Neoadjuvant Zoledronate and Atorvastatin in Triple Negative Breast Cancer
Neoadjuvant Zoledronate and Atorvastatin in Triple Negative Breast Cancer
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. The ABCSG-12 clinical trial have reported
 improved Disease Free Survival (DFS) and Overall Survival (OS) in mostly chemotherapy naive
 premenopausal patients after a 3-years of treatment with zoledronate (zol) and
 ovarian-suppression therapy. The ZO-FAST study showed better DFS for immediate use of zol in
 postmenopausal patients receiving adjuvant hormonal treatment. Preliminary evidences support
 the role of zoledronate also in neoadjuvant setting reporting better responses in cases of
 treatment with zol and chemotherapy (cht) compared with cht alone. The 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. Selected cancer
 subtypes may present a more pronounced mevalonate activity able to confer an aggressive
 phenotype. It has been shown that a mutant p53 acts as promoter of MVA upregulation. 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. Due to the strong interplay between the MVA and 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 suggest that 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. Others medications are able to modulate the MVA pathway. Statins, a
 first-class of lipid-lowering medications that inhibit the enzyme HMG-CoA reductase, inhibit
 the sterol biosynthesis via the mevalonate pathway. A possible anti-tumor effect of statins
 can be predicted with the same mechanism of action described for BPs, through the
 interference with the MVA axis. Actually, the anti-tumor activity of statins have been
 investigated in different retrospective analyses. In breast cancer a more robust signal has
 been retrospectively reported and prospective studies have enquired the exquisite antitumor
 activity of statins in pre-operative breast cancer setting. 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
 triple-negative breast cancer (TNBC). 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.

 The primary clinical objective is to assess the anti-tumor activity of the combination of
 neoadjuvant standard cht associated with zol and atorvastatin, measured by the proportion of
 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 zol and atorvastatin according to high/low p53 levels 2)
 to address the efficacy of neoadjuvant cht associated or not with zol/atorvastatin combo in
 terms of disease free survival and overall survival); 3) to study the safety profile of study
 treatments; 4) to investigate the treatment modulation 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 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 cht (ARM A) or the combination of zol and
 atorvastatin associated with the above mentioned neoadjuvant cht (ARM B).
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.
Inclusion Criteria:

 1. Histologically confirmed diagnosis of non-metastatic operable TNBC subjected to
 diagnostic core biopsy

 2. TNBC defined as HER2/ER/PgR negative receptors

 3. Female, aged ≥ 18 years

 4. ECOG (Eastern Cooperative Oncology Group) performance status ≤ 1

 5. Clinical indication for a neoadjuvant approach according to the investigator's
 judgment. The standard chemotherapy will consist of a complete pre-operative treatment
 with anthracyclines and taxanes (in sequence or combination), including platinum
 derivatives and dose-dense schedules, according to the best physician choice (BPC)

 6. Availability of paraffin-embedded tumor block (FFPE) taken at diagnostic biopsy for
 IHC and RNA-Seq molecular determinations

 7. Patients with reproductive potential must have a negative serum pregnancy test within
 7 days prior to study entry. They must agree to use a medically acceptable method of
 contraception throughout the treatment period and for 3 months after discontinuation
 of treatment

 8. Written informed consent signed prior to enrolment according to ICH/GCP.

 Exclusion Criteria:

 1. Presence of metastatic disease

 2. Previous investigational treatment for any condition within four weeks prior to study
 registration

 3. Treatment with bisphosphonates, denosumab or other drug that, in the investigator's
 judgment, affects bone metabolism

 4. Treatment with statins or other drugs that, in the investigator's judgment,
 potentially affect the mevalonate pathway

 5. Any previous treatment for the currently diagnosed breast cancer, including radiation
 therapy, chemotherapy, biotherapy and/or hormonal therapy

 6. Inadequate bone marrow, hepatic or renal function including the following:

 1. Hb< 9.0 g/dL, absolute neutrophil count < 1.5 x 109/L, platelets <100 x 109/L

 2. Total bilirubin > 1.5 x ULN, excluding cases where elevated bilirubin can be
 attributed to Gilberts Syndrome

 3. AST (SGOT), ALT (SGPT) > 2.5 x ULN

 4. Creatinine > 1.2 x ULN, calcium < 8.6 mg/dL

 7. Co-existing active infection or concurrent illness that, at the judgment of the
 investigator, contra-indicate the inclusion of the patient in the study

 8. Active liver disease or unexplained persistent elevations of serum transaminases
 exceeding 3 times the upper limit of normal

 9. Co-existing dental diseases that form a contraindication to the use of zol

 10. Any medical or other condition that in the Investigator's opinion renders the patient
 unsuitable for this study due to unacceptable risk

 11. Psychiatric disorders or altered mental status precluding understanding of the
 informed consent process and/or completion of the necessary study assessment and
 procedures

 12. Known hypersensitivity to the active substance, to other bisphosphonates or to any
 excipients of zoledronate

 13. Known hypersensitivity to the active substance or to any excipients of atorvastatin.
 Conditions of rare hereditary problems of galactose intolerance, Lapp lactose
 deficiency or glucose-galactose malabsorption

 14. Anticipation of need for major surgical procedure during the course of the trial

 15. Pregnant or breast feeding women.