Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer.

BACKGROUND: Tertiary Lymphoid Structures (TLS) correlate with positive outcomes in patients with NSCLC and the efficacy of immune checkpoint blockade (ICB) in cancer. The actin regulatory protein hMENA undergoes tissue-specific splicing, producing the epithelial hMENA11a linked to favorable prognosis in early NSCLC, and the mesenchymal hMENAΔv6 found in invasive cancer cells and pro-tumoral cancer-associated fibroblasts (CAFs). This study investigates how hMENA isoforms in tumor cells and CAFs relate to TLS presence, localization and impact on patient outcomes and ICB response. METHODS: Methods involved RNA-SEQ on NSCLC cells with depleted hMENA isoforms. A retrospective observational study assessed tissues from surgically treated N0 patients with NSCLC, using immunohistochemistry for tumoral and stromal hMENA isoforms, fibronectin, and TLS presence. ICB-treated patient tumors were analyzed using Nanostring nCounter and GeoMx spatial transcriptomics. Multiparametric flow cytometry characterized B cells and tissue-resident memory T cells (TRM). Survival and ICB response were estimated in the cohort and validated using bioinformatics pipelines in different datasets. FINDINGS: Findings indicate that hMENA11a in NSCLC cells upregulates the TLS regulator LTßR, decreases fibronectin, and favors CXCL13 production by TRM. Conversely, hMENAΔv6 in CAFs inhibits LTßR-related NF-kB pathway, reduces CXCL13 secretion, and promotes fibronectin production. These patterns are validated in N0 NSCLC tumors, where hMENA11ahigh expression, CAF hMENAΔv6low, and stromal fibronectinlow are associated with intratumoral TLS, linked to memory B cells and predictive of longer survival. The hMENA isoform pattern, fibronectin, and LTßR expression broadly predict ICB response in tumors where TLS indicates an anti-tumor immune response. INTERPRETATION: This study uncovers hMENA alternative splicing as an unexplored contributor to TLS-related Tumor Immune Microenvironment (TIME) and a promising biomarker for clinical outcomes and likely ICB responsiveness in N0 patients with NSCLC. FUNDING: This work is supported by AIRC (IG 19822), ACC (RCR-2019-23669120), CAL.HUB.RIA Ministero Salute PNRR-POS T4, "Ricerca Corrente" granted by the Italian Ministry of Health.

Association of hearing loss with patient-reported functional outcomes in adult survivors of childhood cancer.

BACKGROUND: Hearing loss is prevalent following ototoxic therapy for childhood cancer. Associations between hearing loss, self-perceived hearing handicap, and functional outcomes have not been examined in survivors. METHODS: Adult survivors treated with platinum or head and neck radiotherapy with hearing loss were recruited. A total of 237 survivors (median age at survey = 37.0 years [range = 30.0-45.0 years]; median = 29.1 years [range = 22.4-35.0 years] since diagnosis; median = 4.0 years [range = 2.9-7.7 years] from last audiogram to survey) completed the Hearing Handicap Inventory for Adults and questionnaires on social and emotional functioning and hearing aid use. Hearing loss severity was defined according to Chang criteria. Multivariable logistic regression models estimated odds ratios (ORs) and 95% confidence intervals (CIs) for associations between hearing loss, hearing handicap, functional outcomes, and hearing aid use with adjustment for sex, race, age at hearing loss diagnosis, and age at survey. RESULTS: Two-thirds of survivors had severe hearing loss, which was associated with increased likelihood of hearing handicap (mild-moderate handicap: OR = 2.72, 95% CI = 1.35 to 5.47; severe handicap: OR = 5.99, 95% CI = 2.72 to 13.18). Survivors with severe hearing handicap had an increased likelihood of social isolation (OR = 8.76, 95% CI = 3.62 to 21.20), depression (OR = 9.11, 95% CI = 3.46 to 24.02), anxiety (OR = 17.57, 95% CI = 3.77 to 81.84), reduced personal income (OR = 2.82, 95% CI = 1.46 to 5.43), and less than full-time employment (OR = 2.47, 95% CI = 1.30 to 4.70). Survivors who did not use a recommended hearing aid were twice as likely to have less than full-time employment (OR = 2.26, 95% CI = 1.10 to 4.61) and reduced personal income (OR = 2.24, 95% CI = 1.08 to 4.63) compared with survivors who wore a hearing aid. CONCLUSION: Self-perceived hearing handicap beyond measured hearing loss is associated with reduced functional outcomes. Assessment of hearing handicap may facilitate targeted interventions in adult survivors with hearing loss.

Organ-specific immunity: A tissue analysis framework for investigating local immune responses to SARS-CoV-2.

Local immune activation at mucosal surfaces, mediated by mucosal lymphoid tissues, is vital for effective immune responses against pathogens. While pathogens like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can spread to multiple organs, patients with coronavirus disease 2019 (COVID-19) primarily experience inflammation and damage in their lungs. To investigate this apparent organ-specific immune response, we develop an analytical framework that recognizes the significance of mucosal lymphoid tissues. This framework combines histology, immunofluorescence, spatial transcript profiling, and mathematical modeling to identify cellular and gene expression differences between the lymphoid tissues of the lung and the gut and predict the determinants of those differences. Our findings indicate that mucosal lymphoid tissues are pivotal in organ-specific immune response to SARS-CoV-2, mediating local inflammation and tissue damage and contributing to immune dysfunction. The framework developed here has potential utility in the study of long COVID and may streamline biomarker discovery and treatment design for diseases with differential pathologies at the organ level.

CD39 and LDHA affects the prognostic role of NLR in metastatic melanoma patients treated with immunotherapy.

BACKGROUND: Identifying response markers is highly needed to guide the treatment strategy in patients with metastatic melanoma. METHODS: A retrospective study was carried out in patients with unresectable/metastatic melanoma (stage IIIb-IV), treated with anti-PD-1 in the first line setting, to better explore the role and the timing of neutrophil/lymphocyte ratio (NLR) as potential biomarker of response. The relationship of NLR with inflammation-immune mediators and the underlying negative effect of raising NLR during immunotherapy, have been investigated with transcriptomic gene analysis. RESULTS: The results confirmed previous findings that a high baseline NLR is associated with a poorer prognosis and with higher serum level of lactate dehydrogenase (LDH), regardless of the presence of brain metastases. The transcriptomic analysis showed that high baseline NLR is associated with a characteristic gene signature CCNA1, LDHA and IL18R1, which correlates with inflammation and tumorigenesis. Conversely, low baseline NLR is associated with the signature CD3, SH2D1A, ZAP70 and CD45RA, linked to the immune-activation. The genes positively associated with NLR (CD39 (ENTPD1), PTEN, MYD88, MMP9 and LDH) are involved in processes of immunosuppression, inflammation and tumor-promoting activity. Increased expression of CD39 correlated with TGFß2, a marker of the N2 neutrophils with immunosuppressive activity. CONCLUSIONS: These results suggest that increasing NLR is associated with an increased neutrophil population, with polarization to the N2 phenotype, and this process may be the basis for the negatively prognostic role of NLR.

Building smart CAR T cell therapies: The path to overcome current challenges.

Successful implementation of adoptive cell therapy (ACT) of cancer requires comprehensively addressing biological and practical challenges. This approach has been largely overlooked, resulting in a gap between the potential of ACT and its actual effectiveness. We summarize the most promising technical strategies in creating an "ideal" ACT product, focusing on chimeric antigen receptor (CAR)-engineered cells. Since many requirements for effective ACT are common to most cancers, what we outline here might have a broader impact.

hMENA isoforms regulate cancer intrinsic type I IFN signaling and extrinsic mechanisms of resistance to immune checkpoint blockade in NSCLC.

BACKGROUND: Understanding how cancer signaling pathways promote an immunosuppressive program which sustains acquired or primary resistance to immune checkpoint blockade (ICB) is a crucial step in improving immunotherapy efficacy. Among the pathways that can affect ICB response is the interferon (IFN) pathway that may be both detrimental and beneficial. The immune sensor retinoic acid-inducible gene I (RIG-I) induces IFN activation and secretion and is activated by actin cytoskeleton disturbance. The actin cytoskeleton regulatory protein hMENA, along with its isoforms, is a key signaling hub in different solid tumors, and recently its role as a regulator of transcription of genes encoding immunomodulatory secretory proteins has been proposed. When hMENA is expressed in tumor cells with low levels of the epithelial specific hMENA11a isoform, identifies non-small cell lung cancer (NSCLC) patients with poor prognosis. Aim was to identify cancer intrinsic and extrinsic pathways regulated by hMENA11a downregulation as determinants of ICB response in NSCLC. Here, we present a potential novel mechanism of ICB resistance driven by hMENA11a downregulation. METHODS: Effects of hMENA11a downregulation were tested by RNA-Seq, ATAC-Seq, flow cytometry and biochemical assays. ICB-treated patient tumor tissues were profiled by Nanostring IO 360 Panel enriched with hMENA custom probes. OAK and POPLAR datasets were used to validate our discovery cohort. RESULTS: Transcriptomic and biochemical analyses demonstrated that the depletion of hMENA11a induces IFN pathway activation, the production of different inflammatory mediators including IFNß via RIG-I, sustains the increase of tumor PD-L1 levels and activates a paracrine loop between tumor cells and a unique macrophage subset favoring an epithelial-mesenchymal transition (EMT). Notably, when we translated our results in a clinical setting of NSCLC ICB-treated patients, transcriptomic analysis revealed that low expression of hMENA11a, high expression of IFN target genes and high macrophage score identify patients resistant to ICB therapy. CONCLUSIONS: Collectively, these data establish a new function for the actin cytoskeleton regulator hMENA11a in modulating cancer cell intrinsic type I IFN signaling and extrinsic mechanisms that promote protumoral macrophages and favor EMT. These data highlight the role of actin cytoskeleton disturbance in activating immune suppressive pathways that may be involved in resistance to ICB in NSCLC.

Evaluation of a clinical method for selective electrode deactivation in cochlear implant programming.

Background: Cochlear implants are a neural prosthesis used to restore the perception of hearing in individuals with severe-to-profound hearing loss by stimulating the auditory nerve with electrical current through a surgically implanted electrode array. The integrity of the interface between the implanted electrode array and the auditory nerve contributes to the variability in outcomes experienced by cochlear implant users. Strategies to identify and eliminate poorly encoding electrodes have been found to be effective in improving outcomes with the device, but application is limited in a clinical setting. Objective: The purpose of this study was to evaluate a clinical method used to identify and selectively deactivate cochlear implants (CI) electrodes related to poor electrode-neural interface. Methods: Thirteen adult CI users participated in a pitch ranking task to identify indiscriminate electrode pairs. Electrodes associated with indiscriminate pairs were selectively deactivated, creating an individualized experimental program. Speech perception was evaluated in the baseline condition and with the experimental program before and after an acclimation period. Participant preference responses were recorded at each visit. Results: Statistically significant improvements using the experimental program were found in at least one measure of speech perception at the individual level in four out of 13 participants when tested before acclimation. Following an acclimation period, ten out of 13 participants demonstrated statistically significant improvements in at least one measure of speech perception. Statistically significant improvements were found with the experimental program at the group level for both monosyllabic words (p = 0.006) and sentences in noise (p = 0.020). Additionally, ten participants preferred the experimental program prior to the acclimation period and eleven preferred the experimental program following the acclimation period. Conclusion: Results from this study suggest that electrode deactivation may yield improvement in speech perception following an acclimation period. A majority of CI users in our study reported a preference for the experimental program. This method proved to be a suitable clinical strategy for identifying and deactivating poorly encoding electrodes in adult CI users.

Distinct spatial immune microlandscapes are independently associated with outcomes in triple-negative breast cancer.

The utility of spatial immunobiomarker quantitation in prognostication and therapeutic prediction is actively being investigated in triple-negative breast cancer (TNBC). Here, with high-plex quantitative digital spatial profiling, we map and quantitate intraepithelial and adjacent stromal tumor immune protein microenvironments in systemic treatment-naïve (female only) TNBC to assess the spatial context in immunobiomarker-based prediction of outcome. Immune protein profiles of CD45-rich and CD68-rich stromal microenvironments differ significantly. While they typically mirror adjacent, intraepithelial microenvironments, this is not uniformly true. In two TNBC cohorts, intraepithelial CD40 or HLA-DR enrichment associates with better outcomes, independently of stromal immune protein profiles or stromal TILs and other established prognostic variables. In contrast, intraepithelial or stromal microenvironment enrichment with IDO1 associates with improved survival irrespective of its spatial location. Antigen-presenting and T-cell activation states are inferred from eigenprotein scores. Such scores within the intraepithelial compartment interact with PD-L1 and IDO1 in ways that suggest prognostic and/or therapeutic potential. This characterization of the intrinsic spatial immunobiology of treatment-naïve TNBC highlights the importance of spatial microenvironments for biomarker quantitation to resolve intrinsic prognostic and predictive immune features and ultimately inform therapeutic strategies for clinically actionable immune biomarkers.

Multiplatform Analysis of Intratumoral PTEN Heterogeneity in Melanoma.

Loss of protein expression of the tumor suppressor PTEN is associated with increased cancer aggressiveness, decreased tumor immune infiltration, and resistance to immune and targeted therapies in melanoma. We assessed a unique cohort of eight melanoma samples with focal loss of PTEN protein expression to understand the features and mechanisms of PTEN loss in this disease. We compared the PTEN-negative (PTEN[-]) areas to their adjacent PTEN-positive (PTEN[+]) areas using DNA sequencing, DNA methylation, RNA expression, digital spatial profiling, and immunohistochemical platforms. Variations or homozygous deletions of PTEN were identified in PTEN(-) areas that were not detected in the adjacent PTEN(+) areas in three cases (37.5%), but no clear genomic or DNA methylation basis for loss was identified in the remaining PTEN(-) samples. RNA expression data from two independent platforms identified a consistent increase in chromosome segregation gene expression in PTEN(-) versus adjacent PTEN(+) areas. Proteomic analysis showed a relative paucity of tumor-infiltrating lymphocytes in PTEN(-) versus adjacent PTEN(+) areas. The findings add to our understanding of potential molecular intratumoral heterogeneity in melanoma and the features associated with the loss of PTEN protein in this disease.

Highly Multiplexed Spatially Resolved Proteomic and Transcriptional Profiling of the Glioblastoma Microenvironment Using Archived Formalin-Fixed Paraffin-Embedded Specimens.

Glioblastoma is a heterogeneous tumor for which effective treatment options are limited and often insufficient. Few studies have examined the intratumoral transcriptional and proteomic heterogeneity of the glioblastoma microenvironment to characterize the spatial distribution of potential molecular and cellular therapeutic immunooncology targets. We applied an integrated multimodal approach comprised of NanoString GeoMx Digital Spatial Profiling, single-cell RNA-seq (scRNA-seq), and expert neuropathologic assessment to characterize archival formalin-fixed paraffin-embedded glioblastoma specimens. Clustering analysis and spatial cluster maps highlighted the intratumoral heterogeneity of each specimen. Mixed cell deconvolution analysis revealed that neoplastic and vascular cells were the prominent cell types throughout each specimen, with macrophages, oligodendrocyte precursors, neurons, astrocytes, and oligodendrocytes present in lower abundance and illustrated the regional distribution of the respective cellular enrichment scores. The spatial resolution of the actionable immunotherapeutic landscape showed that robust B7H3 gene and protein expression was broadly distributed throughout each specimen and identified STING and VISTA as potential targets. Lastly, we uncovered remarkable variability in VEGFA expression and discovered unanticipated associations between VEGFA, endothelial cell markers, hypoxia, and the expression of immunoregulatory genes, indicative of regionally distinct immunosuppressive microdomains. This work provides an early demonstration of the ability of an integrated panel-based spatial biology approach to characterize and quantify the intrinsic molecular heterogeneity of the glioblastoma microenvironment.

Survey of selective electrode deactivation attitudes and practices by cochlear implant audiologists.

OBJECTIVES: The purpose of this study was to explore clinician attitudes regarding selective electrode deactivation and to investigate the primary methodology used to identify poorly encoded electrodes, deactivate identified electrodes, and measure outcomes. METHODS: An online survey consisting of 32 questions was administered to certified clinical and research cochlear implant (CI) audiologists. Questions asked participants about their demographic information, device programming patterns, and attitudes regarding selective electrode deactivation. RESULTS: Fifty-four audiologists completed the survey. When asked whether they believed selectively deactivating poorly encoded electrodes could improve speech perception outcomes, 43% of respondents selected 'Probably Yes,' 39% selected 'Definitely Yes,' and 18% selected 'Might or Might Not.' Of those who reported deactivating electrodes as part of CI programming, various methodology was reported to identify and deactivate poorly encoding electrodes and evaluate effectiveness of deactivation. General reasons against deactivation were also reported. DISCUSSION: CI audiologists generally believed selective electrode deactivation could be used to improve speech perception outcomes for patients; however, few reported implementing selective electrode deactivation in practice. Among those who do perform selective electrode deactivation, the reported methodology was highly variable. CONCLUSION: These findings support the need for clinical practice guidelines to assist audiologists in performing selective electrode deactivation.

Mutagenicity of the organic fraction of World Trade Center dust.

Most studies of the health effects and chemical characterization of the dust resulting from the catastrophic collapse of the World Trade Center (WTC) on September 11, 2001, have focused on the large inorganic fraction of the dust; however, chemical analyses have identified mutagens and carcinogens in the smaller organic fraction. Here, we determined the mutagenicity of the organic fraction of WTC dust in Salmonella. Only 0.74% of the mass of the particulate matter (PM) <53 µm in diameter was extractable organic matter (EOM). Because the EOM was 10 times more mutagenic in TA100 +S9 than in TA98 +S9 and was negative in TA98 -S9, we inferred, respectively, that polycyclic aromatic hydrocarbons (PAHs) played a role in the mutagenicity and not nitroarenes. In TA98 +S9, the mutagenic potency of the EOM (0.1 revertant/µg EOM) was within the range of EOMs from air and combustion emissions. However, the EOM-based mutagenic potency of the particles (0.0007 revertants/µg PM) was 1-2 orders of magnitude lower than values from a review of 50 combustion emissions and various air samples. We calculated that 37 PAHs analyzed previously in WTC EOM were 5.4% of the EOM mass and 0.04% of the PM mass; some air contained 0.3 µg WTC EOM/m3 (0.02 µg PAHs/m3 ). Populations exposed to WTC dust have elevated levels of prostate and thyroid cancer but not lung cancer. Our data support earlier estimates that PAH-associated cancer risk among this population, for example, PAH-associated lung cancer, was unlikely to be significantly elevated relative to background PAH exposures.

Audiologists' attitudes and practice toward referring for psychosocial intervention with cochlear implant patients.

Background: Hearing loss is associated with a range of poor psychosocial outcomes. Cochlear implants (CI) are an available treatment option for significant hearing loss and have been linked to improved quality of life in patients. Evidence suggests that audiologists lack the skills to appropriately detect, address, and refer for psychosocial needs among patients with hearing loss. The objective of this study is to examine the attitudes and practice patterns related to psychosocial care among audiologists who work with CI users. Methods: A cross-sectional survey was administered to clinical audiologists who work with CI recipients in the United States. The survey evaluated participants' attitudes toward psychosocial services and factors that contribute to their abilities to address the psychosocial needs of their patients. Additionally, participants were surveyed about their practice patterns including the use of psychosocial screeners, clinical protocols regarding psychosocial care, and referral patterns for coordinated psychosocial services. Descriptive statistics were used to summarize survey responses. Results: Sixty-eight audiologists completed the survey. Of these audiologists, a majority (73.6%) held the attitude that most or all CI patients would benefit from psychosocial intervention. Despite clinicians' recognition of psychosocial needs in this population, over 90% of participants reported never screening for psychosocial symptoms. Additionally, a majority of respondents indicated that they seldom refer their patients for psychosocial services, with referrals occurring less than half the time (58%) or never (27%). Additionally, few audiologists reported utilizing protocols or resources for guiding psychosocial practices. Audiologists indicated that the primary factors that influence their psychosocial practices include time available to spend with the patient and their comfort level in counseling. Conclusion: Audiologists working with CI patients recognize the potential benefit of psychosocial intervention in this population. Nevertheless, audiologists encounter barriers in clinical practice which limit their ability to identify and address the psychosocial needs of their patients. Strategies designed to enhance audiologists' capacity to recognize the psychosocial needs of CI users, in addition to improved interprofessional practice on CI teams, implies significant opportunities to improve the provision of patient-centered hearing care.

Baseline gene expression profiling determines long-term benefit to programmed cell death protein 1 axis blockade.

Treatment with immune checkpoint inhibitors has altered the course of malignant melanoma, with approximately half of the patients with advanced disease surviving for more than 5 years after diagnosis. Currently, there are no biomarker methods for predicting outcome from immunotherapy. Here, we obtained transcriptomic information from a total of 105 baseline tumor samples comprising two cohorts of patients with advanced melanoma treated with programmed cell death protein 1 (PD-1)-based immunotherapies. Gene expression profiles were correlated with progression-free survival (PFS) within consecutive clinical benefit intervals (i.e., 6, 12, 18, and 24 months). Elastic net binomial regression models with cross validation were utilized to compare the predictive value of distinct genes across time. Lasso regression was used to generate a signature predicting long-term benefit (LTB), defined as patients who remain alive and free of disease progression at 24 months post treatment initiation. We show that baseline gene expression profiles were consistently able to predict long-term immunotherapy outcomes with high accuracy. The predictive value of different genes fluctuated across consecutive clinical benefit intervals, with a distinct set of genes defining benefit at 24 months compared to earlier outcomes. A 12-gene signature was able to predict LTB following anti-PD-1 therapy with an area under the curve (AUC) equal to 0.92 and 0.74 in the training and validation set, respectively. Evaluation of LTB, via a unique signature may complement objective response classification and characterize the logistics of sustained antitumor immune responses.

Nivolumab serum concentration in metastatic melanoma patients could be related to outcome and enhanced immune activity: a gene profiling retrospective analysis.

BACKGROUND: Nivolumab is an anti-PD-1 antibody approved for treating metastatic melanoma (MM), for which still limited evidence is available on the correlation between drug exposure and patient outcomes. METHODS: In this observational retrospective study, we assessed whether nivolumab concentration is associated with treatment response in 88 patients with MM and if the patient's genetic profile plays a role in this association. RESULTS: We observed a statistically significant correlation between nivolumab serum concentration and clinical outcomes, measured as overall and progression-free survival. Moreover, patients who achieved a clinical or partial response tended to have higher levels of nivolumab than those who reached stable disease or had disease progression. However, the difference was not statistically significant. In particular, patients who reached a clinical response had a significantly higher concentration of nivolumab and presented a distinct genetic signature, with more marked activation of ICOS and other genes involved in effector T-cells mediated proinflammatory pathways. CONCLUSIONS: In conclusion, these preliminary results show that in patients with MM, nivolumab concentration correlates with clinical outcomes and is associated with an increased expression of ICOS and other genes involved in the activation of T effectors cells.

A single-nucleus and spatial transcriptomic atlas of the COVID-19 liver reveals topological, functional, and regenerative organ disruption in patients.

The molecular underpinnings of organ dysfunction in acute COVID-19 and its potential long-term sequelae are under intense investigation. To shed light on these in the context of liver function, we performed single-nucleus RNA-seq and spatial transcriptomic profiling of livers from 17 COVID-19 decedents. We identified hepatocytes positive for SARS-CoV-2 RNA with an expression phenotype resembling infected lung epithelial cells. Integrated analysis and comparisons with healthy controls revealed extensive changes in the cellular composition and expression states in COVID-19 liver, reflecting hepatocellular injury, ductular reaction, pathologic vascular expansion, and fibrogenesis. We also observed Kupffer cell proliferation and erythrocyte progenitors for the first time in a human liver single-cell atlas, resembling similar responses in liver injury in mice and in sepsis, respectively. Despite the absence of a clinical acute liver injury phenotype, endothelial cell composition was dramatically impacted in COVID-19, concomitantly with extensive alterations and profibrogenic activation of reactive cholangiocytes and mesenchymal cells. Our atlas provides novel insights into liver physiology and pathology in COVID-19 and forms a foundational resource for its investigation and understanding.

RNA profiling of circulating tumor cells systemically captured from diagnostic leukapheresis products in prostate cancer patients.

The application of circulating tumor cells (CTCs) in both clinical practice and research has been continuously limited by the rare number of targets that can be found in a tube of peripheral blood. Diagnostic leukapheresis (DLA) was used to increase the sampling volume. AdnaTest was used to process the whole leukopak, and the RNAs of captured CTCs was then profiled by NanoString nCounter platform. Spike-in experiments and leukopaks from patients with metastatic prostate cancer were used to validate this new strategy. The whole leukopak was further concentrated five times to reduce the total volume from 150 â€‹mL to 30 â€‹mL, which enabled it to be processed by 3 separate AdnaTest kits. The spike-in experiment demonstrated a reliable capture when there were more than 100 cancer cells/10 â€‹mL of concentrated leukopak. In 1 out of 5 real patient samples, CTCs were only detected in the leukopak, but not in peripheral blood. The RNA profiling of DLA CTCs indicated a more aggressive phenotype of CTCs occurred when the patient was experiencing a disease relapse, even when the serum prostate specific antigen (PSA) level was still relatively low and CTCs in peripheral blood were not detectable. We established a new protocol, integrating DLA, AdnaTest and NanoString nCounter technology, to profile RNAs from CTCs captured from a large blood screening volume. The new protocol can process the whole leukopak with sensitive CTC capture. The RNA profiling of CTCs can provide valuable information for disease monitoring.

Use of High-Plex Data Reveals Novel Insights into the Tumour Microenvironment of Clear Cell Renal Cell Carcinoma.

Although immune checkpoint inhibitors (ICIs) have significantly improved the oncological outcomes, about one-third of patients affected by clear cell renal cell carcinoma (ccRCC) still experience recurrence. Current prognostic algorithms, such as the Leibovich score (LS), rely on morphological features manually assessed by pathologists and are therefore subject to bias. Moreover, these tools do not consider the heterogeneous molecular milieu present in the Tumour Microenvironment (TME), which may have prognostic value. We systematically developed a semi-automated method to investigate 62 markers and their combinations in 150 primary ccRCCs using Multiplex Immunofluorescence (mIF), NanoString GeoMx® Digital Spatial Profiling (DSP) and Artificial Intelligence (AI)-assisted image analysis in order to find novel prognostic signatures and investigate their spatial relationship. We found that coexpression of cancer stem cell (CSC) and epithelial-to-mesenchymal transition (EMT) markers such as OCT4 and ZEB1 are indicative of poor outcome. OCT4 and the immune markers CD8, CD34, and CD163 significantly stratified patients at intermediate LS. Furthermore, augmenting the LS with OCT4 and CD34 improved patient stratification by outcome. Our results support the hypothesis that combining molecular markers has prognostic value and can be integrated with morphological features to improve risk stratification and personalised therapy. To conclude, GeoMx® DSP and AI image analysis are complementary tools providing high multiplexing capability required to investigate the TME of ccRCC, while reducing observer bias.

Concomitant medication of cetirizine in advanced melanoma could enhance anti-PD-1 efficacy by promoting M1 macrophages polarization.

BACKGROUND: The clinical observation showed a potential additive effect of anti-PD-1 agents and cetirizine in patients with advanced melanoma. METHODS: Clinical outcomes of concomitant cetirizine/anti-PD-1 treatment of patients with stage IIIb-IV melanoma were retrospectively collected, and a transcriptomic analysis was performed on blood samples obtained at baseline and after 3 months of treatment. RESULTS: Patients treated with cetirizine concomitantly with an anti-PD-1 agent had significantly longer progression-free survival (PFS; mean PFS: 28 vs 15 months, HR 0.46, 95% CI: 0.28-0.76; p = 0.0023) and OS (mean OS was 36 vs 23 months, HR 0.48, 95% CI: 0.29-0.78; p = 0.0032) in comparison with those not receiving cetirizine. The concomitant treatment was significantly associated with ORR and DCR (p < 0.05). The expression of FCGR1A/CD64, a specific marker of macrophages, was increased after the treatment in comparison with baseline in blood samples from patients receiving cetirizine, but not in those receiving only the anti-PD1, and positively correlated with the expression of genes linked to the interferon pathway such as CCL8 (rho = 0.32; p = 0.0111), IFIT1 (rho = 0.29; p = 0.0229), IFIT3 (rho = 0.57; p < 0.0001), IFI27 (rho = 0.42; p = 0.008), MX1 (rho = 0.26; p = 0.0383) and RSAD2 (rho = 0.43; p = 0.0005). CONCLUSIONS: This retrospective study suggests that M1 macrophage polarization may be induced by cetirizine through the interferon-gamma pathway. This effect may synergize with the immunotherapy of advanced melanoma with anti-PD-1 agents.