIRB796 and dasatinib. In addition (c), since both agents function in large part to suppress p38 MAPK activation future, our data point to the biochemical roles played by FANCA in modulating upstream pathways that govern p38 activation. Moreover (d), given that in FA patients, TNF hypersensitive stem cells are over-exposed to TNF-alpha, particularly during inflammatory events and that exposure to TNF was shown not only to suppress hematopoiesis in FA but also to favor the emergence of neoplastic clones, these results point to these two agents as potential candidates for preclinical trials seeking to enhance hematopoiesis and suppress clonal evolution. Disclosures:No relevant conflicts of interest to declare.

Correction of the Fanconi Anemia Phenotype at the Cellular Level by S Phase Delay: Implications for Etiopathogenesis and Treatment

AbstractAbstract 879Fanconi Anemia (FA) is a rare, complex, recessively inherited disease, with 13 known complementation groups, in which many patients develop bone marrow failure and/or leukemia, especially myelogenous leukemia, at an early age. Although bone marrow transplantation (BMT) has been beneficial, a high proportion of these patients go on to develop head and neck cancers and other solid malignancies, regardless of whether BMT has been carried out. Our laboratory, and others, have shown that FA is associated with a cell cycle defect in which FA cells fail to slow or arrest their rate of replicative, S-phase DNA synthesis as do normal cells following treatment with a DNA interstrand cross-linking (ICL) agent. FA cells are also markedly hypersensitive to the clastogenic and cytotoxic effects of such ICL agents, characteristics used to define the disease in the classic diepoxybutane, or DEB test. We wished to test whether, if the cell cycle defect were corrected by treating the cells with an inhibitor of DNA synthesis, the cytoclastic and cytotoxic effects of an ICL agent, psoralen plus ultraviolet A light (PUVA), would be affected in FA cells. Among DNA synthesis inhibitors, we were particularly interested in examining hydroxyurea and 5-fluorouracil (HU and 5-FU), because they have been used successfully to treat other blood diseases and cancers, respectively, such as sickle cell anemia and colon adenocarcinomas. Following treatment with PUVA, normal lymphoblastoid cells (two cell lines), FA lymphoblastoid cells (two FA-A, one FA-C and one FA-G lines), and two genetically corrected FA lymphoblastoid cell lines (FA-A and FA-C) were either mock treated or treated with an inhibitor of DNA synthesis (high dose thymidine, methotrexate, or HU) for 24 hours. Chromosome breaks and both short term (trypan blue exclusion) and long term cell viability (colony forming ability) were then measured. Except for one FA-A cell line that had low thymidine kinase activity, and therefore did not respond to thymidine, all uncorrected FA cell lines, but not corrected FA cell lines, showed dramatic reductions in clastogenicity and increases in viability following PUVA and treatment with any of the DNA synthesis inhibitors. Normal cells and corrected FA cells failed to show a comparable response.These results indicate that the S-phase cell cycle defect in FA is important in its etiopathogenesis. The stalled DNA replication forks documented in FA cells containing DNA ICLs may actually be due, in part or entirely, to a prior defect in cell cycle regulation when damaged FA cells enter S phase. These results also suggest at least two possible modes of therapeutic intervention, treatment of FA patients with HU or 5-FU to prevent or delay onset of complications including bone marrow failure, leukemia, or, particularly in patients who have undergone BMT, head, neck, and other tumors. Disclosures:No relevant conflicts of interest to declare.

Therapy Tolerance in Selected Patients With Androgen-Independent Prostate Cancer Following Strontium-89 Combined With Chemotherapy

Clinicians may have reservations about using strontium-89 for the treatment of bone metastases because of concerns that it may limit future use of chemotherapy. We assessed the rate of bone marrow failure in patients with prostate cancer who had received a dose of strontium-89. This subgroup analysis involved 34 patients with androgen-independent prostate cancer who had been given a dose of strontium-89 and six weekly doses of doxorubicin after response to induction chemotherapy. We assessed subsequent hematotoxicity in terms of bone marrow failure and the ability to tolerate additional treatments during a median of 25 months (range, 7 to 76 months) after the strontium-89 was administered. No patients developed bone marrow failure within 6 months of receiving strontium-89. Five (15%) of 34 patients developed bone marrow failure at a median 23 months (range, 6 to 53 months) after the strontium-89 treatment. Bone marrow biopsy performed in two of these five patients showed complete replacement of the marrow by tumor. Thirty-one patients (91%) received subsequent cytotoxic treatments at a median 11 months (range, 1 to 33 months) after the strontium-89 treatment. This analysis demonstrated that a single dose of strontium-89 combined with chemotherapy did not affect the delivery of subsequent courses of chemotherapy in a select group of patients. However, a majority of these therapies were given off protocol and were administered at a dose schedule that might be considered inappropriate or inadequate. The clinical role and safety profile of radiopharmaceuticals combined with chemotherapy in prostate cancer therapy deserve further exploration.

Molecular diagnosis of Fanconi anemia and dyskeratosis congenita.

AbstractThe inherited bone marrow (BM) failure syndromes Fanconi anemia (1) and dyskeratosis congenita (2) are genetic disorders in which patients develop BM failure at a high frequency, usually in association with a number of somatic abnormalities. They are the best characterized and the most common of this group of disorders.KeywordsFanconi AnemiaDyskeratosis CongenitaMyelin Protein ZeroMultiplex PCRsFanconi Anemia PatientThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The Inherited Bone Marrow Failure Syndromes: Fanconi Anemia, Dyskeratosis Congenita and Diamond‐Blackfan Anemia

The inherited bone marrow (BM) failure syndromes, Fanconi anemia (FA), dyskeratosis congenita (DC) and Diamond–Blackfan anemia (DBA), are genetic disorders in which patients develop BM failure at a high frequency, usually in association with a number of somatic abnormalities. The recent identification of four FA genes (FANCA, FANCC, FANCF, FANCG), one DC gene (DKC1) and one DBA gene (RPS19) has confirmed their genetic heterogeneity and has provided new methods of diagnosis; this is particularly useful where clinical presentation is atypical, as in the Hoyeraal–Hreidarsson syndrome, a severe variant of X‐linked DC. Recent data suggest that the FA proteins function in a novel cell pathway which has an important role in maintaining genomic stability; the DKC1 encoded nucleolar protein, dyskerin, is predicted to have an important role in ribosomal RNA (rRNA) processing and the RPS19 protein is a structural ribosomal protein. These syndromes therefore provide important information about novel cell pathways which may lead to a better understanding of normal hematopoiesis and of the poorly understood idiopathic aplastic anemia (AA). In turn, this may lead to new treatments, not only for FA, DC and DBA, but also for some types of idiopathic AA.