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Comparison of written reports of mammography, sonography and magnetic resonance mammography for preoperative evaluation of breast lesions, with special emphasis on magnetic resonance mammography | <p>Patients with abnormal breast findings (<italic>n</italic> = 413) were examined by mammography, sonography and magnetic resonance (MR) mammography; 185 invasive cancers, 38 carcinoma <italic>in situ</italic> and 254 benign tumours were confirmed histologically. Sensitivity for mammography was 83.7%, for sonography it was 89.1% and for MR mammography it was 94.6% for invasive cancers. In 42 patients with multifocal invasive cancers, multifocality had been detected by mammography and sonography in 26.2%, and by MR mammography in 66.7%. In nine patients with multicentric cancers, detection rates were 55.5, 55.5 and 88.8%, respectively. Carcinoma <italic>in situ</italic> was diagnosed by mammography in 78.9% and by MR mammography in 68.4% of patients. Combination of all three diagnostic methods lead to the best results for detection of invasive cancer and multifocal disease. However, sensitivity of mammography and sonography combined was identical to that of MR mammography (ie 94.6%).</p> | <contrib id="A1" contrib-type="author"><name><surname>Malur</surname><given-names>Sabine</given-names></name><xref ref-type="aff" rid="I1">1</xref></contrib><contrib id="A2" contrib-type="author"><name><surname>Wurdinger</surname><given-names>Susanne</given-names></name><xref ref-type="aff" rid="I2">2</xref></contrib><contrib id="A3" contrib-type="author"><name><surname>Moritz</surname><given-names>Andreas</given-names></name><xref ref-type="aff" rid="I1">1</xref></contrib><contrib id="A4" contrib-type="author"><name><surname>Michels</surname><given-names>Wolfgang</given-names></name><xref ref-type="aff" rid="I1">1</xref></contrib><contrib id="A5" contrib-type="author"><name><surname>Schneider</surname><given-names>Achim</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>aschneider@med.uni-jena.de</email></contrib> | Breast Cancer Research | <sec><title>Introduction</title><p>Mammography and sonography are the standard imaging techniques for detection and evaluation of breast disease [<xref ref-type="bibr" rid="B1">1</xref>]. Mammography is the most established screening modality [<xref ref-type="bibr" rid="B2">2</xref>]. Especially in young women and women with dense breasts, sonography appears superior to mammography, and differentiation between solid tumours and cysts is easier. Sensitivity and specificity of sonography or mammography are higher if sonography and mammography are combined [<xref ref-type="bibr" rid="B3">3</xref>].</p><p>It is generally accepted that MR mammography is the most sensitive technique for diagnosis of breast cancer, whereas the reported specificity of MR mammography varies [<xref ref-type="bibr" rid="B4">4</xref>,<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B8">8</xref>,<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B10">10</xref>,<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B12">12</xref>]. In those studies, MR mammography was performed and evaluated by highly specialized radiologists in a research setting. It was therefore the purpose of the present prospective study to compare the validity of MR mammography with mammography and sonography in clinical routine practice. Findings for the three diagnostic methods documented on routine reports that were available to the surgeon preoperatively formed the basis of this comparison. Special emphasis was placed on the identification of multifocal and multicentric invasive disease.</p></sec><sec sec-type="methods"><title>Patients and methods</title><sec><title>Patients</title><p>Between September 1995 and September 1998, 413 patients with abnormal breast findings were referred for histological evaluation to the Department of Gynecology of the Friedrich-Schiller University, Jena, Germany. Patients had been selected and referred because of the presence of breast lesions detected by palpation and/or mammography and/or sonography. In addition, MR mammography was performed in all patients. We excluded five patients with invasive cancer who had a history of core-needle or fine-needle biopsy cancer within 2 weeks before referral, because the presence of haematoma may mimic false-positive findings on MR mammography. In addition, five patients who did not keep still during MR mammography were excluded.</p></sec><sec><title>Imaging</title><p>Analysis of the sonograms taken in patients with histologically confirmed carcinoma <italic>in situ</italic> were excluded from analysis because the value of sonography for detection of premalignant disease is limited. Mammography was not performed in 32 patients who were younger than 30 years or who had had a mastectomy with suspected local recurrence. For all patients, written reports of mammographic, sonographic and dynamic MR mammographic findings were available preoperatively.</p><p>The majority of mammograms (68%) were performed at the Institute for Diagnostic and Interventional Radiology, Friedrich-Schiller-University, using a senograph DMR (GE Medical Systems, Milwaukee, Wisconsin, USA) with standard craniocaudal and mediolateral oblique projections. Mammograms obtained at other institutions that were considered to match the quality standards of our institution were also accepted for evaluation.</p><p>All sonography and MR examinations were carried out at the Department of Gynecology and the Institute for Diagnostic Interventional Radiology, Friedrich-Schiller-University, respectively. Sonography was done using a 7.5-MHz linear array probe with a Sonoline Versa Pro (Siemens, Erlangen, Germany). MR mammography was performed exclusively at the Institute for Diagnostic Interventional Radiology, using a Gyroscan ACSII (Philips, Nijmegen, The Netherlands) with a field strength of 1.5 T using a double-breast coil. Dynamic T1-weighted images were aquired using a multislice two-dimensional fast-field-echo (FFE) sequence. We used the following parameters: TR 97, TE 5.0, flip angle 80°, slice thickness 4.0 mm, gap 0.4 mm, field of view 350 mm and transverse orientation. In addition T2-weighted images (4000/300/90°/4.0 mm/0.4/350 mm) were obtained. As contrast medium, 0.1 mmol gadolinium-DTPA/kg body weight (Magnevist; Schering, Berlin, Germany) was used and injected as a bolus. One unenhanced and seven enhanced studies were acquired with an acquisition time of 1 min. Criteria for malignancy were signal enhancement of 90% or more within the first 2 min after bolus injection and signal plateau or washout phenomena afterward. Additional criteria were irregular borders of the lesion and low signal intensity in the T2-weighted images.</p><p>Mammograms were read by three different radiologists, sonography was done by three different gynaecologists and MR mammography was interpreted by a total of six different radiologists.</p><p>Definition for multifocal carcinoma was a distance of less than 3 cm and for multicentric carcinoma a distance over 3 cm between various lesions.</p></sec><sec><title>Statistical analysis</title><p>Interpretation of the various diagnostic procedures was compared with the histological examination with regard to sensitivity, specificity, accuracy, and positive and negative predictive value. Criteria for suspected malignancy in the written reports were the terms 'cancer', 'malignant lesion or tumor', or 'suspicious for cancer'. Sensitivity, specificity, negative and positive predicitive value, and accuracy were evaluated as follows:</p><p>Sensitivity = patients with suspected breast cancer/patients with histologically confirmed breast cancer</p><p>Specificity = patients with suspected benign disease/patients with histologically confirmed benign disease</p><p>Positive predictive value = patients with histologically confirmed breast cancer/patients with suspected breast cancer</p><p>Negative predictive value = patients with histologically confirmed benign disease/patients with suspected benign disease</p><p>Accuracy = patients with true-positive and true-negative detected disease/patients with histologically confirmed breast cancer</p><p>A result was classified as false-negative when a diagnostic method classified a histologically confirmed cancer as benign. A result was classified as false-positive when a diagnostic method classified a histologically confirmed benign lesion as cancer. We compared the preformance of all diagnostic methods individually and in combination using the results from all patients. Statistical analysis was performed for all variables with Fisher's exact test and Pearson's χ<sup>2</sup> test.</p></sec></sec><sec><title>Results</title><p>All patients underwent breast surgery and all abnormal lesions identified by mammography, sonography or MR mammography were surgically removed. A total of 477 breast lesions were examined histologically, revealing the presence of 185 invasive cancers, 38 carcinomata <italic>in situ</italic> and 254 benign lesions (fibroadenoma, papilloma, intraductal or adenoid ductal hyperplasia, cystic mastopathia). There were four patients with malignant lesions in both breasts. In 42 patients multifocal tumours and in nine patients multicentric tumors were found on histological examination. Among the 185 invasive lesions, 178 were primary cancers, five were recurrences, one was metastatic and one was an angiosarcoma. The majority of invasive breast cancers were staged as pT1c (44%). Six per cent of tumors were detected in stage pT1a, 18% in stage pT1b, 25% in stage pT2, 3% in stage pT3 and 4% in stage pT4. The distribution of histopathological tumour types is shown in Table <xref ref-type="table" rid="T1">1</xref>. The mean age of patients was 58 years (range 19-85 years).</p><p>The sensitivity of MR mammography was significantly higher than those of mammography and sonography (<italic>P</italic> < 0.005 and <italic>P</italic> < 0.05; Table <xref ref-type="table" rid="T2">2</xref>). The specificity of sonography was significantly higher than those of mammography and MR mammography (<italic>P</italic> < 0.05 and <italic>P</italic> < 0.005; Table <xref ref-type="table" rid="T2">2</xref>). The negative predictive values for sonography and MR mammography were significantly higher than that of mammography (<italic>P</italic> < 0.05 and <italic>P</italic> < 0.005; Table <xref ref-type="table" rid="T2">2</xref>). With regard to accuracy, no significant difference between the three modalities was found (Table <xref ref-type="table" rid="T2">2</xref>). Combining of all three diagnostic methods yielded the best results for detection of cancer (<italic>P</italic> < 0.005; Table <xref ref-type="table" rid="T3">3</xref>). The sensitivity and negative predictive value for the combination of mammography and MR mammography, and the combination of sonography and MR mammography were significantly higher than those for the combination of mammograpy and sonography (<italic>P</italic> < 0.05; Table <xref ref-type="table" rid="T3">3</xref>). The highest result for accuracy was seen for a combination of all three methods (<italic>P</italic> < 0.05; Table <xref ref-type="table" rid="T3">3</xref>).</p><p>Mammography was false-negative in 30 out of 184 invasive cancers, sonography was false-negative in 20 out of 185 cancers, and 10 out of 185 invasive cancers were missed by MR mammography. The majority of false-negative findings was found in stage1 disease, ductal carcinoma and grade 3 tumors (Table <xref ref-type="table" rid="T4">4</xref>). Of 10 invasive cancers missed by MR mammography, eight were found by mammography and sonography. By all three techniques, one invasive ductal carcinoma (pT1b) was misinterpreted as fibroadenoma. In another patient, a microinvasive lobular carcinoma of 5 mm diameter was not detected with mammography and MR mammography, whereas sonography detected a solid, benign tumour. MR mammography identified 10 invasive cancers (5.2%) that were missed by mammography and sonography, whereas one invasive cancer was found by mammography alone. By sonography alone, not a single case of invasive disease was detected when MR mammography or mammography were nonsuspected.</p><p>The highest detection rate for multifocal invasive disease was seen with MR mammography, which identified 28 out of 42 (66.7%) histologically confirmed multifocal invasive cancers, whereas mammography and sonography both identified 11 (26.2%) of these cancers (<italic>P</italic> < 0.05). The combination of all three diagnostic methods leads to the best result for detection of multifocality (76.2%; <italic>P</italic> < 0.05), whereas the detection rate with the combination of mammography and sonography was 35.7%, with the combination of sonography and MR mammography it was 69% (<italic>P</italic> < 0.05 versus mammography + sonography), and with the combination of mammography and MR mammography it was 73.8% (<italic>P</italic> < 0.05 versus mammography + sonography). Multifocal invasive disease was suspected in 12 patients by mammography, in 13 patients by sonography, and in 16 patients by MR mammography, but only unifocal disease was confirmed by histology. Out of nine patients with histologically confirmed multicentric invasive cancer, eight (88.8%) of these cancers were detected by MR mammography and five (55.5%) by mammography or sonography. One patient was diagnosed with multicentric invasive-lobular carcinoma stage pT2G2, which had been misinterpreted as benign tumour by sonography and mammography, and as haematoma by MR mammography.</p><p>Out of 38 patients with carcinoma <italic>in situ</italic>, mammography (suspicious microcalcifications, exclusively) identified 30 cases (78.9%) and MR mammography identified 26 cases (68.4%). When combining mammography and MR mammography, sensitivity for detection of carcinoma <italic>in situ</italic> increased to 87% (not significant).</p></sec><sec><title>Discussion</title><p>When the validity of individual diagnostic methods for detection of invasive breast cancer was analyzed, the sensitivity and specificity of mammography ranged from 79.9 to 89% and from 64 to 93.5%, respectively [<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B13">13</xref>]; for sonography from 67.6 to 96% and from 93 to 97.7%, respectively [<xref ref-type="bibr" rid="B13">13</xref>,<xref ref-type="bibr" rid="B14">14</xref>]; and for MR mammography from 91 to 98.9 and from 20 to 97.4%, respectively [<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B8">8</xref>,<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B10">10</xref>,<xref ref-type="bibr" rid="B15">15</xref>,<xref ref-type="bibr" rid="B16">16</xref>,<xref ref-type="bibr" rid="B17">17</xref>].</p><p>The performance of mammography, sonography and MR mammography was compared in three large series (Table <xref ref-type="table" rid="T5">5</xref>). The present results are similar with regard to sensitivity and specificity for the detection of malignant breast lesions, with MR mammography reaching the highest sensitivity of all imaging procedures.</p><p>When combinations of all three technique were analyzed, mammography and sonography (standard method) had a sensitivity of 83% and a specificity of 92% for detection of malignant disease [<xref ref-type="bibr" rid="B3">3</xref>]. A combination of mammography, sonography and MR mammography (combined method) showed a sensitivity of 95% and a specificity of 64% [<xref ref-type="bibr" rid="B3">3</xref>]. For nonpalpable lesions, sensitivity increased from 73% by the standard method to 82% for the combined method. Specificity for the standard method (89%) was higher than that for the combined method (71%). For palpable lesions a sensitivity of 85% for the standard and 98% for the combined method was achieved, whereas specificity for the standard method was 100% compared with 45% for the combined methods [<xref ref-type="bibr" rid="B3">3</xref>]. The positive predictive value was 94% for the standard and 80% for the combined methods, and the negative predictive values were 78 and 89%, respectively [<xref ref-type="bibr" rid="B3">3</xref>]. In the present study, we also found the highest sensitivity, specificity, and positive and negative predictive values for the combination of all three methods. Combination of mammography and sonography was as sensitive as MR mammography alone (94.6% versus 94.6%).</p><p>The majority of false-positive results for invasive cancer by MR mammography (80 out of 439) were caused by papillomas, intraductal hyperplasia grade 2 or 3, or fibroadenomas in the present series. These lesions have a good blood supply and may mimic invasive cancer [<xref ref-type="bibr" rid="B16">16</xref>,<xref ref-type="bibr" rid="B18">18</xref>].</p><p>Ten out of 185 (5.4%) malignant lesions were classified as false-negative by MR mammography. On histology, the majority of false-negative invasive cancers were lobular cancers (four out of 10). Bone <italic>et al</italic> [<xref ref-type="bibr" rid="B18">18</xref>] reported false-negative results in 11 out of 155 readings, with the majority being lobular cancers on histology. Lack of tumour-induced neovascularity may explain such findings. In particular, invasive lobular cancers infiltrate the normal tissue with columns of single cells, and receive adequate oxygenation without the requirement for increased vascularization [<xref ref-type="bibr" rid="B19">19</xref>]. Buadu <italic>et al</italic> [<xref ref-type="bibr" rid="B11">11</xref>] found that lobular and mucinous carcinomas had a low microvessel density.</p><p>Multifocality of breast cancers can be recognized adequately by MR mammography [<xref ref-type="bibr" rid="B20">20</xref>,<xref ref-type="bibr" rid="B21">21</xref>]. Boetes <italic>et al</italic> [<xref ref-type="bibr" rid="B22">22</xref>] reported that all 61 multifocal cancers were detected by MR mammography, compared with 31% by mammography and 38% by sonography. Esserman <italic>et al</italic> [<xref ref-type="bibr" rid="B20">20</xref>] detected multifocality by MR mammography in 100% (10 out of 10) versus 44% (four out of nine) by mammography. Relevant changes in therapy due to additional multicentric and contralateral tumour findings by MR mammography occur in 18% of patients as compared with conventional imaging [<xref ref-type="bibr" rid="B23">23</xref>]. We found a detection rate of multifocality of 66.7% by MR mammography, as compared with 26.2% by mammography and sonography. However, in 16 patients multifocal invasive disease as diagnosed by MR mammography was shown to be unifocal by histology.</p><p>Kramer <italic>et al</italic> [<xref ref-type="bibr" rid="B24">24</xref>] reported that MR mammography yielded the highest sensitivity for detection of multicentricity as compared with mammography and sonography (89, 66 and 79%, respectively) in 38 patients. These findings are comparable with the present results, in which eight out of nine multicentric cancers were diagnosed correctly.</p><p>Carcinoma <italic>in situ</italic> is identified by mammography through the presence of suspicious microcalcifications. Suspicious microcalcifications are more frequent in intraductal than in infiltrating cancers [<xref ref-type="bibr" rid="B25">25</xref>], which was also observed in the present series. Mammography showed a detection rate for carcinoma <italic>in situ</italic> of 78.9%, as compared with 65.8% by MR mammography; the combination of mammography and MR mammography lead to a detection rate of 86.4%. Fischer <italic>et al</italic> [<xref ref-type="bibr" rid="B26">26</xref>] reported that carcinoma <italic>in situ</italic> was identified by MR mammography in 25 out of 35 patients (72%); three ductal carcinomata <italic>in situ</italic> were detected by MR mammography exclusively. Sittek <italic>et al</italic> [<xref ref-type="bibr" rid="B27">27</xref>] reported that 14 out of 20 carcinomata <italic>in situ</italic> (70%) were correctly diagnosed by MR mammography on the basis of focal increase of signal intensity. Those authors concluded that carcinoma <italic>in situ</italic> is not reliably detected by MR mammography because of lack of a uniform pattern of enhancement. Esserman <italic>et al</italic> [<xref ref-type="bibr" rid="B20">20</xref>] reported a detection rate of 43% for ductal carcinoma <italic>in situ</italic> by MR mammography. Among 36 woman with carcinoma <italic>in situ</italic>, Gilles <italic>et al</italic> [<xref ref-type="bibr" rid="B28">28</xref>] demonstrated two cases without early contrast enhancement.</p><p>The present study showed that, for detection of breast cancer, MR mammography is not superior to a combination of sonography and mammography. For identification of multifocal or multicentric disease, MR mammography proved to be the most accurate technique.</p></sec> |
BRCA1 and BRCA2 protein expressions in an ovotestis of a 46, XX true hermaphrodite | <p><italic>BRCA1</italic> and <italic>BRCA2</italic> breast cancer susceptibility genes encode proteins, the normal cellular functions of which are complex and multiple, and germ-line mutations in individuals predispose both to breast and to ovarian cancer. There is nevertheless substantial evidence linking BRCA1 and BRCA2 to homologous recombination and DNA repair, to transcriptional control and to tissue proliferation. There is controversy regarding the localization of BRCA1 and BRCA2 proteins to either nucleus or cytoplasm and whether the expression is present in premeiotic germ cells or can still be expressed in mitotic spermatogonia. We report herein an immunohistochemical study of BRCA1 and BRCA2 distribution in a rather unsual tissue (an ovotestis), which addresses this issue.</p> | <contrib id="A1" contrib-type="author"><name><surname>Bernard-Gallon</surname><given-names>Dominique J</given-names></name><xref ref-type="aff" rid="I1">1</xref></contrib><contrib id="A2" contrib-type="author"><name><surname>Déchelotte</surname><given-names>Pierre</given-names></name><xref ref-type="aff" rid="I2">2</xref></contrib><contrib id="A3" contrib-type="author"><name><surname>Vissac</surname><given-names>Cécile</given-names></name><xref ref-type="aff" rid="I1">1</xref></contrib><contrib id="A4" contrib-type="author"><name><surname>Aunoble</surname><given-names>Bénédicte</given-names></name><xref ref-type="aff" rid="I1">1</xref></contrib><contrib id="A5" contrib-type="author"><name><surname>Cravello</surname><given-names>Laetitia</given-names></name><xref ref-type="aff" rid="I1">1</xref></contrib><contrib id="A6" contrib-type="author"><name><surname>Malpuech</surname><given-names>Georges</given-names></name><xref ref-type="aff" rid="I3">3</xref></contrib><contrib id="A7" contrib-type="author"><name><surname>Bignon</surname><given-names>Yves-Jean</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>Yves-Jean.Bignon@cjp.u-clermont1.fr</email></contrib> | Breast Cancer Research | <sec><title>Introduction</title><p>Hereditary predisposition to breast cancer can be attributed to germline mutations in the <italic>BRCA1</italic> or <italic>BRCA2</italic> breast cancer susceptibility genes [<xref ref-type="bibr" rid="B1">1</xref>]. Germline mutations in the <italic>BRCA1</italic> and <italic>BRCA2</italic> genes are associated with the development of breast and ovarian cancers [<xref ref-type="bibr" rid="B2">2</xref>]. <italic>BRCA2</italic> is associated the development of breast cancer in both women and men [<xref ref-type="bibr" rid="B3">3</xref>], and a moderate increased risk for the development of ovarian cancer.</p><p>Zabludoff <italic>et al</italic> [<xref ref-type="bibr" rid="B4">4</xref>] investigated the tissue distribution of <italic>Brca1</italic> mRNA in adult mouse tissues and reported that <italic>Brca1</italic> mRNA levels were most abundant in the testis and the ovary. They also found that high level <italic>Brca1</italic> mRNA expression in the testis of mice was detected in meiotic cells and postmeiotic round spermatids and, in contrast, little or no <italic>Brca1</italic> mRNA was expressed in premeiotic germ cells. A low level of <italic>Brca1</italic> mRNA was also detected in Sertoli cells. Blackshear <italic>et al</italic> [<xref ref-type="bibr" rid="B5">5</xref>], on the contrary, demonstrated in the mouse that <italic>Brca1</italic> and <italic>Brca2</italic> mRNA are expressed in mitotic spermatogonia in addition to early meiotic prophase spermatocytes; Sertoli cells and Leydig interstitial cells were found consistently negative for <italic>Brca1</italic> and <italic>Brca2</italic> transcripts. In the normal mouse adult ovary, <italic>Brca1</italic> and <italic>Brca2</italic> transcripts were localized specifically to granulosa cells, thecal cells and oocytes of developing follicles as well as luteal cells of recently formed corpora lutea and surface epithelium.</p><p>Considering these results, we further investigated the presence of human BRCA1 and BRCA2 proteins in an ovotestis by immunochemical analysis with a different panel of antibodies against BRCA1 and BRCA2.</p></sec><sec sec-type="materials|methods"><title>Materials and methods</title><p>True hermaphroditism is a rare cause of atypical genitalia that presents significant diagnostic and management challenges. The patient (male, 6 months old, karyotype of 46, XX; analysis of a sex-determining region of the Y chromosome [SRY] was negative) had a testis on the left side and an ovotestis on the right side. Hematoxylin eosin saffron (HES) demonstrated male and female compartments of the ovotestis (Fig. <xref ref-type="fig" rid="F1">1a</xref>). This paper presents our laboratory findings concerning the BRCA1 and BRCA2 protein expression in this particular gonad.</p><p>All antibodies are described in Table <xref ref-type="table" rid="T1">1</xref>.</p></sec><sec><title>Results and discussion</title><p>The specificity of the polyclonal antibody against BRCA1 (K-18) has been demonstrated elsewhere for BRCA1 [<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B7">7</xref>], ascertained by Western blotting, and the 220 kDa band corresponding to BRCA1 was detected in both HBL-100 and MCF-7 breast cell lines. Moreover, other major bands appeared around 100 kDa in the two cell lines, which may correspond to different variants to BRCA1 [<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B8">8</xref>].</p><p>Chen <italic>et al</italic> [<xref ref-type="bibr" rid="B9">9</xref>] previously reported that the monoclonal antibodies anti-BRCA1 8F7 and 17F8 always exhibited a nuclear staining pattern in breast epithelial cells. Lee <italic>et al</italic> [<xref ref-type="bibr" rid="B10">10</xref>] had already reported that nuclear staining of the BRCA1 protein with 17F8 monoclonal antibody was also seen in tumor cells that were not of breast or ovarian origin. Wilson <italic>et al</italic> [<xref ref-type="bibr" rid="B11">11</xref>] also reported recently that 17F8 antibody detected BRCA1 nuclear and cytoplasmic stainings in breast specimens according to antibody concentration. We have also reported a nuclear staining pattern with different tissues, notably in child mammary gland [<xref ref-type="bibr" rid="B12">12</xref>] and in lung from a fetus at 19 weeks' gestation [<xref ref-type="bibr" rid="B13">13</xref>], with the 8F7 and 17F8 anti-BRCA1 monoclonal antibodies raised against a glutathione-<italic>S</italic>-transferase (GST)-BRCA1 fusion protein containing amino acids encoded by a 3´ portion of BRCA1 exon 11 and by a 5´ portion of BRCA1 exon 11, respectively. These antibodies seemed to remain more specific, and they exhibited nuclear staining.</p><p>We used anti-BRCA1 antibodies raised against amino acids 768-793 (66046N) and amino acids 1847-1863 (66056N). Characterization of these antibodies in MCF-7 human breast tumor cell lysates by Western blotting (data not shown) resulted in the detection of a 220 kDa band corresponding to BRCA1, and one other band around 100 kDa that may correspond to the BRCA1 protein missing exon 11. The 66036N antibodies elicited against amino acids 2-20 of human BRCA1 recognized a 220 kDa BRCA1 protein in HBL-100 breast cell lysates [<xref ref-type="bibr" rid="B14">14</xref>].</p><p>The antibody for BRCA2 (66066E) recognizes epitopes between amino acids 1323-1346 of human BRCA2. The antibody 66076E recognizes epitopes between amino acids 2586-2600. Antibodies were purchased from PharMingen (San Diego, CA, USA) and tested by Western blotting in HBL-100 human breast cells to ensure they recognized the 390 kDa BRCA2 protein. Both BRCA2 antibodies also cross-reacted with smaller proteins, which could be degradation products.</p><p>The 3E6 and 5F6 anti-BRCA2 monoclonal antibodies were generated using two bacterially expressed and purified GST-BRCA2 fusion proteins, containing amino acids 188-563 and 2336-2478 as antigens, respectively [<xref ref-type="bibr" rid="B15">15</xref>]. Using the 3E6 antibodies, we detected a protein of 390 kDa, in MCF-7 human tumor breast cell lysates, which corresponds to the predicted size of the 3418 amino acid BRCA2 sequence. We also detected, in CCL 221 colorectal adenocarcinoma cell lysates, a single band at 390 kDa using the 5F6 antibodies (data not shown).</p><p>As shown in Fig. <xref ref-type="fig" rid="F1">1</xref> and Table <xref ref-type="table" rid="T1">1</xref> for the BRCA1 protein expression study with K-18 antibodies, only cytoplasmic stainings in male germ cells and in oocytes were obtained (Fig. <xref ref-type="fig" rid="F1">1b</xref>). Predominant nuclear stainings of Sertoli cells and oocytes were obtained with 8F7 antibodies (Fig. <xref ref-type="fig" rid="F1">1c</xref>), and cytoplasmic staining of oocytes was also exhibited. Staining patterns with 17F8 antibodies varied from exclusively nuclear staining in Sertoli cells (Fig. <xref ref-type="fig" rid="F1">1d</xref>) to exclusively cytoplasmic staining in oocytes (Fig. <xref ref-type="fig" rid="F1">1e</xref>). Exclusively cytoplasmic staining was seen in male and female germ cells with 66046N (Fig. <xref ref-type="fig" rid="F1">1f</xref>) and 66056N antibodies (data not shown). In contrast, cytoplasmic staining with 66036N antibodies (Fig. <xref ref-type="fig" rid="F1">1g</xref>) was exhibited in male and female germ cells, and low nuclear staining was found in Sertoli cells.</p><p>For BRCA2 protein expression in Sertoli cells and oocytes, exclusively cytoplasmic staining was seen with 66066E antibodies (Fig. <xref ref-type="fig" rid="F1">1h</xref>). Cytoplasmic staining (data not shown) was exhibited with 66076E antibodies in oocytes and in male germ cells. Cytoplasmic staining was obtained in male and female compartments with 3E6 antibodies (Fig. <xref ref-type="fig" rid="F1">1i</xref>). With 5F6 antibodies (data not shown), exclusively cytoplasmic staining was obtained for BRCA2 protein in the male compartment, and low intensive nuclear and cytoplasmic stainings were obtained in oocytes and follicles.</p><p>The differences of staining patterns for the same protein in the same tissue may be explained by the choice of the antibodies. The monoclonal antibodies raised against GST-BRCA1 or GST-BRCA2 fusion proteins seem more specific than antibodies raised against a 20 amino acid peptide.</p></sec><sec><title>Conclusion</title><p>In conclusion, we show using different antibodies that BRCA1 proteins, like BRCA2, are widely expressed in two varieties of non-embryogenic human tissues associated with the cell cycle. BRCA1 and BRCA2 proteins are expressed during growth and differentiation in the ovary. Moreover, they are expressed beyond the spermatogenesis. This is consistent with proposed functions for <italic>BRCA1</italic> and <italic>BRCA2</italic> genes.</p></sec> |
Transforming growth factors-β are not good biomarkers of chemopreventive efficacy in a preclinical breast cancer model system | <p>Using a carcinogen-initiated rat model of mammary tumorigenesis, we tested the hypothesis that transforming growth factor (TGF)-βs are useful biomarkers of chemopreventive efficacy in the breast. The chemopreventive agents tested were tamoxifen and the retinoids 9-<italic>cis</italic>-retinoic acid (9cRA) and <italic>N</italic>-(4-hydroxyphenyl)retinamide (4-HPR), because both antiestrogens and retinoids have previously been shown to upregulate TGF-βs <italic>in vitro</italic>. Despite demonstrable chemopreventive efficacy in this model, none of these agents, alone or in combination, had any significant impact on the expression of TGF-βs in the mammary ductal epithelium or periductal stroma as determined by immunohistochemistry. These data suggest that TGF-βs are not likely to be useful biomarkers of chemopreventive efficacy in a clinical setting.</p> | <contrib id="A1" contrib-type="author"><name><surname>Zujewski</surname><given-names>JoAnne</given-names></name><xref ref-type="aff" rid="I2">2</xref></contrib><contrib id="A2" contrib-type="author"><name><surname>Vaughn-Cooke</surname><given-names>Anika</given-names></name><xref ref-type="aff" rid="I2">2</xref></contrib><contrib id="A3" contrib-type="author"><name><surname>Flanders</surname><given-names>Kathleen C</given-names></name><xref ref-type="aff" rid="I1">1</xref></contrib><contrib id="A4" contrib-type="author"><name><surname>Eckhaus</surname><given-names>Michael A</given-names></name><xref ref-type="aff" rid="I3">3</xref></contrib><contrib id="A5" contrib-type="author"><name><surname>Lubet</surname><given-names>Ronald A</given-names></name><xref ref-type="aff" rid="I4">4</xref></contrib><contrib id="A6" contrib-type="author"><name><surname>Wakefield</surname><given-names>Lalage M</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>wakefiel@dce41.nci.nih.gov</email></contrib> | Breast Cancer Research | <sec><title>Synopsis</title><sec><title>Introduction:</title><p>Chemoprevention has been defined as the use of noncytotoxic nutrients or pharmacologic agents to enhance intrinsic physiologic mechanisms that protect the organism against the development of mutant clones and their progression to malignant cancer. In a recent landmark trial, tamoxifen, a hormonally active selective estrogen receptor modulator (SERM), was shown to decrease the risk of invasive breast cancer by 49% in asymptomatic, but at-risk women [<xref ref-type="bibr" rid="B1">1</xref>]. The search is now on for agents with improved risk-benefit profiles, and for agents that will prevent the subclass of estrogen receptor-negative tumors, the incidence of which was unaffected by the SERMS. Retinoids have already shown potential in this regard [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B4">4</xref>,<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B7">7</xref>]. Because it will not be possible to test many agents in large randomized clinical trials, efforts are underway to develop useful tissue-based surrogate end-point biomarkers that can be used to select only the most promising agents (and doses) for large-scale trials.</p><p>Provocative mechanistic connections have been made between the steroid hormone superfamily, including the SERMS and retinoids, and the TGF-β family of multifunctional growth factors [<xref ref-type="bibr" rid="B8">8</xref>]. The TGF-β system has tumor suppressor activity, and loss of TGF-β response is associated with advanced disease in many human tumor types, including the breast [<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B10">10</xref>]. Conversely, experimental overexpression of TGF-β in the mammary gland protects against tumorigenesis [<xref ref-type="bibr" rid="B11">11</xref>]. This strongly suggests that interventions that enhance TGF-β function early in tumorigenesis could delay or prevent the course of the disease. SERMs such as tamoxifen can upregulate TGF-β production and activation by many cell types, including human breast cancer cell lines [<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B13">13</xref>,<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B15">15</xref>]. Similarly, retinoids can upregulate TGF-β production and activation, both in cell culture and in rats <italic>in vivo</italic> [<xref ref-type="bibr" rid="B16">16</xref>,<xref ref-type="bibr" rid="B17">17</xref>]. It is plausible, therefore, that upregulation of endogenous TGF-β could contribute to the chemopreventive efficacy of SERMs and retinoids.</p><p>In the present study we used a carcinogen-induced rat mammary carcinogenesis model to test the hypothesis that chemoprevention by tamoxifen and retinoids is associated with local upregulation of TGF-βs in the mammary gland, and that TGF-βs might therefore be useful as potential surrogate end-point biomarkers of chemopreventive efficacy in clinical trials.</p></sec><sec><title>Materials and methods:</title><p>A standard protocol for induction of breast cancer in female Sprague-Dawley rats using a single dose of <italic>N</italic>-nitroso-<italic>N</italic>-methylurea (NMU) at 8 weeks of age was used [<xref ref-type="bibr" rid="B4">4</xref>,<xref ref-type="bibr" rid="B18">18</xref>]. Chemopreventive agents were incorporated into powdered lab chow [<xref ref-type="bibr" rid="B18">18</xref>] and fed <italic>ad libitum</italic>, beginning 1 week after injection with NMU. The rats were fed 9cRA (Kuraray Company, Osaka, Japan) at 120 mg/kg of diet, tamoxifen (Sigma Chemical Co, St Louis, MO, USA) at 1.0 mg/kg of diet, and 4-HPR (RW Johnson Pharmaceutical Research Unit, Spring House, PA, USA) at 782 mg/kg of diet.</p><p>Rats were weighed and palpated for the presence of mammary tumors weekly, and six rats in each experimental group were sacrificed after 6 and 12 weeks of treatment with chemopreventive agent. For experiments to determine the effect of high doses of tamoxifen administered over shorter periods of time, rats were given 10 mg tamoxifen/kg body weight per day intragastrically, or 1 mg tamoxifen/kg in the diet, and were sacrificed after 1 day or 3 weeks of treatment. All palpated tumors were confirmed at necropsy, and mammary glands were fixed in neutral buffered formalin and embedded in paraffin. The number 2 (first thoracic) mammary gland was sectioned for histology and immunohistochemistry.</p><p>Immunohistochemical staining was done using rabbit polyclonal antibodies raised against synthetic peptides that correspond to regions in the mature forms of TGF-β<sub>1</sub>, TGF-β<sub>2</sub> and TGF-β<sub>3</sub>: anti-TGF-β<sub>1</sub>-LC and anti-TGF-β<sub>1</sub>-CC [<xref ref-type="bibr" rid="B19">19</xref>], anti-TGF-β<sub>2</sub> (sc-90; Santa Cruz Biotechnologies Inc, Santa Cruz, CA, USA), and anti-50-60-β<sub>3</sub>-LC [<xref ref-type="bibr" rid="B20">20</xref>], respectively. Anti-latent TGF-β-binding protein (LTBP; Ab39) was raised against the purified full-length platelet LTBP [<xref ref-type="bibr" rid="B21">21</xref>]. The antibodies were affinity purified against the immunizing peptide (anti-TGF-β<sub>3</sub>) or against protein A sepharose (anti-TGF-β<sub>1</sub>-LC, anti-TGF-β<sub>1</sub>-CC and anti-TGF-β<sub>2</sub>). Immunohistochemical staining was performed using an indirect immunoperoxidase detection protocol (Vectastain Elite kit, Vector Laboratories, Burlingame, CA, USA). Staining intensity was scored on a scale of 0-4+, using the mouse embryo control section as a reference standard for each run. Ducts and periductal stroma were scored independently. Staining was scored in a blinded manner by two independent observers, and discrepancies were rescored by consensus. Staining intensity was plotted as the mean ± standard deviation for each experimental group.</p></sec><sec><title>Results:</title><p>Palpable mammary tumors were first detected after approximately 35 days following initiation of NMU, and by 70 days incidence had reached 100% in rats not treated with chemopreventive agents (Fig. <xref ref-type="fig" rid="F1">1a</xref>). Tamoxifen, alone or in combination with retinoids, decreased tumor incidence by more than 70% by the end of the study, whereas 9cRA alone decreased it by 50%. 4-HPR alone had a relatively modest effect on tumor incidence in the present study. However, it significantly decreased tumor multiplicity (Fig. <xref ref-type="fig" rid="F1">1b</xref>), indicating that the dose used was efficacious. There was minimal toxicity associated with the chemopreventive intervention, except in the tamoxifen + 4-HPR group, in which mild toxicity was observed, as judged by the weights of the experimental animals (Fig. <xref ref-type="fig" rid="F1">1c</xref>).</p><p>All three TGF-β isoforms and the LTBP (part of the naturally occurring latent TGF-β complex) showed broadly similar immunostaining patterns in the mammary glands of untreated rats at 15 weeks of age (Fig. <xref ref-type="fig" rid="F2">2</xref>). They were present both in the ductal epithelium and in the periductal stroma, suggesting that the TGF-βs are synthesized by the epithelial cells, and possibly stromal cells, and are sequestered in the extracellular matrix. This staining pattern is consistent with a role for the TGF-βs in the maintenance of normal mammary homeostasis.</p><p>None of the chemopreventive agents used, alone or in combination, were found to affect expression of any of the TGF-β isoforms or the LTBP in either ductal epithelium or periductal stroma after 6 weeks of chemopreventive intervention (Fig. <xref ref-type="fig" rid="F3">3</xref>). The 6-week time point was chosen as representative of the period of preneoplasia, as the majority of the animals had no palpable tumors at this time (Fig. <xref ref-type="fig" rid="F1">1</xref>). In the study set, eight out of 36 (22%) of the slides showed histologic evidence of hyperplasia, one out of 36 had a ductal carcinoma <italic>in situ</italic> (mammary intraepithelial neoplasia [<xref ref-type="bibr" rid="B22">22</xref>]), and one out of 36 had a carcinoma. We further investigated the effect of tamoxifen at higher doses and earlier time points. In rats that received tamoxifen at 10 mg/kg per day intragastrically (equivalent to 600 mg/day for a human) or 1 mg/kg per day intragastrically (equivalent to 60 mg/day for a human) for either 1 day or 3 weeks, again no consistent changes were seen in TGF-β expression, using either the TGF-β<sub>1</sub>-CC or the TGF-β<sub>2</sub> antibodies (data not shown).</p><p>After 6 weeks of treatment, we noticed that mammary glands from tamoxifen-treated rats were less developed than those of untreated control animals, having fewer tertiary ducts and terminal end buds, and they could consistently be identified from a blind data set (Fig. <xref ref-type="fig" rid="F4">4</xref>). By 12 weeks of treatment, all three chemopreventive agents had a significant effect on glandular histology, with tamoxifen and 9cRA showing the greatest suppression of ductal development and lobule formation, and 4-HPR showing a relatively mild effect.</p></sec><sec><title>Discussion:</title><p>One major goal in the field of prevention is the identification of surrogate biomarkers that might rapidly predict the effect of a given agent on the primary end-point of cancer incidence. The most informative markers are those with modulation that is likely to be directly related to the preventive effect, and a compelling argument can be made that TGF-βs may fall into this category. However, the present data in a well-established preclinical model of breast cancer, employing a variety of highly effective chemopreventive regimens, suggest that this is not the case.</p><p>Most of the previous studies on the regulation of TGF-βs by tamoxifen and retinoids have been done in tissue culture [<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B13">13</xref>,<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B17">17</xref>]. The lack of effect on TGF-β expression in the present <italic>in vivo</italic> study may reflect the dependence of the response on contextual cues that are only present in the artificial <italic>in vitro</italic> environment. In an <italic>in vivo</italic> study [<xref ref-type="bibr" rid="B16">16</xref>], all-<italic>trans</italic>-retinoic acid was shown to cause an upregulation of TGF-β isoforms in rats, with kinetics and isoform selectivity that varied with the target tissue. However, the rats were vitamin A-deficient, and it is not known whether the same effects would be seen in vitamin A-replete animals such as were used in the present study. In a small study in humans [<xref ref-type="bibr" rid="B23">23</xref>] tamoxifen treatment was shown to cause a consistent induction in extracellular TGF-β in breast cancer biopsies, when compared with pretreatment biopsies from the same patients, and complex effects of tamoxifen on induction of TGF-β<sub>2</sub> in the plasma of patients with metastatic breast cancer have been described [<xref ref-type="bibr" rid="B24">24</xref>]. It is possible that tamoxifen is only effective in inducing TGF-β in the context of a tumor, and not in the normal or initiated tissue that was the subject of the present study. However, an optimal surrogate end-point biomarker in a prevention setting needs to be modulated in normal or premalignant tissues. Although we cannot eliminate the possibility of more subtle effects of chemopreventive agents on TGF-β bioavailability or cellular responsiveness, in our preliminary analyses we have seen no effects on the expression of type I and type II TGF-β receptors (data not shown).</p><p>There is considerable evidence to suggest that, at late stages in tumorigenesis, TGF-βs can actually promote the tumorigenic process, particularly if the epithelial cells have lost responsiveness to the growth inhibitory effects of TGF-β by this time [<xref ref-type="bibr" rid="B9">9</xref>]. While the present work was in progress, a study was reported [<xref ref-type="bibr" rid="B25">25</xref>] that showed that loss of the type II TGF-β receptor can already be seen in a significant fraction of hyperplasias without atypia in the human breast. Furthermore, loss of the receptor correlated with increased risk of subsequent development of invasive breast cancer. Thus, loss of TGF-β response may be a very early event in the development of human breast cancer. Because locally elevated TGF-β levels could select for TGF-β-resistant cells, and because TGF-βs can have oncogenic effects on the stroma, it may actually be important for the safety profile of chemopreventive agents to demonstrate that they do not increase TGF-β levels in the at-risk breast. In this regard, this demonstration that the expression of TGF-βs in the preclinical rat model is unaffected by tamoxifen, 9cRA, or 4-HPR may actually have positive implications, because all three agents are already in clinical use.</p><p>The NMU-induced rat model of mammary tumorigenesis is widely used for chemoprevention studies, and yields rapid development of hormonally responsive mammary tumors with 100% incidence [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B4">4</xref>,<xref ref-type="bibr" rid="B18">18</xref>]. To do this, the initiating agent is given at 8 weeks of age and the chemopreventive agent is started a week later, during the period of active development of the mammary gland. We observed that the histology of the tamoxifen-treated mammary glands differed significantly from control glands when examined after 6 weeks of tamoxifen treatment, showing fewer terminal end-buds and less tertiary branching. Part of the chemopreventive efficacy of antiestrogens and retinoids in this model may therefore be due to a generalized decrease in ductal development. Since chemopreventive agents are unlikely to be given to humans during the pubertal period, this form of preclinical model may not accurately reflect the degree of chemopreventive benefit that could be achieved in humans. Although the accelerated time course and high penetrance of disease reduces the costs of this model, it may be advisable to confirm efficacy of promising agents in a model that delays application of the chemopreventive agent until the mammary gland is fully developed.</p><p>In conclusion, we have shown that treatment of rats with tamoxifen or retinoids results in effective chemoprevention of mammary tumorigenesis, without any detectable effect on local expression of TGF-βs. Although we cannot rule out more subtle effects on TGF-β activity, such as the activation of latent forms, the data suggest that TGF-βs are not involved in the underlying molecular mechanism of chemoprevention induced by these agents. This agrees with <italic>in vitro</italic> work [<xref ref-type="bibr" rid="B26">26</xref>] that showed that blockade of TGF-β signaling did not abrogate the growth inhibitory effect of tamoxifen on breast cancer cells. Given the very limited breast tissue available in clinical trials, we do not recommend testing for TGF-βs as a surrogate end-point biomarkers at this time.</p></sec></sec><sec><title>Full article</title><sec><title>Introduction</title><p>Chemoprevention has been defined as the use of noncytotoxic nutrients or pharmacologic agents to enhance intrinsic physiologic mechanisms that protect the organism against the development of mutant clones and their progression to malignant cancer [<xref ref-type="bibr" rid="B27">27</xref>]. Members of the nuclear receptor superfamily are considered to be particularly promising targets for chemoprevention, because of their pivotal role in the regulation of metabolic, developmental, and differentiation pathways [<xref ref-type="bibr" rid="B28">28</xref>]. In a recent landmark trial [<xref ref-type="bibr" rid="B1">1</xref>], tamoxifen, a hormonally active SERM, was shown to decrease the risk of invasive breast cancer by 49% in asymptomatic, but at-risk women. Another SERM, raloxifene, also shows promise [<xref ref-type="bibr" rid="B29">29</xref>]. These studies validate the concept of using pharmacologic agents for prevention of human breast cancer in apparently healthy individuals.</p><p>The search is now on for agents with improved risk-benefit profiles, and for agents that will prevent the subclass of estrogen receptor-negative tumors, the incidence of which was unaffected by the SERMS. Retinoids, a family of compounds structurally related to vitamin A, have already shown potential in this regard [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B4">4</xref>,<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B7">7</xref>]. Since it will not be possible to test many agents in large randomized clinical trials, efforts are underway to develop useful tissue-based surrogate end-point biomarkers that can be used to select only the most promising agents (and doses) for large-scale trials.</p><p>Provocative mechanistic connections have been made between the steroid hormone superfamily, including the SERMS and retinoids, and the TGF-β family of multifunctional growth factors. TGF-βs are potent inhibitors of the growth of many epithelial cell types [<xref ref-type="bibr" rid="B8">8</xref>]. Recent work has implicated the TGF-β system as an important tumor suppressor pathway, and loss of TGF-β response is associated with advanced disease in many human tumor types, including the breast [<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B10">10</xref>,<xref ref-type="bibr" rid="B30">30</xref>,<xref ref-type="bibr" rid="B31">31</xref>]. In mouse models, over-expression of TGF-β<sub>1</sub> in the mammary gland protects against tumorigenesis [<xref ref-type="bibr" rid="B11">11</xref>], whereas local inactivation of the type II TGF-β receptor enhances tumorigenesis [<xref ref-type="bibr" rid="B32">32</xref>]. This strongly suggests that interventions that enhance TGF-β function early in tumorigenesis could delay or prevent the course of the disease.</p><p>Antiestrogens such as tamoxifen have been shown to upregulate TGF-β production and activation by many cell types, including human breast cancer cell lines [<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B13">13</xref>,<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B15">15</xref>]. Similarly, retinoids can upregulate TGF-β production and activation, both in cell culture and in rats <italic>in vivo</italic> [<xref ref-type="bibr" rid="B16">16</xref>,<xref ref-type="bibr" rid="B17">17</xref>]. Therefore, it is reasonable to propose that some of the chemopreventive efficacy of these agents against breast cancer <italic>in vivo</italic> could be mediated via a local upregulation of TGF-βs, with concomitant enhancement of tumor suppressor activity.</p><p>In the present study, we used a carcinogen-induced rat model of mammary carcinogenesis to test whether chemoprevention by tamoxifen and by two different retinoids (4-HPR, also known as fenretinide; and 9-cRA) is associated with local upregulation of TGF-βs in the initiated mammary gland. If this were the case, TGF-βs might be useful as potential surrogate end-point biomarkers in clinical trials. However, the results show that TGF-β levels, as detected immunohistochemically, are not affected by tamoxifen or retinoids in this preclinical model of early-stage breast cancer.</p></sec></sec><sec><title>Materials and method</title><sec><title>Mammary carcinogenesis studies</title><p>A standard protocol for induction of breast cancer in female Sprague-Dawley rats was used [<xref ref-type="bibr" rid="B4">4</xref>,<xref ref-type="bibr" rid="B18">18</xref>], with initiation induced by a single intravenous dose of NMU (50 mg/kg body weight) at 8 weeks of age. Retinoids and tamoxifen were incorporated into powdered laboratory chow as described previously [<xref ref-type="bibr" rid="B18">18</xref>] and fed <italic>ad libitum</italic>, beginning 1 week after injection with NMU. Rats were fed 9cRA (Kuraray Company, Osaka, Japan) at 120 mg/kg of diet, tamoxifen (Sigma Chemical Co, St Louis, MO, USA) at 1.0 mg/kg of diet, and 4-HPR (RW Johnson Pharmaceutical Research Institute) at 782 mg/kg of diet.</p><p>Rats were weighed weekly and palpated for the presence of mammary tumors. Six rats in each experimental group were sacrificed after 6 and 12 weeks of treatment with chemopreventive agent. The 6-week sacrifice time was chosen for the immunohistochemical studies to represent the period of premalignancy, because the incidence of palpable tumors is less than 20% for all experimental groups at that time. By 12 weeks all rats that have not received a chemopreventive agent have tumors, so the primary purpose of the 12-week sacrifice time was to allow an accurate determination of chemopreventive efficacy for the particular experiment.</p><p>For experiments to determine the effect of high doses of tamoxifen administered over shorter periods of time, rats were given 10 mg tamoxifen/kg body weight per day intragastrically or 1 mg tamoxifen/kg in the diet, and were sacrificed after 1 day or 3 weeks of treatment.</p><p>All palpated tumors were confirmed at necropsy, and mammary glands were fixed in neutral buffered formalin and embedded in paraffin. The number 2 (first thoracic) mammary gland was sectioned for histology and immunohistochemistry.</p></sec><sec><title>Immunohistochemistry of TGF-βs</title><p>Immunohistochemical staining was done using rabbit polyclonal antibodies raised against synthetic peptides that correspond to regions in the mature forms of TGF-β<sub>1</sub>, TGF-β<sub>2</sub>, and TGF-β<sub>3</sub>. Antibodies to TGF-β<sub>1</sub> were raised against a synthetic peptide corresponding to residues 1-30 of the mature protein in either the Laboratory of Chemoprevention (anti-TGF-β<sub>1</sub>-LC; NIH, Bethesda, MD, USA) or the Collagen Corporation (anti-TGF-β<sub>1</sub>-CC; Palo Alto, CA, USA). These antibodies were raised against different preparations of the 1-30 peptide, and they recognize different epitopes of this peptide [<xref ref-type="bibr" rid="B19">19</xref>]. The LC antibody usually stains intracellular TGF-β<sub>1</sub>, whereas the CC antibody stains extracellular TGF-β<sub>1</sub>. Anti-TGF-β<sub>2</sub> (sc-90; Santa Cruz Biotechnologies Inc) was raised to a peptide corresponding to residues 72-99 of the mature TGF-β<sub>2</sub>. Anti-TGF-β<sub>3</sub> (anti-50-60-β<sub>3</sub>-LC) was raised against residues 50-60 of mature TGF-β<sub>3</sub> [<xref ref-type="bibr" rid="B20">20</xref>]. Anti-LTBP (Ab39) was raised against the purified full-length platelet LTBP [<xref ref-type="bibr" rid="B21">21</xref>].</p><p>The antibodies were affinity purified against the immunizing peptide (anti-TGF-β<sub>3</sub>) or against protein A sepharose (anti-TGF-β<sub>1</sub>-LC, anti-TGF-β<sub>1</sub>-CC, and anti-TGF-β<sub>2</sub>), and have been assayed for specificity by Western blot analysis [<xref ref-type="bibr" rid="B19">19</xref>,<xref ref-type="bibr" rid="B20">20</xref>,<xref ref-type="bibr" rid="B33">33</xref>]. All antibodies reacted with the appropriate TGF-β isoform except anti-TGF-β<sub>1</sub>-CC, which showed some cross-reactivity with TGF-β<sub>3</sub> on Western blots.</p><p>Immunohistochemical staining was performed using an indirect immunoperoxidase detection protocol (Vectastain Elite kit; Vector Laboratories) following treatment of sections with hyaluronidase to improve antibody penetration. Optimal antibody concentrations were determined by titration on select samples before analysis of the full experimental set. Staining was shown to be specific in control experiments in which either the primary antibody was preincubated with a 50-fold molar excess of immunizing peptide before being applied to the section (anti-TGF-β<sub>2</sub>, anti-TGF-β<sub>3</sub>), or the section was stained with an equivalent concentration of nonimmune rabbit immunoglobulin (anti-TGF-β<sub>1</sub>-LC, anti-TGF-β<sub>1</sub>-CC, and anti-LTBP). In analysis of the full experimental set, for any given antibody all sections were stained at the same time so as to be directly comparable, and a normal mouse embryo section was included as a positive control. A normal rabbit immunoglobulin control was also run for the whole set.</p></sec><sec><title>Quantitation of immunostaining</title><p>Two different systems were used to grade immunostaining. For all samples, staining of the ducts and periductal stroma were scored independently. For samples after 6 weeks of chemopreventive treatment, staining intensity was scored on a scale of 0–4+, using the mouse embryo control section as a reference standard for each run. Staining was scored in a blinded manner by two independent observers, and scores never differed by more than one point. Discrepancies were rescored by consensus. Staining intensity was plotted as the mean ± standard deviation for each experimental group.</p></sec></sec><sec><title>Results</title><sec><title>Chemopreventive efficacy</title><p>Palpable mammary tumors were first detected after approximately 35 days following initation with NMU, and by 70 days incidence had reached 100% in rats not treated with chemopreventive agents (Fig. <xref ref-type="fig" rid="F1">1a</xref>). Tamoxifen, alone or in combination with retinoids, decreased tumor incidence by more than 70% by the end of the study, whereas 9cRA alone decreased it by 50%. 4-HPR alone had a relatively modest effect on tumor incidence in the present study. However, it significantly decreased tumor multiplicity (Fig. <xref ref-type="fig" rid="F1">1b</xref>) indicating that the dose used was efficacious. These incidence and multiplicity data are consistent with previously published studies using this model [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B4">4</xref>,<xref ref-type="bibr" rid="B34">34</xref>]. The mean weights of the rats in the various experimental groups differed by less than 10% from those of rats not treated with a chemopreventive agent, except for the rats treated with a combination of tamoxifen and 4-HPR, in which the mean weight was approximately 15% lower than the that in untreated group by the end of the study (Fig. <xref ref-type="fig" rid="F1">1c</xref>). This suggests that there was minimal toxicity associated with the chemopreventive intervention, except in the tamoxifen + 4-HPR group, in which mild toxicity was observed.</p></sec><sec><title>Effect of chemopreventive agents on TGF-β expression in initiated mammary gland</title><p>Figure <xref ref-type="fig" rid="F2">2</xref> shows the typical immunohistochemical staining pattern for the TGF-βs and the LTBP (part of the naturally occurring latent TGF-β complex) in initiated mammary glands of 15-week-old rats that had not been treated with chemopreventive agents. All three TGF-β isoforms and the LTBP showed broadly similar staining patterns. They were present both in the ductal epithelium and in the periductal stroma, suggesting that the TGF-βs are synthesized by the epithelial cells, and possibly stromal cells, and are sequestered in the extracellular matrix. This staining pattern is consistent with a role for the TGF-βs in the maintenance of normal mammary homeostasis.</p><p>None of the chemopreventive agents used, alone or in combination, were found to affect expression of any of the TGF-β isoforms or the LTBP in either ductal epithelium or periductal stroma after 6 weeks of chemopreventive intervention (Fig. <xref ref-type="fig" rid="F3">3</xref>). The 6-week time point was chosen as representative of the period of preneoplasia, because the majority (>80%) of the rats had no palpable tumors at this stage (Fig. <xref ref-type="fig" rid="F1">1</xref>). Human clinical material for biomarker analysis in primary chemoprevention studies is also likely to comprise normal and initiated, at-risk epithelium with some early preneoplastic changes, but without evidence of major neoplastic change.</p><p>In the 6-week study set, eight out of 36 (22%) of the glands sampled showed histologic evidence of hyperplasia. In addition, one out of 36 glands sampled had a lesion with the appearance of a ductal carcinoma <italic>in situ</italic> (focal necrosis, marked atypia and abundant mitoses, placing it in the category of 'mammary intraepithelial neoplasia' by the Annapolis naming convention [<xref ref-type="bibr" rid="B22">22</xref>]), and an additional one had an invasive carcinoma. Both the <italic>in situ</italic> and the invasive carcinomas were in the control group. However, both samples also had histologically normal ducts on the same slide, which were scored for the analysis. There was no difference in staining between the ducts that were proximal to the tumor and those that were more distal, and neither were there any differences in staining observed between histologically normal and hyperplastic ducts in any of the samples analyzed (data not shown). Since the focus of the present study was on TGF-β expression changes in the preneoplastic gland, staining of the tumors was not scored for the analysis, but in the two cases present, the staining did not differ significantly from that of the surrounding normal-appearing ducts (not shown).</p><p>Assuming that a rat weighs approximately 250 g and eats 10 g of chow each day, the equivalent human doses to those used in this study would be approximately 2.5 mg/day tamoxifen, 2 g/day 4-HPR, and 290 mg/day 9cRA. The human doses that are currently being used in clinical trials are 20 mg/day for tamoxifen, 0.2-0.4 g/day for 4-HPR, and 100-250 mg/day for 9cRA. The different doses of tamoxifen and 4-HPR used in humans and rats probably reflect interspecies differences in pharmacokinetics. However, because the efficacious dose for tamoxifen was 10 times lower in the rat than the dose that has previously been shown to upregulate TGF-β<sub>1</sub> expression in human breast tumor tissue [<xref ref-type="bibr" rid="B23">23</xref>], we looked at the effect of a higher dose of tamoxifen in the rats. Furthermore, because one study [<xref ref-type="bibr" rid="B35">35</xref>] showed that TGF-β may be transiently upregulated early after administration of tamoxifen, we also looked at earlier time points. In rats receiving tamoxifen at 10 mg/kg per day intragastrically (equivalent to 600 mg/day for a human) or 1 mg/kg per day (equivalent to 60 mg/day for a human) for either 1 day or 3 weeks, again no consistent changes were seen in TGF-β expression, using either the TGF-β<sub>1</sub>-CC or the TGF-β<sub>2</sub> antibodies (data not shown).</p></sec><sec><title>Effect of chemopreventive agents on the histology of the mammary gland</title><p>While scoring the immunohistochemical slides, we noticed that the mammary histology appeared to be altered in rats treated with chemopreventive agents. Hematoxylin and eosin stained sections from the samples after 6 weeks of treatment were analyzed by a veterinary pathologist (MAE). The mammary glands from tamoxifen-treated rats were less developed than those of untreated control animals, having fewer tertiary ducts and terminal end-buds, and could consistently be identified from a blind data set (Fig. <xref ref-type="fig" rid="F4">4</xref>). By 12 weeks of treatment, all three chemopreventive agents had a significant effect on glandular histology, with tamoxifen and 9cRA showing the greatest suppression of ductal development and lobule formation, and 4-HPR showing a relatively mild effect.</p></sec></sec><sec><title>Discussion</title><sec><title>TGF-βs as candidate biomarkers</title><p>One major goal in the field of prevention is the identification of surrogate biomarkers that might rapidly predict the effect of a given agent on the primary end-point of cancer incidence. There is an extensive literature showing that steroid hormone superfamily members, such as antiestrogens and retinoids, can upregulate TGF-β activity in a variety of systems [<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B13">13</xref>,<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B15">15</xref>,<xref ref-type="bibr" rid="B16">16</xref>,<xref ref-type="bibr" rid="B17">17</xref>,<xref ref-type="bibr" rid="B23">23</xref>,<xref ref-type="bibr" rid="B24">24</xref>,<xref ref-type="bibr" rid="B35">35</xref>]. This suggested that the chemopreventive action of these agents against breast cancer could be mediated in part through enhancing the tumor suppressor activity of the endogenous TGF-β system, and thus that changes in TGF-β expression might serve as useful surrogate end-point biomarkers of chemopreventive efficacy. However, here we used the NMU-induced rat model of mammary carcinogenesis to show that the chemopreventive effect of tamoxifen and two retinoids is not associated with any consistent changes in TGF-β levels, at least as determined immunohistochemically.</p></sec><sec><title>Comparison with earlier studies</title><p>Most of the previous studies on the regulation of TGF-βs by tamoxifen and retinoids [<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B13">13</xref>,<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B17">17</xref>] have been done in tissue culture. Thus, the lack of effect on TGF-β expression in the present <italic>in vivo</italic> study might reflect the dependence of the response on contextual cues that are only present in the artificial <italic>in vitro</italic> environment. In one previous <italic>in vivo</italic> study [<xref ref-type="bibr" rid="B16">16</xref>] all-<italic>trans</italic>-retinoic acid upregulated TGF-β expression in rats, with kinetics and isoform selectivity that varied with the target tissue. However, the rats were vitamin A-deficient, and it is not known whether the same effects would be seen in vitamin A-replete animals, such as were used in the present study, or whether the response would vary with the specific retinoid used.</p><p>There are some data that support an effect of tamoxifen on upregulation of TGF-βs <italic>in vivo</italic> in humans. Three months of tamoxifen treatment was shown to cause a consistent induction in extracellular TGF-β in breast cancer biopsies, when compared with pretreatment biopsies from the same patients [<xref ref-type="bibr" rid="B23">23</xref>]. Furthermore, complex effects of tamoxifen on induction of TGF-β<sub>2</sub> in the plasma of patients with metastatic breast cancer have been described [<xref ref-type="bibr" rid="B24">24</xref>]. It is possible that tamoxifen is only effective in inducing TGF-β in the context of a tumor, and not in normal or initiated tissue, which was the subject of the present study. This issue could be reassessed in preclinical models using the same agents to treat established proliferative intraepithelial neoplasia. However, for ease of tissue acquisition, an optimal surrogate end-point biomarker in a prevention setting needs to be modulated in normal or premalignant tissues.</p></sec><sec><title>Alternative levels of regulation of the bioefficacy of TGF-βs</title><p>Interestingly, in cell culture, both tamoxifen and all-<italic>trans</italic>-retinoic acid have been shown [<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B13">13</xref>,<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B17">17</xref>] to increase the fraction of TGF-β in its biologically active, as opposed to its latent form. The current method for discriminating between active and latent TGF-β in tissue samples requires the use of frozen sections and immunofluorescence techniques, which are not practical for routine clinical use [<xref ref-type="bibr" rid="B36">36</xref>]. As simpler assays become available, however, the issue of possible changes in TGF-β activation status should be readdressed. Retinoids can also affect cellular responsiveness to TGF-βs at the level of receptor expression and downstream events [<xref ref-type="bibr" rid="B37">37</xref>,<xref ref-type="bibr" rid="B38">38</xref>]. To date, expression of TGF-β receptors and downstream signaling components such as the Smads have not been well-characterized in this rat model, but in our preliminary analyses we saw no effect of retinoids on type I and type II TGF-β receptor expression in the mammary gland (data not shown). At this time, however, we certainly cannot rule out the possibility that tamoxifen and retinoids may be having subtle effects on the TGF-β system at levels other than the regulation of TGF-β expression.</p></sec><sec><title>Lack of effect of chemopreventive agents on TGF-β expression may have positive implications</title><p>There is considerable evidence to suggest that, at late stages in tumorigenesis, TGF-βs can actually promote the tumorigenic process, particularly if the epithelial cells have lost responsiveness to the growth regulatory effects of TGF-β by this time [<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B39">39</xref>,<xref ref-type="bibr" rid="B40">40</xref>,<xref ref-type="bibr" rid="B41">41</xref>]. Thus, advanced human tumors show increased levels of TGF-β expression [<xref ref-type="bibr" rid="B42">42</xref>,<xref ref-type="bibr" rid="B43">43</xref>,<xref ref-type="bibr" rid="B44">44</xref>], and TGF-βs are known to suppress the immunosurveillance system, to enhance angiogenesis, invasion and metastasis, and to increase drug resistance [<xref ref-type="bibr" rid="B45">45</xref>,<xref ref-type="bibr" rid="B46">46</xref>,<xref ref-type="bibr" rid="B47">47</xref>,<xref ref-type="bibr" rid="B48">48</xref>].</p><p>In the colon, loss of the type II TGF-β receptor occurs at the late adenoma to carcinoma transition [<xref ref-type="bibr" rid="B49">49</xref>], suggesting that early premalignant lesions retain TGF-β responsiveness and would be amenable to interventions that enhance TGF-β activity. However, while the present work was in progress, a study was reported [<xref ref-type="bibr" rid="B25">25</xref>] showing that loss of the type II TGF-β receptor can already be seen in a significant fraction of hyperplasias without atypia in the human breast. Furthermore, loss of the receptor correlated with increased risk of subsequently developing invasive breast cancer. Thus, unlike in the colon, loss of TGF-β response may be a very early event in the development of human breast cancer.</p><p>Since locally elevated TGF-β levels may select for TGF-β-resistant cells, and because TGF-βs can have oncogenic effects on the stroma, it may actually be important for the safety profile of chemopreventive agents to demonstrate that they do not increase TGF-β levels in the at-risk breast. For example, tamoxifen resistance in a xenograft model of advanced human breast cancer, was recently shown [<xref ref-type="bibr" rid="B50">50</xref>] to be associated with an increase in TGF-βs and concomitant immunosuppressive effects on natural killer cells. In this regard, our demonstration that the expression of TGF-βs in the preclinical rat model is unaffected by tamoxifen, 9cRA, and 4-HPR may actually have positive implications, because these agents are already in clinical use.</p></sec><sec><title>Limitations of the NMU-induced rat model of mammary carcinogenesis</title><p>The NMU-induced rat model of mammary tumorigenesis is widely used for chemoprevention studies and yields rapid development of hormonally responsive mammary tumors with 100% incidence [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B4">4</xref>,<xref ref-type="bibr" rid="B18">18</xref>]. To do this, the initiating agent is given at 8 weeks of age, during early puberty, and the chemopreventive agent is typically given continuously, starting 1 week later. Since sexual maturity is achieved at approximately 11 weeks of age in rats, this means that the chemopreventive agent is given during a period of active development of the mammary gland.</p><p>In the present study we observed that the histology of the tamoxifen-treated mammary gland differed significantly from control glands when examined after 6 weeks of tamoxifen treatment. Specifically, there were fewer terminal end-buds and less tertiary branching, which are indicative of a delay or arrest in normal mammary development. This is consistent with the known requirement for estrogen for proper mammary development [<xref ref-type="bibr" rid="B51">51</xref>]. We saw a lesser effect with 4-HPR, although this type of phenomenon has also been described in the literature for retinoids [<xref ref-type="bibr" rid="B4">4</xref>]. Thus, part of the chemopreventive efficacy of antiestrogens and retinoids in this model may be due to a generalized decrease in ductal development. Because chemopreventive agents are unlikely to be given to humans during the pubertal period, this form of preclinical model may not accurately reflect the degree of chemopreventive benefit that could be achieved in humans. Although the accelerated time course and high penetrance of disease reduces the costs of this model, it may be advisable to confirm efficacy of promising agents in a model that delays application of the chemopreventive agent until the mammary gland is fully developed.</p></sec></sec><sec><title>Conclusion</title><p>We have shown that treatment of rats with tamoxifen or retinoids results in effective chemoprevention of mammary tumorigenesis, without any detectable effect on local expression of TGF-βs. Although we cannot rule out more subtle effects on TGF-β activity, such as the activation of latent forms, the data suggest that the underlying molecular mechanism of chemoprevention by these agents does not involve increases in TGF-β expression. This agrees with <italic>in vitro</italic> work showing that blockade of TGF-β signalling did not abrogate the growth inhibitory effect of tamoxifen on breast cancer cells [<xref ref-type="bibr" rid="B26">26</xref>]. Given the very limited breast tissue available in clinical chemoprevention trials, we do not recommend testing for TGF-βs as surrogate end-point biomarkers at this time.</p></sec> |
Smoking and high-risk mammographic parenchymal patterns: a
case-control study | <sec><title>Introduction:</title><p>Overall, epidemiological studies [<xref ref-type="bibr" rid="B1">1</xref>,<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B4">4</xref>] have
reported no substantial association between cigarette smoking and the risk of
breast cancer. Some studies [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B7">7</xref>] reported a significant increase of
breast cancer risk among smokers. In recent studies that addressed the
association between breast cancer and cigarette smoking, however, there was
some suggestion of a decreased risk [<xref ref-type="bibr" rid="B8">8</xref>,<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B10">10</xref>], especially among current smokers,
ranging from approximately 10 to 30% [<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B10">10</xref>]. Brunet <italic>et al</italic> [<xref ref-type="bibr" rid="B11">11</xref>]
reported that smoking might reduce the risk of breast cancer by 44% in carriers
of <italic>BRCA1</italic> or <italic>BRCA2</italic> gene mutations. Wolfe [<xref ref-type="bibr" rid="B12">12</xref>] described four different mammographic patterns created by
variations in the relative amounts of fat, epithelial and connective tissue in
the breast, designated N1, P1, P2 and DY. Women with either P2 or DY pattern
are considered at greater risk for breast cancer than those with N1 or P1
pattern [<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B13">13</xref>,<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B15">15</xref>]. There are no published studies
that assessed the relationship between smoking and mammographic parenchymal
patterns.</p></sec><sec><title>Aims:</title><p>To evaluate whether mammographic parenchymal patterns as
classified by Wolfe, which have been positively associated with breast cancer
risk, are affected by smoking. In this case-control study, nested within the
European Prospective Investigation on Cancer in Norfolk (EPIC-Norfolk) cohort
[<xref ref-type="bibr" rid="B16">16</xref>], the association between smoking habits and
mammographic parenchymal patterns are examined. The full results will be
published elsewhere.</p></sec><sec><title>Methods:</title><p>Study subjects were members of the EPIC cohort in Norwich who also
attended the prevalence screening round at the Norwich Breast Screening Centre
between November 1989 and December 1997, and were free of breast cancer at that
screening. Cases were defined as women with a P2/DY Wolfe's mammographic
parenchymal pattern on the prevalence screen mammograms. A total of 203 women
with P2/DY patterns were identified as cases and were individually matched by
date of birth (within 1 year) and date of prevalence screening (within 3
months) with 203 women with N1/P1 patterns who served as control
individuals.</p><p>Two views, the mediolateral and craniocaudal mammograms, of both
breasts were independently reviewed by two of the authors (ES and RW) to
determine the Wolfe mammographic parenchymal pattern.</p><p>Considerable information on health and lifestyle factors was
available from the EPIC Health and Lifestyle Questionnaire [<xref ref-type="bibr" rid="B16">16</xref>]. In the present study we examined the subjects' personal
history of benign breast diseases, menstrual and reproductive factors, oral
contraception and hormone replacement therapy, smoking, and anthropometric
information such as body mass index and waist:hip ratio.</p><p>Odds ratios (ORs) and their 95% confidence intervals (CIs) were
calculated by conditional logistic regression [<xref ref-type="bibr" rid="B17">17</xref>], and
were adjusted for possible confounding factors.</p></sec><sec><title>Results:</title><p>The characteristics of the cases and controls are presented in
Table <xref ref-type="table" rid="T1">1</xref>. Cases were leaner than controls. A larger
percentage of cases were nulliparous, premenopausal, current hormone
replacement therapy users, had a personal history of benign breast diseases,
and had had a hysterectomy. A larger proportion of controls had more than three
births and were current smokers.</p><p>Table <xref ref-type="table" rid="T2">2</xref> shows the unadjusted and adjusted OR
estimates for Wolfe's high-risk mammographic parenchymal patterns and smoking
in the total study population and in postmenopausal women separately. Current
smoking was strongly and inversely associated with high-risk patterns, after
adjustment for concomitant risk factors. Relative to never smokers, current
smokers were significantly less likely to have a high-risk pattern (OR 0.37,
95% CI 0.14-0.94). Similar results were obtained when the analysis was confined
to postmenopausal women. Past smoking was not related to mammographic
parenchymal patterns. The overall effect in postmenopausal women lost its
significance when adjusted for other risk factors for P2/DY patterns that were
found to be significant in the present study, although the results were still
strongly suggestive. There was no interaction between cigarette smoking and
body mass index.</p></sec><sec><title>Discussion:</title><p>In the present study we found a strong inverse relationship
between current smoking and high-risk mammographic parenchymal patterns of
breast tissue as classified by Wolfe [<xref ref-type="bibr" rid="B12">12</xref>]. These
findings are not completely unprecedented; Greendale <italic>et al</italic> [<xref ref-type="bibr" rid="B18">18</xref>] found a reduced risk of breast density in association with
smoking, although the magnitude of the reduction was unclear. The present
findings suggest that this reduction is large.</p><p>Recent studies [<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B10">10</xref>]
have suggested that breast cancer risk may be reduced among current smokers. In
a multicentre Italian case-control study, Braga <italic>et al</italic> [<xref ref-type="bibr" rid="B10">10</xref>] found that, relative to nonsmokers, current smokers had a
reduced risk of breast cancer (OR 0.84, 95% CI 0.7-1.0). These findings were
recently supported by Gammon <italic>et al</italic> [<xref ref-type="bibr" rid="B9">9</xref>], who
reported that breast cancer risk in younger women (younger than 45 years) may
be reduced among current smokers who began smoking at an early age (OR 0.59,
95% CI 0.41-0.85 for age 15 years or younger) and among long-term smokers (OR
0.70, 95% CI 0.52-0.94 for those who had smoked for 21 years or more).</p><p>The possible protective effect of smoking might be due to its
anti-oestrogenic effect [<xref ref-type="bibr" rid="B1">1</xref>,<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B19">19</xref>]. Recently there has been renewed interest in the potential
effect of smoking on breast cancer risk, and whether individuals may respond
differently on the basis of differences in metabolism of bioproducts of smoking
[<xref ref-type="bibr" rid="B20">20</xref>,<xref ref-type="bibr" rid="B21">21</xref>]. Different relationships
between smoking and breast cancer risk have been suggested that are dependent
on the rapid or slow status of acetylators of aromatic amines [<xref ref-type="bibr" rid="B20">20</xref>,<xref ref-type="bibr" rid="B21">21</xref>]. More recent studies [<xref ref-type="bibr" rid="B22">22</xref>,<xref ref-type="bibr" rid="B23">23</xref>], however, do not support these
findings.</p><p>The present study design minimized the opportunity for bias to
influence the findings. Because subjects were unaware of their own case-control
status, the possibility of recall bias in reporting smoking status was
minimized. Systematic error in the assessment of mammograms was avoided because
reading was done without knowledge of the risk factor data. Furthermore, the
associations observed are unlikely to be explained by the confounding effect of
other known breast cancer risk factors, because we adjusted for these in the
analysis. We did not have information on passive smoking status, however, which
has recently been reported to be a possible confounder [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B21">21</xref>,<xref ref-type="bibr" rid="B24">24</xref>].</p><p>The present data indicate that adjustment for current smoking
status is important when evaluating the relationship between mammographic
parenchymal pattern and breast cancer risk. They also indicate smoking as a
prominent potential confounder when analyzing effects of other risk factors
such as obesity-related variables. It seems that parenchymal patterns may act
as an informative biomarker of the effect of cigarette smoking on breast cancer
risk.</p></sec> | <contrib id="A1" contrib-type="author"><name><surname>Sala</surname><given-names>Evis</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>evis.sala@srl.cam.ac.uk</email></contrib><contrib id="A2" contrib-type="author"><name><surname>Warren</surname><given-names>Ruth</given-names></name><xref ref-type="aff" rid="I1">1</xref><xref ref-type="aff" rid="I2">2</xref></contrib><contrib id="A3" contrib-type="author"><name><surname>McCann</surname><given-names>Jenny</given-names></name><xref ref-type="aff" rid="I1">1</xref></contrib><contrib id="A4" contrib-type="author"><name><surname>Duffy</surname><given-names>Stephen</given-names></name><xref ref-type="aff" rid="I1">1</xref><xref ref-type="aff" rid="I3">3</xref></contrib><contrib id="A5" contrib-type="author"><name><surname>Luben </surname><given-names>Robert</given-names></name><xref ref-type="aff" rid="I1">1</xref></contrib><contrib id="A6" contrib-type="author"><name><surname>Day</surname><given-names>Nicholas</given-names></name><xref ref-type="aff" rid="I1">1</xref><xref ref-type="aff" rid="I3">3</xref></contrib> | Breast Cancer Research | <sec><title>Introduction</title><p>Overall, epidemiological studies [<xref ref-type="bibr" rid="B1">1</xref>,<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B4">4</xref>] have
reported no substantial association between cigarette smoking and the risk of
breast cancer. Some studies [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B7">7</xref>] reported a significant increase of
breast cancer risk among smokers. It has been suggested [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B21">21</xref>,<xref ref-type="bibr" rid="B24">24</xref>] that passive exposure to cigarette smoking may alter prior
associations seen when only active smoking was assessed, with increased risk
being observed for passive smoking exposure. Furthermore, there is a
possibility of heterogeneity in the response to the carcinogenic effect of
smoking, which might explain inconsistent findings for cigarette smoking as a
risk factor for breast cancer [<xref ref-type="bibr" rid="B20">20</xref>].</p><p>In recent studies that addressed the association between breast cancer
and cigarette smoking, however, there was some suggestion of a decreased risk
[<xref ref-type="bibr" rid="B8">8</xref>,<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B10">10</xref>],
especially among current smokers, ranging from approximately 10 to 30% [<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B10">10</xref>]. Brunet <italic>et al</italic> [<xref ref-type="bibr" rid="B11">11</xref>] reported that smoking might reduce the risk of breast
cancer by 44% in carriers of <italic>BRCA1</italic> or <italic>BRCA2</italic> gene
mutations.</p><p>Wolfe [<xref ref-type="bibr" rid="B21">21</xref>] described four different
mammographic patterns that are created by variations in the relative amounts of
fat, epithelial and connective tissue in the breast, designated N1, P1, P2 and
DY. Women with either P2 or DY patterns are considered to be at greater risk
for breast cancer than those with N1 or P1 pattern [<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B13">13</xref>,<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B15">15</xref>].</p><p>There are no published studies that assessed the relationship between
smoking and mammographic parenchymal patterns.</p><p>The aim of the present study was to evaluate whether mammographic
parenchymal patterns as classified by Wolfe [<xref ref-type="bibr" rid="B12">12</xref>], which
have been positively associated with breast cancer risk, are affected by
smoking. In the present case-control study, nested within the European
Prospective Investigation on Cancer in Norfolk (EPIC-Norfolk) cohort [<xref ref-type="bibr" rid="B16">16</xref>],
the association between smoking habits and mammographic parenchymal patterns
are examined. The full results will be published elsewhere.</p></sec><sec sec-type="materials|methods"><title>Materials and methods</title><p>Study subjects were members of the EPIC cohort in Norwich [<xref ref-type="bibr" rid="B16">16</xref>], who also attended the prevalence screening round at the
Norwich Breast Screening Centre between November 1989 and December 1997 and
were free of breast cancer at that screening. A case-control study was designed
within this cohort.</p><p>Cases were defined as women with a P2/DY Wolfe's mammographic
parenchymal pattern on the prevalence screen mammogram. Assuming a 2.5-fold
increase in risk of P2/DY mammographic patterns from the lowest quintile of a
quantitative factor to the highest, 200 cases and 200 controls will yield a
power of approximately 90%. A total of 203 women with P2/DY patterns were
identified as cases and were individually matched by date of birth (within 1
year) and date of prevalence screening (within 3 months) to 203 women with
N1/P1 patterns who served as controls. Additional information regarding case
selection is presented elsewhere [<xref ref-type="bibr" rid="B25">25</xref>].</p><p>We examined the screening records of each woman. Mammograms of both
breasts were collected. Two views, the mediolateral and craniocaudal
mammograms, of both breasts were independently reviewed by two of the authors
(ES and RW) to determine the Wolfe mammographic parenchymal pattern. The
inter-reader agreement in the classification of mammographic parenchymal
patterns was 95% on the four pattern categories, and 99% when the P2 and DY
categories were combined, but for the purposes of the present study we used
only the films in which we agreed on the patterns.</p><p>Considerable information on health and lifestyle factors was available
from the EPIC Health and Lifestyle Questionnaire [<xref ref-type="bibr" rid="B16">16</xref>].
In the present study we examined the subjects' personal and family history of
benign breast diseases and cancer, menstrual and reproductive factors, oral
contraception and hormone replacement therapy, physical activity, smoking, and
anthropometric information such as body mass index and waist:hip ratio.</p><sec><title>Statistical methods</title><p>Odds ratios (ORs) and their 95% confidence intervals (CIs) were
calculated by conditional logistic regression, which takes into account the
matching of controls to cases [<xref ref-type="bibr" rid="B17">17</xref>]. Adjustment was
performed for those variables that were previously found to be associated with
high-risk mammographic parenchymal patterns [<xref ref-type="bibr" rid="B25">25</xref>].</p></sec></sec><sec><title>Results</title><p>The characteristics of the cases and controls are presented in Table
<xref ref-type="table" rid="T1">1</xref>. The mean age of cases and controls was similar
(because they were matched on date of birth). Cases were leaner than controls.
A larger percentage of cases were nulliparous, similar proportions of cases and
controls had between one and three births, and a larger proportion of controls
had more than three births. A larger proportion of cases were pre-menopausal,
current hormone replacement therapy users, had a personal history of benign
breast diseases, and had had a hysterectomy, whereas a larger proportion of
controls were current smokers. The cases and controls were similar with respect
to age at menarche and age at menopause.</p><p>Table <xref ref-type="table" rid="T2">2</xref> shows the unadjusted and adjusted OR
estimates for Wolfe's high-risk mammographic parenchymal patterns and smoking
in the total study population and in post-menopausal women separately. Current
smoking was strongly and inversely associated with high-risk patterns, after
adjustment for concomitant risk factors. Relative to never smokers, current
smokers were significantly less likely to have a high-risk pattern (OR 0.37,
95% CI 0.14-0.94). Similar results were obtained when the analysis was confined
to postmenopausal women. Past smoking was not related to the mammographic
parenchymal patterns. The overall effect in postmenopausal women lost its
statistical significance when adjusted for other risk factors for P2/DY
patterns that were found to be significant in this study, although the results
are still strongly suggestive. There was no interaction between cigarette
smoking and body mass index (<italic>P</italic> =0.73 and 0.72 in the whole study
population and in postmenopausal women, respectively).</p></sec><sec><title>Discussion</title><p>In the present study, we found a strong inverse relationship between
current smoking and mammographic parenchymal patterns of breast tissue as
classified by Wolfe [<xref ref-type="bibr" rid="B12">12</xref>]. These findings are not
completely unprecedented; Greendale <italic>et al</italic> [<xref ref-type="bibr" rid="B18">18</xref>]
found a reduced risk of breast density in association with smoking, although
the magnitude of the reduction was unclear. Our findings suggest that this
reduction is large.</p><p>Recent studies [<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B10">10</xref>]
suggest that breast cancer risk may be reduced among current smokers. In a
multicentre Italian case-control study, Braga <italic>et al</italic> [<xref ref-type="bibr" rid="B10">10</xref>] found that, relative to nonsmokers, current smokers had a
reduced risk of breast cancer (OR 0.84, 95% CI 0.7-1.0). These findings were
recently supported by Gammon <italic>et al</italic> [<xref ref-type="bibr" rid="B9">9</xref>], who
reported that breast cancer risk in younger women (younger than 45 years) may
be reduced among current smokers who began smoking at an early age (OR 0.59,
95% CI 0.41-0.85 for age 15 years or younger) and among long-term smokers (OR
0.70, 95% CI 0.52-0.94 for those who had smoked for 21 years or longer).</p><p>The possible protective effect might be due to the anti-oestrogenic
effect of smoking [<xref ref-type="bibr" rid="B1">1</xref>,<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B19">19</xref>]. Exposure to cigarette smoking causes an earlier menopause
[<xref ref-type="bibr" rid="B1">1</xref>,<xref ref-type="bibr" rid="B26">26</xref>]. Smoking appears to alter
the metabolism of oestradiol leading to enhanced formation of the inactive
catechol estrogens [<xref ref-type="bibr" rid="B1">1</xref>]. Furthermore, smoking increases
circulating androgens through adrenal cortical stimulation [<xref ref-type="bibr" rid="B2">2</xref>], but the conversion rates of androgens to oestrogens are
lower in those who smoke [<xref ref-type="bibr" rid="B27">27</xref>]. There has been a recent
resurgence of interest in the potential effect of smoking on breast cancer
risk, and whether individuals may respond differently on the basis of
differences in metabolism of bioproducts of smoking [<xref ref-type="bibr" rid="B20">20</xref>,<xref ref-type="bibr" rid="B21">21</xref>]. Different relationships between
smoking and breast cancer risk have been suggested that are dependent on the
rapid or slow status of acetylators of aromatic amines [<xref ref-type="bibr" rid="B20">20</xref>,<xref ref-type="bibr" rid="B21">21</xref>], rapid acetylators being better
able to inactivate the potential carcinogenic tobacco compounds. More recent
studies [<xref ref-type="bibr" rid="B22">22</xref>,<xref ref-type="bibr" rid="B23">23</xref>] do not support
these findings, however.</p><p>The present study design minimized the opportunity for bias to
influence the findings. Systematic error in the assessment of mammograms was
avoided because reading was done without knowledge of the risk factor data.
Because subjects were unaware of their own case-control status, the possibility
of recall bias in reporting smoking status was minimized. Furthermore, the
associations observed are unlikely to be explained by the confounding effect of
other known breast cancer risk factors, because we adjusted for these in the
analysis. We did not have information on passive smoking status, however, which
has recently been reported as a possible confounder [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B21">21</xref>,<xref ref-type="bibr" rid="B24">24</xref>].</p><p>Although, ideally we would have liked to evaluate the relationship
between intensity and duration of smoking and mammographic parenchymal patterns
among current smokers, the numbers were too small to perform the analysis.
Trends for intensity and duration of smoking were not monotonic, and <italic>P</italic>
values were inconclusive (between 0.05 and 0.1). Age at menopause and time
since menopause were not related to mammographic patterns in the present study
(data not shown). Although current smokers were likely to have an early
menopause (70% of current smokers were postmenopausal before age 50 years),
there was no difference among mean age at menopause in the three smoking
categories (<italic>P</italic> = 0.15). There was no difference in time since menopause
among current smokers.</p><p>These data indicate that adjustment for current smoking status is
important when evaluating the relationship between mammographic parenchymal
patterns and breast cancer risk. They also indicate smoking to be a prominent
potential confounder when analyzing effects of other risk factors, such as
obesity-related variables. It appears that parenchymal patterns may act as an
informative biomarker of the effect of cigarette smoking on breast cancer
risk.</p></sec> |
Altered expression of estrogen receptor-α variant messenger RNAs
between adjacent normal breast and breast tumor tissues | <sec><title>Introduction:</title><p>Estrogen receptor (ER)-α and ER-β are believed to
mediate the action of estradiol in target tissues. Several ER-α and
ER-β variant messenger RNAs have been identified in both normal and
neoplastic human tissues. Most of these variants contain a deletion of one or
more exons of the wild-type (WT) ER messenger RNAs. The putative proteins that
are encoded by these variant messenger RNAs would therefore be missing some
functional domains of the WT receptors, and might interfere with WT-ER
signaling pathways. The detection of ER-α variants in both normal and
neoplastic human breast tissues raised the question of their possible role in
breast tumorigenesis.</p><p>We have previously reported an increased relative expression of
exon 5 deleted ER-α variant (ERD5) messenger RNA and of another ER-α
variant truncated of all sequences following the exon 2 of the WT ER-α
(ERC4) messenger RNA in breast tumor samples versus independent normal breast
tissues. In contrast, a decreased relative expression of exon 3 deleted
ER-α variant (ERD3) messenger RNA in tumor tissues and cancer cell lines
versus independent normal reduction mammoplasty samples has recently been
reported. These data were obtained in tissues from different individuals and
possible interindividual differences cannot be excluded.</p></sec><sec><title>Aims:</title><p>The goal of this study was to investigate the expressions of ERC4,
ERD5 and ERD3 variant messenger RNAs in normal breast tissues and their matched
adjacent primary breast tumor tissues.</p></sec><sec><title>Materials and methods:</title><p>Eighteen cases were selected from the Manitoba Breast Tumor Bank,
which had well separated and histopathologically characterized normal and
adjacent neoplastic components. All tumors were classified as primary invasive
ductal carcinomas. Six tumors were ER-negative/progesterone receptor
(PR)-negative, nine were ER-positive/PR-positive, two were
ER-positive/PR-negative, and one was ER-negative/PR-positive, as measured by
ligand-binding assay. For each specimen, total RNA was extracted from frozen
normal and tumor tissue sections and was reverse transcribed. The expressions
of ERC4, ERD3 and ERD5 messenger RNAs relative to WT ER-α messenger RNA
were investigated by previously validated semiquantitative reverse
transcription polymerase chain reaction (PCR) assays performed using three
different sets of primers.</p></sec><sec><title>Results:</title><p>As shown Figure <xref ref-type="fig" rid="F2">1a</xref>, two PCR products were
obtained that corresponded to WT ER and ERC4 messenger RNAs. For each case, the
mean of the ratios obtained in at least three independent PCR experiments is
shown for both normal and tumor compartments (Fig <xref ref-type="fig" rid="F2">1b</xref>). A
statistically higher ERC4 messenger RNA relative expression was found in the
neoplastic components of ER-positive/PR-positive tumors, as compared with
matched adjacent normal tissues (<italic>n</italic> = 9; <italic>P</italic> = 0.019, Wilcoxon
signed-rank test).</p><p>Two PCR products were obtained that corresponded to WT ER and ERD3
messenger RNAs (Fig <xref ref-type="fig" rid="F2">2a</xref>). A significantly higher
expression of ERD3 messenger RNA was observed in the normal compared with the
adjacent neoplastic components of ER-positive subset (<italic>n</italic> =8; <italic>P</italic>
=0.023, Wilcoxon signed-rank test; Fig <xref ref-type="fig" rid="F2">2b</xref>).</p><p>Two PCR products were obtained that corresponded to WT ER and ERD5
complementary DNAs (Fig <xref ref-type="fig" rid="F2">3a</xref>). As shown in Figure
<xref ref-type="fig" rid="F3">3b</xref>, a statistically significant higher relative
expression of ERD5 messenger RNA was observed in tumor components when this
expression was measurable in both normal and adjacent tumor tissues (<italic>n</italic>
=15; <italic>P</italic> =0.035, Wilcoxon signed-rank test).</p></sec><sec><title>Discussion:</title><p>A statistically significant higher ERC4 messenger RNA expression
was found in ER-positive/PR-positive tumors as compared with matched normal
breast tissues. ERC4 variant messenger RNA has previously been demonstrated to
be more highly expressed in ER-positive tumors that showed poor as opposed to
tumors that showed good prognostic characteristics. Interestingly, we also have
reported similar levels of expression of ERC4 messenger RNA in primary breast
tumors and their concurrent axillary lymph node metastases. Taken together,
these data suggest that the putative role of the ERC4 variant might be
important at different phases of breast tumorigenesis and tumor progression;
alteration of ERC4 messenger RNA expression and resulting modifications in ER
signaling pathway probably occur before breast cancer cells acquire the ability
to metastasize. Transient expression assays revealed that the protein encoded
by ERC4 messenger RNA was unable to activate the transcription of an
estrogen-responsive element-reporter gene or to modulate the wild-type ER
protein activity. The biologic significance of the changes observed in ERC4
messenger RNA expression during breast tumorigenesis remains to be
determined.</p><p>A higher relative expression of ERD3 messenger RNA in the normal
breast tissue components compared with adjacent neoplastic tissue was found in
the ER-positive subgroup. These data are in agreement with the recently published report of
Erenburg <italic>et al</italic>, who showed a decreased relative expression of ERD3
messenger RNA in neoplastic breast tissues compared with independent reduction
mammoplasty and breast tumor. Transfection experiments showed that the
activation of the transcription of the pS2 gene by estrogen was drastically
reduced in the presence of increased ERD3 expression. The authors hypothesized
that the reduction in ERD3 expression could be a prerequisite for breast
carcinogenesis to proceed.</p><p>We observed a significantly higher relative expression of ERD5
messenger RNA in breast tumor components compared with matched adjacent normal
breast tissue. These data confirm our previous observations performed on
unmatched normal and neoplastic human breast tissues. Upregulated expression of
this variant has already been reported in ER-negative/PR-positive tumors, as
compared with ER-positive/PR-positive tumors, suggesting a possible correlation
between ERD5 messenger RNA expression and breast tumor progression. Even though
it has been suggested that ERD5 could be related to the acquisition of
insensitivity to antiestrogen treatment (ie tamoxifen), accumulating data
refute a general role for ERD5 in hormone-resistant tumors. Only ER-positive
pS2-positive tamoxifen-resistant tumors have been shown to express
significantly higher levels of ERD5 messenger RNA, as compared with control
tumors. Taken together, these data suggest that the exact biologic significance
of ERD5 variant expression during breast tumorigenesis and breast cancer
progression, if any, remains unclear.</p><p>In conclusion, we have shown that the relative expressions of ERC4
and ERD5 variant messenger RNAs were increased in human breast tumor tissue, as
compared with normal adjacent tissue, whereas the expression of ERD3 variant
messenger RNA was decreased in breast tumor tissues. These results suggest that
the expressions of several ER-α variant messenger RNAs are deregulated
during human breast tumorigenesis. Further studies are needed to determine
whether these changes are transposed at the protein level. Furthermore, the
putative role of ER-α variants in the mechanisms that underlie breast
tumorigenesis remains to be determined.</p></sec> | <contrib id="A1" contrib-type="author"><name><surname>Leygue</surname><given-names>Etienne</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>eleygue@cc.umanitoba.ca</email></contrib><contrib id="A2" contrib-type="author"><name><surname>Dotzlaw</surname><given-names>Helmut</given-names></name><xref ref-type="aff" rid="I1">1</xref></contrib><contrib id="A3" contrib-type="author"><name><surname>Watson</surname><given-names>Peter H</given-names></name><xref ref-type="aff" rid="I1">1</xref></contrib><contrib id="A4" contrib-type="author"><name><surname>Murphy</surname><given-names>Leigh C</given-names></name><xref ref-type="aff" rid="I1">1</xref></contrib> | Breast Cancer Research | <sec><title>Introduction</title><p>Estrogen receptor (ER)-α and ER-β are believed to mediate
the action of estradiol in target tissues [<xref ref-type="bibr" rid="B1">1</xref>,<xref ref-type="bibr" rid="B2">2</xref>]. These two receptors, which belong to the steroid/retinoic
acid/thyroid receptor superfamily [<xref ref-type="bibr" rid="B3">3</xref>], contain several
structural and functional domains [<xref ref-type="bibr" rid="B4">4</xref>] that are encoded by
two messenger RNAs that contain eight exons [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B6">6</xref>]. Upon ligand binding, ER-α and ER-β proteins
recognize specific estrogen-responsive elements located in DNA in the proximity
of target genes, and through interactions with several coactivators modulate
the transcription of these genes [<xref ref-type="bibr" rid="B7">7</xref>].</p><p>Several ER-α and ER-β variant messenger RNAs have been
identified in both normal and neoplastic human tissues [<xref ref-type="bibr" rid="B8">8</xref>,<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B10">10</xref>,<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B12">12</xref>]. Most of these variants contain
a deletion of one or more exons of the wild-type (WT)-ER messenger RNA. The
putative proteins encoded by these variant messenger RNAs would therefore be
missing some functional domains of the WT receptors and might interfere with WT
ER signaling pathways. Indeed, <italic>in vitro</italic> functional studies have shown
that some recombinant ER-α variant proteins can affect estrogen-regulated
gene transcription. For example, the variant protein encoded by exon 3 deleted
ER-α variant (ERD3) messenger RNA, which is missing the second zinc finger
of the DNA binding domain, has been shown [<xref ref-type="bibr" rid="B13">13</xref>] to have a
dominant negative activity on WT ER-α receptor action. A similar dominant
negative activity has been observed for ERD5 variant protein (encoded by an
ER-α variant messenger RNA deleted in exon 5 sequences), which is missing
a part of the hormone-binding domain of the WT molecule [<xref ref-type="bibr" rid="B14">14</xref>]. Interestingly, a constitutive hormone-independent
activity [<xref ref-type="bibr" rid="B15">15</xref>] and a WT enhancing activity [<xref ref-type="bibr" rid="B16">16</xref>] have also been attributed to ERD5 variant protein in
different systems. The relevance of the levels achieved in these transfection
experiments to <italic>in vivo</italic> expression remains unclear. It should also be
noted that these functional activities are likely to be cell-type and promoter
specific [<xref ref-type="bibr" rid="B8">8</xref>].</p><p>The discovery that these ER-α variants are expressed in both
normal and neoplastic human breast tissues, however, raised the question of
their possible role in breast tumorigenesis [<xref ref-type="bibr" rid="B8">8</xref>]. We have
previously reported an increased relative expression of ERD5 messenger RNA and
of ERC4 messenger RNA, another ER-α variant messenger RNA that is
truncated of all sequences following the exon 2 of the WT ER-α [<xref ref-type="bibr" rid="B17">17</xref>], in breast tumor samples versus independent normal breast
tissues [<xref ref-type="bibr" rid="B18">18</xref>,<xref ref-type="bibr" rid="B19">19</xref>]. In contrast,
Erenburg <italic>et al</italic> [<xref ref-type="bibr" rid="B20">20</xref>] recently reported a
decreased relative expression of ERD3 messenger RNA in tumor tissues and cancer
cell lines versus independent normal reduction mammoplasty samples. Those data,
which suggested that alteration in ERD5, ERD3 and clone 4 messenger RNA
expression might occur during breast tumorigenesis, were obtained in tissues
from different individuals, and possible interindividual differences cannot be
excluded.</p><p>In order to clarify this issue, we investigated the expression of
these three variant messenger RNAs in normal breast tissues and their matched
adjacent primary breast tumor tissues.</p></sec><sec sec-type="materials|methods"><title>Materials and methods</title><sec><title>Human breast tissues and reverse transcription</title><p>In order to investigate the expressions of ERC4, ERD3 and ERD5
messenger RNA relative to WT-ER messenger RNA within matched normal and breast
tumor tissues, eighteen cases were selected in the National Cancer Institute of
Canada Manitoba Breast Tumor Bank (Winnipeg, Manitoba, Canada), which had well
separated and histopathologically characterized normal and adjacent neoplastic
components. The Tumor Bank, which serves as a national Tumor Bank and is funded
by the National Cancer Institute of Canada, has been reviewed and received
approval from the Ethics Review Committee, Faculty of Medicine, University of
Manitoba.</p><p>The processing of specimens collected in the Manitoba Breast Tumor
Bank has already been described [<xref ref-type="bibr" rid="B21">21</xref>]. Briefly, each
specimen had been rapidly frozen as soon as possible after surgical removal. A
portion of the frozen tissue block was processed to create a paraffin-embedded
tissue block that was matched and oriented relative to the remaining frozen
block. These paraffin blocks provide high quality histologic sections, which
are used for pathologic interpretation and assessment, and are mirror images of
the frozen sections used for RNA extractions.</p><p>For each case, tumor and adjacent normal tissues from the same
individual were histologically characterized by observation of paraffin
sections. The presence of normal ducts and lobules, as well as the absence of
any atypical lesion, were confirmed in all normal tissue specimens. All tumor
components were classified as primary invasive carcinomas. Seven tumors were
ER-negative (ER < 3 fmol/mg protein), with progesterone receptor (PR) values
ranging from 2.2 to 11.2f mol/mg protein, as measured using ligand-binding assay
[<xref ref-type="bibr" rid="B22">22</xref>]. Axillary nodal metastases were observed in five of
these cases. Eleven tumors were ER-positive (ER values ranged from 3.5 to
159 fmol/mg protein), with PR values ranging from 5.8 to 134 fmol/mg protein.
These tumors spanned a wide range of grades (grades 5-9, median 7.5), which
were determined using the Nottingham grading system [<xref ref-type="bibr" rid="B23">23</xref>]. Axillary nodal metastases were observed in one of these
cases. Patients were from 39 to 86 years old (median 54 years). Total RNA was
extracted from frozen tissue sections and reverse-transcribed in a final volume
of 25 μ l as previously described [<xref ref-type="bibr" rid="B18">18</xref>]. The quality
of complementary DNAs obtained was assessed by amplification of the
ubiquitously expressed glyceraldehyde-3-phosphate dehydrogenase complementary
DNA, as described previously [<xref ref-type="bibr" rid="B18">18</xref>].</p></sec><sec><title>Triple primer polymerase chain reaction</title><p>A previously described triple primer polymerase chain reaction (PCR)
assay has been used to coamplify ERC4 and WT-ER-α complementary DNAs
[<xref ref-type="bibr" rid="B19">19</xref>,<xref ref-type="bibr" rid="B24">24</xref>]. Primers used consisted
of ERU primer (5' -TGTGCAATGACTATGCTTCA-3', sense, located in WT-ER
exon 2, position 792-811), ERL primer (5' -GCTCTTCCTCCTGTTTTTAT-3',
antisense, located in WT-ER exon 3, position 940-921), and C4L primer (5'
-TTTCAGTCTTCAGATACCCCAG-3', antisense, located in ERC4 sequence, position
1336-1315). The given positions correspond to the published sequences for WT-ER
[<xref ref-type="bibr" rid="B1">1</xref>] and ERC4 [<xref ref-type="bibr" rid="B17">17</xref>].</p><p>PCR amplifications were performed as previously described [<xref ref-type="bibr" rid="B18">18</xref>,<xref ref-type="bibr" rid="B24">24</xref>]. Briefly, 0.2 μ l reverse transcription mixture was
amplified in a final volume of 15 μ l, in the presence of 1.5 μ Ci of
[α-<sup>32</sup>P] deoxycytidine triphosphate (dCTP; 3000 Ci/mmol),
4 ng/μl of each primer and 0.3 unit of Taq DNA polymerase. Each cycle
consisted of 1min at 94°C, 30s at 60°C and 1min at 72°C. PCR
products were then separated on 6% polyacrylamide gels containing 7mol/l urea
(polyacrylamide gel electrophoresis). After electrophoresis, the gels were
dried and autoradiographed. Two PCR products were obtained, which were
identified by subcloning and sequencing, performed as previously described
[<xref ref-type="bibr" rid="B18">18</xref>]. PCR products migrating with the apparent size of
149 and 536 base pairs were shown to correspond to WT-ER and ERC4 complementary
DNAs, respectively.</p></sec><sec><title>Polymerase chain reaction</title><p>Two different primer sets, ERD3 and ERD5, were used to coamplify
WT-ER and ERD3 complementary DNAs, and WT-ER and ERD5 complementary DNAs,
respectively. ERD3 primer set consisted of D3U primer (5'
-TGTGCAATGACTATGCTTCA-3', sense, located in WT-ER exon 2, position
792-811) and D3L primer (5' -TGTTCTTCTTAGAGCGTTTGA-3', antisense,
located in WT-ER exon 4, position 1145-1125). ERD5 primer set consisted of D5U
primer (5' -CAGGGGTGAAGTGGGGTCTGCTG-3', sense, located in WT-ER
exon 4, position 1060-1082) and D5L primer (5'-α
TGCGGAACCGAGATGATGTAGC-3', anti-sense, located in WT-ER exon 6, position
1542-1520). The given positions correspond to published sequences for WT-ER
[<xref ref-type="bibr" rid="B1">1</xref>].</p><p>PCR amplifications were performed and PCR products analyzed as
previously described [<xref ref-type="bibr" rid="B18">18</xref>]. Briefly, 0.2 μ l reverse
transcription mixture was amplified in a final volume of 15 μ l, in the
presence of 1.5 μ Ci of [α-<sup>32</sup>P] dCTP (3000 Ci/mmol),
4ng/μ l of each primer of the primer set considered (ERD3 or ERD5 primer
set) and 0.3 unit of Taq DNA polymerase. Each cycle consisted of 30s at
94°C, 30s at 60°C and 30s at 72°C. PCR products were then
separated on 6% polyacrylamide gels containing 7mol/l urea (polyacrylamide gel
electrophoresis). Following electrophoresis, the gels were dried and
autoradiographed. For each PCR, two PCR products were obtained, which were
identified by subcloning and sequencing. PCR products migrating with the
apparent size of 354 and 483 base pairs, using ERD3 and ERD5 primer set,
respectively, were shown to correspond to WT-ER complementary DNA. PCR products
migrating with the apparent size of 237 and 344 base pairs, using ERD3 and ERD5
primer set, were shown to correspond to ERD3 and ERD5 complementary DNAs,
respectively.</p></sec><sec><title>Quantitation and statistical analysis</title><p>For each experiment, bands corresponding to the variant messenger
RNA (ie ERC4, ERD3 or ERD5) and to WT-ER were excised from the gel and counted
in a scintillation counter. For each set of primers (ie ERC4, ERD3 and ERD5
primer set) and for each sample, four independent PCR assays were performed.
The ratios between ERC4, ERD3 or ERD5 signals and corresponding WT-ER signal
were calculated. For each experiment, in order to correct for overall
interassay variations (due to different batches of radiolabelled [α
-<sup>32</sup>P] dCTP or of Taq DNA polymerase), the ratio observed in the same
particular tumor (case number 12) was arbitrarily given the value of one and
all other ratios expressed relatively. Under our experimental conditions, some
samples did not have measurable levels (ie signal lower than twice the
background value) of ERD3 or ERD5 variant messenger RNAs (see Figs
<xref ref-type="fig" rid="F2">2a</xref> and <xref ref-type="fig" rid="F3">3a</xref>) in any of the four
repetitions performed. Only cases that had detectable levels in at least three
of the replicates in both their normal and tumor compartments were included in
the statistical analysis. The significance of the differences in the relative
levels of expression of ERC4, ERD3 and ERD5 messenger RNAs between matched
normal and tumor components was determined using the Wilcoxon signed-rank
test.</p></sec></sec><sec><title>Results</title><sec><title>Relative expression of ERC4 messenger RNA in matched normal and
breast tumor tissues</title><p>A recently described triple-primer PCR assay was used to compare the
relative expressions of ERC4 messenger RNA between adjacent normal and tumor
components [<xref ref-type="bibr" rid="B19">19</xref>,<xref ref-type="bibr" rid="B24">24</xref>]. In this
assay, three primers are used simultaneously during the PCR: the upper primer
is able to recognize both WT-ER and ERC4 complementary DNA sequences, whereas
the two lower primers are specific for each complementary DNA. Competitive
amplification of two PCR products occurs, giving a final PCR product ratio
related to the initial input of target complementary DNAs. This approach has
been validated previously both by competitive amplification of spiked
complementary DNA preparations [<xref ref-type="bibr" rid="B19">19</xref>] and by comparison to
RNAse protection assays [<xref ref-type="bibr" rid="B24">24</xref>].</p><p>As shown Figure <xref ref-type="fig" rid="F1">1a</xref>, two PCR products were
obtained, which migrated at the apparent size of 149 and 536 base pairs. These
products have been shown to correspond to WT-ER and ERC4 messenger RNAs,
respectively [<xref ref-type="bibr" rid="B24">24</xref>]. One should note the presence, in
samples where WT-ER and ERC4 signals are high (Fig <xref ref-type="fig" rid="F1">1a</xref>,
lane 5), of minor additional bands, one of which has been previously identified
as corresponding to exon 2-duplicated ER-α variant complementary DNA
[<xref ref-type="bibr" rid="B24">24</xref>]. The presence of these minor PCR products did not
interfere with the quantitative aspect of the triple-primer PCR assay [<xref ref-type="bibr" rid="B24">24</xref>]. For each case, the mean of the ratios obtained in at
least three independent PCR experiments, expressed in arbitrary units, is shown
for both normal and tumor compartments (Fig <xref ref-type="fig" rid="F1">1b</xref>). A higher
clone 4 messenger RNA relative expression in the tumor compartment was observed
in 12 out of 18 cases. This difference did not, however, reach statistical
significance (<italic>P</italic> = 0.47, Wilcoxon signed-rank test). When considering
only the ER-positive/PR-positive subset (<italic>n</italic> = 9), as measured by the
ligand-binding assay, a statistically higher ERC4 messenger RNA relative
expression was found in the neoplastic components, as compared with matched
adjacent normal tissues (<italic>P</italic> = 0.019, Wilcoxon signed-rank test).</p></sec><sec><title>Relative expression of ERD3 messenger RNA in matched normal and
breast tumor tissues</title><p>A PCR assay, performed using primers annealing to sequences in exons
2 and 4, was used to investigate ERD3 messenger RNA expression relative to
WT-ER in these 18 matched cases. We [<xref ref-type="bibr" rid="B18">18</xref>] and others
[<xref ref-type="bibr" rid="B25">25</xref>] have previously shown that the coamplification of
WT-ER and an exon-deleted ER-α variant complemetary DNA resulted in the
amplification of two PCR products, the relative signal intensity of which
provided a previously validated measurement of exon-deleted ER-α variant
expression.</p><p>Two PCR products were obtained, that migrated with an apparent size
of 354 and 237 base pairs (Fig <xref ref-type="fig" rid="F2">2a</xref>). These fragments were
shown by subcloning and sequencing to correspond to WT-ER and ERD3 messenger
RNAs (data not shown). The relative ERD3 signal was measurable in the normal
and in the tumor compartments of 13 cases (Fig <xref ref-type="fig" rid="F2">2b</xref>). Out
of these 13 cases, ERD3 messenger RNA expression was higher in the normal
compartment in 10 cases. This difference, however, did not reach statistical
significance (<italic>P</italic> = 0.057, Wilcoxon signed-rank test). A significantly
higher expression of ERD3 messenger RNA in the normal compared with the
adjacent neoplastic components was found when only the ER-positive subset was
considered, however (<italic>n</italic> = 8; <italic>P</italic> = 0.023, Wilcoxon signed-rank
test).</p></sec><sec><title>Relative expression of ERD5 messenger RNA in matched normal and
breast tumor tissues</title><p>Using primers annealing to sequences in exons 4 and 6 of WT-ER, we
also investigated the relative expression of ERD5 messenger RNA in these 18
matched cases. Two PCR products were detected, that migrated at an apparent
size of 483 and 344 base pairs, and that have previously been shown to
correspond to WT-ER and ERD5 complementary DNAs, respectively (Fig
<xref ref-type="fig" rid="F3">3a</xref>). As shown in Fig <xref ref-type="fig" rid="F3">3b</xref>, a
statistically significant higher relative expression of ERD5 messenger RNA was
observed in tumor components when this expression was measurable in both normal
and adjacent tumor tissues (<italic>n</italic> = 15; <italic>P</italic> = 0.035, Wilcoxon
signed-rank test).</p></sec></sec><sec><title>Discussion</title><p>The expression of ERC4, ERD3 and ERD5 variant messenger RNAs relative
to WT-ER messenger RNA expression within adjacent normal and neoplastic human
breast tissues was investigated using previously described semi-quantitative
reverse transcription PCR assays [<xref ref-type="bibr" rid="B18">18</xref>,<xref ref-type="bibr" rid="B19">19</xref>,<xref ref-type="bibr" rid="B24">24</xref>]. These assays allow the
determination of the expression of ER-α variant messenger RNA relative to
WT-ER messenger RNA using a very small amount of starting material, and offer
the advantage of allowing investigators to work with histopathologically well
characterized human breast tissue regions. It should be noted, however, that
the sensitivities of the assays used in this study differed from each other.
The triple-primer PCR assay has previously been set up to allow the
determination of ERC4 relative expression in tumor samples with very low ER
levels, as measured by ligand-binding assay [<xref ref-type="bibr" rid="B24">24</xref>].</p><p>We showed that, in samples with a detectable level of ERC4 messenger
RNA using a standardized RNAse protection assay, the relative expression of
this variant to WT-ER messenger RNA expression was similar to the relative
expression of ERC4 PCR product obtained after triple-primer PCR [<xref ref-type="bibr" rid="B24">24</xref>]. Triple-primer PCR assay applied to the detection of ERC4
messenger RNA in 18 matched normal and tumor breast tissues gave a measurable
value of expression in 36 out of the 36 samples studied. This contrasts with
the detection of 30 out of 36 and 33 out of 36 obtained using ERD3-specific and
ERD5-specific primers, respectively. These differences in sensitivity probably
result from different primer set efficiencies under our experimental
conditions.</p><p>A higher ERC4 messenger RNA relative expression in tumor components
compared with the normal adjacent tissue component has been observed in the
ER-positive/PR-positive subgroup. This result is in agreement with our previous
data [<xref ref-type="bibr" rid="B19">19</xref>] obtained by comparing ERC4 messenger RNA
expression between independent normal reduction mammoplasty samples and a group
of ER-positive/PR-positive breast tumors. Even though a higher ERC4 messenger
RNA relative expression was observed in the tumor component of 12 out of 18
cases, this difference did not reach statistical significance. This absence of
statistically significant differences might result from the low number of
matched cases studied or from the different biology of ER-negative cases.
Further studies are needed to clarify this issue and to draw any conclusion
regarding the expression of ERC4 messenger RNA in ER-negative samples.</p><p>ERC4 variant messenger RNA has previously been shown [<xref ref-type="bibr" rid="B26">26</xref>] to be more highly expressed in ER-positive tumors that
show poor prognostic characteristics (presence of more than four axillary lymph
nodes, tumor size >2 cm, aneuploid, high percentage S-phase cells) than in
ER-positive tumor with good prognostic characteristics (absence of axillary
lymph node, tumor size <2 cm, diploid, low percentage S-phase cells).
Moreover, in that previous study, a higher ERC4 messenger RNA expression was
also observed in ER-positive/PR-negative tumors, as compared with
ER-positive/PR-positive tumors. interestingly, we have also recently reported
similar levels of expression of ERC4 messenger RNA in primary breast tumors and
their concurrent axillary lymph node metastases [<xref ref-type="bibr" rid="B24">24</xref>].
Taken together, these data suggest that the putative role of the ERC4 variant
might be important at different phases of breast tumorigenesis and tumor
progression; alteration in ERC4 messenger RNA expression and resulting
modifications in ER signaling pathway probably occur before breast cancer cells
acquire the ability to metastasize. Transient expression assays revealed that
the protein encoded by ERC4 messenger RNA was unable to activate the
transcription of an estrogen responsive element-reporter gene or to modulate
WT-ER protein activity [<xref ref-type="bibr" rid="B17">17</xref>]. The biologic significance
of the changes observed in ERC4 messenger RNA expression during breast
tumorigenesis and tumor progression therefore remains unclear.</p><p>A trend toward a higher relative expression of ERD3 messenger RNA in
the normal breast tissue components compared with adjacent neoplastic tissue
was found (10 out of 13 cases), which reached statistical significance when the
ER-positive subgroup only was considered. These data are in agreement with the
recently published report of Erenburg <italic>et al</italic> [<xref ref-type="bibr" rid="B20">20</xref>] who showed a decreased relative expression of ERD3
messenger RNA in neoplastic breast tissues and breast cancer compared with
independent reduction mammoplasty and breast tumor. Transfection experiments
performed by those investigators showed that the activation of the
transcription of the pS2 gene by estrogen was drastically reduced in the
presence of increased ERD3 expression. Moreover, ERD3 transfected MCF-7 human
breast cancer cells had a reduced saturation density, exponential growth rate
and <italic>in vivo</italic> invasiveness, as compared with control cells. These data
led the authors to hypothesize that the reduction of ERD3 expression could be a
prerequisite for breast carcinogenesis to proceed. They suggested that if high
levels of ERD3 could attenuate estrogenic effects in normal breast tissue, low
levels might lead to an excessive and unregulated mitogenic action of
estrogen.</p><p>We observed a significantly higher relative expression of ERD5
messenger RNA in breast tumor components compared with matched adjacent normal
breast tissue. These data confirm our previous observations [<xref ref-type="bibr" rid="B18">18</xref>] performed on unmatched normal and neoplastic human breast
tissues. Upregulated expression of this variant has already been reported in
ER-negative/PR-positive tumors, as compared with ER-positive/PR-positive tumors
[<xref ref-type="bibr" rid="B15">15</xref>,<xref ref-type="bibr" rid="B27">27</xref>], suggesting a possible
correlation between ERD5 messenger RNA expression and breast tumor progression.
Interestingly, ERD5 messenger RNA can be detected in human pituitary adenomas,
but not in normal pituitary samples [<xref ref-type="bibr" rid="B28">28</xref>]. This
underscores the putative involvement of this ER variant in other tumor systems.
Even though it has been suggested that ERD5 could be related to the acquisition
of insensitivity to antiestrogen treatment (ie tamoxifen) [<xref ref-type="bibr" rid="B29">29</xref>,<xref ref-type="bibr" rid="B30">30</xref>], accumulating data refute a
general role for ERD5 in hormone-resistant tumors [<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B25">25</xref>,<xref ref-type="bibr" rid="B31">31</xref>,<xref ref-type="bibr" rid="B32">32</xref>]. Only ER-positive pS2-positive tamoxifen resistant tumors
have been shown to express significantly higher levels of ERD5 messenger RNA,
as compared with control tumors [<xref ref-type="bibr" rid="B33">33</xref>]. Taken together,
these data suggest that the exact biologic significance of ERD5 variant
expression during breast tumorigenesis and breast cancer progression, if any,
remains unclear.</p><p>Among all the articles published so far on ER variants, only one has
investigated ER variant expression between normal and neoplastic matched
samples. Okada <italic>et al</italic> [<xref ref-type="bibr" rid="B33">33</xref>] recently reported a
study performed on 15 cases. They observed an apparent difference in ER variant
messenger RNA expression between adjacent normal and tumor samples. That study
was performed using a less sensitive PCR approach, however, because PCR
products were stained using ethidium bromide, and no attempt was made to
quantify ER variant messenger RNA expression relative to WT-ER messenger RNA
expression.</p><p>In conclusion, we have shown that the relative expression of ERC4 and
ERD5 variant mRNAs was increased in human breast tumor tissue, as compared with
normal adjacent tissue, whereas the expression of ERD3 variant messenger RNA
was decreased in breast tumor tissues. These results, which confirm previous
data obtained on independent human breast tissue samples [<xref ref-type="bibr" rid="B18">18</xref>,<xref ref-type="bibr" rid="B19">19</xref>], suggest that the expressions of
several ER-α variant messenger RNAs are deregulated during human breast
tumorigenesis. Further studies are needed to determine whether these changes
are transposed at the protein level. Only the use of specific antibodies that
are able to recognize specifically the different ER variant proteins putatively
encoded by these variant messenger RNAs will allow this issue the be addressed.
Furthermore, the putative role of ER-α variants in the mechanisms that
underlie breast tumorigenesis remain to be determined.</p></sec> |
The Million Women Study: design and characteristics of the study population | <sec><title>Objectives:</title><p>To describe the design of the Million Women Study and the characteristics of the study population.</p></sec><sec><title>Study design:</title><p>Population-based cohort study of women aged 50-64 in the UK.</p></sec><sec><title>Setting:</title><p>Women are asked to join the Million Women Study when they are invited to routine screening for breast cancer at 61 of the screening centres of the UK National Health Service Breast Screening Programme (NHSBSP). An estimated 71% of women screened by the NHSBSP return a completed questionnaire.</p></sec><sec><title>Participants:</title><p>800 000 women were recruited between May 1996 and June 1999, and it is planned that an additional 200 000 will be recruited by the year 2000.</p></sec><sec><title>Results:</title><p>The characteristics of the first 121 000 women recruited into the Million Women Study are described here. At recruitment 33% of the study population were currently using hormone replacement therapy and 47% had used it at some time. Over half (54%) had used oral contraceptives, and 18% were current smokers at the time of recruitment. Before they were screened 1.4% of the women had been diagnosed with breast cancer in the past, 6% had a mother with a history of breast cancer and 3.7% had a sister with a history of breast cancer. It is estimated that 1 million women will have been recruited by early in the year 2000, and that by the end of the year 2002 there will be 5000 screen-detected breast cancers and 23 000 deaths in the cohort, the majority of which will be attributed to cancer (12 600 deaths) and circulatory disease (8000 deaths).</p></sec><sec><title>Conclusions:</title><p>By the end of the year 2002, the Million Women Study will have sufficient statistical power to detect relative risks of 0.8 or less, or of 1.2 or more in current users compared with never users of hormone replacement therapy for mortality from breast cancer, colorectal cancer, lung and ovarian cancer, ischaemic heart disease and stroke.</p></sec> | <contrib id="A1" contrib-type="author"><collab>The Million Women Study Collaborative Group</collab><xref ref-type="aff" rid="I1">1</xref></contrib> | Breast Cancer Research | <sec><title>Introduction</title><p>The Million Women Study is a nationwide collaborative research project in the UK, the chief aim of which is to describe the relationship between use of hormone replacement therapy (HRT) and the risk of various conditions, particularly breast cancer. The study began in May 1996 and the plan is to recruit and follow-up a cohort of 1 million women invited to attend the UK National Health Service Breast Screening Programme (NHSBSP).</p><p>The NHSBSP was set up in 1988. Once every 3 years each woman in the UK aged between 50 and 64 years who is registered with the NHS is sent a letter by the NHSBSP, offering her routine screening for breast cancer by mammography. About 1 million women are screened annually by the NHSBSP and about 5000 of them have a breast cancer detected on mammography [<xref ref-type="bibr" rid="B1">1</xref>]. The day-to-day organization and screening activities are performed by about 100 separate screening offices throughout the UK, and the work is monitored and statistics gathered centrally by a national co-ordinating centre.</p><p>The present paper describes the design of the Million Women Study and the characteristics of the study population.</p></sec><sec sec-type="methods"><title>Methods</title><p>The Million Women Study is a population-based cohort study. Women are recruited when they are invited for routine breast cancer screening, and the main outcomes to be examined at follow-up are the incidence of screen-detected breast cancer and cause-specific mortality.</p><sec><title>Attendance at screening</title><p>About three-quarters of the women who are invited for screening by the NHSBSP subsequently attend for mammography [<xref ref-type="bibr" rid="B1">1</xref>]. Before the study could be launched, it was necessary to demonstrate that inviting women to join the Million Women Study would not reduce uptake of screening offered by the NHSBSP. During 1994 and 1995 a total of 6000 women who were due to be invited for breast cancer screening in Oxford and West London were randomly divided into two groups. One group was sent the usual invitation for screening and the other group was sent the study questionnaire, accompanying the usual invitation to screening. Attendance rates for screening were similar, at 71%, among those who were and were not sent an accompanying questionnaire [<xref ref-type="bibr" rid="B2">2</xref>].</p></sec><sec><title>Recruitment procedures</title><p>Women are asked to join the Million Women Study by participating NHSBSP screening centres at the time that or just before they are sent their usual invitation for routine breast cancer screening. A questionnaire is included with each woman's invitation and, if the woman wishes to join the study, she is asked to complete the questionnaire, to give signed permission for follow-up, and to return the questionnaire at the time she is screened. A freephone number is provided for women who have any questions or problems filling out the questionnaire. The questionnaire is four pages long (A4 size) and includes questions about lifestyle and sociodemographic factors, reproductive history, past use of oral contraceptives, use of HRT, past medical history and family history of breast cancer. Completed questionnaires are transferred periodically from the participating screening centres to the study co-ordinating centre at the Imperial Cancer Research Fund Cancer Epidemiology Unit (CEU), Oxford, UK.</p></sec><sec><title>Data storage, entry and checking</title><p>The confidential completed questionnaires are stored securely at all times. Once they reach the CEU they are checked and coded by trained staff and then scanned electronically. The scanned data are 'captured' using computerized intelligent character recognition and optical mark reading software (Eyes and Hands<sup>®</sup>; Readsoft Inc, Slough, UK). Range and logical checks are performed at the time of data entry. Any inconsistency or information that is not recognised by the data capture software is verified manually by trained data entry staff, who also validate computer-interpreted data and check each questionnaire to confirm whether signed consent for follow up has been granted. Each week the verified data for about 50 individuals are checked against the original questionnaires and the error rate is consistently below 1%. This partially automated process thus permits data to be entered rapidly and with high accuracy.</p></sec><sec><title>Follow up for breast cancer</title><p>Each screening centre of the NHSBSP is required to compile annual statistics on its activities, which include details of all breast cancers detected at mammography [<xref ref-type="bibr" rid="B1">1</xref>]. A list of women enrolled into the Million Women Study at each centre is cross-checked at regular intervals against the list of the women diagnosed with screen-detected breast cancer at that centre. If a breast cancer has been diagnosed at screening in a study participant, routinely recorded details of the cancer are abstracted, including tumour location, histology, size, grade, invasive status and involvement of axillary lymph nodes. Information on hormone receptor status and treatment is abstracted when it is available. Several approaches are being used to identify breast cancers diagnosed subsequent to screening. One will involve record linkage with cancer registry data. Also, women will be contacted directly 2-3 years after they were screened, and asked about new illnesses, including any new breast cancers, that may have been diagnosed (see Additional follow up, below). This will permit the identification of both screen-detected and interval cancers.</p></sec><sec><title>Follow-up for deaths</title><p>Deaths are identified annually by computerized matching of name, date of birth and NHS number of the women who gave signed consent for follow up in the Million Women Study, with the national death files held by the Office of National Statistics. For each death thus identified the date of death and underlying and associated causes of death are provided by the Office of National Statistics.</p></sec><sec><title>Additional follow up</title><p>Participants will be sent a follow-up questionnaire about 2-3 years after recruitment, to ascertain changes in use of HRT and incident morbidity, for example breast cancers, diagnosed outside the screening programme.</p></sec><sec><title>Validation</title><p>The most important variables for this study are the subjects' identification details, their use of HRT, any diagnosis of breast cancer and the recording of deaths. To assess the accuracy of the subjects' identification details and of the recording of deaths, a random sample of 5000 women recruited in 1996 has been selected for flagging on the NHS Central Register (NHSCR). Identification details recorded for the study (name, address, date of birth and NHS number) enabled all but 10 (0.2%) of the 5000 women to be identified on the NHSCR. The completeness and accuracy of the reported deaths will be validated in the future against those recorded in the NHSCR for these 5000 women.</p><p>The reliability of diagnosis of screen-detected breast cancers is monitored by various quality control procedures within the NHSBSP. Screen-detected breast cancers are verified according to defined procedures, and the invasive status, size and type of cancer are recorded for virtually 100% of the cancers.</p><p>The validity of reported information on use of HRT, including the type and dose, is being examined and a full report will be published in due course. Preliminary comparisons with the prescription records from one general practice in Oxfordshire indicate at least 95% agreement for reported current use of HRT, including the hormonal constituents of the preparation used most recently (Banks <italic>et al</italic>, unpublished data).</p></sec></sec><sec><title>Results</title><p>After it was demonstrated that the Million Women Study questionnaire did not alter attendance rates [<xref ref-type="bibr" rid="B2">2</xref>], each screening centre in England, Scotland and Wales was invited to participate in the study. Almost all the centres expressed enthusiasm for the study, although practical problems precluded the involvement of some centres. The most frequent reason for screening centres not participating was that the Million Women Study questionnaire could not readily be packaged together with the letters and other information normally posted to women when they are invited for screening.</p><sec><title>Accrual of the cohort</title><p>Recruitment of women into the study began in May 1996. Most of the participating screening centres began recruitment during 1997, and 61 centres were taking part by late 1998. The locations of these centres are shown in Figure <xref ref-type="fig" rid="F1">1</xref>. Before recruitment could begin at any centre, local ethical committee approval was required, and this often entailed contacting more than one ethical committee for each centre. In total, 126 local ethical committees were approached and approval for the study was obtained without exception.</p><p>Figure <xref ref-type="fig" rid="F2">2</xref> shows the numbers of questionnaires returned to the CEU between May 1996 and June 1999. More than 800000 questionnaires had been returned by the middle of 1999, and according to this accrual rate it is estimated that a cohort of 1 million women will have been recruited by early in the year 2000.
</p></sec><sec><title>Response rate</title><p>Statistics presented here are based on the first 227 000 questionnaires, which were printed between May 1996 and February 1997. This represented a convenient point in the accrual of women to assess response rate, because the layout and colour of the questionnaire were modified at this stage. Table <xref ref-type="table" rid="T1">1</xref> shows the numbers of questionnaires dispatched and returned, and whether the respondents also gave signed permission for follow up. Overall 121 000 (53%) of the questionnaires sent out were returned to the CEU. Women who returned a questionnaire are referred to as 'respondents', and it is estimated that they comprise about 71% of the women screened at the participating centres. Not all respondents can be included in the cohort to be followed, however, because 7% of them did not give signed consent or gave insufficient personal details for follow up. The remaining 93% of the respondents who can be followed are referred to as 'the cohort' or as 'participants'.</p></sec><sec><title>Characteristics of 121 000 respondents</title><p>Table <xref ref-type="table" rid="T2">2</xref> summarizes certain characteristics of the first 121 000 respondents, including details of their age, use of HRT, reproductive history, past use of oral contraceptives and consumption of cigarettes. It can be seen that most women are aged between 50 and 64 at recruitment (a small number of women are screened just before their 50th birthday and women aged over 65 can be screened by the NHSBSP if they specifically request it). It can also be seen that for most variables there is little missing data. One-third (33%) of the women reported currently using HRT, and almost half (47%) had used it at some time. More than half (54%) had used oral contraceptives and 18% were current smokers.</p><p>Table <xref ref-type="table" rid="T3">3</xref> summarizes the history and family history of breast disease, including breast cancer, in the respondents: 1.4% of the women had breast cancer diagnosed before recruitment and 9% reported that their mother and/or sister(s) had breast cancer diagnosed in the past. Table <xref ref-type="table" rid="T4">4</xref> summarizes the respondents' history of various other illnesses and operations. It can be seen that a substantial proportion of women have had hypertension diagnosed or are being treated for it, that one in four women have had a hysterectomy, one in five have been sterilized and one in 14 have had a bilateral oophorectomy.</p></sec><sec><title>Comparison of participants and nonparticipants</title><p>The overwhelming reason for nonparticipation in the Million Women Study is not attending for breast cancer screening, having been invited to do so. Women are asked to bring the completed questionnaire with them when they are screened, and thus far over 99% of the respondents were recruited in this way. Although no envelope or pre-paid postage is provided, a small number of the respondents posted their questionnaire back to the screening or co-ordinating centre, and virtually all of them also attended for breast cancer screening.</p><p>A direct comparison of those who agreed to participate in the study with those who did not has been performed in one general practice in Oxfordshire and similar comparisons are planned for other areas. A full report of these findings will be published in the future, but preliminary results suggest that there are few substantial differences between participants and nonparticipants. At this stage the main difference between the groups appears to be that nonparticipants are more likely than participants to be prescribed medications for the treatment of hypertension (Banks <italic>et al</italic>, unpublished data).</p><p>About 7% of the respondents returned the study questionnaire but did not give sufficient information and/or signed permission for follow up. Table <xref ref-type="table" rid="T5">5</xref> compares their characteristics with those of the 93% who can be followed. It can be seen that the main difference between these two groups is that the women who gave consent and sufficient information to be followed were more likely to be current users of HRT (33 versus 25%) and to have ever used oral contraceptives (54 versus 45%) than the women who cannot be followed.</p></sec><sec><title>Expected numbers and statistical power</title><p>At the present accrual rate it is expected that a cohort of 1 million women will have been recruited by the year 2000. Based on national statistics from the NHSBSP [<xref ref-type="bibr" rid="B1">1</xref>], about 5000 screen-detected breast cancers would be expected in this cohort. Given these numbers, and the expected proportion of current and never users of HRT at recruitment, the study should have 80% power to detect a relative risk of 1.1 in both current users and in current users of durations of at least 5 years, compared with never users.</p><p>Another aim of the study is to examine the relationship between use of HRT and mortality from various causes, the objective being to present findings with respect to the most important causes of death within 5 years. Table <xref ref-type="table" rid="T6">6</xref> shows the expected numbers of deaths from various causes by the end of 2002, assuming that 1 million women are recruited by the beginning of the year 2000. As with other cohort studies of women taking hormonal agents [<xref ref-type="bibr" rid="B3">3</xref>], it is likely that mortality in these women will be somewhat lower than that the general population because of self-selection of relatively healthy subjects into the study. The expected numbers in Table <xref ref-type="table" rid="T6">6</xref> have, therefore, been calculated assuming that death rates from causes other than breast cancer are 20% lower than the national rate and that breast cancer death rates are 30% lower than the national rate, thus taking into account the additional expected benefit of screening [<xref ref-type="bibr" rid="B4">4</xref>]. It can be seen that by the end of 2002 about 23 000 deaths will have occurred, with the majority being attributed to cancer (12 600 deaths) or to diseases of the circulatory system (8000).</p><p>Previous studies have suggested that both recency and duration of HRT use are important in determining its effect on breast cancer, and perhaps on other diseases
[<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B6">6</xref>]. However, because women tend to stop taking HRT when they become ill, there are problems in interpreting differences in mortality according to HRT use at the time of death. One way of overcoming these problems is to examine mortality in relation to use of HRT before diagnosis of any serious illness. Analyses of cause-specific mortality in relation to use of HRT within the Million Women Study will, therefore, exclude women with serious illnesses at the time of recruitment and be based on use as recorded at the time of entry into the cohort.</p><p>Table <xref ref-type="table" rid="T6">6</xref> shows the least extreme detectable relative risks for each of the main causes of death to be examined for various patterns of HRT use as compared with never users. These power calculations show that for a common cancer, such as colorectal cancer, there should be sufficient power to detect an increase or decrease in mortality of as little as about 20% in current users compared with in nonusers, and of about 25% in current users of long duration compared with nonusers. Even for endometrial cancer, which is the least common of the causes listed, it should be possible to detect quite modest increases or decreases in mortality of around 40% in current users compared with never users, and of about 45% in current users of long durations compared with never users.</p><p>By the end of the year 2002, the largest numbers of expected deaths among these women will be due to breast cancer and ischaemic heart disease. Thus, the effect of HRT use on deaths from these two causes will be particularly important in determining the net benefit or risk to mortality in HRT users as compared with nonusers. For both of these conditions relative risks of greater than 1.1 or less than 0.9 would be detectable among current versus never users. The corresponding figures among current users with durations of use of 5 or more years are 1.2 and 0.8, respectively.</p></sec><sec><title>Other questions</title><p>Many other questions about women's health can also be answered by this study. The cohort is sufficiently large to provide reliable data on the health effects of many lifestyle factors, including the consumption of tobacco and alcohol, and on the effects of past use of other hormonal agents, such as oral contraceptives. In addition, the Medical Research Council is supporting an extension of the study to evaluate the effect of HRT on the efficacy of mammography. In that study, women recalled for further assessment after screening and women with interval cancers are being identified. Information on interval cancers is being sought from cancer registries and also directly by sending women a follow-up questionnaire 2-3 years after their initial screen and asking about recent morbidity, including diagnosis of breast cancer. This will allow estimation of how HRT affects the sensitivity and specificity of mammography.</p></sec></sec><sec><title>Discussion</title><p>The main purpose of the Million Women Study is to examine the relationship between breast cancer and use of HRT, in a context where use of hormonal therapy is recorded as reliably as possible and breast cancers are diagnosed as uniformly and consistently as possible. Obtaining details of use of HRT before any breast cancer is diagnosed will minimise possible reporting biases of use of such therapy. Moreover, studying screen-detected breast cancers overcomes the potential bias that women who are taking HRT may be more likely to be screened than women who do not use such therapy. The limitation of examining screen-detected cancers alone, however, is that use of HRT may itself reduce the efficacy of mammographic screening. The plan, therefore, is to follow the women screened for interval breast cancer, and to include those cancers in the analyses of the relation between use of HRT and breast cancer.</p><p>Because the entire cohort is being followed up for deaths, it will also be possible to look at the relationship between use of HRT and mortality from various causes. Women prescribed HRT tend to be healthier than those who are not, however, and so it is crucial that analyses take proper account of the so-called 'healthy user effect' [<xref ref-type="bibr" rid="B7">7</xref>]. In designing the study attention has been given to the recording of detailed information about illnesses present at the time of recruitment. It can be seen in Tables <xref ref-type="table" rid="T3">3</xref> and <xref ref-type="table" rid="T4">4</xref> that a substantial proportion of women recruited have had illnesses such as hypertension and other cardiovascular disease in the past that would affect their risk of death from circulatory disease and other causes. The plan is to analyse results separately according to history of previous illness, and most weight will be given to the findings in women who had no previous illness.</p><p>Randomized clinical trials of HRT are now underway. These trials will have sufficient statistical power to detect a substantial reduction in ischaemic heart disease, but will not be able to pick up important, but modest, changes in the risk of cancer [<xref ref-type="bibr" rid="B7">7</xref>]. Thus, there will be a continued need for observational data to look at the effects of HRT on disease.</p></sec><sec><title>Conclusion</title><p>The Million Women Study is one of the largest cohort studies ever devised. Recruitment is proceeding rapidly and the study is on target to accrue a cohort of 1 million women by the year 2000. Preliminary results indicate that the women joining the Million Women Study do not differ substantially from women of a similar age in the general population.</p><p>It is expected that, within 5 years, the study will have sufficient statistical power to answer questions about the role of HRT in mortality from breast cancer and other specific conditions of interest.</p><p>This cohort may ultimately include about one women in every five aged between 50 and 64 years in the UK. This excellent co-operation at a national level reflects the efficient organization of the NHSBSP. It is also indicative, perhaps, of concern by women at the lack of reliable knowledge about the long-term effects of HRT and the fact that in the UK today there is substantial use of this type of therapy.</p></sec><sec><title>Appendix</title><p>NHS Breast Screening Centres that began recruitment before December 1998 (in alphabetical order) are as follows: Avon, Aylesbury, Barnsley, Basingstoke, Bedfordshire & Hertfordshire, Cambridge & Huntingdon, Chelmsford & Colchester, Chester, Cornwall, Crewe, Cumbria, Doncaster, Dorset, East Berkshire, East Cheshire, East Devon, East of Scotland, East Suffolk, Gateshead, Gloucestershire, Great Yarmouth, Hereford & Worcester, Kings Lynn, Leicestershire, Liverpool, Manchester, Milton Keynes, Newcastle, North Birmingham, North East Scotland, North Lancashire, North Middlesex, North Nottingham, North of Scotland, North Tees, North Yorkshire, Nottingham, Oxford, Portsmouth, Rotherham, South Birmingham, South East Scotland, South East Staffordshire, Sheffield, Shropshire, Somerset, South Derbyshire, South Essex, South Lancashire, South West Scotland, Surrey, Warrington Halton St Helens & Knowsley, Warwickshire Solihull & Coventry, West Berkshire, West Devon, West of London, West Suffolk, West Sussex, Wiltshire, Winchester and Wycombe.</p><p>The Million Women Study Co-ordinating Centre staff are as follows: Emily Banks, Valerie Beral, Anna Brown, Diana Bull, Becky Cameron, Barbara Crossley, Diane Deciacco, Dave Ewart, Laura Gerrard, Julie Hall, Sally Hall, Elizabeth Hilton, Ann Hogg, Carol Keene, Nikki Langley, Nicky Langston, Gillian Reeves, Moya Simmonds.</p><p>The Steering Committee members are Joan Austoker, Emily Banks, Valerie Beral, Ruth English, Julietta Patnick, Richard Peto, Gillian Reeves, Martin Vessey and Matthew Wallis.</p><p>The Writing Committee members are Emily Banks, Valerie Beral and Gillian Reeves.</p></sec> |
"Glutathione S-transferase M1 null genotype: lack of association with tumour characteristics and sur(...TRUNCATED) | "<sec><title>Background:</title><p>Glutathione S-transferase (GST)M1, a member of the μ clas(...TRUNCATED) | "<contrib id=\"A1\" contrib-type=\"author\"><name><surname>Lizard-Nacol</surname><given-names>Sarab<(...TRUNCATED) | Breast Cancer Research | "<sec><title>Introduction</title><p>The human glutathione S-transferases (GSTs) are a multigene, iso(...TRUNCATED) |
"Cyclin D<sub>1</sub> expression during rat mammary tumor development\n\t\t and its potential role (...TRUNCATED) | "<sec><title>Background:</title><p>Resistance to mammary tumorigenesis in Copenhagen rats is\n\t\t\t(...TRUNCATED) | "<contrib id=\"A1\" contrib-type=\"author\"><name><surname>Korkola</surname><given-names>James E</gi(...TRUNCATED) | Breast Cancer Research | "<sec><title>Introduction</title><p>Most strains of rats develop multiple mammary tumors when initia(...TRUNCATED) |
"Increased cell survival by inhibition of BRCA1 using an antisense approach in an estrogen responsiv(...TRUNCATED) | "\n <sec>\n <title>Introduction:</title>\n <p>Germline mutations in the bre(...TRUNCATED) | "\n <contrib id=\"A1\" contrib-type=\"author\">\n <name>\n <surname>Annab(...TRUNCATED) | Breast Cancer Research | "\n <sec>\n <title>Introduction</title>\n <p>Germline mutations in the breast and ovari(...TRUNCATED) |
A novel cell culture model for studying differentiation and apoptosis in the mouse mammary gland | "<sec><title>Background:</title><p>This paper describes the derivation and characterization of a nov(...TRUNCATED) | "<contrib id=\"A1\" contrib-type=\"author\"><name><surname>Gordon</surname><given-names>Katrina E</g(...TRUNCATED) | Breast Cancer Research | "<sec><title>Introduction</title><p>With each successive pregnancy, the mammary gland completes a cy(...TRUNCATED) |
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