Cytogenetic and molecular studies in patients with chronic myeloid leukemia and variant Philadelphia translocations

Cytogenetic and Molecular Studies in Patients with Chronic Myeloid Leukemia and Variant Philadelphia Translocations Alfonso Zaccaria, Nicoletta Testoni, Angela Tassinari, Bommina Celso, Feyruz Rassool, Giuseppe Saglio, Angelo Guerrasio, Gianantonio Rosti, and Sante Tura

ABSTRACT: Out of 105 Philadelphia (Ph) positive chronic myeloid leukemia patients analyzed, six (5.7%) carried a variant Ph translocation, namely t(6;9;9;10;22)(q24;p13;q34;p15;q11); t(9;t3:22)(q'~4:q21:qlt):der(2]12pler~2q31::gq21--~gq34::22qll~22qter) and (b~r(9)t(2:9) (gpter-~9q21 ::2q31-~2qt(;r);t17:9:221((tl 1 :q34;ql 11,~4(t+ :t(7:9:22)((135:q;~4:ql t ]. and tfg:l 1:22) (q34;ql 3;ql 1 ), respectively. Five of these patients were analyzed with Southern blotting. Three of them showed an atypical molecular pattern; namely, the patient with t(9;13;22) showed no rearrangement in the breakpoint cluster region (bcr), the patient with t(7;9;22)(q35:q34:q11) showed a 3' deletion, and the patient with t(7;9;22),14q+ showed a bcr rearrangement 3' to the exon 4 of the M-BCR. Chromosome in situ hybridization studies demonstrated that in patient one, a two-step translocation occurred: the first step moved the 3' bcr from chromosome 22 to chromosome 9, and the second moved the terminal part of 22q, carrying the e-sis protooncogene, to lop. Variant Ph translocations appear to be associated with atypical molecular breakpoints.

INTRODUCTION N i n e t y p e r c e n t of t h e p a t i e n t s w i t h c h r o n i c m y e l o i d l e u k e m i a (CML) are c h a r a c t e r ized by a s p e c i f i c c h r o m o s o m e a b n o r m a l i t y s u c h as t h e P h i l a d e l p h i a c h r o m o s o n l e (Ph). T h e Ph o r i g i n a t e s f r o m a r e c i p r o c a l t r a n s l o c a t i o n b e t w e e n c h r o m o s o m e 9 a n d c h r o m o s o m e 22 [1, 2]. As a c o n s e q u e n c e of t h i s t r a n s l o c a t i o n , t h e c-abl p r o t o o n c o g e n e , h o m o l o g o u s to t h e A b e l s o n m o u s e l e u k e m i a v i r u s o n c o g e n e , m o v e s f r o m its n o r m a l l o c a t i o n o n b a n d q34 of c h r o m o s o m e 9 to b a n d q l l of c h r o m o s o m e 22. T h e b r e a k p o i n t s o n c h r o m o s o m e 22 o c c u r in a very r e s t r i c t e d area (:ailed t h e m a j o r b r e a k p o i n t c l u s t e r r e g i o n (M-BCR) [31. T h i s area is part of a g e n e c o n s t i t u t e d hy a large n u m b e r of e x o n s a n d i n t r o n s , h o m o l o g o u s l y n a m e d the bcr or P h i l a d e l p h i a {phl) gene. T h e t r a n s l o c a t i o n r e s u l t s in a c h i m e r i c g e n e c o n s i s t i n g of t h e 5' part of t h e bcr g e n e a n d t h e 3' part of t h e c-abl. T h e m a j o r i t y of t h e b r e a k p o i n t s o c c u r w i t h i n t h e From the Centro di Genetica e Citogenetica Oncologi(:a, lstiluto di Ematologia l,orenzo ~ Ariosto Seragnoli, Policlinico S. Orsola, Universita' (ti Bologna, )laly (A. Z., N. T., A. T.. B, C., G. R., S. T.], the MRC Leukaemia Unit, Hammersmith Hospital l,ondon, England (F. R.I. and the Dil)artimento di Scienze Biomediche e Oncologia Umana, Sezione Clinica, Universita' di Torino. Italy [(;. S., A. G.}.

Address reprint requests to: Dr. AlJonso Zaccaria. Istituto di Ematologia l,.e A. Seragm)li, Universita' di Bologna, Policlinico S. Orsola. Via Massarenti 9, 40138 Bologna, Italy. Received March 7, 1989: accepted April 18, 1989.

191 © 1989 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas. New York, NY 10010

Cancer Genet Cytogenet 42:191 201 (1989) 0165-4608/89/$03.50

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A. Zaccaria et al. first c-abl intron on c h r o m o s o m e 9 and between exon 2 and exon 3 of the M-BCR on c h r o m o s o m e 22, thus leading to a novel 8.5-kb hybrid mRNA and, in turn, to a 210kd protein instead of the normal 7-kb mRNA and 145-kd protein, respectively I4l. The a d d i t i o n of the a m i n o t e r m i n a l part, c o d e d by the bcr gene, to the c-abl protein, highly increases its n o r m a l l y low tyrosine kinase (TK) activity [5[. Variations in both the cytogenetic and m o l e c u l a r patterns may occur. Variant Ph translocations have been described in 2 - 8 % of CML patients I6I. Their incidence could be increased by e m p l o y i n g high-resolution banding techniques I7]. Traditionally, they are d i v i d e d into two groups: simple and c o m p l e x translocations. S i m p l e translocations are c o m m o n l y considered to occur between c h r o m o s o m e 22 and a c h r o m o s o m e other than 9. Complex translocations are characterized by i n v o l v e m e n t of one or more chromosomes besides chromosomes 9 and 22. Nevertheless, m o l e c u l a r biology studies have clearly demonstrated that c h r o m o s o m e 9 is always i n v o l v e d in variant translocations (including simple variants), even w h e n subtle alterations are not detectable with conventional cytogenetic techniques [8]. It is generally accepted that variant Ph translocations have no unfavorable prognostic value in CML patients I1, 6[. However, some authors do not share this view I9, 10I. Moreover, recent reports have pointed out the heterogeneity of the breakpoints on c h r o m o s o m e 22 in patients with a standard t(9;22) [11-13[. Translocations with different breakpoints might be responsible for production of different types of hybrid proteins, as has been demonstrated for Ph-positive acute l y m p h o b l a s t i c leukemia [14[. In situ hybridization studies might be useful both in confirming b a n d i n g patterns in Ph variants and in detecting variant translocations at a s u b m i c r o s c o p i c level. In this series of 105 patients with Ph-positive CML in the chronic phase studied at diagnosis with a high-resolution banding technique, we found six patients with variant translocations. They have been analyzed with cytogenetic, molecular, and, when possible, c h r o m o s o m e in situ h y b r i d i z a t i o n techniques. The bands at which the breakpoints occur are generally involved in n o n r a n d o m translocations in several hemopoietic disorders, and some of them carry specific gene sequences and fragile sites. Because three of our five patients showed atypical molecular patterns, we suggest that the involvement of other c h r o m o s o m e s in variant Ph translocations may enhance the occurrence of atypical bcr breakpoints.

MATERIALS AND METHODS One h u n d r e d five consecutive adult (ages 3 5 - 6 7 years) patients with Ph-positive CML were the object of this study. All were studied at diagnosis, and all were in chronic phase.

Chromosome Studies and Banding Technique Metaphases were prepared with a methotrexate s y n c h r o n i z a t i o n technique [7[. 20 × 106 marrow cells were cultured for at least 24 hours at 37°C in 10 ml RPMI-1603 m e d i u m s u p p l e m e n t e d with 30% fetal calf serum, p e n i c i l l i n - s t r e p t o m y c i n (50 IU, 50 p~g/ml), and L-glutamine (200 raM). Cultures were s y n c h r o n i z e d with 10 7 M MTX at 37°C for 17 hours, released from the MTX block with 20/xg/ml of b r o m o d e o x y u r i dine (BrdU), and e x p o s e d to Colcemid (0.05 ~g/ml) for the last 10 minutes of the culture. Following incubation at 37°C in 8 ml KCI (0.075 M) for 10 minutes, cells were fixed in 3 : 1 absolute methanol : glacial acetic acid. Chromosome preparations were made by d r o p p i n g the cell s u s p e n s i o n onto microscope slides wet with distilled water. All preparations were stained directly with Giemsa stain. The b a n d i n g techniques used were R b a n d i n g with acridine orange and G b a n d i n g with Wright

193

Variant Ph Translocations in CML

m-bcr

[ kBo BoOc B k t

II

I

I

I

~

k Bo k I

1

|

Bo i l l H I

i i

H B H B.g !B?B H H BQ • B

l

im •

eel •

•

II •

2

3

4

5

i

probe B

probe

I

J

I

m

A pt. 3 ( N R ) ,,

pt. 5 ( R )

Figure 1 Restriction map of the M-BCR region on chromosome 22 and representation of the probes employed in the study. The regions in which no rearrangement was found (in patient 3) and the region in which a rearrangement was found (in patient 5) are also shown.

stain. In each case, analysis was performed on 15-20 cells, and at least five metaphases were karyotyped. Chromosome identification was performed in accordance with ISCN [15].

Southern Blotting DNA purified from bone marrow or peripheral blood cells was digested with Bglll, BclI, and EcoRI restriction endonucleases, r u n in 0.8% agarose gel, and transferred to nitrocellulose paper by the Southern technique [16]. Two probes were used: a 1.2-kb HindIll-BgllI genomic probe, which recognized the 3' part of the M-BCR region (probe A), and a 0.450-kb EcoRI-PstI cDNA fragment recognizing 4 bcr exons mapping 5' to the site where the breakpoint on chromosome 22 occurs (probe B). The restriction map of the bcr gene and the probes employed in this study are s h o w n in Figure 1. The probes were labeled with dCT 32p by nick translation and hybridized for 24 hours at 42°C in 50% formamide [17]. Then, the filters, washed at the appropriate stringency, were autoradiographed with Kodak or Amersham films for 3 7 days at - 70°C.

In Situ Hybridization In situ hybridization was carried out according the method of Boyd et al. [18]. Slides were treated with RNase (100 gg/ml)/2 x SSC at 37°C for 60 minutes. Chromosomal DNA was denatured in 70% formamide/0.1 mM EDTA/2 xSSC (pH 7.0] at 65°C for 4 minutes. The probe was labeled to a specific activity of 50 x 108 dpm/gg, lyophilized, and resuspended in hybridization buffer containing 50% formamide, 5 x Denhardt's solution, 5 x SSPE, 10% dextran sulphate, and 20 gg/ml salmon sperm DNA. We used two probes; one was a 4-kb cDNA probe (kindly provided by Dr. Canaani), w h i c h corresponded to 95% of bcr and lacked exon 5 (according to the n o m e n c l a t u r e reported in [18]). The other probe was a 2.7-kb cDNA sis probe [19, 20]. The probe was denatured by boiling for 5 minutes, and 30 gl of probe mixture at a concentration of 0.004 ng/ml was hybridized with denatured DNA overnight at 42°C. The slides were rinsed thereafter in 5 x SSC, washed in 2 x SSC (2 hours at room temperature and 1 hour at 65°C), and then in 0.2 x SSC for 30 m i n u t e s at 60°C. After d i p p i n g in Ilford L4 nuclear emulsion, the slides were stored in the dark for 7 14 days at 4°C, developed in D19 for 8 m i n u t e s at 15°C, and then banded with Wright

194

A. Zaccaria et al.

stain according to Harper and Saunders [21]. Hybridization of c o m p l e m e n t a r y DNA sequences in situ was seen as one or more black grains over a particular chromosome. The n u m b e r of grains per unit length of c h r o m o s o m e was scored in 3 5 - 5 0 metaphases.

RESULTS Cytogenetics In our series of 105 Ph-positive CML patients, six carried a variant Ph translocation. Detailed data are shown in Table 1. Patient 1 had a 46,XY,t(6;9;9;10;22) (q24;p13;q34;p15;q11), patient 2 had an unusual picture consisting of a 46,XX, t(9;22)(q34;q11) in 50% of the cells and a c o m p l e x translocation involving chrom o s o m e s 2, 9, and 22 in the other 50% of the cells. Patient 3 had a t(9;13;22)(q34;q21;q11), patient 4 had a 46,XX,t(7;9;22)(q35;q34;q11), patient 5 had a t(7;9;22)(q11;q34;q11), and patient 6 showed a t(9:11;22)(q34;q13:q11) (Figs. 2-4). In none of these patients did the c o m p l e x Ph variant translocations result in a " m a s k e d " Ph chromosome.

Chromosome In Situ Hybridization Studies In situ hybridization to metaphase was performed in patients 1 and 3. Analysis by R banding in patient 1 indicated the i n v o l v e m e n t of five c h r o m o s o m e s in this c o m p l e x translocation, but the exact c h r o m o s o m a l exchanges were ambiguous. Analysis of 50 metaphases by in situ hybridization using the sis probe with a total number of 265 grains s h o w e d a clustering of grains on the normal c h r o m o s o m e 22 (15%) and on 10p (13.5%). Analysis of 50 metaphases using the bcr probe with a total n u m b e r of 301 grains s h o w e d a clustering of grains on the normal 22 (16%) and 9q (12%). In patient three, analysis of 35 metaphases using the sis probe with a total number of 235 grains s h o w e d a clustering of grains on the normal c h r o m o s o m e 22 (15.3%) and on 13q (16%). The clustering of grains was significant, with p < 0.001.

Table 1

C h r o m o s o m e and m o le c u la r characteristics of the six patients with variant Ph translocations

Patient

Karyotype

1. V. G. 2. F. F.

46,XY,t{6;9;9;10;22)(q24;pl 3;q34:p15;q 11) 46,XX,t(9;22)(q34;q11) (50%)/46,XX,- 2,- 9, + der(2)t(2;9;22) (2pter-+2q31::::9q21--,9q34::22q11-+22qter), + der(9) t(2;9)(9pter--,9q21::2q31-+2qter) (50%) 46,XY,t(9;13;22)(q34;q21;q11] 46,XX,t{7;9;22)(q35;q34;q 11) 46,XY,t(7;9;22)(q11;q34;q11),14q + 46,XX,t(9:l 1;22)(q34;q13;ql 1)

3. 4. 5. 6.

G. G. O. A. B. R. L. C.

bcr rearrangement

R R

NR R" R~, ND

Abbreviations: R, rearranged; NR, not rearranged between the EcoRI and HindIll restriction sites located 5' and 3' to the M-BCR,respectively: ND. not done. " Rearranged in the M-BCR,but tile 3' part is de|eted. ~'Rearranged within EcoRl and B(:llrestriction site located between exnns 4 and 5 of M-BCR.

195

Variant Ph T r a n s l o c a t i o n s in CML

(7,t.-;o n . 1

6 6q-

2

9p-

tier(2)

9q+

10

9

10p+

der(9)

22

22

22¢ 1-

22q-

F i g u r e 2 Partial karyotypes of patients I and 2. In patient 1 five chromosomes were involved, and the 22q was translocated on 10p, as confirnled by in silu hybridization studies (R bands)

Molecular Studies S o u t h e r n blot a n a l y s e s w e r e p e r f o r m e d in five patients. Patients 1 a n d 2 w e r e characterized by a classic r e a r r a n g e m e n t in the M-BCR region. In patient 3, no rearrangem e n t was f o u n d e m p l o y i n g two probes (A and B) and several restriction e n z y m e s (EcoRI, BgllI, BclI, BamHI, HindlII). T h u s no r e a r r a n g e m e n t was f o u n d w i t h i n the EcoRI site located 5' to the M-BCR and the H i n d l l l site located 3' to the M-BCR. In contrast, p a t i e n t 4 s h o w e d no r e a r r a n g e m e n t w i t h p r o b e A after d i g e s t i o n w i t h BgllI, EcoRI, and H i n d l l I r e s t r i c t i o n e n z y m e s . A rearranged band was f o u n d after Bcll

196

A. Zaccaria et al.

Cas("

n . 3

•

9

C,~ist~

9q+

13

13q-

22

22q-

tl . 4

~u 7 Figure 3

7q+

9

9q+

22

22q-

Partial karyotypes of patients 3 and 4 {R bands]

digestion and hybridization with 5' probe B. So, we hypothesize that there was a 3' bcr deletion in this patient. Patient 5 was found to have a rearrangement with 3' (A) and 5'(B) probes employing Bcll as restriction enzyme. No rearrangement was found with probe A and BglII or EcoRI restriction enzymes. The breakpoint was then localized within the short EcoRI-Bcll region sited between exons 4 and 5 of the M-BCR.

DISCUSSION Variant translocations occur in 1.8 8% of patients with Ph-positive CML [61. All c h r o m o s o m e s may be involved, with the exception of c h r o m o s o m e Y. The breakpoints are n o n r a n d o m because a preferential i n v o l v e m e n t of 28 bands, belonging particularly to c h r o m o s o m e s 3, 11, 12, 14, and 17, has been observed [61. In six out of

197

Variant Ph Translocations in CML

~:

i~i¸

ii 7

7q-

9

9q+

ii 9

9q+

= 22

22q-

C.as~, n . 0

Figure 4

1 1 1 lq-

22 2 2 q -

Partial karyotypes of patient 5 (G bands) and patient 6 (R bands)

105 patients in the present study, a variant translocation was detected, thus confirming the incidence reported in the literature. All the breakpoints reported here, except 13q21, have already been described in variant translocations. Recently, some authors have pointed out that fragile sites could enhance the occurrence of variant translocations [221. Three out of the nine breakpoints reported in this paper were found to be associated with fragile sites; namely, the 13q21 band is a c o m m o n BrdU fragile site (FRA13B), and 11q13 and 2q31 are c o m m o n aphidilcolin fragile sites ( F R A l l H and FRA2G, respectively) [23]. No k n o w n protooncogene is localized at the level of one of the bands cited above, apart from c-myb, which is mapped at 6q23, very close to 6q24, which was one of the breakpoints of patient 1. Nevertheless, the breakpoints reported here occur at bands n o n r a n d o m l y involved in hematologic malignancies [24]: band 6q24 is involved in translocations and deletions in lymphomas and acute lymphoblastic leukemias [25, 26]; band 9p13 is involved in the t(8;9)(q24;p13) in a case of B-cell malignant l y m p h o m a [271; band 10p15 in the t(10;ll)(p15;q22-q23) described in acute lymphocytic leukemia (ALL) and acute n o n l y m p h o c y t i c leukemia (ANLL), particularly of the M5 subtype [28, 29], and 13q21 in chronic and subacute

198

A. Zaccaria et al. myeloproliferative disorders: in these cases, a del(13}(q12-q23) has been described [30[. Breakpoints at the 7ql I level have been described in the t(7:12) observed in ALL J3t I, while breakpoints at the 11(t13 level have been ohserved in M4-M5 ANI,IJ [32, 33J and in l y m p h o p r o l i l e r a t i v e disorders J25, 34-37I. Interestingly, breakpoints at band 7q35 have been described in T-AI,L and baw~ been found to be strictly associated with involvement of the T-cell receptor gene 138]. Moreover, at the same band level, a large group of tbe mdr family genes is m a p p e d J39I. This fact nlay have importaut prognostic implications. Finally, breakpoints at the 2q31 lew,q have been observed in secondary leukemias by Paietta et al. and O s b i m u r a et al., who described a t(2;3) [40, 41 ], and deletions at band 9q21 have been described in ANLL [421. In the majority of the studies reported in the literature, no correlation between variant translocations and clinical outcome was found, even if some authors reported a shorter survival I9, 10]. In particular, Przepiorka and Thomas included the variant translocations within tire unfawrrable prognostic factors of patients with Pb-positiw,' CML eligible for allogeneic bone lnarrow transplantation [10]. Three of our five patients had both wtriant Ph translocations and atypical nmlecular patterns. Patient 3, for example, showed no rearrangement in spite of the presence of a cytogenetically typical Ph c h r o m o s o m e and the use of several restriction enzymes and probes. Selleri et al. found a similar pattern in three of 20 patients analyzed [13]. In that study, Northern blot analysis did not allow detection of any 8.5-kb chimeric mRNA in two patients, while in the third a chimeric transcript was observed e m p l o y i n g a c-abl cDNA probe, but not a bcr cDNA probe. The biologic significance of these findings is at present obscure. More data regarding expression of hybrid genes in these patients are needed in order to verily whether or not they constitute a molecularly homogenous group with similar prognostic features. Patient 4 had a 3' deletion of the M-BCR, similar to what has been observed in the CMLderived K562 cell line [431, A 3' deletion was identified by the presence of a typical bcr rearrangement e m p l o y i n g the 5' probe (probe B) and not the 3' probe (probe A). The presence of a typical 5' bcr rearrangement suggests that there must be no difference in terms of hybrid 5' bcr/c-abl formation, m-RNA expression, and p210 production in these patients with respect to 3' n o n d e l e t e d rearrangement. In patients with a typical rearrangement, a hybrid 5'c-abl/3'bcr is also formed on c h r o m o s o m e 9. This h y b r i d gene is expressed, but it is not considered of crucial importance in the pathogenesis of the disease [44]. Thus, the deletion of the 3' part of the M-BCR should not interfere with major pathogenetic mechanisms. Patient 5 had a breakpoint in the 3' part of the M-BCR, n a m e l y between exons 4 and 5. In our series, this was a rare event, as only three patients out of 105 i n c l u d e d in the study showed a translocation 3' to exon 4. A still obscure field is represented by the fate of the 3' part of the ber gene after its translocation to c h r o m o s o m e 9 and, subsequently, to other chromosomes. In this respect, the identification of the exact breakpoints on the involved c h r o m o s o m e s s h o u l d allow us to h y p o t h e s i z e about the formation of other hybrid genes with possible transcriptional activity and protein production. A few data have been reported in the literature regarding this point. In some cases, studied with in situ c h r o m o s o m e hybridization, 3' bcr sequences have been found on the involved additional chromosomes, n a m e l y 8q and 12p in the cases reported by Ohyashiki [45], w h i l e in another case with a t(5;9;22) reported by Ohyashiki and in a case by Ishihara with t(2;9;13;22), 3' bcr sequences were detected on chromosome 9q [46]. A similar pattern was detected in patient 1 of the present study. On the contrary, the c-sis oncogene is constantly moved to one of the further involved chromosomes, as we d e m o n s t r a t e d in patients I and 3. Variant translocations are considered to be a two-step p h e n o m e n o n : a typical t(9;22) may be followed by another translocation with involvement of other chromosome(s). As a matter of fact, patient 1 is particularly interesting because of this. The c-

Variant Ph T r a n s l o c a t i o n s in CML

199

sis p r o b e h y b r i d i z e d to b o t h the n o r m a l 22 and to 10p, w h i l e the bcr probe h y b r i d ized to 9q, t h u s d e m o n s t r a t i n g that a s e c o n d breakage o c c u r r e d 3' to the first bcr t r a n s l o c a t i o n . T h i s is a c l e a r - c u t d e m o n s t r a t i o n that the variant t r a n s l o c a t i o n occ u r r e d in t w o s u c c e s s i v e steps. In several patients, Ishihara et al. was able to detect the p r o g r e s s i v e a p p e a r a n c e of c o m p l e x t r a n s l o c a t i o n s in p r e v i o u s l y t(9122) s t a n d a r d c l o n e s [46]. H o w e v e r , in o u r p a t i e n t s the c o m p l e x t r a n s l o c a t i o n s d e s c r i b e d above w e r e p r e s e n t at diagnosis, and no e v o l u t i o n was d e t e c t e d later. If the t w o - s t e p transl o c a t i o n h y p o t h e s i s is correct, the s e c o n d t r a n s l o c a t i o n c o u l d i n f l u e n c e the integrity of the 22q, in particular, of the 3' BCR region. The 3' d e l e t i o n of the bcr g e n e o b s e r v e d in p a t i e n t 4 c o u l d reflect s u c h an event. H o w e v e r , the 5' part of the bcr is f r e q u e n t l y a t y p i c a l l y r e a r r a n g e d (cases 3 and 5). In these cases, a direct i n v o l v e m e n t of the a d d i t i o n a l c h r o m o s o m e ( s ) in the t r a n s l o c a t i o n m i g h t be postulated. H o w e v e r , m o l e c u l a r a b n o r m a l i t i e s o c c u r f r e q u e n t l y in patients w i t h no c o m p l e x t r a n s l o c a t i o n [13,47]. Thus, c o m p l e x t r a n s l o c a t i o n s m a y be regarded as a p o t e n t i a l i n d u c i n g factor of a t y p i c a l b r e a k p o i n t s , w h e n t h e y occur, but not as the m o s t i m p o r t a n t factor.

This work was supported by Italian National Resear(:h Council, Rnma, Fimdized Prc~ject ()m:ologia, Contra(:t no. 87.02.805.44 and the Asso(:iazione ltaliana per la Ricer(:a suI (]an(:ro, Milan().

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