Anne F. Schott

ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome

Application of stem cell biology to breast cancer research has been limited by the lack of simple methods for identification and isolation of normal and malignant stem cells. Utilizing in vitro and in vivo experimental systems, we show that normal and cancer human mammary epithelial cells with increased aldehyde dehydrogenase activity (ALDH) have stem/progenitor properties. These cells contain the subpopulation of normal breast epithelium with the broadest lineage differentiation potential and greatest growth capacity in a xenotransplant model. In breast carcinomas, high ALDH activity identifies the tumorigenic cell fraction, capable of self-renewal and of generating tumors that recapitulate the heterogeneity of the parental tumor. In a series of 577 breast carcinomas, expression of ALDH1 detected by immunostaining correlated with poor prognosis. These findings offer an important new tool for the study of normal and malignant breast stem cells and facilitate the clinical application of stem cell concepts.

Activating ESR1 mutations in hormone-resistant metastatic breast cancer

Breast cancer is the most prevalent cancer in women, and over two-thirds of cases express estrogen receptor-α (ER-α, encoded by ESR1). Through a prospective clinical sequencing program for advanced cancers, we enrolled 11 patients with ER-positive metastatic breast cancer. Whole-exome and transcriptome analysis showed that six cases harbored mutations of ESR1 affecting its ligand-binding domain (LBD), all of whom had been treated with anti-estrogens and estrogen deprivation therapies. A survey of The Cancer Genome Atlas (TCGA) identified four endometrial cancers with similar mutations of ESR1. The five new LBD-localized ESR1 mutations identified here (encoding p.Leu536Gln, p.Tyr537Ser, p.Tyr537Cys, p.Tyr537Asn and p.Asp538Gly) were shown to result in constitutive activity and continued responsiveness to anti-estrogen therapies in vitro. Taken together, these studies suggest that activating mutations in ESR1 are a key mechanism in acquired endocrine resistance in breast cancer therapy.

Circulating Tumor Cells and Response to Chemotherapy in Metastatic Breast Cancer: SWOG S0500

PURPOSE Increased circulating tumor cells (CTCs; five or more CTCs per 7.5 mL of whole blood) are associated with poor prognosis in metastatic breast cancer (MBC). A randomized trial of patients with persistent increase in CTCs tested whether changing chemotherapy after one cycle of first-line chemotherapy would improve the primary outcome of overall survival (OS). PATIENTS AND METHODS Patients with MBC who did not have increased CTCs at baseline remained on initial therapy until progression (arm A). Patients with initially increased CTCs that decreased after 21 days of therapy remained on initial therapy (arm B). Patients with persistently increased CTCs after 21 days of therapy were randomly assigned to continue initial therapy (arm C1) or change to an alternative chemotherapy (arm C2). RESULTS Of 595 eligible and evaluable patients, 276 (46%) did not have increased CTCs (arm A). Of those with initially increased CTCs, 31 (10%) were not retested, 165 were assigned to arm B, and 123 were randomly assigned to arm C1 or C2. No difference in median OS was observed between arm C1 and C2 (10.7 and 12.5 months, respectively; P = .98). CTCs were strongly prognostic. Median OS for arms A, B, and C (C1 and C2 combined) were 35 months, 23 months, and 13 months, respectively (P < .001). CONCLUSION This study confirms the prognostic significance of CTCs in patients with MBC receiving first-line chemotherapy. For patients with persistently increased CTCs after 21 days of first-line chemotherapy, early switching to an alternate cytotoxic therapy was not effective in prolonging OS. For this population, there is a need for more effective treatment than standard chemotherapy.

Sentinel Lymph Node Biopsy Performed After Neoadjuvant Chemotherapy is Accurate in Patients with Documented Node-Positive Breast Cancer at Presentation

BackgroundThe optimal strategy for incorporating lymphatic mapping and sentinel lymph node biopsy into the management of breast cancer patients receiving neoadjuvant chemotherapy remains controversial. Previous studies of sentinel node biopsy performed following neoadjuvant chemotherapy have largely reported on patients whose prechemotherapy, pathologic axillary nodal status was unknown. We report findings using a novel comprehensive approach to axillary management of node-positive-patients receiving neoadjuvant chemotherapy.MethodsWe evaluated 54 consecutive breast cancer patients with biopsy-proven axillary nodal metastases at the time of diagnosis that underwent lymphatic mapping with nodal biopsy as well as concomitant axillary lymph node dissection after receiving neoadjuvant chemotherapy. All cases were treated at a single comprehensive cancer center between 2001 and 2005.ResultsThe sentinel node identification rate after delivery of neoadjuvant chemotherapy was 98%. Thirty-six patients (66%) had residual axillary metastases (including eight patients that had undergone resection of metastatic sentinel nodes at the time of diagnosis), and in 12 cases (31%) the residual metastatic disease was limited to the sentinel lymph node. The final, post-neoadjuvant chemotherapy sentinel node was falsely negative in three cases (8.6%). The negative final sentinel node accurately identified patients with no residual axillary disease in 17 cases (32%).ConclusionsSentinel lymph node biopsy performed after the delivery of neoadjuvant chemotherapy in patients with documented nodal disease at presentation accurately identified cases that may have been downstaged to node-negative status and can spare this subset of patients (32%) from experiencing the morbidity of an axillary dissection.

Sentinel node biopsy prior to neoadjuvant chemotherapy

BACKGROUND Several studies have explored sentinel lymph node biopsy (SLNB) after neoadjuvant chemotherapy, but false negative rates and the loss of pretreatment nodal staging are limitations. Sentinel lymph node biopsy prior to induction chemotherapy may address both. METHODS Sentinel lymph node biopsy was performed in clinically node negative patients prior to initiating chemotherapy. Standard level I/II axillary lymph node dissection (ALND) was performed at the time of surgery in those patients who had metastases in the sentinel lymph node (SLN). RESULTS Twenty-five patients had 26 SLNB prior to the initiation of chemotherapy. The SLN was identified in all cases (100%). Twelve patients (48%) were found to be node negative and did not require axillary node dissection after chemotherapy. Of the patients who were SLN positive and underwent completion ALND, residual nodal disease was identified in 60%. There were no surgical complications or delay of chemotherapy. CONCLUSIONS Sentinel lymph node biopsy prior to neoadjuvant chemotherapy can avoid the morbidity of ALND without compromising the accuracy of axillary staging. It allows for identification of node positive patients subsequently rendered disease free in the regional nodes, which can assist in planning additional chemotherapy or radiation.

Preclinical and Clinical Studies of Gamma Secretase Inhibitors with Docetaxel on Human Breast Tumors

Purpose: Accumulating evidence supports the existence of breast cancer stem cells (BCSC), which are characterized by their capacity to self-renew and divide indefinitely and resistance to conventional therapies. The Notch pathway is important for stem cell renewal and is a potential target for BCSC-directed therapy. Experimental Design: Using human breast tumorgraft studies, we evaluated the impact of gamma secretase inhibitors (GSI) on the BCSC population and the efficacy of combining GSI with docetaxel treatment. The mouse experimental therapy paralleled a concurrent clinical trial in patients with advanced breast cancer, designed to determine the maximum-tolerated dose of the GSI, MK-0752, administered sequentially with docetaxel, and to evaluate BCSC markers in serial tumor biopsies. Results: Treatment with GSI reduced BCSCs in MC1 and BCM-2147 tumorgrafts by inhibition of the Notch pathway. GSI enhanced the efficacy of docetaxel in preclinical studies. In the clinical trial, 30 patients with advanced breast cancer were treated with escalating doses of MK-0752 plus docetaxel. Clinically, meaningful doses of both drugs were possible with manageable toxicity and preliminary evidence of efficacy. A decrease in CD44+/CD24−, ALDH+, and mammosphere-forming efficiency were observed in tumors of patients undergoing serial biopsies. Conclusions: These preclinical data show that pharmacologic inhibition of the Notch pathway can reduce BCSCs in breast tumorgraft models. The clinical trial shows feasibility of combination GSI and chemotherapy, and together these results encourage further study of Notch pathway inhibitors in combination with chemotherapy in breast cancer. Clin Cancer Res; 19(6); 1512–24. ©2012 AACR.

Comprehensive Axillary Evaluation in Neoadjuvant Chemotherapy Patients With Ultrasonography and Sentinel Lymph Node Biopsy

BackgroundThere is ongoing debate regarding the optimal sequence of sentinel lymph node (SLN) biopsy and neoadjuvant chemotherapy (CTX) for breast cancer. We report the accuracy of comprehensive pre–neoadjuvant CTX and post–neoadjuvant CTX axillary staging via ultrasound imaging, fine-needle aspiration (FNA) biopsy, and SLN biopsy.MethodsFrom 2001 to 2004, 91 neoadjuvant CTX patients at the University of Michigan Comprehensive Cancer Center underwent axillary staging by ultrasonography, ultrasound-guided FNA biopsy, SLN biopsy, or a combination of these.ResultsAxillary staging was pathologically negative by pre–neoadjuvant CTX SLN biopsy in 53 cases (58%); these patients had no further axillary surgery. In 38 cases (42%), axillary metastases were confirmed at presentation by either ultrasound-guided FNA or SLN biopsy. These 38 patients underwent completion axillary lymph node dissection (ALND) after delivery of neoadjuvant CTX. Follow-up lymphatic mapping was attempted in 33 of these cases, and the SLN was identified in 32 (identification rate, 97%). One third of these cases were completely node negative on ALND. Residual metastatic disease was identified in 22 cases, and the SLN was falsely negative in 1 (4.5%).ConclusionsPatients receiving neoadjuvant CTX can have accurate axillary nodal staging by ultrasound-guided FNA or SLN biopsy. In cases of documented axillary metastasis at presentation, repeat axillary staging with SLN biopsy can document the post–neoadjuvant CTX nodal status. This strategy optimizes pre–neoadjuvant CTX and post–neoadjuvant CTX staging information by distinguishing the patients who are node negative at presentation from those who have been downstaged to node negativity and offers the potential for avoiding unnecessary ALNDs in both of these patient subsets.

Prospective Early Response Imaging Biomarker for Neoadjuvant Breast Cancer Chemotherapy

Purpose: The American Cancer Society estimates that in 2006, 212,920 women will be diagnosed with breast cancer and that 40,970 women will die from the disease. The development of more efficacious chemotherapies has improved outcomes, but the rapid assessment of clinical benefit from these agents remains challenging. In breast cancer patients receiving neoadjuvant chemotherapy, treatment response is traditionally assessed by physical examination and volumetric-based measurements, which are subjective and require macroscopic changes in tumor morphology. In this study, we evaluate the feasibility of using diffusion magnetic resonance imaging (MRI) as a reliable and quantitative measure for the early assessment of response in a breast cancer model. Experimental Design: Mice implanted with human breast cancer (MX-1) were treated with cyclophosphamide and evaluated using diffusion MRI and growth kinetics. Histologic analyses using terminal nucleotidyl transferase–mediated nick end labeling and H&E were done on tumor samples for correlation with imaging results. Results: Cyclophosphamide treatment resulted in a significant reduction in tumor volumes compared with controls. The mean apparent diffusion change for treated tumors at days 4 and 7 posttreatment was 44 ± 5% and 94 ± 7%, respectively, which was statistically greater (P < 0.05) than the control tumors at the same time intervals. The median time-to-progression for control and treated groups was 11 and 32 days, respectively (P < 0.05). Conclusion: Diffusion MRI was shown to detect early changes in the tumor microenvironment, which correlated with standard measures of tumor response as well as overall outcome. Moreover, these findings show the feasibility of using diffusion MRI for assessing treatment response of a breast tumor model in a neoadjuvant setting.

Defining the Benefits of Neoadjuvant Chemotherapy for Breast Cancer

Preoperative or neoadjuvant chemotherapy is an option in patients with early-stage breast cancer. Neoadjuvant treatment has been compared with standard, postoperative adjuvant chemotherapy with the dual goals of improving survival and facilitating local therapies. Unfortunately, neoadjuvant chemotherapy does not seem to improve overall survival, as demonstrated in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B18 trial, among others. Neoadjuvant chemotherapy may convert a previously unresectable, locally advanced breast cancer to an operable tumor, and in primarily operable tumors, downstaging results in a small increase (7% to 12%) in breast conservation rates. However, many patients experience insufficient response or are not candidates for breast preservation, irrespective of response, because of either skin or chest wall involvement or multicentric or multifocal disease. Furthermore, an estimated 10% of American women choose mastectomy as a personal preference despite their surgeons’ recommendation of breast-conserving therapy. Aside from the potential clinical benefits that are achieved by downstaging, neoadjuvant therapy allows direct and early observation of the response to treatment, which in theory could lead to modifications of the treatment plan in the event of poor response. However, clinical and radiographic monitoring during neoadjuvant chemotherapy to predict pathologic complete response (pCR) is notoriously inaccurate. Furthermore, although it is clear that those patients with a poor initial response have a worse prognosis, modification of chemotherapy after observed poor response has not resulted in clinically meaningful improvements in outcome. On the basis of the limited clinical advantages of neoadjuvant chemotherapy, postoperative adjuvant systemic therapy is considered the standard of care. The preoperative setting could provide an opportunity to study the impact of systemic therapies on breast cancer biology. As a research tool, neoadjuvant chemotherapy is useful because of the availability of tumor response as an end point and the availability of tissue for biopsy and biomarker development. To facilitate neoadjuvant trials, empirical definitions of pCR have been developed with the goal of using pCR as a surrogate for long-term outcomes. Numerous studies have demonstrated that the burden of pathologically detected residual disease after neoadjuvant chemotherapy is associated with long-term prognosis. However, there has been little if any agreement regarding the precise definition of pCR. In the article that accompanies this editorial, von Minckwitz et al address this controversy in a review of their experience with more than 6,000 patients treated in a series of seven prospectively conducted clinical trials of neoadjuvant anthracycline and taxane–based chemotherapy (some including trastuzumab). They compared multiple existing definitions of pCR to determine how robustly each definition serves as a surrogate for patient survival outcomes. Not surprisingly, they found that the pCR definition that allowed the least residual cancer in the breast and nodes was most highly correlated with the best survival. Although some authors have reported that small amounts of residual invasive or in situ cancer do not seem to be prognostic, the data from this highly powered, pooled analysis of prospectively performed neoadjuvant trials suggest otherwise: any finding of residual cancer—in situ, invasive, or in axillary lymph nodes—was associated with a worse prognosis compared with absolutely no evidence of residual disease. This result, by itself, would probably not have warranted publication in such a high-impact journal as Journal of Clinical Oncology. However, the authors have provided clarity with respect to the results of neoadjuvant chemotherapy by further evaluating the prognostic implications of pCR, using their best definition, within the separate intrinsic breast cancer biologic subtypes. In their large data set, von Minckwitz et al clearly demonstrate that pCR to anthracycline and taxane–based chemotherapy (or lack thereof) had no substantial prognostic value in patients with luminal A (estrogen receptor [ER] positive and/or progesterone receptor [PgR] positive, human epidermal growth factor receptor 2 [HER2] negative, grade 1 or 2) tumors. In fact, the pCR rate is so low in this group (6.7%) that residual disease after treatment with cytotoxic chemotherapy should be expected. Perhaps more surprisingly, pCR in luminal B/HER2-positive tumors (ER positive and/or PgR positive, HER2 positive, all grades) was relatively uncommon (11% to 22%), even with the use of trastuzumab, and achievement of pCR in this subgroup was also not prognostic for survival. In contrast, patients whose subtype is typically associated with a worse prognosis (triple negative, or ER negative and HER2 positive) were much more likely to have pCR (28% to 32%), and those who achieved pCR had a much better outcome. Although the reasons for these findings are not entirely clear from these data, a possible explanation is that lack of pCR to chemotherapy in less proliferative JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 30 NUMBER 15 MAY 2

ESR1 mutations in circulating plasma tumor DNA from metastatic breast cancer patients

Purpose: Mutations in the estrogen receptor (ER)α gene, ESR1, have been identified in breast cancer metastases after progression on endocrine therapies. Because of limitations of metastatic biopsies, the reported frequency of ESR1 mutations may be underestimated. Here, we show a high frequency of ESR1 mutations using circulating plasma tumor DNA (ptDNA) from patients with metastatic breast cancer. Experimental Design: We retrospectively obtained plasma samples from eight patients with known ESR1 mutations and three patients with wild-type ESR1 identified by next-generation sequencing (NGS) of biopsied metastatic tissues. Three common ESR1 mutations were queried for using droplet digital PCR (ddPCR). In a prospective cohort, metastatic tissue and plasma were collected contemporaneously from eight ER-positive and four ER-negative patients. Tissue biopsies were sequenced by NGS, and ptDNA ESR1 mutations were analyzed by ddPCR. Results: In the retrospective cohort, all corresponding mutations were detected in ptDNA, with two patients harboring additional ESR1 mutations not present in their metastatic tissues. In the prospective cohort, three ER-positive patients did not have adequate tissue for NGS, and no ESR1 mutations were identified in tissue biopsies from the other nine patients. In contrast, ddPCR detected seven ptDNA ESR1 mutations in 6 of 12 patients (50%). Conclusions: We show that ESR1 mutations can occur at a high frequency and suggest that blood can be used to identify additional mutations not found by sequencing of a single metastatic lesion. Clin Cancer Res; 22(4); 993–9. ©2015 AACR.