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Supplementary Table S1-S12

SUPPLEMENTARY FIGURE (1-7) AND TABLE (1-12) LEGENDS

E2F activation metagene & Fluorescence activated cell sorting

Purpose:To investigate the presence of ESR1 mutations in primary estrogen-receptor–positive (ER+) breast cancer treated with extended (>4 weeks) neoadjuvant (presurgical) aromatase inhibitor (NAI) therapy and to identify patients who may gain less benefit from aromatase inhibition (AI) alone based upon on-treatment changes in gene expression.Experimental Design:We evaluated ER, progesterone receptor, and Ki67 by immunostaining, ESR1 mutations by droplet-digital PCR and expression of over 800 key breast cancer genes in paired pre- and post-NAI tumor samples from 87 ER+ breast cancer patients.Results:Cell proliferation and estrogen-regulated genes (ERG) remained suppressed in most tumors indicative of persistent response to NAI. Enrichment of ESR1 mutations was found in five tumors and predominantly in patients receiving therapy for >6 months. ESR1-mutant tumors showed increased expression of ESR1 transcript and limited suppression of ERGs and proliferation-associated genes in response to NAI. ESR1 wild-type tumors with high residual proliferation (Ki67r ≥10%; 15/87 tumors) showed lower ESR1/ER expression pre- and post-therapy and lower ERGs. Tumors with ESR1 mutations or Ki67r ≥10% showed less inhibition of estrogen response, cell cycle, and E2F-target genes.Conclusions:Ligand-independent ER signaling, as a result of ESR1 mutation or reduced ER dependence, identified after extended NAI therapy, can guide early selection of patients who would benefit from combination therapy.

Supplementary Figures S1-S7

To investigate the presence ofWe evaluated ER, progesterone receptor, and Ki67 by immunostaining,Cell proliferation and estrogen-regulated genes (ERG) remained suppressed in most tumors indicative of persistent response to NAI. Enrichment ofLigand-independent ER signaling, as a result of

For premenopausal women with primary ER + breast cancer, oophorectomy (OvX) is an evidence-based cost-effective option and is standard treatment in many countries. However, there is virtually no data describing the effects of OvX on breast tumour biology. We therefore, characterised the endocrine and genome-wide transcriptional impact of OvX in 56 premenopausal women with ER + breast cancer for 2 weeks prior to mastectomy. Plasma estradiol concentrations decreased from 406 ± 41 to 20.7 ± 2.6 pmol/l (mean ± sem) 24 h after OvX, and to 8.1 ± 0.8 pmol/l 2 weeks later at mastectomy. Ki67 decreased in 33/36 (91.7%) tumours. The expression of 655 genes changed significantly (FDR, Genetics: Ovary removal alters gene activity in breast tumours Surgical removal of the ovaries alters the expression of hundreds of genes in the tumour cells of premenopausal women with breast cancer. Ben Haynes from Royal Marsden Hospital in London, UK and colleagues characterised molecular changes in 56 premenopausal women from Vietnam with oestrogen receptor-positive breast cancer who underwent oophorectomies, a standard treatment for this patient population. They showed that blood levels of the hormone estradiol dropped precipitously following ovary-removal surgery. Levels of a protein that was indicative of tumour growth also went down, as did genes involved in regulating hormone signalling and cell proliferation. The results are consistent with those seen in postmenopausal women following treatment with an oestrogen-blocking drug, but oophorectomy had a more dramatic effect. The data could aid the search for predictive biomarkers of who stands to benefit most from ovary removal.

Introduction: The substantial majority of breast cancers present with early-stage disease, although micro-metastatic disease may be established at the time of diagnosis and may ultimately result in metastatic disease recurrence. Prior studies have shown that analysis of circulating tumour DNA (ctDNA) can detect micro-scopic minimal residual disease (MRD) serving as biomarker to anticipate future cancer recurrence, although sensitivity of current assays is limited. We present a novel molecular fingerprint ctDNA assay that exploits tumour whole genome sequencing (WGS) to develop highly personalised assays. Methods: WGS was conducted on paired tumor and germline DNA from 11 early breast cancer patients to identify tumour specific small indels (SIs) to track in plasma as individual molecular fingerprints. Patient-specific amplicon-based panels were designed and validated to identify somatic reporters with no sequencing background. DNA was extracted from plasma samples taken at pre-surgery (baseline), post-surgery, and every 3 months until 1 year and every 6 months thereafter, and sequenced to 100,000X. Results: Molecular fingerprint assays were demonstrated to detect less than a single cancer genome, with undetectable background in control samples. Using plasma samples from patients with early stage breast cancer, we detected ctDNA in 80% of the baseline plasma time-points with 100% specificity (95% confidence limits (CI) 98.7%-100%). ctDNA was detected in all patients prior to relapse, with an observed lead-time of 15.5 months, substantially improved compared with previous techniques, detecting residual disease that was not detectable by tracking mutations using digital PCR. Conclusions: This proof-of-principle study demonstrates that tumor-specific molecular fingerprints combined with ultra-deep plasma DNA sequencing have the potential to transform MRD detection. Identifying those patients who have undetectable recurrence would pave the way for identifying who is cured by surgery alone and who requires further therapy. Citation Format: Iñaki Comino-Mendez, Ros Cutts, Isaac García-Murillas, Neha Chopra, Maria Afentakis, Abigail Evans, Duncan Wheatley, Anthony Skene, Simon Russell, Mohini Varughese, Mitch Dowsett, Ian E. Smith, Nick Turner. Molecular fingerprint sequencing for minimal residual disease detection in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3608.