Recurrent breast cancer accounts for most disease-associated mortality and can develop decades after primary tumor therapy. Recurrences arise from residual tumor cells (RTCs) that can evade therapy in a dormant state, however the mechanisms are poorly understood. CRISPR-Cas9 screening identified the transcription factors SOX5/6 as functional regulators of tumor recurrence. Loss of SOX5 accelerated recurrence and promoted escape from dormancy. Remarkably, SOX5 drove dormant RTCs to adopt a cartilage-dependent bone development program, termed endochondral ossification, that was confirmed by [18F]NaF-PET imaging and reversed in recurrent tumors escaping dormancy. In patients, osteochondrogenic gene expression in primary breast cancers or residual disease post-neoadjuvant therapy predicted improved recurrence-free survival. These findings suggest that SOX5-dependent mesodermal transdifferentiation constitutes an adaptive mechanism that prevents recurrence by reinforcing tumor cell dormancy.

Supplementary Figure S1 shows sgRNA distribution at each of the time points during disease progression as well as clonal enrichment for sgRNAs targeting Sox5 and Sox6 in MTB/TAN cells

Supplementary Figure S4 shows chondrogenesis and osteogenesis signature enrichment during dormancy in vitro in MTB/TAN cells as well as GFP positivity relative to Alcian blue staining

Supplementary Figure S2 shows Sox5 and Sox6 sgRNA editing efficiency, primary tumor and recurrent tumor growth rate at the orthotopic and metastatic site in MTB/TAN cells

Supplementary Figure S3 shows Sox5 shRNA knockdown efficiency, proliferation and apoptosis rates in vitro and in vivo, and Sox5 sgRNA editing efficiency in nucleofected D2.OR cells

List of targets and sgRNAs used in the custom CRISPR-Cas9 screen. sgRNAs with validated editing efficiency are highlighted

Supplementary Figure S5 shows μCT and [18F]NaF PET in MTB/TAN cells as well as GFP positivity relative to Alcian blue staining

A, PGRMC1 is overexpressed in human breast cancers of the basal subtype using PAM50. TMEM97 has the highest expression in luminal B tumors. B, PGRMC1 is overexpressed in hormone receptor–negative cancers. LDLR expression is highest in ER−/HER2+. TMEM97 has the highest expression in ER+/HER2+ tumors. C, PGRMC1 is overexpressed in smaller higher-grade tumors. LDLR and TMEM97 expression is highest in higher-grade tumors.

This shows the association of TK1 with early breast cancer relapse segregated by molecular subtype.

This shows the association of our proliferation score with established scores like Oncotype Dx and PAM50.

PGRMC1 is associated with early breast cancer relapse in a proliferation-dependent manner. Effect size estimates were aggregated across datasets by meta-analysis to determine the risk of relapse within 5 years from all cancers. A, Association of PGRMC1 with early breast cancer relapse. B, Association of PGRMC1 with early breast cancer relapse within the basal subtype. C, Association of PGRMC1 with early relapse adjusted for estimated proliferation. JRH, John Radcliffe Hospital.

Abstract The ternary complex of progesterone receptor membrane component 1 (PGRMC1)–sigma-2 receptor/transmembrane protein 97 (σ2R/TMEM97)–low-density lipoprotein receptor (LDLR) has recently been discovered and plays a role in cholesterol transport. This study investigated whether individual components of that complex are prognostic breast cancer biomarkers and have defined expression in established molecular subtypes. A total of 4,463 invasive breast cancers were analyzed as a function of molecular and phenotypic markers, estimates of cellular proliferation, and recurrence-free survival. A gene expression signature–based assay was utilized to estimate cellular proliferation. Cox proportional hazards regression estimated relapse-free survival and multivariate Cox analysis adjusted for the association of proliferation with early relapse. PGRMC1–σ2R/TMEM97–LDLR expression was stratified by immunohistochemical (IHC) and molecular subtype, tumor grade, and size. TMEM97 exhibited the strongest correlation with proliferation, highest in estrogen receptor (ER)–positive disease (r = 0.59, P = 8.1−114). TMEM97 and PGRMC1 were associated with a risk of early recurrence, dependent upon their association with proliferation. The risk of early recurrence was highest with TMEM97 and only seen in ER+/HER2− disease [HR = 1.5; 95% confidence interval (CI) = 1.35–1.67; P = 5.4−14] and ER+ malignancies (HR = 1.49; 95% CI = 1.31–1.68; P = 3.1−10). There was no increased risk of recurrence with TMEM97 expression in ER−/HER2− (HR = 1.05; 95% CI = 0.88–1.25; P = 0.63) or ER− disease (HR = 1.02; 95% CI = 0.89–1.17; P = 0.75). Components of a ternary complex associated with rapid internalization of low-density lipoprotein are biomarkers associated with cellular proliferation and early recurrence, which should help guide studies exploring them in the context of additional markers of aggressive disease. Elucidating the role of PGRMC1, TMEM97, and LDLR in breast cancer will facilitate a mechanistic understanding of how proliferation interplays with cholesterol metabolism in malignant transformation or propagation. Significance: This first large-scale analysis of the putative ternary complex responsible for rapid low-density lipoprotein internalization in breast cancer reveals a link between component expression and recurrence, with prognostic implications for identifying patients needing supplemental posttreatment surveillance and/or additional therapeutic approaches.

This shows the trimeric complex correlation with PAM50 ROR.

PGRMC1 is weakly associated with TK1. PGRMC1 exhibits a significant correlation with TK1 when all cancers are combined, although the magnitude of these associations is weak. PGRMC1 vs. TK1

This shows that the association of TK1 with breast cancer relapse is dependent on proliferation.

These are the publicly available microarray datasets combined for this analysis.

The shows the number of samples with RNA-sequencing data available from the TCGA database.

Background: Recurrence after early-stage breast cancer (BC) is a challenge, occurring in ∼30% of patients (pts). Recurrences may arise from reactivation of disseminated tumor cells (DTCs) persisting in a dormant state after primary treatment. The presence of minimal residual disease (MRD) as bone marrow DTCs and/or circulating tumor DNA (ctDNA) in the blood increases the risk of BC recurrence/death. It remains unclear which pts with DTCs will have these reactivate or develop detectable ctDNA before clinical relapse. We evaluated the association and temporal relationship of ctDNA with DTCs in a population of high-risk BC survivors, and the relationship of these markers with subsequent metastatic recurrence. Methods: “PENN SURMOUNT” is a single center, prospective, longitudinal cohort study examining MRD biomarkers among pts within 5 years (y) of BC diagnosis who completed all curative treatment except endocrine therapy. Eligible pts must have had: 1) TNBC, or 2) HER2+ or HR+ BC with positive LN and/or residual disease after neoadjuvant therapy, or 3) HR+ BC with a 21-gene Recurrence score >25 and/or high risk Mammaprint. Pts had annual bone marrow aspirate (BMA) for DTCs by immunohistochemistry (using methods of Naume et al.). DTC+ pts went on therapeutic trial; DTC- pts had up to 5y of annual BMA and blood testing. ctDNA was retrospectively assessed using the RaDaR assay, which targets pt-specific somatic mutations identified by whole-exome sequencing (WES) of primary tumor tissue. Results: Of 184 pts enrolled from 2016 – 2021, 121 had tissue available; 114/121 (94%) had successful WES. A total of 338 plasma samples from 96 pts (median 2 timepoints each, range 1-12) have been successfully tested by RaDaR to date. Overall, ctDNA was detected in 11 samples from 9/96 pts (9.3%) with a median eVAF of 0.009% (range 0.002-0.084%). Two pts were ctDNA+ at baseline (BL), and 7 became positive on surveillance. 87/96 (90.6%) were ctDNA- across all timepoints. 34/96 pts (35%) were DTC+, either at BL (n=24, 25%) or after (n=10, 10%). Considering all timepoints, concordance was 64%. Of 34 ever-DTC+ pts, 4 (12%) were ctDNA+ (of whom 3/4 recurred) and 30 remained ctDNA- (with 1/30 who recurred). Among the 62 pts who remained DTC-, 5 (8%) were ctDNA+ (with 5/5 who recurred), and 57 remained ctDNA- (of whom 5/57 recurred). All ctDNA positivity in DTC+ pts occurred at the time of or after DTC positivity. Over median follow-up (f/u) of 65 months (m), BC recurrence occurred in 14/96 pts (15%), with 2 locoregional-only and 12 distant +/- locoregional recurrences (involving the bone, liver, lung/pleura, and brain); 8/14 pts (57%) were ctDNA+ prior to relapse. 7/12 (58%) with distant recurrences were ctDNA+ prior to metastatic diagnosis, at a median lead time of 15 m (range 0 – 25). Overall, ctDNA+ pts experienced a median lead time from ctDNA positivity to recurrence of 13 m (range 0 – 25). Only 1 of 9 ctDNA+ pts has not recurred; this pt was DTC+ and went on therapeutic trial, without evidence of recurrence over 20 m f/u. 30/34 DTC+ pts (89%) who went on therapeutic trial have not had ctDNA detected during f/u and have not recurred. Overall, ctDNA status was significantly associated with relapse (p<0.01), with a PPV of 89% and NPV of 93%. Of the 24 BL DTC+ pts, 2 became ctDNA+ at subsequent timepoints, an average of 18 m after DTC assessment, and both relapsed (3 and 5 m from ctDNA detection, respectively). Conclusions: In this surveillance study of high-risk BC pts, DTC+ pts were identified who subsequently developed detectable ctDNA and clinical relapse. Where there were discordant results, the timing of DTC and ctDNA positivity revealed a window of opportunity for intervention. A strategy combining both markers for surveillance and intervention to prevent metastatic disease may be of value. Citation Format: Eleanor Taranto, Nicholas J. Seewald, Lauren J. Bayne, Emma Walinsky, Shannon Deluca, Natalie NC. Shih, Pauleen Sanchez, Isoris Nivar, Bana Ambasager, Clodagh Murray, Amber Chevalier, Christopher G. Smith, Igor Makhlin, Killian Rohn, Brooke L. Goodspeed, Jessica Savage, Paul Wileyto, Jianping Wang, George Belka, Elizabeth Chislock, Lindsay R Berry, Don Berry, Anupma Nayak, Michael Feldman, Amy S. Clark, Lewis A. Chodosh, Angela DeMichele. Circulating tumor DNA (ctDNA), dormant disseminated tumor cells (DTCs) and recurrence outcomes in breast cancer survivors on the SURMOUNT Study [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr PS9-03.

This shows the number of samples with available data for analysis.