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Karyotypic changes in phyllodes tumors of the breast
Karyotypic Changes in Phyllodes Tumors of the Breast Claudia U. Dietrich, Nikos Pandis, Georgia Bardi, Manuel R. Teixeira, Tatiana Soukhikh, Catarina Petersson, Johan A. Andersen, and Sverre Heim
ABSTRACT: Cytogenetic analysis of short-term cultures of five phyllodes tumors of the breast-classified
as benign (one tumor), borderline malignant (two tumors removed from the same breast in 1991 and 1993), and malignant (two tumors) - revealed clonal changes with simple structural abnormalities in the benign tumor, the borderline malignant tumors, and one malignant t u m o r in which benign areas and areas of borderline malignancy were also present. In contrast, the malignant tumor without admixed borderline malignant or benign areas had a complex karyotype. The karyotype of the benign phyllodes tumor was 46,XX,del(12)(pllp12)/46,XX,t(8;18)(p11;p11)/46,XX. The first borderline malignant phyllodes t u m o r had t(3;20)(p21;q13) as the sole abnormality. When the t u m o r recurred, this was no longer the only clone detected and the tumor karyotype was now 46,XX,t(3;20)(p21;q13)/46,XX,t(9;10)(p22;q22)/46,XX, t(1;8)(p34; q24)/46,XX,del(11)(q22- 23)/46,XX. The malignant~borderline malignant~benign tumor had t(1;6)(p34;p22) as the sole clonal abnormality. Finally, the karyotype of the malignant phyllodes t u m o r which contained no benign or borderline malignant areas was 42,XX,der(1)t(1;4)(q21;q21),der(3)t(3;17)(q29;q21),-4,i(8) (qlO), - 10, - 13,i(13)(q10),der(14)t(1;14)(q21;p11),der(14)t(4;14)(p12;p11),- 17/80-90,idemx2, +del(1)(q12), +i(1) (plO), +dic(5;5)(p14;p14), +i(6)(p10), +del(7)(p11), +dup(7)(qllq36), +i(15)(q10),inc/46,XX. The findings indicate some cytogenetic similarities between benign~borderline malignant phyllodes tumors and fibroaden o m a s of the breast, presumably reflecting similar pathogenetic mechanisms in the two types of mixedlineage tumors.
INTRODUCTION Phyllodes tumors of the breast are characterized by coproliferation of epithelial and mesenchymal cells. They account for less than 1% of all breast tumors [1]. The histologic evaluation of phyllodes tumors a n d their classification as benign, borderline malignant, or malignant are among the most difficult problems in breast tumor pathology [2]. Only one phyllodes tumor of the breast has been cytogenetically characterized before [3]. We here present the cytogenetic analysis of five karyotypically abnormal phyllodes tumors. PATHOLOGIC EXAMINATION Case 1--Tumor 1
A 2.4-cm cystic tumor removed from the breast of an 81-yearold w o m a n was shown to contain soft, leaf-like intracystic projections. The abundant, hypercellular fibroblast-like
From the Department of Medical Genetics (C. U. D., N. P., G. B., M. R. T., T. S., S. H.), Odense University; Department of Pathology (J. A. A.), University Hospital, Odense, Denmark; and Department of Clinical Genetics (C. P., S. H.), UniversityHospital, Lund, Sweden. Address reprint requests to: Dr. Claudia U. Dietrich, Department of Medical Genetics, Odense University, WinslCwparken15, DK-5000 Odense C, Denmark. Received December 28, 1993; accepted April 19, 1994. 200 Cancer Genet Cytogenet 78:200-206 (1994) 0165-4608/94/$07.00
mesenchymal c o m p o n e n t (Fig. 1A) invaded the surrounding fatty tissue. Mitotic figures were sparse (0 mitosis per high-power field, HPF). The epithelial structures were hyperplastic but clearly benign. The t u m o r was diagnosed as a benign phyllodes tumor. Case 2 - - T u m o r s 2 and 3
In 1991 and 1993, two sharply circumscribed, firm tumors were surgically removed from the left breast of a now 47year-old woman. The first tumor (2) measured 6 x 4 cm, the recurrence (3) was 4 cm in diameter. Both tumors showed leaf-like formations and were dominated by a hypercellular, fibroblast-like mesenchymal c o m p o n e n t (Fig. 1B) in w h i c h the cells had normal to slightly enlarged, dark nuclei. The mean mitotic count was 0.6 mitoses per HPE In both tumors, the mesenchymal c o m p o n e n t invaded the surrounding tissue. The epithelial c o m p o n e n t was hyperplastic and benign w i t h o u t any carcinoma in situ lesions. Both the original tumor and the recurrence were diagnosed as borderline malignant phyllodes tumors. Case 3 - - T u m o r 4
A 26- x - 2 4 - c m t u m o r that filled out the right breast of a 56year-old woman was shown to be partly cystic, partly lobular, with a dominating mesenchymal c o m p o n e n t and a benign, slightly hyperplastic epithelium. The connective tissue was
© 1994 Elsevier Science Inc. 655 Avenue of the Americas, New York. NY 10010
201
F i g u r e 1 Histologic sections of 4 phyllodes tumors of the breast (H&E x 80, Fig. 1A-1C; H&E x 32, Fig. 1D). A) Benign phyllodes tumor (case 1). B) Borderline malignant phyllodes tumor (case 2 - tumor 3). C) Malignant phyllodes tumor (case 3). D) Malignant phyllodes tumor (case 4).
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generally of moderate cellularity with few if any mitoses (0 per HPF), but some areas were more or even highly cellular, with moderate to severe nuclear pleomorphism (Fig. 1C) and many mitotic figures (up to 6 per HPF). Although the diagnosis was malignant phyllodes tumor, areas of borderline malignancy and, especially, areas with benign phyllodes tumor histology were quantitatively dominant.
ing. The cultures were exposed to demecolcine, the ceils were dislodged by trypsinization, subjected to hypotonic shock in 0.05 M KC1, and fixed in methanol:acetic acid (3:1). G-banding was obtained with Wright stain. The criteria for clonality and karyotype description followed the recommendations of the ISCN [7].
Case 4 - T u m o r 5
RESULTS
A 5 × 3 x 2-cm breast tumor of a 62-year-old woman showed leaf-like projections typical of phyllodes tumor of the breast. The tumor connective tissue component was extremely hypercellular with marked nuclear pleomorphism and a high mitotic activity (5 mitoses per HPF). The tumor epithelium showed the growth pattern of a poorly differentiated adenocarcinoma (10 mitoses per HPF) and a rather highly differentiated squamous cell carcinoma (0-1 mitosis per HPF) in different areas (Fig. 1D). Remnants of highly differentiated glandular and partly microcystic structures were found. In 14 of 17 axillary lymph nodes, metastases were found; they too displayed all three malignant components. The diagnosis was malignant phyllodes tumor with biphasic malignancy. According to Azzopardi [4], this tumor may also be classified as carcinosarcoma with remnants of phyllodes tumor, as it displayed both epithelial and connective tissue malignancy.
The cytogenetic analysis detected clonal chromosomal abnormalities in all five tumors (Table 1). Four tumors--the benign, the two borderline malignant, and the malignant one that also comprised areas of borderline malignant and benign histology-had simple structural abnormalities only (Fig. 2). The first of the two borderline malignant phyllodes tumors removed from the same breast in the course of 2 years had a clone with t(3;20)(p21;q13) as sole abnormality. In the recurrence, the t(3;20) was still present but now also three other, cytogenetically unrelated clones were found. Multiple, cytogenetically unrelated clones were also seen in the benign phyllodes tumor. The fifth tumor, classified as malignant and without admixture of benign or borderline malignant areas, differed from the others by being karyotypically characterized by a complex hypodiploid clone (Fig. 3). It also exhibited clonal evolution with a derivative subclone arisen through polyploidization of the mother clone.
CYTOGENETIC EXAMINATION All tumor specimens were mechanically and enzymatically disaggregated and the cells plated in plastic flasks. Tumors 1 and 2 were short-term cultured and processed for cytogenetic analysis as described by Pandis et al. [5]. The procedures for tissue disaggregation and culturing were modified to include differential sedimentation of the epithelial and mesenchymal tumor fractions [6] in tumors 3-5 because we knew beforehand in these cases that the tumors probably were of mixed lineage. All harvests were completed within 3 to 7 days after plat-
DISCUSSION The finding of a karyotypically identical clone with t(3;20) (p21;q13) in the primary and recurrent phyllodes tumors (case 2) removed from the same breast in the course of 2 years indicates that this clone was pathogenetically important. The finding of three additional, cytogenetically unrelated clones in the second sample (tumor 3) may be attributable to the technical improvements then introduced [6] in the processing of the tumor tissue, which allowed us to culture mesenchymal and epithelial cells separately. Thus, all 50 cells ana-
Table 1 Cytogenetic data on the five phyllodes tumors of the breast
Case
No. of mitoses analyzed
No. of abnormal mitoses
Karyotype a
1
100
8
46,XX,de|(12)(pllp12)[5]/46,XX,t(8;
2b
41
7
46,XX,t(3;20)(p21;q13)[7]/46,XX[34]
2
200
68
3
73
4
200
18)(pll;p11)[3]/46,XX[92]
46,XX,t(3;20)(p21;q13)[55]/46,XX,t(9; 10)(p22;q22)[8]/46,XX, t(1;8)(p34;q24)[3]/46,XX,del(11)(q22-23)[2]/46,XX[132]
9c 175
46,XX,t(1;6)(p34;p22)[3]/46,XX[64] 42,XX,der(1)t(1;4)(q21;q21),der(3)t(3;17)(q29;q21),
- 4,i(8)
( q l 0 ) , - 10, - 1 3 , i ( 1 3 ) ( q 1 0 ) , d e r ( 1 4 ) t ( 1 ; 1 4 ) ( q 2 1 ; p 1 1 ) , d e r ( 1 4 ) t(4;14)(p12;p11), - 171165]/80-90,idemx2,
+ d e l ( 1 ) ( q 1 2 ) , + i(1)
(pl0), + dic(5;5)(p14;p14), + i(6)(p10), + del(7)(p11), + dup(7) (qllq36), + i(15)(q10),inc[10]/46,XX[25] ° Numbers in square brackets denote the n u m b e r of mitoses in each clone, inc: incomplete karyotype description because of n u m e r o u s unidentified c h r o m o s o m e s . h In the course of two years, two tumors were removed from the same breast of patient 2. c: Nonclonal c h a n g e s were seen in six mitoses.
Cytogenetics in Breast Phyllodes Tumors
203
Case I
12
8
18
9
10
Case 2
3
20
II.
1
8
1
6
11
Case 3
Figure 2 Partial karyotypesillustrating the clonal karyotypicabnormalities seen in cases 1-3, namely del(12){pllp12) and t(8;18)(p11;p11) in case 1, t(3;20)(p21;q13), t(9;10)(p22;q22), t(1;8)(p34;q24), and del(11)(q22-23) in case 2, and t(1;6)(p34;p22) in case 3. Arrowheads indicate breakpoints. lyzed from a culture with purely mesenchymal morphology (data not shown) had a t(3;20)(p21;q13). In contrast, the t(3;20) was only very occasionally seen in the predominantly epithelial cultures from this case. Instead, these were cytogenetically characterized by a normal karyotype or the pres-
ence of the rearrangements t(9;10), t(1;8), and del(11)(q22-23) in separate small clones. These findings not only tally with the biphasic phenotype of phyllodes tumors, but also demonstrate cytogenetic monoclanality of the mesenchymal tumor tissue but cytogenetic polyclonality of the epithelial tu-
.i
,¸
20
19
~igure 3
14
13
y
2
9
21
Di
16
10
22
17
11
4
~epresentative karyogram of the hypodiploid clone seen in case 4. Arrowheads indicate breakpoints.
15
B
3
18
12
~X
3.
Cytogenetics in Breast Phyllodes Tumors mor component. Based on restriction fragment length p o l y m o r p h i s m (RFLP) analysis of the X-chromosome-linked PGK (phosphoglycerokinase) gene, Noguchi et al. [8] recently showed that all five benign phyllodes tumors they investigated were c o m p o s e d of a polyclonal epithelial fraction, whereas the mesenchymal component was monoclonal. The cytogenetic and molecular data with regard to tumor clonality are therefore in full accord. As the cultures of both the benign phyllodes tumor (tumor 1) and the malignant/borderline malignant/benign tumor (tumor 4) exhibited a mixture of mesenchymal and epithelial cells, the lineage of the clonal del(12)(pllp12) and t(8;18) in tumor 1 and the t(1;6) in tumor 4 is unknown. Moreover, in tumor 4 we do not know w h i c h histologically different areas p r e d o m i n a t e d in the sample processed for cytogenetic analysis and were later reflected in the karyotype. The cultural m o r p h o l o g y was mixed also in the case of the malignant phyllodes tumor (tumor 5), and here too conclusions about w h i c h cells contained the clonal abnormalities must be tentative. It is h i g h l y suggestive, however, that some of the changes, i.e., i(8)(q10) and i(13)(q10), are very c o m m o n in malignant epithelial tumors. For example, an i(8)(q10) has been reported in 31 adenocarcinomas of the breast and other organs according to Mitelman's Catalog of Chromosome Aberrations in Cancer [9]. In contrast, only two mesenchymal malignant tumors with i(8)(q10) have been described until now [10]. The finding of cytogenetic m o n o c l o n a l i t y in the malignant tumor 5 was somewhat unexpected against the background that it contained no less than three histologically distinct malignant cell populations, corresponding to its sarcomatous, adenocarcinomatous, and squamous cell carcinoma areas. Of course, the most simple explanation would be to accept that the t u m o r was i n d e e d monoclonal in spite of its p h e n o t y p i c heterogeneity. It is also possible, however, that different proliferation rates in vitro may have favored the cytogenetic analysis of one cell type while preventing the study of the two others, and that we hence failed to get a complete picture of the t u m o r cell populations' karyotypic complexity. Of some relevance in this context might be the histologic observation that the a d e n o c a r c i n o m a c o m p o n e n t exhibited twice as many mitotic figures (10 per HPF) as the mesenchymal c o m p o n e n t (5 per HPF), whereas in the squamous cell c o m p o n e n t hardly a single mitosis was seen. None of the clonal aberrations detected in the five tumors was present in more than one case. However, band lp34 was found to be rearranged in one of the borderline malignant tumors as well as in the malignant/borderline malignant/benign phyllodes tumor. The only phyllodes tumor of the breast with karyotypic aberrations previously reported [3] had a complex karyotype but was nevertheless benign; none of the aberrations of that case were seen in the tumors we analyzed. It is perhaps worthy of note that two bands involved in structural abnormalities in cases 2 and 4 - 1 0 q 2 2 and 14p11-were also rearranged in the t u m o r reported by Birdsall et al. [3]. The latter band, as well as lq21, was actually rearranged in both homologs in the malignant tumor of case 4. The derivative chromosomes resulting from these rearrangements were der(1)t(1;4)(q21;q21), der(14)t(1;14)(q21;p11), and der(14)t(4;14) (p12;p11).
205 The only disease entity that combines epithelial and mesenchymal proliferation in a histologic pattern similar to that of benign phyllodes t u m o r is fibroadenoma [11]. One might therefore ask if fibroadenomas of the breast are karyotypically similar to phyllodes tumors. To the best of our knowledge, only 14 breast fibroadenomas with karyotypic abnormalities characterized by chromosome b a n d i n g have been reported [12-14]. Rearrangement of band 12p12, w h i c h in the benign phyllodes t u m o r studied by us was found as del(12)(pllp12), has been reported in two breast fibroadenomas with a t(12 ;21)(p12 ;q22) and a t(8;12)(q24;p12) as sole anomalies [12]. Also, clonal rearrangements of bands 3p2122, 8q24, and 10q21-22, present in our case 2, have been detected in breast fibroadenomas [12-14]. A l t h o u g h the data obviously still are insufficient for reliable comparisons to be made, it seems that some cytogenetic similarities exist between benign/borderline malignant phyllodes tumors and fibroadenomas of the breast, p r e s u m a b l y reflecting similar pathogenetic mechanisms in the two t u m o r types. This work was supported partly by the Danish Cancer Society. C.U.D. is a fellow of the Deutsche Forschungsgemeinschaft (Di 510/2-1). N.P. and G.B. are on leave from the Papanikolaou Research Center, Hellenic Anticancer Institute, Athens, Greece. T.S. is on leave from the Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia.
REFERENCES 1. Kessinger A, Foley JF, Lemon HM, Miller DM (1972): Metastatic cystosarcoma phyllodes: a case report and review of the literature. J Surg Oncol 4:131-14z 2. Page DL, Anderson T], Johnson RL (1987): Sarcomas of the breast. In: Diagnostic Histopathology of the Breast, 1st Ed., DL Page, TJ Anderson, eds. C. Livingstone, Edinburgh, pp. 335-353. 3. Birdsall SH, MacLennan KA, Gusterson BA [1992): t(6;12) (q23;q13) and t(lO;16)(q22;p11) in a phyllodes tumor of breast. Cancer Genet Cytogenet 60:74-77. 4. Azzopardi JG (1979): Problems in Breast Pathology, 1st Ed. W.B. Saunders Company Ltd., London, p. 374. 5. Pandis N, Heim S, Bardi G, Limon J, Mandahl N, Mitelman F (1992): Improved technique for short-term culture and cytogenetic analysis of human breast cancer. Genes Chrom Cancer 5:14-20. 6. Dietrich CU, Pandis N, Andersen ]A, Heim S (1994): Chromosome abnormalities in adenolipomas of the breast: Karyotypic evidence that the mesenchymal component constitutes the neoplastic parenchyma. Cancer Genet Cytogenet 72:146-150. 7. ISCN (1991]: Guidelines for Cancer Cytogenetics, Supplement to An International System for Human Cytogenetic Nomenclature, F. Mitelman, ed. S. Karger, Basel. 8. Noguchi S, Motomura K, Inaji H, Imaoka S, Koyama H (1993): Clonal analysis of fibroadenoma and phyllodes tumor of the breast. Cancer Res 53:4071-4074. 9. Mitelman F (1991): Catalog of Chromosome Aberrations in Cancer, 4th Ed. Wiley-Liss Inc., New York. 10. Molenaar WM, De Jong B, Buist J, Idenburg vJS, Seruca R, Vos AM, Hoekstra JH (1989): Chromosomal analysis and the classification of soft tissue sarcomas. Lab Invest 60:266-274. 11. Fechner RE (1987): Fibroadenoma and related lesions. In: Diagnostic Histopathology of the Breast, 1st Ed., DL Page, TJ Anderson, eds. C. Livingstone, Edinburgh, pp. 72-85.
206 12. Calabrese G, Di Virgilio C, Cianchetti E, Guanciali Franchi P, Stuppia L, Parruti G, Bianchi PG, Palka G (1991): Chromosome abnormalities in breast fibroadenomas. Genes Chrom Cancer 3:202-204. 13. Fletcher JA, Pinkus GS, Weidner N, Morton CC (1991): Lineage-
C . U . Dietrich et al. restricted clonality in biphasic solid tumors. Am J Pathol 138:1199-1207. 14. Stephenson CF, Davis RI, Moore GE, Sandberg AA (1991): Cytogenetic and fluorescence in situ hybridization analysis oi breast fibroadenomas. Cancer Genet Cytogenet 63:32-36.
ABSTRACT: Cytogenetic analysis of short-term cultures of five phyllodes tumors of the breast-classified
as benign (one tumor), borderline malignant (two tumors removed from the same breast in 1991 and 1993), and malignant (two tumors) - revealed clonal changes with simple structural abnormalities in the benign tumor, the borderline malignant tumors, and one malignant t u m o r in which benign areas and areas of borderline malignancy were also present. In contrast, the malignant tumor without admixed borderline malignant or benign areas had a complex karyotype. The karyotype of the benign phyllodes tumor was 46,XX,del(12)(pllp12)/46,XX,t(8;18)(p11;p11)/46,XX. The first borderline malignant phyllodes t u m o r had t(3;20)(p21;q13) as the sole abnormality. When the t u m o r recurred, this was no longer the only clone detected and the tumor karyotype was now 46,XX,t(3;20)(p21;q13)/46,XX,t(9;10)(p22;q22)/46,XX, t(1;8)(p34; q24)/46,XX,del(11)(q22- 23)/46,XX. The malignant~borderline malignant~benign tumor had t(1;6)(p34;p22) as the sole clonal abnormality. Finally, the karyotype of the malignant phyllodes t u m o r which contained no benign or borderline malignant areas was 42,XX,der(1)t(1;4)(q21;q21),der(3)t(3;17)(q29;q21),-4,i(8) (qlO), - 10, - 13,i(13)(q10),der(14)t(1;14)(q21;p11),der(14)t(4;14)(p12;p11),- 17/80-90,idemx2, +del(1)(q12), +i(1) (plO), +dic(5;5)(p14;p14), +i(6)(p10), +del(7)(p11), +dup(7)(qllq36), +i(15)(q10),inc/46,XX. The findings indicate some cytogenetic similarities between benign~borderline malignant phyllodes tumors and fibroaden o m a s of the breast, presumably reflecting similar pathogenetic mechanisms in the two types of mixedlineage tumors.
INTRODUCTION Phyllodes tumors of the breast are characterized by coproliferation of epithelial and mesenchymal cells. They account for less than 1% of all breast tumors [1]. The histologic evaluation of phyllodes tumors a n d their classification as benign, borderline malignant, or malignant are among the most difficult problems in breast tumor pathology [2]. Only one phyllodes tumor of the breast has been cytogenetically characterized before [3]. We here present the cytogenetic analysis of five karyotypically abnormal phyllodes tumors. PATHOLOGIC EXAMINATION Case 1--Tumor 1
A 2.4-cm cystic tumor removed from the breast of an 81-yearold w o m a n was shown to contain soft, leaf-like intracystic projections. The abundant, hypercellular fibroblast-like
From the Department of Medical Genetics (C. U. D., N. P., G. B., M. R. T., T. S., S. H.), Odense University; Department of Pathology (J. A. A.), University Hospital, Odense, Denmark; and Department of Clinical Genetics (C. P., S. H.), UniversityHospital, Lund, Sweden. Address reprint requests to: Dr. Claudia U. Dietrich, Department of Medical Genetics, Odense University, WinslCwparken15, DK-5000 Odense C, Denmark. Received December 28, 1993; accepted April 19, 1994. 200 Cancer Genet Cytogenet 78:200-206 (1994) 0165-4608/94/$07.00
mesenchymal c o m p o n e n t (Fig. 1A) invaded the surrounding fatty tissue. Mitotic figures were sparse (0 mitosis per high-power field, HPF). The epithelial structures were hyperplastic but clearly benign. The t u m o r was diagnosed as a benign phyllodes tumor. Case 2 - - T u m o r s 2 and 3
In 1991 and 1993, two sharply circumscribed, firm tumors were surgically removed from the left breast of a now 47year-old woman. The first tumor (2) measured 6 x 4 cm, the recurrence (3) was 4 cm in diameter. Both tumors showed leaf-like formations and were dominated by a hypercellular, fibroblast-like mesenchymal c o m p o n e n t (Fig. 1B) in w h i c h the cells had normal to slightly enlarged, dark nuclei. The mean mitotic count was 0.6 mitoses per HPE In both tumors, the mesenchymal c o m p o n e n t invaded the surrounding tissue. The epithelial c o m p o n e n t was hyperplastic and benign w i t h o u t any carcinoma in situ lesions. Both the original tumor and the recurrence were diagnosed as borderline malignant phyllodes tumors. Case 3 - - T u m o r 4
A 26- x - 2 4 - c m t u m o r that filled out the right breast of a 56year-old woman was shown to be partly cystic, partly lobular, with a dominating mesenchymal c o m p o n e n t and a benign, slightly hyperplastic epithelium. The connective tissue was
© 1994 Elsevier Science Inc. 655 Avenue of the Americas, New York. NY 10010
201
F i g u r e 1 Histologic sections of 4 phyllodes tumors of the breast (H&E x 80, Fig. 1A-1C; H&E x 32, Fig. 1D). A) Benign phyllodes tumor (case 1). B) Borderline malignant phyllodes tumor (case 2 - tumor 3). C) Malignant phyllodes tumor (case 3). D) Malignant phyllodes tumor (case 4).
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generally of moderate cellularity with few if any mitoses (0 per HPF), but some areas were more or even highly cellular, with moderate to severe nuclear pleomorphism (Fig. 1C) and many mitotic figures (up to 6 per HPF). Although the diagnosis was malignant phyllodes tumor, areas of borderline malignancy and, especially, areas with benign phyllodes tumor histology were quantitatively dominant.
ing. The cultures were exposed to demecolcine, the ceils were dislodged by trypsinization, subjected to hypotonic shock in 0.05 M KC1, and fixed in methanol:acetic acid (3:1). G-banding was obtained with Wright stain. The criteria for clonality and karyotype description followed the recommendations of the ISCN [7].
Case 4 - T u m o r 5
RESULTS
A 5 × 3 x 2-cm breast tumor of a 62-year-old woman showed leaf-like projections typical of phyllodes tumor of the breast. The tumor connective tissue component was extremely hypercellular with marked nuclear pleomorphism and a high mitotic activity (5 mitoses per HPF). The tumor epithelium showed the growth pattern of a poorly differentiated adenocarcinoma (10 mitoses per HPF) and a rather highly differentiated squamous cell carcinoma (0-1 mitosis per HPF) in different areas (Fig. 1D). Remnants of highly differentiated glandular and partly microcystic structures were found. In 14 of 17 axillary lymph nodes, metastases were found; they too displayed all three malignant components. The diagnosis was malignant phyllodes tumor with biphasic malignancy. According to Azzopardi [4], this tumor may also be classified as carcinosarcoma with remnants of phyllodes tumor, as it displayed both epithelial and connective tissue malignancy.
The cytogenetic analysis detected clonal chromosomal abnormalities in all five tumors (Table 1). Four tumors--the benign, the two borderline malignant, and the malignant one that also comprised areas of borderline malignant and benign histology-had simple structural abnormalities only (Fig. 2). The first of the two borderline malignant phyllodes tumors removed from the same breast in the course of 2 years had a clone with t(3;20)(p21;q13) as sole abnormality. In the recurrence, the t(3;20) was still present but now also three other, cytogenetically unrelated clones were found. Multiple, cytogenetically unrelated clones were also seen in the benign phyllodes tumor. The fifth tumor, classified as malignant and without admixture of benign or borderline malignant areas, differed from the others by being karyotypically characterized by a complex hypodiploid clone (Fig. 3). It also exhibited clonal evolution with a derivative subclone arisen through polyploidization of the mother clone.
CYTOGENETIC EXAMINATION All tumor specimens were mechanically and enzymatically disaggregated and the cells plated in plastic flasks. Tumors 1 and 2 were short-term cultured and processed for cytogenetic analysis as described by Pandis et al. [5]. The procedures for tissue disaggregation and culturing were modified to include differential sedimentation of the epithelial and mesenchymal tumor fractions [6] in tumors 3-5 because we knew beforehand in these cases that the tumors probably were of mixed lineage. All harvests were completed within 3 to 7 days after plat-
DISCUSSION The finding of a karyotypically identical clone with t(3;20) (p21;q13) in the primary and recurrent phyllodes tumors (case 2) removed from the same breast in the course of 2 years indicates that this clone was pathogenetically important. The finding of three additional, cytogenetically unrelated clones in the second sample (tumor 3) may be attributable to the technical improvements then introduced [6] in the processing of the tumor tissue, which allowed us to culture mesenchymal and epithelial cells separately. Thus, all 50 cells ana-
Table 1 Cytogenetic data on the five phyllodes tumors of the breast
Case
No. of mitoses analyzed
No. of abnormal mitoses
Karyotype a
1
100
8
46,XX,de|(12)(pllp12)[5]/46,XX,t(8;
2b
41
7
46,XX,t(3;20)(p21;q13)[7]/46,XX[34]
2
200
68
3
73
4
200
18)(pll;p11)[3]/46,XX[92]
46,XX,t(3;20)(p21;q13)[55]/46,XX,t(9; 10)(p22;q22)[8]/46,XX, t(1;8)(p34;q24)[3]/46,XX,del(11)(q22-23)[2]/46,XX[132]
9c 175
46,XX,t(1;6)(p34;p22)[3]/46,XX[64] 42,XX,der(1)t(1;4)(q21;q21),der(3)t(3;17)(q29;q21),
- 4,i(8)
( q l 0 ) , - 10, - 1 3 , i ( 1 3 ) ( q 1 0 ) , d e r ( 1 4 ) t ( 1 ; 1 4 ) ( q 2 1 ; p 1 1 ) , d e r ( 1 4 ) t(4;14)(p12;p11), - 171165]/80-90,idemx2,
+ d e l ( 1 ) ( q 1 2 ) , + i(1)
(pl0), + dic(5;5)(p14;p14), + i(6)(p10), + del(7)(p11), + dup(7) (qllq36), + i(15)(q10),inc[10]/46,XX[25] ° Numbers in square brackets denote the n u m b e r of mitoses in each clone, inc: incomplete karyotype description because of n u m e r o u s unidentified c h r o m o s o m e s . h In the course of two years, two tumors were removed from the same breast of patient 2. c: Nonclonal c h a n g e s were seen in six mitoses.
Cytogenetics in Breast Phyllodes Tumors
203
Case I
12
8
18
9
10
Case 2
3
20
II.
1
8
1
6
11
Case 3
Figure 2 Partial karyotypesillustrating the clonal karyotypicabnormalities seen in cases 1-3, namely del(12){pllp12) and t(8;18)(p11;p11) in case 1, t(3;20)(p21;q13), t(9;10)(p22;q22), t(1;8)(p34;q24), and del(11)(q22-23) in case 2, and t(1;6)(p34;p22) in case 3. Arrowheads indicate breakpoints. lyzed from a culture with purely mesenchymal morphology (data not shown) had a t(3;20)(p21;q13). In contrast, the t(3;20) was only very occasionally seen in the predominantly epithelial cultures from this case. Instead, these were cytogenetically characterized by a normal karyotype or the pres-
ence of the rearrangements t(9;10), t(1;8), and del(11)(q22-23) in separate small clones. These findings not only tally with the biphasic phenotype of phyllodes tumors, but also demonstrate cytogenetic monoclanality of the mesenchymal tumor tissue but cytogenetic polyclonality of the epithelial tu-
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~epresentative karyogram of the hypodiploid clone seen in case 4. Arrowheads indicate breakpoints.
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Cytogenetics in Breast Phyllodes Tumors mor component. Based on restriction fragment length p o l y m o r p h i s m (RFLP) analysis of the X-chromosome-linked PGK (phosphoglycerokinase) gene, Noguchi et al. [8] recently showed that all five benign phyllodes tumors they investigated were c o m p o s e d of a polyclonal epithelial fraction, whereas the mesenchymal component was monoclonal. The cytogenetic and molecular data with regard to tumor clonality are therefore in full accord. As the cultures of both the benign phyllodes tumor (tumor 1) and the malignant/borderline malignant/benign tumor (tumor 4) exhibited a mixture of mesenchymal and epithelial cells, the lineage of the clonal del(12)(pllp12) and t(8;18) in tumor 1 and the t(1;6) in tumor 4 is unknown. Moreover, in tumor 4 we do not know w h i c h histologically different areas p r e d o m i n a t e d in the sample processed for cytogenetic analysis and were later reflected in the karyotype. The cultural m o r p h o l o g y was mixed also in the case of the malignant phyllodes tumor (tumor 5), and here too conclusions about w h i c h cells contained the clonal abnormalities must be tentative. It is h i g h l y suggestive, however, that some of the changes, i.e., i(8)(q10) and i(13)(q10), are very c o m m o n in malignant epithelial tumors. For example, an i(8)(q10) has been reported in 31 adenocarcinomas of the breast and other organs according to Mitelman's Catalog of Chromosome Aberrations in Cancer [9]. In contrast, only two mesenchymal malignant tumors with i(8)(q10) have been described until now [10]. The finding of cytogenetic m o n o c l o n a l i t y in the malignant tumor 5 was somewhat unexpected against the background that it contained no less than three histologically distinct malignant cell populations, corresponding to its sarcomatous, adenocarcinomatous, and squamous cell carcinoma areas. Of course, the most simple explanation would be to accept that the t u m o r was i n d e e d monoclonal in spite of its p h e n o t y p i c heterogeneity. It is also possible, however, that different proliferation rates in vitro may have favored the cytogenetic analysis of one cell type while preventing the study of the two others, and that we hence failed to get a complete picture of the t u m o r cell populations' karyotypic complexity. Of some relevance in this context might be the histologic observation that the a d e n o c a r c i n o m a c o m p o n e n t exhibited twice as many mitotic figures (10 per HPF) as the mesenchymal c o m p o n e n t (5 per HPF), whereas in the squamous cell c o m p o n e n t hardly a single mitosis was seen. None of the clonal aberrations detected in the five tumors was present in more than one case. However, band lp34 was found to be rearranged in one of the borderline malignant tumors as well as in the malignant/borderline malignant/benign phyllodes tumor. The only phyllodes tumor of the breast with karyotypic aberrations previously reported [3] had a complex karyotype but was nevertheless benign; none of the aberrations of that case were seen in the tumors we analyzed. It is perhaps worthy of note that two bands involved in structural abnormalities in cases 2 and 4 - 1 0 q 2 2 and 14p11-were also rearranged in the t u m o r reported by Birdsall et al. [3]. The latter band, as well as lq21, was actually rearranged in both homologs in the malignant tumor of case 4. The derivative chromosomes resulting from these rearrangements were der(1)t(1;4)(q21;q21), der(14)t(1;14)(q21;p11), and der(14)t(4;14) (p12;p11).
205 The only disease entity that combines epithelial and mesenchymal proliferation in a histologic pattern similar to that of benign phyllodes t u m o r is fibroadenoma [11]. One might therefore ask if fibroadenomas of the breast are karyotypically similar to phyllodes tumors. To the best of our knowledge, only 14 breast fibroadenomas with karyotypic abnormalities characterized by chromosome b a n d i n g have been reported [12-14]. Rearrangement of band 12p12, w h i c h in the benign phyllodes t u m o r studied by us was found as del(12)(pllp12), has been reported in two breast fibroadenomas with a t(12 ;21)(p12 ;q22) and a t(8;12)(q24;p12) as sole anomalies [12]. Also, clonal rearrangements of bands 3p2122, 8q24, and 10q21-22, present in our case 2, have been detected in breast fibroadenomas [12-14]. A l t h o u g h the data obviously still are insufficient for reliable comparisons to be made, it seems that some cytogenetic similarities exist between benign/borderline malignant phyllodes tumors and fibroadenomas of the breast, p r e s u m a b l y reflecting similar pathogenetic mechanisms in the two t u m o r types. This work was supported partly by the Danish Cancer Society. C.U.D. is a fellow of the Deutsche Forschungsgemeinschaft (Di 510/2-1). N.P. and G.B. are on leave from the Papanikolaou Research Center, Hellenic Anticancer Institute, Athens, Greece. T.S. is on leave from the Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia.
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