ALBERT

Description: Lymphoblastoid cells from normal subjects and from patients with the bone marrow failure and cancer prone inherited disease, Fanconi anemia (FA) were treated in culture with psoralen plus ultraviolet A radiation (PUVA) in a scheme shown to produce interstrand crosslinks in cellular DNA. Hypersensitivity to DNA interstrand crosslinks, with associated increased clastogenicity, is considered to be a diagnostic hallmark of the disease. Following this cells were treated with hydroxyurea, 5 fluorouracil, or high dose thymidine for 24 hours. Clastogenicity and cytotoxicity, measured as trypan blue exclusion, were then found to be markedly increased in FA cells but not in FA cells subsequently treated with any of these other agents. Similar results were also found when all drugs were removed after these treatments and the cells cultured for 10 days without any drug in colony forming ability assays. We propose that the mechanism is related to decrease in the rate of DNA synthesis, which we have shown occurs in normal but not FA cells following PUVA, and which is also produced by these other drugs in the concentrations used here. Hydroxyurea has been used for many years as a safe and effective treatment for sickle cell anemia. It is now proposed as a possible treatment for Fanconi anemia to delay or even prevent development of bone marrow failure and/or other complications, including leukemogenesis and carcinogenesis, with or without prior bone marrow transplantation.

Description: Abstract 5176 Introduction Telangiectasia macularis eruptiva perstans (TMEP) is a rare form of cutaneous mastocytosis seen commonly in adults. TMEP may show systemic manifestations and may be associated with myelodysplasia, myeloproliferative disorders, acute myeloid leukemia, and/or lymphoproliferative disease, but is not well recognized by hematologists. We present two cases of this rare but fascinating disease that illustrate the wide range of associated findings that may be present. Case 1 A 73-year-old female presented with hyperpigmented patches on her thighs, knees, and ankles/dorsal feet. She had no systemic symptoms. Skin biopsy showed dilated vessels and associated mast cells indicative of TMEP. A toluidine blue stain highlighted increased mast cells around the superficial vessels in the papillary dermis. Some mast cells in the superficial dermis also showed c-kit immunoreactivity. After four years she is still asymptomatic and no further studies have been performed. Case 2 A 28-year-old female presented to a doctor's office with a three day history of oral labial edema with burning and pruritus. She took Benadryl at home and also received Benadryl injection without relief. She had lesions on her forehead that spread to involve the remainder of her body, lasting from five minutes to hours. She also experienced flushing, dizziness, tinnitus, dyspnea, wheezing, cough, arthralgia and daily abdominal cramps with diarrhea. She was referred for bone marrow biopsy and further evaluation, and was diagnosed with TMEP on skin biopsy. The diagnosis was confirmed with positive toluidine blue and Giemsa stains and c-kit immunoreactivity. She had no bone marrow involvement. Case 3 A 36 old female presented with a rash for over a year. It initially started on her face and then spread to the chest, upper arms and hands. It was focally pruritic and painful. She also complained of fatigue, muscle pain on the shoulders and weight gain. Physical examination showed diffuse scattered telengiectasias of the face, upper palate, buccal- labial mucosa, neck, upper chest, upper arms, palms and fingers. A skin biopsy revealed dilated blood vessels and increased mast cells in the superficial dermis. Toluidine blue and Giemsa stains demonstrated the mast cells and the c-kit immunostain was also reactive. Cutaneous mastocytosis is a mast cell proliferative disorder with at least four different clinical forms: urticaria pigmentosa, solitary mastocytoma, diffuse cutaneous mastocytosis, and TMEP. In TMEP, characteristically, lesions are ill defined, non-pruritic, but urticate on rubbing, telengiectatic tan/brown 2–6 mm macules located symmetrically over the trunk and extremities and rarely on the face. Occasionally, urticaria pigmentosa may coexist with this lesion; however TMEP should be distinguished from urticaria pigmentosa with overlying telangiectases. Darier's sign is usually absent or minimal. This is because the lesions are characteristically paucicellular, and the few mast cells may not yield significant degranulation to exhibit Darier's sign and dermographism. Symptoms are the result of degranulation of mast cells with the release of multiple mediators. Flushing, blistering, pruritus, cardiac arrhythmias, dyspnea, asthma exacerbations, hypotension, gastrointestinal upset, acid reflux, peptic ulcer disease, diarrhea, splenomegaly, increased numbers of mast cells in the bone marrow, abnormal skeletal radiographs, irritability and nonspecific neuropsychiatric symptoms can be seen. TMEP is characteristically composed of subtly increased numbers of ovoid to spindle shaped mast cells infiltrating the papillary dermis and surrounding dilated superficial capillaries and venules. To distinguish mast cells from histiocytes, Giemsa and toluidine blue stains are useful. Tissue sections showing more than 5–10 mast cells are confirmatory for the diagnosis. c-Kit immunohistochemistry can be used to confirm the diagnosis. c-Kit is a proto-oncogene that codes for a tyrosine kinase receptor (CD117) present on mast cells and melanocytes. The present cases illustrate the wide diversity of systemic manifestations of mastocytosis that may accompany TMEP. Case one showed no systemic signs at all, whereas cases two and three showed significant systemic disease. In case three lesions started on the face, an unusual location for TMEP. Appropriate work-up is mandatory in cases presenting with TMEP. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4214 Fanconi anemia (FA) is an inherited, cancer-prone bone marrow failure disease. FA is heterogeneous, with 13 complementation groups, but all groups have in common hypersensitivity to agents that produce DNA interstrand cross links (DISCLs), with associated increased clastogenicity, as a diagnostic hallmark. Although progress has been made in treating FA, particularly using bone marrow transplantation (BMT) to prevent bone marrow failure and leukemogenesis, BMT is not a trivial procedure, and treatment remains challenging. Head and neck cancers, which occur in high frequency in FA, are a particular problem that is not well remedied by BMT. Lymphoblastoid cells from normal subjects and from patients with FA were treated in culture with psoralen plus ultraviolet A radiation (PUVA) in a regimen shown to produce DISCLs. Following this, cells were treated with hydroxyurea, 5-fluorouracil, or high dose thymidine, in doses we have shown to produce a marked decrease in rate of DNA synthesis, for 24 hours. We have previously shown that clastogenicity and cytotoxicity, measured as trypan blue exclusion as well as colony forming ability (CFA), are markedly increased in FA cells, complementation groups A, B, C, and E, associated with deficiencies in their corresponding FA core proteins, but these increases are not observed in these FA cells subsequently treated with any of these other, DNA synthesis retarding agents, which effectively correct the FA phenotype in culture. FA A and C cells genetically corrected for the FANC A and G gene, respectively, display normal clastogenicity and cytotoxicity following PUVA, and do not show this correction following subsequent treatment with hydroxyurea, 5-fluorouracil, or high dose thymidine. We now report similar results for short term cell viability, and similar, although less marked, results for clastogenicity in FA complementation group D1 cells, associated with a deficiency in BRCA2. When all drugs were removed after these treatments and the cells cultured for 10 days without any drug in CFA assays, the FA group D1 cells resembled normals, however, and did not show this correction. We propose that the mechanism in FA A, B, C and G cells is related to a decrease in the rate of DNA synthesis, which we have shown occurs in normal but not FA cells following PUVA, and which is also produced by these other agents in the concentrations used here. The partial correction observed in FA group D1 cells may be due to this or a different mechanism. Partial or complete correction appears to apply to multiple FA complementation groups. Hydroxyurea has been used for many years as a safe and effective treatment for sickle cell anemia and other diseases. It is now proposed as a possible treatment for FA to delay or even prevent development of bone marrow failure and/or other complications, including leukemogenesis and carcinogenesis, with or without prior BMT. In some cases it may serve as a viable alternative where BMT is not fungible. Alternatively it may obviate the need for BMT altogether in responsive patients, or be effectively used in combination with other modalities. Complementation group may be important in determining which patients may be less responsive or require modified regimens. Disclosures: Off Label Use: We have obtained laboratory results which show partial or complete restoration of cytotoxicity and clastogenicity, as well as colony forming ability in the absence of drug in FA A, B, C, and G but not D1 cells, following treatment with a DNA cross-linking agent, in Fanconi anemia lymphoblastoid cells, by subsequent application of hydroxyurea, to normal levels. Hydroxyurea has been used for many years as a safe and effective treatment for sickle cell anemia. It is now proposed as a possible treatment for Fanconi anemia to delay or even prevent development of bone marrow failure and/or other complications, including leukemogenesis and carcinogenesis. It may be less effective in FA complementation group D1. Disclosures: Off Label Use: We have obtained laboratory results which show partial or complete restoration of cytotoxicity and clastogenicity, as well as colony forming ability in the absence of drug, following treatment with a DNA cross-linking agent, in Fanconi anemia lymphoblastoid cells, by subsequent application of hydroxyurea, to normal levels. Hydroxyurea has been used for many years as a safe and effective treatment for sickle cell anemia. It is now proposed as a possible treatment for Fanconi anemia to delay or even prevent development of bone marrow failure and/or other complications, including leukemogenesis and carcinogenesis..

Description: One of us (WCL) has previously proposed a mathematical model, Co-Recessive Inheritance, for inherited diseases associated with DNA repair deficiencies (Lambert WC, Lambert MW: Mutat. Res., 1985;145:227–234; Lambert WC: Keynote Address, 21st Anniversary Celebration, MRC Cell Mutation Unit, University of Sussex, UK. Mutat. Res., 1992;273:179–102). The model is also applicable to diseases associated with defective cell cycle modulation following specific types of DNA damage, such as Fanconi Anemia, with or without additional defects in DNA repair. The model proposes that in some complementation groups of these diseases defective alleles at more than one locus are required for the disease phenotype to be expressed. It follows from the model (A readily understandable derivation will be presented.) that the carrier frequencies of the genes involved are very much higher than would be predicted based on classical population genetics. This may impact on recent observations of higher than expected co-inheritance of defective alleles of Fanconi Anemia and Bloom Syndrome genes along with BRCA genes in certain populations (e.g., Koren-Michowitz, M, et al.: Am. J. Hematol., 2005;78:203–206), and provides an explanation for the lower than expected incidence of cancer in these individuals. It also provides an explanation for finding biallelic defects in the same DNA repair genes in more than one complementation group of Fanconi Anemia (Howlett NG, et al.: Science, 2002;297:606–609). The Co-Recessive Model predicts that other findings of this nature are to be expected, and provides some guidelines that may be helpful in the process of gene discovery in Fanconi Anemia. Among the more important of these are 1) that the search for defective genes in each complementation group should not cease when one such gene is found, even if one or more patients in the group is homozygous or compound heterozygous for defective alleles of that gene, and 2) that carrier frequencies for some Fanconi Anemia genes may be much higher than would otherwise be anticipated, with a significant proportion of the normal population being carriers. If the latter hypothesis is correct, it follows that the relevance of these rare diseases and their associated genes to disease, including bone marrow failure, in the general population is dramatically greater than has been generally believed.

Description: Abstract 879 Fanconi 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.

Description: We have previously shown that the primary cell cycle defect in the inherited, cancer-prone, bone marrow failure associated disease, Fanconi anemia (FA), is not in the G2 phase of the cell cycle, as had been thought for many years, but rather in the S phase. FA cells challenged with the DNA cross-linking agent, psoralen coupled with long wavelength, ultraviolet (UVA) radiation (PUVA), fail to slow their progression through the S phase of the subsequent cell cycle, as do normal cells. FA cells are extremely sensitive to the cytotoxic and clastogenic effects of DNA cross-linkers, such as PUVA, so much so that the diagnosis of FA is based on an assay, the “DEB test”, in which cells are examined for clastogenic and cytotoxic effects of diepoxybutane (DEB), a DNA cross-linking agent. More recently, we have shown that artificially slowing the cell cycle of FA cells exposed to PUVA by subsequent treatment with agents which slow their progression through S phase leads to markedly increased viability and reduced chromosome breakage in vitro. We now show that similar results can be obtained in vivo in patients with another DNA repair deficiency disease, xeroderma pigmentosum (XP), a recessively inherited disorder associated with defective repair of sunlight induced adducts in the DNA of sun-exposed tissues followed by development of numerous mutations causing large numbers of cancers in these same tissues. We treated two patients with XP, a light complected black male and a white female, both 14 years of age, in sun-exposed areas with 5-fluorouracil, an inhibitor of DNA synthesis, daily for three months. In contrast to normal patients, who only show clinical results if an inflammatory response is invoked, marked improvement in the clinical appearance of the skin was seen with no inflammation observed. This effect was confirmed histologically by examining epidermis adjacent to excised lesions in sun-exposed areas and further verified by computerized image analysis. Treatment with agents that slow progression through S phase, such as hydroxyurea, may similarly improve clinical outcomes in patients with FA or others who are developing bone marrow failure.