PSMA-positive extracellular vesicles as a predictive biomarker of response to radiotherapy in advanced prostate cancer

Abstract

The present disclosure provides materials and methods for assessing and / or treating subjects having prostate cancer. In some embodiments, materials and methods for assessing a subject's tumor burden using a liquid biomarker, tumor-derived extracellular vesicle expressing a surface antigen, are provided. Provided are materials and methods for using the presence or absence of the biomarker to assess whether a subject should be treated with radiotherapy alone, or whether the subject would benefit from systemic therapy, either alone or in combination with radiotherapy.

Description

PSMA-POSITIVE EXTRACELLULAR VESICLES AS A PREDICTIVE BIOMARKER OF RESPONSE TO RADIOTHERAPY IN ADVANCED PROSTATE CANCERBACKGROUND

[0001] Prostate cancer is the most commonly diagnosed cancer type, and the second leading cause of cancer-related death in American men26. Prostate cancer usually metastasizes to the bone, and metastatic bone disease increases the risk of intractable bone pain, pathological skeletal fracture, spinal-cord compression, and overall decreased survival27. After primary treatment with radical prostatectomy or radiation therapy, a relevant proportion of patients develop metastases. Immediate or delayed androgen deprivation therapy (ADT), chemotherapy, chemohormonal therapy and palliative radiotherapy have traditionally been the mainstay of the management of metastatic prostate cancer (MPC). Sensitive PSA detection and improved imaging are increasingly leading to the diagnosis of oligometastatic disease, which in turn has raised new questions concerning the value of metastasis-directed therapy (MDT) on progression free survival (PFS) and overall survival (OS). The definition of oligometastatic disease is inconsistent and varies from as few as one but up to between three and five metastases. Malignant cells at this state are supposed to have limited metastatic capacity, accompanied with less aggressive behavior28.Accumulating evidence suggests that local MDT could defer disease progression, delay the need of systemic therapies and spare their toxicities. Proper patient selection, as well as the definitions used and relevant endpoints, may be critical to developing optimal approaches to combat oligometastatic disease29.

[0002] Radiotherapy and in particular stereotactic ablative radiotherapy (SABR) presents a logical option for MDT and has been used in many retrospective case series30. Timing of the diagnosis of oligometastatic disease is widely held to be critically important for improved patient outcomes. 68% of expert participants in the Advanced Prostate Cancer Consensus Conference (APCCC) considered it important to distinguish between patients presenting with what is frequently called “synchronous” versus “metachronous” (e.g., appeared later in the course of disease) oligometastatic disease. Further, despite the lack of high-level evidence, 64% of APCCC members voted for an ablative MDT in metachronous oligometastatic PC31.

[0003] Patients with metastatic castration-resistant prostate cancer (mCRPC) who progress after chemotherapy and next-generation antiandrogen therapy experience a meager median survival of 13.6 months32. A subset of these patients with oligometastatic disease are ideal candidates for stereotactic body radiotherapy (SBRT) also known as stereotactic ablative radiotherapy (SABR)28. Two recent phase 2 randomized trials evaluated SABR efficacy oligometastatic castration-sensitive prostate cancer (CSPC); one showed that SABR prolongs progression-free survival with minimal toxicity3, while the otherdemonstrated that SABR prolonged ADT-free survival4. However, distant failure after SABR remains the primary manifestation of disease progression. In a separate phase 2 trial in oligometastatic CRPC (<3 lesions) identified with11C choline positron emission tomography (PET) and computed tomography (CT), SABR was very effective for local control (75% at 2 years)33. Unfortunately, the mean time to distant recurrence was 5.1 months, and 19% of patients experienced distant recurrence within 3 months. This suggests that advanced PET imaging may not be sufficiently sensitive, but a combination of PET imaging and minimally invasive biomarkers could improve the selection of truly oligometastatic CRPC34.

[0004] Emerging randomized trials have determined a clinical benefit for SABR in patients with oligometastatic disease3. In Castration-Sensitive Prostate Cancer (CSPC), SABR improved 5-year ADT-free survival in STOMP and median PFS in ORIOLE34.Oligometastasis-directed SABR is safe and locally effective treatment of metastatic prostate cancer. SABR may delay more toxic systemic therapies as demonstrated in the recent NRG- BR001 phase 1 trial in a spectrum of solid tumor types35, and in the SABR-COMET randomized, phase 2, open-label trial in oligometastatic cancers49 50. Part of the benefit of SABR stems from the induction of a systemic immune response33. Peripheral expansion of clonotypic and tumor-reactive CD8 T cells is a prerequisite for local antitumor response and abscopal effect3637. Thus, there is a critical need to understand the systemic suppression of anti-prostate cancer immunity, particularly in patients treated with SABR.

[0005] Tumor-derived extracellular vesicles (tdEVs) are emerging as promising liquid biomarkers for cancer diagnosis and prognosis and prediction of treatment response38. EVs are nanosized vesicles released by all cell types, including tumor cells. They contain surface molecules and cargo from donor cells and travel in body fluids, including blood and urine. EVs function in intracellular communication by transferring intracellular contents, such as miRNA, mRNA, and proteins39. The clinical utility of EVs in the management of prostate cancer has been an active area of investigation4041. Markers that reliably identify tumor- derived extracellular vesicles (tdEVs) are needed. Clinical data investigating a role for EVs in patient selection and response monitoring have not been documented. Given the observation that clinical interventions affect EV subpopulations in a variable manner, it is essential to identify and validate these markers as they relate to tumor burden, treatment response, and antitumor immunity42.SUMMARY

[0006] This present disclosure provides materials and methods for assessing and / or treating patients with prostate cancer. In one embodiment, the disclosure provides a method of treating prostate cancer in a subject, said method comprising: (a) obtaining a sample fromthe subject, and (b) determining the presence and / or the amount of tumor-derived extracellular vesicles (tdEVs) in said sample and administering a therapy, wherein (i) if the amount of tdEVs is below a threshold measurement, the subject is administered a metastasis-directed therapy (MDT) only; or (ii) if the amount of tdEVs is above a threshold measurement, the subject is administered a systemic therapy and optionally also a MDT. In one embodiment, the threshold measurement is approximately 2x106EVs per ml.

[0007] In another embodiment, the prostate cancer is selected from the group consisting of castration-sensitive prostate cancer (CSPC), oligometastatic prostate cancer (omPC), oligometastatic castration-sensitive prostate cancer (omCSPC), metastatic castration resistant prostate cancer (CRPC), and clinically organ-confined prostate cancer. In one embodiment, the sample is obtained from the subject prior to receiving a therapy. In another embodiment, prior to obtaining the sample, the subject received one or more of a metastasis-directed therapy (MDT) or a systemic therapy or a combination thereof. In still another embodiment, the MDT is stereotactic ablative radiation therapy (SABR) or radiotherapy or a combination thereof. In yet another embodiment, the sample is selected from the group consisting of a blood sample, a plasma sample, a serum sample, and a urine sample.

[0008] In another embodiment, the tdEVs are determined or measured using a technique selected from the group consisting of flow cytometry, high resolution flow cytometry, an enzyme-linked immunosorbent assay (ELISA), mass spectrometry, droplet-based vesicle EV analysis, proximity-extension assay, radioanalytical assay, and ultrasensitive immunoassays (e.g., SimoA, MSD). In one embodiment, the tdEVs are determined or measured using high resolution flow cytometry. In another embodiment, a tdEV measurement in plasma is indicative of tumor burden and / or micrometastatic disease in the subject. In still another embodiment, the tdEVs comprise one or more surface markers selected from the group consisting of PSMA, STEAP1 , PDL1 , B7-H3, CEACAM5, CD56, DLL3, SYP, GPC3, ISM1 , CELSR3 and HMMR. In yet another embodiment, the tdEVs are PSMA-positive extracellular vesicles (PSMA+EVs).

[0009] In an embodiment, the one or more therapies are selected from the group consisting of metastasis-directed therapy (MDT), systemic therapy, surgery, radiation therapy, cryotherapy, high-intensity focused ultrasound (HIFU), transurethral ultrasound ablation (TULSA), focal laser ablation (FLA), photodynamic therapy (PDT), hormone therapy, chemotherapy, immunotherapy, targeted drug therapy, cryotherapy (cryoablation), and radiofrequency ablation. In one embodiment, the therapy is a metastasis-directed therapy (MDT) or a combination therapy comprising MDT and systemic therapy. In another embodiment, the MDT is selected from the group consisting of radiotherapy and surgery. Inyet another embodiment, the radiotherapy is selected from the group consisting of stereotactic ablative radiation therapy (SABR), external beam radiotherapy, hypofractionated radiotherapy, proton particle therapy, unsealed radionucleotide therapy, brachytherapy, and intensity modulated radiotherapy.

[0010] In another embodiment, the systemic therapy is selected from the group consisting of hormone therapy, luteinizing hormone-releasing hormone agonist / antagonist therapy, androgen-receptor targeting agent-based therapy, immunotherapy, PARP inhibitor-based therapy, chemotherapy, second generation androgen inhibitor-based therapy, and theranostic (radionucleotide) therapy. In one embodiment, the therapy does not include androgen deprivation therapy (ADT). In another embodiment, the subject received one or more therapies and the administering step (c) includes continuing or intensifying the previously received therapies.

[0011] In yet another embodiment, the one or more therapies include a checkpoint inhibitor. In one embodiment, the checkpoint inhibitor is selected from an inhibitor of CTLA-4, 4-1 BB (CD137), 4-1 BBL (CD137L), PDL1 , PDL2, PD1 , B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, BTLA, SIGLEC9, and 2B4. In another embodiment, the checkpoint inhibitor is an inhibitor of PDL1 and / or an inhibitor of B7-H3.

[0012] In still another embodiment, the method further comprises the step of determining disease progression in the subject. In one embodiment, disease progression is measured by biochemical progression (bPFS) and / or radiographic progression (rPFS).

[0013] Disclosed herein is a method of detecting PSMA+EVs in a sample from a subject, said method comprising (a) obtaining a sample from the subject; and (b) determining the presence of and / or the amount of tumor-derived extracellular vesicles (tdEVs) in said sample comprising using a technique selected from the group consisting of flow cytometry, high resolution flow cytometry, an enzyme-linked immunosorbent assay (ELISA), mass spectrometry, droplet-based vesicle EV analysis, proximity-extension assay, radioanalytical assay, and ultrasensitive immunoassays (e.g., SimoA, MSD). In one embodiment, the tdEVs are determined or measured using high resolution flow cytometry.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 provides a diagram of the study cohorts.

[0015] Figures 2A-2B provide oncological outcomes of the study cohorts, presenting Kaplan-Meier curves for biochemical progression-free survival (bPFS)(Figure 2A) and radiographic progression-free survival (rPFS)(Figure 2B) in the Ghent University, Iridium Network, and ORIOLE cohorts.

[0016] Figures 3A-3C show a comparative analysis of baseline PSA and PSMA+EV between study cohorts, presenting baseline levels of PSA (Figure 3A) and PSMA+EV (Figure 3B) in the three cohorts. P values were determined by Kruskal-Wallis test. Spearman coefficients and P value for correlation between baseline levels of PSA and PSMA+EV for the three cohorts are presented in Figure 3C.

[0017] Figures 4A-4D show progression-free survival stratified by number of lesions treated, providing Kaplan-Meier curves for biochemical progression-free survival (bPFS) and radiographic progression-free survival (rPFS) in the ORIOLE cohort in Figures 4A-4B, and the STOMP-like pooled cohort in Figures 4C-4D.

[0018] Figures 5A-5B show progression-free survival stratified by baseline PSMA+EV levels, presenting Kaplan-Meier curves for biochemical progression-free survival (bPFS)(Figure 5A) and radiographic progression-free survival (rPFS)(Figure 5B) in the pooled cohorts.

[0019] Figures 6A-6B provide progression-free survival stratified by baseline PSA levels in pooled cohorts, presenting Kaplan-Meier curves for biochemical progression-free survival (bPFS)(Figure 6A) and radiographic progression-free survival (rPFS)(Figure 6B) in the pooled cohort.

[0020] Figures 7A-7D provide progression-free survival stratified by baseline PSA levels in separate study cohorts, presenting Kaplan-Meier curves for biochemical progression-free survival (bPFS) and radiographic progression-free survival (rPFS) in the STOMP-like cohort in Figures 7A-7B, and the ORIOLE cohort in Figures 7C-7D.

[0021] Figures 8A-8D show progression-free survival stratified by baseline PSA doubling time (DT) levels, presenting Kaplan-Meier curves for biochemical progression-free survival (bPFS) and radiographic progression-free survival (rPFS) in the STOMP-like cohort in Figures 8A-8B, and in the ORIOLE cohort in Figures 8C-8D.

[0022] Figures 9A-9B provide progression-free survival stratified by baseline PSA and PSMA+EVs, presenting Kaplan-Meier curves for biochemical progression-free survival (bPFS)(Figure 9A) and radiographic progression-free survival (rPFS)(Figure 9B) in the pooled cohort.

[0023] Figures 10A-10D show progression-free survival stratified by PSMA+EV levels, presenting Kaplan-Meier curves for biochemical progression-free survival (bPFS) and radiographic progression-free survival (rPFS) in the STOMP-like cohort in Figures 10A-10B, and in the ORIOLE cohort in Figures 10C-10D.

[0024] Figures 11A-11 D provide progression-free survival stratified by baseline PSA and PSMA+EVs, presenting Kaplan-Meier curves for biochemical progression-free survival (bPFS) and radiographic progression-free survival (rPFS) in the STOMP-like cohort in Figures 11A-11 B, and in the ORIOLE cohort in Figures 11C-11 D.

[0025] Figures 12A-12B describe the association of baseline PSMA+EV levels with biochemical PFS in ORIOLE SABR and observation arms, presenting Kaplan-Meier curves for biochemical progression-free survival (bPFS) in patients of the ORIOLE SABR and observation arms stratified by baseline levels of PSMA+EV low (Figure 12A) and high (Figure 12B).

[0026] Figures 13A-13B describe the association of baseline PSMA+EV levels with radiographic PFS (rPFS) in ORIOLE SABR and observation arms, presenting Kaplan-Meier curves for radiographic progression-free survival (rPFS) in patients of the ORIOLE SABR and observation arms stratified by baseline levels of PSMA+EV low (Figure 13A) and high (Figure 13B).

[0027] Figures 14A-14B describe the association of baseline PSA levels with biochemical and radiographic progression-free survival in ORIOLE SABR and observation arms, presenting Kaplan-Meier curves for biochemical and radiographic progression-free survival in patients of the ORIOLE SABR observation arms stratified by baseline levels of PSA low (Figure 14A) and high (Figure 14B).DETAILED DESCRIPTION

[0028] Disclosed herein are compositions and methods of treating prostate cancer in a subject. In some cases, the methods and materials provided herein can be used to identify a mammal (e.g., a human) that will experience long-term disease-free survival following treatment with stereotactic ablative radiotherapy. In some cases, the methods and materials provided herein can be used to treat a mammal (e.g., a human) having prostate cancer (e.g., a treatment-resistant prostate cancer). In some cases, a treatment-resistant prostate cancer can be resistant to one or more cancer treatments (e.g., androgen deprivation therapy, antiandrogen therapy, chemotherapy, radiotherapy, or other treatment modalities). For example, a treatment-resistant prostate cancer can be castration-resistant prostate cancer (CRPC, which can also be referred to as a hormone-resistant prostate cancer (HRCP)). In some cases, the materials and methods provided herein can be used to treat a mammal (e.g., a human) having prostate cancer (e.g., CRPC). In some embodiments, prostate cancer can be castration-sensitive prostate cancer (CSPC), oligometastatic prostate cancer (omPC), oligometastatic castration-sensitive prostate cancer (omCSPC), metastatic castration resistant prostate cancer (CRPC), and clinically organ-confined prostate cancer.

[0029] In some cases, the materials and methods provided herein can be used to identify a mammal (e.g., a human) as having oligometastatic prostate cancer. The concept of oligometastatic disease was first suggested in 1995 by Hellman and Weichselbaum as a unique biologic state between localized and widespread metastatic disease1 28. This concept has changed the dogma that all metastatic disease was beyond cure, now proposing that patients with early metastatic disease may still be cured with a combination of local and metastasis-directed therapies (MDT). SABR-COMET was the first randomized trial to evaluate stereotactic body radiotherapy (SABR) in various oligometastatic cancers2. This trial was the first to suggest a survival benefit with SABR; however, prostate cancer only represented 16% of the treatment group. In prostate cancer, two randomized clinical trials have since demonstrated a benefit of SABR in the oligometastatic setting (ORIOLE and STOMP trials)34. Further, a randomized phase II trial compared SABR in combination with abiraterone and abiraterone alone, and showed that the addition of SABR improved PFS and biochemical control (ARTO trial)43.

[0030] Several markers for prostate cancer have been identified, including e.g. prostatespecific antigen (PSA), the six-transmembrane epithelial antigen of the prostate (STEAP)44, the prostate stem cell antigen (PSCA)45and the prostate-specific membrane antigen (PSMA; PSM)46. PSMA is a tumor-associated antigen and type II transmembrane protein, which is expressed on the membrane of prostatic epithelial cells and overexpressed on prostate tumor cells. Human PSMA is highly expressed in the prostate, roughly a hundred times greater than in most other tissues. In some prostate cancers, PSMA is the second-most upregulated gene product, with an 8- to 12-fold increase over levels in noncancerous prostate cells. Because of this high expression, PSMA is being developed as a potential biomarker for therapy and imaging of some cancers. In human prostate cancer, the higher PSMA-expressing tumors are associated with quicker time to progression and a greater percentage of patients suffering relapse. In vitro studies using prostate and breast cancer cell lines with decreased PSMA levels showed a significant decrease in the proliferation, migration, invasion, adhesion, and survival of cells.

[0031] The term “prostate cancer,” as used herein, refers to cancer that occurs in the prostate. The prostate is a small walnut-shaped gland in males that produces the seminal fluid that nourishes and transports sperm. Prostate cancer is one of the most common types of cancer. Many prostate cancers grow slowly and re confined to the prostate gland, where they may not cause serious harm. However, while some types of prostate cancer grow slowly and may need minimal or even no treatment, other types are aggressive and can spread quickly.

[0032] Tumor-derived extracellular vesicles (tdEVs) are microscopic particles released by tumors into the bloodstream and are emerging as non-invasive biomarkers for cancer detection and prognosis5. Two important benefits of circulating tdEV are that 1 ) their concentrations correlate with tumor burden6and 2) unlike circulating tumor cells and circulating tumor DNA, they are detectable across the entire spectrum of the disease from localized prostate cancer to widespread metastatic castration resistant prostate cancer (CRPC)6’7. Association of circulating levels of PSMA+EV with radiographic tumor burden has been demonstrated in prostate cancer6. Furthermore, pre-SABR PSMA+EV can predict risk of disease recurrence in omCRPC patients treated with SABR. An observational study is disclosed herein, evaluating the prognostic and predictive value of PSMA+EV in two cohorts of omCSPC patients: the ORIOLE trial [PI: Phuoc Tran] and the STOMP-like cohort (Ghent University [PI: Piet Ost, Belgium], the Iridium Network [PI: Carole Mercier, Belgium]).

[0033] As demonstrated herein, the presence or absence of tdEVs can be used as a systemic biomarker that can be detected in circulating blood. The presence or absence of an elevated level of tdEVs in a sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having prostate cancer can be used to determine whether or not a prostate cancer has progressed from a localized tumor to metastatic disease. For example, the presence of an elevated level of tdEVs can be detected in the blood of a mammal (e.g., a human) having CSPC, omCSPC, or widely metastatic disease.

[0034] Having the ability to identify a mammal having prostate cancer as having oligometastatic disease as described herein (e.g., based on the presence of an elevated level of tdEVs in a sample (e.g., a blood sample) obtained from the mammal) provides a simple and non-invasive method for diagnosing treatment-resistant oligometastatic disease. An embodiment of the present disclosure also allows for determination of the efficacy of SABR treatment based on the concentration of tdEVs present in the sample taken from a subject suffering from prostate cancer.

[0035] In one embodiment, the present disclosure provides methods for assessing a mammal having prostate cancer. The methods can include, or consist essentially of, (a) obtaining a sample from the subject, (b) determining the presence of and / or the amount of tumor-derived extracellular vesicles (tdEVs) in said sample and administering a therapy, wherein (i) if the amount of tdEVs is below a threshold measurement, the subject is administered a metastasis-directed therapy (MDT) only; or (ii) if the amount of tdEVs is above a threshold measurement, the subject is administered a systemic therapy and optionally also a MDT. In one embodiment, the sample is obtained from the subject prior to receiving a therapy. In one embodiment, the subject is a human. In one embodiment, the blood sample is a plasma sample. In one embodiment, the threshold measurement isapproximately 2x106EVs per ml. In another embodiment, the threshold measurement is below 2x107EVs per ml. In other embodiments, the threshold measurement is approximately 1 x105, 2x105, 3x105, 4x105, 5x105, 6x105, 7x105, 8x105, 9x105, 1 x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 1 x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x107, or 9x107’ EVs per ml.

[0036] In one embodiment, the tdEVs are obtained from a subject suffering from prostate cancer as a sample. The sample may be a blood sample, a plasma sample, a serum sample, a urine sample, or a sample taken directly from a tumor (e.g., a biopsy sample).

[0037] In another embodiment, the presence or absence of tdEVs is evaluated in the sample obtained from a subject suffering from prostate cancer. The tdEVs can be determined or measured using a technique selected from the group consisting of flow cytometry, high resolution flow cytometry, an enzyme-linked immunosorbent assay (ELISA), mass spectrometry, droplet-based vesicle EV analysis, proximity-extension assay, or radioanalytical assay. In yet another embodiment, the tdEVs can be determined or measured using single molecule arrays (SiMoA), assays that are significantly more sensitive than ELISA assays.

[0038] In some embodiments, the circulating tdEVs express cancer associated antigens that were also expressed by the tumor they were derived from. In some embodiments, the cancer associated antigen is a tumor antigen, i.e., a part of a tumor cell such as a protein or peptide expressed in a tumor cell which may be derived from the cytoplasm, the cell surface or the cell nucleus, in particular those which primarily occur intracellularly or as surface antigens of tumor cells. The term “cancer associated antigen” as used herein can be any type of cancer antigen that may be associated with a cancer as is known in the art and includes antigens found on the cell surface, including tumor cells, as well as soluble cancer antigens. Several cell surface antigens on tumors and normal cells have soluble counterparts. A cancer associated antigen can be a cell surface antigen or a soluble cancer antigen located in the tumor microenvironment or otherwise in close proximity to the tumor being treated.

[0039] In an embodiment, the tdEVs comprise one or more surface markers selected from the group consisting of plasma-specific membrane antigen (PSMA), six transmembrane epithelial antigen of the prostate 1 (STEAP1 ), programmed cell death ligand 1 (PDL1 ), B7 homolog 3 protein (B7-H3), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), neural cell adhesion molecule (CD56), delta-like protein 3 (DLL3), synaptophysin (SYP), glypican-3 (GPC3), isthmin 1 (ISM1 ), cadherin EGF LAG seven-passG-type receptor 3 (CELSR3), and hyaluronan-mediated motility receptor (HMMR). In one embodiment, the tdEVs are PSMA-positive extracellular vesicles (PSMA+EV).

[0040] In one embodiment, methods provided herein comprise administration to a subject of one or more therapies selected from the group consisting of metastasis-directed therapy (MDT), systemic therapy, surgery, radiation therapy, cryotherapy, high-intensity focused ultrasound (HIFU), transurethral ultrasound ablation (TULSA), focal laser ablation (FLA), photodynamic therapy (PDT), hormone therapy, chemotherapy, immunotherapy, targeted drug therapy, cryotherapy (cryoablation), and radiofrequency ablation. In a particular embodiment, MDT may comprise radiotherapy and surgery. In another embodiment, the radiotherapy is selected from a group consisting of stereotactic ablative radiation therapy (SABR), external beam radiotherapy, hypofractionated radiotherapy, proton particle therapy, unsealed radionucleotide therapy, brachytherapy, and intensity modulated radiotherapy. In still another embodiment, the systemic therapy is selected from a group consisting of hormone therapy, luteinizing hormone-releasing hormone agonist / antagonist therapy, androgen-receptor targeting agent-based therapy, immunotherapy, PARP inhibitor-based therapy, chemotherapy, second generation androgen inhibitor-based therapy, and theranostic (radionucleotide) therapy.

[0041] SABR is rapidly emerging as a safe and potentially curative treatment option in omCSPC with the potential to avoid the use of non-curative systemic therapy, delaying the negative quality of life impact of androgen deprivation and the development of castration refractory disease. Both the ORIOLE and STOMP trials reported long-term disease-free survival in -20% of patients, while most patients only have a modest delay in progression and were initiated on systemic therapy34. This divergence in outcomes suggests that select omCSPC patients can benefit from SABR alone, while others may require the addition of systemic therapy. This highlights a current unmet need to develop predictive biomarkers that can help select patients who may experience a durable response from SABR without systemic therapy. Unfortunately, to date no effective liquid biomarkers exist to better select patients for SABR alone or in combination with systemic therapy.

[0042] In certain embodiments, the therapy administered to the subject with prostate cancer is a checkpoint blockade inhibitor molecule. The checkpoint blockade inhibitor molecule is selected from a group consisting of an inhibitor of CTLA-4, 4-1 BB (CD137), 4- 1 BBL (CD137L), PDL1 , PDL2, PD1 , B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, BTLA, SIGLEC9, and 2B4.

[0043] Immune checkpoints refer to a variety of inhibitory pathways of the immune system that are crucial for maintaining self-tolerance and for modulating the duration and amplitudeof an immune responses. Tumors use certain immune-checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumor antigens4748. Immune checkpoint inhibitors include any agent that blocks or inhibits in a statistically significant manner, the inhibitory pathways of the immune system. Such inhibitors may include antibodies, or antigen binding fragments thereof, that bind to and block or inhibit immune checkpoint receptors or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. Illustrative immune checkpoint molecules that may be targeted for blocking or inhibition include, but are not limited to, CTLA-4, 4-1 BB (CD137), 4- 1 BBL (CD137L), PDL1 , PDL2, PD1 , B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, BTLA, SIGLEC9, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, yd, and memory CD8+ (op) T cells), CD160 (also referred to as BY55), and CGEN-15049. Immune checkpoint inhibitors include antibodies, or antigen binding fragments thereof, or other binding proteins, that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1 , PDL2, PD1 , B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, BTLA, SIGLEC9, 2B4, CD160, and CGEN-15049.

[0044] In one embodiment, the present disclosure provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a composition described herein, and further comprising administering to the subject a therapeutically effective amount of a checkpoint inhibitor. In another embodiment, the checkpoint inhibitor is selected from the group consisting of an inhibitor of CTLA-4, 4-1 BB (CD137), 4-1 BBL (CD137L), PDL1 , PDL2, PD1 , B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, BTLA, SIGLEC9, and 2B4. In some embodiments, the checkpoint inhibitor is selected from the group consisting of pembrolizumab, avelumab, atezolizumab, cetrelimab, dostarlimab, cemiplimab, spartalizumab, camrelizumab, durvalumab, and nivolumab. In other embodiments, an aforementioned method is provided further comprising administering to the subject an isolated tumor associated antigen (TAA). In one embodiment, provided herein is a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a composition described herein, and further comprising administering to the subject one or more inhibitors selected from the group consisting of inhibitors of ALK, PARP, VEGFRs, EGFR, FGFR1 -3, HIF1 a, PDGFR1 -2, c-Met, c-KIT, Her2, Her3, AR, PR, RET, EPHB4, STAT3, Ras, HDAC1-11 , mTOR, and CXCR4.

[0045] In one embodiment, provided herein is a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a composition provided herein, and further comprising administering to the subject a therapeuticallyeffective amount of radiation therapy. In one embodiment, provided herein is a method of treating cancer in a subject comprising administering a therapeutically effective amount of a composition described herein, and further comprising administering to the patient a cancer treatment surgery. In one embodiment, provided herein is a method of concurrently treating two or more cancers in a subject comprising administering to the subject a therapeutically effective amount of a composition described herein.

[0046] In another embodiment, the aforementioned method is followed by assessment of disease progression. In one embodiment, disease progression is measured by biochemical progression (bPFS). In another embodiment, disease progression is measured by radiographic progression (rPFS).EXAMPLE

[0047] The present Example demonstrates that tdEVs are biomarkers for tumor burden and can be used to predict and guide treatment responsiveness and regimens. For example, as shown herein, PSMA+EV is a novel prognostic biomarker of tumor burden and micrometastatic disease in omCSPC and a predictive biomarker of biochemical progression for SABR MDT.

[0048] Purpose: Two randomized clinical trials (STOMP and ORIOLE) demonstrated that stereotactic ablative radiotherapy (SABR) can prolong ADT-free survival or progression-free survival (PFS) in metachronous oligometastatic castration-sensitive prostate cancer (omCSPC) patients. While most omCSPC patients have an intermediate delay in progression, a small subset experiences a durable response following SABR. Therefore, the present study focuses on finding biomarkers that can identify omCSPC patients who are likely to have durable response to SABR alone. The prognostic and predictive value of circulating PSMA-positive extracellular vesicles (PSMA+EV) and prostate specific antigen (PSA) was investigated in an observational study using blood samples from three independent patient cohorts.

[0049] Methods: Plasma samples of 127 STOMP-like omCSPC patients treated with SABR were obtained from Ghent University (N=80) and Iridium Network (N=47) in Belgium. Samples from the ORIOLE trial were obtained from John Hopkins University, USA (N=30 SABR arm, N=16 observation arm). Pre-SABR PSMA+EV levels (EV / ml) were measured by nanoscale flow cytometry. Biochemical progression (bPFS) and radiographic progression (rPFS) were used as clinical endpoints. Optimal cutoffs for PSA (ng / ml) and PSMA+EV (EVs / ml) low and high were defined as the point with the most significant log-rank test split. Kaplan-Meier curves and univariate Cox regression models were used to determine the association of PSMA+EV and PSA levels with clinical outcomes.

[0050] Results: In the STOMP-like cohorts, median bPFS were 27.9 and 18.0 months for PSMA+EV low and high groups, respectively (p=0.039). Median rPFS were 36.0 and 27.0 months, respectively (p=0.029). In the ORIOLE trial, median bPFS was 24.3 and 5.9 months in PSMA+EV low and high, respectively (p=0.0028). Median rPFS was 36.0 and 8.6 months, respectively (p=0.0031). The combination of pre-SABR low levels of both PSMA+EV and PSA was associated with lower risk of radiographic progression in both STOMP-like (HR=0.25, 95% Cl: 0.09-0.56, p=0.003) and ORIOLE cohorts (HR=0.12, 95% Cl: 0.02-0.44, p=0.005). Furthermore, PSMA+EV remained a significant independent prognostic biomarker of biochemical and radiographic progression on multivariate analysis controlling for the effect of PSA levels, number of lesions, and lesion location. In the ORIOLE cohort, low PSMA+EV was a predictive biomarker of SABR MDT (p=0.012). Specifically, within the low PSMA+EV group, patients in the treatment group had lower risk of biochemical recurrence (HR = 0.194, 95% Cl: 0.065-0.644, p=0.004) while in the high PSMA+EV group, there was not a significant treatment effect on biochemical recurrence (HR = 1.173, 95% Cl: 0.498-2.766, p=0.715).

[0051] Conclusions: PSMA+EV is a novel prognostic biomarker of radiographically occult tumor burden in omCSPC. PSMA+EV may inform clinical decisions regarding which patients achieve a durable benefit from consolidative SABR along.Materials and Methods

[0052] Patient Cohorts: Three cohorts of patients with stored plasma available were obtained from Ghent University (PI: Piet Ost, Belgium)(N=80), the Iridium Network (PI: Carole Mercier, Belgium)(N=47), and the ORIOLE trial (PI: Phuoc Tran)(N=46)3. Study approval was granted by the Mayo Clinic Institutional Review Board (IRB #21 -004451). The Ghent University cohort enrolled patients between 2015 and 2020 under the existing STOMP trial protocol (PI: Piet Ost)4. In the Iridium Network cohort, patients were treated for omCSPC with SABR between 2018 and 2020. Detailed characteristics of the ORIOLE trial has been previously reported3.

[0053] Enumeration of PSMA-Positive Extracellular Vesicles (PSMA+EV): Plateletpoor plasma (PPP) samples were incubated with fluorescent PSMA antibodies (J591 clone) and concentrations of PSMA+EV were measured by nanoscale flow cytometry as previously described68. Based on calibration, PSMA+EV with a minimum diameter of 188 nm and a minimum fluorescence intensity of 460 molecules of equivalent soluble fluorochrome (MESF) were counted. All measurements were performed blinded from clinical data. Each sample was run in three technical replicates and average was used for data analysis.

[0054] Statistical Analysis: Biochemical (bPFS) and radiographic (rPFS) progression were used as clinical endpoints to determine the association of PSMA+EV levels, serum PSAand PSA doubling time (PSA DT) at 3 months with oncological outcomes. Biochemical progression was defined as any of the following: i) for patients who have undergone a radical prostatectomy, PSA rise to > 0.2 ng / mL from nadir after MDT ; or ii) for patients who did not experience a PSA nadir below 0.2 ng / mL after MDT: then first rise in PSA after reaching nadir or ill) for patients treated with primary radiation to the prostate, PSA nadir +2 ng / mL after MDT or iv) initiation of systemic therapy, local recurrence, or distant recurrence prior to reaching numerical definition of PSA as above. Radiographic progression was defined as new nodal lesions, intrapelvic or distant, bone, or visceral lesions on conventional (bone scintigraphy) or molecular (11C Choline PET / CT or PSMA-PET CT) imaging with application of the RECIST version 1.1 criteria. Kaplan-Meier (KM) estimates were used to estimate survival curves. For each KM plot, p values were derived from the log-rank test for difference between groups. The optimal cutoff values of PSMA+EV (2.1 x106EVs per mL for the STOMP-like cohort, 1 .6x106EVs per mL for the ORIOLE cohort) and PSA (1 .0 ng / mL for the STOMP-like cohort, 7.0 ng / mL for the ORIOLE cohort) were defined as the value with the most significant log-rank test split in univariate Cox proportional hazard model using rPFS as clinical endpoint9. The same cut-off values were used for all statistical analyses throughout the study. The hazard ratio (HR) of each biomarker was calculated with each clinical outcome (bPFS and rPFS). The effect of PSMA+EV on biochemical progression and radiographic progression was tested while controlling for the effect of PSA levels, number of lesions, and lesion location in multivariate Cox proportional hazard models. For correlative studies, PSMA+EV and PSA levels were treated as categorical variables. PSA DT was also treated as a categorical variable (< or > 3 months). For predictive biomarker assessment, PSMA+EV predictive value was tested by including PSMA+EV, treatment group, and treatment-by-biomarker interaction term in a Cox proportional hazard model. Hazard ratios (HRs) with 95% confidence intervals were calculated. Statistical software (SAS, version 9.4, SAS Institute Inc, Cary, NC) was used for the multivariate Cox models. Prism v9.0.1 (Graph Pad Software) was used for all other statistical analyses. For association with oncological outcomes, PSMA+EV and PSA levels were converted to categorical variables and used to classify patients as high and low.Results

[0055] Baseline Characteristics, PSMA+EV Levels and Survival Outcomes. One hundred and seventy-three patients with plasma samples available were included (Ghent University n=80, Iridium Network n=47, ORIOLE Trial n=46). The diagram for the study cohorts is included in Figure 1. Sixteen patients with CRPC disease or local recurrence / non- metastatic disease were excluded from the study. For the ORIOLE cohort, 30 patients were treated with SABR and 16 patients were part of the observation arm. Patient characteristicsfor the three cohorts are presented in Table 1. For all patients treated with SABR (n=157), no active systemic therapies were received concomitantly with SABR. Per ESTRO-EORTC classification10, 92% (145 / 157) of patients were diagnosed with metachronous oligorecurrent prostate cancer. Repeat oligorecurrence and synchronous oligometastasis were observed in 5% and 3%, respectively. Oligometastatic disease was diagnosed with advanced PET imaging (80%) and conventional imaging (20%)(Table 1 ). All patients were diagnosed with C11-Choline PET in the Ghent University cohort while most patients (94%) received PSMA- PET in the Iridium Network. The median follow up was 45.7 months (range 41 .2-51 .7). Median bPFS was 21 .5 months in the Ghent University cohort, 17.5 months in the Iridium cohort and 11.1 months in ORIOLE SABR arm (Figure 2A). Median rPFS was 29.0 months in the Ghent University cohort, 32.1 months in the Iridium cohort and 25.3 months in ORIOLE SABR arm (Figure 2B).

[0056] Table 1 : Patient Characteristics

[0057] Comparative analysis between baseline (pre-SABR) PSA levels demonstrates no significant difference between the Ghent University and Iridium Network cohorts (1 .9 and 2.0 ng / ml, p>0.99)(Figure 3A). There was a significant difference in baseline PSA when comparing the Ghent University and Iridium Network cohorts (p<0.0001 ) with the ORIOLE cohort (6.8 ng / ml) which could be explained by the lower sensitivity of conventional imaging (ORIOLE) over PET imaging (both Belgium centers) for detecting radiographic recurrence. Comparative analysis between baseline PSMA+EV demonstrate no significant difference between the Ghent University and Iridium Network cohorts (5.91 x106and 4.98x106EVs / ml, p=0.74). Similar to baseline PSA, there was a significant difference in baseline levels of PSMA+EV in the ORIOLE trial cohort (median: 2.12x106EVs / ml) when compared to the Ghent University (p<0.0001) and Iridium Network (p=0.0004) cohorts (Figure 3B). Nocorrelation was found between baseline levels of PSA and PSMA+EV in the three cohorts (Figure 3C). Given the similarities in patient characteristics, treatment plan and oncological outcomes of the Ghent University and Iridium Network cohorts with the STOMP trial4, both cohorts were combined, and are referred to as “STOMP-like” cohort hereafter. Oncological outcomes of patients stratified by the number of metastatic lesions can be found in Figure 4.

[0058] Baseline PSMA+EV is a prognostic biomarker in oligorecurrent castrationsensitive prostate cancer. Following stratification of patients based on PSMA+EV levels in the pooled cohort of the ORIOLE trial and STOMP-like cohorts, a median bPFS was 26.1 and 15.0 months, respectively (Figure 5A). Median rPFS was 36.0 months and 25.0 months for patients with low and high PSMA+EV (p=0.003)(Figure 5B). Low baseline levels of PSMA+EV were associated with lower risk of biochemical recurrence (HR=0.59, 95% Cl: 0.40-0.85, p=0.005) and radiographic recurrence (HR=0.55, 95% Cl: 0.34-0.81 , p=0.003). Patients stratified based on PSA and PSA doubling time (PSA DT) demonstrated similar differences in PFS outcomes (i.e., low PSA and low PSA DT are both associated with lower risk of biochemical recurrence), but baseline PSA was a superior predictor of outcome compared to PSA DT (Figures 6-8).

[0059] PSMA+EV remained an independent predictor of risk of both biochemical progression and radiographic progression when controlling for the effect of PSA levels, number of lesions, and lesion location in multivariable Cox proportional hazard models. In the pooled cohort, low PSMA+EV was also an independent predictor of bPFS (HR=0.59, 95% Cl: 0.40-0.85, p=0.005) and rPFS (HR=0.55, 95% Cl: 0.34-0.81 , p=0.003)(Table 2).

[0060] Table 2: Multivariate analysis of the risk of progression for baseline PSMA+EV, PSA, and PSA DT in the pooled STOMP-like and ORIOLE cohort157 Observations Read; 145 Observations used

[0061] Combination baseline PSA and PSMA+EV and oncological outcomes. While patient stratification on PSMA+EV alone produced significant differences in PFS, combining PSMA+EVs and PSA resulted in identification of long-term responders to SABR. Patients were stratified as PSA low / PSMA+EV low, PSA high / PSMA+EV high, PSA low / PSMA+EV high and PSA high / PSMA+EV low. In the pooled cohort, 15% (19 / 127) of patients presented with PSA low / PSMA+EV low levels. Median bPFS and rPFS were not reached, and PSA low / PSMA+EV low was a superior prognostic marker of bPFS and rPFS (Figures 9A-9B). Combination of baseline levels of PSMA+EV and PSA was associated with lower risk of biochemical recurrence (HR=0.34, 95% Cl: 0.18-0.58, p=0.0002) and radiographic recurrence (HR=0.22, 95% Cl: 0.09-0.44, p=0.0001 ). Progression-free survival stratified by PSMA+EV and combination of PSMA+EV and PSA for the individual cohorts is presented in Figures 10-11.

[0062] PSMA+EV is a predictive biomarker of response to SABR. Here, the baseline levels of PSMA+EV were analyzed in both SABR and observation arms of the ORIOLE cohort to evaluate their predictive value. Patients who presented with low levels of PSMA+EV showed benefit from SABR compared to patients in the observation arm (Figure 12A). Median bPFS was 24.3 and 5.8 months for SABR and observation arms, respectively (p=0.003). Patients treated with SABR had a significantly lower risk of biochemicalprogression (HR=0.19, 95% Cl: 0.065-0.644, p=0.004). In contrast, SABR did not show any benefit for patients with high baseline levels of PSMA+EV compared to observation (5.9 and 7.1 months, p=0.95)(Figure 12B). Risk of biochemical progression was not statistically different between both groups (HR=1.17, 95% Cl: 0.498-2.766, p=0.715). Baseline levels of PSMA+EV did not significantly affect risk of radiographic progression (Figure 13). There was no significant treatment effect observed in the high PSMA group (HR=1 .39, 95% Cl: 0.50- 3.84, p=0.512) or the low PSMA group (HR=0.57, 95% Cl: 0.14-2.22, p=0.419). In patients with low baseline PSA levels, bPFS benefit from SABR was observed compared to observation, but the dichotomy (p=0.03) was not as significant as the benefit seen in patients with low PSMA+EVs when compared to observation patients (p=0.003)(Figure 14).

[0063] Table 3: Risk of disease progression for baseline PSA and PSMA+EV levels in the pooled cohortDiscussion

[0064] Systemic therapy with ADT in combination with next generation AR targeted therapy has Level 1 evidence for overall survival benefit in patients with low volume metastatic CSPC. The benefit of systemic therapy intensification remains very modest in patients presenting with metachronous low-volume disease (< 3 non-visceral metastases)11. The concept of metastasis-directed therapy (MDT) with SABR or surgery in oligorecurrent prostate cancer is gaining traction following the positive results from the STOMP and ORIOLE randomized trials34. MDT represents a safe and effective treatment option to eliminate visible metastases on imaging without using systemic therapy. Of note, -10-15% of patients achieve prolonged disease control with no sign of recurrence on imaging for at least 4 years post-treatment12-16. While local control remains excellent (-90% at 2 years follow-up) after SABR treatment, oligoprogression remains the major cause of clinical failure post-SABR and it suggests that patients may present at diagnosis with micrometastatic disease below the threshold of detection of both conventional and advanced imaging17. To date, decision of treatment with MDT and / or systemic intensified hormonal therapy forpatients with oligorecurrent CSPC is informed by clinical presentation such as volume and number of metastases, metastasis location, patient comorbidities and physician experience. The integration of tumor biomarkers with image variables better supports treatment decisions and estimation of response to SABR12. The present application demonstrates the clinical value of a pre-treatment PSMA+EV liquid biopsy as a non-invasive biomarker to improve stratification of oligometastatic disease and prioritize the use of SABR alone in patients with low volume disease and treatment intensification with systemic therapy alone or combined with SABR in patients with high volume disease.

[0065] 3 potential prognostic biomarkers for SABR were assessed; PSMA+EV, PSA, and PSA doubling time, in a large multi-institutional dataset to date of 157 patients with omCSPC. This assessment revealed that high baseline concentrations of PSMA+EV was a prognostic biomarker of both biochemical and radiographic progression following SABR treatment which is in line with the proof-of-concept in omCRPC6. The combination of baseline PSA and PSMA+EV was superior to all three individual biomarkers. On assessment with multivariable Cox proportional hazard models, controlling for the effect of PSA levels, number of lesions, and lesion location, PSMA+EV remains an independent prognostic biomarker. A total of 29 / 157 (18.5%) patients were identified with PSA low and PSMA+EV low and this combination was associated with long PFS. Similar to ORIOLE and STOMP studies, 30 / 157 patients (19.1%) experienced neither biochemical nor radiographic progression at a median follow-up of 40.3 months (95% Cl: 36.2-48.5). From those, 16 patients (53.3%) were classified as pre-SABR PSA low and PSMA+EV low. Conversely, 109 / 157 patients (69.4%) showed evidence of radiographic progression at a median followup of 20.4 months (95% Cl: 17.0-25.3) and only 7 of them (6.4%) had both PSA low and PSMA+EV low. This work demonstrates that pre-SABR blood levels of PSA and PSMA+EV may serve as a prognostic biomarker to differentiate patients with true omCSPC from those who likely harbor micrometastatic disease at diagnosis, and may be predictive of patient response to SABR alone.

[0066] Comparison of SABR and observation arms in the ORIOLE trial identified PSMA+EV as a predictive biomarker of biochemical progression in response to SABR. SABR demonstrated durable benefit in patients with low PSMA+EV, yet SABR did not provide any benefit in patients with high PSMA+EV compared to observation. Validation of PSMA+EV as a predictive biomarker of both biochemical and radiographic progression is currently ongoing in a prospective trial (DIVINE; NCT06378866) and could provide a promising blood-based predictive biomarker for metastasis directed therapies (MDT) in omCSPC.

[0067] Owing to a low tumor mutational burden and limited metastatic burden, circulating tumor DNA (ctDNA) concentrations are very low in omCSPC compared to metastaticCRPC18, 19. In the ORIOLE trial, 41% of patients had detectable ctDNA (mean 1 .3 mutations per participant) and ctDNA abundance was not associated with clinical outcome3. While studies utilizing ultrasensitive sequencing technologies will validate the potential utility of ctDNA in omCSPC, the abundance and detection rate of PSMA+EV in this setting makes it a suitable biomarker for risk stratification and treatment selection. As EVs carry molecular cargo from donor cells and protect the cargo from enzymatic degradation20, characterizing the DNA content of PSMA+EV via assay can provide further utility to EVs as a liquid biopsy.

[0068] While the study had access to patient plasma samples from the ORIOLE trial, most of patients in the STOMP trial had stored serum samples. Plasma is more suitable for PSMA+EV measurement (data not shown). Therefore, plasma samples from patients recruited outside the STOMP trial but under the same IRB protocol were utilized here, although there is potential for bias and variability due to the samples being collected from a non-randomized cohort. Furthermore, no samples for the STOMP-like observation arm were available to validate PSMA+EV as a predictive biomarker of SABR response.

[0069] Median levels of PSMA+EV were significantly lower in the ORIOLE cohort compared to the STOMP-like cohort, which is counterintuitive considering that ORIOLE trial enrolled patients diagnosed with conventional imaging. While variability in blood collection cannot be excluded, handling and storage conditions which could have impacted PSMA+EV measurement22, including freeze-thaw cycles (unpublished data), demonstrated a low positive predictive value (PPV= 0.43) for bone scans at initial staging of prostate cancer resulting in 57% false positive rate25. This finding suggests that, in addition to lower sensitivity than PSMA and choline PET, bone scans may also overestimate the true oligometastatic burden better reflected by blood PSA and PSMA+EV.

[0070] Deek et al. found several somatic mutations (e.g. TP53, ATM, RB1 , BRCA1 / 2) in omCSPC21that are associated with higher risk of clinical failure following SABR12. This suggests that the definition of oligometastasis should not solely rely on the number of metastases but also on the intrinsic tumor biology. Notably, tumor-derived EVs carry molecular cargo (e.g DNA, RNA, proteins, etc...) of the tumor of origin, and they can provide a snapshot of the tumor’s molecular profile in patients with metastatic prostate cancer without the need for tissue biopsy23 24. In some embodiments, the methods provided herein may leverage EV’s molecular cargo in conjunction with somatic and germline mutational signatures to provide further utility to EVs by improving patient risk stratification and selection of appropriate treatment strategy.

[0071] Thus, PSMA+EV is a novel prognostic biomarker of tumor burden and micrometastatic disease in omCSPC and a predictive biomarker of biochemical progressionfor SABR in omCSPC. Oligometastatic prostate cancer represents a heterogenous patient population, and the combination of PSMA+EV and PSA can help refine selection of patients who can experience durable disease-free survival in response to SABR. This observational study provides the first clinical use of extracellular vesicles on a prospective trial of omCSPC, and strengthens the clinical value of PSMA+EV for personalized radiation therapy The notable divergence in survival curves stratified by PSMA+EV and PSA levels highlights the need for prospective validation of PSMA+EV as a predictive biomarker for SABR.References:1 . Dhondt B, Pinheiro C, Geeurickx E, et al: Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. J Extracell Vesicles 12:e12315, 20232. 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Claims

WHAT IS CLAIMED IS:1 . A method of treating prostate cancer in a subject, said method comprising:(a) obtaining a sample from the subject;(b) determining the presence of and / or the amount of tumor-derived extracellular vesicles (tdEVs) in said sample and administering a therapy, wherein (i) if the amount of tdEVs is below a threshold measurement, the subject is administered a metastasis-directed therapy (MDT) only; or (ii) if the amount of tdEVs is above a threshold measurement, the subject is administered a systemic therapy and optionally also a MDT.

2. The method of claim 1 , wherein the threshold measurement is approximately 2x106EVs per ml.

3. The method of claim 1 or 2, wherein the prostate cancer is selected from the group consisting of castration-sensitive prostate cancer (CSPC), oligometastatic prostate cancer (omPC), oligometastatic castration-sensitive prostate cancer (omCSPC), metastatic castration resistant prostate cancer (CRPC), and clinically organ -confined prostate cancer.

4. The method of any of the preceding claims, wherein the sample is obtained from the subject prior to receiving a therapy.

5. The method of any of the preceding claims, wherein prior to obtaining the sample, the subject received one or more of a metastasis-directed therapy (MDT) or a systemic therapy or a combination thereof.

6. The method of any of the preceding claims, wherein the MDT is stereotactic ablative radiation therapy (SABR) or radiotherapy or a combination of.

7. The method of any of the preceding claims, wherein the sample is selected from the group consisting of a blood sample, a plasma sample, a serum sample, and a urine sample.

8. The method of any of the preceding claims, wherein the tdEVs are determined or measured using a technique selected from the group consisting of flow cytometry, high resolution flow cytometry, an enzyme-linked immunosorbent assay (ELISA), mass spectrometry, droplet-based vesicle EV analysis, proximity-extension assay, radioanalytical assay.

9. The method of claim 7, wherein the wherein the tdEVs are determined or measured using high resolution flow cytometry.

10. The method of any of the preceding claims, wherein a tdEV measurement in plasma is indicative of tumor burden and / or micrometastatic disease in the subject.11 . The method of any of the preceding claims, wherein the tdEVs comprise one or more surface markers selected from the group consisting of PSMA, STEAP1 , PDL1 , B7-H3, CEACAM5, CD56, DLL3, SYP, GPC3, ISM1 , CELSR3 and HMMR.

12. The method of any of the preceding claims, wherein the tdEVs are PSMA-positive extracellular vesicles (PSMA+EV).

13. The method of any of the preceding claims, wherein the one or more therapies are selected from the group consisting of metastasis-directed therapy (MDT), systemic therapy, surgery, radiation therapy, cryotherapy, high-intensity focused ultrasound (HIFU), transurethral ultrasound ablation (TULSA), focal laser ablation (FLA), photodynamic therapy (PDT), hormone therapy, chemotherapy, immunotherapy, targeted drug therapy, cryotherapy (cryoablation), and radiofrequency ablation.

14. The method of any of the preceding claims, wherein the therapy is a metastasis- directed therapy (MDT) or a combination therapy comprising MDT and systemic therapy.

15. The method of claim 13, wherein the MDT is selected from the group consisting of radiotherapy and surgery.

16. The method of any of claim 14, wherein the radiotherapy is selected from the group consisting of stereotactic ablative radiation therapy (SABR), external beam radiotherapy, hypofractionated radiotherapy, proton particle therapy, unsealed radionucleotide therapy, brachytherapy, and intensity modulated radiotherapy.

17. The method of any of the preceding claims, wherein the systemic therapy is selected from the group consisting of hormone therapy, luteinizing hormone-releasing hormone agonist / antagonist therapy, androgen-receptor targeting agent-based therapy, immunotherapy, PARP inhibitor-based therapy, chemotherapy, second generation androgen inhibitor-based therapy, and theranostic (radionucleotide) therapy.

18. The method of any of the preceding claims, wherein the therapy does not include androgen deprivation therapy (ADT).

19. The method of any of the preceding claims, wherein the subject received one or more therapies and the administrating step (c) includes continuing or intensifying the previously received therapies.

20. The method of any of the preceding claims, wherein the one or more therapies include a checkpoint inhibitor.21 . The method of claim 19, wherein the checkpoint inhibitor is selected from an inhibitor of CTLA-4, 4-1 BB (CD137), 4-1 BBL (CD137L), PDL1 , PDL2, PD1 , B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, BTLA, SIGLEC9, and 2B4.

22. The method of claim 20, wherein the checkpoint inhibitor is an inhibitor of PDL1 and / or an inhibitor of B7-H3.

23. The method of any of the preceding claims, further comprising the step of determining disease progression in the subject.

24. The method of claim 23, wherein disease progression is measured by biochemical progression (bPFS) and / or radiographic progression (rPFS).

25. A method of detecting PSMA+EVs in a sample from a subject, said method comprising(a) obtaining a sample from the subject;(b) determining the presence of and / or the amount of tumor-derived extracellular vesicles (tdEVs) in said sample comprising using a technique selected from the group consisting of flow cytometry, high resolution flow cytometry, an enzyme-linked immunosorbent assay (ELISA), mass spectrometry, droplet-based vesicle EV analysis, proximity-extension assay, and radioanalytical assay.

26. The method of claim 24, wherein the wherein the tdEVs are determined or measured using high resolution flow cytometry.

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