Triple-negative breast cancer (TNBC) is a heterogeneous disease defined by the lack of
      expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal
      growth factor receptor 2 (HER2). Roughly, it represents 15% of all breast cancers. Patients
      with TNBC are generally younger than the overall population of breast cancer patients and
      they are more frequently affected by larger and aggressive tumors (i.e. high
      nuclear-grading), associated with a poor prognosis and with a significant risk of relapse in
      the first three years after diagnosis. Since the negative expression of HER2 and
      hormone-receptors, patients affected by TNBC are not candidate for hormonal therapy or
      anti-HER2 agents, leaving cytotoxic chemotherapy as the only option for systemic therapy.

      Despite these common features, TNBCs are characterized by a notable diversity within the
      group. Histologic variability provides one example of such diversity, with invasive ductal,
      metaplastic and medullary breast cancers (two very different subtypes of breast cancer)
      coexisting in this patient population. Furthermore, the TNBC subtype does not directly
      correspond to a single molecular breast cancer subgroup. Though most fit into the category of
      basal-like cancers, these groups are overlapping rather than synonymous, with certain
      populations of ER-positive and HER2-positive tumors also known to express basal-like markers.
      Indeed, molecular evaluation has identified additional subgroups within the TNBC, confirming
      the true heterogeneity and complexity of such subtype of breast cancer.

      Due to this complex picture of histological and molecular characterization, TNBC still
      represents a therapeutic challenge for oncologist with several unmet clinical needs. Clearly,
      there is a need for a better understanding about the biology of TNBC and much more there is
      an urgent need for therapeutic options in TNBC, ideally in the form of targeted agents. Up to
      now, the heterogeneity of TNBC has made the achieving of these goals particularly complex.
      However, the identification of biomarkers able to predict response to systemic therapies is
      of crucial importance, as it will not only allow for better outcomes in responsive subgroups
      of TNBC, but also prevent unnecessary exposure of unresponsive patients to ineffective
      therapy. In this way, predictive biomarkers will facilitate the development of personalized
      medicine for TNBC.

      At present, there is not a clear, proven effective single agent that targets a driving
      vulnerability in TNBC. However, there are a number of potential therapies currently under
      investigation that may eventually improve outcomes in these patients. Deep understanding of
      molecular pathways involved in TNBC carcinogenesis is of paramount importance for identify
      novel therapeutic options, including the optimal repositioning of drugs already available for
      clinical intent and potentially active in TNBC, such as the case of platinum salts,
      PARP-inhibitors (in BRCA mutation) and potentially bisphosphonates and statins, that
      represent the focus of this study.

      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. Clinical trials have shown some positive
      effects of BPs on patients outcome, reporting an improved Disease Free Survival (DFS) and
      Overall Survival (OS) in mostly premenopausal early breast cancer patients after a 3-years of
      treatment with zoledronate and ovarian suppression therapy and a better DFS for immediate use
      of zoledronate in postmenopausal patients receiving adjuvant hormonal treatment. Moreover,
      preliminary evidences support the role of zoledronate also in neoadjuvant setting with
      reported better responses in cases of treatment with zoledronate and chemotherapy compared
      with chemotherapy alone, suggesting a direct antitumor effect of zol in combination with cht.

      In the final analysis of the AZURE trial no improvement in the primary endpoint of DFS was
      observed for the overall patient population. However, subgroup analysis showed that
      zoledronic acid significantly improved DSF (HR=0.76; p<0.005) in women who were at least 5
      years postmenopausal at study entry. Moreover, zoledronate was found to improve overall
      survival including women of unknown menopausal status but with age older than 60 years.

      In the same line of the neoAzure trial a more recently randomized clinical trial (the JONIE
      study) clearly confirmed the benefit of adding zol to neoadjuvant chemotherapy in HER2
      negative early breast cancers. In that study Asian patients with HER2-negative invasive
      breast cancer were randomly assigned to either the CT or CT+ZOL (CTZ) group. One hundred and
      eighty-eight patients were randomized to either the CT group (n = 95) or the CTZ group (n =
      93) from March 2010 to April 2012, and 180 patients were assessed. All patients received four
      cycles of FEC100 (fluorouracil 500 mg/m2, epirubicin 100 mg/m2, and cyclophosphamide 500
      mg/m2), followed by 12 cycles of paclitaxel at 80 mg/m2 weekly. Zol (4 mg) was administered
      three to four times weekly for 7 weeks to the patients in the CTZ group. The primary endpoint
      was the pathological complete response (pCR) rate. The results of this randomized controlled
      trial indicated that the rates of pCR in CTZ group (14.8%) was doubled to CT group (7.7%),
      respectively (one-sided chi-square test, p = 0.068), though the additional efficacy of
      zoledronic acid was not demonstrated statistically. The pCR rate in postmenopausal patients
      was 18.4% and 5.1% in the CTZ and CT groups, respectively (one-sided Fisher's exact test, p =
      0.071), and that in patients with triple-negative breast cancer was 35.3% and 11.8% in the
      CTZ and CT groups, respectively (one-sided Fisher's exact test, p = 0.112). The authors
      concluded the addition of zol to neoadjuvant CT has potential anticancer benefits in
      postmenopausal patients and in particular in the patients with triple-negative breast cancer.
      Actually, other clinical trials analyzed the role of BPs in breast cancer and, according to a
      recent meta-analysis of all randomized controlled trials (13 RCTs including more than 15.000
      patients) that appraised the effects of BPs on survival irrespectively to the types of BPs,
      it seems evident a positive effect in selected patients (HR 0.81(0.69-0.95).

      In line with this observation, Valachis A. et al. published a meta-analysis focusing on the
      specific role of zol as adjuvant treatment in breast cancer. In the meta-analysis fifteen
      studies were considered eligible and were further analyzed. The use of zol resulted in a
      statistically significant better overall survival outcome (five studies, 6,414 patients;
      hazard ratio [HR], 0.81; 95% confidence interval [CI], 0.70-0.94) while no significant
      differences were found for the disease-free survival outcome (seven studies, 7,541 patients;
      HR, 0.86; 95% CI, 0.70-1.06) or incidence of bone metastases (seven studies, 7,543 patients;
      odds ratio [OR], 0.94; 95% CI, 0.64-1.37).

      Even though different explanations have been proposed over-time, the exact anticancer
      mechanism of action of BPs still remains not well understood. Basically, BPs are mevalonate
      (MVA) pathway inhibitors and one of the most intriguing hypothesis supporting their
      anticancer activity relies on the modulation of the mevalonate downstream metabolism.
      Mevalonate (MVA) is synthesized from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) by
      HMG-CoA reductase (HMG-CoAR). MVA is further metabolized to farnesyl pyrophosphate (FPP), a
      precursor of cholesterol and sterols. FPP is also converted to geranylgeranyl pyrophosphate,
      and these lipids are used for post-translational modification of proteins that are involved
      in various aspects of tumor development and progression. Many studies showed that the MVA
      pathway is up-regulated in several cancers such as leukemia, lymphoma, multiple myeloma, as
      well as prostate, hepatic, pancreatic, esophageal and breast cancers. Several mechanisms may
      be involved in dysregulation of this pathway. They include mutation in HMG-CoAR and
      sterol-regulatory element binding protein (SREBP) and more specifically the mutation of p53.
      It has been shown that mevalonate pathway is significantly upregulated in case of mutant p53.
      Sterol biosynthesis intermediates reveal that this pathway is both necessary and sufficient
      for the phenotypic effects of mutant p53 on breast tissue architecture. It has been shown
      that the enzymes of the mevalonate pathways are under transcriptional control of SREBPs. In
      breast cancer cells oncogenic mutant p53 acts as a transcriptional cofactor for SREBPs,
      leading to elevated expression of mevalonate enzymes. One of the most important biological
      implications of MVA pathway upregulation in cancer cells is the aberrant activation of the
      Hippo pathway, a molecular axis with a central role in carcinogenesis. Two Hippo pathway
      related transcriptional coactivators, YAP and TAZ, promote tissue proliferation and the
      self-renewal of normal and cancer stem cells, and incite metastasis. Strikingly, YAP and TAZ
      are controlled by the same architectural features that first inhibit and then foster cancer
      growth, such as ECM elasticity, cell shape, and epithelial-to-mesenchymal transition. Due to
      the strong interplay between the MVA and the Hippo pathways, the modulation of MVA axis has
      deep impact on the function of YAP/TAZ as transcriptional regulators of tumour growth. These
      findings implicate the mevalonate pathway as a therapeutic target for selected tumors with
      up-regulation of these pathways.

      Preclinical and clinical evidences, generated within the AIRC 5x1000 project, suggest that in
      selected breast cancer cases the BPs are able to interfere with YAP/TAZ expression, via MVA
      pathway. This kind of activity may be part of the mechanism of action of BPs as antitumor
      drugs.

      Similarly to BPs, others medications are able to modulate the MVA pathway. Statins, also
      known as HMG-CoA reductase inhibitors, are a first-class of lipid-lowering medications that
      inhibit the enzyme HMG-CoA reductase, which plays a central role in the production of
      cholesterol. Statins inhibit the sterol biosynthesis via the mevalonate pathway. From above,
      a possible anti-tumor effect of statins can be predicted exactly with the same mechanism of
      action already described for BPs, i.e. through the interference with the MVA axis. Actually,
      the anti-tumor activity of statins have been investigated over-time in different
      retrospective analyses with conflicting results. In breast cancer a more robust signal has
      been retrospectively reported and, more recently, prospective studies have enquired the
      exquisite antitumor activity of statins in pre-operative breast cancer setting. Since BPs and
      statins act exactly on the same mevalonate pathway, a synergistic antitumor effect of the BPs
      and statins combination was predicted and reported in preclinical models, especially in cases
      of triple negative breast cancer, enriched in mutant p53 and YAP/TAZ expression.

      From above, the clinical trial herein proposed aims to investigate the antitumoral clinical
      activity of zoledronate (zol) and statins (atorvastatin) combination, in patients receiving
      neoadjuvant chemotherapy for TNBC. The results of this project may eventually contribute to
      unveil a novel combined treatment in TNBC through the repositioning of clinical approved low
      toxic drugs, able to target relevant masterpieces of breast cancer cells metabolism.

      The primary objective of the study is to address in patients with TNBC the antitumor activity
      of pre-operative standard chemotherapy associated or not with zoledronate (zol) and
      atorvastatin measured through its effect on YAP and TAZ immunochemistry (IHC) expressions,
      which are considered co-primary objectives (proof of concept objective).

      The primary clinical objective is to assess the anti-tumor activity of the combination of
      neoadjuvant standard chemotherapy associated with zoledronate and atorvastatin, measured by
      the proportion of pathological complete response (pCR) obtained after neoadjuvant treatment
      in patients with TNBC.

      Secondary objectives are: 1) to evaluate the anti-tumor activity of pre-operative standard
      chemotherapy associated or not with zoledronate (zol) and atorvastatin according to high/low
      p53 levels, measured through its effect on both YAP and TAZ IHC expressions and the
      proportion of pCR; 2) to address the efficacy of neoadjuvant chemotherapy associated or not
      with zoledronate/atorvastatin combo in terms of disease free survival (DFS) and overall
      survival (OS); 3) to study the safety profile of study treatments; 4) to investigate the
      treatment modulation (up/down regulation) of YAP and TAZ gene expression (RNA-Seq) in tumor
      tissues collected at the time of core-biopsy and definitive surgery; 5) to address the
      modulation of Ki67expression by IHC in the formalin fixed paraffin embedded (FFPE) diagnostic
      core biopsy tumor block and in the tumor tissue collected at surgery.

      Patients fulfilling the eligibility criteria will be randomized to receive standard
      anthracyclines/taxanes based neoadjuvant chemotherapy (ARM A) or the combination of
      zoledronate and atorvastatin associated with the above mentioned neoadjuvant chemotherapy
      (ARM B).

      The standard anthracyclines/taxanes based neoadjuvant CT will be administered according to
      the standard care in both arms; in the ARM B the neoadjuvant chemotherapy will be followed by
      zoledronate 4 mg i.v. (every 3/4 wks) and atorvastatin 80 mg/die, for a duration of 6 months
      of treatment (except for patients treated with dose- dense schedule who will receive
      zoledronate and atorvastatin for 4.5 months, according to the duration of neoadjuvant cht).

      Prior to enrolment, the formalin fixed paraffin embedded (FFPE) diagnostic core biopsy
      specimens will be analyzed by investigational site pathologists to determine the presence of
      invasive TNBC and the p53 and Ki67 values by IHC. p53 and Ki67 evaluation will be then
      repeated at the time of definitive surgery. After enrolment, the FFPE diagnostic core biopsy
      will be tested for YAP and TAZ gene and protein expression by RNA-Seq and IHC respectively.
      The same evaluations will be then repeated at the time of definitive surgery.

      The study is composed by two phases. Within the first phase patients will be randomized to
      one of the two study treatment arms described above. The relative reductions of YAP and TAZ
      IHC-expression at surgery with respect to core-biopsy analysis will be evaluated as primary
      proof of concept endpoint. The experimental arm containing zoledronate and atorvastatin will
      be considered deserving further development if a significant difference between arms in terms
      of relative reduction of YAP/TAZ IHC expression at surgery with respect to core-biopsy
      analysis will be identified in at least one of the two proteins. If such reduction in YAP or
      TAZ expression will be observed, at the second phase patients will be recruited only in the
      experimental arm (ARM B) and the control arm (ARM A) will be considered as calibration arm.
      Moreover the anti-tumor activity of the combination of neoadjuvant standard chemotherapy
      associated with zoledronate and atorvastatin measured by the proportion of patients with pCR
      obtained after neoadjuvant treatment will be assessed as primary clinical endpoint.

      Patients will be monitored for AEs using the definitions and criteria for grading provided by
      NCI CTCAE version 4.03. Disease free survival (DFS) and overall survival (OS) will be
      assessed as secondary endpoints. The post treatment follow-up procedure required for all
      patients consists in disease assessment with mammography and breast ultrasound scan,
      according to RECIST criteria version 1.1, for the evaluation of the tumor burden and DFS, and
      visits following definitive surgery planned according to the clinical practice to report any
      new adverse events or changes in existing events in order to collect data on the secondary
      endpoints.

      From 102 (1st phase) to 154 (2nd phase) patients will be registered in this clinical trial.
      The overall duration of the project, is expected to be 36 months, including 20 months for the
      execution of the first phase (recruitment, patients follow-up and data analysis), followed by
      12 months for the running of the second phase. Fifteen experimental centers will take part
      into the study.