Safety and activity of RRx-001 in patients with advanced cancer: a first-in-human, open-label, dose-escalation phase 1 study.

BACKGROUND: Epigenetic alterations have been strongly associated with tumour formation and resistance to chemotherapeutic drugs, and epigenetic modifications are an attractive target in cancer research. RRx-001 is activated by hypoxia and induces the generation of reactive oxygen and nitrogen species that can epigenetically modulate DNA methylation, histone deacetylation, and lysine demethylation. The aim of this phase 1 study was to assess the safety, tolerability, and pharmacokinetics of RRx-001. METHODS: In this open-label, dose-escalation, phase 1 study, we recruited adult patients (aged >18 years) with histologically or cytologically confirmed diagnosis of advanced, malignant, incurable solid tumours from University of California at San Diego, CA, USA, and Sarah Cannon Research Institute, Nashville, TN, USA. Key eligibility criteria included evaluable disease, Eastern Cooperative Group performance status of 2 or less, an estimated life expectancy of at least 12 weeks, adequate laboratory parameters, discontinuation of all previous antineoplastic therapies at least 6 weeks before intervention, and no residual side-effects from previous therapies. Patients were assigned to receive intravenous infusions of RRx-001 at increasing doses (10 mg/m(2), 16·7 mg/m(2), 24·6 mg/m(2), 33 mg/m(2), 55 mg/m(2), and 83 mg/m(2)) either once or twice-weekly for at least 4 weeks, with at least three patients per dose cohort and allowing a 2-week observation period before dose escalation. Samples for safety and pharmacokinetics analysis, including standard chemistry and haematological panels, were taken on each treatment day. The primary objective was to assess safety, tolerability, and dose-limiting toxic effects of RRx-001, to determine single-dose pharmacokinetics, and to identify a recommended dose for phase 2 trials. All analyses were done per protocol. Accrual is complete and follow-up is still on-going. This trial is registered with ClinicalTrials.gov, number NCT01359982. FINDINGS: Between Oct 10, 2011, and March 18, 2013, we enrolled 25 patients and treated six patients in the 10 mg/m(2) cohort, three patients in the 16·7 mg/m(2) cohort, three patients in the 24·6 mg/m(2) cohort, four patients in the 33 mg/m(2) cohort, three patients in the 55 mg/m(2), and six patients in the 83 mg/m(2) cohort. Pain at the injection site, mostly grade 1 and grade 2, was the most common adverse event related to treatment, experienced by 21 (84%) patients. Other common drug-related adverse events included arm swelling or oedema (eight [32%] patients), and vein hardening (seven [28%] patients). No dose-limiting toxicities were observed. Time constraints related to management of infusion pain from RRx-001 resulted in a maximally feasible dose of 83 mg/m(2). Of the 21 evaluable patients, one (5%) patient had a partial response, 14 (67%) patients had stable disease, and six (29%) patients had progressive disease; all responses were across a variety of tumour types. Four patients who had received RRx-001 were subsequently rechallenged with a treatment that they had become refractory to; all four responded to the rechallenge. INTERPRETATION: RRx-001 is a well-tolerated novel compound without clinically significant toxic effects at the tested doses. Preliminary evidence of activity is promising and, on the basis of all findings, a dose of 16·7 mg/m(2) was recommended as the targeted dose for phase 2 trials. FUNDING: EpicentRx (formerly RadioRx).

Novel nitric oxide generating compound glycidyl nitrate enhances the therapeutic efficacy of chemotherapy and radiotherapy.

Selective release of nitric oxide (NO) in tumors could improve the tumor blood flow and drug delivery for chemotherapeutic agents and radiotherapy, thereby increasing the therapeutic index. Glycidyl nitrate (GLYN) is a NO generating small molecule, and has ability to release NO on bioactivation in SCC VII tumor cells. GLYN-induced intracellular NO generation was significantly attenuated by NO scavenger carboxy-PTIO (cPTIO) and NAC. GLYN significantly increases tumor blood flow, but has no effect on the blood flow of normal tissues in tumor-bearing mice. When used with cisplatin, GLYN significantly increased the tumor growth inhibition effect of cisplatin. GLYN also had a modest radiosensitizing effect in vitro and in vivo. GLYN was well tolerated and there were no acute toxicities found at its effective therapeutic doses in preclinical studies. These results suggest that GLYN is a promising new drug for use with chemotherapy and radiotherapy, and provide a compelling rationale for future studies of GLYN and related compounds.

Characterization of direct radiation-induced immune function and molecular signaling changes in an antigen presenting cell line.

Radiation therapy is a widely used cancer treatment and pre-transplantation conditioning regimen that has the potential to influence anti-tumor and post-transplantation immune responses. Although conventionally fractionated radiation doses can suppress immune responses by depleting lymphocytes, single high doses of local tumor radiation can enhance immune responses. Using phospho-flow cytometry analysis of a human monocytic cell line, we identified novel radiation-induced changes in the phosphorylation state of NFκB family members known in other cell types to maintain and regulate immune function. These phosphorylation changes were p53 independent, but were strongly dependent upon ATM activation due to DNA damage. We found that radiation promotes the activation and APC functional maturation through phosphorylation of NFκB Essential Modulator (NEMO). Our results and the analytic methods are especially well suited to the study of functional changes in APC when radiation is used for immune modulation in clinical protocols.

ROCKET: Phase II Randomized, Active-controlled, Multicenter Trial to Assess the Safety and Efficacy of RRx-001 + Irinotecan vs. Single-agent Regorafenib in Third/Fourth Line Colorectal Cancer.

INTRODUCTION: RRx-001 is a novel cysteine-targeted alkylating agent that releases nitric oxide (NO). The primary biological activities of this hybrid molecule include macrophage repolarizing and vascular normalization. The purpose of this clinical trial (ROCKET) (NCT02096354) was to compare the safety and efficacy of the combination therapy RRx-001 + irinotecan vs. regorafenib in third/fourth line colorectal cancer that previously received treatment with irinotecan. PATIENTS AND METHODS: A total of 34 patients were randomized (24 to RRx-001 + irinotecan (RxI) and 10 to single-agent regorafenib (RegI)) and were the basis for the intention-to-treat analysis (ITT, comprising all 34 patients). RRx-001 treatment was administered as an up-to-2-month "primer" followed by irinotecan for patients randomized to the RRx-001 arm (24). The efficacy and safety data are presented for the 34 patients in the (ITT) efficacy analysis. Therapy consisted of intravenous administration of RRx-001 at 4 mg once weekly for up to 2 months, at which point RRx-001 was discontinued, followed by intravenous infusion of irinotecan at 180 mg/m2 on day 1 in a 21-day cycle vs. 160 mg oral regorafenib daily for 3/4 weeks followed at progression, if applicable, by irinotecan 180 mg/m2 on day 1 in a 21-day cycle. There were 3 patients (3/24 = 12.5%) with prior single agent irinotecan on the RRx-001 randomized arm and 2 (2/10 = 20%) on the regorafenib randomized arm. Numerous patients had irinotecan combination therapies prior to randomized treatment. There were 15 patients on RRx-001 arm that received irinotecan post-RRx-001 in the randomized trial. There were 5 PRs on RRx-001 plus irinotecan leading to an overall response of 20.8% (5/24). There were 37.5% (9/24) of RRx-001 randomized patients with KRAS mutant type while 60% (6/10) regorafenib randomized patients were of KRAS type mutant. There were only 4 patients with available QOL and Edmonton Symptom Assessment System, an insufficient sample size to allow for any meaningful analysis. RESULTS: Median patient follow-up was approximately 14.5 months (SD 4.5 months). Median overall survival was 8.6 months for RxI and 4.7 months for RegI. Median progression free survival was 6.1 months for RxI vs. 1.7 months for RegI (a statistically significant result, 2-sided log-rank test, P = .0030). The toxicity profile of RxI was substantially improved compared with RegI. CONCLUSION: The results of this trial demonstrate improved efficacy of RxI compared with RegI in patients with metastatic colorectal cancer after previous treatment with irinotecan, and late-stage clinical development in this indication is planned on the strength of the observed "signal" accompanied by a sufficient safety profile.

ASTRO's Framework for Radiopharmaceutical Therapy Curriculum Development for Trainees.

In 2017, the American Society for Radiation Oncology (ASTRO) board of directors prioritized radiopharmaceutical therapy (RPT) as a leading area for new therapeutic development, and the ASTRO RPT workgroup was created. Herein, the workgroup has developed a framework for RPT curriculum development upon which education leaders can build to integrate this modality into radiation oncology resident education. Through this effort, the workgroup aims to provide a guide to ensure robust training in an emerging therapeutic area within the context of existing radiation oncology training in radiation biology, medical physics, and clinical radiation oncology. The framework first determines the core RPT knowledge required to select patients, prescribe, safely administer, and manage related adverse events. Then, it defines the most important topics for preparing residents for clinical RPT planning and delivery. This framework is designed as a tool to supplement the current training that exists for radiation oncology residents. The final document was approved by the ASTRO board of directors in the fall of 2021.

Clinical Pharmacokinetics of Oral Sodium Selenite and Dosing Implications in the Treatment of Patients with Metastatic Cancer.

BACKGROUND: Selenite is a radiosensitizer and inhibitor of androgen receptor expression and function. In a phase I study (NCT02184533) in 15 subjects with metastatic cancer receiving daily oral sodium selenite with palliative radiation therapy, disease stabilization was observed, as evidenced by tumor regression, marked reduction in pain symptoms, and decreased prostate-specific antigen levels (only patients with castrate-resistant prostate cancer). OBJECTIVE: The aim of this work was to characterize the pharmacokinetics of selenite to suggest dosing strategies and to propose a study design for further investigation. METHODS: With selenium plasma concentrations obtained from five dosing cohorts (5.5, 11, 16.5, 33, and 49.5 mg), a population pharmacokinetic model was constructed using NONMEM. The model described externally administered selenite (inorganic) with a baseline component for endogenous selenium levels. Using the pharmacokinetic model, simulations were performed to suggest dosing regimens that achieved in vitro target selenite levels, and optimal pharmacokinetic sampling times for a subsequent study were proposed using PopED. RESULTS: A one-compartment model characterized selenite pharmacokinetics. Parameter estimates were absorption rate constant (0.64 h-1), apparent clearance (1.58 L/h), apparent volume of distribution (42.3 L), and baseline selenium amount (5270 µg). A logarithmic function characterized the inverse relationship between dose level and bioavailability. Four regimens to reach in vitro target selenite levels were proposed: 33 mg daily, 16.5 mg twice daily (BID), 11 mg BID, and 5.5 mg thrice daily (TID). Optimal sampling times were 1, 2, 6, and 24 h. DISCUSSION: The population model described the pharmacokinetic data well. Three regimens (33 mg daily, 11 mg BID, 5.5 mg TID) achieved in vitro target selenite levels after one dose. The model and optimal sampling times may inform future studies evaluating the efficacy of selenite for metastatic cancer treatment.

The Radiobiology of Radiopharmaceuticals.

Radiopharmaceutical therapy or targeted radionuclide therapy (TRT) is a well-established class of cancer therapeutics that includes a growing number of FDA-approved drugs and a promising pipeline of experimental therapeutics. Radiobiology is fundamental to a mechanistic understanding of the therapeutic capacity of these agents and their potential toxicities. However, the field of radiobiology has historically focused on external beam radiation. Critical differences exist between TRT and external beam radiotherapy with respect to dosimetry, dose rate, linear energy transfer, duration of treatment delivery, fractionation, range, and target volume. These distinctions simultaneously make it difficult to extrapolate from the radiobiology of external beam radiation to that of TRT and pose considerable challenges for preclinical and clinical studies investigating TRT. Here, we discuss these challenges and explore the current understanding of the radiobiology of radiopharmaceuticals.

Harnessing genome-wide association studies to minimize adverse radiation-induced side effects.

Radiotherapy is used as definitive treatment in approximately two-thirds of all cancers. However, like any treatment, radiation has significant acute and long-term side effects including secondary malignancies. Even when similar radiation parameters are used, 5%-10% of patients will experience adverse radiation side effects. Genomic susceptibility is thought to be responsible for approximately 40% of the clinical variability observed. In the era of precision medicine, the link between genetic susceptibility and radiation-induced side effects is further strengthening. Genome-wide association studies (GWAS) have begun to identify single-nucleotide polymorphisms (SNPs) attributed to overall and tissue-specific toxicity following radiation for treatment of breast cancer, prostate cancer, and other cancers. Here, we review the use of GWAS in identifying polymorphisms that are predictive of acute and long-term radiation-induced side effects with a focus on chest, pelvic, and head-and-neck irradiation. Integration of GWAS studies with "omic" data, patient characteristics, and clinical correlates into predictive models could decrease radiation-induced side effects while increasing therapeutic efficacy.

Locally advanced rectal cancer: The past, present, and future.

From a series of clinical trials in the last several decades, current treatment paradigms for locally advanced rectal cancer include: (1) preoperative long-course radiotherapy (RT) combined with radiosensitizing chemotherapy; (2) preoperative short-course RT alone followed by adjuvant postoperative chemotherapy; and (3) total neoadjuvant therapy with induction chemotherapy followed by chemoradiotherapy. Other strategies under active investigation in both institutional and cooperative trials include neoadjuvant chemotherapy alone without RT in select patients, total neoadjuvant therapy, watchful waiting after a clinical complete response as an alternative to surgical resection, and the use of different chemotherapeutic and targeted agents. The focus of this review is on established and novel therapeutic strategies for locally advanced rectal cancer.

A Review of Newly Diagnosed Glioblastoma.

Glioblastoma is an aggressive and inevitably recurrent primary intra-axial brain tumor with a dismal prognosis. The current mainstay of treatment involves maximally safe surgical resection followed by radiotherapy over a 6-week period with concomitant temozolomide chemotherapy followed by temozolomide maintenance. This review provides a summary of the epidemiological, clinical, histologic and genetic characteristics of newly diagnosed disease as well as the current standard of care and potential future therapeutic prospects.

Just eat it: A review of CD47 and SIRP-α antagonism.

The mammalian immune system consists of two distinct arms, nonspecific innate and more specific adaptive, with the innate immune response as the first line of defense and protection, which primes and amplifies subsequent adaptive responses. On the basis of this binary immune interplay, stimulation of T cells through checkpoint inhibitors (CIs), which bypasses innate involvement, seems likely to engender suboptimal or incomplete anticancer immunity, given that the successful induction of effect or responses depends on two-way innate/adaptive coordination. Indeed, the majority of patients-70%-80%, do not respond to CIs, which is potentially problematic if access to more optimal standard therapies is withheld or delayed in favor of ineffective or only marginally effective anti-PD-1/PD-L1 treatment. Therefore, stimulation of the innate immune response in combination with CIs (or other inducers of T cell cytotoxicity) has the potential to make the immune system "whole" and thereby to enhance and broaden the anti-tumor activity of PD-1/PD-L1 inhibitors for example, in relatively nonimmunogenic or "cold" tumor types. A critical innate macrophage immune checkpoint and druggable target is the antiphagocytic and "marker of self" CD47-SIRPα pathway, which is co-opted by cancer cells to mediate escape from immune-mediated clearance and checkpoint inhibition. This review summarizes the status of key CD47 antagonists in clinical trials, including the biologics, Hu5F9-G4 (5F9), TTI-621, and ALX148, as well as the small molecule, RRx-001, now in a Phase 3 clinical trial, which has not been previously included in CD47-SIRPα reviews focused on biologics. Hu5F9-G4 (5F9), TTI-621, ALX148, and RRx-001 are chosen as compounds with potentially promising data that have advanced the farthest in clinical development.

Locally administered heparin-binding epidermal growth factor-like growth factor reduces radiation-induced oral mucositis in mice.

Oral mucositis refers to lesions of the oral mucosa observed in patients with cancer being treated with radiation with or without chemotherapy, and can significantly affect quality of life. There is a large unmet medical need to prevent oral mucositis that can occur with radiation either alone or in combination with chemotherapy. We investigated the efficacy of locally administered heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent epithelial proliferation and migration stimulator of the oral mucosa as a potential therapy to prevent radiation induced oral mucositis. Using a single dose (20 Gy) of radiation to the oral cavity of female C57BL/6 J mice, we evaluated the efficacy of HB-EGF treatment (5 µl of 10 µg/ml) solution. The results show that HB-EGF delivered post radiation, significantly increased the area of epithelial thickness on the tongue (dorsal tongue (42,106 vs 53,493 µm2, p < 0.01), ventral tongue (30,793 vs 39,095 µm2, *p < 0.05)) compared to vehicle control, enhanced new epithelial cell division, and increased the quality and quantity of desmosomes in the oral mucosa measured in the tongue and buccal mucosa. This data provides the proof of concept that local administration of HB-EGF has the potential to be developed as a topical treatment to mitigate oral mucositis following radiation.

Durability of response in metastatic melanoma patients after combined treatment with radiation therapy and ipilimumab.

AIM: We previously reported a prospective trial evaluating the safety and efficacy of combining ipilimumab and radiation therapy in patients with metastatic melanoma. Herein, we provide a long-term update on patients with complete response (CR) or partial response (PR). PATIENTS & METHODS: We continued to follow these patients with serial imaging including computed tomography, PET or MRI. RESULTS: Two of the three patients with CR are still alive and without evidence of melanoma but with chronic treatment-induced hypophysitis. The third patient died of hepatocellular carcinoma, but with no evidence of melanoma. Among the three patients with PR, two achieved CR after pembrolizumab monotherapy. CONCLUSION: This long-term follow up reveals the striking durability of the CRs, which appears to correlate with a grade 2-3 hypophysitis.

TGF-beta: a master immune regulator.

Introduction: Transforming Growth Factor-Beta (TGF-ß) is a master regulator of numerous cellular functions including cellular immunity. In cancer, TGF-ß can function as a tumor promoter via several mechanisms including immunosuppression. Since the immune checkpoint pathways are co-opted in cancer to induce T cell tolerance, this review posits that TGF-ß is a master checkpoint in cancer, whose negative regulatory influence overrides and controls that of other immune checkpoints.Areas Covered: This review examines therapeutic agents that target TGF-ß and its signaling pathways for the treatment of cancer which may be classifiable as checkpoint inhibitors in the broadest sense. This concept is supported by the observations that 1) only a subset of patients benefit from current checkpoint inhibitor therapies, 2) the presence of TGF-ß in the tumor microenvironment is associated with excluded or cold tumors, and resistance to checkpoint inhibitors, and 3) existing biomarkers such as PD-1, PD-L1, microsatellite instability and tumor mutational burden are inadequate to reliably and adequately identify immuno-responsive patients. By contrast, TGF-ß overexpression is a widespread and profoundly negative molecular hallmark in multiple tumor types.Expert Opinion: TGF-ß status may serve as a biomarker to predict responsiveness and as a therapeutic target to increase the activity of immunotherapies.

Development of a biodosimeter for radiation triage using novel blood protein biomarker panels in humans and non-human primates.

Purpose: In a significant nuclear event, hundreds of thousands of individuals will require rapid triage for absorbed radiation to ensure effective medical treatment and efficient use of medical resources. We are developing a rapid screening method to assess whether an individual received an absorbed dose of ≥2 Gy based on the analysis of a specific panel of blood proteins in a fingerstick blood sample.Materials and methods: We studied a data set of 1051 human blood samples obtained from radiotherapy patients, normal healthy individuals, and several special population groups. We compared the findings in humans with those from irradiation studies in non-human primates (NHPs).Results: We identified a panel of three protein biomarkers, salivary alpha amylase (AMY1), Flt3 ligand (FLT3L), and monocyte chemotactic protein 1 (MCP1), which are upregulated in human patients receiving fractionated doses of total body irradiation (TBI) therapy as a treatment for cancer. These proteins exhibited a similar radiation response in NHPs after single acute or fractionated doses of ionizing radiation.Conclusion: Our work provides confidence in this biomarker panel for biodosimetry triage using fingerstick blood samples and in the use of NHPs as a model for irradiated humans.

A Prospective, Phase 1 Trial of Nivolumab, Ipilimumab, and Radiotherapy in Patients with Advanced Melanoma.

PURPOSE: Preclinical data suggest that radiotherapy (RT) is beneficial in combination with immune checkpoint blockade. Clinical trials have explored RT with single-agent immune checkpoint blockade, but no trials have reported RT with the combination of nivolumab and ipilimumab. PATIENTS AND METHODS: We conducted a phase 1 study of patients with stage IV melanoma receiving nivolumab and ipilimumab with two different dose-fractionation schemes of RT. Patients had at least one melanoma metastasis that would benefit from palliative RT and one metastasis that would not be irradiated. Nivolumab 1 mg/kg + ipilimumab 3 mg/kg and extracranial RT with a dose of 30 Gy in 10 fractions was administered in Cohort A, and then 27 Gy in 3 fractions was administered in Cohort B. The primary outcome was safety. RESULTS: Twenty patients were treated (10 in each cohort). The rates of treatment-related grade 3-4 adverse events in Cohort A and B were 40% and 30%, respectively. There were no grade ≥3 adverse events attributed to RT. Patients responded to treatment outside of the irradiated volume (Cohort A 5/10; Cohort B 1/9). No evaluable patients had progression of irradiated metastases. Immunologic changes were seen in the peripheral blood with increases in T-cell receptor diversity in some responding patients. CONCLUSIONS: RT with nivolumab and ipilimumab was safe compared with historical data of nivolumab and ipilimumab alone. Immunologic effects were observed in the peripheral blood. Randomized studies are ongoing to assess whether RT increases the efficacy of nivolumab and ipilimumab.

Anti-integrin monoclonal antibody CNTO 95 enhances the therapeutic efficacy of fractionated radiation therapy in vivo.

Selective targeting of up-regulated integrins on tumor cells is a novel antiangiogenesis strategy for treating solid tumors. CNTO 95 is a fully human anti-alpha(v) integrin monoclonal antibody and has shown antitumor activity when used as a single agent in preclinical studies. We previously showed that radiation combined with an integrin alpha(v)beta(3) antagonist cRGD peptide increased the therapeutic efficacy of radiation in preclinical tumor models. We hypothesized that the combination of radiation and CNTO 95 would synergistically enhance the efficacy of radiation therapy. The in vitro studies showed that CNTO 95 radiosensitized and induced apoptosis in M21 cells in vitronectin-coated dishes. In mice bearing established human cancer xenograft tumors, CNTO 95 alone had only a moderate effect on tumor growth. The combined therapy of CNTO 95 and fractionated radiation significantly inhibited tumor growth and produced the longer tumor growth delay time in multiple tumor models. Maintenance dosing of CNTO 95 following irradiation contributed to efficacy and was important for continued inhibition of tumor regrowth. Immunohistochemistry studies showed that the combined use of CNTO 95 and radiation reduced the alpha(v) integrin and vascular endothelial growth factor receptor expression and the microvessel density and increased apoptosis in tumor cells and the tumor microenvironment. CNTO 95 alone and in combination with radiation did not produce any obvious signs of systemic toxicity. These results show that CNTO 95 can potentiate the efficacy of fractionated radiation therapy in a variety of human cancer xenograft tumor types in nude mice. These findings are very promising and may have high translational relevance for the treatment of patients with solid tumors.

Results from a Phase 1 Study of Sodium Selenite in Combination with Palliative Radiation Therapy in Patients with Metastatic Cancer.

In preclinical studies, selenite had single agent activity and radiosensitized tumors in vivo. Here we report results from a Phase 1 trial in 15 patients with metastatic cancer treated with selenite (5.5 to 49.5 mg) orally as a single dose 2 hours before each radiation therapy (RT) treatment. Patients received RT regimens that were standard of care. The primary objective of the study was to assess the safety of this combination therapy. Secondary objectives included measurement of pharmacokinetics (PK) and evaluation of efficacy. Endpoints included assessment of PK, toxicity, tumor response, and pain before and after treatment. The half-life of selenite was 18.5 hours. There were no adverse events attributable to selenite until the 33 mg dose level, at which the primary toxicities were grade 1 GI side effects. One patient treated with 49.5 mg had grade 2 GI toxicity. Although this was not a DLT, it was felt that the highest acceptable dose in this patient population was 33 mg. Most patients had stabilization of disease within the RT fields, with some demonstrating objective evidence of tumor regression. Most patients had a marked improvement in pain and seven out of nine patients with prostate cancer had a decrease in PSA ranging from 11-78%. Doses up to 33 mg selenite were well tolerated in combination with RT. A randomized, well controlled study is needed at the 33 mg dose level to determine if selenite results in clinically meaningful improvements in the response to palliative RT.

High-fidelity detection and sorting of nanoscale vesicles in viral disease and cancer.

Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to many fields of medicine as biomarkers and mediators of or treatments for disease. However, exosomes and small viruses fall below the detection limits of conventional flow cytometers due to the overlap of particle-associated scattered light signals with the detection of background instrument noise from diffusely scattered light. To identify, sort, and study distinct subsets of EVs and other nanoparticles, as individual particles, we developed nanoscale Fluorescence Analysis and Cytometric Sorting (nanoFACS) methods to maximise information and material that can be obtained with high speed, high resolution flow cytometers. This nanoFACS method requires analysis of the instrument background noise (herein defined as the "reference noise"). With these methods, we demonstrate detection of tumour cell-derived EVs with specific tumour antigens using both fluorescence and scattered light parameters. We further validated the performance of nanoFACS by sorting two distinct HIV strains to >95% purity and confirmed the viability (infectivity) and molecular specificity (specific cell tropism) of biological nanomaterials sorted with nanoFACS. This nanoFACS method provides a unique way to analyse and sort functional EV- and viral-subsets with preservation of vesicular structure, surface protein specificity and RNA cargo activity.