1993 82: 606-612   

 De Felice and G MeloniF Lo Coco, PG Pelicci, F D'Adamo, D Diverio, G Alimena, E Montefusco, W Arcese, G Avvisati, L comments]remission after suppression of specific gene rearrangements [see Polyclonal hematopoietic reconstitution in leukemia patients at

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Polyclonal Hematopoietic Reconstitution in Leukemia Patients at Remission After Suppression of Specific Gene Rearrangements

By Francesco Lo Coco, Pier Giuseppe Pelicci, Francesca D’ Adamo, Daniela Diverio, Giuliana Alimena, Enrico Montefusco, William Arcese, Giuseppe Avvisati, Lidia De Felice, Giovanna Meloni, Clara Nervi, Franco Mandelli, and Giuseppe Saglio

Clonality studies of hematopoietic reconstitution after re- mission were performed in 24 female patients (pts) with leukemias characterized by specific molecular markers. At diagnosis, 13 pts had promyelocytic leukemia (PML) reti- noic acid receptor-a (RAR-a)-rearranged acute promyelo- cytic leukemia (APL), 8 Philadelphia positive (Ph’) break- point cluster region (BCR + ) chronic myeloid leukemia (CML), and 3 Ph+ (BCR + ) acute lymphoblastic leukemia (ALL). All pts were analyzed at presentation and after Southern blot suppression of specific rearrangements after various treatments, including conventional chemotherapy, autologous or allogeneic bone marrow transplant (BMT), all-trans retinoic acid, and a-2b interferon. DNA from BM samples collected at diagnosis and, during remission phases, were subjected to Southern blot analysis with the M27@ probe to detect X chromosome methylation differ- ences, and with BCR, in CML and ALL cases, or PML/RAR- a probes for gene rearrangements, in APL cases. Twenty- one of the 24 pts had polyclonal methylation patterns at

OST HUMAN TUMORS are monoclonal prolifera- M tions originating from a single transformed cell, and consistent genetic defects with potential transforming capa- bility have been identified in a variety of neoplastic dis- ease~.’-~ Among hematologic malignancies, significant ex- amples are the BCR/ABL molecular rearrangement of Philadelphia positive (Ph+) chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL)4 and the more recently reported fusion of promyelocytic leukemia (PML) and retinoic acid receptor-a (RAR-a) genes in the t( 15; 17) translocation of acute promyelocytic leukemia (APL).5-8 Besides their pathogenetic relevance, such rear- rangements provide ideal clonal markers of leukemic cells to be exploited for both diagnosis and monitoring of these diseases.’-14

Studies on glucose 6-phosphate dehydrogenase (G6PD) isoenzyme expressi~n’~ and of methylation differences at X-linked DNA polymorphic loci’6 in heterozygous females also provide methods to assess clonality and to determine

From Hematology, the Department of Human Biopathology and the Institute of Histology, University La Sapienza, Roma; the Clin- ica Medica, University of Perugia; and the Clinica Medica, Univer- sity of Torino, Italy.

Submitted December 29, 1992; accepted March 12, 1993. Supported by CNR PF ACRO Grant No. 92.02263.PF39. Address reprint requests to Francesco Lo Coco, MD, Hematol-

ogy, Department of Human Biopathology, University La Sapienza, Via Benevento 6, 00161 Roma, Italy.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1993 by The American Society of Hematology. 0006-49 71/93/8203-0025$3.00/0

remission, together with disappearance of the specific rear- rangement, whereas 3 pts retained the same single un- methylated DXS255 allele detected at diagnosis despite no evidence of gene rearrangement. Concerning these 3 pts, such an apparently clonal pattern was also observed in one case in T lymphocytes and skin-derived DNA; in a sec- ond case in BM fibroblasts and T lymphocytes; and, in the third case, in blood mononuclear cells obtained from her healthy female BM donor. All these 3 pts are in unmain- tained clinical and cytogenetic remission after more than 20 months off therapy. These data suggest that (1 ) polyclo- nal and presumably normal hematopoiesis occurs in APL, CML, and Ph+ ALL pts once the major burden of leukemic cells carrying a specific rearrangement is suppressed by treatment; and (2) unbalanced X chromosome methylation patterns, or aberrant methylation of X chromosome re- gions may be observed in some cases, most likely reflect- ing constitutional features simulating a clonal picture. 0 1993 by The American Society of Hematology.

the level of stem cell involvement in hematologic disorders like CML and other myeloproliferative syndromes, acute myeloid leukemia (AML), and myelodysplastic syn- dromes. ”-’O

Surprisingly, using the latter methods, several groups have reported that clonal hematopoiesis may also occur in AML patients during the remission phase induced by che- m~therapy.~’-’~ The persistence of clonal hematopoiesis during apparent complete remission has been interpreted according to the theory of a multistep pathogenesis process in human leukemia. In this context, a preleukemic clone maintaining a normal differentiation ability could survive treatment and proliferate once overt leukemia is elimi- nated.z’-24 Alternatively, clonal remissions could be the con- sequence of the regenerative activity of a single normal pre- cursor cell spared by chemotherapy. However, very little is known about the clinical and prognostic significance of clonal remission in le~kemia.’~

To address these issues, we decided to investigate the na- ture of remission after treatment in a series of female pa- tients with Ph + CML, P h + ALL, and t( 15; 17) APL, which were found to be heterozygous at the X-linked DXS255 locus and who achieved suppression at Southern analysis of specific breakpoint cluster region (BCR)/ABL or PML/ RAR-a rearrangements.

MATERIALS AND METHODS

Patient selection. Twenty-four patients (pts), including 13 APL, 8 P h + CML, and 3 P h + ALL cases, were studied. Selection was based on the following criteria: ( I ) female sex; (2) presence of a specific cytogenetic and/or molecular marker at diagnosis in greater than 90% of leukemic cells; (3) response to treatment with cytoge- netic and/or molecular remission, ie, complete cytogenetic conver- sion to normal karyotype and/or disappearance at Southem blot of the specific gene rearrangement detected at diagnosis; and (4) het- erozygosity at the DXS255 locus on the X chromosome.

606 Blood, VOI 82, NO 2 (July 15), 1993: pp 606-61 2

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POLYCLONAL HEMATOPOIESIS IN LEUKEMIA REMISSION 607

Therapy. Individual treatments for each of the 24 pts are re- ported in Table 1. Briefly, 8 pts with APL received as induction therapy conventional chemotherapy (CHT) schedules including an anthracycline (daunorubicin or idarubicin) alone or in combina- tion with cytosine arabinoside (7 + 3 regimen). Following achieve- ment of complete remission (CR), 7 pts received CHT for consolida- tion and maintenance, and 1 pt received high-dose CHT followed by autologous BMT. Five APL pts received all-trans retinoic acid (ATRA) at oral doses of 45 to 50 mg/m2 until the achievement of CR. Of this group, 4 pts received as consolidation conventional CHT and 1 underwent high-dose CHT and autologous BMT.

Table 1. DXS255 Methylation Patterns at Diagnosis and at

All pts with CML were initially treated with a-2b interferon (IFN) (Hoffman-La Roche, Nutley, NJ) according to reported pro- t o c o l ~ ~ ~ for at least 12 months. After hematologic response and partial karyotypic conversion (15% to 75% Ph+ BM metaphases) after 1 year, 3 pts underwent BM harvesting and autologous BMT, whereas the 5 remaining pts achieved a complete Ph’ suppression and were maintained on IFN therapy. Finally, the 3 pts with Ph’ + ALL were treated with CHT according to the GIMEMA 0288 pro- tocoIz6 and, after CR, received allogeneic BMT from HLA-identical siblings.

DNA samples. DNA was obtained from BM aspirates at diag-

Remission Following Suppression of Specific Rearrangements

Methyl Patterns at Remission

Methyl Time of Patient Allele at Sampling Source Methylated

No. Age Disease Diagnosis Therapy (from CR’) of DNA Allele(s) Follow-Up

1 2 3 4 5 6 7 8 9

10 11 12

13

14 15 16 17

18

19

20

21

22

23

24

13 41 30 46

6 18 2

40 30 29 22 51

63

69 67 42 35

54

27

50

40

27

41

33

APL APL APL APL APL APL APL APL APL APL APL APL

APL

CML CML CML CML

CML

CML

CML

CML

Ph+ ALL

Ph+ ALL

Ph+ ALL

BM donor

A B A B A B A A B A A B

A

A A A B

B

B

B

B

B

A

B

CHT ATRA/CHT ATRA/CHT CHT CHT ATRA/CHT CHT CHT CHT Auto BMT CHT ATRA/Auto BMT

ATRA/CHT

IFN IFN IFN IFN

IFN

IFN/Auto BMT

IFN/Auto BMT

IFN/Auto BMT

CHT Allo BMT

CHT Allo BMT CHT Allo BMT

5 mo 7 mo 1 mo 1 mo 5 mo 4 mo 5 mo 4 mo 8 mo

44 mo 11 mo 2 mo 4 mo 8 mo (rel)

15 mo 15 mo 16 mo 14 mo 53 mo 48 mo 39 mo 54 mo 52 mo 52 mo

7 mo 12 mo 3 mo

28 mo 6 mo

14 mo 15 mo 17 mo 17 mo 19 mo 4 mo 2 mo 7 mo (rel) 2 mo 1 mo 2 mo 3 mo

14 mo

BM BM BM BM BM BM BM BM BM BM BM BM BM BM BM T cells Skin biopsy BM BM PB BM BM BM T cells BM BM BM BM BM BM T cells BM Fibroblasts BM BM BM BM BM BM BM BM BM PB

A. B A, B A, B A, B A, B A. B A, B A, B A. B A. B A, B A, B A. B B A A A A. B A. B A. B A. B A. B A. B A. B A. B A. B A. B A. B B B B B B B A. B At, Bt B A. B At, B t A. B A t A t A t

CCR 15+ m CCR 10+ m Rel. (5 m) dead CCR 17+ m CCR 22+ m Rel. (6 m) dead CCR 20+ m CCR 6+ m CCR 30+ m CCR 63+ m CCR 29+ m

Dead

CCR 20+ m CCR 26+ m CCR 60+ m CCR 57+ m

CCR 59+ m

CCR 63+ m

CCR 20+ m

CCR 37+ m

CCR 21+ m

Dead

CCR 22+ m

CCR 34+ m

Abbreviations: PB. peripheral blood; CCR, continuous complete remission; rel, relapse. Also from BMT in patients no. 22, 23, and 24.

t Donor polymorphism.

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608 LO COCO ET AL

nosis and remission. In 9 cases, additional remission samples were collected during the follow-up and in 2 cases at relapse. A T-cell fraction at a purity of 98% as shown by FACS analysis ( W o n Dickinson, Mountain View, CA) was obtained from circulating blood cells in 2 CML pts (no. 18 and 21) and in 1 case of APL (no. 13) after immunomagnetic separation of Ficoll isolated mononu- clear cells coated with an anti-CD3 antibody under the recommen- dations specified by the manufacturer (Dynabeads; Unipath, Mi- lan, Italy). A skin biopsy was performed in pt no. 13 in remission. In pt no. 2 1, a fibroblast culture was established from a BM specimen collected in remission. Briefly, 2 X IO' cells were seeded in flasks containing 8 mL of alpha medium and 20% fetal calf serum (FCS). Fibroblasts were grown to confluence, subcultured at intervals of 2 to 3 weeks, and then harvested and used for DNA extraction. Fi- nally, DNA was also obtained from peripheral blood of the female BM donor of pt no. 24. Informed consent was obtained for all these procedures.

BCR and PML/RAR-a genes rearrangements. Analysis of the major BCR in CML and ALL pts and ofthe RAR-a and PML genes in APLs was performed as previously reported."."

X chromosome methylation analysis. Methylation patterns at the DXS255 polymorphic locus were analyzed in all cases at diag- nosis and during remission phases after suppression at Southern blot of the BCR or PML/RAR-a rearrangements detected at diag- nosis. DNAs were initially digested with BamHI. Two aliquots of the BamHI-digested DNA were separately digested with either Msp I or its methylation-sensitive isoschizomer Hpa 11. After electropho- resis on 0.7% agarose gels, DNA was denatured and blotted onto nitrocellulose filters. Hybridization was performed with the M278 probe, which recognizes the DXS255 locus at cen-p11.2 on chro- mosome X.z7 A partial restriction map of this locus with location of the M27P fragment and variable copy number tandem repeat (VNTR) motif is shown in Fig 1. Filters were washed in 0.1% so- dium dodecyl sulfate (SDS) and 2% SSC at room temperature for I5 minutes and subsequently at 65°C for 30 to 40 minutes and ex- posed for 48 to 72 hours at -80°C using intensifying screens.

Interpretation ofmethylation analysis. Because of the presence of a VNTR sequence flanked by 3' and SMsp I sites (Fig I), mater- nal and paternal X chromosomes are distinguishable at DXS255 on BamHIIMsp I digestion after hybridization with the M278 probe. Whereas Msp I also cuts its methylated CCGG recognition se- quence, the isoschizomer Hpa ll cuts this latter only if unmeth- ylated. Based on the random nature of X chromosome inactivation during early embryogenesis:' in the presence of a monoclonal cell population only one of the parental chromosome is methylated and, hence, only one of the parental bands is cut by Hpa 11, whereas in polyclonal cells, both parental alleles are in active and inactive states; therefore, both bands are partially cut by Hpa 11. BamHI predigestion was performed, as suggested by others:9 to better re- solve higher molecular weight fragments arising in Hpa I1 digests. To facilitate comparison between diagnostic and remission fea- tures, we have conventionally indicated as A and B the higher and lower molecular weight fragments visualized after BamHIIMsp I digestion, corresponding to unmethylated fragments detectable on

BamHIIHpa I1 lanes (Table I and Figs 2 through 4). Autoradio- graphic signals were quantitated using a Ultroscan XL laser densi- tometer (LKB). The percentage of A and B bands detected on BamHIIHpa I1 digestions was determined and expressed as a ratio.

RESULTS

The main clinical and molecular features at diagnosis and remission are shown in Table 1. The results may be summa- rized as follows.

( 1) Methylation analysis at diagnosis showed the presence of a single digested (unmethylated) A or B BumHI/Hpa I1 fragment in all cases, suggesting clonal origin of the cell population. The same DNA specimens showed specific bands of rearrangements of the BCR or RAR-a/PML genes whose intensity was always identical to that of the germline allele.

(2) Disappearance of the gene rearrangements at remis- sion was associated in all but 3 cases (nos. 13, 21, and 24; Table I), with an approximate equal proportion of unmeth- ylated A and B alleles, which suggested the restoration of polyclonal hematopoiesis. However, the ratio of A/B bands, as shown by densitometric scanning, varied in some cases from the expected 5050 proportion, ranging from 1.65 to 0.61 considering all polyclonal cases (in which two A/B bands were visible at remission). Figure 2 shows Southern blot results at diagnosis and remission in 2 representative cases.

(3) In pts analyzed sequentially, polyclonal A/B ratios of DXS255 unmethylated alleles were always maintained dur- ing remission in conjunction with suppression of specific gene rearrangements. By contrast, relapses (cases no. 12 and 22, Table 1) were characterized by reappearance of the ab- normal BCR or PML/RAR-a fragments and of the same unmethylated DXS255 allele as was present at diagnosis. In no instance did a clonal methylation pattern precede the appearance of specific rearrangements in these cases.

(4) Three of the 24 pts showed the same methylation pat- tern in remission as that observed at diagnosis, despite Southern blot suppression of the specific leukemic marker (nos. 13, 2 I , and 24). In the APL pt no. 13, the single un- methylated fragment seen at diagnosis could be detected in two sequential BM samples collected in remission, as well as in DNA from circulating T cells and from cells from a skin biopsy. Pt no. 2 I (with CML) showed the same methylation picture at diagnosis and after treatment in four sequential BM specimens collected at 6, 14, 17 and 19 months after autologous BMT. In this pt, the same DXS255 unmeth- ylated fragment was also detected in DNA from peripheral blood (PB) T lymphocytes and BM fibroblasts (Fig 3).

Fig 1. Partial restriction map-of the DXS255 locus with

M I t i B location of the M278 probe and VNTR sequence giving origin to the polymorphism. B. BamHI: M. Msp I; H. Hpa I).

B M t n

M27g

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POLYCLONAL HEMATOPOIESIS IN LEUKEMIA REMISSION 609

BM DIAGNOSIS

1 2

EM REMISSION

1 2

BY DIAGNOSIS EM REMISSION

2 I . 2 1 ..,,.. _c

BCR

" . L . -

. I

t c

M278

." w

9.0- 9.0- rl)

+-

RH15

Fig 2. Detection of DXS255 methylation patterns and specific reanangements in a CML patient (left side) and an APL patient (right) at diagnosis and remission. Hybridization with the M27B probe shows in lanes 1 (BamHI plus Msp I digestion) hetemzygosity, and in lanes 2 (BamHI plus Hpa II digestion) the assessment of methylation status. A single allele is unmethylated in both cases in diagnostic BM DNA, whereas two BamHI/Hpa II bands of similar intensity are visible in remission DNA. The highest molecular weight bands in lanes 2 arise from digestion at 3 and 5 BamHl sites to DXS255, whereas the additional more faint bands visible in same lanes probably result from digestion at 3 BamHl site and 5 Msp I/Hpa II site, if unmethylated. DNA configuration of the BCR (CML case, left) and PML (APL) gene loci on the same diagnostic and remission DNA is shown below. Allelic abnormal fragments (arrows) are detected at diagnosis, whereas only germline bands are visible in remission controls. Bgl II and Hindlll digestions were performed for BCR and RH1 5 hybridizations, respectively. (For more details on the PML locus gene map and location of the RH1 5 probe see references 8 and 11 .)

( 5 ) In the case of pt 24, who had Ph'+ ALL and under- logically normal hematopoietic cells during remission s u p went allogeneic BMT in first CR, a polyclonal A/B ratio was ports this view. Nevertheless, based on X chromosome inac- observed after chemotherapy before transplantation, tivation studies, several investigators have questioned the whereas a single new unmethylated allele of donor origin nature of this remission, suggesting that in some cases the was documented in two sequential analyses following BMT persistence of clonal hematopoiesis implies the existence of from a female sibling. This latter picture was compared with a preleukemic clone surviving treatment. This clone might the DXS255 methylation status of blood cells from the do- nor, which showed an identical pattern (Fig 4). These 3 pts (nos. 13, 2 I, and 24) are presently in continuous unmain- tained remission at 20, 22, and 34 months from diagnosis with cytogenetic and Southern blot suppression of their spe- cific leukemic markers.

DISCUSSION

Most current strategies used in the treatment of human leukemia are based on the assumption that normal hemato- poietic stem cells, although temporarily suppressed by the tumor, still survive in the BM. The reemergence of morpho-

antedate, in the pathogenetic process, the Occurrence of identifiable gene re arrangement^?^-^^

In this study, we analyzed methylation patterns at the X linked DXS255 locus in conjunction with PML/RAR-a and BCR gene configuration to assess the nature of hemato- poietic reconstitution at remission in APL and Ph'' leuke- mias. Suppression (at Southern blot) of the clone carrying the specific rearrangement was accompanied by restoration of polyclonal hematopoiesis in 21 of 24 cases, following different treatment approaches (conventional chemother- apy, ATRA, a-2b interferon, and BMT). Claxton et alW have reported similar observations in five CML pts who obtained Ph' suppression after interferon therapy. The a p

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610

A

B

E M DIAGNOSIS EM REMISSION

1 2 1 2

5.0- - -0

A- A- -- B, B-

T- LYM PHOC.

LO COCO ET AL

FIBROBLASTS

1 2 I 2

Irrl, c..,

A -

B -

M278 M 278

5.0-

BCR

Fig 3. Methylation assessment and gene rearrangement in pt no. 21, affected by CML. A single unmethylated A fragment, after BamHl plus Hpe II digestion (lanes 2) is detected by M278 probe on diagnostic and remission EM, blood T cells, and cultured BM fibroblasts. A rearranged BCR band present at diagnosis (arrow) and germline configuration of remission BM are shown below.

parent clonal pattern in our remaining 3 cases is likely to be attributable to skewed lyonization with preferential methyl- ation of a single allele. Such a phenomenon has recently been described to occur in a significant proportion of nor- mal female^.^'"^ However, we note that the extent of skewed methylation observed in our cases is unusually high if compared with skewing ratios found by these authors. Therefore, another possibility is that an aberrant methyl- ation of the DXS255 region was present in our 3 cases. Unfortunately, because of the lack of heterozygosity at the phosphoglycerate kinase (PGK) and hypoxantine-guanine phosphoribosyl transferase (HPRT) loci, we were unable to have other informative data in these 3 pts.

In our study, we found that pt no. 13 had an identical pattern in remission marrow, in T cells, and in cells from a skin biopsy; in pt no. 21, cultured BM fibroblasts and T lymphocytes showed the same methylation pattern as that detected in the BM at presentation and in several BM speci- mens at remission (Fig 3). In the third pt (no. 24), the single unmethylated allele found after BMT was identical to that found in PB cells from her healthy donor (Fig 4). Whatever the reasons for the features observed (skewed lyonization or aberrant methylation), we note that comparison with non- malignant tissues is mandatory whenever apparently clonal methylation features are observed. The fact that these 3 pts are free of disease more than 20 months after achievement

of CR further supports our interpretation of apparent clon- ality. After our submission of the present report, Gale et reported similar data in a consistent series ofAML pts. After detection in some cases of skewed lyonization in both T- cells and granulocytes at remission, these authors note that preponderance of a single clone is rather unusual in hemato- poietic reconstitution and emphasize the difficulty of using X chromosome inactivation studies for the assessment of clonality. Finally, Gale et al” did not observe a distinct prognostic outcome in pts with skewed lyonization with re- spect to the median overall survival of AML pts. Although these data and our own support claims against the evidence of frequent clonal remission in leukemia, some instances of true clonal hematopoietic reconstitution might have oc- curred within the cases ofpreviously reported series, particu- larly when constitutive X-inactivation patterns were also a n a l y ~ e d . ” . ~ ” ~ ~ However, the clinical significance of this rare event is still obscure, and the possibility also exists that one or only a few normal progenitors spared by chemother- apy had reconstituted hematopoiesis. Finally, it is worth noting that, with the exception of the few CML cases de- scribed by Claxton et al,” none of the previous studies re- ported the analysis of leukemias characterized by specific markers.

In conclusion, our data indicate that clonal hematopoi- etic reconstitution rarely, if ever, occurs in leukemia pts

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POLYCLONAL HEMATOPOIESIS IN LEUKEMIA REMISSION 61 1

BM DIAGNOSIS

BM REMISSION

PRE BMT

A -

B -

1 2 1 2

A -

B -

M 2 7 p

5.0 - - 5.0-

B C R

A - B -

BM REMISSION POST BMT

1 2

5.0-

B C R

DONOR L Y M P H O C Y T E S

1 2

A - B -

M 278

Fig 4. Methylation pattems and BCR gene configuration in case no. 24 (pt with Ph + ALL). The BCR rearrangement (arrow) seen at diagnosis is not further visible in remission after chemotherapy (before allogeneic BMT). The same postchemotherapy remission sample shows the expected A/B polyclonal methylation ratio, whereas a skewed B pattern was observed in the BM DNA collected after BMT. DNA extracted from lymphocytes of BM donor showed this same B skewed pattern. The faint B unmethylated band resulting from BemHI/Hpa II digestion (lane 2) visible on the diagnostic BM DNA was interpreted as resulting from the presence of 10% normal cells in the sample.

while they persist in cytogenetic remission with suppression of specific tumor markers. Moreover, as shown in cases stud- ied sequentially before and during relapse, it appears un- likely that the expansion of a preleukemic and apparently normal clone may precede the reappearance of specific gene rearrangements.

ACKNOWLEDGMENT

We are grateful to ProfJ. Goldman for kindly reviewing the man- uscript.

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