Enhancing circulatory myokines and extracellular vesicle uptake with targeted exercise in patients with prostate cancer (the MYEX trial): a single-group crossover study.

INTRODUCTION: Physical activity is associated with improved disease progression and cancer-specific survival in patients with prostate cancer (PCa). However, the mechanisms underlying these associations remain unclear, while the relative impact of exercise modes is unknown. This study aims to examine the differential impact of exercise mode on tumour-suppressive skeletal muscle-associated systemic molecules as well as their delivery mechanism. This study will compare the effects of the two main exercise modes, aerobic and resistance, on (1) circulatory myokine levels, (2) skeletal muscle-induced extracellular vesicle abundance and cargo contents, and (3) uptake of extracellular vesicles (EVs) in PCa cells in patients with localised or advanced PCa. METHODS: A single-group cross-over design will be used for patients at opposite ends of the disease spectrum. A total of 32 patients (localised PCa, n = 16; metastatic castrate-resistant PCa, n = 16) will be recruited while capitalising on two ongoing studies. Ethics amendment has been approved for two ongoing trials to share data, implement the acute exercise sessions, and collect additional blood samples from patients. The patients will undertake two exercise sessions (aerobic only and resistance only) in random order one week apart. Blood will be collected before, after, and 30 min post-exercise. Circulating/EV-contained myokine levels (irisin, IL-6, IL-15, FGF-21, and SPARC) and plasma skeletal muscle-induced EVs will be measured using ELISA and flow cytometry. PCa cell line growth with or without collected plasma will be examined using PCa cell lines (LNCaP, DU-145, and PC-3), while evaluating cellular uptake of EVs. Ethics amendments have been approved for two capitalising studies to share data, implement acute exercise sessions and collect additional samples from the patients. DISCUSSION: If findings show a differential impact of exercise mode on the establishment of an anti-cancer systemic environment, this will provide fundamental knowledge for developing targeted exercise prescriptions for patients with PCa across different disease stages. Findings will be reported in peer-reviewed publications and scientific conferences, in addition to working with national support groups to translate findings for the broader community. TRIAL REGISTRATION: The registration for the two capitalising studies are NCT02730338 and ACTRN12618000225213.

Spatial transcriptomics reveals discrete tumour microenvironments and autocrine loops within ovarian cancer subclones.

High-grade serous ovarian carcinoma (HGSOC) is genetically unstable and characterised by the presence of subclones with distinct genotypes. Intratumoural heterogeneity is linked to recurrence, chemotherapy resistance, and poor prognosis. Here, we use spatial transcriptomics to identify HGSOC subclones and study their association with infiltrating cell populations. Visium spatial transcriptomics reveals multiple tumour subclones with different copy number alterations present within individual tumour sections. These subclones differentially express various ligands and receptors and are predicted to differentially associate with different stromal and immune cell populations. In one sample, CosMx single molecule imaging reveals subclones differentially associating with immune cell populations, fibroblasts, and endothelial cells. Cell-to-cell communication analysis identifies subclone-specific signalling to stromal and immune cells and multiple subclone-specific autocrine loops. Our study highlights the high degree of subclonal heterogeneity in HGSOC and suggests that subclone-specific ligand and receptor expression patterns likely modulate how HGSOC cells interact with their local microenvironment.

Meta-Analysis of Circulating Tumor Cell PD-L1 Expression and the Association with Clinical Outcomes in Non-Small Cell Lung Cancer.

BACKGROUND: Programmed death ligand-1 (PD-L1) expression on circulating tumor cells (CTCs) has been suggested to provide prognostic information in non-small cell lung cancer (NSCLC), but consensus relative to treatment outcomes is lacking. We conducted the first comprehensive meta-analysis exploring its potential as a prognostic and predictive marker, and assessed the concordance between PD-L1 + CTCs and paired tumor tissue in NSCLC patients. METHOD: A comprehensive search was applied to PubMed and EMBASE to identify 26 studies that evaluated PD-L1 + CTCs and their association with survival outcomes in 1236 NSCLC patients. RESULTS: The meta-analysis estimated a mean PD-L1 + CTCs detection rate of 61% (95% CI, 49-72). Subgroup analysis based on treatment showed that PD-L1 + CTCs was not significantly associated with better overall survival (OS) in NSCLC patients treated with immune checkpoint inhibitors (ICIs) (Hazard Ratio (HR) = 0.96, 95% CI, 0.35-2.65, P = 0.944), but was predictive of worse OS in those treated with other therapies (HR = 2.11, 95% CI, 1.32-3.36, P = 0.002). Similarly, PD-L1 + CTCs was not significantly associated with superior progressing free survival (PFS) in NSCLCs treated with ICIs (HR = 0.67, 95% CI, 0.41-1.09, P = 0.121), but was significantly associated with shorter PFS in patients treated with other therapies (HR = 1.91, 95% CI, 1.24-2.94, P = 0.001). The overall estimate for the concordance between PD-L1 expression on CTCs and tumor cells was 63% (95% CI, 44-80). CONCLUSION: The average detection rate of PD-L1 + CTCs was comparable to the rate of PD-L1 expression in NSCLC tumors. There was a trend towards better PFS in ICI-treated NSCLC patients with PD-L1 + CTCs. Larger longitudinal studies on the association of PD-L1 + CTCs with clinical outcomes in NSCLC patients treated with ICIs are warranted.

Incidence and Mortality of Conjunctival Melanoma in Australia (1982 to 2014).

Purpose: The purpose of this study was to estimate the incidence and mortality of conjunctival melanoma in Australia from 1982 to 2014. Methods: De-identified unit data for all cases of ocular melanoma were extracted from the Australian Cancer Database from 1982 to 2014. Conjunctival melanoma cases were extracted, and the incidence and mortality were analyzed. Incidence rates were age-standardized against the 2001 Australian Standard Population. Mortality was assessed using log-rank and Cox regression. Results: From 1982 to 2014, there were 299 cases of conjunctival melanoma. The age-standardized incidence rate was 0.48 (95% confidence interval [CI] = 0.41 to 0.54) per million per year. Women (0.52, 95% CI = 0.42 to 0.62) had a higher incidence than men (0.42, 95% CI = 0.33 to 0.51). The incidence of conjunctival melanoma increased in men (+1.46%) and significantly women (+1.41%, P = 0.023) over the study period. The mean 5-, 10-, and 15-year disease-specific survival were 90%, 82%, and 80%, respectively, during the 33-year interval. Comparisons of survival among age, sex, and state revealed no significant differences when tested using log-rank or Cox regression. Conclusions: In conclusion, we found an increase in the rate of conjunctival melanoma diagnoses in Australia from 1982 to 2014. Over the same period, disease survival remained unchanged at a mean of 90%.

Detection of metastases using circulating tumour DNA in uveal melanoma.

BACKGROUND: Approximately 50% of uveal melanoma (UM) patients will develop metastatic disease depending on the genetic features of the primary tumour. Patients need 3-12 monthly scans, depending on their prognosis, which is costly and often non-specific. Circulating tumour DNA (ctDNA) quantification could serve as a test to detect and monitor patients for early signs of metastasis and therapeutic response. METHODS: We assessed ctDNA as a biomarker in three distinct UM cohorts using droplet-digital PCR: (A) a retrospective analysis of primary UM patients to predict metastases; (B) a prospective analysis of UM patients after resolution of their primary tumour for early detection of metastases; and (C) monitoring treatment response in metastatic UM patients. RESULTS: Cohort A: ctDNA levels were not associated with the development of metastases. Cohort B: ctDNA was detected in 17/25 (68%) with radiological diagnosis of metastases. ctDNA was the strongest predictor of overall survival in a multivariate analysis (HR = 15.8, 95% CI 1.7-151.2, p = 0.017). Cohort C: ctDNA monitoring of patients undergoing immunotherapy revealed a reduction in the levels of ctDNA in patients with combination immunotherapy. CONCLUSIONS: Our proof-of-concept study shows the biomarker feasibility potential of ctDNA monitoring in for the clinical management of uveal melanoma patients.

Single-nucleus RNA sequencing of pre-malignant liver reveals disease-associated hepatocyte state with HCC prognostic potential.

Current approaches to staging chronic liver diseases have limited utility for predicting liver cancer risk. Here, we employed single-nucleus RNA sequencing (snRNA-seq) to characterize the cellular microenvironment of healthy and pre-malignant livers using two distinct mouse models. Downstream analyses unraveled a previously uncharacterized disease-associated hepatocyte (daHep) transcriptional state. These cells were absent in healthy livers but increasingly prevalent as chronic liver disease progressed. Copy number variation (CNV) analysis of microdissected tissue demonstrated that daHep-enriched regions are riddled with structural variants, suggesting these cells represent a pre-malignant intermediary. Integrated analysis of three recent human snRNA-seq datasets confirmed the presence of a similar phenotype in human chronic liver disease and further supported its enhanced mutational burden. Importantly, we show that high daHep levels precede carcinogenesis and predict a higher risk of hepatocellular carcinoma development. These findings may change the way chronic liver disease patients are staged, surveilled, and risk stratified.

Feasibility of supervised telehealth exercise for patients with advanced melanoma receiving checkpoint inhibitor therapy.

PURPOSE: To determine the feasibility, safety and preliminary efficacy of a telehealth supervised exercise programme in patients with advanced melanoma receiving checkpoint inhibitor therapy. METHODS: A 8-week non-randomised feasibility pilot trial utilising a telehealth delivered multimodal exercise programme undertaken thrice weekly with assessments at baseline and post-intervention. The study was considered feasible if there were no severe or life-threatening adverse events as a result of exercise, and three or more of the following criteria were met: the recruitment rate was >50%, completion rate was >80%, median programme attendance was >75%, median exercise compliance >75%, and average tolerance was >70%. Preliminary efficacy was assessed for objective measures of physical function (2-min step test, repeated chair stand test, 30-s push-up test, and a modified static balance test) and quality of life (QoL), fatigue and other patient-reported outcomes were assessed using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30. RESULTS: Eleven patients (32-80 years) were included in the study (6 female, 5 male). The recruitment rate was 48%, completion rate 91%, programme attendance 88%, median exercise compliance 82.1% and 84.9% for resistance and aerobic exercise, respectively, and tolerance 88%, with no severe or life-threatening adverse events as a result of exercise. In terms of preliminary efficacy, physical function significantly improved while QoL was maintained following the intervention. CONCLUSION: An 8-week telehealth exercise intervention is feasible and safe for patients with advanced melanoma and appears to improve physical function while preserving QoL during checkpoint inhibitor therapy.

Locally performed postoperative circulating tumour DNA testing performed during routine clinical care to predict recurrence of colorectal cancer.

BACKGROUND: Identifying patients at high risk for colorectal cancer recurrence is essential for improving prognosis. In the postoperative period, circulating tumour DNA (ctDNA) has been demonstrated as a significant prognostic indicator of recurrence. These results have been obtained under the strict rigours of clinical trials, but not validated in a real-world setting using in-house testing. We report the outcomes of locally performed postoperative ctDNA testing conducted during routine clinical care and the association with the recurrence of colorectal cancer. METHODS: We recruited 36 consecutive patients with newly diagnosed colorectal cancer between 2018 and 2020. Postoperative plasma samples were collected at the first outpatient review following resection. Tumour-informed ctDNA analysis was performed using droplet digital polymerase chain reaction or targeted next-generation sequencing. RESULTS: At the time of surgery, there were 24 patients (66.7%) with localized cancer, nine (25%) with nodal spread, and three (8.3%) with metastatic disease. The median time from surgery to plasma sample donation was 22 days (IQR 20-28 days). At least one somatic mutation was identified in primary tumour tissue for 28 (77.8%) patients. Postoperative ctDNA was detected in five patients (13.9%). The median duration of follow-up was 32.0 months (IQR 27.2-38.1 months). Two patients (5.56%) developed metastatic recurrence. However, neither had detectable postoperative ctDNA. There were no instances of loco-regional recurrence. CONCLUSION: Analysis of postoperative ctDNA testing can be performed locally, however this study did not reproduce the adverse association between detectable postoperative ctDNA and the development of colorectal cancer recurrence seen in clinical trials.

Genetic analysis of heterogeneous subsets of circulating tumour cells from high grade serous ovarian carcinoma patients.

Circulating tumour cells (CTCs) are heterogenous and contain genetic information from the tumour of origin. They bear specific intra- and extra-cellular protein markers aiding in their detection. However, since these markers may be shared with other rare cells in the blood, only genetic testing can confirm their malignancy. Herein, we analyse different CTC subsets using single cell whole genome DNA sequencing to validate their malignant origin. We randomly selected putative CTCs identified by immunostaining that were isolated from 4 patients with high grade serous ovarian cancer (HGSOC) and one with benign cystadenoma. We specifically targeted CTCs positive for epithelial (CK/EpCAMpos), mesenchymal (vimentinpos), and pseudoendothelial (CK/EpCAMpos plus CD31pos) markers. We isolated these cells and performed whole genome amplification (WGA) and low-pass whole-genome sequencing (LP-WGS) for analysis of copy number alterations (CNA). Of the CK/EpCAMpos cells analysed from the HGSOC patients, 2 of 3 cells showed diverse chromosomal CNAs. However, the 4 pseudoendothelial cells (CK/EpCAMpos plus CD31pos) observed in the HGSOC cases did not carry any CNA. Lastly, two of the clusters of vimentin positive cells sequenced from those found in the benign cystadenoma case had CNA. Despite the low number of cells analysed, our results underscore the importance of genetic analysis of putative CTCs to confirm their neoplastic origin. In particular, it highlights the presence of a population of CK/EpCAMpos cells that are not tumour cells in patients with HGSOC, which otherwise would be counted as CTCs.

Identification of TP53 mutations in circulating tumour DNA in high grade serous ovarian carcinoma using next generation sequencing technologies.

Plasma circulating tumour DNA (ctDNA) has been suggested to be a viable biomarker of response to treatment in patients with high grade serous ovarian carcinoma (HGSOC). TP53 mutations are present in more than 90% of HGSOCs but somatic variants are distributed across all exonic regions of the gene, requiring next generation sequencing (NGS) technologies for mutational analysis. In this study, we compared the suitability of the Accel (Swift) and Oncomine (ThermoFisher) panels for identification of TP53 mutations in ctDNA of HGSOC patients (N = 10). Only 6 patients (60%) were found to have TP53 mutations using the ACCEL panel but the addition of molecular tags in the Oncomine panel improved ctDNA detection with at least one mutation detected in all cases (100%). Orthogonal validation of the 14 somatic variants found by Oncomine, using droplet digital PCR, confirmed 79% (11/14) of the identified mutations. Overall, the Oncomine panel with unique molecular identifiers (UMI) appears more useful for ctDNA analysis in HGSOC.

Extracellular vesicles as a liquid biopsy for melanoma: Are we there yet?

Melanoma is the most aggressive form of skin cancer owing to its high propensity to metastasise in distant organs and develop resistance to treatment. The scarce treatment options available for melanoma underscore the need for biomarkers to guide treatment decisions. In this context, an attractive alternative to overcome the limitations of repeated tissue sampling is the analysis of peripheral blood samples, referred to as 'liquid biopsy'. In particular, the analysis of extracellular vesicles (EVs) has emerged as a promising candidate due to their role in orchestrating cancer dissemination, immune modulation, and drug resistance. As we gain insights into the role of EVs in cancer and melanoma their potential for clinical use is becoming apparent. Herein, we critically summarise the current evidence supporting EVs as biomarkers for melanoma diagnosis, prognostication, therapy response prediction, and drug resistance. EVs are proposed as a candidate biomarker for predicting therapeutic response to immune checkpoint inhibition. However, to realise the potential of EV analysis for clinical decision-making strong clinical validation is required, underscoring the need for further research in this area.

Incidence and mortality of uveal melanoma in Australia (1982-2014).

AIMS: We aimed to estimate the incidence and mortality of uveal melanoma (UM) in Australia from 1982 to 2014. METHODS: Deidentified unit data for all cases of ocular melanoma were extracted from the Australian Cancer Database from 1 January 1982 to 31 December 2014. UM cases were extracted and trends in incidence and disease-specific mortality were calculated. Incidence rates were age-standardised against the 2001 Australian Standard Population. Mortality was assessed using Cox regression. RESULTS: From 1982 to 2014, there were 5087 cases of ocular melanoma in Australia, of which 4617 were classified as UM. The average age-standardised incidence rate of UM was 7.6 (95% CI 7.3 to 7.9) per million. There was an increase (p=0.0502) in the incidence of UM from 1982 to 1993 with an annual percent change (APC) of +2.5%, followed by a significant decrease in the incidence of UM from 1993 to 2014 (APC -1.2%). The average 5-year survival from 1982 to 2011 did not significantly change from an average of 81%, with an average APC (AAPC) of +0.1%. A multivariate Cox regression revealed that residence in Western Australia (p=0.001) or Tasmania (p=0.05), age ≥60 years (p<0.001) and histological classification as mixed (p<0.001) or epithelioid cells (p<0.001) were significantly associated with reduced survival. CONCLUSION: In conclusion, we found that the incidence of UM peaked in the 1990s. Although treatment for primary UM has improved in the last 30 years, overall survival did not change significantly in the last 30 years.

Acute effect of high-intensity interval aerobic exercise on serum myokine levels and resulting tumour-suppressive effect in trained patients with advanced prostate cancer.

PURPOSE: Although skeletal muscle releases cytokines called myokines during exercise, the kinetics of the acute myokine response to exercise (exercise-induced circulatory myokine level alteration) is unknown in patients with advanced prostate cancer. We measured myokine levels in serum obtained from patients with metastatic castrate-resistant prostate cancer (mCRPC) before and after exercise and assessed the growth-suppressive effect of the serum by applying it to a PCa cell line. METHODS: Nine patients with mCRPC (age = 67.8 ± 10.1 years, time since mCRPC diagnosis 36.2 ± 22.5 months) undertook 34 min of a high-intensity interval exercise session on a cycle ergometer. Blood was collected immediately pre, post and 30 min post. Serum levels of secreted protein acidic and rich in cysteine (SPARC), oncostatin M (OSM), interleukin-6 (IL-6), interleukin-15 (IL-15), decorin, irisin, and IGF-1 were determined. Growth of the androgen-independent PCa cell line DU-145 exposed to serum collected at three points was measured. RESULTS: There was a significant elevation of SPARC (19.9%, P = 0.048), OSM (11.5%, P = 0.001), IL-6 (10.2%, P = 0.02) and IL-15 (7.8%, P = 0.023) in serum collected immediately after exercise compared to baseline, returning to baseline after 30 min rest. A significant reduction in DU-145 Cell growth and the Cell Index area under the curve at 72 h incubation was observed with the presence of serum obtained immediately post-exercise (Cell Index at 72 h: 16.9%, P < 0.001; area under the curve: 15.2%, P < 0.001) and with the presence of serum obtained 30 min post-exercise compared to baseline (Cell Index at 72 h: 6.5%; area under the curve: 8.8%, P < 0.001). CONCLUSION: This study provides preliminary evidence for an acute exercise-induced myokine response and tumour growth suppression in serum after a bout of high-intensity interval exercise in patients with advanced PCa.

Size-Based Method for Enrichment of Circulating Tumor Cells from Blood of Colorectal Cancer Patients.

Circulating tumor cells (CTCs) are precursors of the metastatic cascade, which is responsible for 90% of all cancer-related deaths. CTCs arise from solid tumors and travel through the bloodstream and lymphatic system. Developments in the isolation and analysis of CTCs promise potential biomarker assays for detection and monitoring of cancer through a minimally invasive procedure. Despite this, the precise role of CTCs in metastasis remains poorly characterized, mainly due to the low density of CTCs (1-10 CTCs per 10 mL of blood) present in patient blood and the lack of robust methods for their isolation in a standard laboratory setting. CellSearch is currently the only FDA-approved CTC enrichment protocol, but limitations of this EpCAM-based method include cost, availability, and the use of a single surface marker for capture. To address these limitations, we have optimized an existing method, MetaCell, which exploits the differences in size of CTCs compared to other blood cells for CTC enrichment from blood. MetaCell contains a membrane with 8 µm pores, and blood is filtered through this kit by capillary action and CTCs, which are typically larger than the pores and remain on top of the membrane, while most leukocytes pass through the pores. The membrane along with these CTCs can be detached and transferred to 6-well plates for culturing or directly used for characterization. Here, we provide a detailed protocol for enrichment of CTCs using the filtration device MetaCell and a procedure for characterization of CTCs by immunohistochemical staining.

Powering single-cell genomics to unravel circulating tumour cell subpopulations in non-small cell lung cancer patients.

BACKGROUND: Circulating tumour cells (CTCs) are attractive "liquid biopsy" candidates that could provide insights into the different phenotypes of tumours present within a patient. The epithelial-to-mesenchymal transition (EMT) of CTCs is considered a critical step in tumour metastasis; however, it may confound traditional epithelial feature-based CTC isolation and detection. We applied single-cell copy number alteration (CNA) analysis for the identification of genomic alterations to confirm the neoplastic nature of circulating cells with only mesenchymal phenotypes. METHODS: We isolated CTCs from blood samples collected from 46 NSCLC patients using the Parsortix system. Enriched cells were subjected to immunofluorescent staining for CTC identification using a multi-marker panel comprising both epithelial and mesenchymal markers. A subset of isolated CTCs was subjected to whole genome amplification (WGA) and low-pass whole-genome sequencing (LP-WGS) for the analysis of copy number alterations (CNAs). RESULTS: CTCs were detected in 16/46 (34.8%) patients, inclusive of CK+/EpCAM+ CTCs (3/46, 6.5%) and Vim+ CTCs (13/46, 28.3%). Clusters of Vim+ cells were detected in 8 samples, which constitutes 50% of the total number of NSCLC patients with CTCs. No patients had detectable hybrid CK+/EpCAM+/Vim+ cells. All of the tested CK+/EpCAM+ CTCs and 7/8 Vim+ CTCs or CTC clusters carried CNAs confirming their neoplastic nature. Notably, the Vim+ cluster with no CNAs was characterised by spindle morphology and, therefore, defined as normal mesenchymal circulating cells. CONCLUSION: Our results revealed that CK-negative, vimentin-expressing cells represent a large proportion of CTCs detected in NSCLC patients, which are likely missed by standard epithelial-marker-dependent CTC categorisation.

Detectable ctDNA at the time of treatment cessation of ipilimumab and nivolumab for toxicity predicts disease progression in advanced melanoma patients.

Background: Immune checkpoint inhibition (ICI) has led to unprecedented outcomes for melanoma patients but is associated with toxicity. ICI resumption after high grade irAEs poses a significant challenge in the clinical management of melanoma patients and there are no biomarkers that can help identify patients that might benefit from resuming treatment. This study aims to determine if circulating tumor DNA (ctDNA) levels at the time of treatment-limiting irAE could guide treatment decisions in this clinical context. Methods: This is a retrospective exploratory biomarker study from 34 patients treated with combination ICI for stage IV melanoma. Patients had a treatment-limiting toxicity and a baseline plasma collection prior to commencing ICI and within 6 weeks of stopping therapy. Blood samples were tested for ctDNA at baseline and cessation therapy. Results: Median progression free survival (PFS) and overall survival (OS) have not been reached (24-month PFS rate 54% and OS rate 72.3%). PD occurred in 47% (16/34) of patients. Median PFS with detectable ctDNA from plasma collected at the time of toxicity was 6.5 months while not reached (NR) with undetectable levels (HR: 4.0, 95% CI 0.95-17.5, p=0.0023). Median OS with detectable ctDNA at cessation for toxicity was 19.4 months and NR for undetectable ctDNA (HR: 3.9, 95%CI 20.8-18.6, p=0.024). Positive ctDNA at the time of cessation was highly specific (specificity 0.94, 95% CI 0.74-0.99, PPV 0.88, 95% CI 0.53-0.99). However, ctDNA negativity has low sensitivity as a predictor of ongoing disease control (sensitivity 0.437, 95% CI 0.23-0.67). Notably, 4/9 (44%) ctDNA negative patients who had disease progression had brain only disease progression. Conclusions: Undetectable ctDNA and CR on imaging after stopping immunotherapy for toxicity results in high rates of long-term durable control. For patients with immunotherapy related toxicity, who have persistent ctDNA at 8 - 12 weeks, the risk of disease progression is significant.

Gut microbiota diversity and composition in predicting immunotherapy response and immunotherapy-related colitis in melanoma patients: A systematic review.

BACKGROUND: Gut microbiome (GM) composition and diversity have recently been studied as a biomarker of response to immune checkpoint blockade therapy (ICB) and of ICB-related colitis. AIM: To conduct a systematic review on the role of GM composition and diversity in predicting response and colitis in patients with melanoma treated with ICB. METHODS: The review protocol was registered in PROSPERO: CRD42021228018. From a total of 300 studies, nine studies met inclusion criteria. Two studies were phase I clinical trials, while the remainder were prospective observational studies. All but one study has moderate risk of bias. In addition, we conducted a relevant search by Reference Citation Analysis (RCA) (https://www.referencecitationanalysis.com). RESULTS: Fecal samples enriched in Firmicutes phylum were associated with good response to ICB, whereas the Bacteroidales family was associated with poor response to ICB. Samples with greater GM diversity were associated with more favorable response to ICB [hazard ratio (HR) = 3.57, 95% confidence interval = 1.02-12.52, P < 0.05]. Fecal samples with a higher abundance in Firmicutes were more susceptible to ICB-related colitis (P < 0.01) whereas samples enriched in Bacteroidetes were more resistant to ICB-related colitis (P < 0.05). Overall, there was limited concordance in the organisms in the GM identified to be associated with response to ICB, and studies evaluating GM diversity showed conflicting results. CONCLUSION: This highlights the need for further prospective studies to confirm whether the GM could be used as a biomarker and potential intervention to modulate ICB response in melanoma patients.

Dependence on a variable residue limits the breadth of an HIV MPER neutralizing antibody, despite convergent evolution with broadly neutralizing antibodies.

Broadly neutralizing antibodies (bNAbs) that target the membrane-proximal external region (MPER) of HIV gp41 envelope, such as 4E10, VRC42.01 and PGZL1, can neutralize >80% of viruses. These three MPER-directed monoclonal antibodies share germline antibody genes (IGHV1-69 and IGKV3-20) and form a bNAb epitope class. Furthermore, convergent evolution within these two lineages towards a 111.2GW111.3 motif in the CDRH3 is known to enhance neutralization potency. We have previously isolated an MPER neutralizing antibody, CAP206-CH12, that uses these same germline heavy and light chain genes but lacks breadth (neutralizing only 6% of heterologous viruses). Longitudinal sequencing of the CAP206-CH12 lineage over three years revealed similar convergent evolution towards 111.2GW111.3 among some lineage members. Mutagenesis of CAP206-CH12 from 111.2GL111.3 to 111.2GW111.3 and the introduction of the double GWGW motif into CAP206-CH12 modestly improved neutralization potency (2.5-3-fold) but did not reach the levels of potency of VRC42.01, 4E10 or PGZL1. To explore the lack of potency/breadth, viral mutagenesis was performed to map the CAP206-CH12 epitope. This indicated that CAP206-CH12 is dependent on D674, a highly variable residue at the solvent-exposed elbow of MPER. In contrast, VRC42.01, PGZL1 and 4E10 were dependent on highly conserved residues (W672, F673, T676, and W680) facing the hydrophobic patch of the MPER. Therefore, while CAP206-CH12, VRC42.01, PGZL1 and 4E10 share germline genes and show some evidence of convergent evolution, their dependence on different amino acids, which impacts orientation of binding to the MPER, result in differences in breadth and potency. These data have implications for the design of HIV vaccines directed at the MPER epitope.

Assessment of a Size-Based Method for Enriching Circulating Tumour Cells in Colorectal Cancer.

Circulating tumour cells (CTC) from solid tumours are a prerequisite for metastasis. Isolating CTCs and understanding their biology is essential for developing new clinical tests and precision oncology. Currently, CellSearch is the only FDA (U.S. Food and Drug Administration)-approved method for CTC enrichment but possesses several drawbacks owing to a reliance on the epithelial cell adhesion molecule (EpCAM) and a resource-intensive nature. Addressing these shortcomings, we optimised an existing size-based method, MetaCell, to enrich CTCs from blood of colorectal cancer (CRC) patients. We evaluated the ability of MetaCell to enrich CTCs by spiking blood with CRC cell lines and assessing the cell recovery rates and WBC depletion via immunostaining and gene expression. We then applied MetaCell to samples from 17 CRC patients and seven controls. Recovery rates were >85% in cell lines, with >95% depletion in WBCs. MetaCell yielded CTCs and CTC clusters in 52.9% and 23.5% of the patients, respectively, without false positives in control patients. CTCs and cluster detection did not correlate with histopathological parameters. Overall, we demonstrated that the MetaCell platform enriched CRC cells with high recovery rates and high purity. Our pilot study also demonstrated the ability of MetaCell to detect CTCs in CRC patients.

Human leucocyte antigen genotype association with the development of immune-related adverse events in patients with non-small cell lung cancer treated with single agent immunotherapy.

INTRODUCTION: Biomarkers that predict the risk of immune-mediated adverse events (irAEs) among patients with non-small cell lung cancer (NSCLC) may reduce morbidity and mortality associated with these treatments. METHODS: We carried out high resolution human leucocyte antigen (HLA)-I typing on 179 patients with NSCLC treated with anti-program death (PD)-1/program death ligand (PDL)-1. Toxicity data were collected and graded as per common terminology criteria for adverse event (CTCAE) v5.0. We used 14.8-week for landmark analysis to address lead-time bias to investigate the correlation between HLA-I/II zygosity, supertypes and alleles with irAE. Furthermore, we assessed the association for irAE with clinical benefit rate (CBR), progression-free survival (PFS) and overall survival (OS). RESULTS: Homozygosity at one or more HLA-I loci, but not HLA-II, was associated with a reduced risk of irAE (relative risk (RR) = 0.61, 95% CI 0.33-0.95, P = 0.035) especially pneumonitis or any grade 3 toxicity. Patients with HLA-A03 supertype had a higher risk of developing irAE (RR = 1.42, 95% CI 1.02-2.01, P = 0.039). The occurrence of any irAE was significantly associated with improved CBR (RR = 1.48, P < 0.0001), PFS (HR = 0.45, P = 0.0003) and OS (HR = 0.34, P < 0.0001). CONCLUSIONS: Homozygosity at one or more HLA-I loci may serve as biomarker to predict patients who are unlikely to experience severe irAEs among patients with NSCLC and treated with anti-PD1/PDL1, but less likely to derive clinical benefit. Patients with HLA-I homozygous might benefit from additional therapy.