Markovic, Svetomir N.

Abstract Throughout gestation, the maternal immune system is tightly modulated to allow growth of a semiallogeneic fetus. During the third trimester, the maternal immune system shifts to a proinflammatory phenotype in preparation for labor. What induces this shift remains unclear. Cell-free fetal DNA (cffDNA) is shed by the placenta and enters maternal circulation throughout pregnancy. Levels of cffDNA are increased as gestation progresses and peak before labor, coinciding with a shift to proinflammatory maternal immunity. Furthermore, cffDNA is abnormally elevated in plasma from women with complications of pregnancy, including preterm labor. Given the changes in maternal immunity at the end of pregnancy and the role of sterile inflammation in the pathophysiology of spontaneous preterm birth, we hypothesized that cffDNA can act as a damage-associated molecular pattern inducing an inflammatory cytokine response that promotes hallmarks of parturition. To test this hypothesis, we stimulated human maternal leukocytes with cffDNA from primary term cytotrophoblasts or maternal plasma and observed significant IL-1β and CXCL10 secretion, which coincides with phosphorylation of IFN regulatory factor 3 and caspase-1 cleavage. We then show that human maternal monocytes are crucial for the immune response to cffDNA and can activate bystander T cells to secrete proinflammatory IFN-γ and granzyme B. Lastly, we find that the monocyte response to cffDNA leads to vascular endothelium activation, induction of myometrial contractility, and PGE2 release in vitro. Our results suggest that the immune response to cffDNA can promote key features of the parturition cascade, which has physiologic consequences relevant to the timing of labor.

Background Most patients with advanced melanomas relapse after checkpoint blockade therapy. Thus, immunotherapies are needed that can be applied safely early, in the adjuvant setting. Seviprotimut-L is a vaccine containing human melanoma antigens, plus alum. To assess the efficacy of seviprotimut-L, the Melanoma Antigen Vaccine Immunotherapy Study (MAVIS) was initiated as a three-part multicenter, double-blind, placebo-controlled phase III trial. Results from part B1 are reported here. Methods Patients with AJCC V.7 stage IIB-III cutaneous melanoma after resection were randomized 2:1, with stage stratification (IIB/C, IIIA, IIIB/C), to seviprotimut-L 40 mcg or placebo. Recurrence-free survival (RFS) was the primary endpoint. For an hypothesized HR of 0.625, one-sided alpha of 0.10, and power 80%, target enrollment was 325 patients. Results For randomized patients (n=347), arms were well-balanced, and treatment-emergent adverse events were similar for seviprotimut-L and placebo. For the primary intent-to-treat endpoint of RFS, the estimated HR was 0.881 (95% CI: 0.629 to 1.233), with stratified logrank p=0.46. However, estimated HRs were not uniform over the stage randomized strata, with HRs (95% CIs) for stages IIB/IIC, IIIA, IIIB/IIIC of 0.67 (95% CI: 0.37 to 1.19), 0.72 (95% CI: 0.35 to 1.50), and 1.19 (95% CI: 0.72 to 1.97), respectively. In the stage IIB/IIC stratum, the effect on RFS was greatest for patients <60 years old (HR=0.324 (95% CI: 0.121 to 0.864)) and those with ulcerated primary melanomas (HR=0.493 (95% CI: 0.255 to 0.952)). Conclusions Seviprotimut-L is very well tolerated. Exploratory efficacy model estimation supports further study in stage IIB/IIC patients, especially younger patients and those with ulcerated melanomas. Trial registration number NCT01546571.

Ovarian cancer and pregnancy are two states in which the host immune system is exposed to novel antigens. Indeed, both the tumor and placenta must invade tissues, remodel vasculature to establish a robust blood supply, and evade detection by the immune system. Interestingly, tumor and placenta tissue use similar mechanisms to induce these necessary changes. One mediator is emerging as a key player in invasion, vascular remodeling, and immune evasion: extracellular vesicles (EVs). Many studies have identified EVs as a key mediator of cell-to-cell communication. Specifically, the cargo carried by EVs, which includes proteins, nucleic acids, and lipids, can interact with cells to induce changes in the target cell ranging from gene expression to migration and metabolism. EVs can promote cell division and tissue invasion, immunosuppression, and angiogenesis which are essential for both cancer and pregnancy. In this review, we examine the role of EVs in ovarian cancer metastasis, chemoresistance, and immune modulation. We then focus on the role of EVs in pregnancy with special attention on the vascular remodeling and regulation of the maternal immune system. Lastly, we discuss the clinical utility of EVs as markers and therapeutics for ovarian cancer and pre-eclampsia.

AbstractProblemCell‐free fetal DNA (cffDNA) shed from the placenta can be detected in maternal blood and increases incrementally during gestation. Concentrations are further elevated with pregnancy complications. Specific activators of cffDNA release in such complications have not been identified. Here, we use trophoblast cells from early and term placenta to examine cffDNA release following apoptosis, infection, and sterile inflammatory stress.Method of StudyHTR8/SVneo cells were used to model first‐trimester trophoblasts, and term cytotrophoblasts (CTBs) were isolated from placentae collected after uncomplicated deliveries. Trophoblasts were treated with varying concentrations of doxorubicin (DOX), lipopolysaccharide (LPS), or high‐mobility group box protein 1 (HMGB1) for 18 h. Cells or supernatants were quantified for caspase‐3/7 cleavage, pro‐inflammatory cytokine secretion, and cffDNA release.ResultsBoth HTR8/SVneo and CTBs underwent caspase‐3/7 cleavage following DOX treatment, with HTR8/SVneo cells more sensitive to apoptosis than term CTBs. Apoptotic cells released more cffDNA in a dose‐dependent manner. Treatment with LPS resulted in an increase in pro‐inflammatory IL‐6 release, particularly in term CTBs compared to early trophoblasts; however, LPS did not affect cffDNA release. Lastly, while neither cell released more TNF‐α following stimulation with HMGB1, both HTR8/SVneo and CTBs released significantly more cffDNA in the presence of HMGB1.ConclusionsThese data show that apoptosis and sterile inflammation induced by DOX and HMGB1, respectively, cause an increase in cffDNA concentrations in both first‐trimester and term trophoblasts. Understanding physiologic release of cffDNA during healthy and complicated pregnancy can identify new targets for the diagnosis and treatment of gestational complications.

Type I hypersensitivity reactions (HSR) to dabrafenib are rare but have been previously described. We present a case where a 72-year-old woman with recurrent, metastatic BRAF-mutated melanoma developed a type I HSR to dabrafenib. We, therefore, developed a desensitization protocol with encorafenib, a similar class agent, to allow the patient to continue with treatment. Patients with a history of HSR to dabrafenib may be considered for encorafenib desensitization when other therapeutic options are limited.

Abstract Malignant melanoma is the most aggressive form of skin cancer, accounting for 10,000 deaths annually. Largely resistant to conventional cytotoxic chemotherapy and radiation, immune checkpoint blockade (ICB) therapies have revolutionized patient care, accounting for partial or complete responses in up to 70% of patients. An important aspect of cancer elimination is activation of the cytotoxic T lymphocyte (CTL) response. Recent data suggest that ICB mediated broadening of the peripheral blood CTL repertoire correlates with good clinical outcomes. Thus, recent studies in cancer immunotherapy have focused on targeting tumor neoantigens (tNeoAg) with cancer vaccines for use in combination with ICB. Computational algorithm-driven predictions of immunogenic tNeoAgs yield vast numbers of potential peptide targets, only a fraction of which may be immunogenic in patients. This likely contributed to the modest success of cancer vaccines targeting predicted, high-avidity tNeoAgs, emphasizing the importance of identifying true tumor rejection antigens, including sub-dominant antigens, outside the scope of predictive models. In addition to promoting T cell-mediated tumor rejection, ICB often comes at the expense of treatment-induced immune-related adverse effects (irAE) that frequently require discontinuation of treatment. Modulation of ICB towards antitumor immunity and away from autoimmunity may dramatically improve the therapeutic index of modern cancer therapy. This appears feasible considering the clinical observation that antitumor efficacy does not correlate with type/severity of irAE, suggesting that the mechanisms of both processes, though likely related (CTL mediated), may not be identical. We aim to identify and separate non-cross-reacting antigenic targets mediating tumor rejection from those mediating irAEs to enable therapeutic interventions that maximize the efficacy of ICB, expanding tumor-specific CTLs with vaccines while minimizing irAEs through desensitization. We have access to samples from responding/nonresponding melanoma patients subjected to ICB with varying degrees of irAEs. We have designed an experimental approach that combines established mass spectrometry and sequencing techniques to identify peptides and matching TCR clones with a novel strategy that targets the TCR-associated CD3 complex to allow inclusion of subdominant antigens in our studies. T-cell “co-potentiation” is achieved when anti-CD3 monovalent Fabs induce a conformational change in the CD3 complex that sustains the T-cell response to weak antigenic stimulation. We have successfully used anti-human CD3 Fabs to co-potentiate in vitro the activation and subsequent response to weak peptide-HLA/TCR interactions of human T cells found in PBMCs isolated from healthy donors. Our preliminary data suggest T-cell co-potentiation may allow identification of ICB-induced CTL clones specific for subdominant tNeoAgs and irAE targets in patients with melanoma undergoing active immunotherapy. Citation Format: Laura Elsbernd, Alfreda Nelson, Hien Huynh, Michele Hoffmann, Christopher Parks, Wendy Nevala, Shari Sutor, Larry Pease, Adam Schrum, Diana Gil, Svetomir N. Markovic. Co-potentiation of human T cells to identify subdominant tumor neoantigens from melanoma patients responding to immune checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B78.

Abstract The most common adverse event (AE) of dabrafenib and trametinib (DT) is pyrexia, which has been reported to occur in up to 71% of patients. Pyrexia resulted in therapy discontinuation in up to 26% of patients who otherwise were benefiting from the treatment. Little is known about the pathogenesis and optimal management of this common AE. We hypothesized that the etiology of pyrexia in patients treated with DT could be extrapolated from DT-related cutaneous adverse effects in which a paradoxical MAPK pathway activation has been identified. We hypothesized that the mechanism of MAPK pathway activation with DT therapy is similar to that reported in patients with familial Mediterranean fever (FMF), a rare, inherited condition characterized by episodes of fever and rash that responds exceedingly well to colchicine-based therapy. Based on this association, our group explored the use of oral colchicine for the treatment of DT-associated pyrexia in five patients with metastatic melanoma. Citation Format: Jesus Vera, Jonas Paludo, Lisa Kottschade, Matthew Block, Yiyi Yan, Charles Loprinzi, Svetomir Markovic. Dabrafenib-trametinib-induced pyrexia successfully treated with colchicine [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B15.

AbstractThrough our involvement in KEYNOTE‐059, we unexpectedly observed durable responses in two patients with metastatic gastroesophageal adenocarcinoma (mGEA) who received ramucirumab (anti‐VEGFR‐2)/paclitaxel after immune checkpoint inhibition (ICI). To assess the reproducibility of this observation, we piloted an approach to administer ramucirumab/paclitaxel after ICI in more patients, and explored changes in the immune microenvironment. Nineteen consecutive patients with mGEA received ICI followed by ramucirumab/paclitaxel. Most (95%) did not respond to ICI, yet after irRECIST‐defined progression on ICI, all patients experienced tumor size reduction on ramucirumab/paclitaxel. The objective response rate (ORR) and progression‐free survival (PFS) on ramucirumab/paclitaxel after ICI were higher than on the last chemotherapy before ICI in the same group of patients (ORR, 58.8% vs 11.8%; PFS 12.2 vs 3.0 months; respectively). Paired tumor biopsies examined by imaging mass cytometry showed a median 5.5‐fold (range 4‐121) lower frequency of immunosuppressive forkhead box P3+ regulatory T cells with relatively preserved CD8+ T cells, post‐treatment versus pre‐treatment (n = 5 pairs). We then compared the outcomes of these 19 patients with a separate group who received ramucirumab/paclitaxel without preceding ICI (n = 68). Median overall survival on ramucirumab/paclitaxel was longer with (vs without) immediately preceding ICI (14.8 vs 7.4 months) including after multivariate analysis, as was PFS. In our small clinical series, outcomes appeared improved on anti‐VEGFR‐2/paclitaxel treatment when preceded by ICI, in association with alterations in the immune microenvironment. However, further investigation is needed to determine the generalizability of these data. Prospective clinical trials to evaluate sequential treatment with ICI followed by anti‐VEGF(R)/taxane are underway.

Introduction Day 15 absolute lymphocyte count (ALC-15) recovery after autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT) is recognized as a prognostic factor for survival. ALC-15 recovery is directly dependent on the amount of collected and infused autograft-absolute lymphocyte count (A-ALC). Our group published a double-blind phase III study which showed that patients infused with an A-ALC ≥ 0.5 x 109 cells/kg experienced superior survival post-APBHSCT. Based on the results from our phase III study, on April 1, 2017, our Bone Marrow Transplant Program changed our standard clinical practice to collect an A-ALC ≥ 0.5 x 109 cells/kg in addition to stem cells for lymphoma patients undergoing APBHSCT. To continue to assess the prognostic ability of A-ALC, we evaluated the overall survival (OS) and progression-free survival (PFS) of diffuse large B-cell lymphoma (DLBCL) patients that underwent APBHSCT after April 1, 2017 compared with a matched-control group in a 1:1:1 ratio with DLBCL patients infused with an A-ALC &lt; 0.5 x 109 cells/kg or an A-ALC ≥ 0.5 x 109 cells/kg prior to April 1, 2017. Methods Eight-five consecutive DLBCL patients after our practice changed on April 1, 2017 received an A-ALC ≥ 0.5 x 109 cells/kg. For each DLBCL patient infused with an A-ALC ≥ 0.5 x 109 cells/kg after April 1, 2017, the set of potential controls was restricted to those DLBCL patients infused with an A-ALC &lt; 0.5 x 109 cells/kg and an A-ALC ≥ 0.5 x 109 cells/kg prior to April 1 2017 . Within this group of potential controls, the subjects were chosen who best matched for the cases in terms of clinical risk variables, including gender, age, stage, lactate dehydrogenase (LDH), performance status, extra-nodal disease, International prognostic index (IPI), and disease status prior to APBHSCT (complete or partial response), using the GREEDY algorithm. Cases were defined as DLBCL patients infused with an A-ALC ≥ 0.5 x 109 cells/kg starting from April 1 2017. Control 1 was defined as DLBCL patients infused with an A-ALC &lt; 0.5 x 109 cells/kg prior to April 1 2017. Control 2 was defined as DLBCL patients infused with an A-ALC ≥ 0.5 x 109 cells/kg prior to April 1 2017. We truncated the survival follow-up to 3 years starting from the date of transplant to match follow-up time between cases and controls. Both matched cases and control groups patients received BEAM conditioning. This retrospective study was approved by the Mayo Clinic Institutional Review Board according to the regulation of the Declaration of Helsinki. Results Cases, control 1 and control 2 were balanced to the clinical risk variables; age (p = 0.8); gender (p = 0.9); LDH (p = 0.6); stage (p = 0.3); performance status (p = 0.5); extra-nodal disease (p = 0.2); IPI (p = 0.6); and clinical status prior to transplant (p = 0.2). Figure 1A showed the OS and figure 1B the PFS. Both the cases and control 2 showed superior OS and PFS in comparison to control 1. Cases and control 2 showed overall similar OS and PFS. Multivariate analysis including all patients continue to depict the A-ALC ≥ 0.5 x 109 cells/kg as an independent predictor for OS (HR = 0.382, 95%CI=0.241-0.605, p &lt; 0.0001) and PFS (HR = 0.437, 95%CI = 0.279-0.629, p &lt;0.0001). Subgroup analysis, including all patients, shows superior OS and PFS for patients infused with an A-ALC ≥ 0.5 x 109 cells/kg for each of the following clinical variables: age [&gt; 60 years: OS (p &lt;0.0001); PFS (p &lt;0.0001) or ≤ 60 years: OS (p &lt;0.01); PFS (p , 0.02)]; extra-nodal disease [&gt; 1: OS (p &lt;0.008); PFS (p &lt;0.01) or ≤1: OS (p &lt; 0.001): PFS (p &lt;0.0005)]; IPI [&gt; 2: OS (p &lt;0.01); PFS (p &lt;0.02) or ≤ 2: OS (p &lt;0.0008); PFS (p &lt;0.0006)]; LDH [abnormal: OS (p &lt;0.006); PFS (p &lt;0.005) or normal: OS (p &lt;0.002); PFS (p &lt;0.002)]; performance status [&gt; 1: OS (p &lt;0.04); PFS (p &lt;0.03) or ≤ 1: OS (p &lt;0.0001); PFS (p &lt;0.0001)]; stage [I/II: OS (p &lt;0.006); PFS (p &lt;0.006) or III/IV: OS (p &lt;0.002); PFS (p &lt;0.001)]; response prior to transplant [complete response: OS (p &lt;0.0007); PFS (p &lt;0.0001) or partial response: OS (p &lt;0.01); PFS (p &lt;0.02)]. Conclusion Our match-control study continues to support the results of our Phase III study showing that the infusion of A-ALC is a prognostic factor for survival of DLBCL patients undergoing APBHSCT. Our studies support the practice of not only collecting enough stem cells (i.e., CD34) for hematologic engraftment, but also enough immune effector cells (i.e., A-ALC) to improve clinical outcomes in DLBCL patients post-APBHSCT. Figure 1 Disclosures Ansell: Regeneron: Research Funding; Bristol Myers Squibb: Research Funding; Trillium: Research Funding; ADC Therapeutics: Research Funding; Takeda: Research Funding; AI Therapeutics: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding.