The authors declare no potential conflicts of interest. Running title: Stochastic profiling of luminal breast cancer by 10cRNA-seqThe heterogeneous composition of solid tumors is known to impact disease progression and response to therapy. Malignant cells coexist in different regulatory states that can be accessed transcriptomically by single-cell RNA sequencing, but these methods have many caveats related to sensitivity, noise, and sample handling. We revised a statistical fluctuation analysis called stochastic profiling to combine with 10-cell RNA sequencing, which was designed for laser-capture microdissection (LCM) and extended here for immuno-LCM. When applied to a cohort of late-onset, early-stage luminal breast cancers, the integrated approach identified thousands of candidate regulatory heterogeneities. Intersecting the candidates from different tumors yielded a relatively stable set of 710 recurrent heterogeneously expressed genes (RHEGs) that were significantly variable in >50% of patients. RHEGs were not confounded by dissociation artifacts, cell cycle oscillations, or driving mutations for breast cancer. Rather, we detected RHEG enrichments for epithelial-to-mesenchymal transition genes and, unexpectedly, the latest pan-cancer assembly of driver genes across cancer types other than breast.Heterogeneous transcriptional regulation conceivably provides a faster, reversible mechanism for malignant cells to sample the effects of potential oncogenes or tumor suppressors on cancer hallmarks. Statement of significanceProfiling intratumor heterogeneity of luminal breast carcinoma cells identifies a recurrent set of genes suggesting sporadic activation of pathways known to drive other types of cancer.

Objective New breast screening modalities are being investigated to address the need for more sensitive breast cancer screening in women with dense breasts. We investigated the preferences and attitudes of these patients regarding adjunct screening modalities to help evaluate the acceptability of these exams. Methods In this institutional review board–approved prospective study, patients with dense breasts on their prior mammogram were invited to complete a survey. Patients were asked to estimate their personal breast cancer risk compared with peers, indicate their level of concern related to screening callbacks, radiation exposure, and intravenous (IV) contrast allergies, and identify which factors might deter them from getting adjunct screening exams. Results Five hundred eight patients with dense breasts presenting for screening mammography completed surveys. While most patients (304/508, 59.9%) felt it was likely or very likely that cancer could be missed on their mammogram, only 8.9% (45/508) had undergone adjunct screening exams in the past 3 years. The most commonly cited deterrents to adjunct screening were cost (340/508, 66.9%), pain (173/508, 34.1%), and concern that adjunct screening could lead to additional procedures (158/508, 31.1%). When asked to select among three hypothetical breast cancer screening modalities, patients strongly preferred the more sensitive examination, even if this involved greater cost (162/508, 31.9%) or IV-contrast administration (315/508, 62.0%). Conclusion Our data suggest that patients with dense breasts prefer adjunct screening exams that are both sensitive and inexpensive, although an increase in sensitivity could outweigh additional cost or even IV-line placement.

IntroductionFor women of the same age and body mass index, increased mammographic density is one of the strongest predictors of breast cancer risk. There are multiple methods of measuring mammographic density and other features in a mammogram that could potentially be used in a screening setting to identify and target women at high risk of developing breast cancer. However, it is unclear which measurement method provides the strongest predictor of breast cancer risk.Methods and analysisThe measurement challenge has been established as an international resource to offer a common set of anonymised mammogram images for measurement and analysis. To date, full field digital mammogram images and core data from 1650 cases and 1929 controls from five countries have been collated. The measurement challenge is an ongoing collaboration and we are continuing to expand the resource to include additional image sets across different populations (from contributors) and to compare additional measurement methods (by challengers). The intended use of the measurement challenge resource is for refinement and validation of new and existing mammographic measurement methods. The measurement challenge resource provides a standardised dataset of mammographic images and core data that enables investigators to directly compare methods of measuring mammographic density or other mammographic features in case/control sets of both raw and processed images, for the purposes of the comparing their predictions of breast cancer risk.Ethics and disseminationChallengers and contributors are required to enter a Research Collaboration Agreement with the University of Melbourne prior to participation in the measurement challenge. The Challenge database of collated data and images are stored in a secure data repository at the University of Melbourne. Ethics approval for the measurement challenge is held at University of Melbourne (HREC ID 0931343.3).

The heterogeneous composition of solid tumors is known to impact disease progression and response to therapy. Malignant cells coexist in different regulatory states that can be accessed transcriptomically by single-cell RNA sequencing, but these methods have many caveats related to sensitivity, noise, and sample handling. We revised a statistical fluctuation analysis called stochastic profiling to combine with 10-cell RNA sequencing, which was designed for laser-capture microdissection (LCM) and extended here for immuno-LCM. When applied to a cohort of late-onset, early-stage luminal breast cancers, the integrated approach identified thousands of candidate regulatory heterogeneities. Intersecting the candidates from different tumors yielded a relatively stable set of 710 recurrent heterogeneously expressed genes (RHEG), which were significantly variable in >50% of patients. RHEGs were not strongly confounded by dissociation artifacts, cell-cycle oscillations, or driving mutations for breast cancer. Rather, RHEGs were enriched for epithelial-to-mesenchymal transition genes and, unexpectedly, the latest pan-cancer assembly of driver genes across cancer types other than breast. These findings indicate that heterogeneous transcriptional regulation conceivably provides a faster, reversible mechanism for malignant cells to evaluate the effects of potential oncogenes or tumor suppressors on cancer hallmarks. Significance: Profiling intratumor heterogeneity of luminal breast carcinoma cells identifies a recurrent set of genes, suggesting sporadic activation of pathways known to drive other types of cancer. See related articles by Schaff and colleagues, p. 1853 and Sutcliffe and colleagues, p. 1868

The publication of this Accepted Manuscript is provided to give early visibility to the contents of the article, which will undergo additional copyediting, typesetting, and review before it is published in its final form. During the production process, errors may be discovered that could affect the content of the Accepted Manuscript. All legal disclaimers that apply to the journal pertain. The reader is cautioned to consult the definitive version of record before relying on the contents of this document.