J. Michael Dixon

Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features

Some breast cancers have been shown to contain a small fraction of cells characterized by CD44+/CD24−/low cell-surface antigen profile that have high tumor-initiating potential. In addition, breast cancer cells propagated in vitro as mammospheres (MSs) have also been shown to be enriched for cells capable of self-renewal. In this study, we have defined a gene expression signature common to both CD44+/CD24−/low and MS-forming cells. To examine its clinical significance, we determined whether tumor cells surviving after conventional treatments were enriched for cells bearing this CD44+/CD24−/low-MS signature. The CD44+/CD24−/low-MS signature was found mainly in human breast tumors of the recently identified “claudin-low” molecular subtype, which is characterized by expression of many epithelial-mesenchymal-transition (EMT)-associated genes. Both CD44+/CD24−/low-MS and claudin-low signatures were more pronounced in tumor tissue remaining after either endocrine therapy (letrozole) or chemotherapy (docetaxel), consistent with the selective survival of tumor-initiating cells posttreatment. We confirmed an increased expression of mesenchymal markers, including vimentin (VIM) in cytokeratin-positive epithelial cells metalloproteinase 2 (MMP2), in two separate sets of postletrozole vs. pretreatment specimens. Taken together, these data provide supporting evidence that the residual breast tumor cell populations surviving after conventional treatment may be enriched for subpopulations of cells with both tumor-initiating and mesenchymal features. Targeting proteins involved in EMT may provide a therapeutic strategy for eliminating surviving cells to prevent recurrence and improve long-term survival in breast cancer patients.

Neoadjuvant Treatment of Postmenopausal Breast Cancer With Anastrozole, Tamoxifen, or Both in Combination: The Immediate Preoperative Anastrozole, Tamoxifen, or Combined With Tamoxifen (IMPACT) Multicenter Double-Blind Randomized Trial

PURPOSE The Immediate Preoperative Anastrozole, Tamoxifen, or Combined With Tamoxifen (IMPACT) trial was designed to test the hypothesis that the clinical and/or biologic effects of neoadjuvant tamoxifen compared with anastrozole and with the combination of tamoxifen and anastrozole before surgery in postmenopausal women with estrogen receptor (ER) -positive, invasive, nonmetastatic breast cancer might predict for outcome in the Arimidex, Tamoxifen Alone or in Combination (ATAC) adjuvant therapy trial. PATIENTS AND METHODS Postmenopausal women with ER-positive, invasive, nonmetastatic, and operable or locally advanced potentially operable breast cancer were randomly assigned to neoadjuvant tamoxifen (20 mg daily), anastrozole (1 mg daily), or a combination of tamoxifen and anastrozole for 3 months. The tumor objective response (OR) was assessed by both caliper and ultrasound. Comparisons were also made of clinical response with ultrasound response, actual and feasible surgery with feasible surgery at baseline, OR in human epidermal growth factor receptor 2 (HER2)-positive cancers, and tolerability. RESULTS There were no significant differences in OR in the intent-to-treat population between patients receiving tamoxifen, anastrozole, or the combination. In patients who were assessed as requiring mastectomy at baseline (n = 124), 44% of patients received breast-conserving surgery (BCS) after anastrozole compared with 31% of patients after tamoxifen (P = .23); this difference became significant for patients who were deemed feasible for BCS by their surgeon (46% v 22%, respectively; P = .03). The OR for patients with HER2-positive cancer (n = 34) was 58% for anastrozole compared with 22% for tamoxifen (P = .18). All treatments were well tolerated. CONCLUSION Neoadjuvant anastrozole is as effective and well tolerated as tamoxifen in ER-positive operable breast cancer in postmenopausal women, but the hypothesis that clinical outcome might predict for long-term outcome in adjuvant therapy was not fulfilled.

Accuracy and Surgical Impact of Magnetic Resonance Imaging in Breast Cancer Staging: Systematic Review and Meta-Analysis in Detection of Multifocal and Multicentric Cancer

PURPOSE We review the evidence on magnetic resonance imaging (MRI) in staging the affected breast to determine its accuracy and impact on treatment. METHODS Systematic review and meta-analysis of the accuracy of MRI in detection of multifocal (MF) and/or multicentric (MC) cancer not identified on conventional imaging. We estimated summary receiver operating characteristic curves, positive predictive value (PPV), true-positive (TP) to false positive (FP) ratio, and examined their variability according to quality criteria. Pooled estimates of the proportion of women whose surgery was altered were calculated. Results Data from 19 studies showed MRI detects additional disease in 16% of women with breast cancer (N = 2,610). MRI incremental accuracy differed according to the reference standard (RS; P = .016) decreasing from 99% to 86% as the quality of the RS increased. Summary PPV was 66% (95% CI, 52% to 77%) and TP:FP ratio was 1.91 (95% CI, 1.09 to 3.34). Conversion from wide local excision (WLE) to mastectomy was 8.1% (95% CI, 5.9 to 11.3), from WLE to more extensive surgery was 11.3% in MF/MC disease (95% CI, 6.8 to 18.3). Due to MRI-detected lesions (in women who did not have additional malignancy on histology) conversion from WLE to mastectomy was 1.1% (95% CI, 0.3 to 3.6) and from WLE to more extensive surgery was 5.5% (95% CI, 3.1 to 9.5). CONCLUSION MRI staging causes more extensive breast surgery in an important proportion of women by identifying additional cancer, however there is a need to reduce FP MRI detection. Randomized trials are needed to determine the clinical value of detecting additional disease which changes surgical treatment in women with apparently localized breast cancer.

Phase II Randomized Study of Neoadjuvant Everolimus Plus Letrozole Compared With Placebo Plus Letrozole in Patients With Estrogen Receptor-Positive Breast Cancer

PURPOSE Cross-talk between the estrogen receptor (ER) and the phosphoinositide-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways is a mechanism of resistance to endocrine therapy, and blockade of both pathways enhances antitumor activity in preclinical models. This study explored whether sensitivity to letrozole was enhanced with the oral mTOR inhibitor, everolimus (RAD001). PATIENTS AND METHODS Two hundred seventy postmenopausal women with operable ER-positive breast cancer were randomly assigned to receive 4 months of neoadjuvant treatment with letrozole (2.5 mg/day) and either everolimus (10 mg/day) or placebo. The primary end point was clinical response by palpation. Mandatory biopsies were obtained at baseline and after 2 weeks of treatment (ie, day 15). Samples were assessed for PI3K mutation status (PIK3CA) and for pharmacodynamic changes of Ki67, phospho-S6, cyclin D1, and progesterone receptor (PgR) by immunohistochemistry. RESULTS Response rate by clinical palpation in the everolimus arm was higher than that with letrozole alone (ie, placebo; 68.1% v 59.1%), which was statistically significant at the preplanned, one-sided, alpha = 0.1 level (P = .062). Marked reductions in progesterone receptor and cyclin D1 expression occurred in both treatment arms, and dramatic downregulation of phospho-S6 occurred only in the everolimus arm. An antiproliferative response, as defined by a reduction in Ki67 expression to natural logarithm of percentage positive Ki67 of less than 1 at day 15, occurred in 52 (57%) of 91 patients in the everolimus arm and in 25 (30%) of 82 patients in the placebo arm (P < .01). The safety profile was consistent with historical results of everolimus monotherapy; grades 3 to 4 adverse events occurred in 22.6% of patients who received everolimus and in 3.8% of patients who received placebo. CONCLUSION Everolimus significantly increased letrozole efficacy in neoadjuvant therapy of patients with ER-positive breast cancer.

Meta-analysis of the impact of surgical margins on local recurrence in women with early-stage invasive breast cancer treated with breast-conserving therapy

PURPOSE There is no consensus on what constitutes adequate negative margins in breast-conserving therapy (BCT). We review the evidence on surgical margins in BCT for early-stage invasive breast cancer. METHODS Meta-analysis of studies reporting local recurrence (LR) relative to quantified final microscopic margin status and the threshold distance for negative margins. The proportion of LR was modelled using random effects logistic meta-regression. RESULTS Based on 21 studies (LR in 1,026 of 14,571 subjects) the odds of LR were associated with margin status [model 1: odds ratio (OR) = 2.02 for positive/close versus negative; model 2: OR = 1.80 for close versus negative, 2.42 for positive versus negative (P<0.001 both models)] but not with margin distance [1mm versus 2mm versus 5mm (P > 0.10 both models)], adjusting for median follow-up time. However, there was weak evidence in both models that the odds of LR decreased as the threshold distance for declaring negative margins increased. This bordered significance in model 2 [OR for 1mm, 2mm, 5mm: 1.0, 0.75, 0.51 (P = 0.097 for trend)], and was not significant in model 1 [OR for 1mm, 2mm, 5mm: 1.0, 0.85, 0.58 (P = 0.11 for trend)] but was evident when one study (of women ≤ 40 years) was excluded from this model [OR for 1mm, 2mm, 5mm: 1.0, 0.72, 0.52 (P = 0.058 for trend)]: this trend was rendered insignificant by adjustment for the proportion of subjects receiving a radiation boost or the proportion of subjects receiving endocrine therapy. CONCLUSIONS Margin status has a prognostic effect in all women treated for invasive breast cancer; increasing the threshold distance for declaring negative margins is weakly associated with reduced odds of LR, however adjustment for covariates (adjuvant therapy) removes the significance of this effect. Adoption of wider margins, relative to narrower widths, for declaring negative margins is unlikely to a have substantial additional benefit for long-term local control in BCT.

Biomarker Changes During Neoadjuvant Anastrozole, Tamoxifen, or the Combination: Influence of Hormonal Status and HER-2 in Breast Cancer—A Study from the IMPACT Trialists

PURPOSE To investigate the relationships between biomarker changes in breast cancer during neoadjuvant (preoperative) endocrine therapy. PATIENTS AND METHODS The IMPACT trial compared the preoperative use of tamoxifen with anastrozole alone or in combination in postmenopausal women (n = 330) with primary breast cancer. Biomarkers were measured in tumor biopsy specimens taken at baseline, and after 2 and 12 weeks of treatment. RESULTS 52 (93%) of 56, 46 (85%) of 54, and 37 (84%) of 44 patients in the anastrozole, tamoxifen, and combination groups, respectively. There was a significantly greater suppression of Ki67 in the anastrozole-treated group than in the tamoxifen- or combination-treated groups, which is parallel to the greater efficacy seen for anastrozole over these two treatments in the Arimidex, Tamoxifen, Alone or in Combination adjuvant trial. A positive relationship was noted between estrogen-receptor level and Ki67 suppression in all patients. Ki67 was reduced to a greater extent in progesterone receptor-positive tumors compared with progesterone receptor-negative tumors. HER-2-negative tumors tended to show a greater reduction in Ki67 compared with HER-2-positive tumors, but the difference was only significant in the tamoxifen group after 2 weeks, and in the anastrozole group after 12 weeks. CONCLUSION These results confirm the value of Ki67 as a molecular marker, and provide information regarding the relationships between treatment-induced changes in Ki67 and other important biomarkers. Studies such as this should help integrate agents targeted at growth factor signaling with endocrine agents in breast cancer.

Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes

The discovery of substantial amounts of 5-hydroxymethylcytosine (5hmC), formed by the oxidation of 5-methylcytosine (5mC), in various mouse tissues and human embryonic stem (ES) cells has necessitated a reevaluation of our knowledge of 5mC/5hmC patterns and functions in mammalian cells. Here, we investigate the tissue specificity of both the global levels and locus-specific distribution of 5hmC in several human tissues and cell lines. We find that global 5hmC content of normal human tissues is highly variable, does not correlate with global 5mC content, and decreases rapidly as cells from normal tissue adapt to cell culture. Using tiling microarrays to map 5hmC levels in DNA from normal human tissues, we find that 5hmC patterns are tissue specific; unsupervised hierarchical clustering based solely on 5hmC patterns groups independent biological samples by tissue type. Moreover, in agreement with previous studies, we find 5hmC associated primarily, but not exclusively, with the body of transcribed genes, and that within these genes 5hmC levels are positively correlated with transcription levels. However, using quantitative 5hmC-qPCR, we find that the absolute levels of 5hmC for any given gene are primarily determined by tissue type, gene expression having a secondary influence on 5hmC levels. That is, a gene transcribed at a similar level in several different tissues may have vastly different levels of 5hmC (>20-fold) dependent on tissue type. Our findings highlight tissue type as a major modifier of 5hmC levels in expressed genes and emphasize the importance of using quantitative analyses in the study of 5hmC levels.

Magnetic Resonance Imaging Screening of the Contralateral Breast in Women With Newly Diagnosed Breast Cancer: Systematic Review and Meta-Analysis of Incremental Cancer Detection and Impact on Surgical Management

PURPOSE Preoperative magnetic resonance imaging (MRI) is increasingly used for staging women with breast cancer, including screening for occult contralateral cancer. This article is a review and meta-analysis of studies reporting contralateral MRI in women with newly diagnosed invasive breast cancer. METHODS We systematically reviewed the evidence on contralateral MRI, calculating pooled estimates for positive predictive value (PPV), true-positive:false-positive ratio (TP:FP), and incremental cancer detection rate (ICDR) over conventional imaging. Random effects logistic regression examined whether estimates were associated with study quality or clinical variables. RESULTS Twenty-two studies reported contralateral malignancies detected only by MRI in 131 of 3,253 women. Summary estimates were as follows: MRI-detected suspicious findings (TP plus FP), 9.3% (95% CI, 5.8% to 14.7%); ICDR, 4.1% (95% CI, 2.7% to 6.0%), PPV, 47.9% (95% CI, 31.8% to 64.6%); TP:FP ratio, 0.92 (95% CI, 0.47 to 1.82). PPV was associated with the number of test positives and baseline imaging. Few studies included consecutive women, and few ascertained outcomes in all subjects. Where reported, 35.1% of MRI-detected cancers were ductal carcinoma in situ (mean size = 6.9 mm), 64.9% were invasive cancers (mean size = 9.3 mm), and the majority were stage pTis or pT1 and node negative. Effect on treatment was inconsistently reported, but many women underwent contralateral mastectomy. CONCLUSION MRI detects contralateral lesions in a substantial proportion of women, but does not reliably distinguish benign from malignant findings. Relatively high ICDR may be due to selection bias and/or overdetection. Women must be informed of the uncertain benefit and potential harm, including additional investigations and surgery.

Breast-conserving surgery with or without irradiation in women aged 65 years or older with early breast cancer (PRIME II): a randomised controlled trial.

BACKGROUND For most older women with early breast cancer, standard treatment after breast-conserving surgery is adjuvant whole-breast radiotherapy and adjuvant endocrine treatment. We aimed to assess the effect omission of whole-breast radiotherapy would have on local control in older women at low risk of local recurrence at 5 years. METHODS Between April 16, 2003, and Dec 22, 2009, 1326 women aged 65 years or older with early breast cancer judged low-risk (ie, hormone receptor-positive, axillary node-negative, T1-T2 up to 3 cm at the longest dimension, and clear margins; grade 3 tumour histology or lymphovascular invasion, but not both, were permitted), who had had breast-conserving surgery and were receiving adjuvant endocrine treatment, were recruited into a phase 3 randomised controlled trial at 76 centres in four countries. Eligible patients were randomly assigned to either whole-breast radiotherapy (40-50 Gy in 15-25 fractions) or no radiotherapy by computer-generated permuted block randomisation, stratified by centre, with a block size of four. The primary endpoint was ipsilateral breast tumour recurrence. Follow-up continues and will end at the 10-year anniversary of the last randomised patient. Analyses were done by intention to treat. The trial is registered on ISRCTN.com, number ISRCTN95889329. FINDINGS 658 women who had undergone breast-conserving surgery and who were receiving adjuvant endocrine treatment were randomly assigned to receive whole-breast irradiation and 668 were allocated to no further treatment. After median follow-up of 5 years (IQR 3·84-6·05), ipsilateral breast tumour recurrence was 1·3% (95% CI 0·2-2·3; n=5) in women assigned to whole-breast radiotherapy and 4·1% (2·4-5·7; n=26) in those assigned no radiotherapy (p=0·0002). Compared with women allocated to whole-breast radiotherapy, the univariate hazard ratio for ipsilateral breast tumour recurrence in women assigned to no radiotherapy was 5·19 (95% CI 1·99-13·52; p=0·0007). No differences in regional recurrence, distant metastases, contralateral breast cancers, or new breast cancers were noted between groups. 5-year overall survival was 93·9% (95% CI 91·8-96·0) in both groups (p=0·34). 89 women died; eight of 49 patients allocated to no radiotherapy and four of 40 assigned to radiotherapy died from breast cancer. INTERPRETATION Postoperative whole-breast radiotherapy after breast-conserving surgery and adjuvant endocrine treatment resulted in a significant but modest reduction in local recurrence for women aged 65 years or older with early breast cancer 5 years after randomisation. However, the 5-year rate of ipsilateral breast tumour recurrence is probably low enough for omission of radiotherapy to be considered for some patients. FUNDING Chief Scientist Office (Scottish Government), Breast Cancer Institute (Western General Hospital, Edinburgh).

Estrogen-Independent Proliferation Is Present in Estrogen-Receptor HER2-Positive Primary Breast Cancer After Neoadjuvant Letrozole

PURPOSE To investigate the impact of human epidermal growth factor receptor (HER) 1 and HER2 gene amplification on endocrine therapy responsiveness, a fluorescence in situ hybridization (FISH) study was conducted on tumor samples from 305 postmenopausal patients with stage II and III estrogen receptor (ER) -positive (ER > or = 10%) breast cancers treated on two independent neoadjuvant endocrine therapy trials. PATIENTS AND METHODS FISH analysis focused on HER1 and/or HER2 immunohistochemistry (IHC) -positive patients and a random selection of HER1/2 IHC-negative patients. HER2 FISH status was correlated with response and changes in the proliferation marker Ki67. RESULTS HER1 was rarely amplified (< 1%), and HER2 amplification was observed in 9.2% of patients. Letrozole response by clinical measurement (71% HER2 FISH positive v 71% HER2 FISH negative), mammogram (44% HER2 FISH positive v 47% HER2 FISH negative), or ultrasound (47% HER2 FISH positive v 54% HER2 FISH negative) was not impaired by HER2 FISH-positive status. In contrast, HER2 FISH-positive tumors showed higher histologic grade (P = .009), higher pretreatment Ki67 (P = .005), and less Ki67 suppression after letrozole when compared with HER2 FISH-negative tumors (P = .0001). Similar observations regarding Ki67 were made in a smaller cohort of tamoxifen-treated tumors. CONCLUSION Neoadjuvant letrozole is clinically effective in ER-positive HER2 FISH-positive tumors, indicating sensitivity to short-term estrogen deprivation. However, continued proliferation despite ongoing letrozole or tamoxifen treatment in the majority of ER-positive HER2 FISH-positive samples (88%) could imply therapeutic resistance that may manifest later in the clinical course of the disease. Discordance between clinical and biomarker findings in this study serves to emphasize the need for surrogate end point validation in neoadjuvant endocrine trials through correlation with information on long-term outcomes.