Sporadic Breast Cancers Exhibit Lossof Heterozygosity on ChromosomeSegment 10q23 Close to the Cowden Disease Locus

Baljit Singh,1 Michael M. Ittmann,2 and John J. Krolewski1*1Department of Pathology and Irving Comprehensive Cancer Center, Columbia University, College of Physicians and Surgeons,New York, New York2Department of Pathology, New York Department of Veterans Affairs Medical Center, New York, New York

Cowden disease, a dominantly inherited syndrome characterized by a variety of proliferative lesions and predisposition tobreast and thyroid cancer, has recently been linked to the polymorphic marker D10S215 on chromosome segment 10q23. Lossof heterozygosity in prostate cancer is linked to the same marker, whereas loss of heterozygosity in glioblastoma, endometrialcancer, and other malignancies also localizes to this region. Most recently, a putative tumor suppressor gene (PTEN/MMAC1)has been identified in the region between D10S215 and an adjacent, more telomeric marker (D10S541) and was found to bealtered in breast cancers, prostate cancers, and glioblastomas. We examined 22 invasive breast cancers for loss ofheterozygosity in the 10q23 region and found loss in 41% (9/22). There were two distinct regions of loss, including one near theD10S541 marker, with an approximately equal frequency of deletion in each. The observed pattern of deletion is consistentwith the presence of a tumor suppressor gene between D10S215 and D10S541. Most of the poorly differentiated carcinomas inthe case collection showed loss of heterozygosity in the region near D10S215, suggesting that this loss correlates with a poorprognosis. Genes Chromosomes Cancer 21:166–171, 1998. r 1998 Wiley-Liss, Inc.

Neoplastic transformation is a multiple step processinvolving the alteration of genes that positively(proto-oncogenes) and negatively [tumor suppres-sor genes (TSGs)] regulate cellular proliferation.The two hit model for genetic inactivation of TSGsstipulates that inactivation requires separate eventsaffecting both alleles of a given gene (reviewed byKnudsen, 1993). One event is usually a chromo-somal alteration and the other a small mutation.Typically, there is a set of individuals who havegermline mutations affecting a single allele of thesegenes. These individuals are predisposed to cancer,because only a single mutational event is requiredin any somatic cell that will subsequently give riseto a particular cancer. The identification of familiespredisposed to particular cancers has facilitated theidentification of some TSGs because genetic link-age analysis can identify the chromosomal regioninvolved. Furthermore, in many cases, the samegenes involved in inherited cancers have also beenshown to be mutated in sporadic cancers.

Loss of heterozygosity (LOH) analysis is widelyemployed to search for putative TSGs. In breastcancer, the LOH observed on chromosome seg-ments 17p13 and 16q23 has been correlated withmutation of the P53 (Eeles et al., 1993) andE-cadherin genes (Berx et al., 1995, 1996), respec-tively. However, in the case of E-cadherin, mutationhas been observed in only a minor histologic variant

of breast cancer, suggesting that the LOH observedin most breast cancers is targeted at another locus(Berx et al., 1995, 1996). Other chromosome armsthat show deletions in breast cancers and aresuspected of harboring TSGs include 1p, 1q, 3p, 6q,7q, 8p, 11p, 11q, 13q, 17q, 18q, and 22q (summa-rized in Devilee and Cornelisse, 1994; Bieche andLidereau, 1995).

Recent data indicate that a TSG involved in thepathogenesis of human breast cancer, as well asother cancers, lies on chromosome segment 10q23.Specifically, three independent studies have docu-mented LOH in this region in 25–50% of prostatecancers (Gray et al., 1995; Ittmann, 1996a,b; Trybuset al., 1996). All groups observed that the region ofmost frequent LOH lies at or very near the D10S215marker. Cowden disease, an autosomal dominantdisorder, is characterized by pathognomonic muco-cutaneous lesions, hamartomas of various organs,abnormalities of the central nervous system, and,most importantly, carcinomas of the breast and

Contract grant sponsor: NIH; Contract grant number: CA56862.Michael M. Ittmann is now at the Department of Pathology,

Houston Veterans Affairs Medical Center, 2002 Holcombe Blvd.,Houston, TX 77030.

*Correspondence to: John J. Krolewski, Department of Pathology,Columbia University, College of Physicians and Surgeons, 630 West168th Street, New York, NY 10032. E-mail: jjk5@columbia.edu

Received 19 May 1997; Accepted 14 August 1997

GENES, CHROMOSOMES & CANCER 21:166–171 (1998)

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r 1998 Wiley-Liss, Inc.

thyroid. The incidence of breast carcinoma in thesekindreds is about 30% (Starink et al., 1986). Cowdendisease has also been linked to 10q23, with one ofthe two highest LOD scores observed for theD10S215 marker (Nelen et al., 1996). Positionalcloning in this region has recently identified a novelgene (PTEN/MMAC1), which appears to encode aphosphatase (Li et al., 1997; Steck et al., 1997). Inaddition, glioblastomas show frequent loss of chro-mosome 10 (Rasheed et al., 1995). Most losses areextensive, but in a subset of glioblastomas withpartial 10q loss, the D10S215 marker is included inthe region of LOH in most such cases, particularlyif non-informative regions adjacent to LOH areconsidered to be lost (Rasheed et al., 1995). Anumber of other tumors, including endometrialcarcinoma (Peiffer et al., 1995), melanoma (Healyet al., 1996), meningioma (Simon et al., 1996), andsmall cell carcinoma of the lung (Petersen et al.,1997), also show loss of 10q.

The possibility that a TSG on 10q23 is involvedin both sporadic and inherited cancers enhances thepotential value and significance of this gene. Tostudy the role of the locus implicated in Cowdendisease in sporadic breast cancer, we examined 22cases of sporadic breast cancer from our institution(summarized in Table 1) for LOH in the 10q23region, using 10 polymorphic microsatellite mark-

ers. Frozen section analysis was performed to en-sure at least 70% carcinoma in the tissue used forisolation of tumor DNA and the absence of anytumor in the accompanying normal breast tissuesample. DNA was ethanol precipitated from ten 10µm thick sections after proteinase K digestion andphenol extraction (Sambrook et al., 1989). TheDNA surrounding microsatellite CA repeat se-quences was amplified using commercial primers(Research Genetics, Huntsville, AL) and matchedpairs of tumor and normal DNA in the presence of[a32P]-dCTP, electrophoresed on denaturing poly-acrylamide gels, and autoradiographed. The mark-ers were ordered using data from the WhiteheadInstitute Center for Genome Research web site(Hudson et al., 1995) in combination with unpub-lished mapping data. The region spanned by ourset of markers is ,12 cM in length.

Figure 1 summarizes the results for the completeset of markers (with the most centromeric markerlisted on the left). For each matched normal-tumorpair that displayed heterozygosity at a particularlocus, we compared the ratio of band intensities andscored as LOH those pairs showing allelic imbal-ance. Although allelic imbalance could representlow-level gene amplification, we have assumed thatit represents allele loss. Of the 22 cases, 9 (41%)showed allelic imbalance at one or more loci in this

TABLE 1. Clinical Data for Cases Studieda

No. Age (years) Size (cm) Pathological Diagnosis Differentiation Metastases? 10q LOH?

1 64 2.5 Invasive ductal carcinoma Moderate 3/20 L.N. 22 41 0.9 Invasive ductal carcinoma Well 0/19 L.N. 23 60 1.5 Invasive ductal carcinoma Moderate 0/19 L.N. 24 75 0.6 Invasive ductal carcinoma Moderate 0/10 L.N. 25 52 1.6 Colloid carcinoma Well 0/16 L.N. 26 76 3.6 Invasive lobular carcinoma Poor 1/5 L.N. 17 43 1.6 Invasive ductal carcinoma Poor 4/24 L.N. 18 94 2.4 Mucinous carcinoma Well N.D. 29 60 7.4 Invasive ductal carcinoma Poor 0/26 L.N. 1

10 68 2.5 Invasive ductal carcinoma Poor 4/18 L.N. 111 96 10.0 Invasive ductal carcinoma Poor 12/13 L.N. 112 64 2.2 Invasive ductal carcinoma Moderate 4/15 L.N. 213 59 2.7 Invasive ductal carcinoma Poor 4/15 L.N. 214 77 1.1 Mucinous carcinoma Well 0/19 L.N. 215 52 1.6 Invasive ductal carcinoma Moderate 0/26 L.N. 116 38 1.2 Invasive ductal carcinoma Poor 5/19 L.N. 217 83 7.0 Invasive ductal carcinoma Poor 2/12 L.N. 118 71 2.0 Invasive lobular carcinoma Moderate 2/26 L.N. 219 40 1.9 Mucinous carcinoma Well 0/17 L.N. 220 67 3.0 Invasive lobular carcinoma Poor Bone, brain 1b

21 51 1.5 Invasive ductal carcinoma Moderate 6/20 L.N. 122 45 2.0 Invasive ductal carcinoma Moderate 0/12 L.N. 2

aAge indicates age in years at diagnosis. Size is the largest dimension of the tumor in cm. L.N. indicates lymph node status, with the numerator denotingthe positive lymph nodes and the denominator the total number of lymph nodes dissected. N.D. indicates lymph node dissection not done.bLOH restricted to the region centered about the D10S583 marker, telomeric to the PTEN/MMAC1 region.

167LOSS OF HETEROZYGOSITY ON 10q23

region. The overall rate of fractional loss (numberwith LOH/informative cases) was 22% and dis-played a bimodal distribution, with one peak cen-tered around 10S541 and a second, more telomericpeak centered around D10S583. Because a numberof tumors (7, 9, 11, 15) retained markers betweenthe two regions, the most likely interpretation ofthe data is that these two regions of loss representindependent deletions. The distance between thesetwo markers is large (,10 cM), further supportingthe concept that these regions are distinct. Theobservation that six tumors show coordinate loss ofboth regions may indicate a pathogenetic linkagebetween genes in the two regions or might correlatewith a greater degree of general chromosome dele-tion in this group of tumors.

One or more markers are lost from the morecentromeric region in 36% (8/22) of tumors, whichcorrelates closely with the region lost in prostate

cancer and the PTEN/MMAC1 locus. Five cases (7,10, 11, 15, 21) lost more than one marker in themore centromeric region. Figure 2 shows represen-tative autoradiographs of amplified DNAs surround-ing polymorphic sequence markers for these fivecases. Densitometry was performed on the autora-diograms for those markers showing loss in Figure2, and the allelic imbalance ratios were determined.Table 2 lists these values and summarizes thecalculations employed, as previously described(MacGrogan et al., 1994). Retention of both allelesyields a ratio of 1.0. MacGrogan et al. (1994) haveempirically determined that ratios .1.50 (for loss ofthe smaller allele) or ,0.67 (for loss of the largerallele) indicate allelic imbalance. For four of thecases in Figure 2 (7, 10, 11, and 15) allelic imbal-ance ratios met these criteria (Table 2). In one case(21), the ratios for three of the four markers fell justshort of these criteria. However, because four

Figure 1. LOH at microsatellite loci on chromosome 10 in sporadicbreast cancer. The top line indicates the D10S designation for eachpolymorphic marker studied. The most centromeric marker is at theleft. Case numbers correspond to those in Table 1. Allelotyping wasperformed as described in the legend to Figure 2. LOH was determinedby comparison of band numbers and intensities in the normal vs. tumor

samples. Open ovals indicate heterozygosity, cross-hatched ovals indi-cate non-informative (homozygous) loci, and black ovals indicate LOH.The bottom line shows the fractional loss (FL). Samples that could notbe amplified are indicated as NA. Each allelotyping reaction wasdetermined at least twice.

168 SINGH ET AL.

Figure 2. Loss of D10S1687-D10S185 in sporadic breast carcinoma.The sequence surrounding polymorphic CA repeat markers on chromo-some 10 was amplified by polymerase chain reaction (PCR) in thepresence of [a32P]-dCTP and 2.5 mM MgCl2, with annealing at 58°C.

Representative autoradiographs of the PCR products displaying allelicimbalance (N, normal; T, tumor) in this region are shown along withsome adjacent heterozygous loci. The locus analyzed is indicated aboveeach autoradiograph. The lost allele is indicated by an arrow.

169LOSS OF HETEROZYGOSITY ON 10q23

consecutive markers displayed a similar extent ofloss in this case, it is likely that the tumor samplecontained a larger amount of contaminating normaltissue than did the other samples examined, ratherthan indicating that the sample actually retainedheterozygosity at these markers. Two of the fivecases (7 and 10) demonstrated relatively restrictedareas of loss. Comparison of the markers retainedand lost in these two cases suggests that thesegment between D10S215 and D10S541 repre-sents the minimal region of deletion.

The putative TSG in the D10S215-541 regiondeleted in these samples is very likely to be thenewly identified PTEN/MMAC1 gene. This genemaps almost precisely midway between these twopolymorphic markers. Homozygous deletions andsmall mutations in PTEN/MMAC1 were identifiedin cell lines and/or xenografts derived from prostatecancers, glioblastomas, and breast cancers, as wellas in primary breast cancers, renal cell carcinomas,and glioblastomas. The actual frequency of PTEN/MMAC1 gene inactivation in any of these tumortypes is not yet known, but our results suggest that40% or more of human breast cancers may bealtered. This is also in line with the 50% rate ofLOH cited, but not shown, in Li et al. (1997).However, in a recent report, Kerangueven et al.(1997) could not clearly identify a region of in-creased LOH on chromosome 10 in sporadic breastcancers. Thus, mutational analyses will be requiredto confirm the extent of involvement. Recently, it

has been shown that PTEN/MMAC1 is indeedaltered in the germline of at least some Cowdendisease patients, strongly implying that this TSGplays a role in cancer predisposition (Liaw et al.,1997).

Finally, there is a strong correlation of LOH withthe grade of the breast cancers in our sample.Specifically, six of nine poorly differentiated can-cers but only two of eight moderately differentiatedcancers displayed LOH in the D10S215 to D10S541region (Table 1). No allelic imbalance was seen infive well-differentiated tumors. The correlationbetween high tumor grade (poorly differentiatedcancers) and loss at the D10S215-D10S541 locus isstatistically significant [P , 0.02; Fisher’s exact testfor 2 3 2 tables (Huntsberger and Leaverton, 1970)].In this analysis, we compared poorly differentiatedcancers with those graded as either moderately orwell differentiated. Of the 11 cases with lymphnode metastases, 6 showed LOH, as did the onlycase with distant metastases. LOH was seen in bothinvasive ductal and invasive lobular cancers. Thesedata are in agreement with similar observationsmade for the involvement of this region in ad-vanced prostate cancers (Ittmann, 1996a) and glialtumors (Rasheed et al., 1995). PTEN/MMAC1 hassignificant homology to tensin, a protein that inter-acts with actin filaments at focal adhesions, thussuggesting that it may have a regulatory role intumor cell invasion and metastases. If this proposedfunction for PTEN/MMAC1 is substantiated, it mightexplain the observation that loss in the 10q23region is observed in advanced tumors.

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10 D10S608 0.6610 D10S215 0.5011 D10S579 2.1511 D10S541 0.6211 D10S583 0.6615 D10S608 0.4615 D10S541 0.6115 D10S583 0.6215 D10S185 0.5821 D10S608 0.6621 D10S579 0.7121 D10S215 0.7421 D10S541 1.37

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