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Detection of Monosomy 7 in Bone Marrow by Fluorescence In Situ Hybridization
Detection of Monosomy 7 in Bone Marrow by Fluorescence In Situ Hybridization A Study of Fanconi Anemia Patients and Review of the Literature Virginia C. Thurston, Tina M. Ceperich, Gail H. Vance, and Nyla A. Heerema
ABSTRACT: Monosomy 7 is frequently found in the bone marrow of patients with Fanconi anemia (FA), marrow myelodysplasia, or acute myelogenous leukemia and is associated with poor prognosis. In our laboratory, cytogenetic analysis of bone marrow from an FA patient found 2 of 30 cells with monosomy 7, but the results of fluorescence in situ hybridization (FISH) indicated that 83 of 207 cells (40%) had monosomy 7. FISH was then used to analyze two earlier samples from the index case, neither of which had monosomy 7 as determined by standard cytogenetics. The FISH analysis determined that the first sample, taken 19 months earlier, had 8 of 200 cells (4%) with monosomy 7 and the second sample, taken 7 months later, contained 43 of 200 cells (21.5%) with monosomy 7. These results indicate a slow evolution toward monosomy 7 in the patient’s bone marrow. Standard metaphase chromosome analysis represents only spontaneously dividing cells, leading us to hypothesize that FISH was detecting monosomy 7 in nondividing cells and that it might be useful in the early detection of abnormal clones. To test this hypothesis, FISH was performed on 13 bone marrow samples from nine patients with FA who did not exhibit monosomy 7 by cytogenetic analysis. Monosomy 7 was detected in 3.44% of nuclei in FA patients and in 3% of nuclei in normal controls. To date, none of these nine FA patients have developed monosomy 7 or leukemia. They are being monitored by standard cytogenetics and by FISH to determine whether monosomy 7 develops and whether it can be detected by FISH prior to its detection by standard cytogenetics. As standard practice, we have adopted FISH analysis for monosomy 7 in all patients with FA. © Elsevier Science Inc., 1999. All rights reserved.
INTRODUCTION Fanconi anemia (FA) is a rare autosomal recessive disease characterized by multiple congenital abnormalities, bone marrow failure, and cancer susceptibility. FA is found in all races and groups and has been reported to have a carrier frequency of 1:300 [1]. Patients with FA may have growth retardation and abnormalities of the skin, upper extremities, kidneys, and gastrointestinal system [2]. Fanconi cells exhibit an abnormally high level of baseline chromosomal breakage [3] and are hypersensitive to both the cytotoxic and the clastogenic effects of DNA crosslinking agents, such as mitomycin C and diepoxybutane
From the Department of Medical and Molecular Genetics, Indiana University School of Medicine (V. C. T., G. H. V.), Indianapolis, Indiana, USA; and the Wayne Hughes Institute (T. M. C., N. A. H.), Roseville, Minnesota, USA. Address reprint requests to: Dr. V. C. Thurston, Department of Medical and Molecular Genetics, Indiana University School of Medicine, 975 Walnut Street, Indianapolis, IN 46202. Received March 18, 1998; accepted July 10, 1998. Cancer Genet Cytogenet 109:154–160 (1999) Elsevier Science Inc., 1999. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
(DEB) [4], which is used as a diagnostic indicator of the disease. FA patients also suffer from a progressive bone marrow failure and increased risk of cancer, predominately acute myelogenous leukemia (AML) [5]. Clonal cytogenetic abnormalities have been reported in the bone marrow of some FA patients with AML, as well as in some without cytological evidence of leukemia. The latter are said to be in the preleukemic phase of the disease. One of the most frequent cytogenetic findings in the bone marrow of patients with FA and AML is monosomy 7, the loss of one chromosome 7 [6]. This cytogenetic abnormality has been associated with poor prognosis and progression to leukemia [7]. Recently, we used fluorescence in situ hybridization (FISH) to study a FA patient in whom standard metaphase chromosome analysis showed monosomy 7 in 2 of 30 bone marrow cells. FISH analysis detected monosomy 7 in 83 of 207 (40%) of bone marrow cells. Eventually, the patient developed monosomy 7 as defined by standard cytogenetic practice in all metaphases analyzed. These observations are similar to those of Kadam et al. [8], who used
0165-4608/99/$–see front matter PII S0165-4608(98)00167-8
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Monosomy 7 in Bone Marrow of Fanconi Anemia Table 1 Karyotypes of patients with FA Sample
Age
P1 P1a P1b P2 P2a P3 P4 P4a P5
10 12 13 16 17 4 8 8 12
P6 P7 P8 P9
7 4 13 11
MATERIALS AND METHODS Age at diagnosis
Karyotype 46,XY[20] 46,XY[20] 46,XY[20] 46,XX[20] 46,XX[20] 46,XY[20] 46,XY[20] 46,XY[20] 46,XX,dup(1)(p22p34), add(18)(p11.3)[15] 46,XY[20] 46,XX[20] 46,XX[20] 46,XY[20]
9
14 3 7 11 6 4 11 8
a
Second sample.
b
Third sample.
FISH to examine the bone marrow cells of two AML patients in whom monosomy 7 had not been detected by standard cytogenetic analysis. FISH analysis detected monosomy 7 in 17% of cells from one patient and in 39.8% of cells from the other. The investigators hypothesized that the monosomy 7 clones detected by FISH were unable to enter mitosis in vitro and therefore could not be detected by metaphase chromosome analysis. According to this hypothesis, our observations would indicate that the dividing bone marrow cells from our FA patient, analyzed by standard metaphase chromosome analysis, were not representative of the patient’s marrow. If this is the case, it might be possible to use FISH for the early detection of abnormal clones. To explore this possibility, we used FISH to examine bone marrow cells from karyotypically normal FA patients in an effort to detect monosomy 7.
Case Report The index case was a 10-year-old white girl when, on December 16, 1991, she was diagnosed with Fanconi anemia by DEB testing for chromosome breakage. She presented with microcephaly, absent left thumb, and a nonfunctional dysmorphic right thumb. She had bilateral abnormal middle-ear anatomy with significant loss of hearing and was noted to have other dysmorphic features, including webbed toes, malposition of the vaginal and rectal openings, and multiple cafe-au-lait spots. At the time of diagnosis, she was noted to be pancytopenic. A bone marrow aspirate demonstrated a hypocellular marrow of all three cell lineages consistent with aplastic anemia. Cytogenetic analysis of a bone marrow aspirate at diagnosis demonstrated a normal karyotype of 46,XX. Three years after diagnosis, cytogenetic analysis of a bone marrow aspirate demonstrated 1 of 30 cells with random loss of chromosome 7. The remainder of the karyotype was normal 46,XX. Another aspirate 11 months later demonstrated loss of chromosome 7 in 2 of 30 cells, but FISH analysis of this aspirate revealed monosomy 7 in 83 of 207 cells (40%) examined. This finding led us to perform FISH analysis on a stored cell pellet from the aspirate taken 3 years after diagnosis. FISH analysis revealed monosomy 7 in 43 of 200 cells (22%). Bone marrow transplantation was recommended. Histological examination of a marrow aspirate taken just before transplantation showed myeloid dysplasia and chromosomal analysis of this specimen showed 20 of 20 cells with monosomy 7. Twenty-six days after bone marrow transplantation, the patient succumbed to a diffuse necrotizing fungal infection of her lungs. Patients Bone marrow cells from normal controls and patients with FA or monosomy 7 were obtained at the Indiana University School of Medicine for use in this study. The cells were fixed in methanol:acetic acid (3:1) and stored at 2208C until use. The test group comprised 13 cell pellets
Table 2 Diagnosis and karyotypes of patients with monosomy 7 Sample
Age
Diagnosis
M1
40
AML
M2
3
MDS/AML
M3 M4 M4a
49 2 2
NHL MDS/AML MDS/AML
M5
,1
Childhood monosomy 7
Karyotype 45,XY,inv(3)(q21q26), 27,t(7;12)(p15;p13)[20] 45,XY,27[1]/45, idem,t(10;12)(q26;q22)[4]/ nonclonal 27[2]/46,XY[13] 45,XX,27[16]/46,XX[4] 45,XY,27[29]/nonclonal[1] 45,XY,27[4]/ 45,idem,t(10;12)(q26;q22)[4]/ 45,idem,t(2;11)(p21;q23), add(8)(q24.1)[2]/nonclonal 27[8]/ 46,XY[2] 45,XY,27[1]/45,idem,26,1r[2]/ 46,XY[27]
Abbreviations: AML, acute myelogenous leukemia; MDS, marrow myelodysplastic syndrome; NHL, non-Hodgkin lymphoma. a
Second sample.
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from nine patients with FA but without monosomy 7 by cytogenetic analysis. The cytogenetic characteristics of these nine patients are listed in Table 1. The positive controls consisted of 6 cell pellets from five patients with monosomy 7 determined by cytogenetic analysis (Table 2). Cell pellets from nine karotypically normal bone marrow donors also were used to determine the hybridization efficiency of the FISH probe.
at room temperature in three changes of 1 3 PBD (Oncor). Signal detection was with anti-dig-FITC antibody with the use of a commercially prepared signal detection and amplification kit (Oncor). One layer of signal detection was generally sufficient for clear signal with very little background. The cells were counterstained with propidium iodide, mounted in antifade, cover slipped, and sealed with nail polish.
FISH Slides were prepared by standard laboratory procedures with the use of cell pellets stored in 3:1 methanol:acetic acid fixative. Cell pellets were dropped onto glass slides and dehydrated in an ethanol series (70–100%) at 2208C. Cells were denatured in 70% formamide/2 3 SSC (pH 7.0) at 728C for 2 min, dehydrated in a cold ethanol series, and placed on a 428C slide warmer. A chromosome 7 a-satellite DNA probe (1.5 mL) was obtained from Oncor (Gaithersburg, MD) and mixed with 15 mL of Hybrisol VI (50 ng/ mL; Oncor), denatured at 708C for 5 min, dispensed onto slides, and allowed to hybridize at 378C overnight. After hybridization, the slides were washed at 458C for 5 min in each of three changes of 50% formamide/2 3 SSC, 2 min at 458C in three changes of 2 3 SSC, and 2 min
FISH Analysis FISH signals were analyzed by using a Leitz Aristoplan microscope equipped with epifluorescence and were photographed by using Kodak film (Ektachrome ASA 400). Images were also obtained with a Photometrics CCD Camera and processed with Quips FISH Imaging software from Vysis (Downers Grove, IL). FISH analysis of all specimens was carried out in a blinded fashion to prevent observer bias. Two hundred cells were read for each patient, the number of chromosome 7 signals for each nucleus was scored by two readers, and the data were exported into Excel for further analysis. To determine whether FISH was able to detect monosomy 7 in cytogenetically normal specimens, the FISH results were compared with the standard metaphase cytogenetic analyses.
Figure 1 Average number of bone marrow cells with one chromosome 7 signal in normal controls.
157
Monosomy 7 in Bone Marrow of Fanconi Anemia
Figure 2 Number of bone marrow cells with one chromosome 7 signal in monosomy 7 patients.
RESULTS Control Cases To determine hybridization efficiency, we employed FISH analysis by using the chromosome 7 centromere probe to
examine bone marrow samples from nine bone marrow donors with normal karyotypes (Fig. 1). Signals were demonstrated in an average of 6 of 200 (3.0%) cells examined, giving a hybridization efficiency of 97%.
Table 3 Summary of cytogenetic and FISH results from index case Date
Sample
Cytogenetics
12/91 12/91 3/92 5/93 3/94 4/94 5/94
Bone marrow Blood Bone marrow Bone marrow Bone marrow Bone marrow Bone marrow
46,XX[24] (no random loss (RL) of 7) Fanconi anemia positive by DEB 46,XX[19] (no RL of 7) 46,XX[20] (no RL of 7) 46,XX[20] (no RL of 7) 46,XX[20] (no RL of 7) 46,XX[20] (1 cell with RL of 7)
1/95
Bone marrow
46,XX[20] (1 cell with RL of 7)
12/95
Bone marrow
46,XX[30] (2 cells with RL of 7)
4/96 7/96 8/96
Bone marrow Bone marrow Bone marrow
47,XX,27,121,122[7]/46,XX[12] 45,XX,27,add(18)(p11.2)[20]
FISH
nuc ish 7cen(D7Z132)[191]/ 7cen(D7Z131)[8] (within normal laboratory parameters) nuc ish 7cen(D7Z131)[43]/ 7cen(D7Z132)[157] nuc ish 7cen(D7Z131)[83]/ 7cen(D7Z132)[124]
nuc ish 7cen(D7Z131)[164]/ 7cen(D7Z132)[2]
158
V. C. Thurston et al. Four years and 7 months after diagnosis, standard cytogenetics revealed a karyotype of 45,XX,27,add(18)(p11.2) in 20 bone marrow cells examined. The next month, FISH analysis revealed monosomy 7 in 98.7% of the bone marrow nuclei examined. Figure 3A and B shows a metaphase spread and interphase nucleus from bone marrow of the index case hybridized with a chromosome 7 centromere probe, demonstrating the presence of a single chromosome 7. A bone marrow nucleus from a normal individual demonstrates two chromosome 7 signals by FISH analysis (Fig. 3C). Test Cases With the use of the same chromosome 7 centromere probe, FISH analysis was performed on 13 bone marrow specimens from nine patients with Fanconi anemia (Fig. 4). Single signals for the chromosome 7 centromere were seen in an average of only 6.88 of 200 cells (3.44%) examined.
DISCUSSION
Figure 3 Metaphase and interphase of bone marrow cells hybridized with chromosome 7 centromere probe. (A) Metaphase spread from index case. Notice that spread contains only one signal. (B) Interphase cells from index case showing one centromere signal. (C) Interphase bone marrow cells from a normal male 46,XX[20], with two centromere signals indicating two copies of chromosome 7.
The positive control group consisted of five patients with monosomy 7 as determined by cytogenetic analysis. When FISH was used to analyze bone marrow from these patients, single signals were seen in an average of 151 of 200 (75.5%) cells examined (Fig. 2). Index Case In the first FISH-analyzed bone marrow specimen (taken 3 years after diagnosis), a low level (22%, or 43 of 200 cells) of monosomy 7 was detected (Table 3). Standard cytogenetic analysis performed at the same time revealed a karyotype of 46,XX[20] with only a single cell showing random loss of chromosome 7. Within 12 months, 83 of 200 cells (40%) examined by FISH demonstrated monosomy 7 despite a normal bone marrow karyotype with 2 of 30 cells showing apparent random loss of chromosome 7 (6.6%).
Two recent studies [9, 10] demonstrate an actuarial risk of 52% for developing MDS or AML or both in patients with Fanconi anemia, which is much higher than the 10% rate cited in most studies that were not actuarial [11–13]. Many FA patients were monosomic for chromosome 7 in their bone marrow prior to or coincident with the development of leukemia [13]. Bone marrow clonal cytogenetic abnormalities have been reported in some FA patients without leukemia, and these patients are said to be in a preleukemic phase [6]. The most frequent chromosome abnormalities are monosomy 7 and translocations or duplications involving 1q [6, 11, 14]. Early detection of these abnormalities could lead to earlier intervention and treatment of these cases. Patients with myelodysplastic syndromes (MDS) may also display monosomy 7 clones in their bone marrow [15]. Yunis et al. [15] found monosomy 7 to be an indicator of poor prognosis and a high probability of evolution to AML. In a much larger study, Yunis et al. [16] again found that, in both MDS and AML, the presence of monosomy 7 was an indicator of poor prognosis and survival times of less than 1 year. None of these studies utilized FISH in the detection of monosomy 7. Monosomy 7 detected by FISH, but not by conventional cytogenetics, has been reported in patients with acute leukemia or MDS. For example, Kadam et al. [8] used FISH to detect monosomy 7 clones in the bone marrow of two patients with AML who did not have monosomy 7 by standard cytogenetics. The investigators hypothesized that the monosomy 7 clone detected by FISH was unable to enter mitosis in vitro and was therefore not detected by conventional cytogenetics. A later study by Kadam et al. [17] used FISH to examine 21 patients with acute lymphoblastic leukemia and detected monosomy 7 in five cases. A separate study using FISH to detect monosomy 7 in MDS [18] found that 5 of 67 patients had monosomy 7 (.6.3 cells with single signals/patient) detected by FISH, but the anomaly was not detected by conventional cytogenetics.
159
Monosomy 7 in Bone Marrow of Fanconi Anemia
Figure 4 Average number of bone marrow cells with one chromosome 7 signal in Fanconi anemia patients.
The percentage of interphase cells with only one signal ranged from 14.4 to 39% (normal was 2.9–5.3%). Another study reported by Arif et al. [19] examined bone marrow aspirates from 25 patients with MDS and 25 patients with AML, none of whom exhibited monosomy 7 by standard cytogenetic analysis. FISH detected monosomy 7 in 9 of 25 MDS patients and 11 of 25 AML patients. Among those who showed monosomy 7 by FISH, 5 MDS and 3 AML patients had normal karyotypes by standard cytogenetic analysis. The rest of the patients with monosomy 7 by FISH had abnormal karyotypes but did not have abnormalities involving chromosome 7. Four of the 5 MDS patients who went on to develop AML had monosomy 7 by standard cytogenetics, indicating that the presence of a monosomy 7 clone in metaphase cells is indicative of a higher rate of progression to AML. Our study contributes to the growing body of evidence that the presence of a monosomy 7 clone in bone marrow aspirates can go undetected by standard cytogenetic analysis. In our index case, the first bone marrow specimen analyzed by FISH contained a low level (22%) of cells with monosomy 7. Standard cytogenetic analysis performed at the same time revealed a normal karyotype with only a single cell showing random loss of chromosome 7. Within the next 12 months, consecutive bone marrow samples dem-
onstrated progressively higher percentages of monosomy 7 by FISH analysis without a corresponding increase by standard cytogenetics. Nineteen months after the first FISH-analyzed specimen, standard cytogenetics revealed a karyotype of 45,XX,27,add(18)(p11.2) in 20 bone marrow cells examined; and, in the next month, FISH analysis revealed monosomy 7 in 98.7% of the 200 bone marrow nuclei examined. In our study of 13 samples from nine other patients with FA, monosomy 7 was not detected by standard karyotype or by FISH. To date, none of these patients have developed leukemia. Continued monitoring of these patients by standard cytogenetics and FISH will determine whether any abnormal clones with monosomy 7 develop and whether they can be detected by FISH before they are detected by standard metaphase cytogenetics. If monosomy 7 is evidence of bone marrow transformation, early detection of a monosomy 7 clone by FISH would allow for earlier intervention, such as bone marrow transplantation, to help prevent or delay the onset of leukemia in these patients. As standard practice, we have adopted FISH analysis with the use of chromosome 7 centromere probes for all patients with FA.
This study was supported by the Fanconi Anemia Research Fund.
160 REFERENCES 1. Swift M (1971): Fanconi’s anaemia in the genetics of neoplasia. Nature 230:370–373. 2. Alter BP, Young NS (1993): The bone marrow failure syndromes. In: Hematology of Infancy and Childhood. DG Nathan, FA Oski, eds. WB Saunders, Philadelphia, pp. 216– 316. 3. Schroeder TM, Anschults R, Knoff A (1964): Spontane Chromosomenaberrationen bei familiarer Panmyelopathie. Humangenetik 1:194. 4. Auerbach AD, Wolman SR (1976): Susceptibility of Fanconi anaemia fibroblasts to chromosome damage by carcinogens. Nature 261:494. 5. Liu JM, Buchwald M, Walsh CE, Young NS (1994): Fanconi anemia and novel strategies for therapy. Blood 84:3995– 4007. 6. Auerbach AD, Allen RG (1991): Leukemia and preleukemia in Fanconi anemia patients: a review of the literature and report of the International Fanconi Anemia Registry. Cancer Genet Cytogenet 51:1–12. 7. Yunis JJ, Brunning RD (1986): Prognostic significance of chromosomal abnormalities in acute leukaemias and myelodysplastic syndromes. Clin Haematol 15:597–620. 8. Kadam P, Umerani A, Srivastava A, Masterson M, Lampkin B, Raza A (1993): Combination of classical and interphase cytogenetics to investigate the biology of myeloid disorders: detection of masked monosomy 7 in AML. Leuk Res 17:365– 374. 9. Butturini A, Gale RP, Verlander PC, Alder-Brecher B, Gillio A, Auerbach A (1994): Hematologic abnormalities in Fanconi anemia: an International Fanconi Anemia Registry study. Blood 84:1650–1655. 10. Gillio AP, Verlander PC, Batish SD, Giampietro PF, Auerbach A (1997): Phenotypic consequences of mutations in the Fan-
V. C. Thurston et al. coni anemia FAC gene: an International Fanconi Anemia Registry study. Blood 90:105–110. 11. Auerbach AD (1992): Fanconi anemia and leukemia: tracking the genes. Leukemia 6 (Suppl. 1):1–4. 12. Berger R, Le Coniat M, Schaison G (1993): Chromosome abnormalities in bone marrow of Fanconi anemia patients. Cancer Genet Cytogenet 65:47–50. 13. Stivrins TJ, Davis RB, Sanger W, Fritz J, Purtilo DT (1984): Transformation of Fanconi’s anemia to acute nonlymphocytic leukemia associated with emergence of monosomy 7. Blood 64:173–176. 14. Young NS, Alter BP (1994): Clinical features of Fanconi’s anemia. In: Aplastic Anemia: Acquired and Inherited. NS Young, BP Alter, eds. WB Saunders, Philadelphia, pp. 275–309. 15. Yunis JJ, Rydell RE, Oken MM, Arnesen MA, Mayer MG, Lobell M (1986): Refined chromosome analysis as an independent prognostic indicator in de novo myelodysplastic syndromes. Blood 67:1721–1730. 16. Yunis JJ, Lobell M, Arnesen MA, Oken MM, Mayer MG, Rydell RE, Brunning RD (1988): Refined chromosome study helps define prognostic subgroups in most patients with primary myelodysplastic syndrome and acute myelogenous leukaemia. Br J Haematol 68:189–194. 17. Kadam P, Masterson M, Suokup S, Morre C, Raza A, Lampkin BC (1994): Detection of unexpected clones of monosomy 7 in childhood acute lymphoblastic leukemia using fluorescence in situ hybridization. Anticancer Res 14:545–548. 18. Flactif M, Lau JL, Preudhomme C, Fenaux P (1994): Fluorescence in situ hybridization improves the detection of monosomy 7 in myelodysplastic syndromes. Leukemia 8:1012–1018. 19. Arif M, Tanaka K, Camodaran C, Asou H, Kyo T, Dohy H, Kamada N (1996): Hidden monosomy 7 in acute myeloid leukemia and myelodysplastic syndrome detected by interphase fluorescence in situ hybridization. Leuk Res 20:709–716.
ABSTRACT: Monosomy 7 is frequently found in the bone marrow of patients with Fanconi anemia (FA), marrow myelodysplasia, or acute myelogenous leukemia and is associated with poor prognosis. In our laboratory, cytogenetic analysis of bone marrow from an FA patient found 2 of 30 cells with monosomy 7, but the results of fluorescence in situ hybridization (FISH) indicated that 83 of 207 cells (40%) had monosomy 7. FISH was then used to analyze two earlier samples from the index case, neither of which had monosomy 7 as determined by standard cytogenetics. The FISH analysis determined that the first sample, taken 19 months earlier, had 8 of 200 cells (4%) with monosomy 7 and the second sample, taken 7 months later, contained 43 of 200 cells (21.5%) with monosomy 7. These results indicate a slow evolution toward monosomy 7 in the patient’s bone marrow. Standard metaphase chromosome analysis represents only spontaneously dividing cells, leading us to hypothesize that FISH was detecting monosomy 7 in nondividing cells and that it might be useful in the early detection of abnormal clones. To test this hypothesis, FISH was performed on 13 bone marrow samples from nine patients with FA who did not exhibit monosomy 7 by cytogenetic analysis. Monosomy 7 was detected in 3.44% of nuclei in FA patients and in 3% of nuclei in normal controls. To date, none of these nine FA patients have developed monosomy 7 or leukemia. They are being monitored by standard cytogenetics and by FISH to determine whether monosomy 7 develops and whether it can be detected by FISH prior to its detection by standard cytogenetics. As standard practice, we have adopted FISH analysis for monosomy 7 in all patients with FA. © Elsevier Science Inc., 1999. All rights reserved.
INTRODUCTION Fanconi anemia (FA) is a rare autosomal recessive disease characterized by multiple congenital abnormalities, bone marrow failure, and cancer susceptibility. FA is found in all races and groups and has been reported to have a carrier frequency of 1:300 [1]. Patients with FA may have growth retardation and abnormalities of the skin, upper extremities, kidneys, and gastrointestinal system [2]. Fanconi cells exhibit an abnormally high level of baseline chromosomal breakage [3] and are hypersensitive to both the cytotoxic and the clastogenic effects of DNA crosslinking agents, such as mitomycin C and diepoxybutane
From the Department of Medical and Molecular Genetics, Indiana University School of Medicine (V. C. T., G. H. V.), Indianapolis, Indiana, USA; and the Wayne Hughes Institute (T. M. C., N. A. H.), Roseville, Minnesota, USA. Address reprint requests to: Dr. V. C. Thurston, Department of Medical and Molecular Genetics, Indiana University School of Medicine, 975 Walnut Street, Indianapolis, IN 46202. Received March 18, 1998; accepted July 10, 1998. Cancer Genet Cytogenet 109:154–160 (1999) Elsevier Science Inc., 1999. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
(DEB) [4], which is used as a diagnostic indicator of the disease. FA patients also suffer from a progressive bone marrow failure and increased risk of cancer, predominately acute myelogenous leukemia (AML) [5]. Clonal cytogenetic abnormalities have been reported in the bone marrow of some FA patients with AML, as well as in some without cytological evidence of leukemia. The latter are said to be in the preleukemic phase of the disease. One of the most frequent cytogenetic findings in the bone marrow of patients with FA and AML is monosomy 7, the loss of one chromosome 7 [6]. This cytogenetic abnormality has been associated with poor prognosis and progression to leukemia [7]. Recently, we used fluorescence in situ hybridization (FISH) to study a FA patient in whom standard metaphase chromosome analysis showed monosomy 7 in 2 of 30 bone marrow cells. FISH analysis detected monosomy 7 in 83 of 207 (40%) of bone marrow cells. Eventually, the patient developed monosomy 7 as defined by standard cytogenetic practice in all metaphases analyzed. These observations are similar to those of Kadam et al. [8], who used
0165-4608/99/$–see front matter PII S0165-4608(98)00167-8
155
Monosomy 7 in Bone Marrow of Fanconi Anemia Table 1 Karyotypes of patients with FA Sample
Age
P1 P1a P1b P2 P2a P3 P4 P4a P5
10 12 13 16 17 4 8 8 12
P6 P7 P8 P9
7 4 13 11
MATERIALS AND METHODS Age at diagnosis
Karyotype 46,XY[20] 46,XY[20] 46,XY[20] 46,XX[20] 46,XX[20] 46,XY[20] 46,XY[20] 46,XY[20] 46,XX,dup(1)(p22p34), add(18)(p11.3)[15] 46,XY[20] 46,XX[20] 46,XX[20] 46,XY[20]
9
14 3 7 11 6 4 11 8
a
Second sample.
b
Third sample.
FISH to examine the bone marrow cells of two AML patients in whom monosomy 7 had not been detected by standard cytogenetic analysis. FISH analysis detected monosomy 7 in 17% of cells from one patient and in 39.8% of cells from the other. The investigators hypothesized that the monosomy 7 clones detected by FISH were unable to enter mitosis in vitro and therefore could not be detected by metaphase chromosome analysis. According to this hypothesis, our observations would indicate that the dividing bone marrow cells from our FA patient, analyzed by standard metaphase chromosome analysis, were not representative of the patient’s marrow. If this is the case, it might be possible to use FISH for the early detection of abnormal clones. To explore this possibility, we used FISH to examine bone marrow cells from karyotypically normal FA patients in an effort to detect monosomy 7.
Case Report The index case was a 10-year-old white girl when, on December 16, 1991, she was diagnosed with Fanconi anemia by DEB testing for chromosome breakage. She presented with microcephaly, absent left thumb, and a nonfunctional dysmorphic right thumb. She had bilateral abnormal middle-ear anatomy with significant loss of hearing and was noted to have other dysmorphic features, including webbed toes, malposition of the vaginal and rectal openings, and multiple cafe-au-lait spots. At the time of diagnosis, she was noted to be pancytopenic. A bone marrow aspirate demonstrated a hypocellular marrow of all three cell lineages consistent with aplastic anemia. Cytogenetic analysis of a bone marrow aspirate at diagnosis demonstrated a normal karyotype of 46,XX. Three years after diagnosis, cytogenetic analysis of a bone marrow aspirate demonstrated 1 of 30 cells with random loss of chromosome 7. The remainder of the karyotype was normal 46,XX. Another aspirate 11 months later demonstrated loss of chromosome 7 in 2 of 30 cells, but FISH analysis of this aspirate revealed monosomy 7 in 83 of 207 cells (40%) examined. This finding led us to perform FISH analysis on a stored cell pellet from the aspirate taken 3 years after diagnosis. FISH analysis revealed monosomy 7 in 43 of 200 cells (22%). Bone marrow transplantation was recommended. Histological examination of a marrow aspirate taken just before transplantation showed myeloid dysplasia and chromosomal analysis of this specimen showed 20 of 20 cells with monosomy 7. Twenty-six days after bone marrow transplantation, the patient succumbed to a diffuse necrotizing fungal infection of her lungs. Patients Bone marrow cells from normal controls and patients with FA or monosomy 7 were obtained at the Indiana University School of Medicine for use in this study. The cells were fixed in methanol:acetic acid (3:1) and stored at 2208C until use. The test group comprised 13 cell pellets
Table 2 Diagnosis and karyotypes of patients with monosomy 7 Sample
Age
Diagnosis
M1
40
AML
M2
3
MDS/AML
M3 M4 M4a
49 2 2
NHL MDS/AML MDS/AML
M5
,1
Childhood monosomy 7
Karyotype 45,XY,inv(3)(q21q26), 27,t(7;12)(p15;p13)[20] 45,XY,27[1]/45, idem,t(10;12)(q26;q22)[4]/ nonclonal 27[2]/46,XY[13] 45,XX,27[16]/46,XX[4] 45,XY,27[29]/nonclonal[1] 45,XY,27[4]/ 45,idem,t(10;12)(q26;q22)[4]/ 45,idem,t(2;11)(p21;q23), add(8)(q24.1)[2]/nonclonal 27[8]/ 46,XY[2] 45,XY,27[1]/45,idem,26,1r[2]/ 46,XY[27]
Abbreviations: AML, acute myelogenous leukemia; MDS, marrow myelodysplastic syndrome; NHL, non-Hodgkin lymphoma. a
Second sample.
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from nine patients with FA but without monosomy 7 by cytogenetic analysis. The cytogenetic characteristics of these nine patients are listed in Table 1. The positive controls consisted of 6 cell pellets from five patients with monosomy 7 determined by cytogenetic analysis (Table 2). Cell pellets from nine karotypically normal bone marrow donors also were used to determine the hybridization efficiency of the FISH probe.
at room temperature in three changes of 1 3 PBD (Oncor). Signal detection was with anti-dig-FITC antibody with the use of a commercially prepared signal detection and amplification kit (Oncor). One layer of signal detection was generally sufficient for clear signal with very little background. The cells were counterstained with propidium iodide, mounted in antifade, cover slipped, and sealed with nail polish.
FISH Slides were prepared by standard laboratory procedures with the use of cell pellets stored in 3:1 methanol:acetic acid fixative. Cell pellets were dropped onto glass slides and dehydrated in an ethanol series (70–100%) at 2208C. Cells were denatured in 70% formamide/2 3 SSC (pH 7.0) at 728C for 2 min, dehydrated in a cold ethanol series, and placed on a 428C slide warmer. A chromosome 7 a-satellite DNA probe (1.5 mL) was obtained from Oncor (Gaithersburg, MD) and mixed with 15 mL of Hybrisol VI (50 ng/ mL; Oncor), denatured at 708C for 5 min, dispensed onto slides, and allowed to hybridize at 378C overnight. After hybridization, the slides were washed at 458C for 5 min in each of three changes of 50% formamide/2 3 SSC, 2 min at 458C in three changes of 2 3 SSC, and 2 min
FISH Analysis FISH signals were analyzed by using a Leitz Aristoplan microscope equipped with epifluorescence and were photographed by using Kodak film (Ektachrome ASA 400). Images were also obtained with a Photometrics CCD Camera and processed with Quips FISH Imaging software from Vysis (Downers Grove, IL). FISH analysis of all specimens was carried out in a blinded fashion to prevent observer bias. Two hundred cells were read for each patient, the number of chromosome 7 signals for each nucleus was scored by two readers, and the data were exported into Excel for further analysis. To determine whether FISH was able to detect monosomy 7 in cytogenetically normal specimens, the FISH results were compared with the standard metaphase cytogenetic analyses.
Figure 1 Average number of bone marrow cells with one chromosome 7 signal in normal controls.
157
Monosomy 7 in Bone Marrow of Fanconi Anemia
Figure 2 Number of bone marrow cells with one chromosome 7 signal in monosomy 7 patients.
RESULTS Control Cases To determine hybridization efficiency, we employed FISH analysis by using the chromosome 7 centromere probe to
examine bone marrow samples from nine bone marrow donors with normal karyotypes (Fig. 1). Signals were demonstrated in an average of 6 of 200 (3.0%) cells examined, giving a hybridization efficiency of 97%.
Table 3 Summary of cytogenetic and FISH results from index case Date
Sample
Cytogenetics
12/91 12/91 3/92 5/93 3/94 4/94 5/94
Bone marrow Blood Bone marrow Bone marrow Bone marrow Bone marrow Bone marrow
46,XX[24] (no random loss (RL) of 7) Fanconi anemia positive by DEB 46,XX[19] (no RL of 7) 46,XX[20] (no RL of 7) 46,XX[20] (no RL of 7) 46,XX[20] (no RL of 7) 46,XX[20] (1 cell with RL of 7)
1/95
Bone marrow
46,XX[20] (1 cell with RL of 7)
12/95
Bone marrow
46,XX[30] (2 cells with RL of 7)
4/96 7/96 8/96
Bone marrow Bone marrow Bone marrow
47,XX,27,121,122[7]/46,XX[12] 45,XX,27,add(18)(p11.2)[20]
FISH
nuc ish 7cen(D7Z132)[191]/ 7cen(D7Z131)[8] (within normal laboratory parameters) nuc ish 7cen(D7Z131)[43]/ 7cen(D7Z132)[157] nuc ish 7cen(D7Z131)[83]/ 7cen(D7Z132)[124]
nuc ish 7cen(D7Z131)[164]/ 7cen(D7Z132)[2]
158
V. C. Thurston et al. Four years and 7 months after diagnosis, standard cytogenetics revealed a karyotype of 45,XX,27,add(18)(p11.2) in 20 bone marrow cells examined. The next month, FISH analysis revealed monosomy 7 in 98.7% of the bone marrow nuclei examined. Figure 3A and B shows a metaphase spread and interphase nucleus from bone marrow of the index case hybridized with a chromosome 7 centromere probe, demonstrating the presence of a single chromosome 7. A bone marrow nucleus from a normal individual demonstrates two chromosome 7 signals by FISH analysis (Fig. 3C). Test Cases With the use of the same chromosome 7 centromere probe, FISH analysis was performed on 13 bone marrow specimens from nine patients with Fanconi anemia (Fig. 4). Single signals for the chromosome 7 centromere were seen in an average of only 6.88 of 200 cells (3.44%) examined.
DISCUSSION
Figure 3 Metaphase and interphase of bone marrow cells hybridized with chromosome 7 centromere probe. (A) Metaphase spread from index case. Notice that spread contains only one signal. (B) Interphase cells from index case showing one centromere signal. (C) Interphase bone marrow cells from a normal male 46,XX[20], with two centromere signals indicating two copies of chromosome 7.
The positive control group consisted of five patients with monosomy 7 as determined by cytogenetic analysis. When FISH was used to analyze bone marrow from these patients, single signals were seen in an average of 151 of 200 (75.5%) cells examined (Fig. 2). Index Case In the first FISH-analyzed bone marrow specimen (taken 3 years after diagnosis), a low level (22%, or 43 of 200 cells) of monosomy 7 was detected (Table 3). Standard cytogenetic analysis performed at the same time revealed a karyotype of 46,XX[20] with only a single cell showing random loss of chromosome 7. Within 12 months, 83 of 200 cells (40%) examined by FISH demonstrated monosomy 7 despite a normal bone marrow karyotype with 2 of 30 cells showing apparent random loss of chromosome 7 (6.6%).
Two recent studies [9, 10] demonstrate an actuarial risk of 52% for developing MDS or AML or both in patients with Fanconi anemia, which is much higher than the 10% rate cited in most studies that were not actuarial [11–13]. Many FA patients were monosomic for chromosome 7 in their bone marrow prior to or coincident with the development of leukemia [13]. Bone marrow clonal cytogenetic abnormalities have been reported in some FA patients without leukemia, and these patients are said to be in a preleukemic phase [6]. The most frequent chromosome abnormalities are monosomy 7 and translocations or duplications involving 1q [6, 11, 14]. Early detection of these abnormalities could lead to earlier intervention and treatment of these cases. Patients with myelodysplastic syndromes (MDS) may also display monosomy 7 clones in their bone marrow [15]. Yunis et al. [15] found monosomy 7 to be an indicator of poor prognosis and a high probability of evolution to AML. In a much larger study, Yunis et al. [16] again found that, in both MDS and AML, the presence of monosomy 7 was an indicator of poor prognosis and survival times of less than 1 year. None of these studies utilized FISH in the detection of monosomy 7. Monosomy 7 detected by FISH, but not by conventional cytogenetics, has been reported in patients with acute leukemia or MDS. For example, Kadam et al. [8] used FISH to detect monosomy 7 clones in the bone marrow of two patients with AML who did not have monosomy 7 by standard cytogenetics. The investigators hypothesized that the monosomy 7 clone detected by FISH was unable to enter mitosis in vitro and was therefore not detected by conventional cytogenetics. A later study by Kadam et al. [17] used FISH to examine 21 patients with acute lymphoblastic leukemia and detected monosomy 7 in five cases. A separate study using FISH to detect monosomy 7 in MDS [18] found that 5 of 67 patients had monosomy 7 (.6.3 cells with single signals/patient) detected by FISH, but the anomaly was not detected by conventional cytogenetics.
159
Monosomy 7 in Bone Marrow of Fanconi Anemia
Figure 4 Average number of bone marrow cells with one chromosome 7 signal in Fanconi anemia patients.
The percentage of interphase cells with only one signal ranged from 14.4 to 39% (normal was 2.9–5.3%). Another study reported by Arif et al. [19] examined bone marrow aspirates from 25 patients with MDS and 25 patients with AML, none of whom exhibited monosomy 7 by standard cytogenetic analysis. FISH detected monosomy 7 in 9 of 25 MDS patients and 11 of 25 AML patients. Among those who showed monosomy 7 by FISH, 5 MDS and 3 AML patients had normal karyotypes by standard cytogenetic analysis. The rest of the patients with monosomy 7 by FISH had abnormal karyotypes but did not have abnormalities involving chromosome 7. Four of the 5 MDS patients who went on to develop AML had monosomy 7 by standard cytogenetics, indicating that the presence of a monosomy 7 clone in metaphase cells is indicative of a higher rate of progression to AML. Our study contributes to the growing body of evidence that the presence of a monosomy 7 clone in bone marrow aspirates can go undetected by standard cytogenetic analysis. In our index case, the first bone marrow specimen analyzed by FISH contained a low level (22%) of cells with monosomy 7. Standard cytogenetic analysis performed at the same time revealed a normal karyotype with only a single cell showing random loss of chromosome 7. Within the next 12 months, consecutive bone marrow samples dem-
onstrated progressively higher percentages of monosomy 7 by FISH analysis without a corresponding increase by standard cytogenetics. Nineteen months after the first FISH-analyzed specimen, standard cytogenetics revealed a karyotype of 45,XX,27,add(18)(p11.2) in 20 bone marrow cells examined; and, in the next month, FISH analysis revealed monosomy 7 in 98.7% of the 200 bone marrow nuclei examined. In our study of 13 samples from nine other patients with FA, monosomy 7 was not detected by standard karyotype or by FISH. To date, none of these patients have developed leukemia. Continued monitoring of these patients by standard cytogenetics and FISH will determine whether any abnormal clones with monosomy 7 develop and whether they can be detected by FISH before they are detected by standard metaphase cytogenetics. If monosomy 7 is evidence of bone marrow transformation, early detection of a monosomy 7 clone by FISH would allow for earlier intervention, such as bone marrow transplantation, to help prevent or delay the onset of leukemia in these patients. As standard practice, we have adopted FISH analysis with the use of chromosome 7 centromere probes for all patients with FA.
This study was supported by the Fanconi Anemia Research Fund.
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