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The infusion autograft absolute number of inhibitory killer immunoglobulin-like receptor (KIR) 2DL2 and activating natural killer (NK)p30 cells are predictors of clinical outcomes in lymphoma patients undergoing autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT). To assess if the long-term recovery of these NK cell subsets still holds clinical relevance, we set up to investigate their prognostic ability at day 100 post-APBHSCT. This was a retrospective single-institution study including 107 patients from our prior phase III trial who had a clinical assessment at day 100 post-APBHSCT. The median follow-up from day 100 was 168.19 months (interquartile range: 156.85-181.28 months). Patients with day 100 inhibitory KIR2DL2 < 0.08 cells/µL and activating NKp30 ≥ 0.19 cells/µL experienced superior overall survival (OS) and progression-free survival (PFS). A multivariate analysis revealed both the day 100 inhibitory KIR2DL2 [OS: HR = 1.449, 95%CI, 1.231-1.895, p < 0.013; and PFS: HR = 2.069, 95%CI, 1.134-3.775, p < 0.021] and activating NKp30 [OS: HR = 4.985, 95%CI, 2.614-9.506, p < 0.0001; and PFS: HR = 4.661, 95%CI, 2.598-8.393, p < 0.0001] were independent predictors for OS and PFS. Inhibitory KIR2DL2 and activating NKp30 NK cells at day 100 are prognostic immune biomarkers in lymphoma patients treated with APBHSCT.
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CD36 expression in both immune and non-immune cells is known to be directly involved in cancer metastasis. Extracellular vesicles (EVs) secreted by malignant melanocytes play a vital role in developing tumor-promoting microenvironments, but it is unclear whether this is mediated through CD36. To understand the role of CD36 in melanoma, we first analyzed the SKCM dataset for clinical prognosis, evaluated the percentage of CD36 in lymphatic fluid-derived EVs (LEVs), and tested whether melanoma-derived EVs increase CD36 expression and induce M2-macrophage-like characteristics. Furthermore, we performed a multiplex immunofluorescence (MxIF) imaging analysis to evaluate the CD36 expression and its colocalization with various other cells in the lymph node (LN) of patients and control subjects. Our findings show that cutaneous melanoma patients have a worse clinical prognosis with high CD36 levels, and a higher percentage of CD36 in total LEVs were found at baseline in melanoma patients compared to control. We also found that monocytic and endothelial cells treated with melanoma EVs expressed more CD36 than untreated cells. Furthermore, melanoma-derived EVs can regulate immunosuppressive macrophage-like characteristics by upregulating CD36. The spatial imaging data show that cells in tumor-involved sentinel LNs exhibit a higher probability of CD36 expression than cells from control LNs, but this was not statistically significant. Conclusively, our findings demonstrated that CD36 plays a vital role in controlling the immunosuppressive microenvironment in the LN, which can promote the formation of a protumorigenic niche.
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Antígenos CD36 , Vesículas Extracelulares , Melanoma , Microambiente Tumoral , Humanos , Vesículas Extracelulares/metabolismo , Melanoma/metabolismo , Melanoma/patología , Antígenos CD36/metabolismo , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Línea Celular Tumoral , Macrófagos/metabolismo , Macrófagos/patología , Pronóstico , Femenino , Melanoma Cutáneo Maligno , Ganglios Linfáticos/patología , Ganglios Linfáticos/metabolismo , MasculinoRESUMEN
PURPOSE: AB160 is a 160-nm nano-immunoconjugate consisting of nab-paclitaxel (ABX) nanoparticles noncovalently coated with bevacizumab (BEV) for targeted delivery into tissues expressing high levels of VEGF. Preclinical data showed that AB160 resulted in greater tumor targeting and tumor inhibition compared with sequential treatment with ABX then BEV. Given individual drug activity, we investigated the safety and toxicity of AB160 in patients with gynecologic cancers. PATIENTS AND METHODS: A 3+3 phase I trial was conducted with three potential dose levels in patients with previously treated endometrial, cervical, and platinum-resistant ovarian cancer to ascertain the recommended phase II dose (RP2D). AB160 was administered intravenously on days 1, 8, and 15 of a 28-day cycle (ABX 75-175 mg/m2, BEV 30-70 mg/m2). Pharmacokinetic analyses were performed. RESULTS: No dose-limiting toxicities (DLT) were seen among the three dose levels tested. Grade 3/4 toxicities included neutropenia, thromboembolic events, and leukopenia. DL2 (ABX 150 mg/m2, BEV 60 mg/m2) was chosen as the RP2D. Seven of the 19 patients with measurable disease (36.8%) had confirmed partial responses (95% confidence interval, 16.3%-61.6%). Pharmacokinetic analyses demonstrated that AB160 allowed 50% higher paclitaxel dosing and that paclitaxel clearance mirrored that of therapeutic antibodies. CONCLUSIONS: The safety profile and clinical activity of AB160 supports further clinical testing in patients with gynecologic cancers; the RP2D is DL2 (ABX 150 mg/m2, BEV 60 mg/m2).
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Albúminas , Protocolos de Quimioterapia Combinada Antineoplásica , Bevacizumab , Neoplasias de los Genitales Femeninos , Paclitaxel , Humanos , Femenino , Paclitaxel/administración & dosificación , Paclitaxel/efectos adversos , Paclitaxel/farmacocinética , Persona de Mediana Edad , Albúminas/administración & dosificación , Albúminas/efectos adversos , Anciano , Neoplasias de los Genitales Femeninos/tratamiento farmacológico , Neoplasias de los Genitales Femeninos/patología , Bevacizumab/administración & dosificación , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Inmunoconjugados/administración & dosificación , Inmunoconjugados/efectos adversos , Inmunoconjugados/farmacocinética , Inmunoconjugados/uso terapéutico , Resultado del Tratamiento , Dosis Máxima ToleradaRESUMEN
Both targeted therapies and immunotherapies provide benefit in resected Stage III melanoma. We hypothesized that the combination of targeted and immunotherapy given prior to therapeutic lymph node dissection (TLND) would be tolerable and drive robust pathologic responses. In NeoACTIVATE (NCT03554083), a Phase II trial, patients with clinically evident resectable Stage III melanoma received either 12 weeks of neoadjuvant vemurafenib, cobimetinib, and atezolizumab (BRAF-mutated, Cohort A, n = 15), or cobimetinib and atezolizumab (BRAF-wild-type, Cohort B, n = 15) followed by TLND and 24 weeks of adjuvant atezolizumab. Here, we report outcomes from the neoadjuvant portion of the trial. Based on intent to treat analysis, pathologic response (≤50% viable tumor) and major pathologic response (complete or near-complete, ≤10% viable tumor) were observed in 86.7% and 66.7% of BRAF-mutated and 53.3% and 33.3% of BRAF-wild-type patients, respectively (primary outcome); these exceeded pre-specified benchmarks of 50% and 30% for major pathologic response. Grade 3 and higher toxicities, primarily dermatologic, occurred in 63% during neoadjuvant treatment (secondary outcome). No surgical delays nor progression to regional unresectability occurred (secondary outcome). Peripheral blood CD8 + TCM cell expansion associated with favorable pathologic responses (exploratory outcome).
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Anticuerpos Monoclonales Humanizados , Azetidinas , Melanoma , Piperidinas , Neoplasias Cutáneas , Humanos , Melanoma/tratamiento farmacológico , Melanoma/etiología , Vemurafenib/uso terapéutico , Terapia Neoadyuvante , Proteínas Proto-Oncogénicas B-raf/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/etiología , MutaciónRESUMEN
Introduction: Proton craniospinal irradiation (pCSI) is a treatment option for leptomeningeal disease (LMD), which permits whole neuroaxis treatment while minimizing toxicity. Despite this, patients inevitably experience progression. Adding systemic therapy to pCSI may improve outcomes. Methods: In this single-institution retrospective case series, we present the feasibility of treatment with pCSI (30Gy, 10 fractions) and an immune checkpoint inhibitor (ICI) in two sequential patients with LMD from melanoma. Results: The first patient developed LMD related to BRAF V600E-mutant melanoma after prior ICI and BRAF-targeted therapy. After pCSI with concurrent nivolumab, the addition of relatlimab, and BRAF-targeted therapy, he remained alive 7 months after LMD diagnosis despite central nervous system progression. The second patient developed LMD related to BRAF-wildtype melanoma after up-front ICI. He received pCSI with concurrent ipilimumab and nivolumab, then nivolumab maintenance. Though therapy was held for ICI hepatitis, the patient remained progression-free 5 months after LMD diagnosis. Conclusion: Adding an ICI to pCSI is feasible for patients with LMD and demonstrates a tolerable toxicity profile. While prospective evaluation is ultimately warranted, pCSI with ICI may confer survival benefits, even after prior ICI.
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OBJECTIVE: The objective of this study was to analyze the hemorrhagic risk of melanoma brain metastases after Gamma Knife radiosurgery (GKRS). METHODS: A prospective institutional database was retrospectively queried to identify patients who underwent GKRS for melanoma brain metastases between 1990 and 2021. Lesional hemorrhage was defined as definite or possible based on radiologists' readings, and severity was graded according to Common Terminology Criteria for Adverse Events. RESULTS: Two hundred ninety-one patients with 1083 lesions treated in 419 sessions were identified. The mean (± SD) patient age was 60 ± 15 years, and 61% were male. The median follow-up period for overall survival (OS) was 11 (range 0-214) months with 581 patient-years. Definite/possible lesional hemorrhages occurred in 13% of lesions, with grade 3 hemorrhages observed in 4% of lesions. Surgical intervention was required in 2% of cases (5% of patients), and all resected lesions were pathologically consistent with melanoma. A decreased risk of definite/possible lesional hemorrhage was associated with a later time period between 2015 and 2021 (OR 0.45, 95% CI 0.266-0.75, p = 0.0021), increased marginal dose (OR 0.91, 95% CI 0.83-0.99, p = 0.037), antiplatelet use post-GKRS (OR 0.195, 95% CI 0.083-0.46, p < 0.001), and whole-brain radiotherapy (WBRT; OR 0.53, 95% CI 0.344-0.82, p = 0.0042). After 2015, more patients received anticoagulation, B-Raf proto-oncogene inhibitors, and immune checkpoint inhibitors, and fewer received bevacizumab (p < 0.001). The cumulative risk of lesional hemorrhage was 17%-20% at 36 months from GKRS, with 95%-96% of cases occurring within 12 months. The median patient OS was 11 (95% CI 9-13) months, and multivariate Cox regression analysis revealed that antiplatelet agents (hazard ratio [HR] 0.66, 95% CI 0.45-0.96, p = 0.031) and immune checkpoint inhibitors (HR 0.35, 95% CI 0.26-0.48, p < 0.001) were associated with longer OS, while WBRT (HR 1.36, 95% CI 1.02-1.81, p = 0.037) and definite/possible hemorrhage (HR 1.39, 95% CI 1.04-1.85, p = 0.024) were associated with shorter OS. CONCLUSIONS: The definite hemorrhage risk of melanoma brain metastases after GKRS was 17% in the first 3 years and 95% of the lesional hemorrhage occurred within the 1st year. Surgical intervention was needed in 5% of patients. Antiplatelet agents and immune checkpoint inhibitors were associated with improved OS, while definite/possible hemorrhage was associated with worse OS.
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Neoplasias Encefálicas , Melanoma , Radiocirugia , Humanos , Masculino , Persona de Mediana Edad , Anciano , Femenino , Radiocirugia/efectos adversos , Resultado del Tratamiento , Estudios Retrospectivos , Melanoma/patología , Inhibidores de Puntos de Control Inmunológico , Inhibidores de Agregación Plaquetaria , Estudios Prospectivos , Neoplasias Encefálicas/cirugía , Hemorragia/etiología , Estudios de SeguimientoRESUMEN
INTRODUCTION: The infusion of autograft Natural Killer Cells (NKC)/CD14+ HLA-DRDIM ratio is a predictor of survival in lymphoma patients undergoing autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT). This study evaluated if the Day 100 NKC/CD14+ HLA-DRDIM ratio still functions as a prognostic immune-biomarker. METHODS: This was a retrospective, single-institution, cohort analysis including 107 patients in this study that had clinical assessment at Day 100 post-APBHSCT from our prior phase III trial. We evaluated the prognostic ability of the Day 100 NKC/CD14+ HLA-DRDIM ratio to predict overall survival (OS) and progression-free survival (PFS) using Cox regression model for outcome analysis and survival by Kaplan-Meier method. RESULTS: The median follow-up from day 100 was 94.7 months (range 4.83-158.1 months) for the entire cohort. Patients with a Day 100 NKC/CD14+ HLA-DRDIM ratio ≥1.67 experienced better OS and PFS versus those with a Day 100 NKC/CD14+ HLA-DRDIM ratio <1.67: median OS was not reached versus 49.7 months, the 5-year OS rates were 91% (95% CI, 81%-96%) versus 40% (95% CI, 27%-55%), p < .0001, respectively; and median PFS was not reached versus 23.5 months, the 5-year PFS rates were 66% (95% CI, 55%-81%) versus 21% (95% CI, 15%-40%), p < .0001, respectively. Day 100 NKC/CD14+ HLA-DRDIM ratio was an independent predictor for OS and PFS in the multivariate analysis. CONCLUSIONS: Day 100 NKC/CD14+ HLA-DRDIM ratio is a prognostic immune-biomarker in lymphoma patients post- APBHSCT.
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Trasplante de Células Madre Hematopoyéticas , Linfoma , Humanos , Estudios Retrospectivos , Trasplante de Células Madre Hematopoyéticas/métodos , Linfoma/terapia , Antígenos HLA-DR , Células Asesinas Naturales , Trasplante Autólogo/métodos , Biomarcadores , Supervivencia sin EnfermedadRESUMEN
Introduction: Metastatic uveal melanoma (MUM) has a poor prognosis and treatment options are limited. These patients do not typically experience durable responses to immune checkpoint inhibitors (ICIs). Oncolytic viruses (OV) represent a novel approach to immunotherapy for patients with MUM. Methods: We developed an OV with a Vesicular Stomatitis Virus (VSV) vector modified to express interferon-beta (IFN-ß) and Tyrosinase Related Protein 1 (TYRP1) (VSV-IFNß-TYRP1), and conducted a Phase 1 clinical trial with a 3 + 3 design in patients with MUM. VSV-IFNß-TYRP1 was injected into a liver metastasis, then administered on the same day as a single intravenous (IV) infusion. The primary objective was safety. Efficacy was a secondary objective. Results: 12 patients with previously treated MUM were enrolled. Median follow up was 19.1 months. 4 dose levels (DLs) were evaluated. One patient at DL4 experienced dose limiting toxicities (DLTs), including decreased platelet count (grade 3), increased aspartate aminotransferase (AST), and cytokine release syndrome (CRS). 4 patients had stable disease (SD) and 8 patients had progressive disease (PD). Interferon gamma (IFNγ) ELIspot data showed that more patients developed a T cell response to virus encoded TYRP1 at higher DLs, and a subset of patients also had a response to other melanoma antigens, including gp100, suggesting epitope spreading. 3 of the patients who responded to additional melanoma antigens were next treated with ICIs, and 2 of these patients experienced durable responses. Discussion: Our study found that VSV-IFNß -TYRP1 can be safely administered via intratumoral (IT) and IV routes in a previously treated population of patients with MUM. Although there were no clear objective radiographic responses to VSV-IFNß-TYRP1, dose-dependent immunogenicity to TYRP1 and other melanoma antigens was seen.
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Viroterapia Oncolítica , Virus Oncolíticos , Estomatitis Vesicular , Animales , Humanos , Interferón beta/metabolismo , Antígenos Específicos del Melanoma , Monofenol Monooxigenasa/metabolismo , Viroterapia Oncolítica/efectos adversos , Virus Oncolíticos/genética , Linfocitos T/metabolismo , Virus de la Estomatitis Vesicular IndianaRESUMEN
PURPOSE: Surveillance after primary melanoma treatment aims to detect early signs of low-volume systemic disease. The current standard of care, surveillance imaging, is costly and difficult to access. We therefore sought to develop methylated DNA markers (MDMs) as promising alternatives for disease surveillance. METHODS: We used reduced representation bisulfite sequencing (RRBS) to identify MDMs in DNA samples obtained from metastatic melanoma, benign nevi, and normal skin tissues. The identified MDMs underwent validation in an independent cohort of tissue and buffy coat DNA samples. Subsequently, we tested the validated MDMs in the plasma DNA of patients with metastatic melanoma undergoing surveillance with total body imaging and compared them with cancer-free controls. To estimate the overall predictive accuracy of the MDMs, we used random forest modeling with bootstrap cross-validation. RESULTS: Forty MDMs demonstrated discrimination between melanoma cases and controls consisting of benign nevi and normal skin. Nine MDMs passing biological validation in tissue were run on 77 plasma samples from individuals with a history of metastatic melanoma, 49 of whom had evidence of disease detected by imaging at the time of blood draw, and 100 cancer-free controls. The cross-validated sensitivity of the panel for imaging-positive disease was 80% with a specificity of 100% in cancer-free controls, resulting in an overall AUC of 0.88 (95% CI, 0.81 to 0.96). The survival estimates for patients with melanoma who tested positive for the panel at 6 months and 1 year were 67% and 56%, respectively, while those who tested negative had survival rates of 100% and 92%. CONCLUSION: MDMs identified by RRBS demonstrate a high degree of concordance with imaging results in the plasma of patients with metastatic melanoma. Further prospective studies in larger intended use cohorts are needed to confirm these findings.
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Melanoma , Nevo , Humanos , Marcadores Genéticos , Estudios Prospectivos , Melanoma/diagnóstico , Melanoma/genética , ADNRESUMEN
The natural history of advanced malignant melanoma demonstrates that, in most cases, widespread tumor dissemination is preceded by regional metastases involving tumor-draining lymph nodes [sentinel lymph nodes (SLNs)]. Under physiological conditions, LNs play a central role in immunosurveillance to non-self-antigens to which they are exposed via afferent lymph. The dysfunctional immunity in SLNs is mediated by tumor secretory factors that allow the survival of metastatic melanoma cells within the LN by creating a premetastatic niche (PMN). Recent studies outline the altered microenvironment of LNs shaped by melanoma mediators. Here, we discuss tumor secretory factors involved in subverting tumor immunity and remodeling LNs and highlight emerging therapeutic strategies to reinvigorate antitumoral immunity in SLNs.
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Melanoma , Neoplasias Cutáneas , Humanos , Metástasis Linfática/patología , Melanoma/patología , Neoplasias Cutáneas/patología , Ganglios Linfáticos , Microambiente TumoralRESUMEN
A 74-year-old male presented with rectal pain; workup uncovered an anal mass, and a diagnosis of melanoma was rendered via histologic examination and immunohistochemical (IHC) studies. Droplet digital PCR (ddPCR)-based BRAF testing was performed and revealed the presence of BRAF V600E, which is a common targetable genetic alteration in melanoma. Interestingly, the ratio of mutant to wild-type copy number was low (0.3%), whereas tumor cell percentage on tissue slides was 90%. With additional workup, BRAF V600E IHC confirmed a very small subset of BRAF V600E-positive cells, and a next-generation sequencing (NGS) panel revealed a pathogenic KIT variant, p.L576P, with an allele frequency of 63%. It was initially hypothesized that the main driver of the melanoma was the KIT alteration, whereas a small subclone (not detected by NGS, which has a 5% limit of detection) was driven by the BRAF V600E detected by ddPCR. To determine whether there were morphologic differences between the 2 clones, a careful review of the histology was performed and revealed distinct morphology of the BRAF V600E-positive cells, including pale cytoplasm, nuclear grooves, and infiltrating eosinophils. Additional IHC workup of the BRAF V600E-positive cells showed coexpression of CD1a, Langerin, and S100, diagnostic of Langerhans cell histiocytosis (LCH). This diagnosis was unexpected and would have been missed without highly sensitive molecular testing; yet it is of clinical importance for the patient. This case raises interesting biology questions regarding the relationship between melanoma and LCH; moreover, it highlights the importance of integrating quantitative information in molecular data interpretation.
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Histiocitosis de Células de Langerhans , Melanoma , Masculino , Humanos , Anciano , Proteínas Proto-Oncogénicas B-raf/genética , Mutación , Melanoma/diagnóstico , Melanoma/genética , Histiocitosis de Células de Langerhans/diagnóstico , Histiocitosis de Células de Langerhans/genéticaRESUMEN
BACKGROUND: SEA-CD40 is an investigational, non-fucosylated, humanized monoclonal IgG1 antibody that activates CD40, an immune-activating tumor necrosis factor receptor superfamily member. SEA-CD40 exhibits enhanced binding to activating FcγRIIIa, possibly enabling greater immune stimulation than other CD40 agonists. A first-in-human phase 1 trial was conducted to examine safety, pharmacokinetics, and pharmacodynamics of SEA-CD40 monotherapy in patients with advanced solid tumors and lymphoma. METHODS: SEA-CD40 was administered intravenously to patients with solid tumors or lymphoma in 21-day cycles with standard 3+3 dose escalation at 0.6, 3, 10, 30, 45, and 60 µg/kg. An intensified dosing regimen was also studied. The primary objectives of the study were to evaluate the safety and tolerability and identify the maximum tolerated dose of SEA-CD40. Secondary objectives included evaluation of the pharmacokinetic parameters, antitherapeutic antibodies, pharmacodynamic effects and biomarker response, and antitumor activity. RESULTS: A total of 67 patients received SEA-CD40 including 56 patients with solid tumors and 11 patients with lymphoma. A manageable safety profile was observed, with predominant adverse events of infusion/hypersensitivity reactions (IHRs) reported in 73% of patients. IHRs were primarily ≤grade 2 with an incidence associated with infusion rate. To mitigate IHRs, a standardized infusion approach was implemented with routine premedication and a slowed infusion rate. SEA-CD40 infusion resulted in potent immune activation, illustrated by dose dependent cytokine induction with associated activation and trafficking of innate and adaptive immune cells. Results suggested that doses of 10-30 µg/kg may result in optimal immune activation. SEA-CD40 monotherapy exhibited evidence of antitumor activity, with a partial response in a patient with basal cell carcinoma and a complete response in a patient with follicular lymphoma. CONCLUSIONS: SEA-CD40 was tolerable as monotherapy and induced potent dose dependent immune cell activation and trafficking consistent with immune activation. Evidence of monotherapy antitumor activity was observed in patients with solid tumors and lymphoma. Further evaluation of SEA-CD40 is warranted, potentially as a component of a combination regimen. TRIAL REGISTRATION NUMBER: NCT02376699.
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Antineoplásicos , Carcinoma Basocelular , Linfoma Folicular , Neoplasias Cutáneas , Humanos , Anticuerpos Monoclonales , Antígenos CD40 , Anticuerpos Monoclonales HumanizadosRESUMEN
Multiplex immunofluorescence (MxIF) images provide detailed information of cell composition and spatial context for biomedical research. However, compromised data quality could lead to research biases. Comprehensive image quality checking (QC) is essential for reliable downstream analysis. As a reliable and specific staining of cell nuclei, 4',6-diamidino-2-phenylindole (DAPI) signals were used as references for tissue localization and auto-focusing across MxIF staining-scanning-bleaching iterations and could potentially be reused for QC. To confirm the feasibility of using DAPI as QC reference, pixel-level DAPI values were extracted to calculate signal fluctuations and tissue content similarities in staining-scanning-bleaching iterations for identifying quality issues. Concordance between automatic quantification and human experts' annotations were evaluated on a data set consisting of 348 fields of view (FOVs) with 45 immune and tumor cell markers. Cell distribution differences between subsets of QC-pass vs QC-failed FOVs were compared to investigate the downstream effects. Results showed that 87.3% FOVs with tissue damage and 73.4% of artifacts were identified. QC-failed FOVs showed elevated regional gathering in cellular feature space compared with the QC-pass FOVs. Our results supported that DAPI signals could be used as references for MxIF image QC, and low-quality FOVs identified by our method must be cautiously considered for downstream analyses.
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Indoles , Neoplasias , Humanos , Técnica del Anticuerpo FluorescenteRESUMEN
Introduction: Immune cell infiltration into the tumor microenvironment is generally associated with favorable clinical outcomes in solid tumors. However, the dynamic interplay among distinct immune cell subsets within the tumor-immune microenvironment as it relates to clinical responses to immunotherapy remains unresolved. In this study, we applied multiplex immunofluorescence (MxIF) to spatially characterize tumor-immune interactions within the metastatic melanoma lymph node. Methods: Pretreatment, whole lymph node biopsies were evaluated from 25 patients with regionally metastatic melanoma who underwent subsequent anti-PD1 therapy. Cyclic MxIF was applied to quantitatively and spatially assess expression of 45 pathologist-validated antibodies on a single tissue section. Pixel-based single cell segmentation and a supervised classifier approach resolved 10 distinct tumor, stromal and immune cell phenotypes and functional expression of PD1. Results: Single cell analysis across 416 pathologist-annotated tumor core regions of interest yielded 5.5 million cells for spatial evaluation. Cellular composition of tumor and immune cell subsets did not differ in the tumor core with regards to recurrence outcomes (p>0.05) however spatial patterns significantly differed in regional and paracrine neighborhood evaluations. Specifically, a regional community cluster comprised of primarily tumor and dendritic cells was enriched in patients that did not experience recurrence (p=0.009). By an independent spatial approach, cell-centric neighborhood analyses identified an enrichment for dendritic cells in cytotoxic T cell (CTL) and tumor cell-centric neighborhoods in the no recurrence patient response group (p<0.0001). Further evaluation of these neighborhoods identified an enrichment for CTL-dendritic cell interactions in patients that did not experience recurrence (p<0.0001) whereas CTL-macrophage interactions were more prevalent in CTL-centric neighborhoods of patients who experienced recurrence (p<0.0001). Discussion: Overall, this study offers a more comprehensive evaluation of immune infiltrates and spatial-immune signatures in the metastatic tumor-immune microenvironment as it informs recurrence risk following immunotherapy.
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Melanoma , Neoplasias Primarias Secundarias , Humanos , Melanoma/tratamiento farmacológico , Linfocitos T Citotóxicos , Inmunoterapia , Ganglios Linfáticos/patología , Microambiente TumoralRESUMEN
Direct interactions between tumor and immune cells mediate the antitumor effect of all modern cancer immunotherapeutic agents. Simultaneously, tumor cells have evolved mechanisms of evasion including the downregulation of HLA-I potentially disrupting the mechanism of action employed by many immune checkpoint inhibitors. And yet the in situ interplay between these cells within the tumor immune microenvironment (TIME) remains elusive. Recent advances in histologic multiplex bioimaging platforms have enabled in-depth molecular characterization of single cells within spatially-preserved and clinically archived tumor tissues. Herein, we applied multiplex immunofluorescence (MxIF) to excisional lymph node biopsies from 14 patients with metastatic melanoma who experienced clear objective responses to immunotherapy (7 complete response; 7 progressive disease) to determine distinguishing features of the TIME in the pretreatment setting. Distinct regions of the TIME were evaluated using 35 proteins probing tumor, immune and vasculature components across 323 fields of view. Single cell compositional analysis confirmed established prognostic immune cell types including increased prevalence of cytotoxic T cells within the tumor core FOVs of responders. Integrating single cell quantification with the spatial arrangement of cellular neighborhoods surrounding tumor cells revealed novel, spatial immune signatures capable of stratifying TIME based on clinical response. Our analysis revealed dynamic cellular composition of the TCCN based on anatomical subregion, functional expression of HLA-I by the index tumor cell and ultimately clinical response to immunotherapy. Overall, this study provides an analytical framework to resolve the cellular complexity of the TIME, increasingly relevant to the outcomes of modern cancer immunotherapy.
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Inhibidores de Puntos de Control Inmunológico , Melanoma , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Melanoma/terapia , Inmunoterapia/métodos , Linfocitos T Citotóxicos/metabolismo , Microambiente TumoralRESUMEN
Approximately 50% of melanomas contain BRAF mutations; the effects on survival are unclear. We aimed to determine whether mutant BRAF expression in melanoma differs according to age, sex, and melanoma-specific survival. A total of 638 patients who resided in Olmsted County, Minnesota, with a first lifetime diagnosis of melanoma between 1970 and 2009 were identified from the Rochester Epidemiology Project (REP). Available tissue was analyzed for a BRAF V600E mutation with immunohistochemistry.