Complex hypodiploidy in acute myeloid leukaemia: A United Kingdom Cancer Cytogenetics Group study

Leukemia Research Vol. 19, No. 12. pp. 905-913. 1995. Copyright 0 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0145-2126195 $9.50 + 0 00

Pergamon 0145-2126(95)00089-S

COMPLEX UNITED

HYPODIPLOIDY IN ACUTE MYELOID LEUKAEMIA: A KINGDOM CANCER CYTOGENETICS GROUP STUDY

Jacqueline M. Hawkins, Barbara Bain, Atul B. Mehta and Anthony V. Moorman on behalf of the UKCCG (Received 6 March 1995. Accepted 27 May 1995) Abstract-Forty cases of acute myeloid leukaemia with 41 hypodiploid clones were investigated in a collaborative study. Cases with -5, -7, -X or -Y either alone or in association with an established translocation were excluded. Karyotypes were reviewed in all cases and bone marrow or blood morphology was reviewed in 22 cases. Twenty-six cases were very complex (more than five abnormal chromosomes), 14 cases were complex (two to five abnormal chromosomes) and only one case was simple (one abnormal chromosome). Chromosomes 5, 7, 17 and 18 were involved in a significantly greater number of cases than expected. Only five cases had normal chromosomes 5 and 7. Chromosomes 10, X and Y were involved in significantly fewer cases than expected. Ten cases had ring chromosomes and 18 showed clonal evolution. Patients were aged between 19 and 90, median 61 years. Evidence of myelodysplasia was found on morphological review in 18/22 cases, and on clinical features in a further five cases. There was a high proportion of cases with FAB M6, ‘erythroleukaemia’ (9/ 31 cases). Only 4/16 patients treated with cytotoxic therapy achieved remission. Median survival was 2.5 months (35 patients). Survival was slightly better for patients with normal chromosomes 5 and 7, and for those with simpler karyotypes, but this was not statistically significant. This study confirms the association between hypodiploidy, complex karyotype, abnormalities of chromosomes 5 and 7 and a poor prognosis. Key words: Acute erythroleukaemia.

myeloid

leukaemia,

hypodiploidy,

Introduction The cytogenetic classification of patients with acute leukaemia is based either on the clonal number of chromosomes (ploidy) or on the specific cytogenetic abnormalities acquired by the leukaemic cells. Hypodiploidy is the clonal loss of one or more chromosomes which results in a cell line with < 46 chromosomes. This was first identified as an adverse prognostic indicator in adult acute leukaemia in 1971 [l]. In that study hypodiploidy was found in 9% of patients with ‘lymphatic leukaemia’ and in 15% of patients with Abbreviations: ALL, acute lymphoblastic leukaemia; IWCL, International Workshops on Chromosomes in Leukaemia; AML, acute myeloid leukaemia; UKCCG, United Kingdom Cancer Cytogenetics Group; MVPP, mustine, vinblastine, procarbazine and prednisone; MDS, myelodysplasia. Correspondence too: Dr M. Martineau, Secretary to the UKCCG, Department of Haematology, The Royal Free Hospital School of Medicine, Pond Street, London NW3 2QG, U.K. (Tel: 0171 794 0500, Ext. 4267; Fax: 0171 794 1417).

complex

karyotype,

myelodysplasia,

‘granulocytic leukaemia’. The incidence of 10% for hypodiploidy in acute lymphoblastic leukaemia is now well established, but the variable prognosis of this group in acute lymphoblastic leukaemia (ALL) reflects the heterogeneity of chromosomal abnormalities which contribute to it [2]. Hypodiploidy, in acute myeloid leukaemia (AML), is also a heterogeneous category in terms of chromosomal involvement and complexity. It comprises the major proportion of the good risk t(8;21) cases which include clonal loss of a sex chromosome [3], as well as cases with monosomy 5 or monosomy 7, known to be associated with a poor prognosis [4]. The prognostic impact of hypodiploidy has been difficult to judge in recent studies because classification by ploidy alone has not been applied. The International Workshops on Chromosomes in Leukaemia (IWCL) for AML, IWCL4 and IWCL6 used hierarchical groupings, the Chicago (IWCL4) or modified Chicago (IWCL6) classifications in which ploidy was applied only to cases not having an established structural change or established chromosome loss [4,5]. The Chicago hypodiploid category excluded cases with clonal loss of chromo-

J. M. Hawkins

et al.

Fig. 1. The G-banded karyogram of Case 18, showing five abnormalities. Possible karyotype 44,XY,-5,ider(13)del(13) (q12q14),der(15)t(15;18)(15pter+q11: :18qll+q21::15q26+ -18,der(22)t(15;18;22)(22pter-+ql3::15qll~q26:: qter), 18q21-+qter).

somes 5 and or 7 as well as those with the established translocations t&21) and t(15;17), the former of which frequently shows clonal loss of a sex chromosome. A further feature considered at IWCL6 was the complexity of the clonal karyotype. A clone with a single chromosomal loss or translocation was described as simple; complex involved two to five numerical and/ or structural changes; very complex involved five or more chromosomal changes. The present study looked at chromosome loss and where possible at morphology in patients with AML and hypodiploid clones. Cases with -5, - 7, -X or -Y as the only loss, whether this occurred alone or in association with an established translocation, such as t&21) or t(15;17), were excluded. The aims were to discover which other chromosomes contributed to both simple and complex loss in hypodiploid AML, the extent to which chromosomes 5, 7, X and Y were involved in complex hypodiploid karyotypes and whether the karyotype related to prognosis.

Materials

and Methods

For this retrospective study, cases with AML and appropriate cytogenetics analysed over the previous 8 years were contributed by nine member laboratories of the UKCCG. Haematological and clinical data were collected from the referring haematologists.

Chromosome preparations were made using standard bone marrow or unstimulated blood cultures and G-banding methods [6]. Eligible hypodiploid cases had a stem line with clonal loss of one or more chromosomes, but cases with loss of chromosomes 5, 7, X or Y either alone or together with the established AML-associated t(8;21) or t(15;17) were excluded. Karyotypes from every case were reviewed and clonality confirmed. Cases 21, 22 and 33 have appeared in previous publications [7, 81. The involvement or lack of involvement of certain chromosomes in clonal abnormalities was investigated using the chi-squared test. The initial clinical diagnosis in each case was made by the referring haematologist. Bone marrow and/or blood smears where available were reviewed for the UKCCG by a panel of two morphologists.

Results

Cytogenetics The 40 cases each had one (39 cases) or two (Case 19) hypodiploid clones. All but one of the 41 clones were complex, with two to five abnormal chromosomes (14 cases) or very complex, with more than five abnormal chromosomes (26 cases). The karyotype of the single case with simple loss was 45,XY, - 17. The commonest modal number of the stem line clones was 45 (15 cases), followed by modal number 44 (12 cases), 43 (nine cases), 42 (two cases), 40 and 39 (one case of each). The complexity of the karyotype (the number of chromosomes involved in changes) ranged from one to 14 with a

Hypodiploidy

in AML

Fig. 2. The G-banded karyogram of Case 39, showing 14 abnormalities. Possible karyotype 40,X,-Y,dic(4;17) der(5)t (5;ll)(qls;q13),der(5) t (5;16)(q35;q13), (@l;pll), -7,der(7) t (7;19)(p22;pll), -8,der(ll) t (11;?14)(q13;?q24), der(l2) t (5;12)(q15;p13), - 14, - 16,dup(17)(q21q23),- 18, - 19,- 21,+ mar (-8 and - 21 random loss).

peak at six. Complexity of the clone was not necessarily related to the modal number. Even clones with a modal number of 45 involved up to seven chromosomes (median = 4) (see Figs 1 and 2). Because of their complexity, complete analysis of the abnormalities was frequently impossible. Rather than attempting an educated guess at the composition of the derived chromosomes, each chromosome was scored as ‘involved’ or ‘not involved’ (Table 1). Chromosomes ‘involved’ included those apparently totally lost, partially lost, or involved in any structural rearrangement. Recognised structural abnormalities detected included inv(3)(q21q26) (Case 12), de1(3)(q25) (Case 14), de1(20)(qllq13) (Case 15) and t(8;21)(q22;q22) (Case

20). The involvement of six chromosomes showed distributions significantly different from those expected (P(O.05 using the chi-squared test with Yates’ correc-

tion). Chromosomes 5, 7, 17 and 18 were involved in a greater number of cases (31, 28, 25 and 19 cases, respectively) (P < 0.001, P < 0.001, P < 0.001, P < 0.05), and chromosomes 10, X and Y were involved in fewer cases (three, two and one cases, respectively) than expected (P
908

J. M. Hawkins et al.

Table 1. Results of cytogenetic analysis of 40 patients with hypodiploidy and acute myeloid leukaemia Case Ploidy 1 2 3 4 5 6 7 8 9 10 11

12 13 14 15 16 17 18 19 19A 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 44 44 44 44 44 44 44 44 44 44 44 44 43 43 43 43 43 43 43 43 43 42 42 40 40 39

Involved chromosomes 17 16 12 5 7 17 5 4 5 1 X 3 5 2 5 5 5 5 2 7 5 x 2 5 4 4 3 5 5 3 5 3 1 3 3 1 1 5 2 Y 2

17 18 18 12 18 7 5 7 7 3 5 11 3 7 13 11 13 4 9 7 1 5 7 5 6 5 7 7 5 7 5 5 5 4 4 5 6 3 4 3

21 22 21 15 7 19 20 5 7 11 4 12 16 15 15 5 12 8 3

17 20 20 21 6 9 15 5 13 17 17 16 12 16 13 11

11

22 7 17 16 7 16 18 18

18 18 17 17

18

22

18 17 17 13

20 20 21 17

12

15

17

17

12 7 7 7 11 16

16 9 11 8 13 17

18 12 13 9

20 17 14 9

22 19 16 14

22 22 15

11

17

18

20

8 7 7 6 6 5 7 7 5 5 5

14 8 8 7 7 7 10

16 12 11

17 13 14

18 15 18

17 21

10

11

16

22

10 7 13

11 9 15

12 11 17

11

16

16

7 5 7

8 7 9

9 7 13

18 20

21

17

20

15 12

17 15

18 17

17

17

17

18

19

20

20

22

12 11 14

13 12 16

13 14 17

16 16 18

17 17 22

18 17

22 18

19

19

Evolution

Clonal comolexitv

N* N N N Y N N N Y Y N N N Y Y Y Y Y N N N Y Y N Y N Y N N N N Y Y Y Y N Y N N N Y

S C C C C C C C C C C vc vc vc vc C C vc vc vc vc vc vc vc vc vc vc C C vc vc vc vc vc vc vc vc vc vc vc vc

*Abbreviations: N = no evolution; S = simple; Y = at least one sideline seen; C = complex, 2-5 abnormal chromosomes;VC = very complex, > 5 abnormal chromosomes.

chromosome 7 (two cases), of chromosome 5, 9, 11, 13, 16 and 20 (one case of each). Eighteen cases (45%) showed clonal evolution including the gaining of a ring in two cases and polyploidization in two cases. Clinical

Clinical data are presented in Table 2. Patients’ ages (known in 39 cases) ranged from 19 to 90, median 61 years. Eighty-five per cent of the patients were over 50 years. The ratio of male to female cases was 11:9. No patient had a known history of exposure to occupational mutagens, but four had a history of iatrogenic mutagenic

exposure; Case 18 had received a renal transplant 7 years previously, Case 14 had been treated with four courses of 32P for polycythaemia rubra vera l-3 years previously, and Cases 7 and 25 had received MVPP (mustine, vinblastine, procarbazine and prednisone) and radiotherapy for Hodgkin’s Disease 3 years and 7 years previously. At least nine cases had clinical evidence of possible myelodysplasia (MDS) prior to diagnosis of AML (Table 2). In 23 of our cases where information was available, there was clinical and/or haematological evidence of prior or underlying MDS. Only one case could be

Hypodiploidyin AML

909

n q

1

2

3

4

5

6

7

8

9

Key

-- 7

Both homologuesabnormal One homologueabnormal

10 11 12 13 14 15 16 17 18 19 20 21 22 ChromosomeNumber

X

Y

Fig. 3. Diagram showing distribution of chromosomes involved in 40 hypodiploid patients with acute myeloid leukaemia.

confirmed as having no evidence of MDS, and this patient (Case 21) was both the youngest in our study (19 years) and had an unusual karyotype, including abnormal chromosomes 1, 21 and X, and normal 5 and 7. Details of treatment were obtained in 23 cases. Sixteen of these patients were treated with cytotoxic therapy. In the remainder, therapy was not given for the following reasons: patient choice, advanced age, or poor physical condition. Only four of the treated patients achieved complete remission. Survival data was obtained for 35 patients - range 1-14 months (median 2.5 months). Haernatology Bone marrow and/or peripheral blood morphology was reviewed in 22 cases and some haematological data were available in a further nine cases. These results are presented in Table 2, and examples of typical morphology are shown in Fig. 4. A high proportion of cases with FAB type M6 was noted (g/31, or 29%). In six cases there was unequivocal evidence of trilineage MDS, and in a further 13 cases there was definite bilineage MDS, with clinical or other evidence of MDS. In the three remaining cases, the morphological findings were

incomplete. In one case (Case 37), the panel amended the diagnosis on the basis of FAB criteria to RAEBT rather than AML. Cytogenetics and clinical correlations Female patients had significantly fewer chromosomal abnormalities than male patients (chi-squared test, P
03

Fig. 4. Peripheral blood and bone marrow appearances of platient 24. (A) Basophilic stippling in red cells. (B) Erythroid hyperplasia, a binucleate erythroblast and a blast cell. Over 50% of nucleated cells were erythroid and there were mar‘e than 30% blast cells among the non-erythroid cells; this patient was therefore diagnosed as AML FAB type M6. (C) Ringe:d sideroblasts in a Perl’s (iron) stain of the bone marrow aspirate.

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911

Hypodiploidy in AML Table 2. Clinical and morphological data on 40 patients with hypodiploidy Case No.

Age (years)

Sex

MDS$

FAB

Treated

CR (months)

1 2 3 4 5 6 7 8* 9 10 11 12 13 14 15* 16 17 18* 19 20 21 22 23 24 25* 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

61 33 56 66 75 72 72 30 62 77 55 46 78 82 60 67 60 69 71 51 19 90 37 68 58 45 61 80 63 NK 59 67 74 61 57 84 59 54 63 59

M F F F F M F F M F M F F M F F M M M F F M M F M M F M M F M M M M F F M M M M

NK NK Y NK Y Y Y NK Y NK NK Y NK Y Y NK NK Y Y NK N Y Y Y Y Y Y Y Y NK NK NK Y NK Y Y Y NK Y Y

NK NK M4 M2 M2 M2 Ml NK M6 M7 NK M4 Ml M2 M2 M2 NK M6 M6 Ml Ml M4 M6 M6 M4 M6 Ml M6 Ml NK NK NK M6 MO Ml Ml t M5A u M6

NK NK Y NK N Y Y NK NK NK NK Y Y NK NK NK Y NK Y NK Y N NK N Y Y Y Y Y NK NK NK N Y Y N N N Y NK

NK NK 12 NK NA N N NK NK NK NK N N NK NK NK N NK N NK 9 NA NK NA N N N N 7 NK NK NK NA N 4 NA NA NA N NK

and acute myeloid leukaemia

Survival (months) Evidence for MDS 1 NK 14 5 3 5 1 3 11 4 1 5 2 2 1 1 1 1 1 NK 9+ 1 2 1 1 3 2 3 12 NK 4 NK 4 1 7 5 1 1 1 NK

NK NK MR NK MR, Clinician’s comment MR MR NK M6 NK NK MR, 6 week history of fatigue and infection NK MR MR, polycythemia rubra vera NK NK M6 MR,M6, refractory anaemia 7 months NK MR negative, history negative MR M6 MR, M6, history of anaemia RAEB 2 months MR, M6 MR, history of RAEB MR, M6 MR inconclusive, anaemia (several months) NK NK NK MR, M6 MR inconclusive MR, 4 week history of infections MR MR MR inconclusive MR M6

*Patient with leukaemia secondary to iatrogenic mutagen exposure, see text. TPatient assessed as RAEBT on review of material, but still included in study. UtAbbreviations: MDS = myelodysplasia; CR = complete remission; NA = not applicable; NK = not known; MR = morphology review; MR inconclusive = morphology of one or more lineages not assessable; N = No; Y = Yes; U = unclassifiable; RAEB = refractory anaemia with excess blasts.

Discussion This study has revealed the overwhelming involvement of chromosomes 5 and 7 in hypodiploid AML. The study, which deliberately excluded cases with simple loss of chromosomes 5 or 7, nevertheless showed the involvement of these chromosomes in 3.5140 cases and revealed only one case with simple change, namely monosomy 17. In addition, chromosomes 17 and 18 were also heavily involved. The IWCL4 noted an

association of - 17 with - 7. Loss of the sex chromosomes and of chromosome 10 were very rare in our study. In addition, we have demonstrated the remarkable complexity of hypodiploid clones with over half the cases showing a very complex karyotype. A remarkable finding of this study was the high incidence of underlying MDS (at least 58%). This contrasts sharply with unselected series of de nova AML in which not more than 15% have underlying MDS.

912

.I. M. Hawkinsef al.

Such cases are known to have a poor prognosis as seen in our study [9]. Survival of our patients was almost without exception extremely short. Although median survival was slightly shorter for those with very complex karyotypes, and conversely survival was slightly longer for those without involvement of chromosomes 5 or 7, none of these differences was statistically significant. A larger cohort of cases is needed to establish whether the trends represent real differences in survival between groups. Two surveys using the modified Chicago classification have both shown that groups with ‘abnormalities of 5 and 7’ and ‘very complex’ clones have a very poor survival (3 months in each case for the former [4] and 4.4 months in each case for the latter [lo]). The prognosis for ‘hypodiploidy’ however, may be poor (5 months [4]) or less poor (28.9 months [lo]). Treatment was shown to be an important component of survival in AML at IWCL4 and IWCL6. This cannot be considered in this study because a number of patients were not treated. The patients in our series have a high proportion of FAB type M6 (29%). This contrasts with the 3-5% of FAB M6 in AML as a whole [ll] and with the IWCL series which included 1% M6 cases, none of which were hypodiploid. However, 13% of the secondary AML cases in IWCL had this FAB type. The FAB M6 is known to be strongly associated with underlying MDS [12] and also with very poor survival [4]. It has also been reported that M6 is associated with an unstable karyotype, including ring chromosomes, polyploidization and double minutes [13]. This association was not apparent in our study. The two cases with polyploidization (doubling of the hypodiploid clone) were MO and Ml FAB types, while ring chromosomes were frequent both among M6 cases and those of other FAB types. A more recent report of the karyotypes of 26 patients with AML M6 [14] included one with a ring, two with fragments, two with polyploidization and five (19%) with complex hypodiploidy. The association between FAB M6 and hypodiploidy therefore seems stronger than that between M6 and an unstable karyotype. A very high proportion of M6 cases in the above study also had abnormalities of chromosomes 5 and/or 7 (77%). In conclusion, we have analysed a relatively large number of cases belonging to a sub-group of hypodiploid AML, i.e. from which simple loss of chromosomes 5, 7, X and Y were excluded. We have demonstrated primarily that karyotypes in these cases tend to be very complex, and that hypodiploid karyotypes without involvement of chromosomes 5 and/or 7 or 17 are extremely rare. It appears that the poor prognosis of this selected hypodiploid group may be related to the high incidence of 5 and 7 involvement rather than to the complexity of the karyotypes. Future studies with greater

numbers of patients might clarify the relative importance of modal number, karyotypic complexity and involvement of chromosomes 5 and 7. Future studies should also include the use of fluorescent in situ hybridization to determine how chromosomes 5 and 7 are involved and to identify other ambiguous chromosomal segments in complex hypodiploid clones. Acknowledgements-The UKCCG thanks the many haematologists who provided the clinical information and bone marrow slides, gratefully acknowledges the support of the Leukaemia ResearchFund and thanks Professor A.V. Hoffbrand for his support.

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2. Seeker-Walker L. M. (1990) Prognostic and biological importance of chromosomefindings in acute lymphoblastic leukemia. 10th anniversary article. Cancer Genet. Cytogenet. 49, 1. 3. Swansbury G. J., Lawler S. D., Alimena G., Arthur D., Berger R., Van den Berghe H., Bloomfield C. D., De la Chapelle A., Dewald G., Garson 0. M., Hagemeijer A., Mitelman F., Rowley J. D. & Sakurai M. (1994) Long-term survival in acute myelogenousleukemia: a secondfollowup of the Fourth International Workshop on Chromosomes in Leukemia. Cancer Genet. Cytogenet. 73, 1. 4. Arthur D. C., Berger R., Golomb H. M., Swansbury G. J., ReevesB. R., Alimena G., Van den Berghe H., Bloomfield C. D., De la Chapelle A., Dewald G. W., Garson 0. M., Hagemeijer A., Kaneko Y., Mitelman F., Pierre R. V., Ruutu T., Sakurai M., Lawler S. D. & Rowley J. D. (1989) The clinical significance of karyotype in acute myelogenous leukaemia. Cancer Genet. Cytogenet. 40, 203. 5. lWCL4 (1984) Fourth International Workshop on Chromosomesin Leukemia - clinical significance of chromosomal abnormalities in acute nonlymphoblastic leukemia. Cancer Genet. Cytogenet. 11, 332.

6. Rooney D. E. & Czepulkowski B. H. (1992) Human Cytogenetics. A Practical Approach. Vol II. Malignancy Abnormalities. Oxford University Press,

and Acquired

New York. 7. Betts D. R., Rohatiner A. Z. S., Evans M. L., RassamS. M. B., Lister T. A. & Gibbons B. (1992) Abnormalities of chromosome 16q in myeloid malignancy: 14 new cases and a review of the literature. Leukemia 6, 1250. 8. Hawkins J. M., Moorman A. V., Hoffbrand A. V., Martineau M., Wright F. S., Mehta A. B., Prentice H. G. & Seeker-Walker L. M. (1994) Association of 17~ loss with late stage or refractory disease in hematological malignancy. Cancer Genet. Cytogenet. 77, 134. 9. Catovsky D. & Hoffbrand A. V. (1989) Acute leukaemia. In Postgraduate Haematology (Hoffbrand A. V. and Lewis S. M., Eds), pp. 379417. Heinemann, Oxford. 10. Palka G., CalabreseG., Fioritoni G., Stuppia L., Franchi P. G., Marino M., Antonucci A., SpadanoA. & Torlontano G. (1992) Cytogenetic survey of 80 patients with acute nonlymphocytic leukemia. Cancer Genet. Cytogenet. 59, 45.

Hypodiploidy in AML 11. Bain B. J. (1990) Leukuemia Diagnosis: a Guide to the FAB Clussifcution. Gower Medical Publishing, London. 12. Brito Babapulle F., Catovsky D. & Galton D. A. G. (1987) Clinical and laboratory features of de nova acute myeloid leukaemia with trilineage myelodysplasia. Br. J. Haematol. 66, 445. 13. Kadam P. R., Balsara B. R., Zafaraullah K. Z., Dadabhoy K. D., Bhisey A. N. & Advani S. H. (1990) Cytogenetic features of erythroleukemia (EL). A study of 11 cases. Cancer Genet. Cytogenet. 50, 89. 14. Olopade 0. I., Thangavelu M., Larson R. A., Mick R., Kowal-Vern A., Schumacher, H. R., Le Beau M. M., Vardiman J. W. & Rowley J. D. (1992) Clinical, morphologic and cytogenetic characteristics of 26 patients with acute erythroblastic leukemia. Blood 80, 2873.

United Kingdom

APPENDIX Cancer Cytogenetics

Group (UKCCG)

Chairman: Professor L.M. Seeker-Walker. Secretary: M. Martineau, Department of Haematology, Royal Free Hospital School of Medicine, Pond Street, London, NW3 2QG, U.K. A full list of UKCCG members is available from the Secretary.

UKCCG

913

laboratories

and participants

Cytogenetics Laboratory

in this study

(No. cases) Participants

Duncan Guthrie Institute of Medical Genetics, Glasgow

(8)

J. Stewart

ICRF Department of Medical Oncology, St Bartholomew’s Hospital, London

(1)

D. Lillington

Cytogenetics Department, Hammersmith Hospital, London

(2)

J. Bungey

Academic Haematology and Cytogenetics, Royal Marsden Hospital, Surrey

(2)

J. Swansbury

Department of Haematology, Royal Free Hospital School of Medicine, London

(6)

J. Hawkins

NW Regional Cytogenetics Service for Oncology, Christie Hospital, Manchester

(15)

C. Harrison

Department of Human Genetics, University of Newcastle

(4)

N. Bown

Regional Genetics Service, Churchill Hospital, Oxford

(1)

H. Sheridan

Genetics Department, BNF PLC, Sellafield

(1)

C. Shippey