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C-anaphase in a case of acute nonlymphocytic leukemia
C-anaphase in a Case of Acute Nonlymphocytic Leukemia P. W. Thompson, S. V. Davies, and J. A. Whittaker
ABSTRACT: C-anaphase was seen in approximately 50% of bone marrow cells from a patient with acute n o n l y m p h o c y t i c leukemia (ANLL). The abnormality acting as a marker for the disease, being present at diagnosis, disappearing during remission and returning at relapse.
INTRODUCTION Although data is accumulating on the structure and functions of the centromere, little is known of the pathogenic role of centromeric abnormalities in human disease (1, 2). A "puffing apart" of the sister chromatids in the centromeric region of the metaphase chromosomes is seen in the autosomal disorder Roberts' syndrome (3). A similar phenomenon termed 'centromere spreading', was reported in the bone marrow cells of cases of megaloblastic anemia with B12 and folate deficiency as early as 1966 (4). More recently, it has been suggested that premature spreading of the centromeres in acute nonlymphocytic leukemia (ANLL) be regarded as a diagnostic criterion (5, 6, 7). The presence of additional rod-shaped or 'acentric' X chromosomes in elderly females is caused by premature separation of the X centromeres in mitosis. This finding was termed premature centromere division (PCD) by Fitzgerald in 1975 (8). The term PCD was also used to describe an increased frequency of cells with chromatid and centromere separation affecting all chromosomes (9, 10, 11, 12). The phenomenon was originally reported in h u m a n lymphocyte cultures by Chamla who described the cells as being in C-anaphase (13). We present data on a patient with ANLL whose bone marrow metaphases showed C-anaphase at diagnosis under several different culture conditions. The anomaly disappeared during remission, and was present again at relapse 12 months later. CASE REPORT
A 76-year-old man presented in April 1991 with a 3 month history of increasing fatigue and dyspnoea. He had under-
gone transurethral resection of the prostate 10 years earlier and received medication for closed angle glaucoma. There was no history of occupational exposure to carcinogens or radiation. Physical examination was normal. Hemoglobin was 7 g/dl, total white cell count 4.5 x 109/1 and platelets 24 x 109/1. The blood film showed 80% blast forms, many having an unusual appearance with bilobed, binucleolate nuclei. A bone marrow aspirate was consistent with acute nonlymphocytic leukemia of FAB M1 type. There was no evidence of megaloblastic change in the erythroid series. He received daunorubicin and cytosine arabinoside as induction chemotherapy and achieved complete remission after consolidation chemotherapy with mitozantrone and high dose cytosine arabinoside. Twelve months later he relapsed and died during attempted re-induction chemotherapy. MATERIALS AND METHODS Cytogenetic studies were performed on a series of bone marrow aspirate samples from the patient. The samples were cultured at 37°C in McCoy's 5A m e d i u m plus 20% fetal calf serum with Colcemid being added to a final concentration of 0.1 ~g/ml. At diagnosis four cultures were established: (1) a direct culture was exposed to Colcemid for 1-hour before harvesting; (2) one culture was given a 17-hour exposure to colcemid; (3) another culture was incubated for 24-hours before the addition of Colcemid for 1 hour; and (4) a further 24-hour culture was blocked with methotrexate (MTX) (10 7 mol/l) and released with thymidine (THY) (10 s tool/l) for the last 5 hours of culture before a short 15 minute exposure to colcemid. In the post-treatment samples the direct culture was not used. RESULTS
From the Institute of Medical Genetics (P. W. T.) and the Department of Haematology (S. V. D., J. A. W.), University of Wales College of Medicine, Cardiff, United Kingdom. Address reprint requests to: E W. Thompson, Institute of Medical Genetics, University of Wales College of Medicine, Heath Park, CARDIFE United Kingdom. Received January 20, 1993; accepted July 16, 1993. 148 Cancer Genet Cytogenet 71:148-150 (1993) 0165-4608/93/$06.00
The results of the cytogenetic findings at diagnosis are shown in Table 1. In most of the C-anaphase cells the characteristic separated centromeres and splayed chromatids were seen in all of the chromosomes, in the remainder varying numbers were affected. These cells appeared to have an otherwise normal karyotype. No other cytogenetic abnormality was found.
© 1993 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York. NY 10010
C-anaphase in ANLL
Table 1
149
Table 2
Frequency of C-anaphase at diagnosis Culture conditions
Percentage of cells showing C-anaphasea
Direct 24-hour culture MTX-synchronized 24-hour culture Extended 16-hour colcemid exposure
52 55 12 60
Comparison of C-anaphase in 24-hour cultures from the patient, a group of ANLL cases and a group of normal controls
Subject group Patient ANLL cases (n = 7)
a 100 cells were examinedfrom each culture.
Normal controls (n = 7)
The n u m b e r of cells i n C-anaphase was similar in the direct, 24 hour and extended colcemid exposure cultures, however, there was a marked reduction in the MTX-synchronized culture. The abnormality remained in 4% of cells i n a second marrow sample before a complete remission was attained. It was absent from 4 remission marrows, but subsequently returned i n 40% of cells at relapse. Table 2 compares the percentage of cells showing C-anaphase i n 24 hour cultures of bone marrow aspirate from the patient, 7 consecutive new ANLL case referrals and 7 consecutive allogeneic bone marrow transplant donors.
DISCUSSION The separated centromeres and splayed chromatids seen in the cells from our patient appear identical to the C-anaphases
Percentage of cells showing C-anaphase a 55 1.3 range 0-4 0.9 range 0-2
a 100 ceils were examinedfrom each culture.
(13) or PCD cells (9) previously reported in lymphocyte and fibroblast culture (Fig. 1). The abnormality was present i n approximately 50% of cells at diagnosis, disappeared as remission was achieved, and returned at relapse. Thus, C-anaphase was a marker for ANLL in this case. This is the first example of C-anaphase associated with ANLL. In previously documented cases the p h e n o m e n o n occurs as an autosomal d o m i n a n t trait, usually with no clinical significance (12). In our patient the absence of any C-anaphases in four remission marrow samples excludes the possibility of the anomaly resulting from a constitutional disorder. Some event i n the neoplastic process in our patient
Figure 1 Metaphase spreads from the patient at time of diagnosis. (a) Normal chromosome morphology, (b-d) various manifestations of C-anaphase.
r
a
i
W
c j
d
150 must have caused a similar type of mutation in the leukaemic
cells. In a study on patients with megaloblastic anemia with B 12 and folate deficiency Menzies et al. (14) reported that in addition to finding chromatid breakage and centromeric spreading, some metaphases showed chromosomes with separated chromatids and other pronounced degeneration of the chromosomes. Morphologically these appear similar to the C-anaphases described herein. A further report by Fitzgerald and Hamer (15) described a patient with RAEB and a Bq-chromosome in w h o m some mitoses in bone marrow cells showed "a degenerate appearance with small eroded chromosomes". Again it is possible that these were also C-anaphases. A n analysis of marrow from normal controls gives a background level for C-anaphase in bone marrow cells of 0.9% (Table 2). This is similar to the figures reported for l y m p h o cytes (9, 10, 11) and fibroblasts (9). The frequency of C-anaphase in our series of 7 cases of ANLL in w h i c h the abnormality was specifically sought was only 1.3% (Table 2), a value w h i c h does not differ significantly from that in normal controls. This result, together with the absence of previously p u b l i s h e d reports, indicate that an increase in cells showing C-anaphase is not a feature of ANLL in general. The cause of C-anaphase in the patient is unknown, as is what role, if any, it has on the neoplastic process. The report by Bamezai et at. (16) on centromeric spreading in megaloblastic anemia provides evidence that it can be "cured" by exogenous t h y m i d i n e being present in the culture m e d i u m , and they suggested that failure of proper DNA synthesis led to the abnormalities seen. The morphological abnormalities found by Menzies et al. (14) were also "cured" by vitamin replacement, and both they and Fitzgerald and Hamer (15) found abnormalities of the cell cycle caused by a defect in DNA metabolism. In a recent p a p e r Fuster et al. (17) report an induction of C-anaphase in approximately 7% of stimulated lymphocytes u n d e r culture conditions that utilize folate- or thymidine- deficient m e d i a to show chromosome fragile sites. In our patient, the MTX-synchronized culture showed only 12 % of cells in C-anaphase c o m p a r e d to over 50% in the other cultures. As release of the block in the culture was by the a d d i t i o n of t h y m i d i n e (10-5 M), it can be postulated that the a d d i t i o n of t h y m i d i n e led to the relative reduction in the n u m b e r of C-anaphases u n d e r these conditions. Alternatively, fewer leukemic metaphases may have been present in the MTX-synchronized culture. All of the above findings suggest that t h y m i d i n e metabolism plays an important role in maintaining centromeric structure during mitosis. Long term culture of leukemic cells which show C-anaphase may help in determining how thymidine concentration is involved in this particular chromosome anomaly.
P . W . Thompson et al. REFERENCES 1. Vig BK (1983): Sequence of centromeric separation: occurrence, possible significance, and control. Cancer Genet Cytogenet 8:249-274. 2. Vig BK, Rattner JB (1989): Centromere, kinetochore, and cancer. Crit Rev Oncogen 1:343-371. 3. Tomkins D, Hunter A, Roberts M (1979): Cytogenetic findings in Roberts'-SC phocomelia syndrome(s). Am J Med Genet 4:17-26. 4. Heath CW (1966): Cytogenetic observations in vitamin B12 and folate deficiency. Blood 27:800-815. 5. Shirashi Y, Taguchi H, Niiya K, Shiomi F, Kikukawa K, Ohmura T, Hamawaki M, Ueda N (1982): Diagnostic and prognostic significance of chromosome abnormalities in marrow and mitogen response of lymphocytes of acute nonlymphocytic leukemia. Cancer Genet Cytogenet 5:1-24. 6. Gallo JH, Misawa S, Testa JR (1984): Centromere spreading in acute nonlymphocytic leukemia. Cancer Genet Cytogenet 12:105-109. 7. Littlefield LG, Joiner EE, Sayer AM (1985): Premature separation of centromeres in marrow chromosomes from an untreated patient with acute myelogenous leukemia. Cancer Genet Cytogenet 16:109-115. 8. Fitzgerald PH (1975): A mechanism of X chromosome aneuploidy in lymphocytes of aging women. Humangenetik 28: 153-158. 9. Rudd NL, Teshima IE, Martin RH, Sisken JE, Weksberg R (1983): A dominantly inherited cytogenetic anomaly: a possible cell division mutant. Hum Genet 65:117-121. 10. Gabarron J, Jimenez A, Glover G (1986): Premature centomere division dominantly inherited in a subfertile family. Cytogenet Cell Genet 43:69-71. 11. Madan K, Lindhout D, Palan A (1987): Premature centromere division (PCD): a dominantly inherited cytogenetic anomaly. Hum Genet 77:193-196. 12. Chamla Y (1988): C-anaphases in lymphocyte cultures versus premature centromere division syndromes. Hum Genet 78: 111-114. 13. Chamla Y, Roumy M, Lass~gues M, Battin J (1980): Altered sensitivity to colchicine and PHA in human cultured cells. Hum Genet 53:249-253. 14. Menzies RC, Crossen PE, Fitzgerald PH, Gunz FW (1966): Cytogenetic and cytochemical studies on marrow cells in B 12 and folate deficiency. Blood 28:581-594. 15. Fitzgerald PH, Hamer JW (1971): Primary acquired red cell hypoplasia associated with clonal chromosomal abnormality and disturbed erythroid proliferation. Blood 38:325-335. 16. Bamezai R, Shiraishi Y, Taguchi H (1986): Centromere spreading in a case of megaloblastic anemia "cured" under TC 199 culture conditions. Cancer Genet Cytogenet 20:341-343. 17. Fuster C, Miro R, Barrios L, Egozcue J (1992): Induction of premature centromere division affecting all chromosomes under culture conditions of fragile site expression. Cancer Genet Cytogenet 58:152-154.
ABSTRACT: C-anaphase was seen in approximately 50% of bone marrow cells from a patient with acute n o n l y m p h o c y t i c leukemia (ANLL). The abnormality acting as a marker for the disease, being present at diagnosis, disappearing during remission and returning at relapse.
INTRODUCTION Although data is accumulating on the structure and functions of the centromere, little is known of the pathogenic role of centromeric abnormalities in human disease (1, 2). A "puffing apart" of the sister chromatids in the centromeric region of the metaphase chromosomes is seen in the autosomal disorder Roberts' syndrome (3). A similar phenomenon termed 'centromere spreading', was reported in the bone marrow cells of cases of megaloblastic anemia with B12 and folate deficiency as early as 1966 (4). More recently, it has been suggested that premature spreading of the centromeres in acute nonlymphocytic leukemia (ANLL) be regarded as a diagnostic criterion (5, 6, 7). The presence of additional rod-shaped or 'acentric' X chromosomes in elderly females is caused by premature separation of the X centromeres in mitosis. This finding was termed premature centromere division (PCD) by Fitzgerald in 1975 (8). The term PCD was also used to describe an increased frequency of cells with chromatid and centromere separation affecting all chromosomes (9, 10, 11, 12). The phenomenon was originally reported in h u m a n lymphocyte cultures by Chamla who described the cells as being in C-anaphase (13). We present data on a patient with ANLL whose bone marrow metaphases showed C-anaphase at diagnosis under several different culture conditions. The anomaly disappeared during remission, and was present again at relapse 12 months later. CASE REPORT
A 76-year-old man presented in April 1991 with a 3 month history of increasing fatigue and dyspnoea. He had under-
gone transurethral resection of the prostate 10 years earlier and received medication for closed angle glaucoma. There was no history of occupational exposure to carcinogens or radiation. Physical examination was normal. Hemoglobin was 7 g/dl, total white cell count 4.5 x 109/1 and platelets 24 x 109/1. The blood film showed 80% blast forms, many having an unusual appearance with bilobed, binucleolate nuclei. A bone marrow aspirate was consistent with acute nonlymphocytic leukemia of FAB M1 type. There was no evidence of megaloblastic change in the erythroid series. He received daunorubicin and cytosine arabinoside as induction chemotherapy and achieved complete remission after consolidation chemotherapy with mitozantrone and high dose cytosine arabinoside. Twelve months later he relapsed and died during attempted re-induction chemotherapy. MATERIALS AND METHODS Cytogenetic studies were performed on a series of bone marrow aspirate samples from the patient. The samples were cultured at 37°C in McCoy's 5A m e d i u m plus 20% fetal calf serum with Colcemid being added to a final concentration of 0.1 ~g/ml. At diagnosis four cultures were established: (1) a direct culture was exposed to Colcemid for 1-hour before harvesting; (2) one culture was given a 17-hour exposure to colcemid; (3) another culture was incubated for 24-hours before the addition of Colcemid for 1 hour; and (4) a further 24-hour culture was blocked with methotrexate (MTX) (10 7 mol/l) and released with thymidine (THY) (10 s tool/l) for the last 5 hours of culture before a short 15 minute exposure to colcemid. In the post-treatment samples the direct culture was not used. RESULTS
From the Institute of Medical Genetics (P. W. T.) and the Department of Haematology (S. V. D., J. A. W.), University of Wales College of Medicine, Cardiff, United Kingdom. Address reprint requests to: E W. Thompson, Institute of Medical Genetics, University of Wales College of Medicine, Heath Park, CARDIFE United Kingdom. Received January 20, 1993; accepted July 16, 1993. 148 Cancer Genet Cytogenet 71:148-150 (1993) 0165-4608/93/$06.00
The results of the cytogenetic findings at diagnosis are shown in Table 1. In most of the C-anaphase cells the characteristic separated centromeres and splayed chromatids were seen in all of the chromosomes, in the remainder varying numbers were affected. These cells appeared to have an otherwise normal karyotype. No other cytogenetic abnormality was found.
© 1993 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York. NY 10010
C-anaphase in ANLL
Table 1
149
Table 2
Frequency of C-anaphase at diagnosis Culture conditions
Percentage of cells showing C-anaphasea
Direct 24-hour culture MTX-synchronized 24-hour culture Extended 16-hour colcemid exposure
52 55 12 60
Comparison of C-anaphase in 24-hour cultures from the patient, a group of ANLL cases and a group of normal controls
Subject group Patient ANLL cases (n = 7)
a 100 cells were examinedfrom each culture.
Normal controls (n = 7)
The n u m b e r of cells i n C-anaphase was similar in the direct, 24 hour and extended colcemid exposure cultures, however, there was a marked reduction in the MTX-synchronized culture. The abnormality remained in 4% of cells i n a second marrow sample before a complete remission was attained. It was absent from 4 remission marrows, but subsequently returned i n 40% of cells at relapse. Table 2 compares the percentage of cells showing C-anaphase i n 24 hour cultures of bone marrow aspirate from the patient, 7 consecutive new ANLL case referrals and 7 consecutive allogeneic bone marrow transplant donors.
DISCUSSION The separated centromeres and splayed chromatids seen in the cells from our patient appear identical to the C-anaphases
Percentage of cells showing C-anaphase a 55 1.3 range 0-4 0.9 range 0-2
a 100 ceils were examinedfrom each culture.
(13) or PCD cells (9) previously reported in lymphocyte and fibroblast culture (Fig. 1). The abnormality was present i n approximately 50% of cells at diagnosis, disappeared as remission was achieved, and returned at relapse. Thus, C-anaphase was a marker for ANLL in this case. This is the first example of C-anaphase associated with ANLL. In previously documented cases the p h e n o m e n o n occurs as an autosomal d o m i n a n t trait, usually with no clinical significance (12). In our patient the absence of any C-anaphases in four remission marrow samples excludes the possibility of the anomaly resulting from a constitutional disorder. Some event i n the neoplastic process in our patient
Figure 1 Metaphase spreads from the patient at time of diagnosis. (a) Normal chromosome morphology, (b-d) various manifestations of C-anaphase.
r
a
i
W
c j
d
150 must have caused a similar type of mutation in the leukaemic
cells. In a study on patients with megaloblastic anemia with B 12 and folate deficiency Menzies et al. (14) reported that in addition to finding chromatid breakage and centromeric spreading, some metaphases showed chromosomes with separated chromatids and other pronounced degeneration of the chromosomes. Morphologically these appear similar to the C-anaphases described herein. A further report by Fitzgerald and Hamer (15) described a patient with RAEB and a Bq-chromosome in w h o m some mitoses in bone marrow cells showed "a degenerate appearance with small eroded chromosomes". Again it is possible that these were also C-anaphases. A n analysis of marrow from normal controls gives a background level for C-anaphase in bone marrow cells of 0.9% (Table 2). This is similar to the figures reported for l y m p h o cytes (9, 10, 11) and fibroblasts (9). The frequency of C-anaphase in our series of 7 cases of ANLL in w h i c h the abnormality was specifically sought was only 1.3% (Table 2), a value w h i c h does not differ significantly from that in normal controls. This result, together with the absence of previously p u b l i s h e d reports, indicate that an increase in cells showing C-anaphase is not a feature of ANLL in general. The cause of C-anaphase in the patient is unknown, as is what role, if any, it has on the neoplastic process. The report by Bamezai et at. (16) on centromeric spreading in megaloblastic anemia provides evidence that it can be "cured" by exogenous t h y m i d i n e being present in the culture m e d i u m , and they suggested that failure of proper DNA synthesis led to the abnormalities seen. The morphological abnormalities found by Menzies et al. (14) were also "cured" by vitamin replacement, and both they and Fitzgerald and Hamer (15) found abnormalities of the cell cycle caused by a defect in DNA metabolism. In a recent p a p e r Fuster et al. (17) report an induction of C-anaphase in approximately 7% of stimulated lymphocytes u n d e r culture conditions that utilize folate- or thymidine- deficient m e d i a to show chromosome fragile sites. In our patient, the MTX-synchronized culture showed only 12 % of cells in C-anaphase c o m p a r e d to over 50% in the other cultures. As release of the block in the culture was by the a d d i t i o n of t h y m i d i n e (10-5 M), it can be postulated that the a d d i t i o n of t h y m i d i n e led to the relative reduction in the n u m b e r of C-anaphases u n d e r these conditions. Alternatively, fewer leukemic metaphases may have been present in the MTX-synchronized culture. All of the above findings suggest that t h y m i d i n e metabolism plays an important role in maintaining centromeric structure during mitosis. Long term culture of leukemic cells which show C-anaphase may help in determining how thymidine concentration is involved in this particular chromosome anomaly.
P . W . Thompson et al. REFERENCES 1. Vig BK (1983): Sequence of centromeric separation: occurrence, possible significance, and control. Cancer Genet Cytogenet 8:249-274. 2. Vig BK, Rattner JB (1989): Centromere, kinetochore, and cancer. Crit Rev Oncogen 1:343-371. 3. Tomkins D, Hunter A, Roberts M (1979): Cytogenetic findings in Roberts'-SC phocomelia syndrome(s). Am J Med Genet 4:17-26. 4. Heath CW (1966): Cytogenetic observations in vitamin B12 and folate deficiency. Blood 27:800-815. 5. Shirashi Y, Taguchi H, Niiya K, Shiomi F, Kikukawa K, Ohmura T, Hamawaki M, Ueda N (1982): Diagnostic and prognostic significance of chromosome abnormalities in marrow and mitogen response of lymphocytes of acute nonlymphocytic leukemia. Cancer Genet Cytogenet 5:1-24. 6. Gallo JH, Misawa S, Testa JR (1984): Centromere spreading in acute nonlymphocytic leukemia. Cancer Genet Cytogenet 12:105-109. 7. Littlefield LG, Joiner EE, Sayer AM (1985): Premature separation of centromeres in marrow chromosomes from an untreated patient with acute myelogenous leukemia. Cancer Genet Cytogenet 16:109-115. 8. Fitzgerald PH (1975): A mechanism of X chromosome aneuploidy in lymphocytes of aging women. Humangenetik 28: 153-158. 9. Rudd NL, Teshima IE, Martin RH, Sisken JE, Weksberg R (1983): A dominantly inherited cytogenetic anomaly: a possible cell division mutant. Hum Genet 65:117-121. 10. Gabarron J, Jimenez A, Glover G (1986): Premature centomere division dominantly inherited in a subfertile family. Cytogenet Cell Genet 43:69-71. 11. Madan K, Lindhout D, Palan A (1987): Premature centromere division (PCD): a dominantly inherited cytogenetic anomaly. Hum Genet 77:193-196. 12. Chamla Y (1988): C-anaphases in lymphocyte cultures versus premature centromere division syndromes. Hum Genet 78: 111-114. 13. Chamla Y, Roumy M, Lass~gues M, Battin J (1980): Altered sensitivity to colchicine and PHA in human cultured cells. Hum Genet 53:249-253. 14. Menzies RC, Crossen PE, Fitzgerald PH, Gunz FW (1966): Cytogenetic and cytochemical studies on marrow cells in B 12 and folate deficiency. Blood 28:581-594. 15. Fitzgerald PH, Hamer JW (1971): Primary acquired red cell hypoplasia associated with clonal chromosomal abnormality and disturbed erythroid proliferation. Blood 38:325-335. 16. Bamezai R, Shiraishi Y, Taguchi H (1986): Centromere spreading in a case of megaloblastic anemia "cured" under TC 199 culture conditions. Cancer Genet Cytogenet 20:341-343. 17. Fuster C, Miro R, Barrios L, Egozcue J (1992): Induction of premature centromere division affecting all chromosomes under culture conditions of fragile site expression. Cancer Genet Cytogenet 58:152-154.