Benefit of systemic therapy in MINDACT patients with small, ER-positive, HER2-negative breast cancers.

Small, hormone receptor-positive (HR+), HER2-negative (HER2-), lymph node-negative breast cancers are associated with relatively low rates of disease recurrence and have therefore been underrepresented in clinical trials assessing the effects of systemic therapy. Consequently, it remains uncertain if this patient population derives benefit from these treatments. For this exploratory analysis, we selected MINDACT (NCT00433589) patients with a HR+, HER2-, T1ab (≤1 cm) tumor and negative lymph nodes. Patients with discordant clinical risk and MammaPrint genomic risk classification were randmomized to receive chemotherapy based on either the clinical or the genomic risk assessment. Endocrine therapy treatment was based on local guidelines. 715/6693 (10.7%) MINDACT patients had HR+, HER2-, T1abN0 breast cancer and were included in this analysis. All were clinically low-risk, 124/715 (17.3%) were genomic high-risk. For genomic high-risk tumors, 8-year distant metastasis-free survival (DMFS) was 92.9% (95% CI 86.2-96.4%) compared to 95.0% (95% CI 92.8-96.6%) for genomic low-risk tumors. For genomic high-risk tumors treated with or without chemotherapy, 8-year DMFS was 89.2% (95% CI 73.6-95.8%) and 94.1% (95% CI 82.9-98.1%), respectively. For genomic low-risk tumors, the 8-year DMFS and disease-free survival (DFS) were 96.1% (95% CI 93.4-97.6%) and 89.3% (95% CI 85.5-92.2%) when treated with endocrine therapy and 92.9% (95% CI 87.9-95.9%) and 79.4% (95% CI 72.5-84.8%) without. In conclusion, although the number of randomized patients is small, patients with small, genomic high-risk breast cancer did not seem to derive benefit from chemotherapy. Endocrine therapy was associated with improved outcomes even in genomic low-risk breast cancers.

Targeting the Epidermal Growth Factor Receptor Pathway in Chemotherapy-Resistant Triple-Negative Breast Cancer: A Phase II Study.

PURPOSE: Epidermal growth factor receptor (EGFR) pathway activation causes chemotherapy resistance, and inhibition of the EGFR pathway sensitizes triple-negative breast cancer (TNBC) cells to chemotherapy in preclinical models. Given the high prevalence of EGFR overexpression in TNBC, we conducted a single-arm phase II study of panitumumab (anti-EGFR monoclonal antibody), carboplatin, and paclitaxel as the second phase of neoadjuvant therapy (NAT) in patients with doxorubicin and cyclophosphamide (AC)-resistant TNBC (NCT02593175). PATIENTS AND METHODS: Patients with early-stage, AC-resistant TNBC, defined as disease progression or ≤80% reduction in tumor volume after four cycles of AC, were eligible for this study and received panitumumab (2.5 mg/kg i.v., every week × 13), paclitaxel (80 mg/m2 i.v. every week × 12), and carboplatin (AUC = 4 i.v., every 3 weeks × 4) as the second phase of NAT. A two-stage Gehan-type design was used to detect an improvement in the pathological complete response (pCR)/residual cancer burden class I (RCB-I) rate from 5% to 20%. Whole-exome sequencing was performed on diagnostic tumor biospecimens, where available. RESULTS: From November 3, 2016, through August 23, 2021, 43 patients with AC-resistant TNBC were enrolled. The combined pCR/RCB-I rate was 30.2%. The most common treatment-related adverse events were neutropenia (72%) and anemia (61%), with 7 (16%), 16 (37%), and 8 (19%) patients experiencing grade 4 neutropenia, grade 3 neutropenia, and grade 3 anemia, respectively. No new safety signals were observed. CONCLUSIONS: This study met its primary endpoint (pCR/RCB-I = 30.2% vs. 5% in historical controls), suggesting that panitumumab should be evaluated as a component of NAT in patients with chemotherapy-resistant TNBC in a larger, randomized clinical trial. SIGNIFICANCE: The epidermal growth factor receptor (EGFR) pathway has been implicated as a driver of chemotherapy resistance in triple-negative breast cancer (TNBC). Here, we evaluate the combination of panitumumab, carboplatin, and paclitaxel as the second phase of neoadjuvant therapy (NAT) in patients with AC-resistant TNBC. This study met its primary efficacy endpoint, and molecular alterations in EGFR pathway genes did not seem to influence response to the study regimen.

Single cell genome and epigenome co-profiling reveals hardwiring and plasticity in breast cancer.

Understanding the impact of genetic alterations on epigenomic phenotypes during breast cancer progression is challenging with unimodal measurements. Here, we report wellDA-seq, the first high-genomic resolution, high-throughput method that can simultaneously measure the whole genome and chromatin accessibility profiles of thousands of single cells. Using wellDA-seq, we profiled 22,123 single cells from 2 normal and 9 tumors breast tissues. By directly mapping the epigenomic phenotypes to genetic lineages across cancer subclones, we found evidence of both genetic hardwiring and epigenetic plasticity. In 6 estrogen-receptor positive breast cancers, we directly identified the ancestral cancer cells, and found that their epithelial cell-of-origin was Luminal Hormone Responsive cells. We also identified cell types with copy number aberrations (CNA) in normal breast tissues and discovered non-epithelial cell types in the microenvironment with CNAs in breast cancers. These data provide insights into the complex relationship between genetic alterations and epigenomic phenotypes during breast tumor evolution.

Disease-specific survival outcomes for patients after locoregional treatment for ductal carcinoma in situ: observational cohort study.

BACKGROUND: Breast-conserving surgery alone, breast-conserving surgery with adjuvant radiation treatment, and mastectomy are guideline-concordant treatments for ductal carcinoma in situ. The aim of this study was to compare survival outcomes between these treatment options. METHODS: A stratified random sample of patients diagnosed with pure ductal carcinoma in situ between 2008 and 2014 was selected from 1330 sites in the USA. Data on diagnosis, treatment, and follow-up were abstracted by local cancer registrars. Population-averaged marginal estimates of disease-specific survival and overall survival for breast-conserving surgery alone, breast-conserving surgery with radiation treatment, and mastectomy were obtained by combining sampling and overlap weights. RESULTS: A total of 18 442 women were included, with a median follow-up of 67.8 (interquartile range 46.1-93.5) months. A total of 35 women died from breast cancer, at a median age of 62 (interquartile range 50-74) years. Population-averaged 8-year rates of disease-specific survival were 99.6% or higher for all treatment groups, with no significant differences between groups (breast-conserving surgery alone versus breast-conserving surgery with radiation treatment, HR 1.19 (95% c.i. 0.29 to 4.85); and mastectomy versus breast-conserving surgery with radiation treatment, HR 1.74 (95% c.i. 0.53 to 5.72). There was no difference in overall survival between the patients who underwent a mastectomy and the patients who underwent breast-conserving surgery with radiation treatment (HR 1.09 (95% c.i. 0.83 to 1.43)). Patients who underwent breast-conserving surgery alone had lower overall survival compared with the patients who underwent breast-conserving surgery with radiation treatment (HR 1.29 (95% c.i. 1.00 to 1.67)). This survival difference vanished for all but one subgroup, namely patients less than 65 years (HR 1.86 (95% c.i. 1.15 to 3.00)). CONCLUSION: There was no statistically significant difference in disease-specific survival between women operated with breast-conserving surgery alone, breast-conserving surgery with radiation treatment, or mastectomy for ductal carcinoma in situ. Given the low absolute risk of disease-specific mortality, these results provide confidence in offering individualized locoregional treatment without fear of compromising survival.

Exploring locoregional treatment reporting in neoadjuvant systemic breast cancer treatment studies: A systematic review.

Accurate information about locoregional treatments in breast cancer neoadjuvant systemic therapy (NST) trials is vital to support surgical decision-making and allow meaningful interpretation of long-term oncological outcomes. This systematic review (PROSPERO registration CRD42023470891) aimed to describe the current practice of outcome reporting in NST studies. A systematic search identified primary research studies published 01/01/2018-08/09/2023 reporting outcomes in patients receiving NST for breast cancer followed by locoregional treatment. Included were randomised controlled trials (RCTs) and non-randomised studies (NRS) with >250 participants reporting at least one locoregional treatment outcome. Outcomes were extracted verbatim and categorised using content analysis. Descriptive statistics were used to summarise results. Of the 3111 abstracts screened, 137 studies (22 RCTs and 115 NRS) reporting at least one locoregional outcome in 575,531 patients were included. The 137 studies reported a total of 510 surgical outcomes with a median of 3 (range 1-12) per study. No single outcome was reported in all studies. Type of breast (n = 129, 94.2 %) and axillary (n = 86, 62.8 %) surgery were reported most frequently. Only 34 % (n = 47) studies reported how treatment response was assessed and if/how this informed surgical decision-making. Only a fifth (n = 28) reported outcomes relating to surgical de-escalation. Only 72 studies (52.6 %) reported any radiation therapy (RT)-related outcome, most frequently whether RT had been received (n = 63/72, 87.5 %). Current reporting of locoregional treatment outcomes in NST studies is poor, inconsistent and urgently needs to be improved. A core outcome set and reporting guidelines may improve the quality and value of future research.

Phase 2 study of neoadjuvant enzalutamide and paclitaxel for luminal androgen receptor-enriched TNBC: Trial results and insights into "ARness".

Luminal androgen receptor (LAR)-enriched triple-negative breast cancer (TNBC) is a distinct subtype. The efficacy of AR inhibitors and the relevant biomarkers in neoadjuvant therapy (NAT) are yet to be determined. We tested the combination of the AR inhibitor enzalutamide (120 mg daily by mouth) and paclitaxel (80 mg/m2 weekly intravenously) (ZT) for 12 weeks as NAT for LAR-enriched TNBC. Eligibility criteria included a percentage of cells expressing nuclear AR by immunohistochemistry (iAR) of at least 10% and a reduction in sonographic volume of less than 70% after four cycles of doxorubicin and cyclophosphamide. Twenty-four patients were enrolled. Ten achieved a pathologic complete response or residual cancer burden-I. ZT was safe, with no unexpected side effects. An iAR of at least 70% had a positive predictive value of 0.92 and a negative predictive value of 0.97 in predicting LAR-enriched TNBC according to RNA-based assays. Our data support future trials of AR blockade in early-stage LAR-enriched TNBC.

Epigenetic activation of SOX11 is associated with recurrence and progression of ductal carcinoma in situ to invasive breast cancer.

BACKGROUND: Risk of recurrence and progression of ductal carcinoma in situ (DCIS) to invasive cancer remains uncertain, emphasizing the need for developing predictive biomarkers of aggressive DCIS. METHODS: Human cell lines and mouse models of disease progression were analyzed for candidate risk predictive biomarkers identified and validated in two independent DCIS cohorts. RESULTS: RNA profiling of normal mammary and DCIS tissues (n = 48) revealed that elevated SOX11 expression correlates with MKI67, EZH2, and DCIS recurrence score. The 21T human cell line model of DCIS progression to invasive cancer and two mouse models developing mammary intraepithelial neoplasia confirmed the findings. AKT activation correlated with chromatin accessibility and EZH2 enrichment upregulating SOX11 expression. AKT and HER2 inhibitors decreased SOX11 expression along with diminished mammosphere formation. SOX11 was upregulated in HER2+ and basal-like subtypes (P < 0.001). Longitudinal DCIS cohort (n = 194) revealed shorter recurrence-free survival in SOX11+ than SOX11- patients (P = 0.0056 in all DCIS; P < 0.0001 in HER2+ subtype) associated with increased risk of ipsilateral breast event/IBE (HR = 1.9, 95%CI = 1.2-2.9; P = 0.003). DISCUSSION: Epigenetic activation of SOX11 drives recurrence of DCIS and progression to invasive cancer, suggesting SOX11 as a predictive biomarker of IBE.

Protocol for the development of a core outcome set and reporting guidelines for locoregional treatment in neoadjuvant systemic breast cancer treatment trials: the PRECEDENT project.

INTRODUCTION: Neoadjuvant systemic anticancer therapy (neoSACT) is increasingly used in the treatment of early breast cancer. Response to therapy is prognostic and allows locoregional and adjuvant systemic treatments to be tailored to minimise morbidity and optimise oncological outcomes and quality of life. Accurate information about locoregional treatments following neoSACT is vital to allow the translation of downstaging benefits into practice and facilitate meaningful interpretation of oncological outcomes, particularly locoregional recurrence. Reporting of locoregional treatments in neoSACT studies, however, is currently poor. The development of a core outcome set (COS) and reporting guidelines is one strategy by which this may be improved. METHODS AND ANALYSIS: A COS for reporting locoregional treatment (surgery and radiotherapy) in neoSACT trials will be developed in accordance with Core Outcome Measures in Effectiveness Trials (COMET) and Core Outcome Set-Standards for Development guidelines. Reporting guidance will be developed concurrently.The project will have three phases: (1) generation of a long list of relevant outcome domains and reporting items from a systematic review of published neoSACT studies and interviews with key stakeholders. Identified items and domains will be categorised and formatted into Delphi consensus questionnaire items. (2) At least two rounds of an international online Delphi survey in which at least 250 key stakeholders (surgeons/oncologists/radiologists/pathologists/trialists/methodologists) will score the importance of reporting each outcome. (3) A consensus meeting with key stakeholders to discuss and agree the final COS and reporting guidance. ETHICS AND DISSEMINATION: Ethical approval for the consensus process will be obtained from the Queen's University Belfast Faculty Ethics Committee. The COS/reporting guidelines will be presented at international meetings and published in peer-reviewed journals. Dissemination materials will be produced in collaboration with our steering group and patient advocates so the results can be shared widely. REGISTRATION: The study has been prospectively registered on the COMET website (https://www.comet-initiative.org/Studies/Details/2854).

eEF1A2 promotes PTEN-GSK3ß-SCF complex-dependent degradation of Aurora kinase A and is inactivated in breast cancer.

The translation elongation factor eEF1A promotes protein synthesis. Its methylation by METTL13 increases its activity, supporting tumor growth. However, in some cancers, a high abundance of eEF1A isoforms is associated with a good prognosis. Here, we found that eEF1A2 exhibited oncogenic or tumor-suppressor functions depending on its interaction with METTL13 or the phosphatase PTEN, respectively. METTL13 and PTEN competed for interaction with eEF1A2 in the same structural domain. PTEN-bound eEF1A2 promoted the ubiquitination and degradation of the mitosis-promoting Aurora kinase A in the S and G2 phases of the cell cycle. eEF1A2 bridged the interactions between the SKP1-CUL1-FBXW7 (SCF) ubiquitin ligase complex, the kinase GSK3ß, and Aurora-A, thereby facilitating the phosphorylation of Aurora-A in a degron site that was recognized by FBXW7. Genetic ablation of Eef1a2 or Pten in mice resulted in a greater abundance of Aurora-A and increased cell cycling in mammary tumors, which was corroborated in breast cancer tissues from patients. Reactivating this pathway using fimepinostat, which relieves inhibitory signaling directed at PTEN and increases FBXW7 expression, combined with inhibiting Aurora-A with alisertib, suppressed breast cancer cell proliferation in culture and tumor growth in vivo. The findings demonstrate a therapeutically exploitable, tumor-suppressive role for eEF1A2 in breast cancer.

Intraoperative Evaluation of Breast Tissues During Breast Cancer Operations Using the MasSpec Pen.

Importance: Surgery with complete tumor resection remains the main treatment option for patients with breast cancer. Yet, current technologies are limited in providing accurate assessment of breast tissue in vivo, warranting development of new technologies for surgical guidance. Objective: To evaluate the performance of the MasSpec Pen for accurate intraoperative assessment of breast tissues and surgical margins based on metabolic and lipid information. Design, Setting, and Participants: In this diagnostic study conducted between February 23, 2017, and August 19, 2021, the mass spectrometry-based device was used to analyze healthy breast and invasive ductal carcinoma (IDC) banked tissue samples from adult patients undergoing breast surgery for ductal carcinomas or nonmalignant conditions. Fresh-frozen tissue samples and touch imprints were analyzed in a laboratory. Intraoperative in vivo and ex vivo breast tissue analyses were performed by surgical staff in operating rooms (ORs) within 2 different hospitals at the Texas Medical Center. Molecular data were used to build statistical classifiers. Main Outcomes and Measures: Prediction results of tissue analyses from classification models were compared with gross assessment, frozen section analysis, and/or final postoperative pathology to assess accuracy. Results: All data acquired from the 143 banked tissue samples, including 79 healthy breast and 64 IDC tissues, were included in the statistical analysis. Data presented rich molecular profiles of healthy and IDC banked tissue samples, with significant changes in relative abundances observed for several metabolic species. Statistical classifiers yielded accuracies of 95.6%, 95.5%, and 90.6% for training, validation, and independent test sets, respectively. A total of 25 participants enrolled in the clinical, intraoperative study; all were female, and the median age was 58 years (IQR, 44-66 years). Intraoperative testing of the technology was successfully performed by surgical staff during 25 breast operations. Of 273 intraoperative analyses performed during 25 surgical cases, 147 analyses from 22 cases were subjected to statistical classification. Testing of the classifiers on 147 intraoperative mass spectra yielded 95.9% agreement with postoperative pathology results. Conclusions and Relevance: The findings of this diagnostic study suggest that the mass spectrometry-based system could be clinically valuable to surgeons and patients by enabling fast molecular-based intraoperative assessment of in vivo and ex vivo breast tissue samples and surgical margins.

Atypia detected during breast screening and subsequent development of cancer: observational analysis of the Sloane atypia prospective cohort in England.

OBJECTIVE: To explore how the number and type of breast cancers developed after screen detected atypia compare with the anticipated 11.3 cancers detected per 1000 women screened within one three year screening round in the United Kingdom. DESIGN: Observational analysis of the Sloane atypia prospective cohort in England. SETTING: Atypia diagnoses through the English NHS breast screening programme reported to the Sloane cohort study. This cohort is linked to the English Cancer Registry and the Mortality and Birth Information System for information on subsequent breast cancer and mortality. PARTICIPANTS: 3238 women diagnosed as having epithelial atypia between 1 April 2003 and 30 June 2018. MAIN OUTCOME MEASURES: Number and type of invasive breast cancers detected at one, three, and six years after atypia diagnosis by atypia type, age, and year of diagnosis. RESULTS: There was a fourfold increase in detection of atypia after the introduction of digital mammography between 2010 (n=119) and 2015 (n=502). During 19 088 person years of follow-up after atypia diagnosis (until December 2018), 141 women developed breast cancer. Cumulative incidence of cancer per 1000 women with atypia was 0.95 (95% confidence interval 0.28 to 2.69), 14.2 (10.3 to 19.1), and 45.0 (36.3 to 55.1) at one, three, and six years after atypia diagnosis, respectively. Women with atypia detected more recently have lower rates of subsequent cancers detected within three years (6.0 invasive cancers per 1000 women (95% confidence interval 3.1 to 10.9) in 2013-18 v 24.3 (13.7 to 40.1) in 2003-07, and 24.6 (14.9 to 38.3) in 2008-12). Grade, size, and nodal involvement of subsequent invasive cancers were similar to those of cancers detected in the general screening population, with equal numbers of ipsilateral and contralateral cancers. CONCLUSIONS: Many atypia could represent risk factors rather than precursors of invasive cancer requiring surgery in the short term. Women with atypia detected more recently have lower rates of subsequent cancers detected, which might be associated with changes to mammography and biopsy techniques identifying forms of atypia that are more likely to represent overdiagnosis. Annual mammography in the short term after atypia diagnosis might not be beneficial. More evidence is needed about longer term risks.

Locoregional Breast Cancer Recurrence in the European Organisation for Research and Treatment of Cancer 10041/BIG 03-04 MINDACT Trial: Analysis of Risk Factors Including the 70-Gene Signature.

PURPOSE: A number of studies are currently investigating de-escalation of radiation therapy in patients with a low risk of in-breast relapses on the basis of clinicopathologic factors and molecular tests. We evaluated whether 70-gene risk score is associated with risk of locoregional recurrence (LRR) and estimated 8-year cumulative incidences for LRR in patients with early-stage breast cancer treated with breast conservation. METHODS: In this exploratory substudy of European Organisation for Research and Treatment of Cancer 10041/BIG 03-04 MINDACT trial, we evaluated women with a known clinical and genomic 70-gene risk score test result and who had breast-conserving surgery (BCS). The primary end point was LRR at 8 years, estimated by cumulative incidences. Distant metastasis and death were considered competing risks. RESULTS: Among 6,693 enrolled patients, 5,470 (81.7%) underwent BCS, of whom 98% received radiotherapy. At 8-year follow-up, 189 patients experienced a LRR, resulting in an 8-year cumulative incidence of 3.2% (95% CI, 2.7 to 3.7). In patients with a low-risk 70-gene signature, the 8-year LRR incidence was 2.7% (95% CI, 2.1 to 3.3). In univariable analysis, adjusted for chemotherapy, five of 12 variables were associated with LRR, including the 70-gene signature. In multivariable modeling, adjuvant endocrine therapy and to a lesser extent tumor size and grade remained significantly associated with LRR. CONCLUSION: This exploratory analysis of the MINDACT trial estimated an 8-year low LRR rate of 3.2% after BCS. The 70-gene signature was not independently predictive of LRR perhaps because of the low number of events observed and currently cannot be used in clinical decision making regarding LRR. The overall low number of events does provide an opportunity to design trials toward de-escalation of local therapy.

Evidence-informed recommendations on managing breast screening atypia: perspectives from an expert panel consensus meeting reviewing results from the Sloane atypia project.

Evidence-based clinical guidelines are essential to maximize patient benefit and to reduce clinical uncertainty and inconsistency in clinical practice. Gaps in the evidence base can be addressed by data acquired in routine practice. At present, there is no international consensus on management of women diagnosed with atypical lesions in breast screening programmes. Here, we describe how routine NHS breast screening data collected by the Sloane atypia project was used to inform a management pathway that maximizes early detection of cancer and minimizes over-investigation of lesions with uncertain malignant potential. A half-day consensus meeting with 11 clinical experts, 1 representative from Independent Cancer Patients' Voice, 6 representatives from NHS England (NHSE) including from Commissioning, and 2 researchers was held to facilitate discussions of findings from an analysis of the Sloane atypia project. Key considerations of the expert group in terms of the management of women with screen detected atypia were: (1) frequency and purpose of follow-up; (2) communication to patients; (3) generalizability of study results; and (4) workforce challenges. The group concurred that the new evidence does not support annual surveillance mammography for women with atypia, irrespective of type of lesion, or woman's age. Continued data collection is paramount to monitor and audit the change in recommendations.

ΔNp63 is regulated by insulin/IGF-1 signaling in normal basal/progenitor mammary cells and in luminal-type breast cancer cells.

Breast cancers are a heterogeneous group of tumors classified according to their histological growth patterns and receptor expression characteristics. Intratumor heterogeneity also exists, with subpopulations of cells with different phenotypes found in individual cancers, including cells with stem or progenitor cell properties. At least two types of breast cancer stem cells (CSCs) exist, the epithelial and the basal/mesenchymal subtypes, although how these phenotypes are controlled is unknown. ΔNp63 is a basal cell marker and regulator of stem/progenitor cell activities in the normal mammary gland and is expressed in the basal-like CSC subpopulation in some estrogen receptor-positive (ER+) and/or human epidermal growth factor receptor 2-positive (HER2+) breast adenocarcinomas. Whilst p63 is known to directly impart CSC properties in luminal breast cancer cells, how p63 is regulated and induced in these cells is unknown. We initially confirmed the existence of a small subpopulation of ΔNp63+ cells in lymph node metastases of ER+ human ductal adenocarcinomas, indicating together with previous reports that ΔNp63+ tumor cells are present in approximately 40% of these metastases. Notably, ΔNp63+ cells show a preferential location at the edge of tumor areas, suggesting possible regulation of ΔNp63 by the tumor microenvironment. Subsequently, we showed that the high levels of ΔNp63 in basal non-transformed MCF-10A mammary epithelial cells rely on insulin in their culture medium, whilst ΔNp63 levels are increased in MCF-7 ER+ luminal-type breast cancer cells treated with insulin or insulin-like growth factor 1 (IGF-1). Mechanistically, small molecule inhibitors and siRNA gene knockdown demonstrated that induction of ΔNp63 by IGF-1 requires PI3K, ERK1/2, and p38 MAPK activation, and acts through FOXO transcriptional inactivation. We also show that metformin inhibits ΔNp63 induction. These data reveal an IGF-mediated mechanism to control basal-type breast CSCs, with therapeutic implications to modify intratumor breast cancer cell heterogeneity and plasticity.

Characterization of Cancer Evolution Landscape Based on Accurate Detection of Somatic Mutations in Single Tumor Cells.

Accurate detection of somatic mutations in single tumor cells is greatly desired as it allows us to quantify the single-cell mutation burden and construct the mutation-based phylogenetic tree. Here we developed scNanoSeq chemistry and profiled 842 single cells from 21 human breast cancer samples. The majority of the mutation-based phylogenetic trees comprise a characteristic stem evolution followed by the clonal sweep. We observed the subtype-dependent lengths in the stem evolution. To explain this phenomenon, we propose that the differences are related to different reprogramming required for different subtypes of breast cancer. Furthermore, we reason that the time that the tumor-initiating cell took to acquire the critical clonal-sweep-initiating mutation by random chance set the time limit for the reprogramming process. We refer to this model as a reprogramming and critical mutation co-timing (RCMC) subtype model. Next, in the sweeping clone, we observed that tumor cells undergo a branched evolution with rapidly decreasing selection. In the most recent clades, effectively neutral evolution has been reached, resulting in a substantially large number of mutational heterogeneities. Integrative analysis with 522-713X ultra-deep bulk whole genome sequencing (WGS) further validated this evolution mode. Mutation-based phylogenetic trees also allow us to identify the early branched cells in a few samples, whose phylogenetic trees support the gradual evolution of copy number variations (CNVs). Overall, the development of scNanoSeq allows us to unveil novel insights into breast cancer evolution.

Effect of Metformin Versus Placebo on New Primary Cancers in Canadian Cancer Trials Group MA.32: A Secondary Analysis of a Phase III Randomized Double-Blind Trial in Early Breast Cancer.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned coprimary or secondary analyses are not yet available. Clinical trial updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.Metformin has been associated with lower cancer risk in epidemiologic and preclinical research. In the MA.32 randomized adjuvant breast cancer trial, metformin (v placebo) did not affect invasive disease-free or overall survival. Here, we report metformin effects on the risk of new cancer. Between 2010 and 2013, 3,649 patients with breast cancer younger than 75 years without diabetes with high-risk T1-3, N0-3 M0 breast cancer (any estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2) were randomly assigned to metformin 850 mg orally twice a day or placebo twice a day for 5 years. New primary invasive cancers (outside the ipsilateral breast) developing as a first event were identified. Time to events was described by the competing risks method; two-sided likelihood ratio tests adjusting for age, BMI, smoking, and alcohol intake were used to compare metformin versus placebo arms. A total of 184 patients developed new invasive cancers: 102 metformin and 82 placebo, hazard ratio (HR), 1.25; 95% CI, 0.94 to 1.68; P = .13. These included 48 contralateral invasive breast cancers (27 metformin v 21 placebo), HR, 1.29; 95% CI, 0.72 to 2.27; P = .40 and 136 new nonbreast primary cancers (75 metformin v 61 placebo), HR, 1.24; 95% CI, 0.88 to 1.74; P = .21. Metformin did not reduce the risk of new cancer development in these nondiabetic patients with breast cancer.

Archival single-cell genomics reveals persistent subclones during DCIS progression.

Ductal carcinoma in situ (DCIS) is a common precursor of invasive breast cancer. Our understanding of its genomic progression to recurrent disease remains poor, partly due to challenges associated with the genomic profiling of formalin-fixed paraffin-embedded (FFPE) materials. Here, we developed Arc-well, a high-throughput single-cell DNA-sequencing method that is compatible with FFPE materials. We validated our method by profiling 40,330 single cells from cell lines, a frozen tissue, and 27 FFPE samples from breast, lung, and prostate tumors stored for 3-31 years. Analysis of 10 patients with matched DCIS and cancers that recurred 2-16 years later show that many primary DCIS had already undergone whole-genome doubling and clonal diversification and that they shared genomic lineages with persistent subclones in the recurrences. Evolutionary analysis suggests that most DCIS cases in our cohort underwent an evolutionary bottleneck, and further identified chromosome aberrations in the persistent subclones that were associated with recurrence.

PTEN in triple-negative breast carcinoma: protein expression and genomic alteration in pretreatment and posttreatment specimens.

Background: Recent advances have been made in targeting the phosphoinositide 3-kinase pathway in breast cancer. Phosphatase and tensin homolog (PTEN) is a key component of that pathway. Objective: To understand the changes in PTEN expression over the course of the disease in patients with triple-negative breast cancer (TNBC) and whether PTEN copy number variation (CNV) by next-generation sequencing (NGS) can serve as an alternative to immunohistochemistry (IHC) to identify PTEN loss. Methods: We compared PTEN expression by IHC between pretreatment tumors and residual tumors in the breast and lymph nodes after neoadjuvant chemotherapy in 96 patients enrolled in a TNBC clinical trial. A correlative analysis between PTEN protein expression and PTEN CNV by NGS was also performed. Results: With a stringent cutoff for PTEN IHC scoring, PTEN expression was discordant between pretreatment and posttreatment primary tumors in 5% of patients (n = 96) and between posttreatment primary tumors and lymph node metastases in 9% (n = 33). A less stringent cutoff yielded similar discordance rates. Intratumoral heterogeneity for PTEN loss was observed in 7% of the patients. Among pretreatment tumors, PTEN copy numbers by whole exome sequencing (n = 72) were significantly higher in the PTEN-positive tumors by IHC compared with the IHC PTEN-loss tumors (p < 0.0001). However, PTEN-positive and PTEN-loss tumors by IHC overlapped in copy numbers: 14 of 60 PTEN-positive samples showed decreased copy numbers in the range of those of the PTEN-loss tumors. Conclusion: Testing various specimens by IHC may generate different PTEN results in a small proportion of patients with TNBC; therefore, the decision of testing one versus multiple specimens in a clinical trial should be defined in the patient inclusion criteria. Although a distinct cutoff by which CNV differentiated PTEN-positive tumors from those with PTEN loss was not identified, higher copy number of PTEN may confer positive PTEN, whereas lower copy number of PTEN would necessitate additional testing by IHC to assess PTEN loss. Trial registration: NCT02276443.