ALBERT

Description: Dyskeratosis congenita is a cancer-prone inherited bone marrow failure syndrome caused by telomere dysfunction. A mouse model recently suggested that p53 regulates telomere metabolism, but the clinical relevance of this finding remained uncertain. Here, a germline missense mutation of MDM4, a negative regulator of p53, was found in a family with features suggestive of dyskeratosis congenita, e.g., bone marrow hypocellularity, short telomeres, tongue squamous cell carcinoma, and acute myeloid leukemia. Using a mouse model, we show that this mutation (p.T454M) leads to increased p53 activity, decreased telomere length, and bone marrow failure. Variations in p53 activity markedly altered the phenotype of Mdm4 mutant mice, suggesting an explanation for the variable expressivity of disease symptoms in the family. Our data indicate that a germline activation of the p53 pathway may cause telomere dysfunction and point to polymorphisms affecting this pathway as potential genetic modifiers of telomere biology and bone marrow function.

Description: Background:Reduced fertility and increased fetal and maternal complications during pregnancy have been reported in patients with Fanconi anemia, Diamond-Blackfan anemia and Shwachman-Diamond syndrome. Such high-risk pregnancies benefit from coordinated care by hematologists and maternal-fetal medicine specialists with expertise in inherited bone marrow failure syndromes (IBMFS). There are no data on fertility and pregnancy outcomes in women with dyskeratosis congenita (DC). Objectives: To determine pubertal development, fertility, and pregnancy outcomes in women with DC. Methods: Prospective evaluation and medical record review was performed of women with DC ≥10 years of age enrolled in the NCI IBMFS cohort study. We examined ages at menarche and menopause, details regarding fertility and pregnancy, hematologic and non-hematologic complications of pregnancy, and maternal and fetal outcomes. Results: We evaluated 27 women with DC (median age 31 years, range 10-63), who all attained menarche (median age 12 years, range 9-17). The median age at natural menopause was 51 years, range 50-52. Seventeen women had 46 pregnancies (median 2 pregnancies per person, range 1-6); 1 was unable to conceive, 3 were using contraceptives and 6 were not yet sexually active. Thirty-two of 46 pregnancies (70%) in 17 women who carried to 27-42 weeks gestation (median 39 weeks) resulted in 34 live births (2 sets of twins). Thirty pregnancies (65%) in 11 women were associated with a variety of complications: 13 ended in miscarriages; 1 was an elective abortion. Maternal complications were preeclampsia (n=3), placenta previa with abruption (n=2 pregnancies in the same patient) and cesarean section for failure to progress (n=5; 4 were in 1 patient). Fetal complications were 5 preterm births (3 associated with preeclampsia) and 2 others had fetal distress. Six women had cytopenia during 8 pregnancies: in 2, mild cytopenia worsened during pregnancy but did not need treatment. One progressed to severe aplastic anemia (SAA) and 1 with SAA was transfusion-dependent throughout. Two patients had normal counts at the start of pregnancy, but developed cytopenia with preeclampsia or abruption with placenta previa, respectively. Three others with normal blood counts had macrocytosis. One was post-transplant. Four had only non-hematologic manifestations of DC. And, 3 women, with no DC-associated clinical features, were identified after diagnosis of an affected offspring. Fourteen of 32 pregnancies were cesarean deliveries performed in 8 patients for maternal complications (n=7), fetal distress (n=2) or failure to progress (n=5). Three of the 18 vaginal deliveries were in 2 mothers with mild cytopenia, and 1 preterm birth in a patient with SAA. Only 14/46 pregnancies (30%) in 6 women were uncomplicated, term vaginal deliveries; of these, 2 occurred in 1 post-hematopoietic stem cell transplant recipient, conceived by in vitro fertilization. There were 8 patients with TERC mutations, 3 TERT, 2 TINF2, 2 RTEL1and 2 were gene unknown. Fetal loss or maternal complication did not differ significantly in relation to mutated genes in mothers. Conclusions: This is the first study of fertility and pregnancy outcomes in patients with DC. We show that females with DC attain menarche and menopause at normal ages and have normal fertility. However, women with DC appear to have high rates of maternal and fetal complications resulting in miscarriage, preeclampsia, and worsening cytopenias. These complications may lead to increased likelihood of cesarean section and/or preterm delivery. Thus, similar to Fanconi anemia and Diamond-Blackfan anemia, pregnant women with DC are at high-risk for complications and should be managed by a hematologic and high risk maternal-fetal medicine team. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2361 Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome (IBMFS) classically diagnosed by the triad of dysplastic nails, reticular skin pigmentation and oral leukoplakia. Patients with DC are at very high risk of bone marrow failure (BMF), cancer, pulmonary fibrosis, and other medical problems. Germline mutations in key telomere biology genes cause DC (DKC1, TERC, TERT, TINF2, NOP10, NHP2, WRAP53, or CTC1), although about 50% of patients lack a known mutation. Leukocyte telomere lengths

Description: Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome. The spectrum of cancer susceptibility in this disorder of telomere biology has not been described. There were more than 500 cases of DC reported in the literature from 1910 to 2008; the National Cancer Institute (NCI) prospective DC cohort enrolled 50 cases from 2002 to 2007. Sixty cancers were reported in 52 literature cases, while 7 occurred among patients in the NCI DC cohort. The 2 cohorts were comparable in their median overall survival (42 years) and cumulative incidence of cancer (40%-50% by age 50 years). The most frequent solid tumors were head and neck squamous cell carcinomas (40% of patients in either cohort), followed by skin and anorectal cancer. The ratio of observed to expected cancers (O/E ratio) in the NCI cohort was 11-fold compared with the general population (P 〈 .05). Significantly elevated O/E ratios were 1154 for tongue cancer and 195 for acute myeloid leukemia. Survival after bone marrow transplantation for aplastic anemia or leukemia was poor in both cohorts. The frequency and types of cancer in DC are surpassed only by those in Fanconi anemia (FA), indicating that FA and DC have similarly high risks of adverse hematologic and neoplastic events, and patients with these diseases should be counseled and monitored similarly.

Description: Abstract 4213 INTRODUCTION Fanconi Anemia (FA) is a primarily autosomal recessive disorder with a defective DNA repair pathway associated with mutations in any of 13 genes. The majority of patients reported in the literature have one or multiple congenital anomalies, including low birth weight, short stature, café au lait spots, abnormal radii and/or thumbs, structural renal abnormalities, microcephaly, and deafness, among others. About 25% of reported patients had few or none of these findings. The hazard of severe aplastic anemia peaks at 10 years of age, and patients have very high risks of acute myeloid leukemia and specific solid tumors, such as head and neck and gynecologic squamous cell carcinomas. Only six patients have been reported who were diagnosed between 40 and 50 years of age. However, patients may present as adults with neoplasms, or remain asymptomatic and undiagnosed. METHODS We report the diagnosis of FA in the oldest known patient, an asymptomatic 55 year old woman (Case 2), identified only because she was a potential stem cell donor for her 42 year old sister (Case 1) with severe aplastic anemia. RESULTS Case 1 had a history of thrombocytopenia at 26, and anemia and thrombocytopenia during pregnancy; physical exam was normal except for a slightly deformed thumb; blood lymphocyte chromosome aberrations were increased with both diepoxybutane (DEB) and mitomycin C (MMC). The patient died following an HLA-matched bone marrow transplant from a non-FA brother. Case 2, the other of two siblings of Case 1 who were HLA matches, had higher than normal chromosome breakage in blood (3 cells with multiple radials with MMC) but not in the FA range; skin fibroblasts were diagnostic of FA, confirming hematopoietic somatic mosaicism. She had a normal appearance, a history of hypothyroidism and mitral valve prolapse, five pregnancies with five children (one miscarriage, one set of twins), a near normal blood count (Hb 13.3 g/dl, MCV 99 fl, WBC 3500/ul, and platelets 139,000/ul), and was a regular blood donor. Bone marrow cellularity and morphology were normal, but cytogenetics showed a small clone (46,XX,add(11)(q23)[6]/46,XX[14]). Complementation analysis of Case 1 indicated group A (FA-A), and molecular analysis identified two mutations in the FANCA gene. One mutation, p.S1208S (c.3624C〉T) was a splice site mutation occurring in exon 36 and has been previously described. The second mutation was a novel nonsense mutation in exon 23, p.S674X (c.2021C〉A). Five siblings had normal breakage results; four were heterozygous for the nonsense mutation and one was negative for both mutations. Case 2, with mosaicism for FA, had the familial splice mutation in both blood and fibroblasts, and the familial nonsense mutation in fibroblasts, but was skewed heavily toward wild-type in blood. cDNA studies confirmed that the predicted splice mutation created an alternate splice site resulting in multiple transcripts, including exon skipping, which varied in different tissues. The molecular mechanism for the loss of the nonsense mutation in the blood is most likely due to back mutation at a hot spot, which occurred in a hematopoietic stem cell which then had a selective growth advantage. CONCLUSIONS Reversion of one FANCA mutation probably occurred in a hematopoietic stem cell which was selected for and repopulated the peripheral blood. A plausible explanation for the lack of FA clinical features is the leaky splice mutation which may provide sufficient levels of protein for normal function in DNA repair. Family members should be tested for FA by chromosome breakage analysis in blood (and/or fibroblasts to identify those who may be mosaics). Those who have FA are at risk of syndrome-specific solid tumors, as well as aplastic anemia, myelodysplastic syndrome, and leukemia, if a non-gene corrected hematopoietic stem cell were to emerge. Even if asymptomatic, they should not be used as stem cell transplant donors for siblings with FA, because they may fail to repopulate the recipient marrow. FA is undoubtedly underdiagnosed in adults at this time. Disclosures: No relevant conflicts of interest to declare.

Description: It is now recognized that Fanconi anemia (FA) is both an inherited bone marrow failure syndrome (IBMFS) and also a highly penetrant cancer susceptibility syndrome associated with leukemias and specific solid tumors. However, the spectrum of cancer susceptibility in other IBMFS remains unclear. The National Cancer Institute (NCI) IBMFS Cohort is the first prospective/retrospective study to follow patients with diverse IBMFS using a comprehensive and unified protocol. During 2002–2007, 66 patients with FA, 55 with Dyskeratosis Congenita (DC), 63 with Diamond-Blackfan anemia (DBA), and 16 with Shwachman-Diamond syndrome (SDS) enrolled in the study and contributed a combined total of 4113 person-years of follow-up. Adverse outcomes ascertained for all patients included bone marrow failure (BMF) leading to death or bone marrow transplant, acute leukemia (AL), myelodysplastic syndrome (MDS), and development of a solid tumor (ST). For each syndrome, we calculated the ratio of observed (O) numbers of cancers versus expected (E) numbers in a demographically matched cohort from the general United States population (O/E ratio). We also calculated cause-specific hazards and cumulative incidence by age of BMF, AL, and ST. For FA, the first adverse event was BMF in 25 patients, AL in 4, and ST in 11; 8 developed MDS. The FA experience was broadly consistent with published reports from our prior North American Survey (NAS) and an independent FA cohort in Germany. In the NCI FA cohort, the O/E ratio was 37 for all ST (versus 48 and 26 respectively in previously analyzed cohorts) and 311 for AL (versus 785 and 868); these increased risks compared with the general population were statistically significant. The O/E ratio for MDS was 4910 (also significant). In time-dependent analysis, the MDS hazard was stable at 0.6%/year. For SDS, no patient has yet developed an adverse event. For DBA, the first adverse event was BMF in 4 and ST in 3 (1 colon and 2 lung cancers); no DBA patient has yet developed AL or MDS. The cumulative incidence of BMF in DBA was 10% by age 30 years. For DC, the first adverse event was BMF in 15, AML in 2, and ST in 5; 5 developed MDS. For DC, the O/E ratio was 10 for all cancers, 7 for all solid tumors, 897 for tongue cancer, and 188 for AL; these increased risks were statistically significant. The O/E ratio was also significantly elevated for MDS (2362). To gain statistical power, we compared DC patients to a pooled cohort of 458 FA from 4 cohorts (NAS, Germany, Israel, and NCI). For DC, the cumulative incidence by age 50 years was 50% for BMF, 10% for AL, and 22% for ST, broadly similar to corresponding values of 56%, 13%, and 30%, respectively, in the pooled FA cohorts. In DC, the cause-specific hazards of ST and AL increased significantly with age, but had a later rise than in FA. The hazard of BMF in DC also increased significantly with age, but lacked the early hazard peak seen in FA. This initial analysis from the NCI IBMFS cohort reveals that as for FA, DC is a highly penetrant bone marrow failure syndrome and a major cancer susceptibility syndrome with numerous events occurring in young adulthood and early middle age. This is the first study to quantify the risk in DC in this way. Continued follow-up of the cohort should clarify the spectrum of cancer susceptibility in each syndrome.

Description: Fanconi anemia (FA) and dyskeratosis congenita (DC) are characterized by bone marrow failure (BMF) and an increased risk of acute myeloid leukemia, myelodysplastic syndrome and epithelial cancers. While cerebellar hypoplasia has been reported in some patients with DC, and small pituitary size and midline brain anomalies in FA, systematic central nervous system (CNS) magnetic resonance imaging (MRI) and correlation with clinical phenotype and aplastic anemia (AA) are lacking. To investigate these associations we evaluated patients with DC and FA enrolled in the National Cancer Institute’s Inherited Bone Marrow Failure Syndrome study. DC: 16 patients, ages 1–23 yrs (median 9 yrs), had CNS MRI as a part of their evaluation. All had peripheral cytopenias; 11 with severe AA were on treatment with androgens or transfusions. 13/16 had at least 2 of the 3 features of the diagnostic triad (dystrophic nails, oral leukoplakia and reticulated skin pigmentation). Among 10 patients with microcephaly, 8 had developmental delay, 7 truncal ataxia and 6 had a severe phenotype with the Hoyeraal-Hreidarsson variant. 7/16 patients had moderate to severe cerebellar hypoplasia. Patients with cerebellar hypoplasia were significantly younger than those without (median age 6 yrs vs. 14 yrs respectively; p=0.01). All patients with cerebellar hypoplasia had truncal ataxia, developmental delay and microcephaly, whereas amongst those without hypoplasia 3 had microcephaly (p=0.01), none had ataxia and one had developmental delay (p=0.001). There was no correlation between the severity of AA and cerebellar hypoplasia (p=0.5). FA: 11 patients, ages 6–42 yrs (median 22 yrs) who were not on current treatment and 13 age and sex-matched controls underwent CNS MRI. 9 patients had cytopenias; 5 with severe AA had received prior treatment (3 bone marrow transplant, 2 transfusions). Pituitary height, width and area in patients with FA were compared with the NIH controls and pituitary heights were compared with published normal values. The mean pituitary height in the FA patients tended to be lower then the NIH or published controls (p=0.06 and p=0.09 respectively). However, the mean pituitary width and the mean pituitary area were significantly lower in patients with FA compared with the NIH controls (p=0.003). In all, 7/11 patients had small pituitary glands; one of these had pituitary stalk interruption and another had an absent septum pellucidum. 7 patients had one or more endocrine abnormalities: 4 growth hormone deficiency, 6 hypothyroidism, 7 hypogenitalia/hypogonadism and 6 short stature. An equal proportion of patients with a small pituitary had short stature, hypothyroidism and growth hormone deficiency when compared with those who had a normal pituitary gland (p=1.0). There was no correlation between small pituitary size and age (p=0.2), any endocrine abnormality (p=0.5) or severe AA (p=0.6). CNS abnormalities were detected by MRI in ∼ 50% of patients with DC or FA. In an age-dependent analysis, the cumulative incidence of AA was the same in those with or without cerebellar hypoplasia in DC and in those with or without a small pituitary in FA. We currently recommend that all patients with DC and FA undergo MRI of the brain and pituitary to more completely characterize the spectrum of CNS anomalies, and to determine whether there is an association with hematologic severity in a larger data set.

Description: Telomeres protect the ends of chromosomes, shorten with age, and are very short in dyskeratosis congenita (DC), an inherited bone marrow failure syndrome (IBMFS) associated with mutations in telomere biology genes. “Short telomeres” were reported in Fanconi Anemia (FA), Diamond-Blackfan Anemia (DBA) and Shwachman-Diamond Syndrome (SDS) using telomere restriction fragment length or Q-FISH assays of total leukocyte or mononuclear cell DNA. These reports focused on group averages, not results from individual patients. Our objective was to determine which categories of IBMFS patients have very short telomeres, and in which leukocyte subsets, using a more sensitive and specific assay. Telomere length was measured in granulocytes, lymphocytes, naïve T-cells, memory T-cells, B-cells, and NK cells using automated multicolor flow fluorescence in situ hybridization (FISH). We previously showed that very short telomeres (