6p+ and 9q− in two chromosomally distinct clones occurring in a case of myelodysplastic syndrome evolving to acute nonlymphocytic leukemia

6p + and 9 q - in Two Chromosomally Distinct Clones Occurring in a Case of Myelodysplastic Syndrome Evolving to Acute Nonlymphocytic Leukemia Alvaro Ringressi, Cristina Mecucci, Alberto Grossi, Pier Antonio Bernabei, Pierluigi Rossi Ferrini, and Herman Van Den Berghe

ABSTRACT:

Different and unrelated chromosome changes were found to occur in a patient with a myelodysplastic syndrome with rapid evolution to acute nonlymphocytic leukemia. A 6p anomaly was found during the chronic phase and a del(9q) characterized the cells in the leukemic phase. Deletions with a breakpoint in 9q31 appeared to be associated with more aggressive disease.

INTRODUCTION Acquired ch r o m o s o m a l abnormalities are frequently found in the bone marrow of myelodysplastic syndrome (MDS) patients, and the appearance of additional chrom o s o m e changes has been observed during clinical evolution [1]. Unrelated clones also have been sporadically reported [2]. We observed such an event in a patient affected by MDS that eventually evolved into acute n o n l y m p h o c y t i c leukemia (ANLL). The examination of the karyotype of the leukemic cell population during the acute phase showed a del(9)(q13q31). This alteration was unrelated with the one observed at the beginning of the disease, characterized by the presence of extra material on the short arm of c h r o m o s o m e 6 (6p +). The literature on 9 q - and the clinical significance of this anomaly will be discussed.

CLINICAL D A T A The patient, a 21-year-old female, white-collar worker, was first seen in October 1983 because of severe thrombocytopenia. Platelets were 25 x 10~/L, hemoglobin (Hb) 11 g/dl, hematocrit (Hct) 35%, mean corpuscular v o l u m e (MCV) 96 fl, white blood cell count (WBC) 10 × 10~/L. Differential count showed 49% neutrophils, 2% From the Department of Hematology, U.S.L. 10D, Universityof Florence (A. R., A. G., P. A, B.. P. R. F,}, Clinica Medica I, University of Perugia (C. M.), ltaly, and the Centre for Human Genetics, University of Leuven, Belgium (C. M., H. V. I3. B.). Address requests for reprints to Herman Van Den Berghe, Center for Human Genetics, Herestraat 49, B-3000 Leuven, Belgium. Received February, 25. 1988; accepted June 23, 1988.

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eosinophils, 1% basophils, 40% lymphocytes, and 8% monocytes. Bone marrow aspiration showed h y p e r p l a s i a of granulocytic lineage (blasts >5%). Binucleated erythroid precursors and small megakaryocytes were noticed. Platelet-associated a n t i b o d y values were borderline; indirect and direct Coombs tests were negative. Survival of nl[In]oxine-labeled platelets was 3 days (normal, 7-10 days), with mild accumulation in the spleen. The LAP score was 23 (normal, 75-100). W h e n bone marrow m o n o n u c l e a r cells were plated in an agar cloning system for CFU-GM, very few clusters and no colonies were observed (normal, 40-100 colonies; observed, 90-400 clusters). A diagnosis was m a d e of refractory anemia with excess blasts (RAEB). A course of steroid therapy resulted in an increase in WBC only, without significant effect on platelets. In February 1985, 2% of blasts with Auer rods and S u d a n black positive a p p e a r e d in the peripheral blood (15% in the bone marrow). A treatment with 1,25-dihydroxyvitamin D 3 ( 0 . 5 0 fxg/day) and danazol was started. Four months later leukemic evolution became evident, with 50% blasts in the bone marrow and 13% in the peripheral blood. Sudan black and chloroacetate esterase were positive in 50% of the immature cells, while the whole blast p o p u l a t i o n stained with a l p h a - n a p h t h y l acetate esterase (ANLL-M4). After treatment with 6m e r c a p t o p u r i n e (6-MP; 25 mg/day on alternate days), the WBC in the peripheral blood normalized. Twelve months later, while the patient was still on 6-MP therapy, the WBC rose to 80 × 109/L (70% blasts). Three courses of therapy (doxorubicin, adriamycin, 6thioguanine) i n d u c e d partial remission. A relapse on May 5, 1987 was treated with mitoxantrone and cytosine arabinoside. The patient died from acute liver and renal failure 1 month later. CYTOGENETIC STUDIES Three karyotypic examinations of blast cells were carried out in this patient. On December 14, 1984, and on August 13, 1985, bone marrow cells were used; the last analysis, on June 20, 1986, was performed on peripheral blood cells. Cells (1.2 × 106/ml) were cultured in RPMI-1640 m e d i u m plus 30% fetal calf serum for 48 hours at 37°C, in a fully h u m i d i f i e d atmosphere with 5% CO2. Cells were treated with colcemid (0.15 ~xg/ml) for the last 30 minutes of culture. Then, they were treated with trypsin to obtain G b a n d i n g [3], heated to 87°C, and stained with acridine orange for R b a n d i n g [4]. Metaphases were photographed with a Agfa-pan 25 film.

RESULTS

In December 1984, three analyzable metaphases were obtained from bone marrow samples. Karyotypes were 46,XX in one cell and 46,XX,6p+ in two. On further analysis, in August 1985, 14 metaphases were obtained from bone marrow cells, with the following pattern: 46,XX (six cells); 46,XX,6p+ (seven cells); 88,XXXX, - 6, - 10, - 17, - 1 7 , - 1 8 , - 18, - 2 0 , - 20,6p + (one cell) (Fig. 1). In June 1986, seven metaphases were obtained from a culture of peripheral blood cells and all had a 4 6 , X X , 9 q - karyotype (Fig. 2). The origin of the extra material on the short arm of chromosome 6 was not identified. Breakpoints were situated distal to band 6p21. The deletion of the long arm of chromosome 9 was interstitial, and our interpretation is del(9)(q13q31). The deleted segment apparently was lost.

DISCUSSION

In hematologic malignancies, when clonal chromosome changes are observed in the proliferating cells, they are, as a rule, persistent throughout further disease evolution (primary chromosome changes). Other chromosome changes may occur (sec-

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MDS Evolving to ANLL

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ondary changes) as additional anomalies, and may vary considerably. The appearance of c h r o m o s o m a l l y unrelated cell proliferations, either s i m u l t a n e o u s l y or successively, is m u c h more rare. It is observed especially in long-standing MDS, as illustrated in the present case. The possible significance of these events with regard to the meaning of c h r o m o s o m e changes in malignant cells and also with regard to disease evolution has been previously discussed [2, 11]. It is clear that clonal chromosome changes may come and go. They probably characterize s u b p o p u l a t i o n s w i t h i n the malignant process that may have started out in one (or more) cell(s) with normal karyotype and may (but not necessarily) correspond to successive steps in tumor evolution. Rearrangements of the short arm of chromosome 6 are frequent in h u m a n neoplastic diseases. Eighty-two cases of involvement of the short arm of chromosome 6 and breakpoint situated distally to band 6p21 are reported in the Mitelman's catalog [5]. A m o n g 49 cases with m y e l o i d neoplasms, ANLL-M2 with t(6;9)(p23;q34) [5-8] are the best represented. Among seven 6p + cases of ANLL and MDS in w h i c h the origin of the extra material could not be identified [5, 9 11], three were in patients who had received c h e m o t h e r a p y for previous neoplasia. Mecucci et al. found 6p-derived markers in ANLL and MDS patients with exposure to w e l l - k n o w n carcinogens [12]. In the present case a previous exposure was not evident. Cytogenetically the 6 p + may have originated from a duplication of segment p21-23 and subsequent trisomy of the d u p l i c a t e d segment. Interestingly, a similar marker has been described in the h u m a n leukemia cell line K562 [13-16[ in which a t(6;6)[p11;p21) was described [16]. The karyotypic analysis performed in our patient after the evolution to ANLL showed a new cytogenetic clone with del(9)(q13q31). Given that the initial alteration in the bone marrow cells is a 6p + marker, the modification could have come about in the same clone through the following steps: loss of 6 p + chromosome, return of # 6 pair to d i p l o i d y by means of nondysjunction, and appearance of chromosome 9 deletion. Although many rearrangements may take place in a neoplastic clone, the combination of all these three events seems unlikely. Because the second clone was identified after 5.5 months of therapy with 6-MP, we cannot rule out that this drug was able either to produce chromosome alterations in h u m a n bone marrow cells or to modify the pattern of growth of different clones [18, 19]. As far as we~ know, however, a del(9q) has not been observed in iatrogenic secondary disorders. Recent data regarding MDS with two cytogenetically distinct cell p o p u l a t i o n s [2] tend to show that the appearance of the second karyotypic alteration is accompanied by the disappearance of the first one and an evolution toward ANLL, and, indeed, this is what h a p p e n e d in our patient. Interstitial deletions of the long arm of chromosome 9 are rather infrequent in hematology, and the cases described so far have been mostly m y e l o i d leukemia, particularly M2 type with t(8:21) [20, 22-26]. In these cases the deleted chromosome 9 was present in a variable portion of the metaphases, which already had p r i m a r y c h r o m o s o m e changes. The new anomaly was not a c c o m p a n i e d by modifications of the cellular morphology, course of the disease, or prognosis. A del(9q) also has been described in association with karyotypic changes other than t(8;21) [21, 23-25, 27, 29]. Moreover, eight cases of ANLL [21, 22, 26, 28-30] and one case of myeloproliferative disorder (MPS) [21] have been described in which the interstitial deletion of the c h r o m o s o m e 9 was the only karyotypic abnormality. In four cases, one MDS, one MPS, one ANLL-M2 t(8;21) [21], plus the present observation, the 9q anomaly became evident when the process entered into a more aggressive stage. Thus, in some patients, a del(9q) may occur as a secondary change, whereas in other cases it seems to be a primary chromosome anomaly. In an attempt to correlate 9q deletions and the clinical appearance of the associated hematologic

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M D S E v o l v i n g to A N L L

Table 1

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Data f r o m six g r o u p s c o m p i l e d f r o m t h e l i t e r a t u r e

1. ANLL-M2: 46,XX,t(8;21)(q22;q22),del(9)(q12q31) / 4 5 , X , - X , the same; Mecucci et al. I21]. 2. ANLL-M2: 45,X,-X,t(8;21)(q22;q22) / the same, del(9)(qllq21); Mecucci et al. [21]. 3. ANLL-M2: 46,XY,t(8;21)(q22;q22) / the same, del(9)(q12q31)/the same, del(9),-Y; Mecucci et al. I21]. 4. ANLL-M2: 45,X,-Y,t(8;21)(q22;q22) / the same, del(9)(q12q21); Mecucci et al. I21]. 5. ANLL-M2: 45,X,-Y,t(8;21)(q22;q22), del(9)(q12q22), Hossfeld et al. [20]. 6. ANLL-M2: 46,XX,t(8;21)(q22;q22),del(9)(q13q22); Hossfeld et al. [20]. 7. ANLL-M2: 46,XX,4q + ,del(7)(q31),t(8;21)(q22;q22),del(9)(q12q21); Hagemeijer et al. [22]. 8. ANLL-M2: 46,XX,t(8;21)(q22;q22),del(9)(qllq13); Hagemeijer et al. [22]. 9. ANLL-M2: 4 5 , X , - Y,t(8;21)(q22;q22),del(9)(q13q22); Yunis et al. [26]. 10. ANLL-M2: 47,XX, + 21,del(9)(q12q21); Mecucci et al. [21]. 11. ANLL-M2: 46,XX,del(5)(qllq35),del(9)(q13q22); Yunis [27]. 12. ANLL-M3: 46,XY,t(15;17)(q22;q21) / the same, del(9)(q21q33); Larson et al. [29]. 13. ANLL-M4: 46,XY,t(12;22)(p12;q11),del(9)(q31q33); Mecucci et al. [21]. 14. ANLL-MI: 46,XY / 43,XY,del(5q), - 7, - 17,( - 20),( 22),( + M1),t(14;?)(p11;?),del(9)(q12q33); Mecucci et al. [21]. 15. ANLL-MI: 46;XX,de|(9)(q12q31); Mecucci et al. [21]. 16. MPS: 46,XY,del(9)(q12q31); Mecucci et al. [21]. 17. ANLL-M6: 46,XY / 46,XY,del(9)(q12q21) / 46,XY, + 8 (this case has been inserted in this group because the del(9q) is single in one of the clones of leukemia); Mecucci et al. [21]. 18. ANLL-M2: 46,XY,del(9)(qllq32); Larson et al. [29]. 19. ANLL-MI: 46,XY,del(9)(q12q21); Kao et al. [28]. 20. ANLL-M4: 46,XX,del(9)(q12q21); Kao et al. [28]. 21. ANLL-M5b: 46,XX,del(9)(q13q22); Yunis [26]. 22. ANLL-M4: 46,XY,del(9)(q12q31); Hagemeijer et al. [22]. 23. ANLL-M2: 46,XX,del(9)(q12q33); Lin et al. [30]. 24. RAEB: 46,XY,del(20)(q11) / 46,XY,del(9)(qllq22),del(11)(q13q23); Davis et al. [23]. 25. RARS: 46,XX,del(9)(q21q31),del(11(q22q24) /47,XX,+8,del(9)(q21q31),del(11)(q22q24); Mecucci et al. [21]. 26. RA: 46,XX / 46,XX,del(9)(q12q31),- 13,t(?;18)(?;q23); Mecucci et al. [21]. 27. RAEB-t: 46,XY,del(9)(q13q22),-17; Yunis et al. [24]. 28. RAEB: 46,XX,del(5)(q13q35),del(9)(q13q22); Yunis et al. [24]. 29. RAEB: 46,XX / 45,XX,-17,del(5)(qllq31),t(6;?)(p25;?),t(12;?)(p13;?),+DM / 45,XX, the same, del(9)(q13q22) / 46,XX, the same, + 8; Jacobs et al. [25]. 30. ANNL-M2: 46,XY,del(9)(q12q31); Mecucci et al. [21]. 31. RAEB-ANLL: RAEB, 47,XY,der(7), + 8; ANLL, the same, del(9)(q21q31); Mecucci et al. [21]. 32. MPS atypical: diagnosis, 46,XX / 45,XX,-5; remission, 46,XX; relapse, 46,XX / 46,XX, del(9)(qllq21); Mecucci et al. [21]. 33. RA-ANLL-M4: RA, 46;XX/46,XX,6p +; ANLL M4, 46,XX,del(9)(q13q31), present case.

d i s o r d e r s , w e s u m m a r i z e d i n T a b l e 1 d a t a f r o m six g r o u p s i s o l a t e d f r o m 32 c a s e s f r o m t h e l i t e r a t u r e p l u s o u r o w n o b s e r v a t i o n . Data r e p o r t e d i n T a b l e 1 s h o w t h a t q12-13 is a c r i t i c a l r e g i o n for t h e p r o x i m a l b r e a k p o i n t s i n 9 q - d e l e t i o n s i n d e p e n d e n t of t h e t y p e of d i s o r d e r (i.e., A N L L or MDS) as w e l l as f r o m t h e t y p e of a c c o m p a n y i n g k a r y o t y p i c a n o m a l i e s [t(8;21) or o t h e r o b s e r v a t i o n s ] (cf. g r o u p s 1, 2, 4, a n d 5). Also, t h e d i s t a l b r e a k p o i n t s a p p e a r e d v e r y v a r i a b l e i n all t h e g r o u p s , b u t i n g r o u p s 3 a n d 6, q33 a n d q31, r e s p e c t i v e l y , w e r e c o n s i s t e n t l y p r e s e n t . A l t h o u g h f u r t h e r s t u d i e s are n e c e s s a r y , it a p p e a r s f r o m g r o u p 6, i n c l u d i n g t h e p r e s e n t case, t h a t a r e a r r a n g e m e n t o n 9q31 m a y a c c o m p a n y s o m e a g g r e s s i v e m y e l o i d p r o l i f e r a tions. Supported by the Inter-university Network for Fundamental Research sponsored by the Belgian Government (1987-1991/.

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