The Association Between Excision Margins and Local Recurrence in 11,290 Thin (T1) Primary Cutaneous Melanomas: A Case-Control Study.

BACKGROUND: At presentation, most primary cutaneous melanomas are "thin" (Breslow thickness ≤1 mm, designated T1 in the American Joint Committee on Cancer staging system) and local recurrence (LR) is rare. Most current management guidelines recommend 1 cm surgical excision margins for T1 melanomas, but evidence to support this recommendation is sparse. We sought to identify clinical and pathologic factors associated with LR in patients with T1 melanomas that might guide primary tumor management. METHODS: From a large, prospectively collected, single-institution database, patients with primary cutaneous melanomas ≤1 mm thick diagnosed between 1970 and 2011 who developed LR were identified and matched with controls. Clinical and pathologic parameters were analyzed for their association with LR. RESULTS: From 11,290 primary melanomas ≤1 mm thick, 176 (1.56 %) cases with LR were identified and 176 controls (without LR) were selected. LR occurred after a median time of 37 months (range 3-306 months) and was associated with narrower excision margins (hazard ratio = 0.95, 95 % confidence interval 0.92-0.98, p = 0.001), desmoplastic, acral, and lentigo maligna melanoma subtypes (p = 0.008), and melanomas composed predominantly of spindle cells (p = 0.005). However, Breslow thickness, Clark level, ulceration, mitotic rate, regression, and lymphovascular invasion were not. CONCLUSIONS: LR was associated with <8 mm histologic excision margins (corresponding to <1 cm margins in vivo) and desmoplastic, acral, and lentigo maligna melanoma subtypes. This study provides evidence that a ≥1 cm clinical excision margin for thin (T1) primary melanomas reduces the risk of LR.

Tumor associated macrophages as key contributors and targets in current and future therapies for melanoma.

INTRODUCTION: Despite the success of immunotherapies for melanoma in recent years, there remains a significant proportion of patients who do not yet derive benefit from available treatments. Immunotherapies currently licensed for clinical use target the adaptive immune system, focussing on Tcell interactions and functions. However, the most prevalent immune cells within the tumor microenvironment (TME) of melanoma are macrophages, a diverse immune cell subset displaying high plasticity, to which no current therapies are yet directly targeted. Macrophages have been shown not only to activate the adaptive immune response, and enhance cancer cell killing, but, when influenced by factors within the TME of melanoma, these cells also promote melanoma tumorigenesis and metastasis. AREAS COVERED: We present a review of the most up-to-date literatureavailable on PubMed, focussing on studies from within the last 10 years. We also include data from ongoing and recent clinical trials targeting macrophages in melanoma listed on clinicaltrials.gov. EXPERT OPINION: Understanding the multifaceted role of macrophages in melanoma, including their interactions with immune and cancer cells, the influence of current therapies on macrophage phenotype and functions and how macrophages could be targeted with novel treatment approaches, are all critical for improving outcomes for patients with melanoma.

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.

Senescence evasion in melanoma progression: uncoupling of DNA-damage signaling from p53 activation and p21 expression.

The best-established function of the melanoma-suppressor p16 is mediation of cell senescence, a permanent arrest following cell proliferation or certain stresses. The importance of p16 in melanoma suggests indolence of the other major senescence pathway through p53. Little or no p53 is expressed in senescent normal human melanocytes, but p16-deficient melanocytes can undergo p53-mediated senescence. As p16 expression occurs in nevi but falls with progression toward melanoma, we here investigated whether p53-dependent senescence occurs at some stage and, if not, what defects were detectable in this pathway, using immunohistochemistry. Phosphorylated checkpoint kinase 2 (CHEK2) can mediate DNA-damage signaling, and under some conditions senescence, by phosphorylating and activating p53. Remarkably, we detected no prevalent p53-mediated senescence in any of six classes of lesions. Two separate defects in p53 signaling appeared common: in nevi, lack of p53 phosphorylation by activated CHEK2, and in melanomas, defective p21 upregulation by p53 even when phosphorylated.

The Cake Flap: a new technique for serial excision of benign cutaneous lesions.

Large congenital melanocytic naevi and other circular benign skin lesions are often excised by serial ellipses in stages. With this conventional technique, normal skin is sacrificed when excising 'dog ears', resulting in an elongated scar. The 'Cake Flap' is a new technique for serial excision of benign skin lesions that allows more than half the area to be excised at the first operation without the appearance the 'dog-ears' that would occur if this was done using the serial excision technique commonly employed. We have used the Cake Flap in four patients with congenital melanocytic naevi to good effect. The design overcomes the shortcomings described of traditional elliptical serial excision without the use of tissue expansion.

Malignancy without immortality? Cellular immortalization as a possible late event in melanoma progression.

Cell senescence is a permanent growth arrest following extended proliferation. Cultured cancer cells including metastatic melanoma cells often appear immortal (proliferate indefinitely), while uncultured benign nevi (moles) show senescence markers. Here, with new explantation methods, we investigated which classes of primary pigmented lesions are typically immortal. Nevi yielded a few proliferating cells, consistent with most nevus cells being senescent. No nevus culture (0/28) appeared immortal. Some thin and thick melanoma cultures proved immortal under these conditions, but surprisingly few (4/37). All arrested cultures displayed three senescence markers in some cells: ß-galactosidase, nuclear p16, and heterochromatic foci/aggregates. However, melanoma cultures also showed features of telomeric crisis (arrest because of ultrashort telomeres). Moreover, crisis markers including anaphase bridges were frequent in uncultured vertical growth-phase (VGP) melanomas. Conversely, all immortal melanoma cultures expressed telomerase reverse transcriptase and telomerase, showing aneuploidy. The findings suggest that primary melanomas are typically precrisis, with immortalization/telomere maintenance as a late event.