CIRCULATING MICRO-RNA (miRNA) AND AR-V7 MUTATIONAL STATUS IN METASTATIC CASTRATION-RESISTANT PROSTATE CANCER (CRPC): PRIMERA+ STUDY (PROSTATE CANCER INNOVATING MARKERS OF EXPECTED RESPONSE TO AGONIST LHRH+ ANDROGEN RECEPTOR INHIBITION

Observational prospective study investigating the plasmatic levels of miRNA according to AR-V7 mutational status in mCRPC patients receiving standard of care therapy. At the time of the enrollment, patients will undergo determination of AR-V7 splice variants on circulating tumor cells and periodic assessment of circulating levels of miRNA at different time points during the treatment course (initiation, 8-weeks assessment, progression); irrespectively of AR-V7 status patients will be allocated to endocrine therapy with enzalutamide or abiraterone plus LHRH agonist (decapeptyl every 3 months) according to standard of care. Integration of local treatment (in particular radiotherapy) will be allowed on oligoprogressive sites of disease and its impact on overall outcome and miRNA levels will be assessed.

Prostate cancer is the most frequent malignancy in male patients; since androgen receptor signalling is capital for cancer progression, androgen deprivation therapy with Luteineizing Hormone Release Hormone Agonists (a LHRH) is the cornerstone of its clinical management until occurrence of castration resistance, resulting in disease relapse following activation of alternative signalling pathways. In order to overcome castration resistance, new generations molecules targeting androgen receptor emerged in the last decade as an alternative to chemotherapy with taxanes: among them Abiraterone and Enzalutamide, a potent androgen receptor inhibitor that restore sensitivity to aLHRH therapy has recently been approved both in the pre-and postchemotherapy setting in metastatic castration-resistant prostate cancer (mCPRC).

Despite promising results, resistance to androgen inhibitors is a critical issue, resulting in treatment failure (primary resistance or adaptative resistance developed during the treatment course), particularly as a result of androgen receptor (AR) amplification or mutations.

This result in a dramatic need to identify predictors of response to treatment in order to choose the correct treatment sequence; nevertheless, this approach is somehow limitated due to scarce avalibility of tissue samples for biomolecular investigation in frail patients with painful bone metastasis.

Among the causes of treatment failure, AR-V7 splice variant of the androgen receptor has been extensively studied by Antonarakis et al, who reported that AR-V7 splice variant in circulating tumor cell (a cost-effective,non invasive assessment performed on blood samples) affects response both to Enzalutamide and to the cytochrome inhibitor Abiraterone, resulting in 0% of PSA response in patients displaying AR-V7(19-39% according to data from Antonarakis et al).

A recent study from Scher et al highlighted that CTC nuclear expression of AR-V7 protein in men with mCRPC as a treatment-specific biomarker was associated with superior survival on taxane therapy over hormone based therapy, advocating for future prospective clinical trials in this setting.

It is therefore most likely that in the next future determination of ARV7 status might become capital in allocating (in almost one third of cases) patients from endocrine therapy to chemotherapy with taxanes;it is also theoretically possible in this scenario that, in case of progression under chemotherapy, those patients considered irreversibly refractory (and thus "lost") to hormone treatment might become eligible to a more and more wide range of genome-driven biologic agents due to increasing availability of genome sequencing techniques that allow to target individual molecular alterations in a tailored perspective.

These future advances might have dramatic consequences on patient management since endocrine therapy as a backbone treatment is a cost-effective, tolerable and safe therapeutic option that should be mantained as long as possible: it is therefore capital to attentively select patient that, despite the presence of an unfavorable biologic profile, maintain a residual sensitivity to hormonal treatment.

It is moreover unclear whether primary resistance could reverse to a more sensitive state: according to recent reports by the same authors AR-V7 positive might revert back to AR-V7 negative status after chemotherapy.

This observation suggest that resistance to endocrine treatment treatment is not an ubiquitous process involving the whole tumor bulk, but arise in most case from clonal expansion of refractory clones in isolated sites of disease progression. In reason of advances in metabolic imaging, these sites could be targeted by locoregional treatment like stereotactic body radiotherapy, a novel radiotherapy modality that allows for focalized delivery of high radiation doses.

In summary, there is an urge to develop new clinical tools to refine patient stratification according to the possibility to extend the use of hormone-based therapies, limiting the resort to chemotherapy and/or biological agents only to selected patients at high risk of progression.

MicroRNA are small non-coding RNAs implicated in protein expression that can be disregulated in various type of cancers. A critical role in prostate cancer progression has been extensively described for:

miRNA 124, a tumor suppressive miRNA, is known to downregulate in vitro the levels of AR transcript variants, as well as alternative signaling networks such as Enhancer of Zeste homolog 2 (EZH2) and Src tyrosine kinase (Src) miR-125b: androgen-independent growth miR-331-3p, miR let-7c, miR-21: transition to castrate-resistant prostate cancer miR-32: androgen-regulated miRNA that is upregulated in CRPC miR-222 : attenuate androgen-induced growth, promote androgen-independent growth miR-30: suppresses ERG expression miR-221, miR-375, and miR-141: overexpressed in metastatic clinically silent cancer MiRNA from peripheral blood samples presents various benefits compared to other techniques, in particular the possibility to assess dynamic changes all along the treatment course to test efficacy of ongoing therapy and the possibility to simultaneously test panels of molecules to identify "signatures" predicting the response to treatment.

HYPOTHESIS Dynamic modifications of circulating miRNA and their correlation with other hallmarks (like presence of AR splice variants) may drive treatment choice and provide valuable informations about response to systemic treatment.

AIM OF THE STUDY To evaluate, in mCRPC patients, a panel of miRNAs involved in various stages of tumor progression and their correlation with AR-V7 mutational status at different time points during the systemic treatment course ( in particular Enzalutamide plus LHRH agonist or Abiraterone plus LHRH agonist or taxane chemotherapy), and their predictive value in terms of response to treatment

STUDY POPULATION Inclusion criteria Castration Resistant Prostate Cancer defined as

* biochemical or clinical progression under therapy with LHRH agonist and
* castrate plasma testosterone levels (\<20 ng/dl or \<1.73 nmol/L) Eligible for medical treatment Age \>18 Informed consentment Exclusion criteria Medical contraindication/refusal to chemotherapy or endocrine therapy Life expectancy inferior to 1 year Previously diagnosed neoplasm

STUDY DESIGN Observational prospective study investigating the plasmatic levels of miRNA according to AR-V7 mutational status in mCRPC patients receiving standard of care therapy. At the time of the enrollment, patients will undergo determination of AR-V7 splice variants on circulating tumor cells and periodic assessment of circulating levels of miRNA at different time points during the treatment course (initiation, 8-weeks assessment, progression); irrespectively of AR-V7 status patients will be allocated to endocrine therapy with enzalutamide or abiraterone plus LHRH agonist (decapeptyl every 3 months) according to standard of care. Integration of local treatment (in particular radiotherapy) will be allowed on oligoprogressive sites of disease and its impact on overall outcome and miRNA levels will be assessed.

STATISTICAL PLAN

Due to the observational nature of the study, we planned a minimum accrual of 45 patients in order to evaluate:

* PSA response (≥50% decline in PSA level from baseline, maintained for ≥4 weeks)
* Best PSA response (maximal percentage decrease in PSA level from baseline )
* Progression-free survival (increase in PSA level ≥ 25% above the nadir )
* Overall survival
* Toxicity (CTCAE V4.0)
* Qol (EORTC QLQ C-30) In the event of disease progression under treatment, in case of oligoprogressive disease (defined as less than 3 sites of disease experiencing progression at restaging imaging ), according to our Institutional practices prosecution of endocrine treatment plus local treatment with radiotherapy will be considered and definitive treatment interruption will be planned in case of further progression 2 months after SBRT.

We envisage a time of accrual of 18 months.

EXPECTED BENEFIT

MiRNA plasma levels might correlate with other predictors of resistance to current standard of care (in particular AR-V7) and may provide real-time information about response to treatment; in the future it may allow to identify subsets of patients that are eligible for integration of systemic therapy with local modalities of treatment to overcome the emergence of refractory clones and maintain durable sensitivity to endocrine treatment and delay the need for chemotherapy even in patients deemed poor-responders.

Eligibility Criteria:
Inclusion Criteria:

* Castration Resistant Prostate Cancer defined as biochemical or clinical progression under therapy with LHRH agonist and castrate plasma testosterone levels (\&lt;20 ng/dl or \&lt;1.73 nmol/L)
* Eligible for medical treatment
* Age \>18 years
* Informed consentment

Exclusion Criteria:

* Medical contraindication/refusal to chemotherapy or endocrine therapy
* Life expectancy inferior to 1 year
* Previously diagnosed neoplasm