Faculty Bibliography

Aberrant glycosylation is a hallmark of cancer biology, and altered glycosylation influences multiple facets of melanoma progression. To identify glycosyltransferases, glycans, and glycoproteins essential for melanoma maintenance, we conducted an in vivo growth screen with a pooled short hairpin RNA library of glycosyltransferases, lectin microarray profiling of benign nevus and melanoma samples, and mass spectrometry-based glycoproteomics. We found that α-2,3-sialyltransferases ST3GAL1 and ST3GAL2 and corresponding α-2,3-linked sialosides are up-regulated in melanoma compared to nevi and are essential for melanoma growth. Glycoproteomics revealed that glycoprotein targets of ST3GAL1 and ST3GAL2 are enriched in transmembrane proteins involved in growth signaling, including the amino acid transporter SLC3A2/CD98hc. CD98hc suppression mimicked the effect of ST3GAL1 and ST3GAL2 silencing, inhibiting melanoma cell proliferation. We found that both CD98hc protein stability and its prosurvival effect on melanoma are dependent upon α-2,3-sialylation mediated by ST3GAL1 and ST3GAL2. Our studies reveal α-2,3-sialosides functionally contributing to melanoma maintenance, supporting ST3GAL1 and ST3GAL2 as therapeutic targets in melanoma.

Skin, hair, and eye (oculocutaneous) color is due to melanin, a pigment produced by melanocytes. This review considers processes required for pigmentation and the complex genetic network that regulates them. The first requisite is migration of neural crest-derived melanoblasts, which populate various embryonic sites, then differentiate into melanocytes or seed stem cell niches. Differentiation is marked by expression of genes essential for melanogenesis, which takes place in melanosomes and involves conversion of tyrosine into melanin. Melanosome biogenesis requires premelanosome maturation through coordinated delivery of melanogenic enzymes such as tyrosinase (TYR), structural proteins, and transporters that establish an intraluminal environment conducive to melanogenesis. Sorting of proteins through endolysosomal pathways and delivery to melanosomes is facilitated by trafficking protein complexes. Finally, melanin is transferred to keratinocytes to protect against ultraviolet light. Numerous pigment-related disorders result from disruption of these pathways, including Waardenburg syndrome caused by melanoblast migration disruption, oculocutaneous albinism presenting with absent/reduced melanogenesis, and melanoma resulting from dysregulation of proliferation/survival. Genetic variants also determine normal color variation, which is pronounced across populations that, historically, lived in different geographical regions. This variation, shaped by genetic factors, environmental influences, and evolutionary pressures, underpins the wide range of pigmentation phenotypes seen today.

UNLABELLED:Melanoma being one of the most common and deadliest skin cancers has been increasing since the past decade. Patients at advanced stages of the disease have very poor prognoses, as opposed to at the earlier stages. Nowadays, the standard of care of advanced melanoma is resection, followed by immune checkpoint inhibition-based immunotherapy. However, a substantial proportion of patients either do not respond or develop resistance. This underscores a need for novel approaches and therapeutic targets as well as a better understanding of the mechanisms of melanoma pathogenesis. Long noncoding RNAs (lncRNA) comprise a poorly characterized class of functional players and promising targets in promoting malignancy. Certain lncRNAs have been identified to play integral roles in melanoma progression and drug resistance; however, systematic screens to uncover novel functional lncRNAs are scarce. In this study, we profile differentially expressed lncRNAs in patient-derived short-term metastatic cultures and BRAF-MEK inhibition-resistant cells. We conduct a focused growth-related CRISPR inhibition screen of overexpressed lncRNAs, validate, and functionally characterize lncRNA hits with respect to cellular growth, invasive capacities, and apoptosis in vitro as well as the transcriptomic impact of our lead candidate the novel lncRNA XLOC_030781. In sum, we extend the current knowledge of ncRNAs and their potential relevance in melanoma. SIGNIFICANCE/UNASSIGNED:LncRNAs have emerged as novel players in regulating many cellular aspects also in melanoma. The number of functional significances of most lncRNAs remains elusive. We provide a comprehensive strategy to identify functionally relevant lncRNAs in melanoma by combining expression profiling with CRISPR inhibition growths screens. Our results broaden the characterized lncRNAs as potential targets for future therapeutic applications.

IMPORTANCE/UNASSIGNED:Tumor-infiltrating lymphocytes (TILs) are a provocative biomarker in melanoma, influencing diagnosis, prognosis, and immunotherapy outcomes; however, traditional pathologist-read TIL assessment on hematoxylin and eosin-stained slides is prone to interobserver variability, leading to inconsistent clinical decisions. Therefore, development of newer TIL scoring approaches that produce more reliable and consistent readouts is important. OBJECTIVE/UNASSIGNED:To evaluate the analytical and clinical validity of a machine learning algorithm for TIL quantification in melanoma compared with traditional pathologist-read methods. DESIGN, SETTING, AND PARTICIPANTS/UNASSIGNED:This multioperator, global, multi-institutional prognostic study compared TIL scoring reproducibility between traditional pathologist-read methods and an artificial intelligence (AI)-driven approach. The study was conducted using retrospective cohorts of patients with melanoma between January 2022 and June 2023 across 45 institutions, with tissue evaluated by participants from academic, clinical, and research institutions. Participants were selected to ensure diverse expertise and professional backgrounds. MAIN OUTCOMES AND MEASURES/UNASSIGNED:Intraclass correlation coefficient (ICC) values were calculated for the manual and AI-assisted arms using log-transformed data. Kendall W values were calculated for Clark scores (brisk = 3, nonbrisk = 2, and sparse = 1). Reliabilities of ICC and W values were classified as moderate (0.40-0.60), good (0.61-0.80), or excellent (>0.80). AI TIL measurements were dichotomized using the 16.6 and median cutoffs. Univariable and multivariable Cox regression analyses assessed the prognostic value of TIL scores adjusted for clinicopathologic variables. RESULTS/UNASSIGNED:There were 111 patients with melanoma in the independent testing cohort (median [range] age at diagnosis, 61.0 [25.0-87.0] years; 56 [50.5%] male) who contributed melanoma whole tissue sections. A total of 98 participants evaluated TILs on 60 hematoxylin and eosin-stained melanoma tissue sections. All 40 participants in the manual arm were pathologists, while the AI-assisted arm included 11 pathologists and 47 nonpathologists (scientists). The AI algorithm demonstrated superior reproducibility, with ICCs higher than 0.90 for all machine learning TIL variables, significantly outperforming manual assessments (ICC, 0.61 for AI-derived stromal TILs vs Kendall W, 0.44 for manual Clark TIL scoring). AI-based TIL scores showed prognostic associations with patient outcomes (n = 111) using the median cutoff approach with a hazard ratio (HR) of 0.45 (95% CI, 0.26-0.80; P = .005), and using the cutoff of 16.6, with an HR of 0.56 (95% CI, 0.32-0.98; P = .04). CONCLUSIONS AND RELEVANCE/UNASSIGNED:In this prognostic study of TIL quantification in melanoma, the AI algorithm demonstrated superior reproducibility and prognostic associations compared with traditional methods. Although the retrospective nature of the cohorts limits demonstration of clinical utility, the publicly available dataset and open-source AI tool offer a foundation for future validation and integration into melanoma management.

Mohs micrographic surgery (MMS) is a tissue-sparing surgical technique that is the standard of care for treatment of several cutaneous malignancies. Current US and international guidelines recommend wide local excision as the first-line surgical therapy for noninvasive melanoma, and use of MMS may be appropriate for melanoma-in-situ, lentigo maligna, and potentially thin invasive malignant melanoma. Based on available literature, MMS can potentially result in lower recurrence rates of melanoma, especially when using immunostaining. This chapter explores the existing evidence supporting MMS for treatment of melanoma as well as its challenges.

Chemotherapy-induced alopecia and radiation-induced alopecia, the thinning or loss of hair due to cytotoxic chemotherapy and radiation therapy, respectively, are distressing adverse effects of cancer treatment. Chemotherapy, targeted therapies, and radiation therapy used in pediatric oncology often lead to alopecia by damaging hair follicles, with varying degrees of severity depending on the specific treatment type, mechanism of action, and damage-response pathway involved. Pediatric chemotherapy-induced alopecia, radiation-induced alopecia, and permanent alopecia, defined as hair regrowth that remains incomplete 6 months or more after treatment, have significant negative impacts on mental health, self-esteem, and social interactions, highlighting the need for further research into supportive care strategies. There are currently no standard interventions for chemotherapy-induced alopecia or radiation-induced alopecia in children, with most recommendations limited to gentle hair care and camouflaging techniques during treatment. Scalp cooling has demonstrated safety and efficacy in reducing chemotherapy-induced alopecia in adults and is currently under investigation in children and adolescents. Topical and low-dose oral minoxidil have been studied in children for other hair loss disorders and may improve hair regrowth after chemotherapy or radiation. Increased awareness and continued research into management strategies for pediatric chemotherapy-induced alopecia and radiation-induced alopecia are necessary to help mitigate its significant negative impact on quality of life.

IMPORTANCE/UNASSIGNED:There exists substantial heterogeneity in outcomes within T stages for patients with cutaneous squamous cell carcinoma (cSCC). OBJECTIVE/UNASSIGNED:To determine whether a customized generative pretrained transformer model, trained on a comprehensive dataset with more than 1 trillion parameters and equipped with relevant focused context and retrieval augmented generation (RAG), could excel in aggregating and interpreting vast quantities of data to develop a novel class-based risk stratification system that outperforms the current standards. DESIGN, SETTING, AND PARTICIPANTS/UNASSIGNED:To build the RAG knowledge base, a systematic review of the literature was conducted that addressed risk factors for poor outcomes in cSCC. Using the RAG-enabled generative pretrained transformer (GPT) model, we developed a novel class-based risk stratification system that assigned point values for risk factors, culminating in a GPT-based prognostication system called the artificial intelligence-derived risk score (AIRIS). The system's performance was validated on a combined prospective and retrospective cohort of 2379 primary cSCC tumors (1996-2023) with at least 36 months of follow-up, against Brigham and Women's Hospital (BWH) and American Joint Committee on Cancer Staging Manual, eighth edition (AJCC8) systems in stratifying risk for locoregional recurrence (LR), nodal metastasis (NM), distant metastasis (DM), and disease-specific death (DSD). MAIN OUTCOMES AND MEASURES/UNASSIGNED:Performance metrics evaluated included distinctiveness, homogeneity, and monotonicity, as defined by the AJCC8, as well as sensitivity, specificity, positive predictive value, negative predictive value, accuracy, the area under the receiver operating characteristic curve, and concordance. RESULTS/UNASSIGNED:The median age at diagnosis was 73 (IQR, 64-81) years, with 38.5% female patients and 61.5% male patients. The AIRIS prognostication system demonstrated superior sensitivity across all outcomes (LR, 49.1%; NM, 73.7%; DM, 82.5%; and DSD, 72.2%) and the highest area under the receiver operating characteristic curve values (LR, 0.69; NM, 0.81; DM, 0.85; and DSD, 0.80), indicating significantly enhanced discriminative capability compared with the BWH and AJCC8 systems. While all systems were comparably distinctive, the AIRIS prognostication system consistently demonstrated the lowest proportion of tumors exhibiting poor outcomes in low-risk categories, suggesting its improved homogeneity and monotonicity. CONCLUSIONS AND RELEVANCE/UNASSIGNED:The results of this diagnostic study suggest that the AIRIS system outperforms the existing BWH and AJCC8 prognostication systems, potentially providing a more effective tool for predicting poor outcomes in cSCC. This study illustrates the potential of large language models in refining prognostic tools, offering implications for treating patients with cancer.

Targeted therapies and immunotherapy have improved treatment outcomes for many melanoma patients. However, patients whose melanomas harbor driver mutations in the neurofibromin 1 (NF1) tumor suppressor gene often lack effective targeted treatment options when their tumors do not respond to immunotherapy. In this study, we utilized patient-derived short-term cultures (STCs) and multiomics approaches to identify molecular features that could inform the development of therapies for patients with NF1 mutant melanoma. Differential gene expression analysis revealed that the epidermal growth factor receptor (EGFR) is highly expressed and active in NF1 mutant melanoma cells, where it hyper-activates ERK and AKT, leading to increased tumor cell proliferation, survival, and growth. In contrast, genetic or pharmacological inhibition of EGFR hindered cell proliferation and survival and suppressed tumor growth in patient-derived NF1 mutant melanoma models but not in NF1 wild-type models. These results reveal a connection between NF1 loss and increased EGFR expression that is critical for the survival and growth of NF1 mutant melanoma cells in patient-derived culture and xenograft models, irrespective of their BRAF and NRAS mutation status.

Response to immune checkpoint inhibitors (ICIs) in metastatic melanoma (MM) varies among patients, and current baseline biomarkers predicting treatment outcomes are limited. As mitochondrial (MT) metabolism has emerged as an important regulator of host immune function, we explored the association of host MT genetics (MT haplogroups) with ICI efficacy in 1,225 ICI-treated patients with MM from the clinical trial CheckMate-067 and the International Germline Immuno-Oncology Melanoma Consortium. We discovered and validated significant associations of MT haplogroup T (HG-T) with resistance to anti-programmed cell death protein-1-based ICI (both single-agent and combination) and have shown that HG-T is independent from established tumor predictors. We also found that patients belonging to HG-T exhibit a unique nivolumab-resistant baseline peripheral CD8⁺ T cell repertoire compared to other MT haplogroups, providing, to our knowledge, the first link between MT inheritance, host immunity and ICI resistance. The study proposes a host blood-based biomarker with stand-alone clinical value predicting ICI efficacy and points to an ICI-resistance mechanism associated with MT metabolism, with clinical relevance in immuno-oncology.

BACKGROUND:Lymphovascular invasion (LVI) is regarded as a high-risk feature of cutaneous squamous cell carcinoma (CSCC) but is currently absent from CSCC staging systems. OBJECTIVE:To assess whether LVI serves as an independent predictor of major poor outcomes in CSCC. METHODS:Twelve centers contributed to a multinational CSCC database. Clinical and pathologic risk factors, treatment, and patient outcomes were retrospectively collected. CSCCs were stratified based on LVI status. Tumors that developed major poor outcomes defined as nodal metastasis, in-transit metastasis, distant metastasis, and disease-specific death were identified. RESULTS:A total of 23,166 CSCCs were identified, 179 were LVI+ tumors (0.8%). LVI+ tumors had a higher cumulative incidence of major poor outcomes than those without LVI (33.5% vs. 3.2% at 3 years; overall cumulative incidence function p < 0.001). In an adjusted analysis, LVI+ tumors had an 82% increase in the rate of developing major poor outcomes when compared to LVI- tumors (subdistribution hazard ratio (SHR) = 1.82; p = 0.002). Notably, LVI+ low-stage BWH tumors (T1 or T2a) had a greater cumulative incidence of major poor outcomes compared to LVI- BWH low-stage tumors (20.7% vs. 1.61% at 3 years, overall cumulative incidence function p < 0.001). LIMITATIONS/CONCLUSIONS:Retrospective study design CONCLUSION: The presence of LVI in CSCC is a high-risk feature that is an independent predictor of metastasis and disease-specific death in both low and high BWH stage tumors.