Pre-operative Zoledronate in Triple Negative Breast Cancer

A Multicenter, Single-arm, Phase II Study to Evaluate the Activity of Pre-operative Zoledronate in Triple Negative Breast Cancer Patients, According to p53 Level

Recent evidences suggest that zoledronate (zol), one of the most used bisphosphonates (BPs) in the clinical setting for the prevention and treatment of bone metastasis in cancer patients, may have antitumor activity in early breast cancer in terms of improved disease free survival, overall survival and better response in BPs treated patients. BPs are mevalonate pathway inhibitors and one of the most intriguing hypothesis supporting their anticancer activity relies on the modulation of the mevalonate downstream metabolism. Biologically active mevalonate metabolites are involved in tumour cell proliferation and invasion and selected cancer subtypes may present a more pronounced mevalonate activity, able of maintaining an aggressive phenotype. The mevalonate pathway has deep impact on the function of YAP/TAZ, transcriptional regulators of tumour growth, and preclinical evidences suggest that BPs are able to interfere with YAP/TAZ expression, via mevalonate pathway. This study addresses the clinical role of BPs in triple negative breast cancer (TNBC) patients selected by the level of mevalonate pathway regulation, namely the p53 expression. This study is a multicenter single-arm, phase II study primarily aimed at assessing the anti-tumor activity of pre-operative zol measured through its effect on the Ki67 proliferative biomarker, in TNBC patients classified according to the p53 expression (high vs low p53 expression). Patients with newly diagnosed, untreated, operable TNBC, intended to definitive breast surgery and suitable for pre-operative therapy with zoledronate will receive a pre-operative, single administration of zol (4mg i.v.), 7 days before definitive breast surgery. Ki67 levels will be assessed in tumor samples collected at the time of diagnosis and after zoledronate treatment at the time of definitive surgery. The secondary objective of the study is to investigate the effect of zoledronate on critical genes/proteins related to p53 and mevalonate pathways, p53/PIN1 and YAP/TAZ, analyzed in the tumor tissue collected at the time of diagnosis and at definitive surgery. Zol safety profile will be evaluated by the NCI-CTCAE scale, version 4.0, and by the occurrence of serious adverse reactions. The total number of patients required is forty. The overall duration of the project is 32 months (30 months for accrual, followed by 2 months of follow-up after the recruitment of the last patient).

Recent evidences suggest that zoledronate, one of the most used bisphosphonates (BPs) in the clinical setting for the prevention and treatment of bone metastasis in cancer patients, may have antitumor activity in early breast cancer. Notwithstanding some conflicting data, the majority of the clinical trials have shown some positive effects of BPs on patients outcome, reporting improved Disease Free Survival (DFS) and Overall Survival in mostly chemotherapy-naive premenopausal patients after a 3-year treatment with zoledronate and better DFS for immediate use of zoledronate in postmenopausal patients receiving adjuvant aromatase inhibitor treatment. Moreover, early breast cancer patients treated with neoadjuvant chemotherapy in combination with zoledronic acid showed better response compared with chemotherapy alone. Even though different explanations have been proposed over-time, the exact mechanism of action of the anti-tumor activity of BPs is still not well understood. Basically, BPs are mevalonate pathway inhibitors and one of the most intriguing hypothesis supporting their anticancer activity relies on the modulation of the mevalonate downstream metabolism. Biologically active mevalonate metabolites are involved in tumour cell proliferation, survival, invasion and metastasis. Moreover, there is evidence that selected cancer subtypes may present a more pronounced mevalonate activity, able of maintaining an aggressive phenotype. Indeed, the mevalonate pathway has deep impact on the function of YAP/TAZ, transcriptional regulators of tumour growth. Preclinical evidences, suggest that BPs are able to interfere with YAP/TAZ expression, via mevalonate pathway. This study addresses the clinical role of BPs in triple negative breast cancer (TNBC) patients selected by the level of mevalonate pathway regulation, namely the p53 expression. This study is a multicenter single-arm, phase II study primarily aimed at assessing the anti-tumor activity of pre-operative zoledronate measured through its effect on the Ki67 proliferative surrogate biomarker, in patients with TNBC classified according to the p53 expression (high vs low p53 expression). An high level of p53 is defined by IHC expression ≥30%, while a low level is defined by IHC expression <30%, as previously described. Patients with newly diagnosed, untreated, operable TNBC, intended to definitive breast surgery and suitable for pre-operative therapy will be registered using a centralized system and will receive a pre-operative, single administration of zoledronate (4mg i.v.), 7 days before definitive breast surgery . Ki67 levels will be assessed in core biopsy tumor samples collected at the time of diagnosis and after zoledronate treatment at the time of definitive surgery. Primary endpoint of the study is the proportion of responder patients, defined as those with at least 30% reduction in Ki67 at surgery with respect to core-biopsy analysis. The secondary objective of the study is to investigate the effect of zoledronate on critical genes/proteins related to p53 and mevalonate pathways, p53/PIN1 and YAP/TAZ, analyzed in the tumor tissue at the time of diagnosis (core biopsy) and at definitive surgery. The safety profile of zoledronate will be evaluated by the National Cancer Institute-Common Terminology Criteria for Adverse Events (NCI-CTCAE) scale, version 4.0 and by the occurrence of serious adverse reactions. The total number of patients required is forty. The overall duration of the project is expected to be 32 months, divided as follows: 30 months for accrual, followed by 2 months of follow-up after the recruitment of the last patient, aimed to collect data on zoledronate safety and tolerability. The primary analysis will be conducted on the PP population which will include all patients registered, who received the dose of study treatment and underwent definitive breast surgery, with no major violations of the eligibility criteria or during study conduction. Proportion of responder patients, defined as those with at least 30% reduction in Ki67 at surgery with respect to core-biopsy analysis, will be presented as point estimate and 95% confidence intervals (95% CIs) for each group. Logistic regression techniques will be used to compare groups in univariate and multivariate model adjusted for ECOG-PS, sex and pre- and postmenopausal status. The results of this project may eventually contribute to unveil novel anticancer mechanism of action of BPs and new therapeutic options for triple negative breast cancer.

Patients fulfilling the eligibility criteria will receive a pre-operative, single administration of zoledronate (4mg i.v.), 7 days before definitive breast surgery

Patients fulfilling the eligibility criteria will be registered using a centralized system and will receive a pre-operative, single administration of zol (4mg i.v.), 7 days before definitive breast surgery .

Assessment of zoledronate effect on Ki67 proliferative surrogate biomarker expression, according to p53 expression

The study is primarily aimed at assessing the anti-tumor activity of pre-operative zoledronate, measured through its effect on the Ki67 proliferative surrogate biomarker, in patients with TNBC selected according to the p53 expression (high vs low p53 expression). Primary endpoint of the study is the proportion of responder patients, defined as those with at least 30% reduction in Ki67 at surgery with respect to core-biopsy analysis. Prior to enrolment, the FFPE diagnostic core biopsy specimens will be analyzed by the study pathologist to determine the presence of invasive TNBC and the Ki67/p53 values. The Ki67/p53 evaluation will be then repeated after treatment at the time of definitive surgery

Effect of zoledronate on critical genes/proteins related to p53 and mevalonate pathways, p53/PIN1 and YAP/TAZ (FFPE core biopsy will be tested for critical genes/proteins expression (by RT-PCR and IHC), including p53/PIN1 and YAP/TAZ)

The secondary endpoint of the study is aimed to investigate the effect of zoledronate on critical genes/proteins related to p53 and mevalonate pathways, p53/PIN1 and YAP/TAZ, analyzed at the time of diagnosis (core biopsy) and at definitive surgery (breast cancer resection). After enrolment, the FFPE core biopsy will be tested for critical genes/proteins expression (by RT-PCR and IHC), including p53/PIN1 and YAP/TAZ. The same evaluations will be performed after treatment, at the time of definitive surgery

Assessment of zoledronate safety (valuated by the National Cancer Institute-Common Terminology Criteria for Adverse Events (NCI-CTCAE) scale, version 4.0 and by the occurrence of serious adverse reactions)

The safety profile of zoledronate will be evaluated by the National Cancer Institute-Common Terminology Criteria for Adverse Events (NCI-CTCAE) scale, version 4.0 and by the occurrence of serious adverse reactions

participants will be followed for AE occurrence from the informed consent signature to 2 months after study drug administration

Inclusion Criteria: Histologically confirmed diagnosis of non-metastatic operable primary invasive TN breast cancer subjected to diagnostic core biopsy TNBC defined as HER2/ER/PgR negative receptors Ki67 and p53 expression determined by IHC Tumour tissue availability at time of diagnosis for IHC evaluation of p53/PIN1, YAP/TAZ and Ki67 protein expression and for RT-PCR molecular testing of critical genes: p53/PIN1, YAP/TAZ Age ≥ 18 years old ECOG (Eastern Cooperative Oncology Group) performance status ≤ 1 Patients with reproductive potential. Female patients must have a negative serum pregnancy test within 7 days prior to start of trial. Patients must agree to use a medically acceptable method of contraception throughout the treatment period and for 3 months (female patients) and 6 months (male patients) after discontinuation of treatment Written informed consent signed prior to enrolment according to ICH/GCP. Exclusion Criteria: Presence of metastatic disease Clinical indication of debulking neo-adjuvant treatment Previous investigational treatment for any condition within 4 weeks from study registration Treatment with bisphosphonates, denosumab or other drug that, in the Investigator's judgment, affects bone metabolism Treatment with statins or other drugs that, in the Investigator's judgment, potentially affect the mevalonate pathway Any previous treatment for the currently diagnosed breast cancer, including radiation therapy, chemotherapy, biotherapy and/or hormonal therapy Inadequate bone marrow, hepatic or renal function including the following Hb< 9.0 g/dL, absolute neutrophil count < 1.5 x 109/L, platelets <100 x 109/L Total bilirubin > 1.5 x ULN, excluding cases where elevated bilirubin can be attributed to Gilberts Syndrome AST (SGOT), ALT (SGPT) > 2.5 x ULN Creatinine > 1.2 x ULN, calcium <8.6mg/dL Co-existing active infection or serious concurrent illness that, at the judgment of the investigator, contra-indicate the inclusion of the patient in the study Co-existing dental diseases that form a contraindication to the use of zol or need for immediate dental work Any medical or other condition that in the investigator's opinion renders the patient unsuitable for this study due to unacceptable risk Psychiatric disorders or altered mental status precluding understanding of the informed consent process and/or completion of the necessary study assessment and procedures Anticipation of need for major surgical procedure during the course of the trial Known hypersensitivity to any excipients of zoledronate Pregnant or breast feeding women.

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