ON-treatment Single-cell Analysis for the Identification of Early Tumor Response Biomarkers on Prospective Collected Serial Tumor Biopsies in Triple Negative Breast Cancer Patient During Standard Neoadjuvant Chemo-immunotherapy (ONSET)

This study explores early breast cancer, focusing on triple-negative and high-risk luminal subtypes. It combines single-cell RNA sequencing and spatial imaging of tumor samples collected at different time points during treatment. The aim is to better understand how cancer cells and immune cells interact and to identify biomarkers that can predict whether a patient will respond to chemo-immunotherapy or develop resistance.

The study assumes that early molecular and spatial changes at the single-cell level can predict treatment response. This knowledge could help doctors adapt therapies, avoiding unnecessary treatment while improving effectiveness. The project seeks to reveal, for the first time, how cellular diversity and spatial relationships contribute to treatment resistance and disease progression.

Tumor samples will be analyzed before treatment, after the first treatment cycle (C1D1), and at surgery. Only the biopsy taken after C1D1 is collected specifically for this study; all other samples come from routine clinical care.

Study Overview

• Status: Not yet recruiting
• Conditions: Breast Cancer Early Stage Breast Cancer (Stage 1-3)
• Intervention / Treatment: Procedure: on treatment biopsy (after C1)

Detailed Description

This exploratory translational study investigates early breast cancer (EBC), focusing on triple-negative (TNBC) and high-risk luminal (ER+/HER2-) subtypes. By integrating single-cell RNA sequencing, using the 10x Genomics platform (or similar technologies if it will be not available at the time of experiment), and spatial imaging analyses from serial tumor biopsies, the project aims to characterize tumor-immune interactions and identify predictive biomarkers of response or resistance to neoadjuvant chemo-immunotherapy.

We hypothesize that early molecular and spatial features, when detected at the single-cell level, can predict treatment sensitivity or resistance. This would enable adaptive therapeutic strategies that minimize overtreatment while maximizing efficacy in EBC. Our goal is to reveal, for the first time, the cellular heterogeneity and spatial interactions that drive therapy resistance and disease progression.

The analysis will be performed on tumor biopsies collected before the start of treatment, after C1D1 and on surgical material. The only tissue sample collected specifically for the study is the biopsy after C1D1.

• Study Type: Interventional
• Enrollment (Estimated): 55
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Giulia Viale, MD; Phone Number: +390226437627; Email: callcenter.mammella@hsronline.it

Study Locations

• Italy
  • Lombardy
    • Milan, Lombardy, Italy, 20132
      • IRCCS Ospedale San Raffaele
      • Contact: Giulia Viale, MD; Phone Number: +390226437627; Email: callcenter.mammella@hsronline.it

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: No

Description

Inclusion Criteria:

1. Female patients aged ≥18 years.
2. ECOG performance status 0-1.

3. Histologically confirmed early breast cancer with one of the following molecular profiles:

   • TNBC: ER and PR negative (IHC <10%) and HER2 negative (IHC 0-1+ or FISH non-amplified).
   • High-risk luminal (ER+/HER2-): ER positive (IHC ≥10%) HER2 negative (IHC 0-1+ or FISH non-amplified), with high-risk features (e.g., Grade 3, PR-negative, high proliferation, high TILS).

4. Clinical indication for neoadjuvant treatment according to standard practice:

   • TNBC: cT1c and/or cN positive, or cT2 (>2 cm) and/or cN positive (stage II,III).
   • High-risk luminal: features as defined above (Grade 3, PR-negative, high proliferation, ER low).
5. Ability to understand and sign written informed consent for participation in the study, approved by the local Ethics Committee.

Exclusion Criteria:

1. HER2-positive tumors.
2. Multifocal tumors - exclusion if a single index lesion cannot be identified and sampled; otherwise allowed if a representative lesion can be biopsied.
3. Known metastatic disease. 4 Clinical contraindications to the planned neoadjuvant therapy.

5. Decision for upfront surgery as determined by the multidisciplinary team. 6. Inability to provide informed consent. 7. Pregnancy or breastfeeding. 8. Prior systemic therapy (chemotherapy, immunotherapy, or endocrine therapy) for the current breast cancer before baseline biopsy 9. On-treatment biopsy clinically not feasible or controindicated

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Other
• Allocation: N/A
• Interventional Model: Single Group Assignment
• Masking: None (Open Label)

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
To characterize the dynamic changes in tumor and immune cell populations in TNBC and to prospectively collect serial samples from high-risk luminal (ER+/HER2-) breast cancer patients.	Gene expression levels per single-cell and relative cell clustering (%) by comparing pre-treatment, on-treatment (after first cycle of standard of care neoadiuvant therapy), and post-treatment (surgery or residual disease)	Pre-treatment, on-treatment (after first cycle of therapy), and post-treatment (surgery or residual disease)

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
To correlate single-cell gene expression data with pCR and RD to identify predictive signatures of therapy response	pCR and RD will be evaluated across all collected timepoints (baseline, after C1 of neadiuvant standard of care therapy and at surgery)	Across all collected timepoints (baseline, after C1 of neadiuvant standard of care therapy and at surgery)

Collaborators and Investigators

• Sponsor: IRCCS San Raffaele

Study record dates

Study Major Dates

• Study Start (Estimated): September 1, 2026
• Primary Completion (Estimated): September 1, 2028
• Study Completion (Estimated): March 1, 2029

Study Registration Dates

• First Submitted: May 8, 2026
• First Submitted That Met QC Criteria: May 18, 2026
• First Posted (Actual): May 19, 2026

Study Record Updates

• Last Update Posted (Actual): May 22, 2026
• Last Update Submitted That Met QC Criteria: May 19, 2026
• Last Verified: May 1, 2026

More Information

Terms related to this study

• Keywords: triple negative breast cancer; high-risk luminal (ER+/HER2neg)
• Additional Relevant MeSH Terms: Neoplasms by Site; Neoplasms; Skin Diseases; Breast Diseases; Breast Neoplasms; Skin and Connective Tissue Diseases; Triple Negative Breast Neoplasms
• Other Study ID Numbers: ONSET

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No