Inherited aplastic anemia with abnormal clones in bone marrow and increased endoreduplication in peripheral lymphocytes

InherRed Aplasfic Anemia wRh Abnormal Clones in Bone Marrow and Increased Endoreduplication in Peripheral Lymphocytes P. Carbone, G. Barbata, S. Mirto, and G. Granata

ABSTRACT: Cytogenetic studies in pe~pheral blood and bone m a ~ o w c~ls ~om a female patient ~ged 31 yea~) with i n h e ~ d aplastic anemia and without other congenital a n o m ~ s are reposed. Endoredupl~ation was increased in stimulated pe~pheral lymphocytes in several investigations. Chromosome breaks were shown to be near the con~ol ~equenc~ ~though chromatid exchange figures and dicent~cs were present. Cytogenetic an~ysis was extended to the three children of our patient. Abnormal clones were d ~ e c ~ d in bone m a ~ o w preparations of our patient in ~1 cytogenetic investigations. At the fi~t examination, two of these clones were prevalent, with their karyotypes b~ng 48,XX,+9,+16 and 46,XX,dup(1)(q24--~q3~,t(1~?)(p12-13;?). The prevailing karyotype after 2 yea~ was 46,XX,t(1~?)(p12-13;?). Inv~vement of chromosomes #1 and #17 is d~cu~ed, ~ n g info account da~ ~ o m the literature concerning several human neopla~as. INTRODUCTION: After the fir~ Mudies carried out by Schroeder et al. [1], m a n y authors have rep o ~ e d high frequencies of chromosomal a b e ~ a ~ o n s in cases of F a n c o n i ' s anemia (FA). Chromatid exchange figures and increased frequency of endoredupHcation were also detected in this autosomal recessive disorder. Absence of chromosome heterogeneity between FA and the E~ren-Dameshek type was recently s h o w n by Dallapiccola et al. [2]. An increase of endoredupHca~ o n wffhont other classic chromosomal abnormalffies was found by Dosik et al. [3] in two MMers with aplasfic anemia wffhout skeletal abnormalities. Abnormal clones in the bone marrow of patients with FA [4-9] had been r e p o s e d in some papers. These abnormal clones were more frequently observed in a d u ~ than in young patients. In this arti~e, we report findings from repeated cytogenetic studies in the peripheral blood and bone marrow cells from one adult pa~ent w ~ h an inherffed aplas~c anemia but w ~ h o u t other congenital anomalies.

~om ~e ~ o • Geneti~ ~. C., G. B., G. G.), U~v~sR~ ~ PM~mo, and ~e ~ v ~ n e ~ EmPoria (S. M.), OspedMe ' ~ v ~ , " PM~mo, ~M~ Addm~ reques~ for rep~n~ to Dr. Paolo Carbone, ~tituto di Genetica, ~ a Archirafi 22, 90123 P~ermo, Raly. Rec~ved Augu~ 8, 1983; accepted O~ober 17, 1983.

259 © 1984 by Elsevier SciencePublishingCo., Inc. 52 Vanderbi~Ave., New Yor~ NY 10017

Cancer Gen~ics and Cytogen~s 13, 259-266 ~986) 0164~60~8~$03~0

260

P. Carbone et al.

CASE REPORT

L. M., a 3 1 ~ v ~ d ~mMe, was first e x a m M e d d ~ e D e p a ~ m e ~ ~ H e m M o ~ g y , " ~ C ~ v M ~ " H o s p H ~ of P M ~ m o , in February 19~1 because of a ~ M ~ y of anemM diagnosed 5 m o t h s before. F a m i l y a n a m n ~ ~ v e ~ e d t h ~ the p ~ e ~ ' s p ~ e ~ s were not m l ~ e d ; an older sister had died at the age of 20 years from aplasfic anemia. The p ~ e ~ ~ d ~ m e h e ~ c~Mmm Physical e x a m ~ a f i o n mveMed a well d e v ~ o p e d , well n o u r i s h e d ~ m M e of s h o ~ stature, w ~ h m i l d cutaneous ~ p ~ p ~ m e ~ (as were all ~ m e m b ~ s ) and w~hont phyMc~ mM~rm~ns. Throe spots ~ ~ o of 1-2 cm w ~ e ~ u n d . The spleen extended 14 cm below the costal m ~ g ~ . There was no h e p ~ o m e g M y or ~ m p h o a d e n o p ~ h y . Hb was found to be 7.5 ~ d l ; packed cell v ~ u m e (PCV) 0.21; ~u~c~ 104.0 × 10~L; WBC 2.4 × 10~L with 40% n e ~ m p ~ ; and p l ~ s 96.0 × 10~L. Bone m ~ m w biopsy was ~ ~ , with m i l d megMob~sfic ~ h m p ~ and no other m ~ u ~ f i o n defects. Iron storage was 1 + ; reficu~n content was 1 + . HbF was 10% and H b ~ 2%. ~ ratio was 0.79, as in ~ M ~ s e m i a ffait. D~ect Coombs test, sugar water test, and acidified serum test were ~ g ~ . S k e l ~ a ] survey and ~ a v e n o u s p y ~ o ~ a m (WP) were n ~ m M . T m ~ m e ~ consisted of W ~ and o x y m ~ h o ~ n e , but d i d not ~ e l d perMsting benefit. The p ~ i e ~ is still M ~ e with severe p a n c ~ o p e ~ on a ~ a n ~ u s i o n m ~ n ~ n a n c e m ~ m e n , and on chelation ~ a p y . She s u r f , s from f f e q u e ~ e p ~ s and ~ c f i o ~ . F a m i l y m e m b ~ s , except ~ e p ~ e ~ c ~ l ~ e n , still m ~ s e h e m ~ o ~ c invesfi~n. CYTOGENETIC STUDIES

Cytogenetic studies were carried out on d ~ e c t bone marrow preparations from the p ~ i e n t and on ~ i m u l ~ e d p e f i p h e r ~ l y m p h o c y t e s ~ o m the p ~ i e n t and her three children. D~afls of the techniques used were described previously [10]. Banding was obtained using the m ~ h o d of Seabright [11], as modified by W h a n ~ P e n g and K n u ~ e n [12]. The patient's bone marrow was examined twice (February 11, 1981; February ~, 1983). Chromosome a b n o r m ~ i t i e s were identified according to the reco m m e n d a t i o n s in the ISCN [13] on good metaphases, with well spread chromosomes, for each p e r i p h e r ~ blood cu~ure. Some 71-15g cells were examined. Te~aploid cells, with or without diplochromosomes, were identified by at least two observers, and their ~ e q u e n c y was d ~ e r m i n e d using the total n u m b e r of metaphases scored, according to the s y ~ e m of Dosik et al. [3]. In each p e f i p h e r ~ blood cu~ure, 497-2411 cells were examined. Data from ~terature [3] and data obtained ~ o m 20 h e a t h y s u p e r s examined in our ~ b o r a t o r y during the same period [14] were utilized as control v ~ u e s . RESULTS The resuhs on chromosome studies in ~ m p h o c ~ e cu~ures from the p ~ i e n t and her three c h i l d r e n are s u m m a r i z e d in Table 1. The frequency of c h r o m o s o m e breaks was shown in all cu~ures to be near that found in c o n s o l s ; h o w e v ~ , c h m m ~ i d exchange figures and dicenffics were increased in the p a t i e ~ . E n d o m d u p l i c a t i o n was ~ e a ~ d both in the p a t i e ~ and in one of her children (S. R., aged 9 years). The frequency of e n d o ~ d u p l ~ e d c ~ l s w ~ ~ g h ~ ~ p e ~ p h e r ~ blood cells of our p a t i e ~ ~ a n in ~ e c ~ M ~ m The cons~u~onM k ~ y o ~ p e was n ~ m M in all s u p e r s exam~ed. The findings in the two c ~ o g e n ~ exam~ns on direct bone m ~ m w preparations from our pa~ent were of g r e ~ interest. A b n ~ m M clones were p r e ~ , with

Inhe~d

AA with AbnormM Clones

TaMe 1

Chmm~ome s ~

261

in ~ m p h o c ~ e c u ~ e s

from ~ e patient and h ~ c ~ n T M ~ # ~ d cells

Subjects (dat~ Pa~ent L.M.--31 years (5-12-81) (3-30-82) ~2-7-82) Children S.R.--8 years (3-12-82) S.R.--9 years (3-12-82) (4-27-82) (12-7-82) S.G.--12 years (3-12~82)

Cells No. cf w~h metaphases breaks counted (%)

CMls wffh gaps (%)

Cells w~h r~r~angements (%)

No. of metaph~ scored

W~h ~#ochr~ m~om~ (%)

Wffho~ ~p~chr~ mo~m~ (%)

8 (1.3311) 10 (0.6477) 2 (0.4024)

5 (0.8319) 8 (0.5181) 1 (0.2012)

100 71 85

3 (3.00) 0 1 (1.18)

2 (2.00) 2 (2.82) 1 (1,18)

1 (1.00)° 3 (4.22)b 3 (3.53)~

601 1544 497

100

1 (1.00)

0

0

1501

0

0

108 100 158

1 (0.92) 2 (2.00) 0

0 1 (1.00) 0

0 0 0

2004 2151 2411

6 (0,2994) I (0.0465) 6 (0.2488)

0 4 (0.1860) 0

100

1 (1.00)

1 (1.00)

0

1500

0

0

°1 dic. bl qr and 2 tr. ~1 dic, 1 tr, and 1 cx.

two of them being the prevailing ones M the fi~t examinMMn. Such abnormM clones were a minority w h e n compared w ~ h calls with a normM karyotype. The karyotype features of one clone were trisomy 9 and trisomy 16 (Fig. 1), whereas chromosome abnormMities in the other clone i n v o D e d chromosomes #1 and #17 Wig. 2). These chromosomes had supernumerary bands: #1 on the long ~ m s and #17 on the sho~ arms. The anomMy of chromosome #1 c o n s i s ~ d in a duplication of lq24 to lq32. We could not eMab~sh the o f i # n of the s u p e r n u m e r a r y segment in the 1 7 p + marker. F u ~ h e r investigations were ca~ied out 2 y e a ~ 1Mer, and cytogenetic anMy~s showed t h d the prevMling clone had 46 chromosomes with a marker 17p +. Cells w ~ h normM karyotype no longer constituted the mMority. In both bone marrow investigations, a c ~ n e w ~ h a lost chromosome # 1 8 and cells w ~ h r a n d o m a n e u p l o i d y were also present. DISCUSSION The s~nificance of cytogenefic investigations on patients w ~ h apparent idiopMhic anemM has been e m p h a s ~ e d by Dosik d M. [3]. D M ~ p ~ c ~ a d al. [2] have shown t h ~ there is no chromosome h ~ e r o g e n e i t y b ~ w e e n FA and the Es~en-Dameshek type. High f e q u e n ~ e s of aberrations, as in FA, have been found in cases with i n h e v Red aplasfic a n e m ~ wffhout congenitM mMformafions [4, 1~]. &n increase of end o r e d u p l ~ a t i o n wRhout other chromosomM abnormMities was found in two cases r e p o s e d by D o c k ~ M. [~]. The frequency of cells w ~ h breaks and gaps in s~mulMed peripherM lymphocytes from our pMient was Mmflar to that found in controls, whereas the frequency of cells with rearrangements was increased. E n d o r e d u p l ~ M n was increased i n

7

2

8

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Figure 1

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G-banded karyotype of a bone marrow c ~ l from the p ~ i e n t showing trisomy 9 and trisomy 16.

20

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20

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--

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9

21

22

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Figure 2 (A) G-banded bone marrow m~aphase and ~s kary~ype ~om the patient, showing l q + and 17p+; (B) Chromosome p~rs #1 and #17 obtained ~om another m~aphase.

- ~

17

17p+

7

6

14

2

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16

13

1

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145~XX,-18 I

~_~.~~,

2 cells

9cells

= 146,X x,dup(q)(q24-q32),t(17;?~12-13;?) I

Figure 3 Abnorm~ clones found in bone marrow of the p~ient. Evolution of the karyotypes, s~rting with the norm~ chromosom~ complement, are schema~zed. (A) First examin~ion (February 11, 1981); (B) second examin~ion (February 8, 1983).

B

3ceils ~

A

~ h ~ R e d A A wRh A b n ~ m M ~ o ~ s

265

c o m p a ~ s o n both with data on healthy subjects from the 1Rerature [3] and from our laboratory ( 0 % - 0 ~ 8 5 7 % on 42,703 metaphases) [14]. E n d o r e d u p l i c a t i o n levels in our patient were Nmilar to those found in 2 cases r e p o s e d b y Dosik et el. [3] and to the average frequency found by Dallapiccola et el. [2] in 7 patients w ~ h F a n c o n i ~ anemia, ENren-Dameshek type. A n increase of e n d o r e d u p l i c a t i o n in one of the patient's c h i l d r e n could be due to a heterozygous ~ete; however, no e n d o r e d u p ~ c a t e d cells were found in the other two children. The interpretation of the cytogenetic resuhs on d ~ e c t bone marrow preparations of our patient is presented in Figure 3. AH clones detected were c o n s i d e r e d in the interpretation; a clone was defined as at least 3 cells wRh the same n u m e ~ c a l chrom o s o m e aberra~on or 2 ce~s wRh the same structural aberration. The detection of intermediate karyotypes at lhe fir~ examination (Fig. 3A) should be p a r t i c u l a ~ y emphaNzed. F u ~ h e r remarks concern the clone wRh + 9 and + 16, w h i c h probably has no selective advantage over 46,XX cells; in fact, no cell wRh a 4 8 , X X , + 9 , + 16 karyotype was detected at the second examination. On the contrary, the clone wRh the marker 1 7 p + was slightly more prevalent over 46,XX cells in the latter investigation. Further studies are necessary for clearer evidence of the selec~ve advantage of this clone. AbnormM clones have been found in the bone marrows from patients wRh FA, and such a finding is mere frequent in adult than young patients [4-9]. Clones wRh c h r o m o s o m e abnormalities were also observed in fibroblast cuhures from patients wRh F A [16, 17]. Clones wRh both numeNcM and structural c h r o m o s o m e abnormalities were present s i m u ~ a n e o u s l y in our patient. We consider the finding, in the same patient, of abnormalities concerning two chromosomes (#1 and #17) frequently involved in several h u m a n neoplasias [18-21] to be of the u t m o ~ interest. Par~al or total trN somy of c h r o m o s o m e #1 was, in fact, observed in several neoplasias [20, 21]; in particular, trisomy of bands lq25 to lq32 was found in m y e l o i d cells obtained from 35 patients wRh myeloproliferative disorders [22]. Chromosome #17 is frequently involved in chronic m y e l o i d leukemia, acute m y e l o i d leukemia, and chronic lymphocytic leukemia [21], ~s long arm being very frequently affected. A few cases of leukemia in w h i c h abnormalRies involve the short arm of c h r o m o s o m e #17 are also k n o w n [21, 23, 24]. A n increased p r e d i s p o s i t i o n to the d e v e l o p m e n t of cancer suggests that R is not unjuNified to consider F A as a p r e n e o p l a s t ~ state. Berger et al. [8] descNbed a case wRh FA evolving into acute leukemia wRh c h r o m o s o m a l l y abnormal clones in the bone marrow. Thus, our results, together wHh those of other authors, concerning the detection of abnormal clones in bone marrow celN of patients wRh i n h e ~ t e d aplasfic anemia, w ~ h or wRhout other congenital abnormalities, should be considered as indicating a stage of disease that m a y develop into overt malignancy. SuppoSed by a grant from the M i n ~ r o Pubb~ca ~tuNone (60%}.

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