The aim of this study is to assess the rates of circulating tumour DNA (ctDNA) in patients treated with surgery for stage 1 breast cancer that is HER2 positive or triple negative.

The study will involve collecting blood samples from patients before and after surgery, if patients are enrolled after surgery, blood samples will be collected after the procedure. On the follow-up visit, the results obtained from the blood tests will serve as a diagnostic method to discern adverse outcomes in the groups of patients with positive and negative ctDNA detection. Also, the results obtained will aid physicians in determining treatment courses for patients, in order to reduce the intensity of adjuvant chemotherapy. By identifying the patients with residual disease with ctDNA analysis, it is possible that this will improve disease prognosis.

Patients with stage 1 breast cancers have a very good prognosis, with the substantial majority cured by local therapy alone. However, the vast majority of HER2 positive and triple negative breast cancers (TNBC) with stage 1 (T1b and T1c) disease receive adjuvant chemotherapy after surgery. To reduce morbidity shorter courses of chemotherapy have become the standard. For HER2 positive breast cancer twelve weeks of weekly taxol and trastuzumab has very good long term outcomes, with only 1% of patients having a distant recurrence at seven year follow-up. Similarly for triple negative breast cancer four cycles of chemotherapy, for example with the docetaxel-cyclophosphamide regimen, may be used. This compares to the use of full adjuvant chemotherapy for HER2 positive breast cancer for example 4 cycles of anthracycline-cyclophosphamide chemotherapy followed twelve weeks of weekly taxol with trastuzumab and pertuzumab, and full (neo)adjuvant chemotherapy for TNBC of 4 cycles of anthracycline-cyclophosphamide chemotherapy followed twelve weeks of weekly taxol with carboplatin.

Detection of circulating tumour DNA (ctDNA) in plasma, after treatment for primary breast cancer, is associated with a very high risk of future relapse. As cancer cells proliferate and die, a small amount of cancer cell DNA is released into blood circulation. Sequencing of the primary cancer to identify the mutations present in the cancer, allows very sensitive detection of these mutations in DNA extracted from plasma. Detection of cancer specific mutations in the plasma DNA implies that there is cancer present in the body. For patients who have already had potentially curative treatment for breast cancer, detection of cancer specific mutations or ctDNA, identifies the presence of molecular residual disease, and that this residual disease is proliferating. The identification of ctDNA in these patients is associated with a very high risk of future relapse.

As most patients with stage I breast cancer are cured by the surgery alone, it is not possible on a population basis to discern adverse outcomes with reducing the intensity of adjuvant chemotherapy, by offering shorter courses of chemotherapy. Yet for those few patients who will relapse without chemotherapy, those with molecular residual disease, short-course chemotherapy increases their risk of relapse compared to full combination adjuvant chemotherapy. Yet in routine clinical practice there is no way of identifying who has molecular residual disease. If we can identify who the patients with residual disease with ctDNA analysis, those patients can escalate treatment to full chemotherapy and thereby improve their outcome.

The Signatera assay identifies the presence of molecular residual disease with high accuracy in many tumour types. Patients with ctDNA detected have a very high risk of future relapse. The assay is available as a routine validated clinical test, of proven clinical validity. The purpose of this study is to allow patients to access the Signatera assay as part of their standard clinical care, and to identify which patients have a high risk of relapse after treatment for stage I breast cancer by detecting the presence of ctDNA after surgery, who would have a higher risk of relapse if treated with short-course chemotherapy.

Patients will be enrolled into the study prior to surgery, in preference, to shorten the time from surgery to the ctDNA testing result being available. Patients will also have a ctDNA test taken prior to surgery, to explore whether this result may also have prognostic significance. The pre-surgery result will not be made available to patients or their treating clinicians in this study, as the full clinical importance of this result is not as clear as detection after surgery.

This protocol describes a study that will be conducted in the United Kingdom, and a parallel protocol will recruit patients in the United States sponsored by Massachusetts General Hospital. The two protocols share the same objectives and statistical design and endpoints, although are tailored to the individual health care systems. The sample size describes the combined recruitment of the protocols, and the databases of the two protocols will be combined for assessment of all endpoints. The study will aim to recruit a minimum of 40% of patients from each country (up to 200 patients from each country).

Inclusion Criteria:

1. Age 18 years or older.

2. Patients with histologically confirmed breast cancer that is either

   A) HER2 positive as defined by 2018 ASCO-CAP guidelines determined by local testing B) Triple negative defined as ER negative (ER staining in <1% tumour cells or ER Allred score <3/8) and PR negative (PR staining in <10% tumour cells or PR Allred score <6/8) and HER2 negative by 2018 ASCO-CAP guidelines determined by local testing. Patients without PR testing results may enrol on the basis of ER and HER2 results.

   Note that patients negative for ER and PR may enrol whilst awaiting HER2 testing results

3. Stage 1 cancer excluding pT1aN0 cancer, defined as

   A) Patients prior to surgery with primary tumour size on imaging 6-20mm and a normal axilla ultrasound or a biopsy negative axilla

   Patients who enrol prior to surgery will only continue further testing in the trial if their pathological staging fits the after surgery criteria.

   B) Patients after surgery with either

   • Primary tumour size pT1b or pT1c (6-20mm) and pN0 or pN1mi (micrometastasis).
   • Primary tumour size pT1a (1-5mm) and pN1mi

   Note that patients consenting after surgery may not enrol with pT1aN0 stage disease

4. Patients should consent prior to surgery (preferred) or within 2 weeks of surgery. Patients who consent after surgery may extend consent to 4 weeks after surgery, although this will delay the receipt of ctDNA results.

5. Planned and fit enough to receive full standard post-operative chemotherapy, with HER2 targeting as appropriate.

6. Ability to give informed consent and comply with study procedures including blood tests and follow-up for five years.

Exclusion Criteria:

1. Distant metastatic disease.
2. Multifocal invasive cancer
3. Diagnosis of alternative cancer within the last 5 years other than resected non-melanoma skin cancer or cervical intraepithelial neoplasia.
4. Any prior treatment (including neo-adjuvant chemotherapy) for the current breast cancer with the exception of surgical resection for patients enrolling after surgery.
5. Known HIV or hepatitis B or hepatitis C infection.