Accelerated telomere shortening and telomerase activation in Fanconi's anaemia

Fanconi's anaemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure that often evolves towards acute leukaemia. FA also belongs to a group of chromosome instability diseases. Because telomeres are directly involved in chromosomal stability and in cell proliferation capacity, we examined telomere metabolism in peripheral blood mononuclear cells (PBMC). Telomere length was significantly shorter in 54 FA patient samples, compared to 51 controls (P<0.0001). In addition, mean telomere terminal restriction fragment lengths (TRF) in nine heterozygous patient samples did not differ from those of controls. In 14 samples from FA patients with severe aplastic anaemia (SFA), telomere length was significantly shorter than in 22 samples of age-matched FA patients with moderate haematological abnormalities (NSFA) (P<0.001). However, no correlation was found between TRF length and the presence of bone marrow clonal abnormalities in 16 additional, separately analysed, patient samples. Sequential measurement of TRF in six FA patients showed an accelerated rate of telomere shortening. Accordingly, telomere shortening rate was inversely correlated with clinical status. Telomerase, the enzyme that counteracts telomere shortening, was 4.8-fold more active in 25 FA patients than in 15 age-matched healthy controls. A model for the FA disease process is proposed.     

GM-CSF in the treatment of Fanconi's anaemia

We have used recombinant human (rh) GM-CSF in two 12-year-old Fanconi's aplastic anaemia patients. They had not received any previous therapy except blood transfusions. Each patient was given three 21 d courses of rh-GM-CSF, the first two at a dose of 3.5μg/kg/d and the third at 7 μg/kg/d s.c. There were significant increases in WBC and absolute neutrophil counts after the first week of rh-GM-CSF which lasted as long as the treatment was continued. Following the cessation of treatment, WBC and ANC dropped rapidly. We conclude that rh-GM-CSF can be used in FAA, especially in severely neutropenic cases.     

Fanconi's anaemia presenting as acute myeloid leukaemia in adulthood

We describe a 28-year-old male patient who presented with apparently de novo acute myeloid leukaemia (AML) who was subsequently found to have Fanconi's anaemia (FA). The gene for complementation group A ( FAA ) has recently been localized to chromosome 16q24.3 and utilizing genetic markers closely linked to this locus we were able to conclude that this patient was likely to belong to complementation group A. FA presenting as AML is an exceptionally rare event and all previously described cases have occurred in patients less than 21 years of age. We conclude that the diagnosis of FA should always be considered in younger patients presenting with AML. It is important that the correct diagnosis is made in these individuals because the administration of conventional chemotherapy may well have devastating consequences for them. Correlations between the specific mutations causing FA and clinical phenotypes are likely to become apparent as more genetic analyses are performed in this group of patients.     

Abnormal telomere metabolism in Fanconi's anaemia correlates with genomic instability and the probability of developing severe aplastic anaemia

Fanconi's anaemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure and a susceptibility to cancer. Haematopoietic stem cell transplantation is the only curative method for restoring normal haematopoiesis, and survival is improved if the transplant is carried out before severe complications occur. However, the evolution of FA is difficult to predict because of the absence of known prognostic factors and the unknown function of the genes involved. In studying 71 FA patients, a correlation was found between severe aplastic anaemia (SAA) and the individual annual telomere-shortening rate (IATSR) in peripheral blood mononuclear cells (P < 10(-3)). Spontaneous apoptosis was highest in SAA patients or patients with high IATSR (> 200 bp/year) (P < 0.01, n = 18). Univariate and multivariate analyses showed that significant relative risks for evolution towards SAA were high IATSR (P < 10(-4)), and that a high number of chromosome breakages occurred in the presence of nitrogen mustard (P < 0.001). A high IATSR was also associated with an increased frequency of malignancy (P < 0.01). Thus, these biological parameters were related to the spontaneous evolution of FA and could be used as prognostic factors. These data indicated that telomeres might play a role in the evolution of bone marrow failure and malignant transformation in FA.     

Follow-up by cytogenetic and fluorescence in situ hybridization analysis of allogeneic bone marrow transplantation in two children with Fanconi's anaemia in transformation

Results of bone marrow transplantation (BMT) in patients with Fanconi's anaemia (FA) in transformation are very poor and only a few cases with favourable outcome have been reported. We present the follow-up of two FA-myelodysplastic syndrome (MDS) patients with monosomy 7 and complex karyotype implicating chromosome 1. Both relapsed with acute myeloid leukaemia (AML) following an allogeneic BMT from an HLA-identical brother. The patients showed clonal cytogenetic evolution coinciding with the leukaemic transformation. In one patient, fluorescence in situ hybridization using X and Y chromosome probes detected an increase of host cells before clinical relapse. Both patients received a successful second allogeneic BMT from the same donor using a more intensive treatment regimen and remain in clinical and cytogenetic remission more than 3 years later.     

Fludarabine-based stem cell transplantation protocol for Fanconi's anaemia in myelodysplastic transformation

Allogeneic stem cell transplantation (SCT) represents the treatment of choice for severe bone marrow (BM) failure in patients with Fanconi's anaemia (FA). However, for FA patients developing leukaemic or myelodysplastic transformation, the results of SCT are much less encouraging. We present a 17-year-old girl with myelodysplastic transformation of FA (refractory anaemia with excess blasts) and oculocutaneous albinism, who was treated by sibling SCT using conditioning with fludarabine, cyclophosphamide (CY) and anti-lymphocyte globulin (ALG). She had rapid engraftment with no toxicity and no graft-versus-host disease (GVHD). Twenty-two months after SCT, she had 100% donor chimaerism on Southern blot analysis.     

Transplantation for Fanconi's anaemia: long-term follow-up of fifty patients transplanted from a sibling donor after low-dose cyclophosphamide and thoraco-abdominal irradiation for conditioning

We describe the long-term follow-up of 50 Fanconi's anaemia patients who were transplanted from a related donor with a median follow-up of >6 years. The survival estimate was 74.4% at 54 months and 58.5% at 100 months. All patients were conditioned with low-dose cyclophosphamide and thoraco-abdominal irradiation. Acute graft-versus-host disease (GvHD) of grade II or more developed in 26 patients and chronic GvHD developed in 30/43 (69.9%) patients. The survival of patients without chronic GvHD ( n  = 13) was 100%. In addition to chronic GvHD, 20 pre-transplant transfusions was shown to have an adverse impact on survival by multivariate analysis (relative risk = 7.08, P  = 0.0003). Prospective follow-up of growth and endocrine function could be performed in 31 patients. Of 20 boys, six have already reached normal puberty within the expected time. Among the 11 girls, three were at the pubertal age at the time of analysis. Growth retardation was common, whereas late complications (e.g. peripheral hypothyroidism, cataract) were rare. However, the most important long-term complication was the occurrence of cancer in seven patients (8-year projected incidence 24%).
Among the 32 survivors, 27 (84.5%) had a normal and four a moderately reduced performance status, and all achieved complete engraftment with donor cells. Therefore transplantation was able to cure these patients who remain at high risk for developing late complications. Clearly, a genetic predisposition and chronic GvHD could have led to the development of these cancers. However, we cannot completely rule out irradiation as a cofactor in the genesis of these cancers, and therefore no longer use irradiation for the conditioning of Fanconi's anaemia patients.     

Positive diepoxybutane test in only one of two brothers found to be compound heterozygotes for Fanconi's anaemia complementation group C mutations

Fanconi's anaemia (FA) is an autosomal recessive disorder characterized by diverse congenital abnormalities, the development of progressive bone marrow failure, and an increased predisposition to malignancy, particularly acute leukaemia. The FA phenotype is so variable that diagnosis on the basis of clinical manifestations alone can be difficult. The modern diagnosis of FA no longer rests entirely on the constellation of clinical and haematological abnormalities first described by Fanconi, but depends on finding elevated chromosomal breakage after incubation of peripheral blood lymphocytes with the chemical clastogens diepoxybutane (DEB) or mitomycin-C (MMC). The cloning of the gene for FA complementation group C [ FAC ] provides an opportunity to test the validity of the 'DEB test' which in recent times has become the main arbiter as to whether a patient is classified as FA or non-FA.
We report on two brothers with similar clinical and haematological features who have both been identified as compound heterozygotes for the FAC mutations L554P and ΔG322, but only one of the brothers has a positive DEB test. On the basis of the DEB test one would be classified as FA and the other as non-FA. The time has come to re-evaluate the diagnostic criteria of 'Fanconi's anaemia'.     

Clinical relevance of cytogenetic abnormalities at diagnosis of acquired aplastic anaemia in adults

The outcome of 81 adult aplastic anaemia patients who had successful cytogenetics at diagnosis and received immunosuppressive therapy was evaluated. Ten patients had an abnormal karyotype, six of which had a trisomy. Four of five evaluable patients with a trisomy responded. One patient with monosomy 7 achieved a complete response and later developed haemolytic paroxysmal nocturnal haemoglobinuria but no recurrence of monosomy 7. None of the patients with a non-numerical karyotypic abnormality responded. No significant differences in survival or later clonal disorders were observed between patients with a normal karyotype and those with an abnormal karyotype.     

Stem cell transplantation from HLA-matched related donor for Fanconi's anaemia: a retrospective review of the multicentric Italian experience on behalf of AIEOP-GITMO

Twenty-seven consecutive Italian patients with Fanconi's anaemia (FA) underwent stem cell transplantation (SCT) from an HLA-matched related donor in 10 Italian centres of the Associazione Italiana Ematologia ed Oncologia Pediatrica (AIEOP), Gruppo Italiano di Trapianto di Midollo Osseo (GITMO). Twenty-two patients (81.5%) were conditioned with low-dose (median 20 mg/kg) cyclophosphamide (Cy) and thoraco-abdominal or total body irradiation (median dose 500 cGy), five patients (18.5%) with high-dose Cy (median 120 mg/kg). Graft-vs.-host disease (GVHD) prophylaxis was carried out with cyclosporin A in 26 cases; methotrexate (MTX) was added in eight cases. One patient received MTX alone. The median follow-up was 36 months. Ninety-two percent of patients (25 out of 27) engrafted, grade II and III acute GVHD occurred in 28% and 8% of patients, respectively, with chronic GVHD in 12.5%. Conditioning-related toxicity was mild: 4% of patients had grade III mucositis, 7.4% had grade II haemorrhagic cystitis, 14.8% had grade III liver toxicity and 11.1% had grade III renal toxicity. Transplant-related mortality at 12 months was 19.2%, survival at 36 months was 81.5%, with a median Karnofsky score of 100%. No late tumours occurred after a mean follow-up of the survivors of 5 years. None of the studied variables significantly affected the survival, including conditioning regimen, acute GVHD and clinical non-haematological phenotype. Among the studied variables, only conditioning regimens containing high-dose Cy and the presence of genital abnormalities were significantly (P < 0.05) associated with an increased rate of acute GVHD. Our study demonstrates that the Italian FA patients undergoing SCT from an HLA-matched related donor have a very good outcome. These patients, when compared with others of different ethnic origin who underwent allogeneic bone marrow transplantation, showed a less severe non-haematological phenotype, raising the possibility that this milder phenotype may have, at least in part, contributed to the outcome. Our data may provide a useful tool for further studies aiming to correlate genotype with phenotype.     

Induction of a hematological and cytogenetic remission in a patient with a myelodysplastic syndrome secondary to Fanconi's anemia employing the S-HAM regimen

 We report on a patient with Fanconi's anemia (FA) who developed a myelodysplastic syndrome (RAEB-T) with complex karyotypic abnormalities (trp 1q23 q 42, monosomy 20, trisomy 13) at the age of 28. The patient achieved a complete hematological and cytogenetic remission after treatment with sequential high-dose cytosine arabinoside/mitoxantrone followed by G-CSF (5 μg/kg). Bone marrow hypoplasia was prolonged with 38 days of granulocytopenia <500/μl and 62 days of platelet transfusion dependency. Nonhematological toxicity did not exceed that of patients without underlying FA. Remission duration was 7 months. This observation shows the feasibility of high-dose Ara C treatment in patients with FA and MDS. Although hematopoiesis remained clonal in remission, the suppression of the cytogenetically abnormal clones transiently reversed the antecedent long-lasting pancytopenia. Received: 16 September 1996 / Accepted: 27 January 1997     

Splenectomy as an adjuvant measure in the treatment of severe aplastic anaemia

The role of splenectomy in aplastic anaemia (AA) is controversial. The hazards of operating on a severely pancytopenic patient, the fear of compromising the patient's immune function, and the improvement of non-surgical treatment have made splenectomy unpopular in this disease.
We have evaluated positive and adverse effects of splenectomy in 80 patients with severe aplastic anaemia (SAA) treated with antilymphocyte globulin (ALG) (group A), using 52 nonsplenectomized ALG patients as controls (group B). All patients survived the operation. Nonfatal complications of surgery occurred in 10 (12.5%). Splenectomy induced a significant increase of peripheral blood neutrophils, reticulocytes and platelets within 2 weeks, followed by a continuous increase of all values over the following weeks. 28/132 patients (21%) developed a late clonal disorder of haemopoiesis, paroxysmal nocturnal haemoglobinuria (PNH) or myelodysplastic syndrome (MDS), or both. Their incidence was identical in groups A and B. 13/28 (59%) died, 10/17 (59%) in group A and 3/11 (27%) in group B (not significant (n.s.)). Overall probability of survival at 18 years after ALG was 51 ± 6% for group A and 61 ± 7% for group B (n.s.).
We conclude that splenectomy in AA is safe. It induces an immediate increase of peripheral blood counts and, thereafter, a continuous improvement of haemopoiesis. It does not increase the incidence of late clonal complications but has a borderline effect on mortality from these disorders. Splenectomy should be reconsidered in selective nontransplanted patients who have prolonged transfusion requirements despite otherwise optimal treatment.     

Splenectomy as an adjuvant measure in the treatment of severe aplastic anaemia

The role of splenectomy in aplastic anaemia (AA) is controversial. The hazards of operating on a severely pancytopenic patient, the fear of compromising the patient’s immune function, and the improvement of non-surgical treatment have made splenectomy unpopular in this disease. We have evaluated positive and adverse effects of splenectomy in 80 patients with severe aplastic anaemia (SAA) treated with antilymphocyte globulin (ALG) (group A), using 52 nonsplenectomized ALG patients as controls (group B). All patients survived the operation. Nonfatal complications of surgery occurred in 10 (12.5%). Splenectomy induced a significant increase of peripheral blood neutrophils, reticulocytes and platelets within 2 weeks, followed by a continuous increase of all values over the following weeks. 28/132 patients (21%) developed a late clonal disorder of haemopoiesis, paroxysmal nocturnal haemoglobinuria (PNH) or myelodysplastic syndrome (MDS), or both. Their incidence was identical in groups A and B. 13/28 (59%) died, 10/17 (59%) in group A and 3/11 (27%) in group B (not significant (n.s.)). Overall probability of survival at 18 years after ALG was 51 ± 6% for group A and 61 ± 7% for group B (n.s.). We conclude that splenectomy in AA is safe. It induces an immediate increase of peripheral blood counts and, thereafter, a continuous improvement of haemopoiesis. It does not increase the incidence of late clonal complications but has a borderline effect on mortality from these disorders. Splenectomy should be reconsidered in selective nontransplanted patients who have prolonged transfusion requirements despite otherwise optimal treatment.     

Aplastic anaemia associated with a Philadelphia chromosome and monosomy 7 during immunosuppressive therapy

We describe a patient who presented with aplastic anaemia associated with the Philadelphia (Ph1) chromosome during immunosuppressive therapy and who subsequently developed myelodysplastic syndrome (MDS) with monosomy 7. Initially the patient had hypocellular fatty marrow without leukaemic blasts or dysplastic features. Chromosome analysis showed 46, XY, t(9;22)(q34;q11) during immunosuppressive therapy, but no leukaemic transformation was detected. The patient showed gradual haematologic improvement and became transfusion independent. Thereafter, bone marrow dysplasia with monosomy 7 progressed following transfusion independence. These findings indicate that multiple cytogenetic evolutions occur in aplastic anaemia during immunosuppressive therapy, and that Ph1 chromosome may play a role in bone marrow suppression rather than development of leukaemia.     

Variable pathogenicity of exon 43del (FAA) in four Fanconi anaemia patients within a consanguineous family

Four Fanconi anaemia group A (FAA) patients within two related consanguineous families are presented: the propositus (male, 13 years, transplanted at age 10), and his three cousins (one male, 8 years, and two female newborns). Assignment of the patients to FAA was based on the functional complementation analysis by somatic cell hybridization and confirmed by mutation screening showing a homozygous deletion of exon 43 (4267-4404del) in the FAA gene to be present in all four patients. The newborn patients had been diagnosed prenatally by DNA analysis. In spite of identical molecular pathology and close familial relationship the clinical phenotypes of the four patients were not concordant. Discordant symptoms included birthweight, pigmentation abnormalities, skeletal, renal and genital abnormalities, whereas microcephaly and possibly the haematological course were concordant. Differences in environmental conditions and/or genetic make-up along with chance effects during development may explain discordant phenotypes despite identical molecular pathology in these patients. However, our results do not rule out the possibility that the exon 43del mutation may have prognostic value for the haematological course of the disease.     

Myelodysplasia as the initial presentation of Fanconi's anaemia in a phenotypically normal child

Patients with Fanconi's anaemia (FA) present with a wide spectrum of congenital abnormalities and develop hypoplastic anaemia. There is also at least a 10% risk of developing acute myeloid leukaemia (Alter 1987). Phenotypically normal patients have also been described who have only shown an increased number of chromosomal breaks (Auerbach, Rogatko & Schroeder-Kurth, 1989). We describe a 7-year-old girl presenting with myelodysplasia (MDS) in whom a diagnoses of FA was made by chromosome analysis after treatment had commenced.     

Hepatocellular carcinoma and squamous cell carcinoma in a patient with Fanconi's anemia

Summary Acute leukemia, hepatocellular carcinoma, and squamous cell carcinoma have been reported in patients with Fanconi's anemia. We report on a 31-year-old woman who developed squamous cell carcinoma of the esophagus and hepatocellular carcinoma. Jaundice and hepatic tumor developed in 1981, after she had received oxymetholone for 10 years. Liver biopsy revealed peliosis hepatis. Androgenic therapy was stopped and the jaundice resolved. However, the hepatic tumor was observed to be unchanged. The patient died of disseminated squamous cell carcinoma, but no metastatic lesions from hepatocellular carcinoma were detected in the autopsy. The association of Fanconi's anemia and squamous cell carcinoma is reviewed, and the malignant potential of androgen-related hepatic tumors is discussed.     

Detection of GPI-anchored protein-deficient cells in patients with aplastic anaemia and evidence for clonal expansion during the clinical course

The incidence of patients with aplastic anaemia (AA) who show a deficiency of glycosylphosphatidylinositol (GPI)-anchored proteins on their peripheral blood (PB) or bone marrow (BM) cells is higher than that previously reported. We analysed the expression of CD55 or CD59 on PB and BM cells and those of colonies and bursts formed in cultures with marrow cells from AA patients. 4/21 (19%) AA cases later developed paroxysmal nocturnal haemoglobinuria (PNH). 7/17 (41. 2%) AA cases showed a subpopulation without GPI-anchored proteins. The defect characteristic for PNH was observed only on the colonies/bursts but not on the PB or BM cells in three cases. We found the affected colonies/bursts in cultures using thawed marrow cells which had been cryopreserved at the diagnosis of aplasia in one patient who developed PNH 3 years later. Two different mutations of the PIG-A gene were found in the colonies/bursts at the time of AA. The nucleotide sequences were identical between the colonies/bursts at the time of AA and those after the transition to PNH. However, one of the mutations was detected only at the haemopoietic progenitor level but not in PB granulocytes. There may be latent subclones with PNH abnormalities which could expand to the level of clinical detection, or which do not progress and remain indolent for a relatively long time, implying the different extents of clonal expansion among the affected progenitors.     

Primary acquired sideroblastic anaemia and myeloproliferative disease: a report on three cases

Summary Three patients who presented with primary acquired sideroblastic anaemia (PASA) later developed myelofibrosis (MF). As both are clonal disorders a second mutation is suggested.     

Chromosome damage and repair in children with sickle cell anaemia and long-term hydroxycarbamide exposure

Hydroxycarbamide (hydroxyurea) provides laboratory and clinical benefits for adults and children with sickle cell anaemia (SCA). Given its mechanism of action and prior reports of genotoxicity, concern exists regarding long-term toxicities and possible carcinogenicity. We performed cross-sectional analyses of chromosome stability using peripheral blood mononuclear cells (PBMC) from 51 children with SCA and 3-12 years of hydroxycarbamide exposure (mean age 13·2 ± 4·1 years), compared to 28 children before treatment (9·4 ± 4·7 years). Chromosome damage was less for children receiving hydroxycarbamide than untreated patients (0·8 ± 1·2 vs. 1·9 ± 1·5 breaks per 100 cells, P = 0·004). There were no differences in repairing chromosome breaks after in vitro radiation; PBMC from children taking hydroxycarbamide had equivalent 2 Gy-induced chromosome breaks compared to untreated patients (30·8 ± 16·1 vs. 31·7 ± 8·9 per 100 cells, P = not significant). Radiation plus hydroxycarbamide resulted in similar numbers of unrepaired breaks in cells from children on hydroxycarbamide compared to untreated patients (95·8 ± 44·2 vs. 76·1 ± 23·1 per 100 cells, P = 0·08), but no differences were noted with longer exposure (97·9 ± 42·8 breaks per 100 cells for 3-6 years of hydroxycarbamide exposure vs. 91·2 ± 48·4 for 9-12 years of exposure). These observations provide important safety data regarding long-term risks of hydroxycarbamide exposure for children with SCA, and suggest low in vivo mutagenicity and carcinogenicity.