Background: Metastatic triple-negative breast cancer (TNBC) has few effective options after multiple lines of chemotherapy and checkpoint inhibitors. We evaluated a multi-component phytochemical platform, AminoTriComplex (ATC), added to metronomic cyclophosphamide and propranolol, while prospectively testing a predefined translational three-marker panel—Survivin (BIRC5), Cystatin C (CST3), and the melatonin receptor MT1 (MTNR1A).

Methods: Prospective, open-label, non-randomized, controlled, multicenter study (January 2023–June 2025). Key eligibility: programmed death-ligand 1 (PD-L1)–positive, refractory stage IV TNBC with progression after pembrolizumab and ≥4 prior chemotherapy lines (including platinum, gemcitabine, vinorelbine, taxane, sacituzumab). Patients received ATC + metronomic backbone (n=147) or metronomic backbone alone (n=119) for 12 weeks. Primary endpoint: objective response rate (ORR; Response Evaluation Criteria in Solid Tumors [RECIST] v1.1). Secondary endpoints: progression-free survival (PFS), blood and paired-biopsy biomarkers (Enzyme-linked immunosorbent assay [ELISA]/ immunohistochemistry [IHC]), Eastern Cooperative Oncology Group (ECOG) status, and safety (Common Terminology Criteria for Adverse Events [CTCAE] v5.0). To control multiplicity for the biomarker triad we used Benjamini–Hochberg false discovery rate (FDR) (q=0.10). Bias-mitigation measures included centralized laboratory workflows, prespecified biomarker thresholds, sensitivity analyses (IPTW), and a blinded independent central radiology review (BICR) on a prespecified 30% subset.

Results: A total of 266 patients were enrolled (147 ATC; 119 control).
•    Efficacy: ORR was 46.3% (68/147) with ATC versus 12.6% (15/119) in control; crude odds ratio 6.64 (95% CI, 3.50–12.58), p<0.001. Median PFS was 8.9 months (95% CI, 7.5–10.4) with ATC versus 3.4 months (95% CI, 2.6–4.2) in control; hazard ratios (HR) 0.42 (95% CI, 0.30–0.60), p<0.001.
•    Translational signal (predefined triad):
√   Survivin↓ ≥50%: 62/68 responders vs 14/79 non-responders.
√    Cystatin C↑ ≥40%: 59/68 vs 11/79.
√    MT1 re-expression (IHC): 58/68 vs 19/79.
√    Full triad (Survivin↓ + Cystatin C↑ + MT1↑): 55/68 responders vs 9/79 non-responders.
All associations were strongly significant under FDR control.
•    Tumor microenvironment (paired re-biopsies at week 12): Ki-67 dropped markedly with ATC (median Δ −41%; 95% CI, −45 to −37; p<0.001) and CD8+ TILs increased (median Δ +34 cells/HPF; 95% CI, 28–40; p<0.001). In the control arm Ki-67 minimally changed (Δ −2%; 95% CI, −5 to +1; p=0.42) and CD8+ rise was small (Δ +2; 95% CI, −1 to +5; p=0.31). IHC showed a coherent pattern: Survivin decreased, Cystatin C increased, and MT1 re-expressed with treatment; imaging responses were consistent with these biological changes.
•    Safety: Toxicities reflected the backbone regimen; no new or disproportionate high-grade signals were attributable to ATC. Exposure-adjusted incidence rate (EAIR) per 100 patient-weeks was 5.0 in both arms.
•    Interpretation: Adding AminoTriComplex to a metronomic regimen was associated with substantially higher ORR and prolonged PFS in heavily pretreated, refractory TNBC. The predefined biomarker triad (Survivin↓ / Cystatin C↑ / MT1↑) tracked with clinical benefit and paralleled favorable shifts in the tumor microenvironment, suggesting a biologically integrated, multi-target mechanism spanning apoptosis re-engagement, invasion/metastasis restraint, and circadian-immune modulation. While compelling, these signals are correlative and arise from a non-randomized, open-label design.
•    Novelty/Significance: (1) A consistent triad-based translational signature aligned with response and PFS in refractory TNBC; (2) circadian biology engagement via MT1 re-expression as a plausible therapeutic axis; (3) a multi-component phytochemical platform showing coherent biomarker-to-clinical concordance.

Limitations: Non-randomized allocation and open assessment introduce potential selection/assessment bias; the 12-week window limits OS readouts; contributions of individual phytochemicals were not isolated; causal mediation was not tested. These are planned for subsequent randomized, blinded trials.

Conclusion: In a hard-to-treat TNBC population, ATC + metronomic therapy produced meaningful improvements in ORR and PFS and a predefined three-marker translational signature consistent with its proposed biology. The triad may serve as a candidate response/prognostic surrogate and warrants confirmation in biomarker-guided phase III studies. These findings identify a promising three-marker triad that is hypothesis-generating and warrants validation in randomized trials.

Triple-Negative breast cancer, AminoTriComplex, Phytotherapy, Metronomic chemotherapy, Propranolol, Survivin, Cystatin C, MT1/MTNR1A, Biomarkers, Circadian oncology, ORR, PFS, ELISA, IHC