Major changes included: 1. Initially, our assumptions for the “TREAT CTC” trial were that after (neo)adjuvant chemotherapy CTC detection rate would be 20% and HER2-positive CTC detection rate would be 15%. A pilot feasibility study was done and we found that CTC detection rate either before or after (neo)adjuvant chemotherapy was 11.9%. We also observed that 40% of the women with HER2-negative early breast cancer and detectable CTC had at least 1 HER2-positive CTC. In two other reported studies CTC detection rate after neoadjuvant chemotherapy was 17% and 10.6%, respectively. As the above mentioned research has shown a lower detection rate of CTC and of HER2-positive CTC, the design was changed to measure CTC rather than HER2-positive CTC. Furthermore in the new “TREAT CTC” design we assume a very conservative CTC detection rate after (neo) adjuvant chemotherapy of 8%. In addition to adjusting the design for the new validated CTC detection rate, strong safeguards have been built into the protocol covering feasibility, patient safety and early stopping mechanisms. Three interim analyses were planned. The first 2 are dedicated to assessing feasibility, the level of detection of CTC, the level of test failures and checking the assumptions. These analyses will be performed after 150 and/or 300 patients (depending on the accrual rate) have been screened. The third interim analysis will be performed at 50% randomisation for assessing test superiority and futility as well as feasibility. 2. The number of patients to be randomized between trastuzumab and observation increased from 80 to 174 in order to increase the power of the study to detect a 15% difference in CTC detection rate at week 18 between the two arms. 3. Several secondary clinical endpoints were added, including the comparison of the recurrence free interval between the trastuzumab and observation arm.

The PS in the selection criteria has been described, selection criteria were expanded to allow the inclusion of male breast cancer and all invasive breast cancer grades and sizes (i.e. we now include grade 1 tumors and tumors < 1cm), unifocal or multifocal unilateral or unifocal or multifocal synchronous bilateral BC if all foci are HER-2 negative and patients with previous history of DCIS because it increases the screening population and leaving We have removed the exclusion for prior use of bisphosphonate treatment or denosumab therapy because some patients could have started the above therapy for ostheoporosis treatment or previously enrolled in a clinical trial, we are however excluding the concomitant administration. We have clarified the confirmation of CTC testing will be done in one national reference lab and image confirmation by two central labs. This is as requested by the original guidelines from Veridex ® decreasing the possibilities of false positives. Timelines have been extended from surgery or last dose of chemotherapy to registration from 12 to 24 weeks to facilitate patient screening and inclusion. We also updated pre-clinical information in the rationale. We added additional information regarding trastuzumab, packaging and reconciliation, as requested by French authorities. We added additional information regarding trastuzumab the duration of administration after the first dose and monitoring in the rationale and changes. The end of study definition is now aligned to our standard wording and SOP. The text of PIS/IC has been clarified and now 4 questions have been summarized in 2 questions. We strongly believe that this amendment will facilitate the accrual of this relative rare population, without jeopardizing their safety. Additional administrative corrections and updates have been implemented.

This amendment is considered scientific because, among other changes, it proposes a modification of the inclusion criteria. In the previous version of the protocol, the patient population was limited to patients who had completed either - adjuvant chemotherapy for node-positive disease (pN ≥ 1, macrometastasis only) or - neoadjuvant chemotherapy; in this case residual invasive disease in breast or lymph nodes is required. These criteria were devised to identify a high risk population, partially to allow sufficient events for a timely analysis of the secondary endpoint recurrence free interval (RFI). Due to increased mammographic screening, node-negative patients are becoming more prevalent as compared to node-positive patients. Furthermore, a considerable proportion of women receiving adjuvant chemotherapy for early breast cancer have high risk node-negative disease. Considering these basically we allow node- negative patients to be included in this study. The implications this amendment may have on the design parameters are the following. Given the currently observed CTC detection rate (approx. 15%) and the results summarized above for the series of 2026 patients, this modification is expected to slightly reduce the overall CTC detection rate, albeit still within the limits expected within the protocol. Therefore, as - the end of accrual is mainly driven by the number of patients randomized and not by the number of patients screened (with upper limit of 2175), - we do not expect that including CTC positive patients with node negative disease will impact the assumed CTC detection rate at week 18 in the observation arm, there would be no need to update the design of the study in terms of number of patients to be randomized or screened.