Combined anti-PD-1 and anti-CTLA-4 checkpoint blockade: Treatment of melanoma and immune mechanisms of action.

Cytotoxic T-lymphocyte associated protein-4 (CTLA-4) and the Programmed Death Receptor 1 (PD-1) are immune checkpoint molecules that are well-established targets of antibody immunotherapies for the management of malignant melanoma. The monoclonal antibodies, Ipilimumab, Pembrolizumab, and Nivolumab, designed to interfere with T cell inhibitory signals to activate immune responses against tumors, were originally approved as monotherapy. Treatment with a combination of immune checkpoint inhibitors may improve outcomes compared to monotherapy in certain patient groups and these clinical benefits may be derived from unique immune mechanisms of action. However, treatment with checkpoint inhibitor combinations also present significant clinical challenges and increased rates of immune-related adverse events. In this review, we discuss the potential mechanisms attributed to single and combined checkpoint inhibitor immunotherapies and clinical experience with their use.

Safeguarding Skin Grafts: An Evidence-Based Summary of Fixation Techniques.

BACKGROUND: Effective skin graft fixation is vital in preventing sheering forces, seroma, and hematoma from compromising graft take. Yet, selecting the ideal technique for securing skin grafts remains a contentious subject, with significant variation in practice existing between surgeons. There is, therefore, benefit to be derived from assessing the literature for evidence-based recommendations to guide the decision-making process. METHODS: A search of Medline and Embase was performed using appropriate key terms, yielding 419 articles. Reference lists were analyzed. Inclusion and exclusion criteria were composed. Level I to III studies, as defined by the Centre for Evidence-Based Medicine, that compared skin graft fixation methods were analyzed. Rayyan QCRI was used for abstract and title screening. After full text screening, 41 studies were included for qualitative analysis. All included randomized control trials (RCTs) were assessed for risk of bias using the Cochrane Risk-of-Bias 2 (ROB2) tool. RESULTS: We identified 4 groups of fixation technique: "tie-over bolster" (TOB), "no TOB," "adhesive glues," and "negative pressure wound therapy" (NPWT). Twelve studies compared TOB with no TOB, with no difference in graft take demonstrated. Sixteen studies compared adhesive glues with traditional methods, with no difference in graft take demonstrated. Thirteen studies compared NPWT with traditional methods, with enhanced graft take demonstrated. Risk of bias was deemed low in 1 of 13 RCTs. CONCLUSIONS: Based on the current evidence, only NPWT is associated with enhanced graft take. However, there is a scarcity of robust level I evidence comparing different fixation techniques, meaning that strong recommendations cannot be made. We propose examples of hypothesis-driven RCTs, in predetermined clinical settings, based on the theoretical benefits of the techniques that would add value to clinical practice.

Anti-cancer pro-inflammatory effects of an IgE antibody targeting the melanoma-associated antigen chondroitin sulfate proteoglycan 4.

Outcomes for half of patients with melanoma remain poor despite standard-of-care checkpoint inhibitor therapies. The prevalence of the melanoma-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4) expression is ~70%, therefore effective immunotherapies directed at CSPG4 could benefit many patients. Since IgE exerts potent immune-activating functions in tissues, we engineer a monoclonal IgE antibody with human constant domains recognizing CSPG4 to target melanoma. CSPG4 IgE binds to human melanomas including metastases, mediates tumoricidal antibody-dependent cellular cytotoxicity and stimulates human IgE Fc-receptor-expressing monocytes towards pro-inflammatory phenotypes. IgE demonstrates anti-tumor activity in human melanoma xenograft models engrafted with human effector cells and is associated with enhanced macrophage infiltration, enriched monocyte and macrophage gene signatures and pro-inflammatory signaling pathways in the tumor microenvironment. IgE prolongs the survival of patient-derived xenograft-bearing mice reconstituted with autologous immune cells. No ex vivo activation of basophils in patient blood is measured in the presence of CSPG4 IgE. Our findings support a promising IgE-based immunotherapy for melanoma.

B cell profiles, antibody repertoire and reactivity reveal dysregulated responses with autoimmune features in melanoma.

B cells are known to contribute to the anti-tumor immune response, especially in immunogenic tumors such as melanoma, yet humoral immunity has not been characterized in these cancers to detail. Here we show comprehensive phenotyping in samples of circulating and tumor-resident B cells as well as serum antibodies in melanoma patients. Memory B cells are enriched in tumors compared to blood in paired samples and feature distinct antibody repertoires, linked to specific isotypes. Tumor-associated B cells undergo clonal expansion, class switch recombination, somatic hypermutation and receptor revision. Compared with blood, tumor-associated B cells produce antibodies with proportionally higher levels of unproductive sequences and distinct complementarity determining region 3 properties. The observed features are signs of affinity maturation and polyreactivity and suggest an active and aberrant autoimmune-like reaction in the tumor microenvironment. Consistent with this, tumor-derived antibodies are polyreactive and characterized by autoantigen recognition. Serum antibodies show reactivity to antigens attributed to autoimmune diseases and cancer, and their levels are higher in patients with active disease compared to post-resection state. Our findings thus reveal B cell lineage dysregulation with distinct antibody repertoire and specificity, alongside clonally-expanded tumor-infiltrating B cells with autoimmune-like features, shaping the humoral immune response in melanoma.

Influencing tumor-associated macrophages in malignant melanoma with monoclonal antibodies.

The application of monoclonal antibodies (mAbs) for the treatment of melanoma has significantly improved the clinical management of this malignancy over the last decade. Currently approved mAbs for melanoma enhance T cell effector immune responses by blocking immune checkpoint molecules PD-L1/PD-1 and CTLA-4. However, more than half of patients do not benefit from treatment. Targeting the prominent myeloid compartment within the tumor microenvironment, and in particular the ever-abundant tumor-associated macrophages (TAMs), may be a promising strategy to complement existing therapies and enhance treatment success. TAMs are a highly diverse and plastic subset of cells whose pro-tumor properties can support melanoma growth, angiogenesis and invasion. Understanding of their diversity, plasticity and multifaceted roles in cancer forms the basis for new promising TAM-centered treatment strategies. There are multiple mechanisms by which macrophages can be targeted with antibodies in a therapeutic setting, including by depletion, inhibition of specific pro-tumor properties, differential polarization to pro-inflammatory states and enhancement of antitumor immune functions. Here, we discuss TAMs in melanoma, their interactions with checkpoint inhibitor antibodies and emerging mAbs targeting different aspects of TAM biology and their potential to be translated to the clinic.

Enriched circulating and tumor-resident TGF-ß+ regulatory B cells in patients with melanoma promote FOXP3+ Tregs.

B cells are emerging as key players of anti-tumor adaptive immune responses. We investigated regulatory and pro-inflammatory cytokine-expressing B cells in patients with melanoma by flow cytometric intracellular cytokine, CyTOF, transcriptomic, immunofluorescence, single-cell RNA-seq, and B:T cell co-culture analyses. We found enhanced circulating regulatory (TGF-ß+ and PD-L1+) and reduced pro-inflammatory TNF-α+ B cell populations in patients compared with healthy volunteers (HVs), including lower IFN-γ+:IL-4+ and higher TGF-ß+:TNF-α+ B cell ratios in patients. TGF-ß-expressing B cells in the melanoma tumor microenvironment assembled in clusters and interacted with T cells via lymphoid recruitment (SELL, CXCL13, CCL4, CD74) signals and with Tregs via CD47:SIRP-γ, and FOXP3-promoting Galectin-9:CD44. While reduced in tumors compared to blood, TNF-α-expressing B cells engaged in crosstalk with Tregs via TNF-α signaling and the ICOS/ICOSL axis. Patient-derived B cells promoted FOXP3+ Treg differentiation in a TGF-ß-dependent manner, while sustaining expression of IFN-γ and TNF-α by autologous T-helper cells and promoting T-helper cell proliferation ex vivo, an effect further enhanced with anti-PD-1 checkpoint blockade. Our findings reveal cytokine-expressing B cell compartments skewed toward regulatory phenotypes in patient circulation and melanoma lesions, intratumor spatial localization, and bidirectional crosstalk between B and T cell subsets with immunosuppressive attributes.

BRAF inhibitors and their immunological effects in malignant melanoma.

INTRODUCTION: The treatment of cutaneous melanoma has been revolutionized by the development of small-molecule inhibitors targeting the MAPK pathway, including inhibitors of BRAF (BRAFi) and MEK (MEKi), and immune checkpoint blockade antibodies, occurring in tandem. Despite these advances, the 5-year survival rate for patients with advanced melanoma remains only around 50%. Although not designed to alter immune responses within the tumor microenvironment (TME), MAPK pathway inhibitors (MAPKi) exert a range of effects on the host immune compartment that may offer opportunities for therapeutic interventions. AREAS COVERED: We review the effects of MAPKi, especially BRAFi, on the TME, focusing on alterations in inflammatory cytokine secretion, recruitment of immune cells and their functions, both during response to BRAFi treatment and as resistance develops. We outline potential combinations of MAPKi with established and experimental treatments. EXPERT OPINION: MAPKi in combination or in sequence with established treatments such as checkpoint inhibitors, anti-angiogenic agents, or new therapies such as adoptive cell therapies, may augment their immunological effects, reverse tumor-associated immune suppression, and offer the prospect of longer-lived clinical responses. Refining therapeutic tools at our disposal and embracing 'old friends' in the melanoma treatment arsenal, alongside new target identification, may improve the chances of therapeutic success.

B Cells in Patients With Melanoma: Implications for Treatment With Checkpoint Inhibitor Antibodies.

The contributions of the humoral immune response to melanoma are now widely recognized, with reports of positive prognostic value ascribed to tumor-infiltrating B cells (TIL-B) and increasing evidence of B cells as key predictors of patient response to treatment. There are disparate views as to the pro- and anti-tumor roles of B cells. B cells appear to play an integral role in forming tumor-associated tertiary lymphoid structures (TLSs) which can further modulate T cell activation. Expressed antibodies may distinctly influence tumor regulation in the tumor microenvironment, with some isotypes associated with strong anti-tumor immune response and others with progressive disease. Recently, B cells have been evaluated in the context of cancer immunotherapy. Checkpoint inhibitors (CPIs), targeting T cell effector functions, have revolutionized the management of melanoma for many patients; however, there remains a need to accurately predict treatment responders. Increasing evidence suggests that B cells may not be simple bystanders to CPI immunotherapy. Mature and differentiated B cell phenotypes are key positive correlates of CPI response. Recent evidence also points to an enrichment in activatory B cell phenotypes, and the contribution of B cells to TLS formation may facilitate induction of T cell phenotypes required for response to CPI. Contrastingly, specific B cell subsets often correlate with immune-related adverse events (irAEs) in CPI. With increased appreciation of the multifaceted role of B cell immunity, novel therapeutic strategies and biomarkers can be explored and translated into the clinic to optimize CPI immunotherapy in melanoma.

Reduced somatosensory cortical activation in experimental models of neuropathic fecal incontinence.

PURPOSE: Alteration in cortical awareness may be the underlying abnormality in patients with neuropathic fecal incontinence. The cortical effects of inferior rectal nerve injury were determined using somatosensory evoked cortical potential recordings in an experimental model of neuropathic fecal incontinence. METHODS: Eighteen female virgin Wistar rats were assigned equally to one of three groups: an unoperated control group, a nerve crush group (positive control), and a nerve balloon compression group. Four weeks following the injury, all animals underwent somatosensory evoked cortical potential recordings. Following this, the inferior rectal nerve was harvested, resin-embedded, sectioned (1 microm thickness), and axonal counts and axonal cross-sectional areas were analyzed using Scion Image software. RESULTS: Somatosensory evoked cortical potentials were reduced in the nerve crush and balloon compression groups compared with controls (P = 0.024, P = 0.03, respectively). The inferior rectal nerve was harvested in 14 of the 18 animals (4 control, 5 nerve crush, 5 balloon compression). There were no differences in median inferior rectal nerve total axonal counts (P = 0.69) or in the frequency distribution of axonal cross-sectional area between groups (control vs. nerve crush and control vs. balloon compression: P = 0.92, P = 0.17, respectively). CONCLUSIONS: Somatosensory evoked cortical potential amplitude is reduced following crush or compression injury to the inferior rectal nerve. In neuropathic fecal incontinence, alteration in cortical awareness may be the result of processing modification at a central and not peripheral level.

A New Approach to an Old Flap: A Technique to Augment Venous Drainage from the Paramedian Forehead Flap.

The paramedian forehead flap is commonly used in nasal reconstruction, and survival of the distal part of the flap is usually essential for a good cosmetic outcome. Venous congestion leading to tissue necrosis is a recognized complication with this flap. The standard paramedian forehead flap is designed with a number of aims. These are to include the supratrochlear artery, to maximize mobility of the flap pedicle, to maximize the reach of the flap, and to minimize cosmetic implications at the donor site. The supratrochlear artery does not possess sizable venae comitantes; thus, the main pathway for venous drainage of the paramedian forehead flap is through superficial veins. The pattern and location of the superficial veins varies and therefore a standard skin pedicle design cannot be expected to always include sufficient veins to prevent venous congestion and subsequent flap necrosis. This article demonstrates the superficial venous anatomy of the forehead using computed tomographic venography, clinical demonstration, and cadaveric dissection, and describes a technique that can be carried out to augment flap venous drainage by performing careful dissection to identify additional superficial veins at the margins of the flap skin pedicle. One or more veins can then be mobilized and included with the flap pedicle to augment its venous drainage. Use of this technique should lead to a lower incidence of flap necrosis secondary to venous congestion.

Combining Immune Checkpoint Inhibitors: Established and Emerging Targets and Strategies to Improve Outcomes in Melanoma.

The immune system employs several checkpoint pathways to regulate responses, maintain homeostasis and prevent self-reactivity and autoimmunity. Tumor cells can hijack these protective mechanisms to enable immune escape, cancer survival and proliferation. Blocking antibodies, designed to interfere with checkpoint molecules CTLA-4 and PD-1/PD-L1 and counteract these immune suppressive mechanisms, have shown significant success in promoting immune responses against cancer and can result in tumor regression in many patients. While inhibitors to CTLA-4 and the PD-1/PD-L1 axis are well-established for the clinical management of melanoma, many patients do not respond or develop resistance to these interventions. Concerted efforts have focused on combinations of approved therapies aiming to further augment positive outcomes and survival. While CTLA-4 and PD-1 are the most-extensively researched targets, results from pre-clinical studies and clinical trials indicate that novel agents, specific for checkpoints such as A2AR, LAG-3, IDO and others, may further contribute to the improvement of patient outcomes, most likely in combinations with anti-CTLA-4 or anti-PD-1 blockade. This review discusses the rationale for, and results to date of, the development of inhibitory immune checkpoint blockade combination therapies in melanoma. The clinical potential of new pipeline therapeutics, and possible future therapy design and directions that hold promise to significantly improve clinical prognosis compared with monotherapy, are discussed.

Routine positron emission tomography and positron emission tomography/computed tomography in melanoma staging with positive sentinel node biopsy is of limited benefit.

Positron emission tomography (PET) is increasingly used for the staging and management of melanoma. The aim of this study was to evaluate the role of PET or PET/ computed tomography (CT) as a routine procedure in patients with positive sentinel node biopsy (SNB). Thirty patients with melanoma of Breslow thickness greater than 1 mm who had PET or PET/CT scans performed within 100 days after a positive SNB were reviewed retrospectively. Two patients (6%) had a positive PET scan, none of which were melanoma related. The first patient had a synchronous neuroendocrine thyroid tumour and the second patient had increased uptake in the chest wall, which proved to be old trauma. Lymph node dissection was positive in five cases (16%). With a median follow-up of 24 months, 21 patients remained disease free. In none of the 30 cases did the early PET scan after a positive SNB alter subsequent melanoma management. The role of PET scanning soon after a positive sentinel node biopsy seems to be of limited benefit. It is questionable whether any imaging is beneficial at this stage. The results of this review suggest that PET scanning might not be indicated for this group of patients.

Assessing response to chemotherapy in metastatic melanoma with FDG PET: Early experience.

OBJECTIVE: The management of metastatic melanoma remains challenging with only modest response rates to chemotherapy but the need to identify the best re-staging techniques remains paramount. This study evaluates our early experience in the use of FDG PET-CT in the assessment of early response to chemotherapy in metastatic melanoma. METHODS: FDG PET-CT was performed at baseline and following two or three cycles of combination or single agent chemotherapy in seven patients. Response was assessed visually as complete, partial metabolic response or progressive disease. RESULTS: There was intense FDG uptake in all metastases at baseline. Following two to three cycles of chemotherapy, there was a complete metabolic response (CMR) in one patient, partial metabolic response (PMR) in two patients and progressive metabolic disease (PMD) in the remaining three patients. Survival was 679 days in the single patient with a CMR, median of 206 and 129 days in the patients with PMR and PMD respectively. CONCLUSION: This pilot study demonstrates the potential use of FDG PET as a biomarker in early response assessment to chemotherapy in metastatic melanoma. PET-CT already plays in integral role in staging high risk melanoma patients and it may also have a promising role in assessing response to current and novel therapies. Further larger studies examining specific therapies and optimal timing are required.

Primary squamous cell carcinoma of the breast: value of positron emission tomography scanning in confirming the diagnosis.

Pure primary squamous cell carcinoma of the breast is rare. Controversy exists as to whether a pure form exists or whether described cases represent extreme squamous metaplasia within an adenocarcinoma. We present a case of pure primary squamous cell carcinoma of the breast confirmed histopathologically with the absence of a distant primary tumor excluded using positron emission tomography. Because of the paucity of guidelines in the literature for this rare tumor, it is difficult to draw firm conclusions regarding the optimal treatment modality or the overall prognosis.

Dual-energy computed tomography: a promising novel preoperative localization study for treatment of primary hyperparathyroidism.

BACKGROUND: The objective of this study was to evaluate dual-energy computed tomography (DE-CT) for preoperative parathyroid tumor (PT) localization in individuals undergoing parathyroidectomy for treatment of primary hyperparathyroidism (PHP). METHODS: DE-CT was evaluated by retrospective review of the clinical and biochemical characteristics, imaging, operative findings, and outcomes for PHP cases undergoing an initial operation at a single center. RESULTS: The accuracy of each preoperative imaging test, based on operative findings and pathological confirmation of removal of a PT from the localized site was: 58% for ultrasound, 75% Tc-99m sestamibi noncontrast single photon emission noncontrast CT, and 75% for DE-CT. DE-CT was able to correctly localize a PT in a 3rd of cases that were nonlocalized. All study patients had normalization of serum calcium and parathyroid hormone levels postoperatively. CONCLUSIONS: DE-CT shows promise for the preoperative PT localization, especially in nonlocalized PHP cases, and warrants further investigation.

PAK4 suppresses PDZ-RhoGEF activity to drive invadopodia maturation in melanoma cells.

Cancer cells are thought to use actin rich invadopodia to facilitate matrix degradation. Formation and maturation of invadopodia requires the co-ordained activity of Rho-GTPases, however the molecular mechanisms that underlie the invadopodia lifecycle are not fully elucidated. Previous work has suggested a formation and disassembly role for Rho family effector p-21 activated kinase 1 (PAK1) however, related family member PAK4 has not been explored. Systematic analysis of isoform specific depletion using in vitro and in vivo invasion assays revealed there are differential invadopodia-associated functions. We consolidated a role for PAK1 in the invadopodia formation phase and identified PAK4 as a novel invadopodia protein that is required for successful maturation. Furthermore, we find that PAK4 (but not PAK1) mediates invadopodia maturation likely via inhibition of PDZ-RhoGEF. Our work points to an essential role for both PAKs during melanoma invasion but provides a significant advance in our understanding of differential PAK function.

IgG subclass switching and clonal expansion in cutaneous melanoma and normal skin.

B cells participate in immune surveillance in human circulation and tissues, including tumors such as melanoma. By contrast, the role of humoral responses in cutaneous immunity is underappreciated. We report circulating skin-homing CD22+CLA+B cells in healthy volunteers and melanoma patients (n = 73) and CD22+ cells in melanoma and normal skin samples (n = 189). Normal and malignant skin featured mature IgG and CD22 mRNA, alongside mRNA for the transiently-expressed enzyme Activation-induced cytidine Deaminase (AID). Gene expression analyses of publically-available data (n = 234 GEO, n = 384 TCGA) confirmed heightened humoral responses (CD20, CD22, AID) in melanoma. Analyses of 51 melanoma-associated and 29 normal skin-derived IgG sequence repertoires revealed lower IgG1/IgGtotal representation compared with antibodies from circulating B cells. Consistent with AID, comparable somatic hypermutation frequencies and class-switching indicated affinity-matured antibodies in normal and malignant skin. A melanoma-associated antibody subset featured shorter complementarity-determining (CDR3) regions relative to those from circulating B cells. Clonal amplification in melanoma-associated antibodies and homology modeling indicated differential potential antigen recognition profiles between normal skin and melanoma sequences, suggesting distinct antibody repertoires. Evidence for IgG-expressing B cells, class switching and antibody maturation in normal and malignant skin and clonally-expanded antibodies in melanoma, support the involvement of mature B cells in cutaneous immunity.