Determination of permissive and restraining cancer-associated fibroblast (DeCAF) subtypes.

Cancer-associated fibroblast (CAF) subpopulations in pancreatic ductal adenocarcinoma (PDAC) have been identified using single-cell RNA sequencing (scRNAseq) with divergent characteristics, but their clinical relevance remains unclear. We translate scRNAseq-derived CAF cell-subpopulation-specific marker genes to bulk RNAseq data, and develop a single- sample classifier, DeCAF, for the classification of clinically rest raining and perm issive CAF subtypes. We validate DeCAF in 19 independent bulk transcriptomic datasets across four tumor types (PDAC, mesothelioma, bladder and renal cell carcinoma). DeCAF subtypes have distinct histology features, immune landscapes, and are prognostic and predict response to therapy across cancer types. We demonstrate that DeCAF is clinically replicable and robust for the classification of CAF subtypes in patients for multiple tumor types, providing a better framework for the future development and translation of therapies against permissive CAF subtypes and preservation of restraining CAF subtypes. Significance: We introduce a replicable and robust classifier, DeCAF, that delineates the significance of the role of permissive and restraining CAF subtypes in cancer patients. DeCAF is clinically tractable, prognostic and predictive of treatment response in multiple cancer types and lays the translational groundwork for the preclinical and clinical development of CAF subtype specific therapies.

Transcriptomic Differences in Peripheral Monocyte Populations in Septic Patients Based on Outcome.

ABSTRACT: Post-sepsis early mortality is being replaced by survivors who experience either a rapid recovery and favorable hospital discharge or the development of chronic critical illness (CCI) with suboptimal outcomes. The underlying immunological response that determines these clinical trajectories remains poorly defined at the transcriptomic level. As classical and non-classical monocytes are key leukocytes in both the innate and adaptive immune systems, we sought to delineate the transcriptomic response of these cell types. Using single-cell RNA sequencing and pathway analyses, we identified gene expression patterns between these two groups that are consistent with differences in TNFα production based on clinical outcome. This may provide therapeutic targets for those at risk for CCI in order to improve their phenotype/endotype, morbidity, and long-term mortality.

Social Determinants of Health and Delivery of Rehabilitation to Older Adults During ICU Hospitalization.

Importance: Older adults with socioeconomic disadvantage develop a greater burden of disability after critical illness than those without socioeconomic disadvantage. The delivery of in-hospital rehabilitation that can mitigate functional decline may be influenced by social determinants of health (SDOH). Whether rehabilitation delivery differs by SDOH during critical illness hospitalization is not known. Objective: To evaluate whether SDOH are associated with the delivery of skilled rehabilitation during critical illness hospitalization among older adults. Design, Setting, and Participants: This cohort study used data from the National Health and Aging Trends Study linked with Medicare claims (2011-2018). Participants included older adults hospitalized with a stay in the intensive care unit (ICU). Data were analyzed from August 2022 to September 2023. Exposures: Dual eligibility for Medicare and Medicaid, education, income, limited English proficiency (LEP), and rural residence. Main Outcome and Measures: The primary outcome was delivery of physical therapy (PT) and/or occupational therapy (OT) during ICU hospitalization, characterized as any in-hospital PT or OT and rate of in-hospital PT or OT, calculated as total number of units divided by length of stay. Results: In the sample of 1618 ICU hospitalizations (median [IQR] patient age, 81.0 [75.0-86.0] years; 842 [52.0%] female), 371 hospitalizations (22.9%) were among patients with dual Medicare and Medicaid eligibility, 523 hospitalizations (32.6%) were among patients with less than high school education, 320 hospitalizations (19.8%) were for patients with rural residence, and 56 hospitalizations (3.5%) were among patients with LEP. A total of 1076 hospitalized patients (68.5%) received any PT or OT, with a mean rate of 0.94 (95% CI, 0.86-1.02) units/d. After adjustment for age, sex, prehospitalization disability, mechanical ventilation, and organ dysfunction, factors associated with lower odds of receipt of PT or OT included dual Medicare and Medicaid eligibility (adjusted odds ratio, 0.70 [95% CI, 0.50-0.97]) and rural residence (adjusted odds ratio, 0.65 [95% CI, 0.48-0.87]). LEP was associated with a lower rate of PT or OT (adjusted rate ratio, 0.55 [95% CI, 0.32-0.94]). Conclusions and Relevance: These findings highlight the need to consider SDOH in efforts to promote rehabilitation delivery during ICU hospitalization and to investigate factors underlying inequities in this practice.

Low dose aspirin prevents endothelial dysfunction in the aorta and foetal loss in pregnant mice infected with influenza A virus.

Influenza A virus (IAV) infection in pregnancy resembles a preeclamptic phenotype characterised by vascular dysfunction and foetal growth retardation. Given that low dose aspirin (ASA) is safe in pregnancy and is used to prevent preeclampsia, we investigated whether ASA or NO-conjugated aspirin, NCX4016, resolve vascular inflammation and function to improve offspring outcomes following IAV infection in pregnant mice. Pregnant mice were intranasally infected with a mouse adapted IAV strain (Hkx31; 104 plaque forming units) and received daily treatments with either 200µg/kg ASA or NCX4016 via oral gavage. Mice were then culled and the maternal lungs and aortas collected for qPCR analysis, and wire myography was performed on aortic rings to assess endothelial and vascular smooth muscle functionality. Pup and placentas were weighed and pup growth rates and survival assessed. IAV infected mice had an impaired endothelial dependent relaxation response to ACh in the aorta, which was prevented by ASA and NCX4016 treatment. ASA and NCX4016 treatment prevented IAV dissemination and inflammation of the aorta as well as improving the pup placental ratios in utero, survival and growth rates at post-natal day 5. Low dose ASA is safe to use during pregnancy for preeclampsia and this study demonstrates that ASA may prove a promising treatment for averting the significant vascular complications associated with influenza infection during pregnancy.

High-Intensity Care for Nursing Home Residents with Severe Dementia Hospitalized at the End of Life: A Mixed Methods Study.

OBJECTIVE: For nursing home residents with severe dementia, high-intensity medical treatment offers little possibility of benefit but has the potential to cause significant distress. Nevertheless, mechanical ventilation and intensive care unit (ICU) transfers have increased in this population. We sought to understand how and why such care is occurring. DESIGN: Mixed methods study, with retrospective collection of qualitative and quantitative data. SETTING: Department of Veterans Affairs (VA) hospitals. METHODS: Using the Minimum Data Set, we identified veterans aged ≥65 years who had severe dementia, lived in nursing homes, and died in 2013. We selected those who underwent mechanical ventilation or ICU transfer in the last 30 days of life. We restricted our sample to patients receiving care at VA hospitals because these hospitals share an electronic medical record, from which we collected structured information and constructed detailed narratives of how medical decisions were made. We used qualitative content analysis to identify distinct paths to high-intensity treatment in these narratives. RESULTS: Among 163 veterans, 41 (25.2%) underwent mechanical ventilation or ICU transfer. Their median age was 85 (IQR, 80-94), 97.6% were male, and 67.5% were non-Hispanic white. More than a quarter had living wills declining some or all treatment. There were 5 paths to high-intensity care. The most common (18 of 41 patients) involved families who struggled with decisions. Other patients (15 of 41) received high-intensity care reflexively, before discussion with a surrogate. Four patients had families who advocated repeatedly for aggressive treatment, against clinical recommendations. In 2 cases, information about the patient's preferences was erroneous or unavailable. In 2 cases, there was difficulty identifying a surrogate. CONCLUSIONS AND IMPLICATIONS: Our findings highlight the role of surrogates' difficulty with decision making and of health system-level factors in end-of-life ICU transfers and mechanical ventilation among nursing home residents with severe dementia.

The impact of continuous calorie restriction and fasting on cognition in adults without eating disorders.

Research into the effects of calorie restriction continues to intrigue those interested in whether it may allow humans to live longer and healthier lives. Animal studies of continuous calorie restriction (CCR) and fasting have demonstrated substantial advantages to health and longevity. However, concerns remain about the impact of restricting calorie intake on human health and cognition. Given the emerging evidence of cognitive impairments in eating disorders, studies investigating restricted calorie intake in healthy humans (in an ethical way) may also have implications for understanding restrictive eating disorders. In this review, the published literature on the impact of CCR and fasting on cognitive function in healthy human participants is synthesized. Of the 33 studies of CCR and fasting in humans identified, 23 demonstrated significant changes in cognition. Despite variation across the cognitive domains, results suggest CCR benefits inhibition, processing speed, and working memory, but may lead to impairments in cognitive flexibility. The results of fasting studies suggest fasting is associated with impairments in cognitive flexibility and psychomotor abilities. Overall, the results of these studies suggest the degree (ie, the severity) of calorie restriction is what most likely predicts cognitive improvements as opposed to impairments. For individuals engaging in sustained restriction, this may have serious, irreversible consequences. However, there are mixed findings regarding the impact of CCR and fasting on this aspect of human functioning, suggesting further research is required to understand the costs and benefits of different types of calorie restriction.

Exploring the role of sporadic BRAF and KRAS mutations during colorectal cancer pathogenesis: A spotlight on the contribution of the endosome-lysosome system.

The highly heterogenous nature of colorectal cancer can significantly hinder its early and accurate diagnosis, eventually contributing to high mortality rates. The adenoma-carcinoma sequence and serrated polyp-carcinoma sequence are the two most common sequences in sporadic colorectal cancer. Genetic alterations in adenomatous polyposis coli (APC), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and tumour protein 53 (TP53) genes are critical in adenoma-carcinoma sequence, whereas v-Raf murine sarcoma viral oncogene homolog B (BRAF) and MutL Homolog1 (MLH1) are driving oncogenes in the serrated polyp-carcinoma sequence. Sporadic mutations in these genes contribute differently to colorectal cancer pathogenesis by introducing distinct alterations in several signalling pathways that rely on the endosome-lysosome system. Unsurprisingly, the endosome-lysosome system plays a pivotal role in the hallmarks of cancer and contributes to specialised colon function. Thus, the endosome-lysosome system might be distinctively influenced by different mutations and these alterations may contribute to the heterogenous nature of sporadic colorectal cancer. This review highlights potential connections between major sporadic colorectal cancer mutations and the diverse pathogenic mechanisms driven by the endosome-lysosome system in colorectal carcinogenesis.

TLR9 Monotherapy in Immune-Competent Mice Suppresses Orthotopic Prostate Tumor Development.

Prostate cancer is ranked second in the world for cancer-related deaths in men, highlighting the lack of effective therapies for advanced-stage disease. Toll-like receptors (TLRs) and immunity have a direct role in prostate cancer pathogenesis, but TLR9 has been reported to contribute to both the progression and inhibition of prostate tumorigenesis. To further understand this apparent disparity, we have investigated the effect of TLR9 stimulation on prostate cancer progression in an immune-competent, syngeneic orthotopic mouse model of prostate cancer. Here, we utilized the class B synthetic agonist CPG-1668 to provoke a TLR9-mediated systemic immune response and demonstrate a significant impairment of prostate tumorigenesis. Untreated tumors contained a high abundance of immune-cell infiltrates. However, pharmacological activation of TLR9 resulted in smaller tumors containing significantly fewer M1 macrophages and T cells. TLR9 stimulation of tumor cells in vitro had no effect on cell viability or its downstream transcriptional targets, whereas stimulation in macrophages suppressed cancer cell growth via type I IFN. This suggests that the antitumorigenic effects of CPG-1668 were predominantly mediated by an antitumor immune response. This study demonstrated that systemic TLR9 stimulation negatively regulates prostate cancer tumorigenesis and highlights TLR9 agonists as a useful therapeutic for the treatment of prostate cancer.

P53 and TLR4 expression are prognostic markers informing progression free survival of advanced stage high grade serous ovarian cancer.

OBJECTIVE: New prognostic biomarkers, and bio-signatures, are urgently needed to facilitate a precision medicine-based approach to more effectively treat patients with high-grade serous ovarian cancer (HGSC). In this study, we analysed the expression patterns of a series of candidate protein biomarkers. METHODS: The panel of markers which included MyD88, TLR4, MAD2, PR, OR, WT1, p53, p16, CD10 and Ki67 was assessed using immunohistochemistry in a tissue microarray (TMA) cohort of n = 80 patients, composed of stage 3-4 HGSCs. Each marker was analysed for their potential to predict both overall survival (OS) and progression-free survival (PFS). RESULTS: TLR4 and p53 were found to be individually predictive of poorer PFS (Log Rank, p = 0.017, p = 0.030 respectively). Cox regression analysis also identified high p53 and TLR4 expression as prognostic factors for reduced PFS (p53; HR=1.785, CI=1.036-3.074, p = 0.037 and TLR4; HR=2.175, CI=1.112-4.253, p = 0.023). Multivariate forward conditional Cox regression analysis, examining all markers, identified a combined signature composed of p53 and TLR4 as prognostic for reduced PFS (p = 0.023). CONCLUSION: Combined p53 and TLR4 marker assessment may help to aid treatment stratification for patients diagnosed with advanced-stage HGSC.

Plasminogen activator inhibitor 1 is associated with high-grade serous ovarian cancer metastasis and is reduced in patients who have received neoadjuvant chemotherapy.

Introduction: High-grade serous ovarian cancer (HGSOC) is the most prevalent and deadliest subtype of epithelial ovarian cancer (EOC), killing over 140,000 people annually. Morbidity and mortality are compounded by a lack of screening methods, and recurrence is common. Plasminogen-activator-inhibitor 1 (PAI-1, the protein product of SERPIN E1) is involved in hemostasis, extracellular matrix (ECM) remodeling, and tumor cell migration and invasion. Overexpression is associated with poor prognosis in EOC. Platelets significantly increase PAI-1 in cancer cells in vitro, and may contribute to the hematogenous metastasis of circulating tumor cells (CTCs). CTCs are viable tumor cells that intravasate and travel through the circulation-often aided by platelets - with the potential to form secondary metastases. Here, we provide evidence that PAI-1 is central to the platelet-cancer cell interactome, and plays a role in the metastatic cascade. Methods: SK-OV-3 cells where PAI-1 had been silenced, treated with healthy donor platelets, and treated with platelet-conditioned medium were used as an in vitro model of metastatic EOC. Gene expression analysis was performed using RNA-Seq data from untreated cells and cells treated with PAI-1 siRNA or negative control, each with and without platelets. Four cohorts of banked patient plasma samples (n = 239) were assayed for PAI-1 by ELISA. Treatment-naïve (TN) whole blood (WB) samples were evaluated for CTCs in conjunction with PAI-1 evaluation in matched plasma. Results and discussion: Significant phenotypic changes occurring when PAI-1 was silenced and when platelets were added to cells were reflected by RNA-seq data, with PAI-1 observed to be central to molecular mechanisms of EOC metastasis. Increased proliferation was observed in cells treated with platelets. Plasma PAI-1 significantly correlated with advanced disease in a TN cohort, and was significantly reduced in a neoadjuvant chemotherapy (NACT) cohort. PAI-1 demonstrated a trend towards significance in overall survival (OS) in the late-stage TN cohort, and correlation between PAI-1 and neutrophils in this cohort was significant. 72.7% (16/22) of TN patients with plasma PAI-1 levels higher than OS cutoff were CTC-positive. These data support a central role for PAI-1 in EOC metastasis, and highlight PAI-1's potential as a biomarker, prognostic indicator, or gauge of treatment response in HGSOC.

TLR7 promotes chronic airway disease in RSV-infected mice.

Respiratory syncytial virus (RSV) commonly infects the upper respiratory tract (URT) of humans, manifesting with mild cold or flu-like symptoms. However, in infants and the elderly, severe disease of the lower respiratory tract (LRT) often occurs and can develop into chronic airway disease. A better understanding of how an acute RSV infection transitions to a LRT chronic inflammatory disease is critically important to improve patient care and long-term health outcomes. To model acute and chronic phases of the disease, we infected wild-type C57BL/6 and toll-like receptor 7 knockout (TLR7 KO) mice with RSV and temporally assessed nasal, airway and lung inflammation for up to 42 days post-infection. We show that TLR7 reduced viral titers in the URT during acute infection but promoted pronounced pathogenic and chronic airway inflammation and hyperreactivity in the LRT. This study defines a hitherto unappreciated molecular mechanism of lower respiratory pathogenesis to RSV, highlighting the potential of TLR7 modulation to constrain RSV pathology to the URT.

Insights into Melanoma Clinical Practice: A Perspective for Future Research.

BACKGROUND: Early diagnosis is the key to improving outcomes for patients with melanoma, and this requires a standardized histological assessment approach. The objective of this survey was to understand the challenges faced by clinicians when assessing melanoma cases, and to provide a perspective for future studies. METHODS: Between April 2022 and February 2023, national and international dermatologists, pathologists, general practitioners, and laboratory managers were invited to participate in a six-question online survey. The data from the survey were assessed using descriptive statistics and qualitative responses. RESULTS: A total of 54 responses were received, with a 51.4% (n = 28) full completion rate. Of the respondents, 96.4% reported ambiguity in their monthly melanoma diagnosis, and 82.1% routinely requested immunohistochemistry (IHC) testing to confirm diagnosis. SOX10 was the most frequently requested marker, and most respondents preferred multiple markers over a single marker. Diagnostic and prognostic tests, as well as therapeutic options and patient management, were all identified as important areas for future research. CONCLUSIONS: The respondents indicated that the use of multiple IHC markers is essential to facilitate diagnostic accuracy in melanoma assessment. Survey responses indicate there is an urgent need to develop new biomarkers for clinical decision making at multiple critical intervention points.

Distinct patterns of biomarker expression for atypical intraductal proliferations in prostate cancer.

High-grade prostatic intraepithelial neoplasia (HGPIN) is a well-characterised precursor lesion in prostate cancer. The term atypical intraductal proliferations (AIP) describes lesions with features that are far too atypical to be considered HGPIN, yet insufficient to be diagnosed as intraductal carcinoma of the prostate (IDCP). Here, a panel of biomarkers was assessed to provide insights into the biological relationship between IDCP, HGPIN, and AIP and their relevance to current clinicopathological recommendations. Tissue samples from 86 patients with prostate cancer were assessed by routine haematoxylin and eosin staining and immunohistochemistry (IHC) with a biomarker panel (Appl1/Sortilin/Syndecan-1) and a PIN4 cocktail (34ßE12+P63/P504S). Appl1 strongly labelled atypical secretory cells, effectively visualising intraductal lesions. Sortilin labelling was moderate-to-strong in > 70% of cases, while Syndecan-1 was moderate-to-strong in micropapillary HGPIN/AIP lesions (83% cases) versus flat/tufting HGPIN (≤ 20% cases). Distinct biomarker labelling patterns for atypical intraductal lesions of the prostate were observed, including early atypical changes (flat/tufting HGPIN) and more advanced atypical changes (micropapillary HGPIN/AIP). Furthermore, the biomarker panel may be used as a tool to overcome the diagnostic uncertainty surrounding AIP by supporting a definitive diagnosis of IDCP for such lesions displaying the same biomarker pattern as cribriform IDCP.

Identifying suicide documentation in clinical notes through zero-shot learning.

Background and Aims: In deep learning, a major difficulty in identifying suicidality and its risk factors in clinical notes is the lack of training samples given the small number of true positive instances among the number of patients screened. This paper describes a novel methodology that identifies suicidality in clinical notes by addressing this data sparsity issue through zero-shot learning. Our general aim was to develop a tool that leveraged zero-shot learning to effectively identify suicidality documentation in all types of clinical notes. Methods: US Veterans Affairs clinical notes served as data. The training data set label was determined using diagnostic codes of suicide attempt and self-harm. We used a base string associated with the target label of suicidality to provide auxiliary information by narrowing the positive training cases to those containing the base string. We trained a deep neural network by mapping the training documents' contents to a semantic space. For comparison, we trained another deep neural network using the identical training data set labels, and bag-of-words features. Results: The zero-shot learning model outperformed the baseline model in terms of area under the curve, sensitivity, specificity, and positive predictive value at multiple probability thresholds. In applying a 0.90 probability threshold, the methodology identified notes documenting suicidality but not associated with a relevant ICD-10-CM code, with 94% accuracy. Conclusion: This method can effectively identify suicidality without manual annotation.

Dynamic interplay between sortilin and syndecan-1 contributes to prostate cancer progression.

Prostate cancer (PCa) development and progression relies on the programming of glucose and lipid metabolism, and this involves alterations in androgen receptor expression and signalling. Defining the molecular mechanism that underpins this metabolic programming will have direct significance for patients with PCa who have a poor prognosis. Here we show that there is a dynamic balance between sortilin and syndecan-1, that reports on different metabolic phenotypes. Using tissue microarrays, we demonstrated by immunohistochemistry that sortilin was highly expressed in low-grade cancer, while syndecan-1 was upregulated in high-grade disease. Mechanistic studies in prostate cell lines revealed that in androgen-sensitive LNCaP cells, sortilin enhanced glucose metabolism by regulating GLUT1 and GLUT4, while binding progranulin and lipoprotein lipase (LPL) to limit lipid metabolism. In contrast, in androgen-insensitive PC3 cells, syndecan-1 was upregulated, interacted with LPL and colocalised with ß3 integrin to promote lipid metabolism. In addition, androgen-deprived LNCaP cells had decreased expression of sortilin and reduced glucose-metabolism, but increased syndecan-1 expression, facilitating interactions with LPL and possibly ß3 integrin. We report a hitherto unappreciated molecular mechanism for PCa, which may have significance for disease progression and how androgen-deprivation therapy might promote castration-resistant PCa.

Altered endosomal-lysosomal biogenesis in melanoma.

Cutaneous melanoma is the deadliest form of skin neoplasm and its high mortality rates could be averted by early accurate detection. While the detection of melanoma is currently reliant upon melanin visualisation, research into melanosome biogenesis, as a key driver of pathogenesis, has not yielded technology that can reliably distinguish between atypical benign, amelanotic and melanotic lesions. The endosomal-lysosomal system has important regulatory roles in cancer cell biology, including a specific functional role in melanosome biogenesis. Herein, the involvement of the endosomal-lysosomal system in melanoma was examined by pooled secondary analysis of existing gene expression datasets. A set of differentially expressed endosomal-lysosomal genes was identified in melanoma, which were interconnected by biological function. To illustrate the protein expression of the dysregulated genes, immunohistochemistry was performed on samples from patients with cutaneous melanoma to reveal candidate markers. This study demonstrated the dysregulation of Syntenin-1, Sortilin and Rab25 may provide a differentiating feature between cutaneous melanoma and squamous cell carcinoma, while IGF2R may indicate malignant propensity in these skin cancers.

Appl1, Sortilin and Syndecan-1 immunohistochemistry on intraductal carcinoma of the prostate provides evidence of retrograde spread.

The presence of intraductal carcinoma of the prostate (IDCP) correlates with late-stage disease and poor outcomes for patients with prostatic adenocarcinoma, but the accurate and reliable staging of disease severity remains challenging. Immunohistochemistry (IHC) has been utilised to overcome problems in assessing IDCP morphology, but the current markers have only demonstrated limited utility in characterising the complex biology of this lesion. In a retrospective study of a cohort of patients who had been diagnosed with IDCP, we utilised IHC on radical prostatectomy sections with a biomarker panel of Appl1, Sortilin and Syndecan-1, to interpret different architectural patterns and to explore the theory that IDCP occurs from retrograde spread of high-grade invasive prostatic adenocarcinoma. Cribriform IDCP displayed strong Appl1, Sortilin and Syndecan-1 labelling patterns, while solid IDCP architecture had high intensity Appl1 and Syndecan-1 labelling, but minimal Sortilin labelling. Notably, the expression pattern of the biomarker panel in regions of IDCP was similar to that of adjacent invasive prostatic adenocarcinoma, and also comparable to prostate cancer showing perineural and vascular invasion. The Appl1, Sortilin, and Syndecan-1 biomarker panel in IDCP provides evidence for the model of retrograde spread of invasive prostatic carcinoma into ducts/acini, and supports the inclusion of IDCP into the five-tier Gleason grading system.

Prediction of Prostate Cancer Biochemical and Clinical Recurrence Is Improved by IHC-Assisted Grading Using Appl1, Sortilin and Syndecan-1.

Gleason scoring is used within a five-tier risk stratification system to guide therapeutic decisions for patients with prostate cancer. This study aimed to compare the predictive performance of routine H&E or biomarker-assisted ISUP (International Society of Urological Pathology) grade grouping for assessing the risk of biochemical recurrence (BCR) and clinical recurrence (CR) in patients with prostate cancer. This retrospective study was an assessment of 114 men with prostate cancer who provided radical prostatectomy samples to the Australian Prostate Cancer Bioresource between 2006 and 2014. The prediction of CR was the primary outcome (median time to CR 79.8 months), and BCR was assessed as a secondary outcome (median time to BCR 41.7 months). The associations of (1) H&E ISUP grade groups and (2) modified ISUP grade groups informed by the Appl1, Sortilin and Syndecan-1 immunohistochemistry (IHC) labelling were modelled with BCR and CR using Cox proportional hazard approaches. IHC-assisted grading was more predictive than H&E for BCR (C-statistic 0.63 vs. 0.59) and CR (C-statistic 0.71 vs. 0.66). On adjusted analysis, IHC-assisted ISUP grading was independently associated with both outcome measures. IHC-assisted ISUP grading using the biomarker panel was an independent predictor of individual BCR and CR. Prospective studies are needed to further validate this biomarker technology and to define BCR and CR associations in real-world cohorts.

Proof of concept nanotechnological approach to in vitro targeting of malignant melanoma for enhanced immune checkpoint inhibition.

Immunotherapies, including immune checkpoint inhibitors, have limitations in their effective treatment of malignancies. The immunosuppressive environment associated with the tumor microenvironment may prevent the achievement of optimal outcomes for immune checkpoint inhibitors alone, and nanotechnology-based platforms for delivery of immunotherapeutic agents are increasingly being investigated for their potential to improve the efficacy of immune checkpoint blockade therapy. In this manuscript, nanoparticles were designed with appropriate size and surface characteristics to enhance their retention of payload so that they can transmit their loaded drugs to the tumor. We aimed to enhance immune cell stimulation by a small molecule inhibitor of PD-1/PD-L1 (BMS202) using nanodiamonds (ND). Melanoma cells with different disease stages were exposed to bare NDs, BMS202-NDs or BMS202 alone for 6 h. Following this, melanoma cells were co-cultured with freshly isolated human peripheral blood mononuclear cells (hPBMCs). The effects of this treatment combination on melanoma cells were examined on several biological parameters including cell viability, cell membrane damage, lysosomal mass/pH changes and expression of γHA2X, and caspase 3. Exposing melanoma cells to BMS202-NDs led to a stronger than normal interaction between the hPBMCs and the melanoma cells, with significant anti-proliferative effects. We therefore conclude that melanoma therapy has the potential to be enhanced by non-classical T-cell Immune responses via immune checkpoint inhibitors delivered by nanodiamonds-based nanoparticles.