Double Minute Chromosomes in the Leukocytes of a Young Girl with Breast Carcinoma Radha Madhavi, Mohan Guntur, Reita Ghosh, and P. K. Ghosh
ABSTRACT: Cy/ogene/it: mmlysis was per(ormed iu (J young girl with breast curcinoma. The most imporhmt finding wos the exishmce of d(mbh! minute (dmin) chromosomes, besides other chromosomal ubnormalities. Removal o( the tumor resulted it] reduction of chr(mlosomal abnormalities in the leukocyte t:ullm'e,~ repe(ded (dter 1 year, suggesting thai lhe presence
o.f tumor may elevate lhe frequency ol chromosom(d abnormalities. The presence o~ dmin chroll]OSOl]]lL% hldJcale,s lhal f(it:lors t-tlusili~ their presence ill IkllllOr cells Ill(IV (I/St) (JiJm'clle it~ I] tllll(lll leukocvtes. INTRODUCTION Breast c a n c e r ~s o n e of t h e m o s t f r e q u e n t f o r m s of n e o p l a s i a in w o m e n arid o c c u r s w i t h a f r e q u e n c y of a r o u n d 14% of all c a n c e r s r e p o r t e d in I n d i a n w o m e n [1 [. T o g e t h e r w i t h carcinoma of the uterine cervix, breast cancer constitutes a major proportion of
all cancers reported in different anatomic sites in h l d i a n women. A l t h o u g h breast cancer is comnlon in elderly women, its occurrence in patients aged less than 20 years is extremely rare. There are occasional reports of breast cancer in young women in the literature [2-4]. O n l y one case of breast cancer was obserw',d in a children's hospital during a 40-year period [5]. A collected series of 7 pediatric cases of breast cancer was reported by M c D i v i t t and Stewart ]6]. Cytogenetic studies in patients w i t h breast cancer have revealed the existenl:e of different types of chromosomal abnormalities [7[. The occurrence of double minute (dnlin) chromosomes, w b i c b is a tmique phenomenon in tumor biology, has been reported in breast tumor cells I81. A l t h o u g h the presence of d m i n chromosomes is w e l l documented in different types of tumors, especially neurogenic tumors [9-13], their presence in peripheral blood lymphocytes is reported rarely in the literature [14, 15[. We report the existence of d n l i n cbromosomes in the peripheral blood lymphocytes of a 14-year-old girl w i t b a malignant growfll in the left breast. MATERIALS AND METHODS Case Report A 1 4 - y e a r - o l d girl n o t i c e d a m o b i l e l u m p in t h e u p p e r o u t e r q u a d r a n t of h e r left breast a b o u t 15 d a y s before s h e w a s e x a m i n e d at our h o s p i t a l . S h e h a d no pain or d i s c h a r g e from t h e n i p p l e a n d h a d r e c e i v e d n o t r e a t n l e n t . T h e m a s s w a s s h a r p l y c i r c u m s c r i b e d
From the Cytogenetics Laboratory. Department of Anthropology, University of Delhi. Delhi, India. Address reprint rcquesls lo: I)1". P. K. Ghosh. Deparllnetfl o( Anthropology, University of Delhi, Delhi 110II07. India. f~.e,ceived March 28. 1989: act:el]ted June, 30, 1989.
2O3 ~5 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas. New York, NY 10Ill0
Cancer Genet Cytogenel 44:203 207 {1990} 0165-4608/90/$03.59
204
R. Madhavi et al.
Figure 1
A lll(!|~l[)h~is(! S]l()Xvs di11ill C}II'OIII()SOII/tL
and was not adherent to the overlying skin. She ba(t a n(;galive bistory of lymt)h no(h; involvement and no signs of ally endocrine dysfun(:tion. She began to menstruat(,' at age 12 years, and had no hist(,'y of previous breast biopsy, estrogen administration, or trauma. None of her relatiw;s bad breast cancer. Tbe tmnor was removed by siml)le excision and was examined histol)athologically. Chromosome Studies Cytogenetic studio's were perfornl(,'d twice: once after the girl's initial admission to the hospital and again 1 year after removal of the tumor. 1,ymphocytes in wboh; blood were (:ultured ill m(xlium T(]-199 SUl)plc'monte(t with 20% fetal calf s c r t n n , phytohemagglutinin, and antibiotics, and the cells w e , r e gr()wn for 56 hours at 37°(2. At 54 hours, Colcemid was added to a final concentration of 0.1 ~g/ml. After llypotonic treatment with KCI an(t fixation in methanol :ace, tic acid (3:1), the slides were prepared by air drying. They were' subsequently stained in 5% Ciemsa. The, ¢:hromosomal aberrations were scor(;(I from 2(1(I metaphases of eat:h cullur(,' at:cording to the criteria of (be International System for Cytogenelic Nomenclalure, [16]. RESULTS Analysis of the data revealed the presence of different types of chrolnosomal abnormalities, the most important of which was the existence of double m i n u t e chromosomes {Fig. 1). The frequency of the chromosomal abnormalities observed in the patient before and after removal of the tumor is shown in Table 1. In both samples analyzed, clmins were found together with other chromosomal abnormalities. Although the types of chromosomal abnormalities observed in both the samples were similar, they varied quantitatively. In the initial sample, 18% of the metaphases
Dmins in Breast Carcinoma Table 1
205
Frequency of c h r o m o s o m a l aberrations in the leukocytes of the patient with breast carcinoma
Date of examination
No. of metaphases
Breaks and gaps
Abnormality
Chromosome
Chromatid
dmins
Others "
Metaphases with abnormalities
4/9/87 15/10/88
200 200
7 (3.5) 5 (2.5)
15 (7.4) 11 (5.5)
8 (4) 5 (2.5)
6 (3) 4 (2)
36 (18) 25 (12.5)
Numbers in parentheses are percentages. Dicentrics, rings, exchanges, and pulverizations.
revealed the presence of chromosomal abnormalities, which i n c l u d e d 8 d m i n s beside other c h r o m o s o m a l abnormalities such as dicentrics, rings, exchanges, and pulverizations. The d m i n s varied in size and were stained similarly to other chromosomes. In the repeat culture of the patient made 1 year after tumor removal, a reduction in the frequency of c h r o m o s o m a l abnormalities was observed. In this sample, 12.5% of the metaphases revealed chromosomal abnormalities, which besides other aberrations i n c l u d e d five dmins. This reduction in frequency of chromosomal a b n o r m a l i t y was, statistically nonsignificant, however (x 2 = 2.34; p > 0.05). DISCUSSION
The most striking cytogenetic feature in the leukocytes of the patient with breast carcinoma observed in the present study is the occurrence of drain chromosomes, w h i c h have been found in a wide variety of h u m a n [17, 18] and animal tumors [19, 20] and rarely in insects/21]. Although dmins are exclusively associated with cancer cells, there are few reports of their presence in the peripheral leukocytes of normal i n d i v i d u a l s [22-24]. The existence of drain chromosomes in both normal and tumor cells indicate that factors p r o d u c i n g them in cancerous cells may also be operative in normal cells. A n o t h e r interesting feature of the present study was the slight d e c l i n e in frequency of c h r o m o s o m a l abnormalities after tumor removal in the patient with breast cancer. Experiments in tumor-bearing mice and fish have suggested that the presence of t u m o r in the body of the animal causes an increase in instability of the karyotype in terms of elevated sister chromatid exchanges (SCEs) [25, 26]. Recently, a r e d u c t i o n in SCE values was observed after surgical removal of tumor in breast cancer patients [27]. Thus removal of cancerous tumor from the host may lead to a decrease in frequency of cytological endpoints such as SCEs and drain chromosomes. The role of drains in the etiology of neoplastic transformation is not k n o w n precisely. Selective gene amplification is often manifested cytogenetically as d m i n s and h o m o g e n e o u s l y staining regions (HSRs), and these structures are generally believed to be cellular responses to tumor challenge [28]. Chromosome versus genetic imbalances resulting from karyotypic abnormalities are often associated with neoplastic transformation. Extra c h r o m o s o m a l material is often present in chromosome 1 of h u m a n cervical carcinomas [29]. No such abnormality, however, could be detected in one such carcinoma; instead d m i n s were present, implying that dmins probably represent the extra DNA necessary to generate genetic imbalance [9, 30]. These cytogenetic equivalents of gene amplification m a y be of fundamental importance for tumor progression since their presence implies poor prognosis (eg, in h u m a n neuroblastoma, amplification of N-myc oncogene was found almost exclusively in tumor from patients with a d v a n c e d stages of the disease [31]). Because amplification of protooncogenes c-myc [32], N-ras [33], and c-neu [34] occur in breast carcinoma, a knowledge of the
206
R. M a d h a v i et al.
D N A s e q u e n c e s a m p l i f i e d in the drains present in breast carcinoma m a y help in u n d e r s l a n d i n g their role in the initiation of m a l i g n a n c y .
REFERENCES 1. National Cancer Registry (1984): Indian (kmncil of Medical Research. New Delhi, India. 2. Thompson WIt (1,908): Case of adenoc:ar(:illOlna of/he breast in a girl aged 11 years. Br Med
] 2:502. 3. Sears IlL Schesing, es M] (1940): Carcinonm of the breast in a 10 ,,,:at old girl. N Engl ] Med 223:760 761. 4. l,ippit WII, Medart WS, Ramasey SN (1970]: Cancer in a hm year old girl. Surgery 6g:395 396. 5. SilnpSOn IS, [:;arson AI (196(`)): Breast tunlt)rs in inhmls and childreu: A 40 year review lif cases at a (:hildre, n's hospital, Can Med Asso(: I 101:100 l[12. 6. M(:Divitt RW. Stewart FW (1961~): Breast car(:in(nna in H~ihlren. lAMA Ig5:388 390. 7. Sandberg AA [1(`]80): The C h r o m o s o m e s in Ilulnan (]iH1GCl"illl(l l,eukemia. Elsevier Science Publishing, New York, pp. 550 -558. 8. Baker PE, tlsu TC (1,q7,q): l)ouble minuh;s in h u m a n carcinoma cell lines with special refe, rmu:e 1o breast 'minors. I Natl Cancer lnst 62:257 2(;2. 9. Atkin NIL Baker MC [1,q801: Cytogel~etic obserwltions on a (:arcinolna of the, cervix uleri with double m i m d e chromatin bodies. F,ur I Cancer 16:793 797. 10. Rahlban-Mahmbaum (;, Gilbert t: [1977]: l)oulfle nfinute chromusonu,*s and honlo,e, eneously s l a i n i n g r e g i o n s i n c h r o m i l s o m e s o f a h u m a n n e u r o b l a s h m m c e l l l i n e . Si:iem:e198:739 742. 11. Cox D, Yuncken C, Si)rigges AI (19[;5}: Minute chromalin bodies in malignanl tumors of
ABSTRACT: Cy/ogene/it: mmlysis was per(ormed iu (J young girl with breast curcinoma. The most imporhmt finding wos the exishmce of d(mbh! minute (dmin) chromosomes, besides other chromosomal ubnormalities. Removal o( the tumor resulted it] reduction of chr(mlosomal abnormalities in the leukocyte t:ullm'e,~ repe(ded (dter 1 year, suggesting thai lhe presence
o.f tumor may elevate lhe frequency ol chromosom(d abnormalities. The presence o~ dmin chroll]OSOl]]lL% hldJcale,s lhal f(it:lors t-tlusili~ their presence ill IkllllOr cells Ill(IV (I/St) (JiJm'clle it~ I] tllll(lll leukocvtes. INTRODUCTION Breast c a n c e r ~s o n e of t h e m o s t f r e q u e n t f o r m s of n e o p l a s i a in w o m e n arid o c c u r s w i t h a f r e q u e n c y of a r o u n d 14% of all c a n c e r s r e p o r t e d in I n d i a n w o m e n [1 [. T o g e t h e r w i t h carcinoma of the uterine cervix, breast cancer constitutes a major proportion of
all cancers reported in different anatomic sites in h l d i a n women. A l t h o u g h breast cancer is comnlon in elderly women, its occurrence in patients aged less than 20 years is extremely rare. There are occasional reports of breast cancer in young women in the literature [2-4]. O n l y one case of breast cancer was obserw',d in a children's hospital during a 40-year period [5]. A collected series of 7 pediatric cases of breast cancer was reported by M c D i v i t t and Stewart ]6]. Cytogenetic studies in patients w i t h breast cancer have revealed the existenl:e of different types of chromosomal abnormalities [7[. The occurrence of double minute (dnlin) chromosomes, w b i c b is a tmique phenomenon in tumor biology, has been reported in breast tumor cells I81. A l t h o u g h the presence of d m i n chromosomes is w e l l documented in different types of tumors, especially neurogenic tumors [9-13], their presence in peripheral blood lymphocytes is reported rarely in the literature [14, 15[. We report the existence of d n l i n cbromosomes in the peripheral blood lymphocytes of a 14-year-old girl w i t b a malignant growfll in the left breast. MATERIALS AND METHODS Case Report A 1 4 - y e a r - o l d girl n o t i c e d a m o b i l e l u m p in t h e u p p e r o u t e r q u a d r a n t of h e r left breast a b o u t 15 d a y s before s h e w a s e x a m i n e d at our h o s p i t a l . S h e h a d no pain or d i s c h a r g e from t h e n i p p l e a n d h a d r e c e i v e d n o t r e a t n l e n t . T h e m a s s w a s s h a r p l y c i r c u m s c r i b e d
From the Cytogenetics Laboratory. Department of Anthropology, University of Delhi. Delhi, India. Address reprint rcquesls lo: I)1". P. K. Ghosh. Deparllnetfl o( Anthropology, University of Delhi, Delhi 110II07. India. f~.e,ceived March 28. 1989: act:el]ted June, 30, 1989.
2O3 ~5 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas. New York, NY 10Ill0
Cancer Genet Cytogenel 44:203 207 {1990} 0165-4608/90/$03.59
204
R. Madhavi et al.
Figure 1
A lll(!|~l[)h~is(! S]l()Xvs di11ill C}II'OIII()SOII/tL
and was not adherent to the overlying skin. She ba(t a n(;galive bistory of lymt)h no(h; involvement and no signs of ally endocrine dysfun(:tion. She began to menstruat(,' at age 12 years, and had no hist(,'y of previous breast biopsy, estrogen administration, or trauma. None of her relatiw;s bad breast cancer. Tbe tmnor was removed by siml)le excision and was examined histol)athologically. Chromosome Studies Cytogenetic studio's were perfornl(,'d twice: once after the girl's initial admission to the hospital and again 1 year after removal of the tumor. 1,ymphocytes in wboh; blood were (:ultured ill m(xlium T(]-199 SUl)plc'monte(t with 20% fetal calf s c r t n n , phytohemagglutinin, and antibiotics, and the cells w e , r e gr()wn for 56 hours at 37°(2. At 54 hours, Colcemid was added to a final concentration of 0.1 ~g/ml. After llypotonic treatment with KCI an(t fixation in methanol :ace, tic acid (3:1), the slides were prepared by air drying. They were' subsequently stained in 5% Ciemsa. The, ¢:hromosomal aberrations were scor(;(I from 2(1(I metaphases of eat:h cullur(,' at:cording to the criteria of (be International System for Cytogenelic Nomenclalure, [16]. RESULTS Analysis of the data revealed the presence of different types of chrolnosomal abnormalities, the most important of which was the existence of double m i n u t e chromosomes {Fig. 1). The frequency of the chromosomal abnormalities observed in the patient before and after removal of the tumor is shown in Table 1. In both samples analyzed, clmins were found together with other chromosomal abnormalities. Although the types of chromosomal abnormalities observed in both the samples were similar, they varied quantitatively. In the initial sample, 18% of the metaphases
Dmins in Breast Carcinoma Table 1
205
Frequency of c h r o m o s o m a l aberrations in the leukocytes of the patient with breast carcinoma
Date of examination
No. of metaphases
Breaks and gaps
Abnormality
Chromosome
Chromatid
dmins
Others "
Metaphases with abnormalities
4/9/87 15/10/88
200 200
7 (3.5) 5 (2.5)
15 (7.4) 11 (5.5)
8 (4) 5 (2.5)
6 (3) 4 (2)
36 (18) 25 (12.5)
Numbers in parentheses are percentages. Dicentrics, rings, exchanges, and pulverizations.
revealed the presence of chromosomal abnormalities, which i n c l u d e d 8 d m i n s beside other c h r o m o s o m a l abnormalities such as dicentrics, rings, exchanges, and pulverizations. The d m i n s varied in size and were stained similarly to other chromosomes. In the repeat culture of the patient made 1 year after tumor removal, a reduction in the frequency of c h r o m o s o m a l abnormalities was observed. In this sample, 12.5% of the metaphases revealed chromosomal abnormalities, which besides other aberrations i n c l u d e d five dmins. This reduction in frequency of chromosomal a b n o r m a l i t y was, statistically nonsignificant, however (x 2 = 2.34; p > 0.05). DISCUSSION
The most striking cytogenetic feature in the leukocytes of the patient with breast carcinoma observed in the present study is the occurrence of drain chromosomes, w h i c h have been found in a wide variety of h u m a n [17, 18] and animal tumors [19, 20] and rarely in insects/21]. Although dmins are exclusively associated with cancer cells, there are few reports of their presence in the peripheral leukocytes of normal i n d i v i d u a l s [22-24]. The existence of drain chromosomes in both normal and tumor cells indicate that factors p r o d u c i n g them in cancerous cells may also be operative in normal cells. A n o t h e r interesting feature of the present study was the slight d e c l i n e in frequency of c h r o m o s o m a l abnormalities after tumor removal in the patient with breast cancer. Experiments in tumor-bearing mice and fish have suggested that the presence of t u m o r in the body of the animal causes an increase in instability of the karyotype in terms of elevated sister chromatid exchanges (SCEs) [25, 26]. Recently, a r e d u c t i o n in SCE values was observed after surgical removal of tumor in breast cancer patients [27]. Thus removal of cancerous tumor from the host may lead to a decrease in frequency of cytological endpoints such as SCEs and drain chromosomes. The role of drains in the etiology of neoplastic transformation is not k n o w n precisely. Selective gene amplification is often manifested cytogenetically as d m i n s and h o m o g e n e o u s l y staining regions (HSRs), and these structures are generally believed to be cellular responses to tumor challenge [28]. Chromosome versus genetic imbalances resulting from karyotypic abnormalities are often associated with neoplastic transformation. Extra c h r o m o s o m a l material is often present in chromosome 1 of h u m a n cervical carcinomas [29]. No such abnormality, however, could be detected in one such carcinoma; instead d m i n s were present, implying that dmins probably represent the extra DNA necessary to generate genetic imbalance [9, 30]. These cytogenetic equivalents of gene amplification m a y be of fundamental importance for tumor progression since their presence implies poor prognosis (eg, in h u m a n neuroblastoma, amplification of N-myc oncogene was found almost exclusively in tumor from patients with a d v a n c e d stages of the disease [31]). Because amplification of protooncogenes c-myc [32], N-ras [33], and c-neu [34] occur in breast carcinoma, a knowledge of the
206
R. M a d h a v i et al.
D N A s e q u e n c e s a m p l i f i e d in the drains present in breast carcinoma m a y help in u n d e r s l a n d i n g their role in the initiation of m a l i g n a n c y .
REFERENCES 1. National Cancer Registry (1984): Indian (kmncil of Medical Research. New Delhi, India. 2. Thompson WIt (1,908): Case of adenoc:ar(:illOlna of/he breast in a girl aged 11 years. Br Med
] 2:502. 3. Sears IlL Schesing, es M] (1940): Carcinonm of the breast in a 10 ,,,:at old girl. N Engl ] Med 223:760 761. 4. l,ippit WII, Medart WS, Ramasey SN (1970]: Cancer in a hm year old girl. Surgery 6g:395 396. 5. SilnpSOn IS, [:;arson AI (196(`)): Breast tunlt)rs in inhmls and childreu: A 40 year review lif cases at a (:hildre, n's hospital, Can Med Asso(: I 101:100 l[12. 6. M(:Divitt RW. Stewart FW (1961~): Breast car(:in(nna in H~ihlren. lAMA Ig5:388 390. 7. Sandberg AA [1(`]80): The C h r o m o s o m e s in Ilulnan (]iH1GCl"illl(l l,eukemia. Elsevier Science Publishing, New York, pp. 550 -558. 8. Baker PE, tlsu TC (1,q7,q): l)ouble minuh;s in h u m a n carcinoma cell lines with special refe, rmu:e 1o breast 'minors. I Natl Cancer lnst 62:257 2(;2. 9. Atkin NIL Baker MC [1,q801: Cytogel~etic obserwltions on a (:arcinolna of the, cervix uleri with double m i m d e chromatin bodies. F,ur I Cancer 16:793 797. 10. Rahlban-Mahmbaum (;, Gilbert t: [1977]: l)oulfle nfinute chromusonu,*s and honlo,e, eneously s l a i n i n g r e g i o n s i n c h r o m i l s o m e s o f a h u m a n n e u r o b l a s h m m c e l l l i n e . Si:iem:e198:739 742. 11. Cox D, Yuncken C, Si)rigges AI (19[;5}: Minute chromalin bodies in malignanl tumors of
