Proteomics of Melanoma Response to Immunotherapy Reveals Mitochondrial Dependence.

Immunotherapy has revolutionized cancer treatment, yet most patients do not respond. Here, we investigated mechanisms of response by profiling the proteome of clinical samples from advanced stage melanoma patients undergoing either tumor infiltrating lymphocyte (TIL)-based or anti- programmed death 1 (PD1) immunotherapy. Using high-resolution mass spectrometry, we quantified over 10,300 proteins in total and ∼4,500 proteins across most samples in each dataset. Statistical analyses revealed higher oxidative phosphorylation and lipid metabolism in responders than in non-responders in both treatments. To elucidate the effects of the metabolic state on the immune response, we examined melanoma cells upon metabolic perturbations or CRISPR-Cas9 knockouts. These experiments indicated lipid metabolism as a regulatory mechanism that increases melanoma immunogenicity by elevating antigen presentation, thereby increasing sensitivity to T cell mediated killing both in vitro and in vivo. Altogether, our proteomic analyses revealed association between the melanoma metabolic state and the response to immunotherapy, which can be the basis for future improvement of therapeutic response.

The macro-metastasis/organ parenchyma interface (MMPI) - A hitherto unnoticed area.

The macro-metastasis/organ parenchyma interface (MMPI) was previously considered an inert anatomical border which sharply separates the affected organ parenchyma from the macro-metastatic tissue. Recently, infiltrative growth of macro-metastases from various primary tumors was described in the brain, liver and lung, with significant impact on survival. Strikingly, the MMPI patterns differed between entities, so that at least nine different patterns were described. The MMPI patterns could be further classified into three major groups: displacing, epithelial and diffuse infiltrating. Additionally, macro-metastases are a source of further tumor cell dissemination in the affected organ; and these intra-organ metastatic dissemination tracks starting from the MMPI also vary depending on the anatomical structures of the colonized organ and influence disease outcome. In spite of their relevance, MMPIs and organ-specific dissemination tracks are still largely overlooked by many clinicians, pathologists and/or researchers. In this review, we aim to address this important issue and enhance our current understanding of the different MMPI patterns and dissemination tracks in the brain, liver and lung.

Inclusion of extranodal extension in the lymph node classification of cutaneous squamous cell carcinoma of the head and neck.

BACKGROUND: The prognostic performance of the recently updated American Joint Committee on Cancer lymph node classification of cutaneous head and neck squamous cell carcinoma (HNSCC) has not been validated. The objective of this study was to assess the prognostic role of extranodal extension (ENE) in cutaneous HNSCC. METHODS: This was a retrospective analysis of 1258 patients with cutaneous HNSCC who underwent surgery with or without adjuvant therapy between 1995 and 2019 at The University of Texas MD Anderson Cancer Center. The primary outcome was disease-specific survival (DSS). Local, regional, and distant metastases-free survival were secondary outcomes. Recursive partitioning analysis (RPA) and a Cox proportional hazards regression model were used to assess the fitness of staging models. RESULTS: No significant differences in 5-year DSS were observed between patients with pathologic lymph node-negative (pN0) disease (67.4%) and those with pN-positive/ENE-negative disease (68.2%; hazard ratio, 1.02; 95% CI, 0.61-1.79) or between patients with pN-positive/ENE-negative disease and those with pN-positive/ENE-positive disease (52.7%; hazard ratio, 0.57; 95% CI, 0.31-1.01). The RPA-derived model achieved better stratification between high-risk patients (category III, ENE-positive with >2 positive lymph nodes) and low-risk patients (category I, pN0; category II, ENE-positive/pN1 and ENE-negative with >2 positive lymph nodes). The performance of the RPA-derived model was better than that of the pathologic TNM classification (Akaike information criterion score, 1167 compared with 1176; Bayesian information criterion score, 1175 compared with 1195). CONCLUSIONS: The number of metastatic lymph nodes and the presence of ENE are independent prognostic factors for DSS in cutaneous HNSCC, and incorporation of these factors in staging systems improves the performance of the American Joint Committee on Cancer lymph node classification.

Elective neck dissection versus observation in patients with head and neck cutaneous squamous cell carcinoma.

BACKGROUND: The survival benefit of elective neck dissection (END) for patients with cutaneous squamous cell carcinoma (cSCC) of the head and neck and no evidence of regional metastasis (cN0) has never been reported. The aim of this study was to determine the effect of END on patient survival. METHODS: The authors included patients with head and neck cSCC who had undergone primary surgery from 1995 to 2017. The primary end point was survival, and the secondary end points were the incidence of occult regional disease and regional disease control. To assess the impact of END on survival, the authors used multivariable Cox proportional hazards models with propensity score and matching techniques for internal validation. RESULTS: A total of 1111 patients presented with no evidence of nodal disease; 173 had END, and 938 were observed. Adjuvant radiotherapy to the neck was administered to 101 patients (9%). END resulted in a 5-year overall survival rate of 52%, whereas the rate was 63% in the observation group (P = .003 [log-rank]). The 5-year disease-free survival rate for patients undergoing END was similar to that for the observation group (73% vs 75%; P = .429). A multivariate regression model showed that the performance of END was not associated with improved rates of overall, disease-specific, or disease-free survival; similarly, among patients with advanced disease (T3-4), those who underwent END did not have improved survival rates. CONCLUSIONS: Among patients with cSCC of the head and neck, observation of the neck nodes resulted in noninferior survival rates in comparison with END at the time of primary surgery. Further studies are required to elucidate the role of END in patients with advanced disease.

Immune-Related Adverse Events (irAEs): Diagnosis, Management, and Clinical Pearls.

PURPOSE OF REVIEW: While immune checkpoint inhibitor (ICI) therapy has improved melanoma patient outcomes, it has also resulted in the rise of unique immune-related adverse events (irAEs). Here, we review and synthesize irAE management recommendations from several oncological societies into a streamlined format to aid in diagnosis and management. We also include clinical pearls highlighting several recent research studies in this field. RECENT FINDINGS: Knowledge of immunotherapy toxicity has continually evolved, and several major oncologic societies have recently released new or updated guidelines. Keeping up with the evolving field of immunotherapy and related toxicities is crucial, because ICI use, in combination with other agents, will only continue to increase and likely result in new and different patterns of irAEs. Providing clear and concise references for clinicians will help ensure proper irAE evaluation and management going forward. We present one such reference here, covering management of common and/or serious irAEs.

TNFSF4 (OX40L) expression and survival in locally advanced and metastatic melanoma.

Immunotherapy with checkpoint inhibitors revolutionized melanoma treatment in both the adjuvant and metastatic setting, yet not all metastatic patients respond, and metastatic disease still often recurs among immunotherapy-treated patients with locally advanced disease. TNFSF4 is a co-stimulatory checkpoint protein expressed by several types of immune and non-immune cells, and was shown in the past to enhance the anti-neoplastic activity of T cells. Here, we assessed its expression in melanoma and its association with outcome in locally advanced and metastatic disease. We used publicly available data from The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE), and RNA sequencing data from anti-PD1-treated patients at Sheba medical center. TNFSF4 mRNA is expressed in melanoma cell lines and melanoma samples, including those with low lymphocytic infiltrates, and is not associated with the ulceration status of the primary tumor. Low expression of TNFSF4 mRNA is associated with worse prognosis in all melanoma patients and in the cohorts of stage III and stage IIIc-IV patients. Low expression of TNFSF4 mRNAs is also associated with worse prognosis in the subgroup of patients with low lymphocytic infiltrates, suggesting that tumoral TNFSF4 is associated with outcome. TNFSF4 expression was not correlated with the expression of other known checkpoint mRNAs. Last, metastatic patients with TNFSF4 mRNA expression within the lowest quartile have significantly worse outcome on anti-PD1 treatment, and a significantly lower response rate to these agents. Our current work points to TNFSF4 expression in melanoma as a potential determinant of prognosis, and warrants further translational and clinical research.

Confidence-Competence Mismatch and Reasons for Failure of Non-Medical Tourniquet Users.

OBJECTIVE: Tourniquet application is a lifesaving skill taught worldwide in first aid bleeding control courses. We observed performance among non-medical users of tourniquets in their confidence, competence, and reasons for failure. METHODS: 179 Israeli military recruits without prior medical training underwent their standard first aid course where they learned Combat Application Tourniquet (CAT; Composite Resources, Rock Hill, SC, USA) use. After course completion, they self-reported confidence in tourniquet use. User performance was assessed 7-14 days later using a HapMed™ mannequin that assessed time, pressure, and blood loss. Competent performance required in aggregate: 1) use with pressure of 200 mmHg or more, 2) hemorrhage volume of less than 638 mL, and 3) correct placement of the tourniquet. For failed performance, a reason for failure was reported independently by both the user and an expert observer. RESULTS: 45 of 179 user performances (25%) were competent. Users who reported high confidence had only a slightly higher chance of achieving competence in tourniquet application (r = 0.17, p = 0.022). The most common reason for failure was excess slack in the CAT's strap (experts 55%, users 39%), and too few turns of the windlass (23% and 31%, respectively) was the second most common reason. Expert and user evaluations had poor agreement (κ = 0.44, 95% CI 0.32-0.56). CONCLUSION: The most common reason for failed use of tourniquets among non-medical users was excess slack in the tourniquet strap. Users self-evaluated their performance inaccurately and demonstrated a confidence-competence mismatch. These pitfalls in performance may help tourniquet instructors improve training of caregivers.

Enhanced sampling of 2D interference patterns.

We propose a simple analysis to improve the resolution of interference patterns which consist of straight fringes. As the pattern is rotated with respect to the detector, each row or column in the camera perceives it in a slightly shifted manner. We support this proposed method by analyzing both simulated and experimental interference patterns, and verify it using an interferogram obtained from a spectrally complex light source. The results imply that this technique could be implemented in different aspects of image analysis common in many fields in physics.

Beyond the diffraction limit via optical amplification.

In a previous article [Astron. Astrophys.561, A118 (2014)], we suggested a method to overcome the diffraction limit behind a telescope. We discuss and extend recent numerical simulations and test whether it is indeed possible to use photon amplification to enhance the angular resolution of a telescope or a microscope beyond the diffraction limit. An essential addition is the proposal to select events with an above-average ratio of stimulated to spontaneous photons. The analysis shows that the diffraction limit of a telescope is surpassed by a factor of 10 for an amplifier gain of 200, if the analysis is restricted to a tenth of the incoming astronomical photons. A gain of 70 is sufficient with a hundredth of the photons. More simulations must be performed to account for the bunching of spontaneous photons.

Does practice make perfect? Prospectively comparing effects of 2 amounts of practice on tourniquet use performance.

INTRODUCTION: Although a lifesaving skill, currently, there is no consensus for the required amount of practice in tourniquet use. We compared the effect of 2 amounts of practice on performance of tourniquet use by nonmedical personnel. METHODS: Israeli military recruits without previous medical training underwent their standard tactical first aid course, and their initial performance in use of the Combat Application Tourniquet (CAT; Composite Resources, Rock Hill, SC) was assessed. The educational intervention was to allocate the participants into a monthly tourniquet practice program: either a single-application practice (SAP) group or a triple-application practice (TAP) group. Each group practiced according to its program. After 3 months, the participants' tourniquet use performance was reassessed. Assessments were conducted using the HapMed Leg Tourniquet Trainer (CHI Systems, Fort Washington, PA), a mannequin which measures time and pressure. RESULTS: A total of 151 participants dropped out, leaving 87 in the TAP group and 69 in the SAP group. On initial assessment, the TAP group and the SAP group performed similarly. Both groups improved their performance from the initial to the final assessment. The TAP group improved more than the SAP group in mean application time (faster by 18 vs 8 seconds, respectively; P = .023) and in reducing the proportion of participants who were unable to apply any pressure to the mannequin (less by 18% vs 8%, respectively; P = .009). CONCLUSION: Three applications per monthly practice session were superior to one. This is the first prospective validation of a tourniquet practice program based on objective measurements.

Extending a Helping Hand: A Comparison of Israel Defense Forces Medical Corps Humanitarian Aid Field Hospitals.

BACKGROUND: During the past 6 years the Israel Defense Forces Medical Corps (IDF-MC) deployed three humanitarian delegation field hospitals (HDFHs) in disaster zones around the globe: Haiti (2010), the Philippines (2013), and Nepal (2015). OBJECTIVES: To compare the activity of these HDFHs and the characteristics of the patients they served. METHODS: This retrospective study was based on the HDFHs' operation logs and patients medical records. The study population included both the staff who participated and the patients who were treated in any of the three HDFHs. RESULTS: The Philippine HDFH was a "hybrid" type, i.e., it was integrated with a local hospital. Both the Haitian and the Nepali HDFHs were the "stand-alone" type, i.e., were completely autonomic in resources and in function. The Nepali HDFH had a larger staff, departed from Israel 4 hours earlier and was active 7 hours earlier as compared to the Haitian one. In total, 5465 patients, 55% of them female, were treated in the three HDFHs. In Haiti, Nepal and the Philippines, disaster-related injuries accounted for 66%, 26% and 2% of the cases, respectively. Disaster-related injuries presented mainly in the first days of the HDFHs' activity. CONCLUSIONS: The next HDFH should be planned to care for a significant proportion of routine medical illnesses. The IDF-MC continuous learning process will enable future HDFHs to save more lives as we "extend a helping hand" to foreign populations in crisis.

Color sorting by retinal waveguides.

Light is being detected by the two distinct types of photoreceptors in the human retina: cones and rods. Before light arrives at the photoreceptors, it must traverse the whole retina, along its array of higher-index Müller cells serving as natural waveguides. Here we analyze this optical process of light propagation through Müller cells by two independent optical methods: numerical beam propagation and analytical modal analysis. We show that the structure and refractive index profile of the Müller cells create a unique spatio-spectral distribution of light. This distribution corresponds to the positions and spectral sensitivities of both cones and rods to improve their light absorption.

MYC Induces Immunotherapy and IFNγ Resistance Through Downregulation of JAK2.

Immunotherapy has revolutionized the treatment of advanced melanoma. Because the pathways mediating resistance to immunotherapy are largely unknown, we conducted transcriptome profiling of preimmunotherapy tumor biopsies from patients with melanoma that received PD-1 blockade or adoptive cell therapy with tumor-infiltrating lymphocytes. We identified two melanoma-intrinsic, mutually exclusive gene programs, which were controlled by IFNγ and MYC, and the association with immunotherapy outcome. MYC-overexpressing melanoma cells exhibited lower IFNγ responsiveness, which was linked with JAK2 downregulation. Luciferase activity assays, under the control of JAK2 promoter, demonstrated reduced activity in MYC-overexpressing cells, which was partly reversible upon mutagenesis of a MYC E-box binding site in the JAK2 promoter. Moreover, silencing of MYC or its cofactor MAX with siRNA increased JAK2 expression and IFNγ responsiveness of melanomas, while concomitantly enhancing the effector functions of T cells coincubated with MYC-overexpressing cells. Thus, we propose that MYC plays a pivotal role in immunotherapy resistance through downregulation of JAK2.

Inflammation induced by tumor-associated nerves promotes resistance to anti-PD-1 therapy in cancer patients and is targetable by interleukin-6 blockade.

While the nervous system has reciprocal interactions with both cancer and the immune system, little is known about the potential role of tumor associated nerves (TANs) in modulating anti-tumoral immunity. Moreover, while peri-neural invasion is a well establish poor prognostic factor across cancer types, the mechanisms driving this clinical effect remain unknown. Here, we provide clinical and mechniastic association between TANs damage and resistance to anti-PD-1 therapy. Using electron microscopy, electrical conduction studies, and tumor samples of cutaneous squamous cell carcinoma (cSCC) patients, we showed that cancer cells can destroy myelin sheath and induce TANs degeneration. Multi-omics and spatial analyses of tumor samples from cSCC patients who underwent neoadjuvant anti-PD-1 therapy demonstrated that anti-PD-1 non-responders had higher rates of peri-neural invasion, TANs damage and degeneration compared to responders, both at baseline and following neoadjuvant treatment. Tumors from non-responders were also characterized by a sustained signaling of interferon type I (IFN-I) - known to both propagate nerve degeneration and to dampen anti-tumoral immunity. Peri-neural niches of non-responders were characterized by higher immune activity compared to responders, including immune-suppressive activity of M2 macrophages, and T regulatory cells. This tumor promoting inflammation expanded to the rest of the tumor microenvironment in non-responders. Anti-PD-1 efficacy was dampened by inducing nerve damage prior to treatment administration in a murine model. In contrast, anti-PD-1 efficacy was enhanced by denervation and by interleukin-6 blockade. These findings suggested a potential novel anti-PD-1 resistance drived by TANs damage and inflammation. This resistance mechanism is targetable and may have therapeutic implications in other neurotropic cancers with poor response to anti-PD-1 therapy such as pancreatic, prostate, and breast cancers.

Adenosine-Deaminase-Acting-on-RNA-1 Facilitates T-cell Migration toward Human Melanoma Cells.

The effect of tumor/T-cell interactions on subsequent immune infiltration is undefined. Here, we report that preexposure of melanoma cells to cognate T cells enhanced the chemotaxis of new T cells in vitro. The effect was HLA class I-restricted and IFNγ-dependent, as it was abolished by ß2M-knockdown, MHC-blocking antibodies, JAK1 inhibitors, JAK1-silencing and IFNgR1-blocking antibodies. RNA sequencing (RNA-seq) of 73 melanoma metastases showed a significant correlation between the interferon-inducible p150 isoform of adenosine-deaminase-acting-on-RNA-1 (ADAR1) enzyme and immune infiltration. Consistent with this, cocultures of cognate melanoma/T-cell pairs led to IFNγ-dependent induction of ADAR1-p150 in the melanoma cells, as visualized in situ using dynamic cell blocks, in ovo using fertilized chick eggs, and in vitro with Western blots. ADAR1 staining and RNA-seq in patient-derived biopsies following immunotherapy showed a rise in ADAR1-p150 expression concurrently with CD8+ cell infiltration and clinical response. Silencing ADAR1-p150 abolished the IFNγ-driven enhanced T-cell migration, confirming its mechanistic role. Silencing and overexpression of the constitutive isoform of ADAR1, ADAR1-p110, decreased and increased T-cell migration, respectively. Chemokine arrays showed that ADAR1 controls the secretion of multiple chemokines from melanoma cells, probably through microRNA-mediated regulation. Chemokine receptor blockade eliminated the IFNγ-driven T-cell chemotaxis. We propose that the constitutive ADAR1 downregulation observed in melanoma contributes to immune exclusion, whereas antigen-specific T cells induce ADAR1-p150 by releasing IFNγ, which can drive T-cell infiltration.

Dynamics and segregation of cell-matrix adhesions in cultured fibroblasts.

Here we use time-lapse microscopy to analyse cell-matrix adhesions in cells expressing one of two different cytoskeletal proteins, paxillin or tensin, tagged with green fluorescent protein (GFP). Use of GFP-paxillin to analyse focal contacts and GFP-tensin to study fibrillar adhesions reveals that both types of major adhesion are highly dynamic. Small focal contacts often translocate, by extending centripetally and contracting peripherally, at a mean rate of 19 micrometers per hour. Fibrillar adhesions arise from the medial ends of stationary focal contacts, contain alpha5beta1 integrin and tensin but not other focal-contact components, and associate with fibronectin fibrils. Fibrillar adhesions translocate centripetally at a mean rate of 18 micrometers per hour in an actomyosin-dependent manner. We propose a dynamic model for the regulation of cell-matrix adhesions and for transitions between focal contacts and fibrillar adhesions, with the ability of the matrix to deform functioning as a mechanical switch.

Idiotypic immunization induces immunity to mutated p53 and tumor rejection.

The p53 molecule might serve as a common tumor-associated antigen, as the tumor suppressor gene p53 is mutated and the p53 protein is often over-expressed in tumor cells. We report that effective immunity to p53 can be induced through an idiotypic network by immunization of mice with a monoclonal antibody (PAb-240) specific for mutated p53, or with a peptide derived from the complementarity determining region (CDR) 3 of the variable domain of the light chain (VL) of this antibody. The immunized mice produced IgG antibodies to p53 and mounted a cytotoxic reaction to a tumor line bearing mutated p53. The idiotypically immunized mice were resistant to challenge with the tumor cells. Thus antibodies to p53 might serve as immunogens for activating resistance to some tumors. At the basic level, these findings indicate that a network of p53 immunity may be organized naturally within the immune system.

Improving retinal image resolution with iterative weighted shift-and-add.

High-resolution retinal imaging requires dilating the pupil, and therefore exposing more aberrations that blur the image. We developed an image processing technique that takes advantage of the natural movement of the eye to average out some of the high-order aberrations and to oversample the retina. This method was implemented on a long sequence of retinal images of subjects with normal vision. We were able to resolve the structures of the size of single cells in the living human retina. The improvement of resolution is independent of the acquisition method, as long as the image is not warped during scanning. Consequently, even better results can be expected by implementing this technique on higher-resolution images.

Gut Microbiota and Antitumor Immunity: Potential Mechanisms for Clinical Effect.

Several landmark preclinical studies have shown an association between the gut microbiota and the effectiveness of immunotherapy for cancer. These studies have sparked clinical trials aimed at modulating the gut microbiota in order to improve clinical response rates to immunotherapy. Despite this, the mechanisms through which the gut microbiota influences the effectiveness of immunotherapy are still incompletely characterized. Preclinical and preliminary clinical findings from numerous types of gut microbiota modulation studies, including fecal transplantation, probiotics, consortia, and diet, demonstrate that favorable microbiota modulation is associated with increased intratumoral infiltration of CD8+ effector T cells. This CD8+ T-cell infiltration is often associated with enhanced intratumoral activity of T-helper type 1 cells and dendritic cells and a lower density of immunosuppressive cells. Herein, we discuss how gut microbiota may affect the activity of immune cells by at least three interlacing mechanisms: activation of pattern recognition receptors, molecular mimicry, and impact of metabolites. We also discuss the therapeutic potential and limitations of the different gut microbiota modulation techniques and their putative mechanisms of immune activation.

Fecal microbiota transplant promotes response in immunotherapy-refractory melanoma patients.

The gut microbiome has been shown to influence the response of tumors to anti-PD-1 (programmed cell death-1) immunotherapy in preclinical mouse models and observational patient cohorts. However, modulation of gut microbiota in cancer patients has not been investigated in clinical trials. In this study, we performed a phase 1 clinical trial to assess the safety and feasibility of fecal microbiota transplantation (FMT) and reinduction of anti-PD-1 immunotherapy in 10 patients with anti-PD-1-refractory metastatic melanoma. We observed clinical responses in three patients, including two partial responses and one complete response. Notably, treatment with FMT was associated with favorable changes in immune cell infiltrates and gene expression profiles in both the gut lamina propria and the tumor microenvironment. These early findings have implications for modulating the gut microbiota in cancer treatment.