Application of interphase cytogenetics to monitor bone marrow transplants

American Journal of Hematology 49:15-20 (1995)

Application of lnterphase Cytogenetics to Monitor Bone Marrow Transplants

Vickie Vrazas, Lisa M. Ooms, Christina Rudduck, Jeffrey Szer, Lynda J. Campbell, and 0. Margaret Garson

Victorian Cancer Cytogenetics Service (V.V., L.J.C., O.M.G.) and University of Melbourne Department of Medicine (L.M.O., C.R.), St. Vincent’s Hospital, and Bone Marrow Transplant Service (J.S.), Royal Melbourne Hospital, Melbourne, Victoria, Australia

Chromosomal in situ hybridization (ISH) has extended the scope of cytogenetic analysis to nondividing cells by the use of chromosome-specific probes detected by nonisotopic techniques. This provides a rapid and sensitive method for identifying chromosomes in interphase cells, and is useful in gauging engraftment following bone marrow transplan- tation, particularly when the number of cells obtained is minimal. We have performed ISH using a Y-heterochromatin-specific probe to monitor patients with malignant hematologi- cal disease who have received a sex-mismatched transplant. The results have been compared with those obtained from concurrently performed standard cytogenetic analy- sis. Host cells were detected by interphase cytogenetics in all patients posttransplant, at times varying from 28-1,825 days, whereas routine analysis detected host cells in only 4 patients, 3 of whom were found to be in relapse. The significance of the persistence of host cells is unknown, but it does not appear to indicate impending relapse.

Key words: transplantation, in situ hybridization, interphase cytogenetics

0 1995 Wiley-Liss, Inc.

INTRODUCTION

~ _ _ _ _ _ ~

The technique of chromosomal in situ hybridization (ISH), using chromosome-specific DNA probes which are detected by nonradioactive means, has increased the scope of cytogenetic analysis. Using this technique it is now possible to derive cytogenetic information from in- terphase and terminally differentiated cells. This applica- tion of ISH is referred to as interphase cytogenetics [ 5 ] , and has the advantage of being rapid to use; it does not necessitate prior culturing of the cells or the production of high quality metaphase spreads; and it is simple to inter- pret.

The application of ISH [6,7] at varying times post- BMT has shown that small numbers of host cells may be detected. Although this would appear to indicate impend- ing relapse, this has not been the case, so that the full significance of finding residual host cells post-BMT is debatable.

Bone marrow transplantation (BMT) is being used more frequently as treatment for various malignant dis- eases because of its increasing rate of success. However, host marrow relapse occurs in approximately 30% of patients who have undergone BMT for acute leukemia, making relapse the single most frequent cause of death following BMT. Thus, the ability to detect the presence of host cells post-BMT may shed some light on the mech- anism of relapse or even graft failure. This can be achieved by different techniques, among which cytoge- netic analysis is the most common, providing that the donor and the recipient are of different sex or that there is a chromosomal abnormality in the leukemic cells. This technique is also limited in that it requires cells to be in division in order to obtain metaphase spreads, which is often difficult in the early stages posttransplant when the marrow is hypocellular. Other techniques used to detect the presence of host cells posttransplantation include

[ 11, restriction fragment length polymorphism (RFLP)

(PCR) at 0.1% “41. Although these techniques are useful, quantification of the results is difficult.

HLA-typing which has a level of sensitivity of 25-50% Received for publication February 14, 1994; accepted December 14, 1994.

Address reprint requests to Dr. O.M. Garson, Victorian Cancer Cyto- genetics Service, St. Vincent’s Hospital, 41 Victoria Parade. Fitzroy, Victoria 3065, Australia.

at ‘-lo% [2731, and polymerase

0 1995 Wiley-Liss, Inc.

16 Vrazas et al.

TABLE I. Clinical Characteristics of the 30 Patients Studied

Clinical characteristics

Total no. of patients Median age in years (range) Sex (M/F) Diagnosis prior to BMT Chronic myelogenous leukemia

Chronic phase

30 28 (5-57)

11/19

I Accelerated phase 1 Blast crisis 2

Remisson 5 Relapse 6

Remission 4

2

Acute nonlymphoblastic leukemia

Acute lymphoblastic leukemia

Relapse 2 Refractory anemia with excess blasts in transformation

Hodgkin’s disease 1 (RAEBT)

HLA match Identical 27 Nonidentical 3

We therefore undertook a study of patients at various stages postsex-mismatched BMT for hematological dis- orders using ISH with a Y-specific probe, and compared these findings with those obtained from standard cytoge- netic studies. All patients were found to have some resid- ual host cells not detected by routine cytogenetic analy- sis, which in most cases appeared unrelated to the occurrence of relapse.

MATERIALS AND METHODS Patients

Thirty patients who received transplants from donors of the opposite sex were studied; bone marrow was uti- lized in 29 cases, and peripheral blood in 1 case (patient no. 7) where bone marrow was unavailable. All patients had malignant hematological disorders but did not belong to any one particular disease subgroup. Patient character- istics are shown in Table I. Conditioning regimens prior to BMT were cyclophosphamide and 12-Gy fractionated total body irradiation in 7 patients (cases 10, 12, 23, 25, 27,28, and 30), and busulphan and cyclophosphamide in the remaining 23. Prophylaxis for graft-vs. -host disease was with cyclosporine and methotrexate, and all patients received T-cell-replete bone marrow [8]. The research protocol for the study was approved by the St. Vincent’s Hospital Research Ethics Committee.

Controls Peripheral blood from 2 healthy volunteers (1 male, 1

female) was mixed and used in a dilution series to deter- mine the hybridization efficiency of the Y probe. Mono- nuclear cells were separated using Ficoll-Paque (Pharrna-

cia, Uppsala, Sweden) by standard techniques and were washed, mixed, and treated with a hypotonic solution (0.075 M KCl) to isolate the nuclei. These were fixed in 3:l methano1:acetic acid, and the solution was dropped onto slides and air-dried. Negative controls using the Y probe on marrows received from female patients trans- planted from female donors were also utilized.

Cell Culture Cells from bone marrow and peripheral blood were

cultured in 1640 RPMI supplemented with NaHCO, (10 mM), Hepes buffer (20 mM), 10% fetal calf serum, pen- icillin (50 IU/ml), streptomycin (50 IU/ml), and L-glu- tamine (2 mM). Chromosome preparations were obtained from short-term fluorodeoxyuridine-synchronized cul- tures [9] after l hr incubation with colcemid (0.8 pg/ml), and harvested using standard procedures. Slides were aged overnight at 60°C and either banded and analyzed for routine cytogenetics, or used for ISH. Cytogenetic data generated from samples yielding 5 or more metaphases are included in this analysis, with a mean of 27 metaphases per patient being examined (range 5-50).

In Situ Hybridization (ISH) The human Y-heterochromatin-specific DNA probe,

2.45 kb Hae 111, (Amersham, RNP-13052), was labeled by nick translation (Boehringer Mannheim, Mannheim, Germany), using biotinylated- 16-dUTP (Boehringer Mannheim, 1093 070) according to the manufacturers instructions.

ISH was performed as described previously by Rud- duck et al. [lo], using a biotin-streptavidin-polyalkaline- phosphatase complex to visualize the biotinylated probes. Slides were counterstained with 2.5% Giemsa, and a minimum of 200 cells per patient were analyzed under a dry X63 objective. For data analysis, Y-chromo- some-positive cells were scored as male, Y-chromo- some-negative cells as female. Positive and negative con- trols from normal male and female individuals were included in all experiments to assess background and hybridization efficiency.

RESULTS

Peripheral blood mononuclear cells from 2 healthy vol- unteers were used in a 10-fold dilution series to establish the sensitivity of male cell detection. The dilution series ranged from 1 : 10-1 : 1,000 (XY:XX), and the undiluted male and female samples served as the positive and neg- ative controls, respectively. The results are shown in Table 11. The criteria for scoring a cell as Y-positive (male) was that the nucleus should contain only 1 solid purple-black dot (Fig. 1). Cells which contained a small or indistinct dot were not considered positive (Fig. 1). The percentage of Y-bearing cells was found to corre-

In TABLE II. Number of Y-Positive Cells in a Dilution Series of Normal Male and Female Lymphocytes (XY:XX) as Determined by ISH Using a Y-Chromosome-Specific Probe

Dilution series No. of Y-bearing cells"

1:l 1:lO 1:loo 1:1,OOo Male control Female control

498 108

11 2

996 0

"For each sample, 1,0oO cells were analyzed. Only cells with one spot were scored as "Y-positive."

Fig. 1. Bone marrow cells after hybridization with a Y-het- erochromatin-specific probe. Large arrows indicate the sin- gle, solid dot observed in a Y-positive cell. Small arrows indicate cells which were not considered positive, due to either a small or an indistinct dot.

spond to the dilution factor, thus illustrating the sensitiv- ity of the technique (0.8%). No Y-bearing cells were observed on the female slide (negative control). Simi- larly, no Y-bearing cells were observed in the female-to- female BMT control.

Cytogenetic studies and ISH on interphase cells were carried out concurrently on the bone marrow from 30 patients who had received sex-mismatched bone marrow transplants at varying intervals post-BMT (21-1,825 days). ISH, using a Y-heterochromatin-specific DNA probe, was performed successfully on all samples. Suc- cessful cytogenetic analysis, however, was dependent on the quality and quantity of sample received, as some man-ows studied soon after BMT were hypoplastic and yielded very few metaphases for analysis.

The results of the cytogenetic and ISH studies are shown in Table 111. Using ISH on interphase cells, we found that host cells were present in all 30 patients,

lterphase Cytogenetics in Marrow Transplants 17

including 1 who had 13% host cells 5 years posttrans- plant. The lowest amount of host cell retention was 5%, which occurred in patient no. 26, at 3 years posttrans- plant. However, no host cells were detected in 27 of the 30 patients studied cytogenetically at initial presentation. When a patient relapsed post-BMT, this was reflected both in the cytogenetic and ISH results. Patient no. 1 (male) at 21 days post-BMT was found on routine cytoge- netics to have all female cells, but ISH detected 10% male cells. He relapsed at day 352 post-BMT, at which time 86% of cells contained a Y-chromosome cytogenet- ically, but only 59% on interphase. The discrepancy in the figures obtained from the two techniques in relapse may be attributed to the difference in the total number of cells studied, as only 21 cells could be analyzed cytoge- netically, whereas over 200 were scored using ISH. Therefore, the result obtained with ISH indicates more accurately the events occurring at the cellular level, as the technique does not require the cells to be in division, so all cells on the preparations can be examined. However, patient no. 17 was found to have 30% host cells present using routine cytogenetics and 62% using ISH on inter- phase cells at day 90 posttransplant when she was consid- ered to be in hematological remission but had graft-vs.- host disease. Approximately 335 days posttransplant, she was found to have no host cells on cytogenetic analysis but showed a 35% host cell retention with ISH.

Using ISH on interphase cells, we were able to obtain a result in 2 patients who had no mitoses on the cytogenetic preparations (patient nos. 12 and 21). Results were also obtained from 2 cases where <5 mitoses were able to be analyzed (patient nos. 15 and 23). Thus, interphase cyto- genetics post-BMT proved to be more informative of the engraftment result than standard cytogenetic techniques.

The percentage retention of host cells as a function of time post-BMT is shown in Figure 2. Each time point represents the first time that the patient was studied with both ISH and routine cytogenetics. Evidence of host cell retention was seen cytogenetically in only 3 cases (patient nos. 13, 17, and 21), 2 of whom were in morphological relapse at the time of study. One further patient (no. 16) showed cytogenetic and ISH evidence of host cell reten- tion when she re-presented in relapse at day 425 post- BMT. Thus, using ISH on interphase cells, host cell retention was seen at all time points post-BMT in all patients with levels varying from 5-20%, and was 20% in 4 cases, 4 years posttransplant, who were considered not to be in relapse.

DISCUSSION

In this study we have shown that interphase cytogenet- ics using a chromosome-specific DNA probe can be used as a means of readily detecting the presence of residual host cells in the bone marrow of sex-mismatched BMT

18 Vrazas et al.

TABLE 111. Results Obtained From Cytogenetic and Interphase Analysis (ISH Using a Y-Chromosome-SDecific Probe)

Patient no. Sex

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

M

F

F M

F M

F

F

F M

M M M F F F

F

F

M

M F

F F F F

F F

M F M

Time of study (days post-BMT)

21 28

300 338 352 21 28 21 21

170 28 28 90

365 2gb 90

365 28 90

210 28 28 90 28 28 39 42 42 90

365 425 90

335 100 180 103 160 120 210 390 420 360 730

1,095 1,095 1,460 1,460 1,460 1,825 1,460 1,460 1,825

No. of Y-bearing cells

__

Cytogenetic analysisa

Interphase analysis

N/M 0/30 (0%) 0/23 (0%) 0135 (0%)

18/21 (86%) 11/11 (100%) 40/40 (1 00%) 40/40 (100%) 0/30 (0%) 0/30 (0%)

35/35 (100%) 0/23 (0%) 0/20 (0%) 0135 (0%) 6/6 (100%)

40/40 (100%) 41/41 (100%) 40140 (100%) 50/50 (100%) 20/20 (100%) 40/40 (100%) 0125 (0%) 0/25 (0%) 0/40 (0%)

N/M 6/10 (60%)

42/42 (100%) 1/1 (100%) 9/9 (100%)

34/34 (100%) 4/16 (25%) 7/10 (70%)

35/35 (100%) 50/50 (100%) 70/70 (100%) 0/30 (0%) 0/42 (0%) 0110 (0%)

16/23 (70%) 19/19 (100%)

N/M 22/22 (100%)

5/5 (100%) 50/50 (100%) 40/40 (100%) 40/40 ( 100%) 20120 (100%) 50/50 (100%) 15/15 (100%) 36/36 (100%) 24/24 (100%) 0/41 (0%)

10% 17% 16% 18% 59% 70% 73% 73% 22% 20% 78% 30% 32%

8% 88% 93% 91% 91% 91% 93% 88% 9%

12% 10% 18% 40% 86% 67% 92 % 82% 29% 38% 65% 85% 89% 14% 17% 19% 59% 17% 37% 89% 79% 82% 95% 93 % 74% 80% 85% 21% 76% 13%

"N/M, no mitoses. bISH study was performed on peripheral blood.

lnterphase Cytogenetics in Marrow Transplants 19

Cytogenetic analysis

ISH analysis

?Am I I

8 u

0 X

& m

2 O - - - - m m m m m m m m m ~ N o o o 0 o o o o ~ ~ o o o o ~ N N N N N N N N N N ~ N ~ ~ ~ ~ ~ O O N - W ~ ~ ~ W W W W N

m C 8 u

* I

- - - N m l . o o q q * * ~ - 4 - -

Days Post-BMT

Fig. 2. Graph showing the percentage of residual host cells at first presentation post- BMT. Patients were studied by both ISH and cytogenetic analysis.

patients. This technique, although limited by the fact that the transplant donors must be of the opposite sex, offers a number of advantages over routine cytogenetic analysis. It is a rapid technique and results can be obtained within 24-48 hr after receiving a specimen, compared to 7-10 days using routine cytogenetics, as prior culturing is not required. As it is not restricted to metaphase spreads, it overcomes the problem of low mitotic indices due to a hypoplastic marrow posttransplant. Using interphase analysis, data can be obtained from a spectrum of cell types including differentiated cells, thus providing us with genotypic information hitherto unavailable. Accord- ing to Hooke’s theorem [ l l ] , 300 metaphases must be scored in order to exclude mosaicism to a level of 1% with a 95% probability. Using interphase cytogenetics, over 200 cells are routinely scored; therefore, the ability to detect the presence of a chimeric state is high. The discrepancy between the cytogenetic and the ISH results (Table 111) is probably due to the fact that the numbers of cells studied by both methods are not comparable. It is also possible that mitotic cells can be lost even in short- term culture [ 121. In order to obtain more accurate infor- mation on cellular interactions, bone marrow smears may need to be studied using ISH.

Our study shows that all of our patients had mixed chimerism, with residual host cells being present in all bone marrows studied for varying periods of time post- BMT (21-1,825 days). We were unable to determine a trend for the loss of host cells as shown previously by Durnam et al. [13], who found that the number of host cells decreased steadily during the first month posttrans- plant, with marrows of patients entirely populated by

donor cells by day 84. Other studies on marrow engraft- ment after transplantation have shown conflicting results. Yam et al. [3], using RFLP analysis, found patients who were still in remission with host/donor mixed chimerism for up to 5 % years posttransplant. These studies are in general agreement with our findings, but an explanation as to the difference between these results and those found by Durnam et al. [13] is not yet apparent. We found mixed chimerism to be universally present, without direct correlations with age, diagnosis, disease status, or condi- tioning regimen. Durnam et al. [13] and van Dekken et al. [14] in their studies also found no direct correlation with any of the above. However, others have reported that host cells are more frequently observed in patients who have received HLA-matched marrow [ 151, and who did [ 161 or did not [ 171 develop acute GVHD.

The presence of residual host cells post-BMT may not be indicative of residual disease. Most of the patients in our study continue to survive and show no signs of clini- cal relapse despite the presence of mixed chimerism. Thus, the relationship of donor-host chimerism to the risk of subsequent relapse or to the occurrence of GVHD post-BMT is as yet unclear, as is the involvement of donor and host cells in the different hemopoietic cell lineages and in the bone marrow microenvironment. De- spite the reported increased sensitivity of DNA diagnostic techniques such as RFLP analysis and PCR in sex- matched and sex-mismatched transplantation studies, both these techniques are difficult to quantitate and re- quire cell lysates, which means that cellular morphology is lost. We plan to extend our studies by combining ISH with immunophenotyping to identify the cell lineage of

20 Vrazas et al.

host and donor cells. In this way we will be able to identify both the phenotype and genotype of cells in the bone marrow, which will permit a more accurate inter- pretation of events occurring posttransplant.

ACKNOWLEDGMENTS

The authors thank Drs. W. Sheridan and K. Tiede- mann for referring several patients for study, and for providing additional clinical details. We acknowledge the support of the Anti-Cancer Council of Victoria (V.V., O.M.G.) and the National Health and Medical Research Council (C.R., O.M.G.), and we thank St. Vincent’s Hospital for permission to publish.

REFERENCES

1. Blazar BR, Soderling CCB, Vallera DA: Assessment of multi-organ system engraftment by genotypic typing using restriction fragment length polymorphisms and by phenotypic typing using a microcytotox- icity assay. J Immunol 137:3338-3345, 1986.

2. Blazar BR, Orr HT, Arthur DC, Kersey JH, Filipovich AH: Restric- tion fragment length polymorphisms as markers of engraftment in allogeneic marrow transplantation. Blood 66: 1436-1444, 1985.

3. Yam PY, Petz LD, Knowlton RG, Wallace RB, Stock AD, Lange GD, Brown VA, Donnis-Keller H, Blume KG: Use of DNA restriction fragment length polymorphisms to document marrow engraftment and mixed hematopoietic chimerism following bone marrow transplanta- tion. Transplantation 43:39!b407, 1987.

4 . Lawler MN, McCann SR, Conneally E, Humphries P: Chimerism following allogeneic bone marrow transplantation: detection of resid- ual host cells using the polymerase chain reaction. Br J Haematol

5. Cremer T, Landegent J, Bruckner A, Scholl HP, Schardin M, Hager HD, DeviIee P, Pearson P, van der Ploeg M: Detection of chromosome aberrations in the human interphase nucleus by visualization of spe- cific target DNAs with radioactive and non-radioactive in situ hybrid- ization techniques: diagnosis of trisomy 18 with probe L1.84. Hum Genet 14:34&352, 1986.

73:205-210, 1989.

6. Petz LD, Yam P, Wallace RB, Stock AD, de LangeG, Knowlton RG, Brown VA, Donnis-Keller H, Hill LR, Forman SJ, Blume KG: Mixed hematopoietic chimerism following bone marrow transplantation for hematological malignancies. Blood 70: 1331-1337, 1987.

7. Singer JW, Keating A, Ramberg R: Long-term stable hematopoietic chimerism following marrow transplantation for acute lymphoblastic leukemia: a case report with in vitro marrow culture studies. Blood 62369, 1983.

8. Szer J, Sheridan WP: Chronic myeloid leukaemia treated by allogeneic bone marrow transplantation from histocompatible sibling donors. An invariably fatal malignancy rendered highly curable. Aust N 2 J Med 21:408-413, 1991.

9. Webber LM, Garson OM: Fluorodeoxyuridine synchronization of bone marrow cultures. Cancer Genet Cytogenet 8: 123-132, 1983.

10. Rudduck C, Lukeis RE, McRobert TL, Chow CW, Garson OM: Chro- mosomal localization of amplified N-myc in neuroblastoma cells using a biotinylated probe. Cancer Genet Cytogenet 5855-59, 1992.

1 I. Hooke EB: Exclusion of chromosomal mosaicism: tables of 90%, 95% and 99% confidence limits and comments on use. Am J Hum Genet 29:94-97, 1977.

12. Rudduck C, Garson OM: The proportion of erythroid mitoses in nor- mal human bone marrow in short-term culture systems. Pathology

13. Durnam DM, Anders KR, Fisher L, O’Quigley J, Bryant EM, Donna11 Thomas E: Analysis of the origin of marrow cells in bone marrow transplant recipients using a Y-chromosome-specific in situ hybridiza- tion assay. Blood 74222C2226, 1989.

4. Van Dekken H, Hagenbeek A, Bauman JGJ: Detection following sex mismatched bone marrow transplant by fluorescence in situ hybridiza- tion with a Y-chromosome specific probe. Leukemia 3:724-728, 1989.

5. Przepiorka D, Donna1 Thomas E, Dumam DM, Fisher L: Use of a probe to repeat sequence of the Y chromosome for detection of host cells in peripheral blood of bone marrow transplant recipients. Am J Clin Pathol95:201-206, 1991.

16. Dorre S, Schneider EM, Haen M, Wallace RB, Wemet P Follow up of chimeric state during early hematopoietic reconstitution after alloge- neic bone marrow transplantation using oligonucleotide probes from minisatellite gene regions. Transplant Proc 21:308%3091, 1989.

17. Korver K, de Lange GG, van den Bergh RL, Schellekens €TA, van Loghem E, van Leeuwen F, Vossen JM: Lymphoid chimerism after allogeneic bone marrow transplantation. Y chromatin staining of pe- ripheral T and B lymphocytes and allotyping of serum immunoglobu- lins. Transplantation 44.643450, 1987.

21:185-188, 1989.