Citations

Abstract Spread to regional lymph nodes is the most common site of metastasis from melanoma and almost all solid tumors. Lymphatic metastases begin in the first lymph nodes draining from the primary tumor, i.e., the sentinel lymph nodes (SLN), preceded by the development of the premetastatic niche (PMN) initiated by primary cutaneous melanoma. Melanoma mediators are known to play a critical role in developing the PMN through the immune subversion in SLN. The DCs are a vital component of LN immunity that directly relates to eliciting T-cell functions. Our multiplex immunofluorescence (MxIF) analysis of SLNs indicates significantly decreased levels of DCs and cytotoxic-T cells in both core and interface areas of tumors in recurred patients treated with immunotherapy compared to non-recurred melanoma patients. The goal of this study is to determine whether the lymphatic extracellular vesicles (L-EVs) of melanoma patients regulate DC tolerization in SLN in order to stimulate the development of PMN. To identify whether L-EVs stimulate DC suppression, we extracted L-EVs from the lymphatic fluid of patients with melanoma downstream of primary cutaneous melanomas, compared it with controls (non-malignant post-operative fluid collected from the lymph node dissection field, and evaluated their effect on DC maturation by challenging these L-EVs in monocytic (CD14+) and hematopoietic stem cells (CD34+) cells. We found that L-EVs derived from melanoma patients suppress differentiation of CD11c+HLA-DR+ DCs and their maturation (defined by CD83 and CD86) compared to control L-EVs in CD14+ based DC maturation assay. We also found a reduction of conventional type 1 DC (cDC1) in CD34+-based DC maturation assay those challenged with patients’ L-EVs compared to control. To understand the mechanism of DC suppression in patients' L-EVs, we also conducted a total RNA sequencing analysis of subsets of DCs from maturation experiments to evaluate differently regulated transcriptomic profiles. The gene ontology analysis indicates that patient-derived L-EVs interfere with tRNA processing, oncostatin M & IL-10 signaling, and other immunoregulatory pathways to alter the hematopoietic cell lineage pathways in the DC maturation. Furthermore, the proteomic analysis of L-EVs also indicated several differentially regulated cargo-identified proteins in patient L-EVs, which are associated with immunomodulation, including regulation of DC function compared to control L-EVs. In conclusion, our study revealed that the patient's L-EVs carry differential proteomic cargoes secreted by cutaneous melanoma, which may be involved in regulating DC differentiation and maturation to diminish their functions and support the developing PMN in SLN. Citation Format: Shankar Suman, Wendy K. Nevala, James W. Jakub, Ray Guo, Alexey A. Leontovich, Raymond M. Moore, Chathu L. Atherton, Liyi Geng, Jeffrey E. Johnson, Sarah A. McLaughlin, Svetomir N. Markovic. Melanoma-derived lymphatic extracellular vesicles suppress dendritic cell differentiation and maturation in the sentinel lymph node [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1503.

AbstractIntroductionThe 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.MethodsThis 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.ResultsThe 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.ConclusionsDay 100 NKC/CD14+HLA‐DRDIM ratio is a prognostic immune‐biomarker in lymphoma patients post‐ APBHSCT.

Introduction: Nanotechnology for cancer therapy aims to increase drug delivery to tumor sites while lowering toxicity. AR160 is a novel 160 nm nano-immunoconjugate consisting of commercial grade nab-paclitaxel (NP) nanoparticles non-covalently coated with rituximab (R) for targeted delivery into tissues expressing CD20. The drug delivery system and clinical grade product was developed in the laboratory of Svetomir N. Markovic, M.D.,Ph.D. Single agent rituximab at a dose of 375 mg/m 2 in relapsed patients with aggressive lymphomas is not utilized and has limited activity in patients with multiple relapses. Taxanes (Younes 1995, Wilson 1995) have modest clinical activity as a single agent with overall response rates of 23% and 17%, respectively, when administered at doses of 200 mg/m 2 and 140 mg/m 2 in previously treated patients with non-Hodgkin lymphoma (NHL). We hypothesized that utilizing the targeted ability of rituximab that the AR160 taxane nanoparticle would have activity in relapsed B-cell NHL and offer a new class of agents for relapsed B-cell NHL. Methods: A 3+3 phase I clinical trial was conducted in patients with relapsed refractory non-Hodgkin lymphoma to determine the maximum tolerated dose of AR160 administered intravenously on days 1, 8, and 15 of a 28-day cycle. Patients were required to have had prior systemic treatment with no other therapy with clinical benefit. Patients with aggressive lymphoma were required to have a stem cell transplant if transplant eligible. AR160 was prepared on-site in the Mayo Clinic Cancer Center Research Pharmacy, and the product was reconstituted by mixing the two agents delivering as one combined infusion at a specific concentration. The starting dose level (DL1) was NP at 100mg/m 2 with R at 40 mg/m 2. These doses were significantly lower than standard doses. Disease evaluations were conducted after every 2 treatment cycles using CT based criteria from the revised 2014 Lugano Classification or PET scans. Patients were treated until disease progression or intolerability. Samples were collected for pharmacokinetic (PK) studies. Results: Nine (7 males and 2 females) patients with a median age of 61 (range 55-84 years) were enrolled (2 follicular lymphoma (FL), 1 previously transformed from FL to diffuse large B-cell lymphoma (DLBCL) followed by FL relapse, 1 high-grade B-cell lymphoma with MYC and BCL2 rearrangements transformed from FL, 1 transformed FL to DLBCL, 2 high-grade B-cell lymphoma with MYC and BCL2 rearrangements, 1 high-grade B-cell lymphoma NOS and 1 mantle cell lymphoma. The median number of prior treatments was 4 (range 3-16). At dose level 1, all 3 of the first 3 patients experienced grade 3 or 4 neutropenia (C1D15, C4D1, and C1D15) with the C4D1 a grade 4 and unlikely related. After the first three patients, de-escalation to dose level -1 was implemented per protocol with NP at 75 mg/m 2 and rituximab at 30 mg/m 2. There was one patient with a grade 3 non-hematological adverse event all possibly related to treatment (anorexia, diarrhea, and enterocolitis). There were 6 partial responses (71%), 1 complete response (14%) with high-grade B-cell lymphoma with MYC and BCL2 rearrangement, 1 stable disease with mantle cell lymphoma and 1 patient had progressive disease. Six of the 9 patients were alive with subsequent therapies. PK evaluation is pending. Conclusion: AR160 therapy is a novel drug delivery system incorporating significantly lower doses of paclitaxel and rituximab that is safe and demonstrates promising clinical activity in patients with low-grade and high-grade previously treated B-cell lymphomas. This principle of treatment is applicable to other disorders. Further clinical testing is warranted in these patient populations.

Introduction: Our group previously published that the infusion autograft inhibitor KIR2DL2 (CD158b+CD337-) and activating NKp30 (CD158b- CD337+) Natural killer (NK) cells receptors were predictors of clinical outcomes in lymphoma patients undergoing autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT) (Porrata et al. Leukemia Research 2019; 81: 1-9). To assess if these subsets of NK cells still hold clinical relevance, we set up to investigate their prognostic ability to predict clinical outcomes at Day 100 post-APBHSCT. Methods: 107 lymphoma patients who participated in our double-blind phase III trail (Porrata et al, Biology of Blood and Marrow Transplantation 2016; 22(6); 1017-1023) and had clinical assessment at Day 100 post-APBHSCT were included in this study. Nkp30 and KIR2DL2 at Day 100 were assessed by flow cytometry analysis. Results: With a median follow-up for the entire cohort from Day 100 was 94.7 months (range: 4.83-158.1 months). Figure 1 shows the overall survival (OS) and progression-free survival (PFS) based on the absolute numbers of KIR2DL2 and Nkp30 NK cells. At Day 100, patients with a KIR2DL2 &lt; 0.08 and Nkp30 ≥0.19 experienced superior OS and PFS compared with patients with a Day 100 KIR2DLD2 ≥0.08 and NKp30 &lt; 0.19. In the multivariate analysis both the KIR2DL2 [OS: HR = 1.767, 95%CI, 1.302-30.31, p &lt; 0.04; and PFS: HR = 2.663, 95%CI, 1.108-6.403, p &lt; 0.03] and Nkp30 [OS: HR = 2.594, 95% CI, 1.239-5.432, p &lt; 0.01; and PFS: HR = 3.676, 95% CI, 1.875-7.206, p &lt; 0.01] were independent predictors for OS and PFS. Conclusion: Day 100 inhibitory KIR2DL2 and activating NKp30 NK cells are prognostic immune-biomarkers in lymphoma patients undergoing APBHSCT.

Abstract BACKGROUND Leptomeningeal disease (LMD) is a devastating complication of cancer with median overall survival (OS) of 3-6 months following diagnosis. Proton craniospinal irradiation (pCSI) is a novel treatment option for LMD, permitting whole neuroaxis treatment with tolerable toxicity and improved efficacy compared to conventional photon whole brain radiation therapy and as needed focal spine radiotherapy. Despite this, patients experience progression and succumb to LMD. Addition of systemic therapy to pCSI may improve outcomes. METHODS In this single institution retrospective case series, we report feasibility of combination treatment with pCSI and immune checkpoint inhibitor (ICI) in patients with LMD from solid tumors. RESULTS Three patients were treated with pCSI (30 Gy in 10 fractions) and ICI. The first patient developed LMD related to large cell neuroendocrine carcinoma of lung primary, diagnosed by imaging and cytology, having previously undergone resection and stereotactic radiosurgery for brain metastases. Clinical and radiographic improvement was noted following pCSI, bevacizumab, and pembrolizumab therapy. Patient experienced disease progression and passed away only after ICI was held due to hypophysitis, with 6-month OS. The second patient developed LMD related to BRAF V600E mutant melanoma, diagnosed by imaging and cytology, having previously undergone ICI and BRAF-targeted therapy. Following pCSI, relatlimab, nivolumab, and BRAF-targeted therapy, the patient remains alive 7 months after LMD diagnosis despite radiographic progression treated with carboplatin, paclitaxel, intrathecal IL-2, ipilimumab, and bevacizumab. The third developed LMD related to BRAF-wildtype melanoma, diagnosed by imaging, having previously received ICI. Patient underwent pCSI followed by ipilimumab and nivolumab, progression-free 5 months after LMD diagnosis. CONCLUSION Addition of systemic therapy to pCSI is feasible for patients with LMD. Combination of ICI and pCSI may confer additional survival benefit, even among patients with prior exposure to ICI. These initial promising results warrant prospective evaluation.

Abstract OBJECTIVE This study aimed to analyze the hemorrhagic risk of melanoma brain metastases following SRS. METHODS A retrospective analysis was conducted using a prospective institutional database between 1990 and 2021. Lesional hemorrhage was categorized as definite or possible, and hemorrhage severity was graded using CTCAE. RESULTS The study identified 291 patients with 1083 lesions. The mean age of patients was 60 ± 15 years, with 61% being male. Median follow-up was 11 months, corresponding to 581 patient-years. Among the patients, 82 (28%) experienced 135 lesional hemorrhages (13%). Of these, 88 lesions in 61 patients were grade 1-2 hemorrhages, 42 lesions in 25 patients were grade 3, and five lesions in five patients progressed from grade 1 to 3. Surgical intervention was required for 17 lesions (2%) in 16 patients (5%). Lesional hemorrhage was associated with radiosurgery performed between 2015 and 2021 (OR, 0.373; 95% CI, 0.231-0.60; p &lt; 0.001), increasing marginal dose (OR, 0.89; 95% CI, 0.82-0.98; p = 0.017), antiplatelet use (OR, 0.192; 95% CI, 0.082-0.45; p &lt; 0.001), and WBRT (OR, 0.51; 95% CI, 0.330-0.77; p = 0.0017). The cumulative lesional hemorrhage risk reached 17–20% at 36 months following radiosurgery, with 95-96% of hemorrhages occurring within the initial year. Median OS was 11 months (95% CI, 9-13 months). Patients who received radiosurgery between 2015 and 2021 exhibited a median OS of 17 months (95% CI, 10-24 months; p &lt; 0.001), while those without hemorrhage had a median OS of 12 months (95% CI, 10-14 months; p = 0.054). CONCLUSIONS Definitive hemorrhage risk was 17% for melanoma brain metastases after SRS within the first three years, with 95% of lesional hemorrhages occurring within the initial year. Surgical intervention was necessary in 5% of patients. Hemorrhage risk and overall survival improved after 2015.