OVERVIEW: What every practitioner needs to know

Are you sure your patient has Fanconi Anemia? What are the typical findings for this disease?

Fanconi anemia (FA) is a rare, recessive (autosomal and X-linked) genetic disease that is characterized by the triad of congenital anomalies, bone marrow failure and a predisposition to cancer, most notably acute myeloid leukemia and squamous cell carcinoma. Fanconi anemia is a clinically heterogenous disease. Children may have the typically described phenotype, or appear entirely normal. Common physicial anomalies include short stature, abnormalities of the thumb and upper extremity, skin findings (cafe-au-lait and hypopigmented spots) and structural kidney anomalies. However, an extensive number of other physical anomalies may occur. Some children with FA have a characteristic facial appearance with microophthalmia, micrognathia and a broad nasal bridge. Many of these anomalies are obvious at the time of birth (e.g., thumb and upper extremity abnormalities).

Many children with FA manifest signs of their disease at birth. Most noticable are infants who are small for gestational age and abnormalities of the thumb and upper extremity. However, approximately one-third of children with FA have no obvious physical anomaly. In these normal appearing children, a diagnosis of FA is most often made after the onset of marrow failure with its resulting cytopenias. Further, as a result of the diverse clinical phenotype, there is a broad range of age at diagnosis, with some children diagnosed in utero and others not diagnosed until adulthood.

One of the first indications of progression of disease to a state of marrow failure is red cell macrocytosis. Clinicians should consider a diagnosis of FA in children with an elevated mean corpuscular volume (MCV), especially in children with short stature and those with physical anomalies.

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The mean age of onset of marrow failure is approximately 8 years with a cumulative incidence of 90% by age 40 years. The cumulative incidence of developing hematologic malignancies (with acute myeloid leukemia (AML) and myelodysplastic syndrome most commonly reported) is approximatley 33% by age 40 years. The cummulative incidence of non-hematologic malignancies (with squamous cell carcinoma of the head and ano-genital region) is approximately 28% by age 40 years. Clinicians should consider a diagnosis of FA in patients diagnosed with squamous cell carcinoma at a young age.

What other disease/condition shares some of these symptoms?

The correct diagnosis of FA is complicated by the rarity of the disease and some phenotypic overlap of FA with other inherited diseases such as Bloom’s syndrome, Nijmengan breakage syndrome and dyskeratosis congenita. All of these diseases may show progressive marrow failure, and to some extent, cancer predispostion, growth failure, congenital anomalies and skin abnormalities. It is important to recognize that lymphocytes from patients with these disease may show increased sensitivity to mitomycin C (MCC) and diepoxybutane (DEB), although rarely with the same high number of chromosome breaks per cell is seen in FA.

What caused this disease to develop at this time?

Fanconi anemia is a genetic disease linked to abnormalities in at least 15 different genes (“FA genes”). Mutations in different FA genes may be associated with varying clinical courses with respect to the mean age of bone marrow failure onset and cancer susceptibility. For example, patients with bi-allelic mutations in the FA gene FANCD1 (BRCA2) have an extremely high incidence of leukemia at an early age (mean age 2.2 years) and brain tumors (medulloblastoma and astrocytomas). These associations have been reported for only a few FA genes with ongoing research in this area of genotype/phenotype associations.

What laboratory studies should you request to help confirm the diagnosis? How should you interpret the results?

  • A diagnosis of FA is traditionally made based on the detection of increased chromsomal breaks in peripheral blood lymphocytes or skin fibroblasts after culture with DNA cross-linking agents, with DEB and MCC most commonly used. These tests are commonly offered by hospital and clinical laboratories, but false positive tests can occur, particularly in laboratories with limited experience and when the assay is only rarely performed by the laboratory. Clinicians are advised to send diagnostic samples to experienced laboratories that routinely perform these technically difficult assays.

  • Confirmation of a diagnosis of FA is based on complementation testing with known FA genes.

  • Recently, another approach to the confirmation of a diagnosis of FA was developed based on correcting the characteristic FA-associated G2/M cell cycle arrest after exposure of cells to DNA-damaging agents. In this test, bicistronic retroviral vectors that each co-express a single FA gene cDNA (for all proteins except FANCD1 and FANCD2) together with the enhanced green flurescent protein (EGFP) are used to transduce FA peripheral blood lymphocytes or fibroblasts. The co-transduction of EGFP allows for the flow cytometric analysis of EGFP+ cells with a determination of whether transduction with an FA-specific cDNA corrects the G2/M arrest seen in FA cells. This testing also guides mutation analysis by DNA sequencing.

  • The spectrum of disease-causing mutations includes single nucleotide changes, insertions, inversions and deletions of various sizes. Large deletions often occur at specific breakpoints and have been shown to arise as a result of Alu-sequence mediated recombination. Small duplications have also been reported.

Would imaging studies be helpful? If so, which ones?

While imaging studies are not diagnostic of FA, they should be utilized to assess physical anomalies associated with the disease. For example, ultrasound studies of the abdomen can be used to identify structural anaomlies of the kidlneys, a frequent finding in patients with FA. Radiographs can help to define the extent of anomalies of the upper extremities.

Confirming the diagnosis

If a diagnosis of FA is suspected based on clinical characteristics, DEB or MMC assays performed by laboratories with demonstrated expertise in performing these assays are the first indicated diagnostic tests.

If you are able to confirm that the patient has Fanconi anemia, what treatment should be initiated?

  • Evaluation and Treatment: Given the rarity of FA, very few pediatric and adult physicians have the experience and expertise to treat this complex disease. Therefore, children with a presumed diagnosis of FA, or who are suspected to have FA, should be referred to a center with FA-specific experts. These centers can provide a comprehensive approach to these patients, with evaluation and care provided by hematologists, hematopoietic stem cell transplant physicians, orthopedic surgeons, cardiologists, gastroenterologists, urologists, endocirinologists, genetic counselors, psychologists and social workers who have FA-specific expertise. These comprehensive centers also provide patients and their parents the opportunity to participate in innovative research studies.

  • Transfusions and Supportive Care: As a result of cytopenias due to progressive marrow failure, children with FA may need supportive care in a manner similar to children with pancytopenia due to other causes. However, unlike many other disease settings, the marrow failure in children with FA is progressive, so a conservative approach to the use of blood products is advised. This conservative approach is recommended to avoid the long-term complications associated with frequent transfusions of red cells and platelets. Red cell transfusions should be used only for symptomatic patients. Platelets are best used prior to surgery or for episodes of bleeding. Patients with thrombocyopenia should avoid medications that inhibit platelet function.Since all children with FA and severe marrow failure should undergo hematopoietic stem cell transplantation, transfusions should be minimized prior to transplantation in a center with significant experience and expertise with this procedure for children with FA. Prior to modern blood banking methods aimed at reducing the transfusion of white blood cells that can result in allosensitization and the development of the powerful immunosuppressant fludarabine as part of the allogeneic transplant preparative regimen, graft failure was a frequent complication limiting the usefulness and benefit of allogeneic transplantation for these patients. There was a direct relationship with the number of transfusions prior to transplant and outcome. However, as a result of improved blood banking techniques and improved preparative regimens, the impact of pre-transplant transfusions is less clear, but fewer transfusions are advised. Patients with neutropenia should be rapidly evaluated for a source ofinfection, including blood cultures to assess for bacteremia, andstarted on broad spectrum antibiotics. Splenectomy as a treatment for cytopenias has been attempted in patients with FA, with limited success. Its routine use is not advised. Finally, immune suppression wtih high dose steroids, anti-thymocyte globulin and cyclosporine, similar to that used to treat the cytopenias assocaited with severe apastic anemia, is not supported by avaialbe data.

  • Hematopoietic Growth Factors: Several studies have evaluated with use of hematopoietic growth factors in children with FA. The growth factors with the greatest potential benefit are those that stimulate granulocytes (sargramostim (GM-CSF) and filgrastim (G-CSF)). In children with FA who have neutropenia, prospective studies have shown the potential for improving the granulocyte count in these patients. Their use is common practice in the care of children with FA. However, treatment with these growth factors is not curative, so they are used on a temporary basis until patients undergo allogeneic hematopoietic stem cell transplantation. There is no data to support the use of growth factors that stimulate red cell production (e.g., erythropoietin) or grwoth factors or cytokines that stimluate platelet production (e.g., thrombopoietin and interleukin-11). These agents should be used only in the context of prospective clinical trials.

  • Androgens: Anabolic steroids alone or in combination with glucocorticoid steroids have been used to treat children with aplastic anemia since the late 1950’s. While the use of anabolic steroids has never been evaluated in prospective studies for children with FA with severe marrow failure, these drugs have been commonly used in the routine clinical care of these children. Anecdotal experience suggest that approximately half of children with FA will have improvements in their blood counts in response to androgen therapy. The steroid most often used is oxymetholone, an oral 17-α alkylated androgen with a chemical structure similar to testosterone. Anecdotal reports confirm a transient improvement in hemoglobin levels in approximately half of treated patients, but with significant toxicities, including virilization, early closure of growth plates, peliosis hepatitis and liver tumors. As a result of these toxicities, children receiving treatment with anabolic steroids should have frequent monitoring of their liver and kidney function, liver ultrasound examinations and monitoring of bone age. Toxicities can be reduced with dose reduction of oxymetholone. Treatment with androgens is not curative and for those children with an improvement in their hemoglobin, the response is transient. Other forms of anabolic steroids are under investigation. Older studies have suggested a worse outcome for children after allogeneic hematopoietic stem cell transplantation who had had exposure to anabolic steroids prior to transplantation. More recent data suggest that androgen exposure may be a surrogate for delayed transplant, often due to an inability to find a well-matched donor. Therefore, many FA specialists will use androgens to treat only patients with severe marrow failure who do not have a matched related donor. For patients with a matched related donor, androgen therapy should be deferred, with these patients proceeding to transplantation when marrow failure is severe.

  • Hematopoietic Stem Cell Transplantation: Allogeneic hematopoietic stem cell transplantation is the only known curative therapy for bone marrow failure associated with FA. However, its successful use does not prevent the occurrence of non-hematologic malignancies in these children and adults. As a result of increased sensitivity of FA cells to ionizing radiation and alkylating forms of chemotherapy, FA patients have excessive toxicity to doses of radiation and chemotherapy that are typically used for teh transpantation of children and adults without FA. Therefore, transplant regimens have evolved over the past several decades to utilize lower doses of both radiation and chemotherapy. While these lower doses have reduced the toxicity associated with allogeneic transplantation, these regimens also resulted in increased rates of graft rejection as a result of inadequate immune suppression. More recently, the inclusion of fludarabine into transplant preparative regimens has increased the probability of engraftment, without over-lapping toxicities. Given the complexities associated with allogeneic hematopoietic stem cell transplantation for patients with FA, these transplants should occur only in institutions with significant expertise in the transplantation of children and adults with FA. In the hands of FA-specific experienced transplant physicians, greater than 90% of FA children with a matched related donor are cured of the hematopoietic manifestations of FA. Further, with the recent use of fludarabine in FA-specific transplant regimens, survival for FA patients receiving grafts from unrelated donors now exceeds 50%, a significant improvement when compared to outcomes for unrelated donor transplants without fludarbine.

What are the adverse effects associated with each treatment option?

Hematopoietic Growth Factors: Treatment of children with hematopoietic growth factors is usualy well tolerated, with few significant side effects and toxicities.

Transfusions: The toxicities associated with red blood cell and platelet transfusions are infrequent given modern blood banking methods. As discussed above, the major concern in children with FA is the risk of graft rejection in heavily transfused patients who subsequently undergo hematopoietic stem cell transplantation.

Anabolic Steroids: The common use of oxymetholone for treatment of marrow failure in children with FA frquently results in unacceptable degrees of virilization, liver tumors, behavioral problems and growth abnormalities.

Hematopoietic Stem Cell Transplantation: Allogeneic transplantation of children with FA results in a large number of potential toxicities, including but not limited to death, graft rejection/failure, bleeding, infections, graft-versus-host disease and an increased risk of secondary malignancies.

What are the possible outcomes of Fanconi anemia?

After the onset of marrow failure, treatment with hematopoietic growth factors, transfusions and anabolic steroid are supportive in nature. They do not used with curative attempt most oftern used only to allow for time to find a suitably matched stem cell donor.

Allogeneic hematopoietic stem cell transplantation is potentially curative for the hematologic manifestations of FA. However, even with successful transplantation, the non-hematopoietic manifestations of the disease are not improved. In patients who develop graft-versus-host disease following transplantation combined with the use of radiation and alkylator chemotherapy agents as part of the preparative regimen, the probability of secondary malignancies is further increased.

What causes this disease and how frequent is it?

Fanconi anemia has an estimated incidence of 1 case per 100,000 live births, a prevalence estimated at 1 to 5 per million with a carrier frequency of approximately 1 in 300. The male to female ratio is 1.24,with males diagnosed earlier than females (median: 6.5 years vs 8 years).

Fanconi anemia cells demonstrate hypersensitivity to DNA cross-linking agents (diepoxybutane (DEB), mitomycin C (MCC), melphalan and cisplatin), oxidative damage and other DNA damaging agents. In response to exposure to these agents, FA cells demonstrate exaggerated G2 cell cycle arrest, chromosomal abnormalities including chromatid breaks and interchanges, enhanced susceptibility to proinflammatory cytookines, defective p53 induction and increased apoptosis. This hypersensitivity of FA cells to DNA damaging agents has suggested a defect in DNA damage surveillance or DNA repair pathways, although direct evidence to support a linkg to DNA repair mechanisms has only recently been identified. Fanconi anemia can be divided into complementation groups based on cell fusion studies with 15 different responsible genes now identified. The fact that some FA patients do not complement with known FA genes suggest the presence of additional, as yet identified genes.

How do these pathogens/genes/exposures cause the disease?

Fanconi anemia is the result of bi-allelic mutation in any of the known FA genes. The mechanism by which the proteins encoded by these genes function and cooperate with other proteins in DNA repair is still under investigation. However, at least some of these FA genes (A, B, C, E, F,G, L, and M) form a complex with nuclear E3 ubiquitin ligase that is required for the monoubiquitination of the downstream FANCD2 protein. FANCD2 is monoubiquitinated during S phase of the cell cycles and in response to DNA damage. Monoubiquitinated FANCD2 localizes to the chromatin where it interacts with FANCD1 /BRCA2 and other DNA repair proteins (RAD51, PCNA, BRCA1, NBS1) to form a second hypothesized complex in DNA damage foci in the nucleus. Another FA protein, FA-I, has also been show to be modified by monoubiquitination in response to DNA damage. The FA genes FANCJ and FANCM have been shown to have DNA interatcing motifs. FA-J possesses DNA helicase activity while FA-M has both helicase and endonuclease domains. Investigations are underway to better understand how these FA proteins are involved in DNA repair mechanisms.

How can Fanconi anemia be prevented?

As a genetic disease, Fanconi anemia cannot be prevented. However, for families who have had a child with FA, recent advances in preimplantation genetic testing and in vitro fertilization now allow for the implantation of blastocysts that are selected for the absence of FA. This technology is now used by families who desire more children without Fa and in families seeking to create a matched related donor for an effected sibling.

What is the evidence?

Valeri, A, Martinez, S, Casado, JA, Bueren, JA. “Fanconi anaemia: from a monogenic disease to sporadic cancer”. Clinical and Translational Oncology. vol. 13. 2011. pp. 215-221.

Rackoff, WR, Orazi, A, Robinson, CA. “Prolonged administration of gransulaocyte colony-stimulating factor (filgrastim) to patients with fanconi anemia: a pilot study”. Blood. vol. 88. 1996. pp. 1588-1593.

Shahidi, NT, Crigler, JF. “Evaluation of growth and endocrine systems in testosterone-corticosteroid-treated patients with aplastic anemia”. Journal of Pediatrics. vol. 70. 1967. pp. 233-242.

Alter, BP. “Cancer in fanconi anemia: 1927-2001”. Cancer. vol. 97. 2003. pp. 425-440.

Kutler, DI, Singh, B, Satagopan, J. “A 2-year perspective on the International Fanconi Anemia Registry”. Blood. vol. 101. 2003. pp. 1249-1256.

Rosenberg, PS, Greene, MH, Alter, BP. “Cancer incidence in persons with Fanconi anemia”. Blood. vol. 101. 2003. pp. 822-826.

Auerbach, AD, Adler, B, Chaganti, RS. “Prenatal and postnatal diagnosis and carrier detection of Fanconi anemia by a cytogenetic method”. Pediatrics. vol. 67. 1981. pp. 128-135.

Auerbach, AD, Rogatko, A, Schroeder-Kurth, TM. “International Fanconi Anemia Registry: Relation of clinical symptoms to diepoxybutane sensitivity”. Blood. vol. 73. 1989. pp. 391-396.

Auerbach, AD. “Fanconi anemia and its diagnosis”. Matuation Research. vol. 668. 2009. pp. 4-10.

Hanenberg, H, Batish, SD, Pollok, KE. “Phenotypic correction of primary Fanconi anemia T-cells with retroviral vectors as a diagnostic tool”. Experimental Hematology. vol. 30. 2002. pp. 410-420.

Bitan, M, Or, R, Shapira, MY. “Fludarabine-based reduced intensity conditioning for stem cell transplantation of Fanconi anemia patients from fully matched related and unrelated donors”. Biology Blood Marrow Transplantation. vol. 12. 2006. pp. 712-718.

Farzin, A, Davies, SM, Smith, FO. “Matched-sibling donor haematopoietic stem cell transplantation in Fanconi anaemia: an update of teh Cincinnati Children's experience”. British Journal of Haematology. vol. 136. 2007. pp. 633-640.

MacMillan, ML, Hughes, MR, Agarwal, S, Daley, GQ. “Cellular therapy for Fanconi anemia: the past, present and future”. Biology of Blood and Marrow Transplantation. vol. 17. 2011. pp. S109-114.

Wagner, JE, Eapen, M, MacMillan, ML. “Unrelated donor bone marrow transplantation for the treatment of Fanconi anemia”. Blood. vol. 109. 2007. pp. 2256-2262.

Zanis-Neto, J, Flowers, ME, Medeiros, CR. “Low-dose cyclophosphamide conditionng for haematopoietic cell transplantation for HLA-matched related donors in patients with Fanconi anemia”. British Journal of Haematogy. vol. 130. 2005. pp. 99-106.

Grewal, SS, Kahn, JP, MacMillan, ML. “Successful hematopoietic stem cell transplantation for Fanconi anemia from an unaffected HLA-genotype-identical sibling selected using preimplantation genetic diagnosis”. Blood. vol. 103. 2004. pp. 1147-1151.

Croop, JM, Cooper, R, Fernandez, C. “Mobilization and collection of peripheral blood CD34+ cells from patients with Fanconi anemia”. Blood. vol. 98. 2001. pp. 2917-2921.

Kelly, PF, Radtke, S, Kalle, C. “Stem cell collection and gene transfer in Fanconi anemia”. Molecular Therapy. vol. 15. 2007. pp. 211-219.

Ongoing controversies regarding etiology, diagnosis, treatment

Several areas of clinical practice and research create ongoing controversy. First, the practice of creating matched sibling donors using preimplantation genetic testing continues to engender debate by many. Second, as a monogenic disease, FA is theoretically correctable using gene therapy technologies. However, while gene therapy has been demonstrated to be potentially curative for several genetic diseases (e.g., severe combined immune deficiency and chronic granulomatous disease), early attempts using gene therapy for FA have not been successful. This is likely due to the very small number of hematopoietic stem cells in the marrow of FA patients (even pre-dating the onset of clinically observable marrow failure) and the poor quality of the available stem cells.