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IntroductionResearch on simplified antibiotic regimens for outpatient treatment of 'Possible Serious Bacterial Infection' (PSBI) and the subsequent World Health Organization (WHO) guidelines provide an opportunity to increase treatment coverage. This multi-country implementation research initiative aimed to learn how to implement the WHO guideline in diverse contexts. These experiences have been individually published; this overview paper provides a summary of results and lessons learned across sites.Methods summaryA common mixed qualitative and quantitative methods protocol for implementation research was used in eleven sites in the Democratic Republic of Congo (Equateur province), Ethiopia (Tigray and Oromia regions), India (Haryana, Himachal Pradesh, Maharashtra, and Uttar Pradesh states), Malawi (Central Region), Nigeria (Kaduna and Oyo states), and Pakistan (Sindh province). Key steps in implementation research were: i) policy dialogue with the national government and key stakeholders, ii) the establishment of a 'Technical Support Unit' with the research team and district level managers, and iii) development of an implementation strategy and its refinement using an iterative process of implementation, programme learning and evaluation.Results summaryAll sites successfully developed and evaluated an implementation strategy to increase coverage of PSBI treatment. During the study period, a total of 6677 young infants from the study catchment area were identified and treated at health facilities in the study area as inpatients or outpatients among 88179 live births identified. The estimated coverage of PSBI treatment was 75.7% (95% CI 74.8% to 78.6%), assuming a 10% incidence of PSBI among all live births. The treatment coverage was variable, ranging from 53.3% in Lucknow, India to 97.3% in Ibadan, Nigeria. The coverage of inpatient treatment ranged from 1.9% in Zaria, Nigeria, to 33.9% in Tigray, Ethiopia. The outpatient treatment coverage ranged from 30.6% in Pune, India, to 93.6% in Zaria, Nigeria. Overall, the case fatality rate (CFR) was 14.6% (95% CI 11.5% to 18.2%) for 0-59-day old infants with critical illness, 1.9% (95% CI 1.5% to 2.4%) for 0-59-day old infants with clinical severe infection and 0.1% for fast breathing in 7-59 days old. Among infants treated as outpatients, CFR was 13.7% (95% CI 8.7% to 20.2%) for 0-59-day old infants with critical illness, 0.9% (95% CI 0.6% to 1.2%) for 0-59-day old infants with clinical severe infection, and 0.1% for infants 7-59 days old with fast breathing.ConclusionImportant lessons on how to conduct each step of implementation research, and the challenges and facilitators for implementation of PSBI management guideline in routine health systems are summarised and discussed. These lessons will be used to introduce and scale-up implementation in relevant Low- and middle-income countries.

Abstract Background The development and spread of artemisinin-resistant Plasmodium falciparum malaria in Greater Mekong Subregion has created impetus for continuing global monitoring of efficacy of artemisinin-based combination therapies (ACTs). This post analyses is aimed to evaluate changes in early treatment response markers 10 years after the adoption of ACTs as first-line treatments of uncomplicated falciparum malaria in Nigeria. Methods At 14 sentinel sites in six geographical areas of Nigeria, we evaluated treatment responses in 1341 children under 5 years and in additional 360 children under 16 years with uncomplicated malaria enrolled in randomized trials of artemether-lumefantrine versus artesunate-amodiaquine at 5-year interval in 2009–2010 and 2014–2015 and at 2-year interval in 2009–2010 and 2012–2015, respectively after deployment in 2005. Results Asexual parasite positivity 1 day after treatment initiation (APPD1) rose from 54 to 62% and 2 days after treatment initiation from 5 to 26% in 2009–2010 to 2014–2015 (P = 0.002 and P 75 000 μl− 1, haematocrit > 27% 1 day post-treatment initiation, treatment with artemether-lumefantrine and enrolment in 2014–2015 independently predicted APPD1. In parallel, Kaplan-Meier estimated risk of recurrent infections by day 28 rose from 8 to 14% (P = 0.005) and from 9 to 15% (P = 0.02) with artemether-lumefantrine and artesunate-amodiaquine, respectively. Mean asexual parasitaemia half-life increased significantly from 1.1 h to 1.3 h within 2 years (P

Abstract Background In acute falciparum malaria, asexual parasite reduction ratio two days post-treatment initiation (PRRD2) ≥ 10 000 per cycle has been used as a measure of the rapid clearance of parasitaemia and efficacy of artemisinin derivatives. However, there is little evaluation of alternative measures; for example, parasite reduction ratio one day after treatment initiation (PRRD1) and its relationship with parasite clearance time (PCT) or PRRD2. This study evaluated the use of PRRD1 as a measure of responsiveness to antimalarial drugs. Methods In acutely malarious children treated with artesunate-amodiaquine (AA), artemether-lumefantrine (AL) or dihydroartemisinin-piperaquine (DHP), the relationships between PRRD1 or PRRD2 and PCT, and between PRRD1 and PRRD2 were evaluated using linear regression. Agreement between estimates of PCT using PRRD1 and PRRD2 linear regression equations was evaluated using the Bland-Altman analysis. Predictors of PRRD1 > 5000 per half cycle and PRRD2 ≥ 10 000 per cycle were evaluated using stepwise multiple logistic regression models. Using the linear regression equation of the relationship between PRRD1 and PCT previously generated in half of the DHP-treated children during the early study phase, PCT estimates were compared in a prospective blinded manner with PCTs determined by microscopy during the later study phase in the remaining half. Results In 919 malarious children, PRRD1 was significantly higher in DHP- and AA-treated compared with AL-treated children (P 15 months, parasitaemia > 10 000/μl and DHP treatment independently predicted PRRD1 > 5000 per half cycle, while age > 30 months, haematocrit ≥31%, body temperature > 37.4 °C, parasitaemia > 100 000/μl, PRRD1 value > 1000 and no gametocytaemia independently predicted PRRD2 ≥ 10 000 per cycle. Using the linear regression equation generated during the early phase in 166 DHP-treated children, PCT estimates and PCTs determined by microscopy in the 155 children in the later phase were similar in the same patients. Conclusions PRRD1 and estimates of PCT using PRRD1 linear regression equation of PRRD1 and PCT can be used in therapeutic efficacy studies. Trial registration Pan African Clinical Trial Registration PACTR201709002064150, 1 March 2017, http://www.pactr.org

Abstract Background Artemisinin-based combination therapies (ACTs) have remained efficacious treatments of acute falciparum malaria in many endemic areas but there is little evaluation of factors contributing to the anaemia of acute falciparum malaria following long term adoption of ACTs as first-line antimalarials in African children. Methods Malarious 4% were evaluated by stepwise multiple logistic regression models. Survival analysis and kinetics of DAFH were evaluated by Kaplan-Meier estimator and non-compartment model, respectively. Results Pre-treatment, 355 of 959 children were anaemic. Duration of illness >2 days and parasitaemia ≤10,000 μL−1 were independent predictors of anaemia pre-treatment. EAA occurred in 301 of 604 children. Predictors of EAA were age ≤ 15 months, history of fever pre-treatment and enrolment haematocrit ≤35%. The probabilities of progression from normal haematocrit to EAA were similar for all treatments. MAFH >4% occurred in 446 of 694 children; its predictors were anaemia pre-treatment, enrolment parasitaemia ≤50,000 μL−1, parasitaemia one day post-treatment initiation and gametocytaemia. DAFH >4% occurred in 334 of 719 children; its predictors were history of fever pre-and fever 1 day post-treatment initiation, haematocrit ≥37%, and parasitaemia >100,000 μL−1. In 432 children, declines in DAFH deficits were monoexponential with overall estimated half-time of 2.2d (95% CI 1.9–2.6). Area under curve of deficits in DAFH versus time and estimated half-time were significantly higher in non-anaemic children indicating greater loss of haematocrit in these children. Conclusion After ten years of adoption of ACTs, anaemia is common pre-and early post-treatment, falls in haematocrit attributable to a single infection is high, and DAFH >4% is common and significantly lower in anaemic compared to non-anaemic Nigerian children. Trial registration Pan African Clinical Trial Registry (PACTR) [ PACTR201709002064150, 1 March 2017 ].

Human activities have dramatically altered reactive nitrogen (N) availability in coastal ecosystems globally. Here we used a gradient of N loading found in a shallow temperate estuary (Waquoit Bay, Massachusetts, USA) to examine how key biogeochemical processes respond to environmental change over time. Using a space-for-time substitution we measured sediment oxygen uptake, dissolved inorganic nitrogen, and di-nitrogen (N2) gas fluxes from sediments collected at four stations. For two stations we compared measurements to those made at the same locations 20 years ago. Spatial variability was not directly correlated to N loading, however the results indicate significant changes in crucial ecosystem processes over time. Sediment oxygen uptake was only 46% of the historic rate and ammonium flux only 34%. The current rate of net denitrification (36 μmol N2-N m-2 h-1) was also lower than the mean historic rate (181 μmol N2-N m-2 h-1). Additionally, at one of the stations we measured a negative average N2 flux rate, indicating that the sediments may be a net source of reactive N. These changes in benthic flux rates are concurrent with a 39% decline in net ecosystem productivity determined from long-term dissolved oxygen data. Although we cannot rule out year-to-year variability we propose that the differences measured between current and historic rates may be explained in part by concurrent changes found in water temperature, precipitation, and freshwater discharge. These regional forcings have the potential to impact N inputs to the estuary, primary producer biomass, and benthic fluxes by altering the supply of organic matter to the sediments. This work highlights the dynamic nature of biogeochemical cycling in coastal ecosystems and underscores the need to better understand long-term changes.

Despite advancements in treating cutaneous melanoma, patients with acral and mucosal (A/M) melanomas still have limited therapeutic options and poor prognoses. We analyzed 156 melanomas (101 cutaneous, 28 acral, and 27 mucosal) using the Foundation One cancer-gene specific clinical testing platform and identified new, potentially targetable genomic alterations (GAs) in specific anatomic sites of A/M melanomas. Using novel pre-clinical models of A/M melanoma, we demonstrate that several GAs and corresponding oncogenic pathways associated with cutaneous melanomas are similarly targetable in A/M melanomas. Other alterations, including MYC and CRKL amplifications, were unique to A/M melanomas and susceptible to indirect targeting using the BRD4 inhibitor JQ1 or Src/ABL inhibitor dasatinib, respectively. We further identified new, actionable A/M-specific alterations, including an inactivating NF2 fusion in a mucosal melanoma responsive to dasatinib in vivo. Our study highlights new molecular differences between cutaneous and A/M melanomas, and across different anatomic sites within A/M, which may change clinical testing and treatment paradigms for these rare melanomas., (© 2024 UICC.)

Introduction: Cutaneous melanoma is among the most common solid tumors to metastasize to the gastrointestinal (GI) tract. Literature summarizing the clinical experience and features of this unique pathology is lacking., Methods: A systematic review of the available literature reporting clinically salient features of melanoma metastases to the small and large intestines was conducted. Additionally, we surveyed our institutional experience of surgically treated melanoma metastasis to the small bowel and colon. A descriptive analysis was performed. Kaplan-Meier curves with log-rank tests were used to analyze time-to-event intervals. Univariable and multivariable Cox logistic regression models were generated to identify predictors of survival., Results: Over 100 studies including 1153 patients were included. GI metastases predominantly affected males, were in the small bowel/jejunum, equally presented as solitary and multiple lesions, and were generally not the first site of distant metastatic disease. The median time from primary lesion diagnosis to GI metastasis was 48 months. Analysis of our institutional cohort suggested that survival in patients receiving complete GI-specific surgical resection and immune checkpoint inhibitors (ICIs) was prolonged compared to palliative resection and without ICI therapy. Positive prognostic factors for survival following GI metastasis included fewer GI metastatic lesions, complete resection, and longer duration between primary tumor diagnosis and GI metastasis., Conclusions: GI metastases are a sign of advanced metastatic melanoma. Clinical suspicion of metastatic involvement in patients with a history of melanoma who develop any abdominal symptoms or anemia should remain high. Receipt of complete surgical resection and ICIs may prolong survival in disseminated melanoma., (© 2024 Wiley Periodicals LLC.)

Study Design: Systematic Review and meta-analysis., Objective: To conduct an updated systematic review and meta-analysis of complications associated with different anterior fusion techniques/approaches and adjuvant resources (i.e., computed tomography angiography (CTA), rhBMP-2, and access surgeons)., Methods: A systematic review was conducted from 1/1/2014-4/1/2024 for studies evaluating the incidence of complications associated with anterior lumbar procedures. Comparisons of complications were made between surgical approach, use of CTA, rhBMP-2, and access surgeons. Meta-analyses were conducted using a generalized linear mixed model., Results: 54 studies were included in the final analysis with 8066 patients and an average follow-up of 31.2 months. The overall complication rate associated with anterior lumbar surgery was 13.1%, including an intraoperative complication rate of 3.8%, postoperative complication rate of 7.4%, infection rate of 1.5%, and reoperation rate of 1.7%. Forest plot analysis showed no significant difference in overall complication rates between open and mini-open techniques, although mini-open techniques were associated with lower overall reoperation rates. The use of CTA was associated with an increase in intraoperative and overall complications, and the use of an access surgeon was associated with a decreased risk of reoperation. The use of rhBMP-2 was not associated with overall complication risk., Conclusions: While anterior lumbar surgery provides numerous benefits, surgeons and patients alike should be aware of the complication and safety profile prior to surgery. High quality studies are warranted to help elucidate the true benefit of certain techniques and adjuvant resources in reducing complications., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

A better understanding of human melanocyte (MC) and MC stem cell biology is essential for treating MC-related diseases. This study employed an inherited pigmentation disorder carrying the SASH1 S519N variant in a Hispanic family to investigate SASH1 function in the MC lineage and the underlying mechanism for this disorder. We used a multidisciplinary approach, including clinical examinations, human cell assays, yeast 2-hybrid screening, and biochemical techniques. Results linked early hair graying to the SASH1 S519N variant, a previously unrecognized clinical phenotype in hyperpigmentation disorders. In vitro, we identified SASH1 as a regulator in MC stem cell maintenance and discovered that TNKS2 is crucial for SASH1's role. In addition, the S519N variant is located in one of multiple tankyrase-binding motifs and alters the binding kinetics and affinity of the interaction. In summary, this disorder links both gain and loss of pigmentation in the same individual, hinting to accelerated aging in human MC stem cells. The findings offer insights into the roles of SASH1 and TNKS2 in MC stem cell maintenance and the molecular mechanisms of pigmentation disorders. We propose that a comprehensive clinical evaluation of patients with MC-related disorders should include an assessment and history of hair pigmentation loss., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We have discovered that human vitiligo patients treated with narrow-band UVB (NBUVB) demonstrated localized resistance to repigmentation in skin sites characterized by distinct cellular and molecular pathways. Using immunostaining studies, discovery-stage RNA-Seq analysis, and confirmatory in situ hybridization, we analyzed paired biopsies collected from vitiligo lesions that did not repigment after 6 months of NBUVB treatment (non-responding) and compared them with repigmented (responding) lesions from the same patient. Non-responding lesions exhibited acanthotic epidermis, had low number of total, proliferative, and differentiated melanocyte (MC) populations, and increased number of senescent keratinocytes (KCs) and of cytotoxic CD8+ T cells as compared with responding lesions. The abnormal response in the non-responding lesions was driven by a dysregulated cAMP pathway and of upstream activator PDE4B, and of WNT/β-catenin repigmentation pathway. Vitiligo-responding lesions expressed high levels of WNT10B ligand, a molecule that may prevent epidermal senescence induced by NBUVB, and that in cultured melanoblasts prevented the pro-melanogenic effect of α-MSH. Understanding the pathways that govern lack of NBUVB-induced vitiligo repigmentation has a great promise in guiding the development of new therapeutic strategies for vitiligo., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Immune checkpoint inhibitors (ICIs) are now the first-line treatment for patients with advanced melanoma. Despite promising clinical results, many patients fail to respond to these therapies. BH3 mimetics, a novel class of small molecule inhibitors that bind and inhibit anti-apoptotic members of the BCL2 family proteins such as BCL2 or MCL1, have been very successful in treating hematologic malignancies. However, there are limited studies on the immunomodulatory role of the BH3 mimetics. Several factors contribute to ICI resistance including myeloid-derived suppressor cells (MDSCs) that exert immunosuppressive effects through direct and indirect inhibition of antitumor immunity. Thus, targeting MDSCs to enhance antitumor immunity has the potential to enhance the efficacy of ICIs. In this study, we show that the MCL1 inhibitor S64315 reduces melanoma tumor growth in an immune cell-dependent manner in mice. Specifically, S64315 enhances antitumor immunity by reducing MDSC frequency and by promoting the activity of CD8+T cells. Additionally, human MDSCs are 10 times more sensitive to S64315 than cutaneous melanoma lines. Further, we found that a higher expression of MCL1 is associated with poor survival for patients treated with anti-PD-1. Finally, combining S64315 and anti-PD-1 significantly slowed tumor growth compared to either agent alone. Together, this proof-of-concept study demonstrates the potential of combining an MCL1 inhibitor with anti-PD-1 in the treatment of melanoma. It justifies the further development of next generation MCL1 inhibitors to improve efficacy of ICIs in treating malignant melanoma., (© 2024. The Author(s).)

Lysophosphatidic acid (LPA) is a bioactive lipid which increases in concentration locally and systemically across different cancer types. Yet, the exact mechanism(s) of how LPA affects CD8 T cell immunosurveillance during tumor progression remain unknown. We show LPA receptor (LPAR) signaling by CD8 T cells promotes tolerogenic states via metabolic reprogramming and potentiating exhaustive-like differentiation to modulate anti-tumor immunity. We found LPA levels predict response to immunotherapy and Lpar5 signaling promotes cellular states associated with exhausted phenotypes on CD8 T cells. Importantly, we show that Lpar5 regulates CD8 T cell respiration, proton leak, and reactive oxygen species. Together, our findings reveal that LPA serves as a lipid-regulated immune checkpoint by modulating metabolic efficiency through LPAR5 signaling on CD8 T cells. Our study offers key insights into the mechanisms governing adaptive anti-tumor immunity and demonstrates LPA could be exploited as a T cell directed therapy to improve dysfunctional anti-tumor immunity., (© 2023. The Author(s).)

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is an immune checkpoint expressed in regulatory T (Treg) cells and activated T lymphocytes. Despite its potential as a treatment strategy for melanoma, CTLA-4 inhibition has limited efficacy. Using data from The Cancer Genome Atlas (TCGA) melanoma database and another dataset, we found that decreased CTLA4 mRNA was associated with a poorer prognosis in metastatic melanoma. To investigate further, we measured blood CTLA4 mRNA in 273 whole-blood samples from an Australian cohort and found that it was lower in metastatic melanoma than in healthy controls and associated with worse patient survival. We confirmed these findings using Cox proportional hazards model analysis and another cohort from the US. Fractionated blood analysis revealed that Treg cells were responsible for the downregulated CTLA4 in metastatic melanoma patients, which was confirmed by further analysis of published data showing downregulated CTLA-4 surface protein expression in Treg cells of metastatic melanoma compared to healthy donors. Mechanistically, we found that secretomes from human metastatic melanoma cells downregulate CTLA4 mRNA at the post-transcriptional level through miR-155 while upregulating FOXP3 expression in human Treg cells. Functionally, we demonstrated that CTLA4 expression inhibits the proliferation and suppressive function of human Treg cells. Finally, miR-155 was found to be upregulated in Treg cells from metastatic melanoma patients compared to healthy donors. Our study provides new insights into the underlying mechanisms of reduced CTLA4 expression observed in melanoma patients, demonstrating that post-transcriptional silencing of CTLA4 by miRNA-155 in Treg cells may play a critical role. Since CTLA-4 expression is downregulated in non-responder melanoma patients to anti-PD-1 immunotherapy, targeting miRNA-155 or other factors involved in regulating CTLA4 expression in Treg cells without affecting T cells could be a potential strategy to improve the efficacy of immunotherapy in melanoma. Further research is needed to understand the molecular mechanisms regulating CTLA4 expression in Treg cells and identify potential therapeutic targets for enhancing immune-based therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Vaddi, Osborne, Nicklawsky, Williams, Menon, Smith, Mayer, Reid, Domenico, Nguyen, Robinson, Ziman, Gao, Zhai and Fujita.)

Melanoma is a cancer of melanocytes, with multiple subtypes based on body site location. Cutaneous melanoma is associated with skin exposed to ultraviolet radiation; uveal melanoma occurs in the eyes; mucosal melanoma occurs in internal mucous membranes; and acral melanoma occurs on the palms, soles, and nail beds. Here, we present the largest whole-genome sequencing study of melanoma to date, with 570 tumors profiled, as well as methylation and RNA sequencing for subsets of tumors. Uveal melanoma is genomically distinct from other melanoma subtypes, harboring the lowest tumor mutation burden and with significantly mutated genes in the G-protein signaling pathway. Most cutaneous, acral, and mucosal melanomas share alterations in components of the MAPK, PI3K, p53, p16, and telomere pathways. However, the mechanism by which these pathways are activated or inactivated varies between melanoma subtypes. Additionally, we identify potential novel germline predisposition genes for some of the less common melanoma subtypes., Significance: This is the largest whole-genome analysis of melanoma to date, comprehensively comparing the genomics of the four major melanoma subtypes. This study highlights both similarities and differences between the subtypes, providing insights into the etiology and biology of melanoma. This article is highlighted in the In This Issue feature, p. 2711., (©2022 The Authors; Published by the American Association for Cancer Research.)

Introduction: Primary hip and knee arthroplasty represent two of the most successful orthopaedic surgical interventions in the past century. Similarly, lumbar fusion (LF) remains a valuable, evidence-based option to relieve pain and disability related to spinal degenerative conditions. This study evaluates the relative improvements in 1-year health-related quality of life (HRQOL) measures among patients undergoing primary single-level LF, primary total hip arthroplasty (THA), and primary total knee arthroplasty (TKA)., Methods: Patients older than 18 years who underwent primary single-level posterior LF (posterolateral decompression and fusion with or without transforaminal lumbar interbody fusion, involving any single lumbar level), TKA, and THA at a single academic institution were retrospectively identified. Patient demographics and surgical characteristics were collected. HRQOL measures were collected preoperatively and at 1-year postoperative time point including Short-Form 12 Physical Component Score (PCS) and Mental Component Score (MCS) along with subspecialty-specific outcomes., Results: A total of 2,563 patients were included (346 LF, 1,035 TKA, and 1,182 THA). Change in MCS-12 and PCS-12 after LF did not vary markedly by preoperative diagnosis. LF patients had a significantly lower preoperative MCS-12 (LF: 50.8, TKA: 53.9, THA: 52.9, P < 0.001), postoperative MCS-12 (LF: 52.5, TKA: 54.8, THA: 54.5, P < 0.001), postoperative PCS-12 (LF: 40.1, TKA: 44.0, THA: 43.9, P < 0.001), ΔPCS-12 (LF: 7.9, TKA: 10.8, THA: 11.9, P < 0.001), and PCS-12 recovery ratio (LF: 10.7%, TKA: 15.1%, THA 16.6%, P < 0.001) compared with TKA and THA patients. In regression analysis, both TKA and LF were found to be independently associated with a smaller ΔPCS-12 improvement (TKA: β = -1.36, P = 0.009; LF: β = -4.74, P < 0.001) compared with THA. TKA (β = -1.42, P = 0.003) was also independently associated with a smaller ΔMCS-12 improvement compared with THA., Conclusions: Patients undergoing single-level LF, TKA, and THA demonstrate notable improvements in HRQOL outcomes at 1 year postoperatively compared with preoperative baseline scores. The greatest improvements were found among THA patients, followed subsequently by TKA and LF patients. Both LF and TKA were independently associated with markedly less improvement in physical disability at 1 year postoperatively compared with THA., Study Design: Retrospective Cohort Study., (Copyright © 2022 by the American Academy of Orthopaedic Surgeons.)

Importance: Treatment with immune checkpoint inhibitors (ICIs) has increased survival in patients with advanced malignant melanoma but can be associated with a wide range of immune-related adverse events (irAEs). The role of human leukocyte antigen (HLA)-DR alleles in conferring irAE risk has not been well studied., Objective: To evaluate the association between irAEs and treatment response, survival, and the presence of HLA-DR alleles after ICI therapy in advanced melanoma., Design, Setting, and Participants: This case-control study used the patient registry and biobanked samples from the tertiary referral University of Colorado Cancer Center. Specimens and clinical data were collected between January 1, 2010, and December 31, 2021. Patients with advanced (stage III unresectable and stage IV) melanoma who received ICI therapy (n = 132) were included in the analysis., Exposures: Immune checkpoint inhibitors (anti-cytotoxic T-lymphocyte antigen 4, anti-programmed cell death protein 1 or its ligand, or the combination) for the treatment of advanced melanoma., Main Outcomes and Measures: The association between irAEs and response to therapy, survival, and HLA-DR alleles., Results: Among the cohort of 132 patients with advanced melanoma (mean [SD] age, 63.4 [7.2] years; 85 men [64%] and 47 women [36%]) treated with ICIs, 73 patients had at least 1 irAE and 59 did not have an irAE. Compared with patients without an irAE, patients with an irAE had higher treatment response rates (50 of 72 [69%] vs 28 of 57 [49%]; P = .02) and increased survival (median, 4.8 [IQR, 0.2-9.6] vs 3.2 [IQR, 0.1-9.2] years; P = .02). Specific HLA-DR alleles were associated with the type of irAE that developed: 7 of 10 patients (70%) who developed type 1 diabetes had DR4; 6 of 12 (50%) who developed hypothyroidism had DR8; 5 of 8 (63%) who developed hypophysitis had DR15; 3 of 5 (60%) who developed pneumonitis had DR1; and 8 of 15 (53%) who developed hepatitis had DR4., Conclusions and Relevance: These findings suggest that IrAEs are associated with treatment response rates and increased survival after ICI therapy for advanced melanoma. Because distinct HLA-DR alleles are associated with given adverse events, HLA genotyping before ICI therapy may aid in identifying risk for specific irAEs that could develop with such treatment.

Study Design: This was a retrospective cohort study., Objective: The objective of this study was to determine if the degree of interbody cage lordosis and cage positioning are associated with changes in postoperative sagittal alignment after single-level transforaminal lumbar interbody fusion (TLIF)., Summary of Background Data: Ideal sagittal alignment and lumbopelvic alignment have been shown to correlate with postoperative clinical outcomes. TLIF is one technique that may improve these parameters, but whether the amount of cage lordosis improves either segmental or lumbar lordosis (LL) is unknown., Methods: A retrospective review was performed on patients who underwent single-level TLIF with either a 5-degree or a 12-degree lordotic cage. LL, segmental lordosis (SL), disk height, center point ratio, cage position, and cage subsidence were evaluated. Correlation between center point ratio and change in lordosis was assessed using the Spearman correlation coefficient. Secondary analysis included multiple linear regression to determine independent predictors of change in SL., Results: A total of 126 patients were included in the final analysis, with 51 patients receiving a 5-degree cage and 75 patients receiving a 12-degree cage. There were no differences in the postoperative minus preoperative LL (∆LL) (12-degree cage: -1.66 degrees vs. 5-degree cage: -2.88 degrees, P =0.528) or ∆SL (12-degree cage: -0.79 degrees vs. 5-degree cage: -1.68 degrees, P =0.513) at 1-month follow-up. Furthermore, no differences were found in ∆LL (12-degree cage: 2.40 degrees vs. 5-degree cage: 1.00 degrees, P =0.497) or ∆SL (12-degree cage: 1.24 degrees vs. 5-degree cage: 0.35 degrees, P =0.541) at final follow-up. Regression analysis failed to show demographic factors, cage positioning, or cage lordosis to be independent predictors of change in SL. No difference in subsidence was found between groups (12-degree cage: 25.5% vs. 5-degree cage: 32%, P =0.431)., Conclusion: Lordotic cage angle and cage positioning were not associated with perioperative changes in LL, SL, or cage subsidence after single-level TLIF., Level of Evidence: Level III., Competing Interests: Dr Schroeder has received funds to travel from AOSpine and Medtronic. Dr Vaccaro has consulted or has done independent contracting for DePuy, Medtronic, Stryker Spine, Globus, Stout Medical, Gerson Lehrman Group, Guidepoint Global, Medacorp, Innovative Surgical Design, Orthobullets, Ellipse, and Vertex. He has also served on the scientific advisory board/board of directors/committees for Flagship Surgical, AO Spine, Innovative Surgical Design, and Association of Collaborative Spine Research. Dr Vaccaro has received royalty payments from Medtronic, Stryker Spine, Globus, Aesculap, Thieme, Jaypee, Elsevier, and Taylor Francis/Hodder and Stoughton. He has stock/stock option ownership interests in Replication Medica, Globus, Paradigm Spine, Stout Medical, Progressive Spinal Technologies, Advanced Spinal Intellectual Properties, Spine Medica, Computational Biodynamics, Spinology, In Vivo, Flagship Surgical, Cytonics, Bonovo Orthopaedics, Electrocore, Gamma Spine, Location Based Intelligence, FlowPharma, R.S.I., Rothman Institute and Related Properties, Innovative Surgical Design, and Avaz Surgical. He has also served as deputy editor/editor of Spine. In addition, Dr Vaccaro has also provided expert testimony. He has also served as deputy editor/editor of Clinical Spine Surgery. The remaining authors declare no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Uveal melanoma (UM) is a subtype of melanoma. Although they share a melanocytic origin with cutaneous melanoma (CM), patients with UM have few treatment options. BCL2 homologous 3 mimetics are small-molecule drugs that mimic proapoptotic BCL2 family members. We compared BCL2 family member expression between UM and CM using immunoblot and The Cancer Genome Atlas transcriptomic analysis. UM has a unique signature of low BFL1 and high PUMA proteins compared with CM and 30 other cancer types, making them an attractive candidate for BCL2 homologous 3 protein mimetics. We tested the efficacy of a BCL2 inhibitor and MCL1 inhibitor (MCL1i) in UM, with viability assays, live-cell imaging, sphere assays, and mouse xenograft models. UM had a higher sensitivity to MCL1i than CM. Overexpression of BFL1 or knockdown of PUMA made the UM more resistant to MCL1i. In contrast, MAPK/extracellular signal‒regulated kinase inhibitor treatment in CM made them more sensitive to MCL1i. However, MCL1i-alone treatment was not very effective to reduce the UM initiating cells; to overcome this, we employed a combination of MCL1i with BCL2 inhibitor that synergistically inhibited UM initiating cell's capacity to expand. Overall, we identify a distinct expression profile of BCL2 family members for UM that makes them susceptible to BCL2 homologous 3 mimetics., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Study Design: This was a retrospective cohort study., Objective: The goal of this study is to evaluate the outcomes of patients with cervical degenerative spondylolisthesis (DS) undergoing anterior cervical discectomy and fusion (ACDF), specifically comparing surgeries that include versus exclude the DS level., Summary of Background Data: DS has been extensively studied in the lumbar spine associated with both back and leg pain leading to worse patient quality of life measures. Conversely, there is a relative dearth of literature regarding surgical and clinical outcomes in the setting of cervical DS., Materials and Methods: A total of 315 patients undergoing ACDF between 2014 and 2018 with minimum of 1-year postoperative patient-reported outcome measures (PROMs) were retrospectively reviewed. Forty-six patients were found to have DS and were categorized based on whether an ACDF was performed at the same level (SL) or at a different level (DL) than the spondylolisthesis. Patient demographics, surgical parameters, preoperative and postoperative radiographs, and PROMs were compared between groups., Results: Of the 315 patients, a total of 46 met the inclusion criteria including 21 SL and 25 DL patients. There were no significant differences in patient demographics between the groups. The SL cohort had a significantly worse preoperative sagittal vertical axis (SL: 34.4 vs. 26.1, P=0.025) but no difference in postoperative or delta sagittal vertical axis. Both patient cohorts reported significant postoperative improvement in all PROMs, except Short-Form 12 Mental Component Score in the SL group. There were no differences between the groups regarding Visual Analog Scale Neck, Visual Analog Scale Arm, Neck Disability Index, or Short-Form 12 Physical Component Score. Regression analysis demonstrated SL to be a significant negative predictor for improvement in Short-Form 12 Mental Component Score (β: -11.27, P=0.10)., Conclusion: Patients treated only at their neurologically symptomatic levels, excluding asymptomatic listhesis in their construct, can expect equivalent radiographic as well as physical function, disability, and pain outcomes 1 year after ACDF compared with patients whose listhetic level was included in their construct., Level of Evidence: Level III., Competing Interests: Dr Schroeder has received funds to travel from AOSpine and Medtronic. Dr Vaccaro has consulted or has done independent contracting for DePuy, Medtronic, Stryker Spine, Globus, Stout Medical, Gerson Lehrman Group, Guidepoint Global, Medacorp, Innovative Surgical Design, Orthobullets, Ellipse, and Vertex. He has also served on the scientific advisory board/board of directors/committees for Flagship Surgical, AO Spine, Innovative Surgical Design, and Association of Collaborative Spine Research. Dr Vaccaro has received royalty payments from Medtronic, Stryker Spine, Globus, Aesculap, Thieme, Jaypee, Elsevier, and Taylor Francis/Hodder and Stoughton. He has stock/stock option ownership interests in Replication Medica, Globus, Paradigm Spine, Stout Medical, Progressive Spinal Technologies, Advanced Spinal Intellectual Properties, Spine Medica, Computational Biodynamics, Spinology, In Vivo, Flagship Surgical, Cytonics, Bonovo Orthopaedics, Electrocore, Gamma Spine, Location Based Intelligence, FlowPharma, R.S.I., Rothman Institute and Related Properties, Innovative Surgical Design, and Avaz Surgical. He has also served as deputy editor/editor of Spine. In addition, Dr Vaccaro has also provided expert testimony. He has also served as deputy editor/editor of Clinical Spine Surgery. The remaining authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

There is increasing evidence that oxidative metabolism and fatty acids play an important role in BRAF-driven tumorigenesis, yet the effect of BRAF mutation and expression on metabolism is poorly understood. We examined how BRAF mutation and expression modulates metabolite abundance. Using the non-transformed NIH3T3 cell line, we generated cells that stably overexpressed BRAF V600E or BRAF WT. We found that cells expressing BRAF V600E were enriched with immunomodulatory lipids. Further, we found a unique transcriptional signature that was exclusive to BRAF V600E expression. We also report that BRAF V600E mutation promoted accumulation of long chain polyunsaturated fatty acids (PUFAs) and rewired metabolic flux for non-Warburg behavior. This cancer promoting mutation further induced the formation of tunneling nanotube (TNT)-like protrusions in NIH3T3 cells that preferentially accumulated lipid droplets. In the plasma of melanoma patients harboring the BRAF V600E mutation, levels of lysophosphatidic acid, sphingomyelin, and long chain fatty acids were significantly increased in the cohort of patients that did not respond to BRAF inhibitor therapy. Our findings show BRAF V600 status plays an important role in regulating immunomodulatory lipid profiles and lipid trafficking, which may inform future therapy across cancers.

Introduction: For patients with stage III melanoma with occult lymph node metastasis, the use of adjuvant therapy is increasing, and completion lymph node dissection (CLND) is decreasing. We sought to evaluate the use of modern adjuvant therapy and outcomes for patients with stage III melanoma who did not undergo CLND., Methods: Patients with a positive SLNB from 2015 to 2020 who did not undergo CLND were evaluated retrospectively. Nodal recurrence, recurrence-free survival (RFS), distant metastasis-free survival (DMFS), and melanoma-specific survival were evaluated., Results: Among 90 patients, 56 (62%) received adjuvant therapy and 34 (38%) underwent observation alone. Patients who received adjuvant therapy were younger (mean age: 53 vs. 65, p < 0.001) and had higher overall stage (Stage IIIb/c 75% vs. 54%, p = 0.041). Disease recurred in 12 of 34 patients (35%) in the observation group and 11 of 56 patients (20%) in the adjuvant therapy group. The most common first site of recurrence was distant recurrence alone (5/34 patients) in the observation group and nodal recurrence alone (8/90 patients) in the adjuvant therapy group. Despite more adverse nodal features in the adjuvant therapy group, 24-month nodal recurrence rate and RFS were not significantly different between the adjuvant and observation cohorts (nodal recurrence rate: 26% vs. 20%, p = 0.68; RFS: 75% vs. 61%, p = 0.39). Among patients with stage IIIb/c disease, adjuvant therapy was associated with a significantly improved 24-month DMFS (86% vs. 59%, p = 0.04)., Conclusions: In this early report, modern adjuvant therapy in patients who forego CLND is associated with longer DMFS among patients with stage IIIb/c disease., (© 2021. Society of Surgical Oncology.)

Objectives: While much of the research concerning factors associated with responses to immune checkpoint inhibitors (ICIs) has focussed on the contributions of conventional peptide-specific T cells, the role of unconventional T cells, such as mucosal-associated invariant T (MAIT) cells, in human melanoma remains largely unknown. MAIT cells are an abundant population of innate-like T cells expressing a semi-invariant T-cell receptor restricted to the MHC class I-like molecule, MR1, presenting vitamin B metabolites derived from bacteria. We sought to characterise MAIT cells in melanoma patients and determined their association with treatment responses and clinical outcomes., Methods: In this prospective clinical study, we analysed the frequency and functional profile of circulating and tumor-infiltrating MAIT cells in human melanoma patients. Using flow cytometry, we compared these across metastatic sites and between ICI responders vs. non-responders as well as healthy donors., Results: We identified tumor-infiltrating MAIT cells in melanomas across metastatic sites and found that the number of circulating MAIT cells is reduced in melanoma patients compared to healthy donors. However, circulating MAIT cell frequencies are restored by ICI treatment in responding patients, correlating with treatment responses, in which patients with high frequencies of MAIT cells exhibited significantly improved overall survival., Conclusion: Our results suggest that MAIT cells may be a potential predictive marker of responses to immunotherapies and provide rationale for testing MAIT cell-directed therapies in combination with current and next-generation ICIs., Competing Interests: The authors declare no potential conflict of interest., (© 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Detection of minimal residual disease in patients with cancer, who are in complete remission with no cancer cells detectable, has the potential to improve recurrence-free survival through treatment selection. Studies analyzing circulating tumor DNA (ctDNA) in patients with solid tumors suggest the potential to accurately predict and detect relapse, enabling treatment strategies that may improve clinical outcomes. Over the past decade, assays for ctDNA detection in plasma samples have steadily increased in sensitivity and specificity. These are applied for the detection of residual disease after treatment and for earlier detection of recurrence. Novel clinical trials are now assessing how assays for "residual disease and recurrence" (RDR) may influence current treatment paradigms and potentially change the landscape of risk classification for cancer recurrence. In this review, we appraise the progress of RDR detection using ctDNA and consider the emerging role of liquid biopsy in the monitoring and management of solid tumors., Competing Interests: Declaration of interests J.C.M.W. and N.R. are inventors/contributors on patents covering methods for ctDNA detection (including WO/2020/104670). P.R. reports institutional research funding from Novartis, Grail/Illumina, ArcherDx/Invitae, Inivata, Epic Sciences, and Tempus; advisory board/honoraria from Novartis, AstraZeneca, Foundation Medicine, Pfizer, Epic Sciences, Inivata, and Natera; and consulting for Tempus. Y.-S.Y. reports honoraria/consultancy from Novartis, Lilly, Pfizer, AstraZeneca, Eisai, MSD, and Specialised Therapeutics and travel support from Pfizer, Eisai, and Roche. S.-J.D. has received research funding from Roche Genentech and Cancer Therapeutics (CTx) CRC and has been an advisory board member for AstraZeneca and Inivata. S.K. serves on the advisory board and is a consultant for Inivata and Natera. N.R. and C.D.M. are officers of Inivata. T.I.M., P.R., E.L.M., R.G., I.T., Y.-S.Y., W.A.R., B.B., A.B., and J.T. served on the advisory board and consulted for Inivata., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Advanced systemic mastocytosis (AdvSM) is a rare hematologic neoplasm driven by the KIT D816V mutation and associated with poor survival. This phase 1 study ( NCT02561988 ) evaluated avapritinib (BLU-285), a selective KIT D816V inhibitor, in patients with AdvSM. The primary endpoints were the maximum tolerated dose, recommended phase 2 dose and safety of avapritinib. Secondary endpoints included overall response rate and changes in measures of mast cell burden. Avapritinib was evaluated at doses of 30-400 mg once daily in 86 patients, 69 with centrally confirmed AdvSM. Maximum tolerated dose was not reached, and 200 mg and 300 mg daily were studied in dose-expansion cohorts. The most frequent adverse events observed were periorbital edema (69%), anemia (55%), diarrhea (45%), thrombocytopenia (44%) and nausea (44%). Intracranial bleeding occurred in 13% overall, but in only 1% of patients without severe thrombocytopenia (platelets <50 × 10 9 /l). In 53 response-evaluable patients, the overall response rate was 75%. The complete remission rate was 36%. Avapritinib elicited ≥50% reductions in marrow mast cells and serum tryptase in 92% and 99% of patients, respectively. Avapritinib induced deep and durable responses, including molecular remission of KIT D816V in patients with AdvSM, and was well tolerated at the recommended phase 2 dose of 200 mg daily., (© 2021. The Author(s).)

Anthropogenic emissions of greenhouse gases are warming the Earth. It is likely that the greatest impacts of climate change on human and natural systems will come from increasingly frequent and severe extreme weather and climate events. Some increases in such extremes are already being detected, and this trend is projected to continue as Earth warms. Here we review the overarching climate drivers of increases in extreme weather and address the context in which extremes occur and the challenges of projecting future changes. The observational evidence for climate-driven increases in extremes and the implications of model projections are reviewed for heat and drought and several types of storms: tropical cyclones, midlatitude storms, and severe local weather, focusing on those changes most relevant to the continental United States. We emphasize the overall observed and modeled trends in extreme weather in which we have the greatest confidence, because they are consistent with our fundamental understanding of weather and climate. Despite remaining uncertainty about many details, especially in model-based projections, the signal of increasing extremes is sufficiently clear that it demands a robust human response, in limiting future emissions of greenhouse gases and in making our human systems more resilient to further changes that are inevitable as Earth continues to warm. Implications : By placing observed and projected changes in extreme weather in the context of our fundamental understanding of physics and statistics, this review makes it clear that these are significant and impactful changes that demand a robust human response.

Background: Adrenal gland metastases (AGMs) are common in advanced-stage melanoma, occurring in up to 50% of patients. The introduction of immune checkpoint inhibitors (ICIs) has markedly altered the outcome of patients with melanoma. However, despite significant successes, anecdotal evidence has suggested that treatment responses in AGMs are significantly lower than in other metastatic sites. We sought to investigate whether having an AGM is associated with altered outcomes and whether ICI responses are dampened in the adrenal glands., Patients and Methods: We retrospectively compared ICI responses and overall survival (OS) in 68 patients with melanoma who were diagnosed with an AGM and a control group of 100 patients without AGMs at a single institution. Response was determined using RECIST 1.1. OS was calculated from time of ICI initiation, anti-PD-1 initiation, initial melanoma diagnosis, and stage IV disease diagnosis. Tumor-infiltrating immune cells were characterized in 9 resected AGMs using immunohistochemical analysis., Results: Response rates of AGMs were significantly lower compared with other metastatic sites in patients with AGMs (16% vs 22%) and compared with those without AGMs (55%). Patients with AGMs also had significantly lower median OS compared with those without AGMs (3.1 years vs not reached, respectively). We further observed that despite this, AGMs exhibited high levels of tumor-infiltrating immune cells., Conclusions: In this cohort of patients with melanoma, those diagnosed with an AGM had lower ICI response rates and OS. These results suggest that tissue-specific microenvironments of AGMs present unique challenges that may require novel, adrenal gland-directed therapies or surgical resection.

Although treatment options for melanoma patients have expanded in recent years with the approval of immunotherapy and targeted therapy, there is still an unmet need for new treatment options for patients that are ineligible for, or resistant to these therapies. BH3 mimetics, drugs that mimic the activity of pro-apoptotic BCL2 family proteins, have recently achieved remarkable success in the clinical setting. The combination of BH3 mimetic ABT-199 (venetoclax) plus azacitidine has shown substantial benefit in treating acute myelogenous leukemia. We evaluated the efficacy of various combinations of BH3 mimetic + azacitidine in fourteen human melanoma cell lines from cutaneous, mucosal, acral and uveal subtypes. Using a combination of cell viability assay, BCL2 family knockdown cell lines, live cell imaging, and sphere formation assay, we found that combining inhibition of MCL1, an anti-apoptotic BCL2 protein, with azacitidine had substantial pro-apoptotic effects in multiple melanoma cell lines. Specifically, this combination reduced cell viability, proliferation, sphere formation, and induced apoptosis. In addition, this combination is highly effective at reducing cell viability in rare mucosal and uveal subtypes. Overall, our data suggest this combination as a promising therapeutic option for some patients with melanoma and should be further explored in clinical trials.

In repigmentation of human vitiligo, the melanocyte (MC) precursors in the hair follicle bulge proliferate, migrate, and differentiate to repopulate the depigmented epidermis. Here, we present a comprehensive characterization of pathways and signals in the bulge that control the repigmentation process. Using biopsies from patients with vitiligo, we have selectively harvested, by laser capture microdissection, MC and keratinocyte precursors from the hair follicle bulge of untreated vitiligo skin and vitiligo skin treated with narrow-band UVB. The captured material was subjected to whole transcriptome RNA-sequencing. With this strategy, we found that repigmentation in the bulge MC precursors is driven by KCTD10, a signal with unknown roles in the skin, and CTNNB1 (encoding β-catenin) and RHO guanosine triphosphatase [RHO GTPase, RHO], two signaling pathways previously shown to be involved in pigmentation biology. Knockdown studies in cultured human MCs of RHOJ, the upmost differentially expressed RHO family component, corroborated with our findings in patients with vitiligo, identified RHOJ involvement in UV response and melanization, and confirmed previously identified roles in melanocytic cell migration and apoptosis. A better understanding of mechanisms that govern repigmentation in MC precursors will enable the discovery of molecules that induce robust repigmentation phenotypes in vitiligo., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Subungual melanomas (SUM) arise beneath the nails of the hands and feet, and account for 0.7-3.5% of all malignant melanomas. Most studies include SUM in the category of acral melanoma, but understanding the specific features of SUM is critical for improving patient care. In this study, we performed a site-specific comparison of the clinical and molecular features between 54 cases of SUM and 78 cases of nonsubungual acral melanoma. Compared to patients with acral melanoma, patients with SUM were younger at diagnosis, had a higher prevalence of primary melanomas on the hand, and had more frequent reports of previous trauma at the tumor site. SUM was deeper than acral melanoma at diagnosis, which correlated with an increased frequency of metastases. Analysis of common melanoma driver genes revealed KIT and KRAS mutations were predominantly found in SUM, whereas BRAF and NRAS mutations occurred almost exclusively in acral melanoma. We also discovered molecular differences in the cell cycle pathway, where CDK4/CCND1 amplifications were more frequent in SUM and CDKN2A/B loss occurred mostly in acral melanoma, and in the PI3K/mTOR pathway, where RICTOR amplification and TSC1 K587R mutations were exclusively in SUM and PTEN loss and AKT1 mutations were exclusively in acral melanoma. Comparison of hand versus foot tumors revealed more frequent ulceration of SUM foot tumors, which correlated with more distal metastases and poorer overall survival. In summary, we find SUM are both clinically and molecularly distinct from acral melanoma, and our data suggest KIT, CDK4/6, and mTOR inhibitors may be particularly relevant and effective treatments for patients with SUM.

To increase understanding of the genomic landscape of acral melanoma, a rare form of melanoma occurring on palms, soles or nail beds, whole genome sequencing of 87 tumors with matching transcriptome sequencing for 63 tumors was performed. Here we report that mutational signature analysis reveals a subset of tumors, mostly subungual, with an ultraviolet radiation signature. Significantly mutated genes are BRAF, NRAS, NF1, NOTCH2, PTEN and TYRP1. Mutations and amplification of KIT are also common. Structural rearrangement and copy number signatures show that whole genome duplication, aneuploidy and complex rearrangements are common. Complex rearrangements occur recurrently and are associated with amplification of TERT, CDK4, MDM2, CCND1, PAK1 and GAB2, indicating potential therapeutic options.

There is an urgent need to develop treatments for patients with melanoma who are refractory to or ineligible for immune checkpoint blockade, including patients who lack BRAF-V600E/K mutations. This is often the case in patients diagnosed with rare melanoma subtypes such as mucosal and acral melanoma. Here, we analyzed data from the cutaneous melanoma The Cancer Genome Atlas Network (TCGA) transcriptomic and proteomic databases for differential expression of apoptosis molecules between melanomas with or without BRAF hotspot mutations. Our data indicated higher B-cell CLL/lymphoma 2 (BCL2) expression in melanoma without BRAF hotspot mutations, suggesting that BH3 mimetics, such as ABT-199 (venetoclax, a small molecule against BCL2), may be a potential therapeutic option for these patients. We explored the efficacy of combining two BH3 mimetics, ABT-199 and a myeloid cell leukemia sequence 1 (MCL1) inhibitor (S63845 or S64315/MIK665) in cutaneous, mucosal and acral melanomas, in vitro and in vivo. Our data indicate this combination induced cell death in a broad range of melanoma cell lines, including melanoma initiating cell populations, and was more potent in melanoma cells without BRAF-V600E/K mutations. Our knockdown/knockout experiments suggest that several pro-apoptotic BCL2 family members, BCL2-like 11 (apoptosis facilitator) (BIM), phorbol-12-myristate-13-acetate-induced protein 1 (NOXA) or BID, play a role in the combination-induced effects. Overall, our study supports the rationale for combining an MCL1 inhibitor with a BCL2 inhibitor as a therapeutic option in patients with advanced melanoma.

Immunotherapy, such as anti-PD1, has improved the survival of patients with metastatic melanoma. However, predicting which patients will respond to immunotherapy remains a significant knowledge gap. In this study we analyzed pre-immunotherapy treated tumors from 52 patients with metastatic melanoma and monitored their response based on RECIST 1.1 criteria. The responders group contained 21 patients that had a complete or partial response, while the 31 non-responders had stable or progressive disease. Whole exome sequencing (WES) was used to identify biomarkers of anti-PD1 response from somatic mutations between the two groups. Variants in codons G34 and G41 in NFKBIE , a negative regulator of NFkB , were found exclusively in the responders. Mutations in NKBIE -related genes were also enriched in the responder group compared to the non-responders. Patients that harbored NFKBIE -related gene mutations also had a higher mutational burden, decreased tumor volume with treatment, and increased progression-free survival. RNA sequencing on a subset of tumor samples identified that CD83 was highly expressed in our responder group. Additionally, Gene Set Enrichment Analysis showed that the TNFalpha signaling via NFkB pathway was one of the top pathways with differential expression in responders vs. non-responders. In vitro NFkB activity assays indicated that the G34E variant caused loss-of-function of NFKBIE , and resulted in activation of NFkB signaling. Flow cytometry assays indicated that G34E variant was associated with upregulation of CD83 in human melanoma cell lines. These results suggest that NFkB activation and signaling in tumor cells contributes to a favorable anti-PD1 treatment response, and clinical screening to include aberrations in NFkB -related genes should be considered.

Current treatment for patients with metastatic melanoma include molecular-targeted therapies and immune checkpoint inhibitors. However, a subset of melanomas are difficult-to-treat. These melanomas include those without the genetic markers for targeted therapy, non-responsive to immunotherapy, and those who have relapsed or exhausted their therapeutic options. Therefore, it is necessary to understand and explore other biological processes that may provide new therapeutic approaches. One of most appealing is targeting the apoptotic/anti-apoptotic system that is effective against leukemia. We used genetic knockdown and pharmacologic approaches of BH3 mimetics to target anti-apoptotic BCL2 family members and identified MCL1 and BCLXL as crucial pro-survival members in melanoma. We then examined the effects of combining BH3 mimetics to target MCL1 and BCLXL in vitro and in vivo. These include clinical-trial-ready compounds such as ABT-263 (Navitoclax) and S63845/S64315 (MIK655). We used cell lines derived from patients with difficult-to-treat melanomas. In vitro, the combined inhibition of MCL1 and BCLXL resulted in significantly effective cell killing compared to single-agent treatment (p < 0.05) in multiple assays, including sphere assays. The combination-induced cell death was independent of BIM, and NOXA. Recapitulated in our mouse xenograft model, the combination inhibited tumor growth, reduced sphere-forming capacity (p < 0.01 and 0.05, respectively), and had tolerable toxicity (p > 0.40). Taken together, this study suggests that dual targeting of MCL1 and BCLXL should be considered as a treatment option for difficult-to-treat melanoma patients.