somatic mutations and human breast cancer. a status report

1582

Somatic Mutations and Human Breast Cancer A Status Report

Robert Callahan, PhD,* Craig S. Cropp, MD,* Giorgio R. Merlo, PhD,* Daniel S. Liscia, M D , t Albert0 P. M . Cappa, M D , t and Rosette Lidereau, PhD$

A systematic study of primay human breast tumor DNA demonstrated that three proto-oncogenes or regions of the genome (c-myc, int-2, and c-erbB2) are frequently amplified and that there is loss of heterozygosity (LOH) on chromosomes lp(37%), lq(20%), 3p(30%), 7(41%), llp(ZO%), 13q(30%), 17p(49%), 17q(29%), and 18q(34%). Specific subsets of tumors can be defined based on the particular collection of mutations they contain. For instance, LOH on chromosomes Ilp, 17p, and 18q frequently occurs in the same tumor. A search for putative tumor suppressor genes within the regions of the genome affected by LOH has been started. In a comprehensive molecular analysis of the p53 gene on chromosome 17p, 46% of the tumors contained a point mutation in the p53 gene. Cancer 1992; 691582-1588.

An important characteristic of human breast cancer is its etiologic heterogeneity due to factors in the patient history and physiologic status.'-3 It seems likely that some of these factors, such as menstrual status, repro- ductive history, family history, long-term treatment with estrogens, diet, and previous atypical benign breast disease, provide a selective environment for the clonal outgrowth of cells which contain somatic mutations. One biological consequence of these mutations may be the development of neoplasia through the un- coupling of normal mammary gland development. Other consequences could be the provision of atypical nonmalignant as well as tumor cells with a selective

Presented at the American Cancer Society and American Joint Committee on Cancer Workshop on Molecular Markers in the Classi- fication and Staging of Cancer, Atlanta, Georgia, December 13-14, 1990.

From the *National Cancer Institute, Bethesda, MD; tAnatomic Pathology Section, S. Giovanni Hospital, Torino, Italy; and the $Centre Rene' Huguenin, St. Cloud, France.

Address for reprints: Robert Callahan, PhD, National Cancer Institute, Bethesda, MD 20892.

Accepted for publication September 10, 1991.

growth advantage, the means to metastasize to distant organ sites, or evasion of host immunosurveillance. Be- cause of this heterogeneity and the multiple events nec- essary for most epithelial tumors, it seems likely that multiple genetic mutations act in concert to produce an invasive breast carcinoma with the ability to metastasize to distant organ sites.

Consistent with this view, cytogenetic analysis of primary human breast tumor cells in culture has demonstrated frequent somatic alterations. These include an- euploidy (either gain or loss of entire chromosomes) and rearrangements affecting chromosomes l q (translocations), 6q (deletions and translocations), 7p (translocations, pericentric inversions, and isochromosomes), and 1 l q trans location^).^ More recently, we and other laboratories have begun to survey the human genome in primary breast tumors at a molecular level to identify frequent somatic mutations of specific genes or alterations of specific regions of the cellular g e n ~ m e . ~ , ~ One goal of these studies has been to determine whether specific mutations are linked to clinical parameters in the patients history, including disease outcome, or whether the gene products can be used as targets for possible therapeutic intervention. In this report we sum- marize the results of our analysis, and discuss their potential implication in understanding the biology of the disease and its clinical management.

Our studies have focused on comparing the DNA of 280 tumors with normal lymphocyte DNA from the same patients. The specimens were collected from two different hospitals and were composed primarily of invasive ductal carcinomas. Currently we have exten- sively surveyed 14 of the 41 nonacrocentric chromosome arms in these tumor DNA samples with recombinant DNA probes which detect either proto-oncogenes or restriction fragment length polymorphisms (RFLP) of anonymous cellular loci. Eleven frequently occurring somatic mutations have been identified as discussed below (Table 1).

Somatic Mutations and Breast Cancer/Callahan et al. 1583

Table 1. Associations Between Genetic Alterations and Clinical Parameters in Patients With Breast Cancer

Frequency Proto-oncogene (YO) Clinical parameters P value Reference no.

c-myc 32 Patient age > 50 yr < 0.02 Escot et a!.' c-erbB2 10 None Ali et al.I7 int-2 16 Local recurrence or distal metastasis < 0.002 Lidereau et al." lp32-pter 26 Decreased survival after relapse < 0.011 Bieche ef a/.34 lq22-q32 20 None Merlo et a/.35 3p21-p25 30 ER-negative tumors Hitopath Grade I11 < 0.019 Ali et ~ 1 . ~ ~

11~11 .2 -~15 .5 20 ER/PR-negative tumors histopath. Grade I11 < 0.002 Ali et a1.,37 Theillet et a1.38

13q14.1(RB-1) 30 Histopath Grade I11 < 0.031 Merlo and Callahad9 1 7 ~ 1 3 47 High proliferative index < 0.022 Merlo et 17q 32 ER-negative tumors < 0.020 Leone et d.'* 18q21-qter 34 Histopath Grade I11 < 0.040 Cropp et al."

ER: estrogen receptor; PR: progesterone receptor; Histopath: histopathologic.

< 0.0001

< 0.006

Amplification of Regions of the Cellular Genome

Amplification of specific regions of the cellular genome is a dominant mutation which is associated with the activation or over-expression of the target gene located within the amplification unit.7 In our study amplification of three regions of the human genome were detected. One region on chromosome 8q, containing the c-myc proto-oncogene, was amplified in 32% of the breast tumors.' This gene is related to the retroviral v- myc on~ogene.~ Frozen sections of primary human breast tumors containing amplification of c-myc were shown by in situ RNA:RNA hybridization to contain high levels of c-myc RNA." In these tumors only the tumor cells contained c-myc RNA. The second affected chromosomal region, located on chromosome 1 lq13, was found to be amplified in 16% of the tumors."*" This region contains the int-2 and hst/K-FGF proto-oncogenes which are members of the fibroblast growth factor (FGF) gene In mouse mammary tumors the mouse mammary tumor virus (MMTV) frequently activates these genes by insertion mutagene- sis.15 Again using in situ RNA:RNA hybridization, we were able to detect int-2 RNA, but not hst/K-FGF RNA, in frozen sections from tumors containing an amplification of these genes.16 This result suggests that int-2 amplification has been selected in these samples during tumor development. A third region on chromosome 17q, containing the c-erbB2 proto-oncogene, was amplified in 10% of the turn or^.'^ The c-erbB2 proto-oncogene is a member of the epidermal growth factor receptor gene family.'8-20 In the rat homologue (neu oncogene), point mutations altering the transmembrane domain of the protein confer on it the capability to ma- lignantly transform tissue culture cells. Although, we have not determined whether amplification of c-erbB2

is associated with elevated levels of the gene product, several other laboratories have found such an association."

The association between the amplification of specific genes and RNA or protein expression, however, is not always p e r f e ~ t . ~ For instance, over-expression of c-myc and c-erbB2 in primary breast tumors has been observed in the absence of gene amplification. In the case of c-myc over-expression, this may in part reflect heavy lymphoplasma-cellular infiltration of the tumor in which the lymphocytes, but not the tumor cells, express c-myc RNA." Among other explanations is the possibility that contaminating normal tissue in the biopsy material has masked the presence of gene amplification in the tumor cells. The association between amplification of sequences on chromosome l l q and int-2 RNA expression is also controversial. For instance, one study found that both genes were expressed when they were amplified in breast tumors." In another study, the expression of neither gene was detected in tumors in which they were am~lified.'~ Instead, a closely linked anonymous gene was coamplified and e~pressed.'~

In vivo models to study how activation of these genes contributes to breast cancer have been devel- oped. Transgenic mouse strains containing as transgenes the c-myc proto-oncogene, the rat neu oncogene, or the in t-2 proto-oncogene showed either altered mammary gland development or a high incidence of spontaneous mammary tumors. For instance, transgenic mouse strains containing the int-2 transgene develop multifocal preneoplastic hyperplasia of the mammary gland which can give rise to focal mammary tu- m o r ~ . ~ ~ Other transgenic mouse strains containing c-myc or the neu transgenes have a high incidence of focal mammary tumor^.^^-^^ However, with the excep-

1584 CANCER Supplement March 15,2992, Volume 69, NO. 6

tion of one unique strain of neu transgenic mice,27 which expresses high levels of the oncoprotein in the mammary gland and develops multifocal mammary tumors, none of these activated transgenes appears to be alone sufficient to induce mammary neoplasia. In this setting, it seems that additional mutations are required for mammary carcinoma development in mice.

Loss of Heterozygosity

In primary human breast tumors loss of heterozygosity (LOH) represents the most frequent type of mutation. LOH appears to occur as a consequence of either inter- stitial deletions, chromosome loss, or aberrant mitotic recombination events and is a common feature of many kinds of malignan~ies.~',~~ It is thought to mask the presence of a "tumor suppressor" gene(s) located within the corresponding affected region on the homologous c h r o m o s ~ m e . ~ ~ , ~ ~ The retinoblastoma gene (RB-1) is re- garded as the paradigm for this type of cellular gene. Inactivation of the RB-1 gene may occur as a consequence of two independent mutation events. Com- monly, one normal allele is lost as a result of LOH, whereas the other allele contains either a small deletion or a point mutation which inactivates the gene product. In our panel of breast tumor DNA, LOH was frequently detected on chromosomes lp,34 lq,35 3 ~ , 3 ~ 1 1p,37*38 13q,39 17p, 17q, and 18q (Table l).40,41

Sat0 et aL4' have "allelotyped" a panel of primary breast carcinoma DNA by examining one RFLP per au- tosomal chromosome arm for LOH. They found that the most frequently affected chromosomes were 13q (21%), 16q (38%), and 17p (52%). The frequency of LOH on the other chromosome arms ranged from 0% to 19%. The low frequency of LOH on some chromosome arms could be interpreted as evidence for nonspecific or "background" genetic alterations sustained during tumor progression. Their findings were similar to other studies on colorectal and renal cell carcinomas, in that most chromosome arms were affected to a varying extent by LOH.43,44 However, the reported frequencies of LOH on these chromosome arms could have been significantly underestimated in these studies since only a single RFLP was tested per chromosome arm. For instance, the apparent frequency of LOH for a particular chromosome arm could be reduced if the target tumor- suppressor gene is located at some distance from the site of the RFLP. For this reason our strategy has been to examine multiple RFLP which are located at regular intervals along each chromosome arm.

Although our study is incomplete, we have consis- tently found that the frequency of LOH varies significantly and in an ordered manner between individual RFLP on the same chromosome arm. This type of pat-

tern of LOH is consistent with the presence of a tumor- suppressor gene near the locus having the highest frequency of LOH. Cellular heterogeneity of the tumor is another factor which could affect the apparent frequency of mutations. The heterogeneity could be a consequence of contaminating normal tissue in the biopsy material or heterogeneity of tumor cells containing the particular mutation. Currently, therefore, it is probably premature to make any definitive conclusions on whether the large number of regions of the genome which are affected by LOH are a consequence of the nonspecific accumulation of mutations during tumor progre~sion,~~ or reflect a progressive selection of mutations which contribute to tumor progression.

Although more extensive and systematic studies will be required to distinguish between these possibilities, several observations suggest to us that the com- plexity of mutations contributing to the evolution of human breast carcinoma will be high. For instance, if the high frequency of LOH at different regions of the cellular genome was a consequence rather than a contributing factor in breast tumor development, the mutations might be expected to occur independent of one another. In fact we have found 11 pairs of mutations which occur together at a statistically significant fre- q~ency .~ ' On this basis two subsets of tumors within our tumor panel can be distinguished: one of these frequently contains LOH on chromosomes l l p , 17p and 18q; whereas in the other subset chromosomes lp, 13q, and 18q are affected by LOH. Similar findings have been reported in other studies of primary breast, colon, and lung carcinoma^.^^*^^-^^ In addition, the activation or inactivation of eight cellular genes by somatic mutations has been found in mouse mammary t ~ m o r s . ' ~ , ~ ' - ~ ~ Taken together these findings suggest that, depending on the history and physiologic status of the patient, groups of collaborating mutations may be selected during tumor development.

Currently, only two of the probable target tumor- suppressor genes affected by LOH in primary breast tumors have been identified. They both are believed to be involved in the normal suppression of cellular proliferation during development. The target for LOH on chromosome 13q appears to be the RB- 1 gene.53-55 This gene encodes a nuclear phosphoprotein which has the capability of binding to DNA.56 Although the level of the protein is constant throughout the cell cycle, the extent of its phosphorylation fluctuate^.^^-^' It is thought that the dephosphorylated form is normally associated with the suppression of cellular proliferation. On chromosome 17p, the p53 gene appears to represent another target tumor-suppressor gene for LOH in primary breast tumors.

Our group41 and others62-66 have found that 10% to


46% of primary breast tumors contain point mutations in the p53 gene. In our study three of 28 tumors contained LOH spanning the p53 gene as well as a point mutation in the remaining p53 allele, as determined by sequencing tumor-derived p53 cDNA clones. More- over, in other studies a germline mutation of the p53 gene has been shown to be linked to the Li-Fraumeni hereditary cancer syndrome,67 in which breast cancer is a major component. The p53 gene also encodes a DNA- binding nuclear pho~phoprotein,~',~~ which is capable of activating the transcription of a reporter gene.70*7' Unlike the RB protein, the levels of the p53 protein do fluctuate during the cell c y ~ l e . ~ * , ~ ~ Some mutant p53 genes have been shown to have transforming activity in ~ i t r o . ~ ~ Two consequences of these mutations are an increase in the half-life of the protein and a loss their ability to stimulate transcription of a reporter gene.71*75 Currently, however, whether the RB and p53 proteins have a direct or indirect involvement in the regulation of the cell cycle or differentiation remains to be determined.

Other cellular genes which may represent targets for LOH in primary breast tumors include the nm23- H176 and deleted in colon carcinoma (DCC)77 genes on chromosomes 17q and 18q, respectively. The nm23-H1 gene is a candidate metastasis suppressor gene, whose expression is down-regulated in highly metastatic tumor ~ e l l s . ~ ~ , ~ ' , ' ~ This gene encodes a 17-kd protein that shares homology with nucleoside diphosphate (NDP) kinases.80,s1 It has been suggested that the nm23-H1 protein may regulate certain G-protein mediated events. LOH at nm23-Hl was detected 64% of the tumor DNA in our tumor panels2 Studies are underway to determine whether loss of expression of the nm23-H1 gene is linked to LOH at this locus. The DCC gene encodes a protein that is structurally related to several neural cell adhesion molecules and cell surface glyco- proteins.77 This gene is expressed in most if not all normal tissues, yet in human colon carcinomas where the gene is frequently affected by LOH, its expression is decreased or absent. A loss of this gene product could contribute to malignancy by altering normal cell-cell or cell-basement membrane interactions. Currently, however, a correlation between LOH at the DCC gene and a decrease or absence of its expression has not been demonstrated in primary human breast tumors. It may also be pertinent that the hereditary cancer syndromes which are associated with a high incidence of either breast and ovarian or only breast carcinomas are linked to chromosomes lps3 and 1 7q,84 respectively. Whether the genes affected by these hereditary mutations are also the targets genes for LOH in sporadic breast cancer remains to be determined.

Potential Usefulness of Mutations or Their Gene Products as Indicators of Disease Outcome

Serious efforts have been made by several laboratories to determine whether specific mutations are associated with particular aspects of the patients history, characteristics of the tumor, or disease o ~ t c o m e . ~ , ~ * ' ~ Particular emphasis has been placed on disease outcome in axillary node-negative patients, since there are few individual breast tumor markers that adequately discriminate patients at high risk for relapse.86 The associations found in our own studies are summarized in Table 1. The only mutation which was associated ( P < 0.002) with local recurrence or distal metastasis was amplification of the int-2 gene. Borg et dS7 have recently reported a similar association between int-2 amplification and poor prognosis in breast cancer patients. In addition, we have found that LOH on chromosome l p is associated ( P < 0.011) with shortened survival for patients after relapse.34 Six other mutations (LOH on chromosomes 3p, l lp , 13q, 17p, 17q, and 18q) were associated with the more aggressive tumors that were histopathologic Grade 3, estrogen receptor negative, or had a high proliferative index.

In general, however, the potential clinical usefulness of the mutations or their gene products as indicators of probable disease outcome is in q u e s t i ~ n . ~ , ~ * ~ ~ There are several underlying factors in the methodol- ogy and analysis of the results used by different laboratories which have contributed to this problem. Currently there is no standardization of reagents or methodologies for detecting mutations or expression of the gene products between laboratories. In addition the care taken to remove normal tissue from the biopsy material before the extraction of DNA, RNA, or protein varies between laboratories. Due to this factor the frequency of particular mutations may be significantly under estimated in some studies. The composition of the various tumor panels with respect to associated clinical parameters and individual characteristics of the tumors can also vary significantly between laboratories. A related variable between different retrospective studies is the treatment the patients received after surgery. In some studies they all received the same treatment, whereas in others it was varied and uncontrolled.

Another problem is the preoccupation with P values in the statistical analysis of the results. In some studies this has led to an over-interpretation of the significance of the data. The P values depend in part on sam- ple size and do not measure the power of an association. Thus, tumor panels which are small in size are restricted in the ability to detect anything but a large effect. Alternatively, a relatively small association with little clinical importance could have an impressive P

1586 CANCER Supplement March 25, 2992, Volume 69, No. 6

value in a sufficiently large panel of tumors. We, therefore, view our studies as well as those of others as pilot studies directed at hypothesis development rather than their confirmation.

Future Possibilities

Clearly this field holds promise for the development of new prognostic indicators that could be useful in the management of the disease. However, before this goal can be reached, a standardization of the reagents and methodologies as well as the development of compara- ble tumor panels must occur. A second area which holds promise for the future is the development of therapeutic approaches based on the over-expressed gene products of mutated cellular genes or the gene products of mutated cellular genes or the gene products of distant cellular genes expressed as a consequence of a particular mutation. For instance, it seems likely that several laboratories will begin therapeutic clinical trials in the near future using monoclonal antibodies prepared against the c-erbB2 protein as a vehicle for radionu- clides or toxins. In the near future it may be possible to develop recombinant vaccinia vectors containing one of the activated genes (i.e., int-2) whose gene product is displayed on the tumor cell surface but not on the surface of normal cells.88 An alternative approach is suggested by the achievement of antiviral protection in vivo by peptide-induced cytotoxic T - ~ e l l s . ~ ~ , ~ ~ A similar approach using a peptide corresponding to a mutated H-rus p21 protein was able to stimulate CD4-positive T-cells to specifically recognize cells expressing the mutated protein sequence.” Thus, it may be possible to develop a therapeutic approach in which class I or class I1 major histocompatibility molecule restricted cytotoxic T-lymphocytes are stimulated with peptides corresponding to the mutated portion of the p53 protein or other potential point mutated tumor-associated and tumor-suppresor gene products.

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