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.

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.

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.

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.

Future perspectives of uveal melanoma blood based biomarkers.

Uveal melanoma (UM) is the most common primary intraocular malignancy affecting adults. Despite successful local treatment of the primary tumour, metastatic disease develops in up to 50% of patients. Metastatic UM carries a particularly poor prognosis, with no effective therapeutic option available to date. Genetic studies of UM have demonstrated that cytogenetic features, including gene expression, somatic copy number alterations and specific gene mutations can allow more accurate assessment of metastatic risk. Pre-emptive therapies to avert metastasis are being tested in clinical trials in patients with high-risk UM. However, current prognostic methods require an intraocular tumour biopsy, which is a highly invasive procedure carrying a risk of vision-threatening complications and is limited by sampling variability. Recently, a new diagnostic concept known as "liquid biopsy" has emerged, heralding a substantial potential for minimally invasive genetic characterisation of tumours. Here, we examine the current evidence supporting the potential of blood circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), microRNA (miRNA) and exosomes as biomarkers for UM. In particular, we discuss the potential of these biomarkers to aid clinical decision making throughout the management of UM patients.

Detection of clinical progression through plasma ctDNA in metastatic melanoma patients: a comparison to radiological progression.

BACKGROUND: The validity of circulating tumour DNA (ctDNA) as an indicator of disease progression compared to medical imaging in patients with metastatic melanoma requires detailed evaluation. METHODS: Here, we carried out a retrospective ctDNA analysis of 108 plasma samples collected at the time of disease progression. We also analysed a validation cohort of 66 metastatic melanoma patients monitored prospectively after response to systemic therapy. RESULTS: ctDNA was detected in 62% of patients at the time of disease progression. For 67 patients that responded to treatment, the mean ctDNA level at progressive disease was significantly higher than at the time of response (P < 0.0001). However, only 30 of these 67 (45%) patients had a statistically significant increase in ctDNA by Poisson test. A validation cohort of 66 metastatic melanoma patients monitored prospectively indicated a 56% detection rate of ctDNA at progression, with only two cases showing increased ctDNA prior to radiological progression. Finally, a correlation between ctDNA levels and metabolic tumour burden was only observed in treatment naïve patients but not at the time of progression in a subgroup of patients failing BRAF inhibition (N = 15). CONCLUSIONS: These results highlight the low efficacy of ctDNA to detect disease progression in melanoma when compared mainly to standard positron emission tomography imaging.

Circulating tumour DNA (ctDNA) as a biomarker in metachronous melanoma and colorectal cancer- a case report.

BACKGROUND: Circulating tumour DNA (ctDNA) has emerged as a promising blood-based biomarker for monitoring disease status of patients with advanced cancers. The presence of ctDNA in the blood is a result of biological processes, namely tumour cell apoptosis and/or necrosis, and can be used to monitor different cancers by targeting cancer-specific mutation. CASE PRESENTATION: We present the case of a 67 year old Caucasian male that was initially treated with BRAF inhibitors followed by anti-CTLA4 and then anti-PD1 immunotherapy for metastatic melanoma but later developed colorectal cancer. The kinetics of ctDNA derived from each cancer type were monitored targeting BRAF V600R (melanoma) and KRAS G13D (colon cancer), specifically reflected the status of the patient's tumours. In fact, the discordant pattern of BRAF and KRAS ctDNA was significantly correlated with the clinical response of melanoma to pembrolizumab treatment and progression of colorectal cancer noted by PET and/or CT scan. Based on these results, ctDNA can be used to specifically clarify disease status of patients with metachronous cancers. CONCLUSIONS: Using cancer-specific mutational targets, we report here for the first time the efficacy of ctDNA to accurately provide a comprehensive outlook of the tumour status of two different cancers within one patient. Thus, ctDNA analysis has a potential clinical utility to delineate clinical information in patients with multiple cancer types.

Correlation between circulating tumour DNA and metabolic tumour burden in metastatic melanoma patients.

BACKGROUND: Circulating tumour DNA (ctDNA) may serve as a measure of tumour burden and a useful tool for non-invasive monitoring of cancer. However, ctDNA is not always detectable in patients at time of diagnosis of metastatic disease. Therefore, there is a need to understand the correlation between ctDNA levels and the patients' overall metabolic tumour burden (MTB). METHODS: Thirty-two treatment naïve metastatic melanoma patients were included in the study. MTB and metabolic tumour volume (MTV) was measured by 18F-fluoro-D-glucose positron emission tomography/computed tomography (FDG PET/CT). Plasma ctDNA was quantified using droplet digital PCR (ddPCR). RESULTS: CtDNA was detected in 23 of 32 patients. Overall, a significant correlation was observed between ctDNA levels and MTB (p < 0.001). CtDNA was not detectable in patients with an MTB of ≤10, defining this value as the lower limit of tumour burden that can be detected through ctDNA analysis by ddPCR. CONCLUSIONS: We showed that ctDNA levels measured by ddPCR correlate with MTB in treatment naïve metastatic melanoma patients and observed a limit in tumour size for which ctDNA cannot be detected in blood. Nevertheless, our findings support the use of ctDNA as a non-invasive complementary modality to functional imaging for monitoring tumour burden.

SIRT1 activation mediates heat-induced survival of UVB damaged Keratinocytes.

BACKGROUND: Exposure to heat stress after UVB irradiation induces a reduction of apoptosis, resulting in survival of DNA damaged human keratinocytes. This heat-mediated evasion of apoptosis appears to be mediated by activation of SIRT1 and inactivation of p53 signalling. In this study, we assessed the role of SIRT1 in the inactivation of p53 signalling and impairment of DNA damage response in UVB plus heat exposed keratinocytes. RESULTS: Activation of SIRT1 after multiple UVB plus heat exposures resulted in increased p53 deacetylation at K382, which is known to affect its binding to specific target genes. Accordingly, we noted decreased apoptosis and down regulation of the p53 targeted pro-apoptotic gene BAX and the DNA repair genes ERCC1 and XPC after UVB plus heat treatments. In addition, UVB plus heat induced increased expression of the cell survival gene Survivin and the proliferation marker Ki67. Notably, keratinocytes exposed to UVB plus heat in the presence of the SIRT1 inhibitor, Ex-527, showed a similar phenotype to those exposed to UV alone; i.e. an increase in p53 acetylation, increased apoptosis and low levels of Survivin. CONCLUSION: This study demonstrate that heat-induced SIRT1 activation mediates survival of DNA damaged keratinocytes through deacetylation of p53 after exposure to UVB plus heat.

Heat-mediated reduction of apoptosis in UVB-damaged keratinocytes in vitro and in human skin ex vivo.

BACKGROUND: UV radiation induces significant DNA damage in keratinocytes and is a known risk factor for skin carcinogenesis. However, it has been reported previously that repeated and simultaneous exposure to UV and heat stress increases the rate of cutaneous tumour formation in mice. Since constant exposure to high temperatures and UV are often experienced in the environment, the effects of exposure to UV and heat needs to be clearly addressed in human epidermal cells. METHODS: In this study, we determined the effects of repeated UVB exposure 1 kJ/m(2) followed by heat (39 °C) to human keratinocytes. Normal human ex vivo skin models and primary keratinocytes (NHEK) were exposed once a day to UVB and/or heat stress for four consecutive days. Cells were then assessed for changes in proliferation, apoptosis and gene expression at 2 days post-exposure, to determine the cumulative and persistent effects of UV and/or heat in skin keratinocytes. RESULTS: Using ex vivo skin models and primary keratinocytes in vitro, we showed that UVB plus heat treated keratinocytes exhibit persistent DNA damage, as observed with UVB alone. However, we found that apoptosis was significantly reduced in UVB plus heat treated samples. Immunohistochemical and whole genome transcription analysis showed that multiple UVB plus heat exposures induced inactivation of the p53-mediated stress response. Furthermore, we demonstrated that repeated exposure to UV plus heat induced SIRT1 expression and a decrease in acetylated p53 in keratinocytes, which is consistent with the significant downregulation of p53-regulated pro-apoptotic and DNA damage repair genes in these cells. CONCLUSION: Our results suggest that UVB-induced p53-mediated cell cycle arrest and apoptosis are reduced in the presence of heat stress, leading to increased survival of DNA damaged cells. Thus, exposure to UVB and heat stress may act synergistically to allow survival of damaged cells, which could have implications for initiation skin carcinogenesis.

Differences in HIV type 1 neutralization breadth in 2 geographically distinct cohorts in Africa.

To investigate whether distinct populations have differing human immunodeficiency virus type 1 (HIV) neutralizing antibody responses, we compared 20 women from Tanzania's HIV Superinfection Study (HISIS) cohort, who were infected multiple HIV subtypes, and 22 women from the Centre for the AIDS Programme of Research in South Africa (CAPRISA) cohort, who were infected exclusively with HIV subtype C. By 2 years after infection, 35% of HISIS subjects developed neutralization breadth, compared with 9% of CAPRISA subjects (P = .0131). Cumulative viral loads between 3 and 12 months were higher in the HISIS group (P = .046) and strongly associated with breadth (P < .0001). While viral load was the strongest predictor, other factors may play a role, as the odds of developing breadth remained higher in HISIS even after correction for viral load.

South African HIV-1 subtype C transmitted variants with a specific V2 motif show higher dependence on α4ß7 for replication.

BACKGROUND: The integrin α4ß7 mediates the trafficking of immune cells to the gut associated lymphoid tissue (GALT) and is an attachment factor for the HIV gp120 envelope glycoprotein. We developed a viral replication inhibition assay to more clearly evaluate the role of α4ß7 in HIV infection and the contribution of viral and host factors. RESULTS: Replication of 60 HIV-1 subtype C viruses collected over time from 11 individuals in the CAPRISA cohort were partially inhibited by antibodies targeting α4ß7. However, dependence on α4ß7 for replication varied substantially among viral isolates from different individuals as well as over time in some individuals. Among 8 transmitted/founder (T/F) viruses, α4ß7 reactivity was highest for viruses having P/SDI/V tri-peptide binding motifs. Mutation of T/F viruses that had LDI/L motifs to P/SDI/V resulted in greater α4ß7 reactivity, whereas mutating P/SDI/V to LDI/L motifs was associated with reduced α4ß7 binding. P/SDI/V motifs were more common among South African HIV subtype C viruses (35%) compared to subtype C viruses from other regions of Africa (<8%) and to other subtypes, due in part to a founder effect. In addition, individuals with bacterial vaginosis (BV) and who had higher concentrations of IL-7, IL-8 and IL-1α in the genital tract had T/F viruses with higher α4ß7 dependence for replication, suggesting that viruses with P/SDI/V motifs may be preferentially transmitted in the presence of BV in this population. CONCLUSIONS: Collectively, these data suggest a role for α4ß7 in HIV infection that is influenced by both viral and host factors including the sequence of the α4ß7 binding motif, the cytokine milieu and BV in the genital tract. The higher frequency of P/SDI/V sequences among South African HIV-1 subtype C viruses may have particular significance for the role of α4ß7 in this geographical region.

Viral escape from HIV-1 neutralizing antibodies drives increased plasma neutralization breadth through sequential recognition of multiple epitopes and immunotypes.

Identifying the targets of broadly neutralizing antibodies to HIV-1 and understanding how these antibodies develop remain important goals in the quest to rationally develop an HIV-1 vaccine. We previously identified a participant in the CAPRISA Acute Infection Cohort (CAP257) whose plasma neutralized 84% of heterologous viruses. In this study we showed that breadth in CAP257 was largely due to the sequential, transient appearance of three distinct broadly neutralizing antibody specificities spanning the first 4.5 years of infection. The first specificity targeted an epitope in the V2 region of gp120 that was also recognized by strain-specific antibodies 7 weeks earlier. Specificity for the autologous virus was determined largely by a rare N167 antigenic variant of V2, with viral escape to the more common D167 immunotype coinciding with the development of the first wave of broadly neutralizing antibodies. Escape from these broadly neutralizing V2 antibodies through deletion of the glycan at N160 was associated with exposure of an epitope in the CD4 binding site that became the target for a second wave of broadly neutralizing antibodies. Neutralization by these CD4 binding site antibodies was almost entirely dependent on the glycan at position N276. Early viral escape mutations in the CD4 binding site drove an increase in wave two neutralization breadth, as this second wave of heterologous neutralization matured to recognize multiple immunotypes within this site. The third wave targeted a quaternary epitope that did not overlap any of the four known sites of vulnerability on the HIV-1 envelope and remains undefined. Altogether this study showed that the human immune system is capable of generating multiple broadly neutralizing antibodies in response to a constantly evolving viral population that exposes new targets as a consequence of escape from earlier neutralizing antibodies.

Multiple pathways of escape from HIV broadly cross-neutralizing V2-dependent antibodies.

Broadly cross-neutralizing (BCN) antibodies are likely to be critical for an effective HIV vaccine. However, the ontogeny of such antibodies and their relationship with autologous viral evolution is unclear. Here, we characterized viral evolution in CAP256, a subtype C-infected individual who developed potent BCN antibodies targeting positions R166 and K169 in the V2 region. CAP256 was superinfected at 3 months postinfection with a virus that was highly sensitive to BCN V2-dependent monoclonal antibodies. The autologous neutralizing response in CAP256 was directed at V1V2, reaching extremely high titers (>1:40,000) against the superinfecting virus at 42 weeks, just 11 weeks prior to the development of the BCN response targeting the same region. Recombination between the primary and superinfecting viruses, especially in V2 and gp41, resulted in two distinct lineages by 4 years postinfection. Although neutralization of some CAP256 clones by plasma from as much as 2 years earlier suggested incomplete viral escape, nonetheless titers against later clones were reduced at least 40-fold to less than 1:1,000. Escape mutations were identified in each lineage, either at R166 or at K169, suggesting that strain-specific and BCN antibodies targeted overlapping epitopes. Furthermore, the early dependence of CAP256 neutralizing antibodies on the N160 glycan decreased with the onset of neutralization breadth, indicating a change in specificity. These data suggest rapid maturation, within 11 weeks, of CAP256 strain-specific antibodies to acquire breadth, with implications for the vaccine elicitation of BCN V2-dependent antibodies. Overall these studies demonstrate that ongoing viral escape is possible, even from BCN antibodies.

Monitoring changes in circulating tumour cells as a prognostic indicator of overall survival and treatment response in patients with metastatic melanoma.

BACKGROUND: New effective treatments for metastatic melanoma greatly improve survival in a proportion of patients. However biomarkers to identify patients that are more likely to benefit from a particular treatment are needed. We previously reported on a multimarker approach for the detection of heterogenous melanoma circulating tumour cells (CTCs). Here we evaluated the prognostic value of this multimarker quantification of CTCs and investigated whether changes in CTC levels during therapy can be used as a biomarker of treatment response and survival outcomes. METHODS: CTCs were captured by targeting the melanoma associated markers MCSP and MCAM as well as the melanoma stem cell markers ABCB5 and CD271. CTCs were quantified in 27 metastatic melanoma patients treated by surgery or with vemurafenib, ipilimumab or dacarbazine. Patients were enrolled prospectively and CTC counts performed at baseline (prior to treatment), during and after treatment. RESULTS: Baseline CTC numbers were not found to be prognostic of overall survival nor of progression free survival. However, a low baseline CTC number was associated with a rapid response to vemurafenib therapy. A decrease in CTCs after treatment initiation was associated with response to treatment and prolonged overall survival in vemurafenib treated patients. CONCLUSIONS: Measuring changes in CTC numbers during treatment is useful for monitoring therapy response in melanoma patients and for providing prognostic information relating to overall survival. Further studies with larger sample sizes are required to confirm the utility of CTC quantification as a companion diagnostic for metastatic melanoma treatment.

HLA-A01 and HLA-B27 Supertypes, but Not HLA Homozygocity, Correlate with Clinical Outcome among Patients with Non-Small Cell Lung Cancer Treated with Pembrolizumab in Combination with Chemotherapy.

INTRODUCTION: Maximal heterozygosity on the human leukocyte antigen (HLA) loci has been found to be associated with improved survival and development of immune-related adverse events (irAEs) among NSCLC patients treated with immunotherapy. Here, we investigated the effect of germline HLA-I/-II on clinical outcomes among NSCLC patients treated with first-line pembrolizumab in combination with chemotherapy. METHOD: We prospectively recruited patients with NSCLC who were commencing first-line pembrolizumab in combination with chemotherapy. DNA from white blood cells was used for high-resolution HLA-I/II typing. RESULTS: Of the 65 patients recruited, 53 complied with the inclusion criteria. We did not find an association between HLA-I/-II homozygosity and clinical outcome among the studied population. However, the presence of HLA-A01 was associated with unfavourable PFS (HR = 2.32, 95%CI 1.13-4.77, p = 0.022) and worsening OS (HR = 2.86, 95%CI 1.06-7.70, p = 0.038). The presence of HLA-B27 was associated with improved PFS (HR = 0.35, 95%CI 0.18-0.71, p = 0.004) and trends toward improving OS. None of the HLA-I supertypes were associated with the development or worsening of irAEs. CONCLUSIONS: The absence of association between genomic HLA-I/-II homozygosity and clinical outcome among patients with advanced NSCLC treated with pembrolizumab in combination with chemotherapy might reflect a diminished role for HLA molecules among patients with low or no PD-L1. HLA-A01 and HLA-B27 might have a role in predicting clinical outcomes among this cohort of patients. Further studies are needed to explore biomarkers for this group of patients.

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.

UCLA1, a synthetic derivative of a gp120 RNA aptamer, inhibits entry of human immunodeficiency virus type 1 subtype C.

Entry of human immunodeficiency virus type 1 (HIV-1) into cells is mediated by the virion surface envelope (Env) glycoproteins, making it a desirable target for antiretroviral entry inhibitors. We previously isolated a family of gp120 binding RNA aptamers and showed that they neutralized the infectivity of HIV-1. In this study, we assessed the activity of a shortened synthetic derivative of the B40 aptamer, called UCLA1, against a large panel of HIV-1 subtype C viruses. UCLA1 tightly bound to a consensus HIV-1 subtype C gp120 and neutralized isolates of the same subtype with 50% inhibitory concentrations (IC(50)s) in the nanomolar range. The aptamer had little toxicity in tests with cell lines and primary cells. Furthermore, it exhibited high therapeutic indices, suggesting that it may be effective at very low doses. Mapping of UCLA1 binding sites on gp120 revealed eight amino acid residues that modulated neutralization resistance. This included residues within the coreceptor binding site, at the base of the V3 loop, and in the bridging sheet within the conserved V1/V2 stem-loop of gp120. The aptamer was also shown to have synergistic effects with T20, a gp41 fusion inhibitor, and IgG1b12 (b12), an anti-CD4 binding site monoclonal antibody. These results suggest that UCLA1 may be suitable for development as a potent HIV-1 entry inhibitor.

The development of CD4 binding site antibodies during HIV-1 infection.

Broadly neutralizing antibodies to the CD4 binding site (CD4bs) of gp120 are generated by some HIV-1-infected individuals, but little is known about the prevalence and evolution of this antibody response during the course of HIV-1 infection. We analyzed the sera of 113 HIV-1 seroconverters from three cohorts for binding to a panel of gp120 core proteins and their corresponding CD4bs knockout mutants. Among sera collected between 99 and 258 weeks post-HIV-1 infection, 88% contained antibodies to the CD4bs and 47% contained antibodies to resurfaced stabilized core (RSC) probes that react preferentially with broadly neutralizing CD4bs antibodies (BNCD4), such as monoclonal antibodies (MAbs) VRC01 and VRC-CH31. Analysis of longitudinal serum samples from a subset of 18 subjects revealed that CD4bs antibodies to gp120 arose within the first 4 to 16 weeks of infection, while the development of RSC-reactive antibodies was more varied, occurring between 10 and 152 weeks post-HIV-1 infection. Despite the presence of these antibodies, serum neutralization mediated by RSC-reactive antibodies was detected in sera from only a few donors infected for more than 3 years. Thus, CD4bs antibodies that bind a VRC01-like epitope are often induced during HIV-1 infection, but the level and potency required to mediate serum neutralization may take years to develop. An improved understanding of the immunological factors associated with the development and maturation of neutralizing CD4bs antibodies during HIV-1 infection may provide insights into the requirements for eliciting this response by vaccination.

Identification of broadly neutralizing antibody epitopes in the HIV-1 envelope glycoprotein using evolutionary models.

BACKGROUND: Identification of the epitopes targeted by antibodies that can neutralize diverse HIV-1 strains can provide important clues for the design of a preventative vaccine. METHODS: We have developed a computational approach that can identify key amino acids within the HIV-1 envelope glycoprotein that influence sensitivity to broadly cross-neutralizing antibodies. Given a sequence alignment and neutralization titers for a panel of viruses, the method works by fitting a phylogenetic model that allows the amino acid frequencies at each site to depend on neutralization sensitivities. Sites at which viral evolution influences neutralization sensitivity were identified using Bayes factors (BFs) to compare the fit of this model to that of a null model in which sequences evolved independently of antibody sensitivity. Conformational epitopes were identified with a Metropolis algorithm that searched for a cluster of sites with large Bayes factors on the tertiary structure of the viral envelope. RESULTS: We applied our method to ID50 neutralization data generated from seven HIV-1 subtype C serum samples with neutralization breadth that had been tested against a multi-clade panel of 225 pseudoviruses for which envelope sequences were also available. For each sample, between two and four sites were identified that were strongly associated with neutralization sensitivity (2ln(BF) > 6), a subset of which were experimentally confirmed using site-directed mutagenesis. CONCLUSIONS: Our results provide strong support for the use of evolutionary models applied to cross-sectional viral neutralization data to identify the epitopes of serum antibodies that confer neutralization breadth.