estrogen receptor mutations in tamoxifen-resistant breast cancer 1

[CANCER RESEARCH 54, 349-353, January I5, I994]

Advances in Brief

Estrogen Receptor Mutations in Tamoxifen-resistant Breast Cancer 1

P r a t i m a S. K a r n i k , 2 S u c h e t a K u l k a r n i , X i a o - P u L i u , G. T h o m a s B u d d , a n d R o n a l d M . B u k o w s k i

The Experimental Therapeutics Program, Cancer Center, Cleveland Clinic Research Foundation, Cleveland, Ohio 44195-5069

Abstract

Clinical resistance to antiestrogens like tamoxifen is a major problem in the treatment of hormone-dependent breast cancers. Since the estrogen receptor plays a central role in mediating the effects of estrogens and antiestrogens, we hypothesized that mutations in the estrogen receptor could be one mechanism by which breast tumors evolve from a hormone- dependent to a hormone-independent phenotype. The eight exons of the estrogen receptor complementary DNA from 20 tamoxifen-resistant and 20 tamoxifen-sensitive tumors were screened by Single Strand Conforma- tion Polymorphism (SSCP), and the variant conformers were sequenced to identify the nucleotide changes. A 42-base pair replacement was found in exon 6 of a tamoxifen-resistant tumor. A single base pair deletion in exon 6 of a tamoxifen-resistant metastatic tumor but not in the primary tumor was detected in another case. If translated, both these mutations could generate truncated receptors with an intact DNA-binding domain and a defective hormone-binding domain that could constitutively activate transcription of previously estrogen-responsive genes. The remaining 18 of 20 tamoxifen-resistant tumors did not contain mutations in any of the 8 exons of the estrogen receptor complementary DNA. These results suggest that mutations in the estrogen receptor occur at a low frequency and do not account for most estrogen-independent, tamoxifen-resistant breast tumors.

Introduct ion

Breast cancer is a major source of morbidity and mortality among women in North America and Europe. The recognition that many breast cancers are dependent on estrogen for growth and progression has been the basis for the continuing interest in the role of ER 3 in the prognosis of this devastating disease (1). A characteristic feature of breast cancer is that the disease evolves into an estrogen-independent growth phenotype, a change that often marks the beginning of a more aggressive growth phase that is nonresponsive to antiestrogen therapy (2). Resistance to antiestrogens like tamoxifen is therefore a major problem in the treatment of hormone-dependent breast cancers. About 30--40% of ER-positive tumors fail, from the outset, to respond to antiestrogen therapy and are considered to be estrogen-independent and tamoxifen-resistant (2). Moreover, the majority of ER + tumors that initially respond to antiestrogen therapy will eventually develop resistance to this treatment without necessarily altering their ER pro- file (2).

The ER is a member of the steroid and thyroid hormone receptor super family that function as ligand-dependent transcription factors. These receptors contain an NH2-terminal domain that affects transcription efficiency, a central DNA-binding domain that binds to the target gene hormone response element and thereby determines target

Received 10/21/93; accepted 11/24/93. 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 Supported by a pilot program of the American Cancer Society. 2 To whom requests for reprints should be addressed, at The Experimental Therapeutics

Program, Cancer Center, Cleveland Clinic Research Foundation, 9500 Euclid Ave., Cleve- land, OH 44195; hy5069.

3 The abbreviations used are: ER, estrogen receptor; PR, progesterone receptor; HBD, hormone binding domain; cDNA, complementary DNA; PCR, polymerase chain reaction; SSCP, single-strand conformation polymorphism.

gene specificity, and a COOH-terminal HBD (3). Studies on the role of ER in breast cancer have focused on measurements of ER by ligand binding assays or immunohistochemistry (4). It is clear from in vitro

mutagenesis that ligand binding and immunoreactivity are not ad- equate indicators of a functional ER since deletion of all or part of the DNA binding domain renders the ER transcriptionally inactive, de- spite its ability to bind estrogen with high affinity. It is remarkable that the mutant ERs that lack the HBD can constitutively activate transcription even in the absence of hormone (3). Indeed, androgen (5, 6), vitamin D 3 (7), and glucocorticoid (8) resistance syndromes are all associated with the inheritance or acquisition of mutant forms of cognate receptors. In contrast, hereditary estrogen resistance syndromes are rare and could prove to be lethal to the organism (9). However, acquired estrogen resistance as seen in human breast tumors could arise due to mutant ERs. Several ER variants with different functional capabilities (9-11) and putative RNA splicing variants of ER (9, 10, 12) are described in breast cancer cell lines and breast tumors. These mutant ERs might confer either dominant positive or dominant negative effects on cancer cells, thereby generating the hormone-independent phenotype of the breast tumors. Intrigued by the possibility that tamoxifen-resistant human breast cancer might contain ER mutations that alter ER function or inactivate it and thereby affect the cancer cell phenotype and its response to antiestrogens, we initiated a study to screen for mutations in the ER cDNA of tamoxifen-resistant and -sensitive breast cancer tissue specimens. The results show that although mutations in ER do occur in tamoxifen- resistant tumors, they are quite rare and do not account for the majority of tamoxifen-resistant breast cancers.

Materials and Methods

Human Breast Tumor Tissue and Clinical Data

Breast tumor tissue was obtained from the tissue procurement core, Depart- ment of Pathology, Cleveland Clinic Research Foundation and characterized by clinical and histopathological features. All the tumors were radioimmuno- logically determined to be estrogen receptor-positive and progesterone receptor positive/negative and were from postmenopausal women with Stages II, III, and IV breast cancer. The classification of tumors based on their response to tamoxifen was as follows:

Group 1: Paired Primary and Metastatic Tumors from Patients Receiv- ingAdjuvant Tamoxifen. These patients received adjuvant tamoxifen therapy after initial surgery and relapsed with metastatic disease in their ipsilateral axillary lymph nodes or in the supraclavicular lymph nodes while still receiving tamoxifen. We have analyzed 5 tumor pairs belonging to this group (10 tumors).

Group 2: Tumor Tissue from Patients with Metastatic Disease (Stage III or IV) Receiving Tamoxifen. The patient's clinical response status is described below.

Tamoxifen Resistant. Patients with Progressive Disease while Receiving Tamoxifen. These tumors were biopsied just prior to initiation or during therapy. We have analyzed 15 tumors belonging to this category.

Tamoxifen Sensitive. Tumors biopsied before initiation of tamoxifen therapy from patients who had an objective response or prolonged disease stability (>4 months) with tamoxifen. We have analyzed 15 tumors belonging to this category.

349

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ER M U T A T I O N S IN TAMOXIFEN-RESISTANT BREAST CANCER

Reverse Transcription-PCR Amplification

Total cellular RNA was extracted from cryostat sections of breast tumors (snap-frozen in liquid N2 and stored frozen at -80~ with RNAzol (Biotexc Laboratory, Houston, TX) according to the recommendations of the supplier. RNA (500 ng) was reverse transcribed at 42~ for 1 h following the instruc- tions of the Gene AmpR RNA PCR system (Perkin Elmer Cetus, Norwalk, CT). First strand cDNA was directly used for 30 cycles of PCR amplification (13) of the 8 ER exons using Gene AmpR PCR reagent kit (Perkin Elmer Cetus). A pair of primers was synthesized (Fig. 1) for each ER exon based on the cDNA sequence (14). To ensure cDNA and not genomic DNA amplification, the primer pairs for each exon were designed on adjacent exons. PCR was carried out in a Thermocycler (Ericomp, Inc., San Diego, CA) in a final volume of 100/~1 containing 250 mM per liter each of dATP, dGTP, and TIP; 20 mM per liter of dCTP; 10/zCi [3ap]dCTP; 50 pmol of each PCR primer; 2.5 units of Taq polymerase; and 10/xl of Taq polymerase buffer. PCR cycles included denaturation for 1 min at 94~ annealing for 1 min at 55~ and polymeriza- tion for 3 min at 72~ followed by one cycle of extension reaction at 72~ for 15 min. We ruled out the possibility of misincorporation errors due to Taq polymerase by performing a minimum of three independent PCR reactions for each exon. Single Strand Conformation Polymorphism (SSCP) (15, 16) of the PCR products was performed to detect possible deletions, insertions, or point mutations.

SSCP

Following PCR, a 5-/xl aliquot of the amplified product was diluted with 45 txl of 0.1% sodium dodecyl sulfate-10 mM EDTA; 2 /xl of this mixture was added to 2/J.1 of Sequenase stop mix (95% formamide-20 mr~ EDTA- 0.05% bromophenol blue- 0.05% xylene cyanol; United States Biochemical Corpo- ration, Cleveland, OH). The cDNA samples were denatured at 95~ for 3 min and immediately placed on ice before being run on 6% polyacrylamide non- denaturing gels with or without 10% glycerol. Electrophoresis was performed in 0.09 M Tris base-0.09 M boric acid-2.5 mM EDTA running buffer either at 40 W at room temperature for 4-6 h, or at 40 W at 4~ for 4--6 h, or at 5 W at room temperature for 16 h. The gels were dried and exposed to XAR-5 film (Kodak) for 24-48 h at room temperature. The plasmid HEGO (cloned ER cDNA; gift from Dr. Geoffrey Greene) served as control on SSCP gels.

DNA Sequencing

The variant SSCP bands were cut out, extracted with 100/xl distilled water for 15 min at room temperature, and reamplified as described above. The PCR products were purified on Magic DNA purification columns and sequenced using the fmol DNA sequencing system from Promega (Madison, WI), according to the manufacturer's protocol. Both the sense and antisense strands were sequenced.

Results

A 42-base Pair Replacement in ER Exon 6 of a Metastatic Breast Tumor. All eight exons of the ER eDNA could be amplified from the total RNA of the 40 breast tumors (described in "Materials

and Methods") with the primers described in Fig. 1. Deletions of entire exons were not observed in any of the 40 tumors examined in this study. PCR amplification of exon 6 from human breast cancer tissue specimens using primers ER 16 (5'-CTGTITGCTCCTAACT- TGCT-3') and ER 17 (5'-GGTGCTGGACAGAAATGTGT-3') re- sulted in the expected fragment length of 191 base pairs in all of the tumors (data not shown). However, PCR-SSCP analysis (Fig. 24) demonstrated variant conformers (indicated by arrows) in the tamoxifen-resistant tumor 17 compared to the wild-type pattern (cloned ER cDNA). The presence of more than one allele in the same specimefi suggested that the tumor contained a heterozygous somatic mutation. Sequence analysis of the variant conformer (Fig. 2B) indicated that 47 nucleotides (1271-1318) of exon 6 were substituted or replaced by 42 nucleotides (1148-1190) of exon 5 (Fig. 2B). Since the 42-nucleotide exon 5 fragment was flanked on either side by exon 6 sequences and the PCR fragment was flanked by the appropriate primers, we ruled out the possibility of this being a PCR artifact. Several independent RNA-PCR and SSCP analyses confirmed the presence of this mutation in tumor 17. As shown in Fig. 2C this 42-base pair substitution generates a 5-base pair deletion, resulting in a frame shift that generates an ER with a translation termination at codon 454 in exon 6. Since all of the other ER exons in this tumor are wild type, on translation this mutation could give rise to a truncated protein with an intact DNA-binding domain and a defective HBD that would be unable to bind hormone. Such mutant proteins could constitutively activate ER-responsive genes and induce ER-independent growth of breast tumors. Tumor 17 ( E R + P R - ) was indeed from a patient who rapidly progressed on tamoxifen and was resistant to the drug from the outset inspite of high ER values (ER value of 500 as determined by

radioimmunoassay). Single Nucleotide Deletion in the Metastatic but not in the

Pr imary Tumor from the Same Patient. SSCP analysis also showed a variant conformer in addition to the wild-type conformer (Fig. 3A) in exon 6 of the metastatic tumor 10. Interestingly, the primary tumor 9 from the same patient contained only the wild-type conformer under two different SSCP conditions (Fig. 3, A1 and A2). Sequence analysis of the variant band (Fig. 3B) revealed a single nucleotide (T) deletion at codon Ser 432 of exon 6 (Fig. 3B). This generates a frame shift and a translation termination codon at 437. Sequencing of the opposite DNA strand confirmed these results. The sequence of remaining exons was identical to the wild-type sequence, indicating that exon 6 of tumor 10 differed from the wild-type sequence by only a single base deletion. Sequence analysis of exon 6 in the primary tumor showed the wild-type sequence with Ser 432. The observation that this patient relapsed while on adjuvant tamoxifen therapy suggests that either the cancer cells acquired the mutation during tumor progression or that tamoxifen therapy selects for breast cancer cells that express mutant

forms of estrogen receptors.

3NI "IORMONE

cDNA

A/B I C - - I -D I E

e9 ~1'

+ ed ERI El:I4 E I~

! . - , ~ r , . i e .

ER8 ~~ o~ ER I2 ER I4 " - ER I6 " - ER I8 " - ER29 " -

IFt- 1 ! ! I !

, , 1 2 1 3 j 4 8 , ER3 ER$ El:iT ER9 ERI ~, ER 13, ERI ~ ER! 7 ERI9 EFI2

ATG TGA Fig. 1. Structure of ER cDNA showing the functional domains the eight exons, and PCR primers (ER1-ER20) for each exon.

350

r3 (o



ER MUTATIONS IN TAMOX1FEN-RESISTANT BREAST CANCER

C Nucleotide and deduced amino acid sequence of Exon-6

Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys Cys Val CTG TTT ~CT CCT AAC TTG CTC TTG GAC AGG AAC CAG GGA AAA TGT GTA CTG TTT GCT CCT AAC TTG CTC TTG GAC AGG AAC CAG GGA AAA TGT GTA Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys Cys Val

Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser Set Arg GAG GGC ATG GTG GAG ATC TTC GAC ATG CTG CTG GCT ACA TCA TCT CGG GAG GGC ATG GTG GAG AGG CTA GAG ATC CTG ATG ATT GGT CTC GTC TGG Glu Gly Met Val Glu Arg Leu Glu Ile Leu Met Ile Gly Leu Val Trp

Phe Arg Met Met Asn Leu Gln Gly GIu Glu Phe Val Cys Leu Lys Ser TTC CGC ATG ATG AAT CTG CAG GGA GAG GAG TTT GTG TGC CTC AAA TCT CGC TCC ATG GAT G CAG GGA GAG GAG TTT GTG TGC CTC AAA TCT Arg Ser Met Asp A laG lyA rgG lyV alC ysV alp toG InI leT

Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Set Ser Thr Leu ATT ATT TTG CTT AAT TCT GGA GTG TAC ACA TTT CTG TCC AGC ACC CTG AT TAT TTT GCT TAATTC TGG AGT GTAC ACA TTT CTG TCC AGC ACC CTG yr Tyr Phe Ala Stop

Fig. 2. A. PCR-SSCP analysis of ER exon 6 of wild-type and from tumors 17-34. The PCR-SSCP analysis and the sequence of the primer pairs ER16 and ER17 are described in "Materials and Methods"; SSCP analysis was performed at 4~ for 4 h. The tamoxifen-resistant tumor 17 shows wild-type and variant SSCP conformers (variant conformers indicated by arrows), whereas tumors 18-34 show a wild-type SSCP pattern. SS and DS, single-stranded and double-stranded DNA, respectively. B. Sequence analysis of ER exon 6 in the tamoxifen-resistant tumor 17 showed that 47 nucleotides of exon 6 were replaced by 42 nucleotides of exon 5 sequences. The primer ER 16, a part of the exon 6 sequence, and 14 replaced nucleotides of exon 5 are shown. C. The complete nucleotide and deduced amino acid sequence of exon 6 in tumor 17 and in wild-type ER eDNA is shown with the sequences of the primers ER16 and ER17 (underlined). The 47 nucleotides of exon 6 when replaced by 42 nucleotides of exon 5 results in a 5 base pair deletion that generates a frame shift and translation termination codon at 454 of exon 6 in the tamoxifen-resistant tumor 17.

Not All ER Mutations Are Associated with the Resistant Phe- notype and Not All Tamoxifen-resistant Tumors Contain ER Mu- tations. Using the same techniques, we have also observed a substi-

tution in ER exon 4 of a tamoxifen-sensi t ive tumor ( tumor 2, f rom a patient with Stage II disease). This substitution alters the amino acid

Glu 352-->Val, a drastic change f rom acidic to basic amino acid (data

not shown). However , this patient responded to adjuvant tamoxifen

therapy and remains disease free. This suggests that not all ER mu-

tations are associated with a tamoxifen-resistant phenotype. Another

such example is the mutat ion Gly 400--~ Val in the ER e D N A isolated

f rom the cell line MCF-7. This mutation only reduces the affinity of

the receptor for estrogen at 37~ but not at 4~ (17). We have also

detected silent mutat ions in the tamoxifen-resistant tumor 6 (exon 2,

Gly 276), in the tamoxifen-resistant tumor 28 (exon 6, Ala 505), in the

tamoxifen-sensi t ive tumor 3 (exon 5, Lys 472) and in the tamoxifen-

sensitive tumor 35 (exon 7, His 577; Table 1).

Surprisingly however, the remaining 18 of 20 E R + , tamoxifen-

resistant tumors that we analyzed did not contain mutat ions in any o f

the eight exons o f the ER e D N A but contain wild-type estrogen

receptors at levels comparable to tamoxifen-sensi t ive tumors. Our

results show that mutat ions in ER do occur in tamoxifen-resistant

tumors; however , they are quite rare and thus do not account for the

majori ty of tamoxifen-resistant breast tumors. Other mechanisms

must therefore be responsible for the tamoxifen-resistant and hor-

mone- independent phenotype seen in these tumors.

Discussion

Estrogen receptor mutat ions have been reported in several breast

cancer cell lines (9) and breast tumors (10-12 and references therein),

al though their correlation with clinical history has not been deter-

mined. Our study of 20 tamoxifen-sensi t ive and 20 tamoxifen-resis-

351



ER MUTATIONS IN TAMOXIFEN-RESISTANT BREAST CANCER

Fig. 3. A. PCR-SSCP analysis of ER exon 6 in tumor 9 (primary) and tumor 10 (tamoxifen-resistant, metastatic tumor) from the same patient. Tu- mor 11 and wild-type (cloned ER cDNA) served as negative controls. The SSCP gels were run at 5 W at room temperature for 16 h and 40 W at 4~ for 4 h. Tumor 10 shows variant SSCP conformers that are indicated by arrows, whereas the primary tumors 9 and 11 show a wild-type SSCP mobility pattern. SS and DS, single-stranded and double- stranded DNA, respectively. B. Sequence analysis of ER exon 6 coding part of HBD in tumors 9 and 10. Sequence analysis was performed as described in "Materials and Methods." A single base pair deletion of T in the codon Ser 432 generates a frame shift and a translation termination codon at 437 in the metastatic tumor 10, whereas the primary tumor 9 from the same patient shows a wild- type sequence.

rant tumors, however, clearly indicates that the frequency of ER mutations is low and does not account for the large number (18 of 20 tamoxifen-resistant tumors analyzed in this study do not contain mutations) of ER-positive tumors that are resistant from the outset or those that eventually develop tamoxifen resistance. A similar trend in

the frequency of mutations of the androgen receptor was observed by Newmarket al. (19) in prostate cancer where they detected a mutation in only 1 of 26 stage B prostate cancer patients. These investigators suggested that the low frequency could be due to early stage disease of the tumors (Stage B) that they analyzed and that if mutations reflect

Table 1 ER mutations in breast tumors

Description of tumor Mutations in the ER cDNA

Response Tumor ER/PR status to tamoxifen Nucleotide change Codon change Consequence

2 ER+/PR + Sensitive Substn a of G A G ~ G T G 3 ER+/PR + Sensitive Substn of AAG--~AAA 6 ER+/PR + Resistant Substn of GGC--+GGT 9 b ER +/PR + Sensitive None

l0 b ER+/PR + Resistant Deletion of T at Ser 432

17 ER+/PR - Resistant Nucleotides 1271-1318 (Inherent) of exon 6 replaced

with exon 5 nucleotides 1148-1190

28 ER+/PR - Resistant Substn of G C C ~ G C G 35 c ER+/PR + Sensitive Substn of CAT--+CAC

Glu 352---~Val Lys 472 (None) Gly 276 (None) None Termination codon at 437 Termination codon at 454

Ala 505 (None) His 577 (None)

Missense Silent Silent None Truncated ER with defective HBD

Truncated ER with defective HBD

Silent Silent

a Substn, substitution. b Primary and metastatic tumors from the same patient. c All other tumors were ER+/PR +, were either tamoxifen-resistant or sensitive, and did not contain any nucleotide changes.

352



ER MUTATIONS IN TAMOX1FEN-RESISTANT BREAST CANCER

t umor progression, the muta t ion f requency could be higher in la.e

stage disease (19). However , in our study this possibil i ty is unlik~ ly

since we analyzed 20 tumors wi th metastat ic disease (progressive or

recurrent disease) and did not f ind a significant increase in f requency

of muta t ions in ER. It is no tewor thy that Mul l ick and C h a m b o n (20)

reported that the ER in the tamoxifen-res is tant cell l ine LY2 is struc-

turally and funct ional ly indis t inguishable f rom that of the parental tamoxifen-sens i t ive M C F- 7 cells. They demons t ra ted that the anties-

t rogen resistance of the LY2 cells cor responds in fact to the estrogen-

independent g rowth of these cells (20). Possible reasons for the failure

to detect muta t ions in the remain ing tamoxifen-res is tant tumors that

we analyzed are: (a) the muta t ion is present in a very small number

o f ceils and cannot be detected by SSCP, or (b) ER muta t ions are rare

and occur at a very low frequency. Our ability to detect muta t ions

depends not only on the use of opt imal condi t ions for SSCP but also

on the propor t ion of sample that contains mutan t c D N A and on the

sensit ivity o f detect ing that cDNA. We rule out the possibil i ty that we

failed to detect muta t ions due to insensi t ivi ty of our techniques be-

cause three different SSCP condi t ions were used to screen each exon.

S S C P has been wide ly applied for de tec t ing muta t ions in var ious

sys tems and has been reported to detect 8 0 - 9 0 % of all muta t ions (16). The other issue concerns the propor t ion of cells in the t umor that

contain the mutat ion. Breast tumors contain nonmal ignan t suppor t ing

s t roma and blood vessels (18). Therefore , the detect ion of a somat ic

muta t ion depends on the propor t ion of t umor and n o n t u m o r cells in

the spec imen and on the propor t ion of t umor cells wi th the mutat ion.

Detec t ion of variant and wi ld- type conformers on an S S C P gel is

d iagnost ic of a somat ic muta t ion but does not indicate whe ther the

wi ld- type allele is present in mal ignant cells that lack the mutat ion, is

present in nonmal ignan t cells, or is present in both. In tumors where

we detected mutat ions , both the wi ld- type and mutant bands were

evident on S S C P gels, indicat ing that the muta t ions are somatic. The

presence of a major wi ld- type band suggests that many cells contain

wi ld- type ER. Whi le some of these cells are nonmal ignant , others

could be mal ignant cells wi thou t the mutat ion.

Inheri ted germ-l ine muta t ions have been extensively descr ibed in

the androgen, g lucocort icoid , and thyroid resistance syndromes , but

germ-l ine muta t ions of es t rogen receptor are not k n o w n and could be

lethal to the o rgan ism (9). Several ER gene muta t ions ( reviewed in

Ref. 10) and even post translat ional modi f ica t ions of ER have been

reported (11). Graham e t al. (9) have descr ibed a f rame-shif t /

te rminat ion mutan t in the ER-posi t ive tamoxifen-res is tant cell line

T47DCO. This mutant was genera ted by a point delet ion in the H B D

just ups t ream of the end of exon 5. This could give rise to an ER prote in t runcated in the midd le of the H B D amino acid 417. Fuqua e t

al. (12) have reported t runcated ER variants lacking exon 5 (10) and

exon 7 (12) that could have been genera ted by transcriptional spl icing errors. A l though several other examples of variant ERs exist in the

literature (10), this report is the first extensive study that has a t tempted

to correlate the clinical his tory and therapeut ic ou t come of breast

tumors wi th E R mutat ions. Our results suggest that ER muta t ions

occur at a low f requency that is probably comparab le to the muta t ion

f requency of any other m a m m a l i a n gene. Whi le mutant es t rogen re-

ceptors could represent a defini t ive m e c h a n i s m for ER- independen t

and tamoxifen-res is tant g rowth of tumors 10 and 17 that we have

described, our s tudy clearly shows that on ly 2 of 20 tamoxifen-

resistant tumors contain ER mutat ions . The remain ing 18 tamoxifen-

resistant tumors contain wi ld- type estrogen receptors. Other mecha-

n isms mus t therefore be responsible for the es t rogen independence

and the clinical resistance to t amoxi fen observed in these tumors .

Acknowledgments

We thank Dr. Geoffrey Greene from the University of Chicago for the generous gift of the ER eDNA plasmid pER35 and Dr. Bryan Williams, Cancer Biology, Cleveland Clinic Research Foundation, for his encouragement and constructive critique.

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1994;54:349-353. Cancer Res Pratima S. Karnik, Sucheta Kulkarni, Xiao-Pu Liu, et al. CancerEstrogen Receptor Mutations in Tamoxifen-resistant Breast

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estrogen receptor mutations in tamoxifen-resistant breast cancer 1

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