[cancer research 54, 513-517, january 15, 1994] allele...

[CANCER RESEARCH 54, 513-517, January 15, 1994]

Allele Loss on Chromosome 16q24.2-qter Occurs Frequently in Breast Cancers

Irrespectively of Differences in Phenotype and Extent of Spread 1

H i t o s h i T s u d a , 2 D a v i d F. C a l l e n , T a k a s h i F u k u t o m i , Y u s u k e N a k a m u r a , a n d S e t s u o H i r o h a s h i

Pathology Division [H. T., S. H.] and Department of Surgery [T. El, National Cancer Center Research Institute and Hospital, Tokyo 104, Japan; Department of Cytogenetics and Molecular Genetics, Adelaide Children's Hospital, North Adelaide, South Australia 5006, Australia [D. E C.]; and Department of Biochemistry, Cancer Institute, Tokyo 170, Japan [Y N.]

A B S T R A C T

Loss of heterozygosity on chromosomal arm 16q has been shown to be a frequent event in sporadic breast cancer and is suggested to be involved in cancer development through inactivation of a tumor-suppressor gene. To specify the commonly deleted region in which the unknown tumor- suppressor gene is located, a deletion map of chromosome 16 was con- structed for 78 breast cancers, using 27 polymorphic DNA markers. Loss of heterozygosity on chromosome 16q was detected in 38 of the tumors. From the deletion map, the incidence of the loss of heterozygosity was deduced to be ->36% in the region distal to 16q12 and was most frequent in the 16q24.2-qter region. Then, association of the loss of heterozygosity in the 16q24.2-qter region with clinicopathological parameters of the tumors was examined for a total of 234 tumors, to reveal its biological significance in breast cancer development. The total incidence of loss of heterozygosity in the 16q24.2-qter region was 52% (118 of 225), and loss of heterozygosity was frequent irrespectively of the presence of invasion and metastasis, differences in clinical stage, tumor size, histological grade, or type, or amounts of estrogen receptor. Inactivation of an unknown tumor-suppressor gene on 16q24.2-qter was thus suggested to be involved commonly in the genesis of sporadic breast cancer, irrespectively of the extent of tumor spread or grade of aggressiveness of the cancer cells. On the other hand, eight cases revealed loss of heterozygosity not at 16q24- qter but in more proximal regions. Therefore, it appears that multiple tumor-suppressor genes are located on chromsome 16q.

also carries mutations in sporadic breast cancer (10); another gene that

causes familial breast cancer is still suggested to exist around 17q21

(11). Frequent LOH of chromosome 16q is reported not only in breast

cancer (4, 5) but also in hepatocellular carcinoma (12) and prostatic cancer (13). We previously specified in hepatocellular carcinoma a commonly deleted region, where an unknown tumor-suppressor gene is considered to be located, on chromosome 16 between the HP locus

on q22.1 and the CTRB locus on q23.2 (12). However, >60% of cases with the LOH were suggested to be due to monosomy of the entire chromosome 16. Sato et al. (5) also identified in breast cancer a commonly deleted region located between the HP and D16S157 loci on 16q24, using five polymorphic DNA markers. To further specify this region, a deletion map of a larger number of cases seemed to be

necessary, employing a larger number of polymorphic DNA markers.

Recently, physical and genetic chromosomal maps have been consid- erably refined using a larger number of novel polymorphic DNA markers (14-21). In the present study, we examined the incidence of LOH and tried to localize more accurately the region commonly deleted on chromosome 16, using polymorphic DNA markers at 27 different loci. Furthermore, association of the LOH with clinical and histological parameters was examined, to reveal its biological role in

breast cancer development.

I N T R O D U C T I O N

In human breast cancer, LOH 3 on chromosomal arms has been

detected frequently by RFLP analysis. LOH in a specific chromosomal region is generally considered to be involved in the genesis and progression of cancer, through inactivation of tumor-suppressor genes located there. Inactivation of tumor-suppressor genes occurs in a recessive manner; usually, one allele containing the gene is deleted, and the other allele contains point mutations or small deletions. Fre- quent LOH is reported for chromosomal arms lp, lq, 3p, 7q, l l p , 13q, 16q, 17p, 17q, and 18q in breast cancer (1-7). A gene associated with susceptibility to breast cancer is suggested to be located on chromosome 17q21 on the basis of linkage analysis of breast cancer families

(8). Although it is impossible to apply such linkage to sporadic cases, several tumor-suppressor genes located on a commonly deleted chromosomal region in sporadic cancer have already been identified, e.g., p53 and prohibitin (9-10). For example, the p53 tumor-suppressor gene located on chromosome 17p13.1, which is a commonly deleted region in diverse types of human cancers, has also been shown to carry mutations frequently (9). The prohibitin gene mapped on 17q12-q21

Received 7/9/93; accepted 11/12/93. The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture and from the Ministry of Health and Welfare of Japan and by a grant-in-aid from the Ministry of Health and Welfare of Japan for Comprehensive 10-Year Strategy for Cancer Control.

2 To whom requests for reprints should be addressed, at National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104, Japan.

3 The abbreviations used are: LOH, loss of heterozygosity; RFLP, restriction fragment length polymorphism.

M A T E R I A L S AND M E T H O D S

DNA Probes and Their Chromosomal Locations. The polymorphic DNA markers used, all localized on chromosome 16, are shown in Table 1. To specify the most frequently deleted region, the loci of these markers were assigned by referring to both genetic and physical maps already published by scientists involved in chromosome 16 mapping (14-21). The order of DNA markers on chromosome 16 according to the genetic map is as follows: pter- HBA1 (JWlO1)-D16S83 (pEKMDA2.1)-D16S131 (VK45C6)-MT2-D16S151 (CJ52.209M1)-[D16S4 (ACH207), D16S38 (CRI-O2)]-HP-CTRB-D16S20 (r 8-9) -D16S157(CJ52.96)- D16S43(CRI-O84)-[D16S7 (p79-2-23), APRT]- -D16S44 (CRI-089)-qter (14-18). By the genetic map, the D16S155 (CJ52. 199) locus is determined to be between D16S43 and D16S7. 4 According to the physical map, the order is as follows: pter-(HBA1, D16S83)-D16S34 (16/55B)- D16S32(16/118)-D16S131-cen- D16S27(p16-5)-(MT2, CETP)- -D16S151-(D16S4, D16S38)-D16S152 (CJ52.1)-(HP, TAT)-CTRB-D16S20- (D16S157, D16S43)-(D16S7, APRT)-D16S44-qter (19). 4 D16S159 (CJ52. 94) and D16S23 (p16-1) are localized in the same physical interval in 16p12.2, and D16S137 (KKA22) maps proximal to D16S159 on the genetic map. 4 Westphal et al. (22) strongly suggested that the position of the TAT locus is proximal to CTRB but distal to liP from 16cen. When these data are combined, the most probable order of the DNA markers is as follows: pter- HBAI-D16S83-D16S34-D16S32-D16S131-(D16S159, D16S23)-(D16S137, cen)-D16S27-(MT2, CETP)-D16S151-(D16S4, D16S38)-D16S152-HP-TAT- C TRB-D16S20-D16S15 7-( D16S4 3 , D16S15 5 )-( D16S T, APR T)-D16S44-qter.

TheD16S35 (16/32) and D16S30 (p16--8) loci are physically mapped to the region pter-pl3 containing D16S32 and D16S131 and to the region q22.1-q24, respectively (14). Additional information on these two loci was not available.

Tissue Samples, DNA Isolation, and Southern Blot Analysis. We collected 234 paired samples of breast cancer and noncancerous tissue from 225 patients with primary disease. In nine patients, bilateral primary lesions were

4 D. E Callen, unpublished observations.

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ALLELE LOSS ON CHROMOSOME 16q IN BREAST CANCER

Table 1 Incidence of loss of heterozygosity at 27 loci on chromosome 16 in breast cancer

Name of locus a (Name of clone) Chromosomal localization Restriction enzyme Total

No. of cases (%)

Heterozygosity

Constitutional Loss in tumor

triBAl (JW101) p13.3 RsaI 64 22 5 (20) i. D16S83 (pEKMDA2.1) p13.3 Rsal 62 31 6 (19) D16S34 (16/55B) p13.2 Pstl 65 21 7 (33) D16S32 (16/118) p13.1 TaqI 48 21 3 (14) D16S35 (16/32) pter-p13 TaqI 77 26 4 (15) D16S131 (pVK45C6) p12.3 Taql 49 8 1 (13)

rD16S159 (CJ52.94) p12.2 Taq 61 34 6 (18) tD16S23 (1916--1) p12.2 PvuIII 70 36 5 (14) D16S137 (pKKA22) 16 PstI 72 37 5 (14) D16S27 (p16-5) q12.1 PvuII 72 26 7 (27)

rMT2 (hMT2) q13 TaqI 76 29 9 (31) tCETP (pCETPll) q13 TaqI 77 36 9 (25) D16S151 (CJ52.209M1) q21 MspI 70 18 6 (33) D16S4 (ACH207) q22.1 Taql 78 28 16 (57) D16S38 (CRI-02) q22.1 BamHI 76 35 14 (40) D16S152 (CJ52.1) q22.1 TaqI 75 21 4 (19) HP (hp2 alpha) q22.3 BamHI 74 27 11 (41) TAT (HP0.4) q22.3 MspI "~

(BB0.4) BamnI~. 68 43 16 (37) (BS0.9) HaelII J

CTRB (pCXP33) q23.2 TaqI 67 34 9 (26) D16S20 (d? 8-9) q24.1 BglII, PvulI 78 45 15 (33) D16S30 (p16-8) q22.1-924 Pst! 71 6 3 (50)

[D16S157 (CJ52.96) q24.2-q24.3 TaqI 78 21 9 (43) tD16S43 (CRI-084) q24.2-q24.3 PstI 71 20 8 (40) D16S155 (CJ52.199) q Mspl 37 11 2 (18) D16S7 (p79-2-23) q24.3 TaqI 76 68 32 (47) APRT (pAT2.3 aprt) @4.3 Taql 78 33 17 (52) D16S44 (CRI-089) q24.3 Bglll 74 10 4 (40)

a Brackets, DNA markers located within same physical interval defined by hybrid breakpoints.

resected synchronously. The cases included 15 intraductal carcinomas and 12 invasive ductal carcinomas with a predominantly intraductal component (predominantly intraductal carcinoma); the other 207 were invasive carcinomas. Information on clinical stage (23), axillary lymph nodal status, histological grade (24), and histological type (25) was available for all of the cases examined. In 194 tumor tissue specimens, the amount of estrogen receptor was measured using an ER-EIA kit (Abbott Laboratory, North Chicago, IL).

Differential diagnosis of intraductal carcinoma of micropapillary or cribri- form type with a low degree of nuclear atypia (grade 1) versus atypical hyperplasia was made based on the criteria described by Azzopardi (26). For intraductal carcinomas, grade 2 corresponded to the former two types with a higher degree of nuclear atypia or the comedo type with a lower degree of nuclear atypia, and grade 3 corresponded to the comedo type with a high degree of atypia.

When the tumor was obviously identified, a portion of fresh tumor tissue in each case was embedded in OCT compound (Miles Inc., Elkhart, IN) and, using frozen sections, the predominant (>50%) distribution of cancer cells was confirmed microscopically. From these tissue samples, high molecular weight DNA was isolated (27). In the 12 predominantly intraductal carcinomas, the DNA was isolated from the intraductal tumor component. In entirely or predominantly intraductal carcinoma cases, the lesions were identified by careful visual observation and multiple portions were sampled from such lesions, embedded in OCT compound, sliced with a cryostat, and stained with hematoxylin and eosin. From these tissue specimens, areas with high proportions (>50%) of cancer cells were collected for DNA preparation (Fig. 1).

For 78 tumors, RFLP analysis using the polymorphic DNA markers at the 27 loci was performed. All of the cases revealed constitutional heterozygosity for at least four of these DNA markers. Five /xg of DNA were digested completely with a restriction enzyme (BamHI, BgllI, HaelII, MspI, PstI, PvulI, RsaI, or TaqI) (from New England Biolabs, Beverly, MA; Toyobo, Tokyo, Japan; or Takara, Kyoto, Japan), subjected to electrophoresis in 0.8% agarose gels (FMC, Rockland, ME), alkali denatured, neutralized, transferred to Ni- troplus membranes (MSI, Westboro, MA) by Southern blotting, and baked at 80~ for 2 h in a vacuum oven.

The membranes were prehybridized, hybridized with [a-32p]-dCTP-labeled DNA probes, and washed as described previously (27). For the CTRB probe, washing was performed under less stringent conditions (28). Autoradiography was performed at -80~ using Kodak XAR-5 film (Eastman-Kodak Co., Rochester, NY) with intensifying screens and an exposure time of 3 h to 3 weeks.

For the other 156 tumors, RFLP analysis was performed using six DNA markers, i.e., CETP, D16S4, HP, TAT (BB0.4), D16S7, and APRT. Constitu- tional heterozygosity for at least one locus was detected in 152 tumors. In total, information on LOH on chromosome 16q was available for 230 tumors.

Statistical analysis was performed by X 2 test.

R E S U L T S

Region on Chromosome 16q Most Frequently Deleted in Breast Cancer. Table 1 shows the incidence of L O H in the tumors at each

po lymorph i c D N A marker locus. Au to rad iograms of six cases show-

ing L O H are presented in Fig. 2. L O H on c h r o m o s o m e 16q was

detected in 38 (49%) of 78 tumors for wh ich delet ion ma pp ing was

performed. No cases revealed rear rangement or h o m o z y g o u s deletion.

For these 38 cases, the m a x i m u m extents o f the deleted regions in one

allele were deduced and are shown in Fig. 2. Fif teen tumors were

sugges ted to have LOH on the entire long arm, whereas the other 23

were sugges ted to have partial allele loss on 16q. F rom the delet ion

map (Fig. 3), the incidence of LOH was sugges ted to be 36% (28 of

78) at the M T 2 locus on 16q13 and 38% or higher (->30 of 78) in the

loci distal to MT2, al though the deleted region differed a m o n g the

cases with partial allele loss. In particular, L O H was mos t f requent in

the 16q24.2-qter region be tween D 1 6 S 4 3 or D 1 6 S 1 5 5 and qter, con-

ta ining the D16S7, A P R T , and D 1 6 S 4 4 loci. LOH on 16q24.2-qter was

detected in 36 of the 38 cases, whereas in the except ional two cases

(cases 22 and 24) the c o m m o n l y deleted regions were sugges ted to be

located be tween the D 1 6 S 3 2 locus at 16p13.1 and the D 6 S 4 locus on

q22.1 (Fig. 3). LOH on the short arm of c h r o m o s o m e 16 was detected in 17 (23%)

of 73 tumors in which const i tut ional he terozygosi ty was seen at one or

more loci. Sixteen of the 17 tumors showing allele loss on the short

arm also showed allele loss on the long arm. Frequent Loss of Heterozygosity on Chromosome 16q24-qter

Irrespectively of Differences in Clinicopathological Parameters. In total, 127 (55%) of 230 tumors , inc luding the cases for which

mapp ing was per formed, revealed L O H on c h r o m o s o m e 16q. Infor-

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not at HP. In six tumors, LOH was detected not at the loci on 16q24.2-qter but at the CETP, D16S4, HP, and/or TAT loci in more proximal regions.

As shown in Table 2, the incidence of LOH on 16q24.2-qter was ->35% in any group with respect to clinical stage, lymph node status, tumor size, histological grade and type, or estrogen receptor status. The incidence of LOH on 16q24.2-qter was relatively lower in the intraductal/predominantly intraductal group (8 of 27, 35%) than in the invasive group (110 of 198, 56%) (P < 0.005). In intraductal/ predominantly intraductal carcinomas, the incidence of LOH was not significantly different among the grade 1 (5 of 16, 31%), grade 2 (1 of 6, 17%), and grade 3 (2 of 5, 40%) subgroups and, thus, between comedo and non-comedo types. The incidence was commonly high both in histological types with a favorable prognosis, i.e., mucinous, papillary, and tubular, and in those with an unfavorable prognosis, i.e., invasive ductal carcinomas of the "not otherwise specified" type and of the atypical medullary type (25) (Table 2). Among the variety of histological types, LOH on chromosome 16q24.2-qter was especially frequent in the papillary (77%) and invasive lobular (75%) types.

The eight cases in total, including cases 22 and 24, that showed LOH on 16q but not at 16q24.2-qter comprised six grade 3 cases and two grade 2 cases. This group did not contain cases of intraductal! predominantly intraductal carcinoma, at stage I, with a tumor size of -<2.0 cm, or of grade 1, but contained two cases without lymph node metastasis.

Fig. 1. Tissue samples and LOH on chromosome 16q in intraductal breast cancers. A, grade 1 case (case 280); B, grade 3 case (case 174). Left, low-power view of cancer tissue samples from which DNA was isolated (hematoxylin-eosin stain). Bar, 2.5 mm. Right, LOH revealed in cancer DNA in these cases (arrows), at D16S7 in A and at D16S4 in B. T, cancer DNA; N, non-cancer DNA.

mation on LOH on 16q24.2-qter was available for 225 tumors, and 118 (52%) of these showed LOH at D16S7, APRT, and/or D16S44.

Among the 89 tumors showing LOH on 16q in the second series, LOH was detected at all informative loci in 67, and the other 22 revealed regional deletion, distal to CETP, D16S4, HP, and TAT in four, six, one, and three cases, respectively. In two tumors, LOH was detected both in regions proximal to HP and in those distal to HP, but

DISCUSSION

In the present study, we analyzed commonly deleted regions in 38 breast cancer tissue specimens which showed deletion of chromosome 16q in at least one locus, using 18 polymorphic DNA markers. The deletion map of chromosome 16 clarified that the region deleted most frequently in breast cancer was q24.2-qter (Fig. 3). This result was confirmed by the study of the second series of cases, in which 118 tumors in total carried LOH on 16q24.2-qter. An as yet undetermined tumor-suppressor gene was thus strongly suggested to be present in this region. To specify further the commonly deleted region and to identify accurately the unknown tumor-suppressor gene locus, it will be necessary to obtain much finer mapping data from the cancer cases showing the partial deletion.

Fig. 2. LOH on chromosome 16q in breast cancer. Autoradiograms of six cases (cases 25, 24, 22, 52, 54, and 20) are presented. T, cancer DNA; N, non- cancer DNA. Names of the clones used as DNA probes are shown to the right of the schematic dia- gram of chromosome 16q. Arrows, allele loss.

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Fig. 3. Deletion map of chromosome 16 in breast cancer. Thirty-nine cases which revealed LOH at one or more loci are presented in decreas- ing order of extent of LOH on 16q (left to right), but there are four exceptions in the last seven cases. Informative loci are indicated (0, LOH; (3, no LOH). Shading, maximum extent of the putative deleted regions. The most frequently deleted region is located on 16q24.2-qter, containing the D16S7, APRT, and D16S44 loci (bar). LOH in this regiol~ was detected in 36 of the 38 tumors showing LOH on 16q. The incidence of LOH was deduced to be consistently high (---36%) not only on 16q24.2-qter but also in the region distal to 16q12, from MT2 to qter.

This chromosomal region seemed to be somewhat different from those reported previously by us for hepatocellular carcinoma (12) and by Sato et al. (5) for breast cancer, in which the commonly deleted chromosomal region was shown to lie on 16q22-q23. The difference was not considered to reflect data inconsistency among the studies. The frequent occurrence (>36%) of LOH at any loci on the long arm distal to 16q12, including the region between the MT2 and D16S44 loci, suggests that multiple tumor-suppressor genes, other than the putative one located on 16q24.2-qter, may exist on 16q.

Table 2 Association of loss of heterozygosity on chromosome 16q24.2-qter with clinical and histological parameters of breast cancer

No. of cases (%)

LOH on chromosome Total 16q24.2-qter

Clinical stage 0--I 53 23 (43)-- 1 II 122 68 (56) 1 III 50 27 (54) - j

No. of metastatic lymph nodes 0 127 64 (50)--- 3 1-3 40 24 ( 6 0 ) [ 4-9 29 15 ( 5 2 ) [ >->-10 26 15 (58) --3 Not resected 3 0 (0)

Tumor size on palpation --<2.0 (cm) 59 26 (44)----q 2.1-5.0 127 74 ( 5 8 ) [ -->5.1 39 18 (46) --2

Histological grade 1 46 23 (50)--- 3 2 83 42 ( 5 1 ) ] 3 96 53 (55) --3

Histologic type Intraductal, predominantly intraductal 27 8 (35)- 5 Invasive ductal, NOS b 125 63 (50)-3[ Invasive ductal, atypical medullary 18 9 (50)]] Invasive lobular 20 15 (75)[[ Papillary 13 10 ( 7 7 ) ~ Tubular 13 8 ( 6 2 ) [ Mucinous 7 3 ( 4 3 ) [ Medullary, pure 2 2(100) 2

Estrogen receptor -->13 (fmol/mg protein) 151 84 (57)-- 3 <13 40 20 (50)--2 Not measured 34 14 (41)

Total 225 118 (52)

<0.005

a Statistical significance was calculated by X 2 test. NS, not significant. b NOS, not otherwise specified.

In hepatocellular carcinoma, LOH on chromosome 16q has been shown to occur frequently in tumors in advanced clinical stages and of poorer differentiation, but not in those in early stages of progression (12). Furthermore, in hepatocellular carcinoma the incidence is significantly higher in highly metastatic tumors (12). However, in breast cancer the incidence of LOH on chromosome 16q was high irrespectively of differences in clinical stage, lymph node status, tumor size, histological grade and type, or estrogen receptor status. LOH on chromosome 16q was frequent even in intraductal/predominantly intraductal carcinomas, in the group where histological grades and types indicated a favorable clinical outcome, and in cases without metastasis. The gene alterations reported previously, i.e., amplification of c-erbB-2, hst-1/int-2, and c-myc genes, mutation of the p53 tumor- suppressor gene and its aberrant protein, and LOH on chromosomal

pa arms 7q, l lp, and 17p, are all reported to be associated with aggressive clinical and/or histological characteristics in breast carcinoma (1, 3, 6, 24, 27, 29-32). We showed previously that alterations of the

NS c-erbB-2 and p53 genes and their proteins were frequently detectable in breast cancers showing the highest histological grade of atypia (grade 3) but rare in those showing low or moderate histological grades of atypia (grades 1 or 2) (24, 27, 31). In contrast, LOH on

NS chromosome 16q was shown to be frequent not only in grade 3 but also in grade 1 and 2 cancers. Therefore, LOH on 16q was strongly suggested to occur commonly in breast cancer at a very early devel- opmental stage, not only among highly aggressive but also in relatively low-grade tumors. Alteration of chromosome 16q is suggested

NS to play a special role in the genesis of breast cancer irrespectively of the grade of biological aggressiveness, whereas other gene alterations, e.g., oncogene amplification, p53 mutation, and LOH on other chro-

NS mosomal arms, are suggested to be involved in the development of various aggressive biological and morphological characteristics, e.g., a high proliferation rate, marked nuclear atypia, and/or dedifferentia- tion.

Eight of the present cases composed a group which showed LOH on 16q but not on 16q24.2-qter. This group consisted mainly of tumors in relatively advanced stages, with aggressive phenotypic characteristics. The region commonly deleted in these eight cases was suggested to be 16cen-q22.1. This result and the consistently frequent occurrence of LOH on the long arm distal to 16q12 suggest that, in addition to the putative gene on 16q24.2-qter, additional multiple tumor-suppressor

NS genes may exist within this region and their inactivation may be involved in the formation of biologically aggressive phenotypes of breast cancer. Particularly, on a percentage basis, the D16S4 locus was most frequently deleted, showing LOH in 57% of the cases and in 16 of 17 tumors with LOH on 16q, with the one tumor (case 24) showing

516




re tent ion at this locus hav ing a b reakpoin t re la t ively near. In the area

o f the D 1 6 S 4 locus, p robab ly in a more p rox imal region, there m a y be

a gene w h i c h is a f fec ted at the same or h igher f requency , c o m p a r e d to

the puta t ive , more distal gene.

L inkage ana lyses have sugges ted the presence o f a gene locus on

c h r o m o s o m e 16q that confers suscept ib i l i ty to breast cancer (8, 33).

Therefore , a l tera t ion o f the u n k n o w n gene on 16q is sugges ted to be

invo lved not on ly in the genes is o f sporad ic breast cancers but also in

suscept ib i l i ty to and/or genes is o f the fami l ia l form.

A C K N O W L E D G M E N T S

We are grateful to Drs. T. Nanasawa and H. Yamamoto, Department of Surgery, National Cancer Center Hospital, for providing surgically resected tissue specimens and to Y. Tanaka for excellent technical assistance. The authors are grateful to Drs. G. Scherer, D. Drayna, J. Arrand, and G. I. Bell for kindly providing DNA probes. DNA probes were also provided by the Ameri- can Type Culture Collection (Rockville, MD) and Collaborative Research Inc. (Bedford, MA).

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1994;54:513-517. Cancer Res Hitoshi Tsuda, David F. Callen, Takashi Fukutomi, et al. and Extent of Spreadin Breast Cancers Irrespectively of Differences in Phenotype Allele Loss on Chromosome 16q24.2-qter Occurs Frequently

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