Early downmodulation of tumor glycolysis predicts response to fasting-mimicking diet in triple-negative breast cancer patients

In preclinical experiments, cyclic fasting-mimicking diets (FMDs) showed broad anticancer effects in combination with chemotherapy. Among different tumor types, triple-negative breast cancer (TNBC) is exquisitely sensitive to FMD. However, the antitumor activity and efficacy of cyclic FMD in TNBC patients remain unclear. Here, we show that a severely calorie-restricted, triweekly, 5-day FMD regimen results in excellent pathologic complete response (pCR) rates (primary endpoint) and long-term clinical outcomes (secondary endpoints) when combined with preoperative chemotherapy in 30 patients with early-stage TNBC enrolled in the phase II trial BREAKFAST. Bulk and single-cell RNA-sequencing analysis revealed that highly glycolytic cancer cells, myeloid cells and pericytes from tumors achieving pCR undergo a significant, early downmodulation of pathways related to glycolysis and pyruvate metabolism. Our findings pave the wave for conducting larger clinical trials to investigate the efficacy of cyclic FMD in early-stage TNBC patients, and to validate early changes of intratumor glycolysis as a predictor of clinical benefit from nutrient restriction. ClinicalTrials.gov Identifier: NCT04248998 The BREAKFAST (clinicaltrial.gov registration code: NCT04248998) trial was a single center, open-label, two-arm, non-comparative, randomized phase II study that enrolled patients with treatment naïve, unilateral, localized (stage I-III, with clinical tumor diameter of at least 1 cm) invasive TNBC (negative HER2, ER, and PgR status according to American Society of Clinical Oncology/College of American Pathologists guidelines 52). The study was conducted at Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. All TNBC pathological diagnoses were centrally reviewed by two expert pathologists. Eligible patients were women aged between 18 and 75 years with Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, adequate hematological, renal, and hepatic function; a BMI equal to or higher than 20 kg/m2, and a LVEF of at least 50% at study enrollment. Patients were excluded if they had experienced significant, non-intentional weight loss during the 3 months before enrollment, if they had a diagnosis of type 1 or 2 diabetes mellitus requiring pharmacologic therapy, or if they had clinically relevant gastrointestinal diseases. Clinical T stage was defined by the longest tumor diameter measured by breast Magnetic Resonance Imaging (MRI) before the initiation of the neoadjuvant treatment. All patients enrolled in the BREAKFAST trial received preoperative chemotherapy, consisting of 4 triweekly cycles of intravenous doxorubicin (60 mg/m2) plus cyclophosphamide (600 mg/m2), followed by 12 cycles of weekly intravenous paclitaxel (80 mg/m2). For scRNA-seq analyses we collected fresh tumor samples at prespecified timepoints from 15 patients enrolled in the trial, finally obtaining scRNA-seq data from n=13 samples collected before treatment initiation (T0), n=11 samples collected after one treatment cycle (T1), and from n=5 samples collected at surgery (T2).