Geoffrey C. Beverstock

Evolution of acquired severe aplastic anaemia to myelodysplasia and subsequent leukaemia in adults

Myelodysplasia (MDS) and leukaemia following acquired aplastic anaemia has been reported as a rare event occurring in about 5% of patients. Improved results in survival of patients with severe aplastic anaemia (SAA) and subsequent prolonged follow‐up created the possibility of evaluating the occurrence of MDS and leukaemia in 38 adult patients with acquired SAA surviving two or more years without bone marrow transplantation. Five patients, age 22, 35, 47, 56, 72 years, two females, three males, all with idiopathic SAA and normal cytogenetic analysis developed a refractory anaemia (RA) 7, 30, 48, 56, 142 months after diagnosis of SAA. In 3/5 RA evolved into an acute myeloid leukaemia (AML) either via a chronic myelomonocytic leukaemia (CMML) (2/3) or via RA with excess of blasts (RAEB) (1/3). Three patients revealed a monosomy 7 during MDS and/or leukaemic phase. One patient died during RA phase without cytogenetic abnormalities. A pattern of evolution could be identified in these patients revealing well‐documented SAA ‐ improvement of bone marrow haematopoiesis ‐ dyshaematopoietic features of one or more cell lines with predominance of dyserythropoiesis ‐ RA ‐ RAEB or CMML ‐ AML. These five patients represent more than 10% of all patients surviving at least 2 years. This implies that the risk of developing MDS and leukaemia in SAA patients surviving with autologous marrow, might increase with longer follow‐up.

Detection of CBP rearrangements in acute myelogenous leukemia with t(8;16)

The CREB-binding protein (CBP) is a large nuclear protein that regulates many signal transduction pathways and is involved in chromatin-mediated transcription. The translocation t(8;16)(p11;p13.3) consistently disrupts two genes: the CBP gene on chromosome band 16p13.3 and the MOZ gene on chromosome band 8p11. Although a fusion of these two genes as a result of the translocation is expected, attempts at detecting the fusion transcript by reverse transcriptase polymerase chain reaction (RT-PCR) have proven difficult; to date, only one in-frame CBP/MOZ fusion transcript has been reported. We therefore sought other reliable means of detecting CBP rearrangements. We applied fluorescence in situ hybridization (FISH) and Southern blot analyses to a series of AML patients with a t(8;16) and detected DNA rearrangements of both the CBP and the MOZ loci in all cases tested. All six cases examined for CBP rearrangements have breakpoints within a 13 kb breakpoint cluster region at the 5′ end of the CBP gene. Additionally, we used a MOZ cDNA probe to construct a surrounding cosmid contig and detect DNA rearrangements in three t(8;16) cases, all of which display rearrangements within a 6 kb genomic fragment of the MOZ gene. We have thus developed a series of cosmid probes that consistently detect the disruption of the CBP gene in t(8;16) patients. These clones could potentially be used to screen other cancer-associated or congenital translocations involving chromosome band 16p13.3 as well.

Rapid detection of chromosome 16 inversion in acute nonlymphocytic leukemia, subtype M4: regional localization of the breakpoint in 16p

The pericentric inversion of chromosome 16 characteristic for acute nonlymphocytic leukemia, subtype M4, was detected in five patients by means of nonradioactive in situ hybridization of complete cosmids. First, five cosmids situated along the short arm of chromosome 16 were used to map the breakpoint of the inversion distal to the rare folate-sensitive fragile site FRA16A. Then, the use of two cosmids on either side of the breakpoint, combined with a probe specific for the centromeric region of chromosome 16, readily detected the inversion, even in poor metaphase spreads.

DETECTION OF TRISOMY 8 IN HEMATOLOGICAL DISORDERS BY IN SITU HYBRIDIZATION

An alphoid repetitive DNA (D8Z2) probe specific for the pericentromeric region of chromosome 8 was used to detect extra copies of chromosome 8 in bone marrow cells obtained from 10 patients with hematological disorders and five controls. Numerical aberrations of chromosome 8 were established by conventional banding techniques. Trisomy 8 was found in four patients with myelodysplastic syndrome (MDS) and three with acute myeloid leukemia (AML). Three additional patients with MDS exhibited an extra chromosome 8 in only one metaphase. In five of the seven trisomy cases, the presence of the trisomy 8 clone was confirmed by in situ hybridization (ISH). In one case of AML with trisomy 8, detected by GTG-banding, no significant numbers of cells containing three spots were found using the alphoid repetitive probe; however, hybridization with a chromosome 8-specific library revealed that the alleged extra chromosome 8 was a translocation chromosome containing only the long arm of chromosome 8. Due to a lack of material, it was not possible to achieve optimal ISH results on the trisomy 8 bone marrow cells of patient 7. In the three MDS patients with a single trisomy 8 metaphase, a slight, albeit significant, increase of trisomy 8 interphase cells was found with ISH. We conclude that this probe is useful for cytogenetic studies. Moreover, ISH, in general, is a powerful tool for precise classification of chromosomal aberrations and can also contribute significantly to the clinical evaluation of patients with hematological disorders.

t(5;12)(q31;p12) A clinical entity with features of both myeloid leukemia and chronic myelomonocytic leukemia

We report two patients with a myeloproliferative disorder (Philadelphia chromosome-negative chronic myeloid leukemia) and t(5;12)(q31;p12). Until now, only three cases of a translocation (5;12)(q31;p12) have been reported. All investigators had problems classifying their patients disease into one of the well-defined entities of either MPD or myelodysplastic disorders. We postulate that this translocation may represent a subgroup of patients with features of both chronic myeloid leukemia and chronic myelomonocytic leukemia (CMMoL).

Effect of subcutaneously administered human recombinant erythropoietin on erythropoiesis in patients with myelodysplasia

Summary In a phase II study, 12 patients with a myelodysplastic syndrome (MDS) and anaemia (nine transfusiondependent) were treated with recombinant human erythropoietin (rHuEpo) to assess the therapeutic effect on erythropoiesis and on transfusion requirement. Patients with a low risk of developing acute leukaemia were included, i.e. refractory anaemia (RA), RA with ringed sideroblasts (RARS) and RA with excess blasts (RAEB), providing the percentage of myeloblasts in the bone marrow did not exceed 10%. Recombinant HuEpo treatment was initated at a dose of 50 units/kg body weight and administered subcutaneously three times weekly. At 3‐week intervals the dose was increased with 50 units/kg per injection, until after 15 weeks a maximum dose of 250 units/kg three times weekly was reached. All patients completed the study. Recombinant HuEpo was well tolerated and no serious side effects were seen. There was no evidence of the emergence of a new malignant clone in response to rHuEpo as shown by sequential karyotyping. In none of the patients was an increase in haemoglobin level or a diminished red blood cell transfusion requirement seen. In four out of 10 evaluable sequential bone marrow smears, an increase in erythropoiesis was seen, suggesting stimulation of ineffective red cell production. One of these patients also showed a rise in reticulocyte count. The number of erythroid progenitor cells (BFU‐E and CFU‐E) in blood and bone marrow was not affected by rHuEpo treatment. Also no change in the number of myeloid progenitor cells (CFU‐GM) in blood and bone marrow was noted. In conclusion, subcutaneous treatment with rHuEpo at dosages up to 250 units/kg body weight (three times weekly) fails to increase the haemoglobin level or to diminish the transfusion requirement in patients with MDS and anaemia. It is unclear whether higher doses of rHuEpo are effective or whether patients with less severe anaemia who are transfusion independent, have a higher likelihood of response.

Fetal cell detection in maternal blood: A study in 236 samples using erythroblast morphology, DAB and HbF staining, and FISH analysis

A protocol to detect fetal nucleated red blood cells (NRBCs) was tested in 217 pregnant women and in 19 nonpregnant controls. All the pregnant women were sampled after chorionic villus sampling (CVS); 20 were also sampled pre-CVS. NRBC recognition was based upon morphology by using staining of hemoglobin with 3,3-diaminobenzidin (DAB) or by immunocytochemical staining for fetal hemoglobin (HbF). This was combined with FISH analysis for both the X- and Y-chromosomes on the same cells. Progressive refinement of the methods increased the number of cases where NRBCs were detected from 53% (DAB) to 75% and 78% for DAB and HbF staining, respectively, with on average 43 NRBCs (range, 1-220). DAB gave a slightly higher yield than HbF in the lower cell count range (<25). In 6 out of 18 controls, NRBCs were detected with DAB, vs. 1 out of 19 (5%) with HbF. FISH analysis in 41 cases resulted in correct sex prediction in 80% (DAB) and 89% (HbF), respectively. Our data demonstrated an increase of cases with NRBCs (30% to 75%), as well as a rise of the mean number of NRBCs (6 to 29 cells), after CVS. We conclude that DAB staining is a straightforward way to screen for the presence of NRBCs in maternal blood, but is not specific for NRBCs of fetal origin. HbF immunophenotyping is a reliable marker for fetal NRBCs, which detected slightly fewer NRBCs than DAB-staining, but improved sex prediction and significantly reduced false-positive results. CVS at 10-13 weeks of gestation causes a significant increase of NRBCs in maternal blood. These data indicate that further refinement of NRBC detection is needed before application of noninvasive prenatal diagnosis using maternal blood is feasible.

Premature menopause because of an inherited deletion in the long arm of the X-chromosome.

A family is described in which both a mother and an infertile daughter had premature menopause at the ages of 31 and 28 years, respectively. Initially, an extensive investigation revealed no apparent cause for their conditions. However, when cytogenetic analysis in the daughter was performed, a terminal deletion in the long arm of one of the X-chromosomes was found. The karyotype was: 46,Xdel(X),(q25-qter). Chromosomal investigation in the mother showed an identical deletion. The karyotype of the patients 35-year-old sister is normal. She has a normal menstrual cycle and two normal children. The presence of such familial cases suggests that chromosomal investigation should be considered in young women with oligomenorrhea, especially those whose mothers have experienced a premature menopause.

Combined GTG-banding and nonradioactive in situ hybridization improves characterization of complex karyotypes

Nonradioactive in situ hybridization (ISH) using biotinylated centromere probes for chromosomes 1, 6, 7, 10, 16, 17, 18, and the X, respectively, was combined with GTG-banding to study cytogenetic changes in two different ovarian cancer cell lines. ISH was performed after GTG-banding on the same metaphase. The use of a low trypsin concentration (0.01%) in the banding procedure was essential for subsequent ISH. This combined approach allows the detection of subtle chromosomal rearrangements and appears to aid the identification of marker chromosomes.

Mosaicism of the 5q deletion as assessed by interphase FISH is a common phenomenon in MDS and restricted to myeloid cells

Interstitial deletion of chromosome 5q is a common cytogenetic abnormality observed in MDS. We have used fluorescence in situ hybridization (FISH) to determine accurately the percentage of cytogenetically abnormal peripheral blood cells. YAC and cosmid probes localized to chromosome 5q were hybridized to interphase nuclei from purified polymorphonuclear cells (PMNs) from six MDS patients with chromosome 5 deletions. Per patient, 25–67% of the cells exhibited one signal for the 5q31-q33 specific probes IL-4, D5S207 and c-fms. This percentage was constant for the various probes utilized for each patient. Hybridization of the same probes to PMNs from healthy individuals and hybridization of probes (D5S39 and D5S498) localized outside the deleted segments to PMNs of the patients, resulted in 90–95% nuclei with two signals. In addition, FACS-purified peripheral blood cells were investigated by FISH using the IL-4 cosmid. This demonstrated that the hybridization pattern in monocytes was similar to that observed in PMNs, whereas T-lymphocytes showed no loss of signals. These results indicate that a subfraction of the myeloid progenitor cells have acquired the 5q deletion.