Citations

Pencil beam scanning delivered with continuous scanning has several advantages over conventional discrete spot scanning. Such advantages include improved beam delivery efficiency and reduced beam delivery time. However, a move dose is delivered between consecutive spots with continuous scanning, and current treatment planning systems do not take this into account. Therefore, continuous scanning and discrete spot plans have an inherent dose discrepancy. Using the operating parameters of the state-of-the-art particle therapy system, we conducted a proof-of-concept study in which we systematically generated 28 plans for cubic targets with different combinations of plan parameters and simulated the dose discrepancies between continuous scanning and a planned one. A nomograph to guide the selection of plan parameters was developed to reduce the dose discrepancy. The effectiveness of the nomograph was evaluated with two clinical cases (one prostate and one liver). Plans with parameters guided by the nomograph decreased dose discrepancy than those used standard plan parameters. Specifically, the 2%/2 mm gamma passing rate increased from 96.3% to 100% for the prostate case and from 97.8% to 99.7% for the liver case. The CTV DVH root mean square error decreased from 2.2% to 0.2% for the prostate case and from 1.8% to 0.9% for the liver case. The decreased dose discrepancy may allow the relaxing of the delivery constraint for some cases, leading to greater benefits in continuous scanning. Further investigation is warranted.

Abstract Current SARS-CoV-2 mRNA vaccines induce robust humoral and cellular immunity in the circulation, but its ability in eliciting respiratory mucosal immunity is less characterized. Here, we demonstrated that systemic mRNA expressing ancestral spike (mRNA-S) vaccination alone induced weak respiratory mucosal neutralizing antibody and cellular immunity, particularly against SARS-CoV-2 Omicron strain. In contrast, an immunization strategy combining systemic mRNA-S administration plus mucosal adenoviral vector expressing ancestral spike (Ad5-S) booster induced strong T cell, B cell, and antibody responses in the respiratory tract, which can last for a long time. Furthermore, we found that Ad5-S mucosal booster promoted robust IgA-producing B cells in the respiratory tract, which were correlated with the levels of mucosal S-specific IgA levels. Strikingly, these local IgA-producing B cells were more cross-reactive to the Delta and Omicron Spike proteins compared to those IgG-producing B cells. We further showed that CD4 T cell help was required for the development of IgA-producing B cell in the respiratory mucosa. Taken together, our study identified a vaccination strategy and its associated mechanisms for the induction of strong cross-reactive IgA responses, which were shown to correlate with optimal protection against breakthrough infection, especially by Omicron sub-lineages. Hence, our data provide insights into the rational design of next-generation SARS-CoV-2 mucosal vaccines required for the protection against infection by SARS-CoV-2 Omicron sub-lineages or future variants.

Abstract Cytotoxic T lymphocytes (CTL) are indispensable in anti-tumor immunity. Although CTLs are prone to exhaustion in patients with advanced cancer, T cell resiliency explains the presence of tumor-reactive CTLs that are less exhausted, capable of cytolytic function, expansion, and rebound in response to immunotherapy to reject metastatic malignancies. However, the features of resilient T cells have not been clearly defined. In this report, we demonstrate that peripheral CX3CR1+ CD8+ T cells with low mitochondrial membrane potential rebounded CTL function quickly after radiation therapy in patients with large tumor burden portraying their functional resiliency. Furthermore, CX3CR1+ CD8+ T cell with low, but not high, mitochondrial membrane potential are highly cytotoxic, accumulate less reactive oxygen species (ROS), and express more Malic enzyme 1 (ME1). ME1 overexpression increases ATP production in a glycolysis-independent manner while concurrently curtailing excessive ROS in activated CD8+ T cells; and expands CX3CR1+NKG7+ effector CD8+ T cells with enhanced cytotoxicity. Importantly, transfection of ME1 mRNA promotes tumoricidal activity in CD8+ T cells from patients with advanced cancers. Our study reveals a mechanism used by CTLs to balance excessive ROS via ME1 to maintain a metabolic and functional resiliency. Modification of ME1 expression in CTLs may be a novel method to improve the efficacy of cancer immunotherapy by preventing T cell exhaustion. Supported by grants from Mayo Clinic Foundation and NIH (R01CA 256927-2)

Abstract Although immune checkpoint blockade that targets the PD-L1 has revolutionized the treatment of human cancers, only a small proportion of patients positively respond to this form of treatment. A limitation of these therapies is that they only target and inhibit PD-L1’s extracellular function, which is to bind its receptor on PD-1 on T cells and inhibit T cell cytotoxicity. Recent discoveries have shown that PD-L1 expressed on tumor cells can signal intrinsically and independently of PD-1 ligation to promote tumor cell survival. Besides tumor cells, host myeloid cells have been shown to have high expression of PD-L1, therefore contributing to the immunosuppressive nature of the tumor. However, PD-L1 intrinsic signaling in human myeloid cells has not been studied. Therefore, the purpose of our work is to determine the intrinsic functions of PD-L1 in myeloid cells and how this impacts anti-tumor immunity. To study this, we used a novel PD-L1 antibody (H1A) that does not block PD-L1’s binding with PD-1, but directs PD-L1 for degradation, thereby decreasing total PD-L1 expression. Additionally, we developed a chimeric mouse model in which the murine PD-1/PD-L1 has been replaced with the human version. This allows us to evaluate PD-L1’s intrinsic function in vivo. In our preliminary studies, the degradation of PD-L1 in human monocytes led to increased chemokine release as well as increased conversion of effector T cells. Additionally, H1A treatment of tumor-bearing mice led to lower tumor burden in a CD8 T cell dependent manner. Based on these results, PD-L1 intrinsic signaling has the potential to regulate the immune-stimulatory response of monocytes, thereby limiting effector T cell responses against cancer. Supported by grants from NIH (R0192389) and Mayo Clinic graduate school of biomedical sciences

Abstract Only a small cohort of patients show durable responses to immune checkpoint inhibitor (ICI) therapy and the mechanisms regulating CD8 T cell responses to ICIs are yet to be elucidated. To that end, we explored T cell intrinsic factors that may be attributed to low ICI therapy response rates. We have identified Dual Specificity Phosphatase 2 (DUSP2) as a potential negative regulator of ICI response within CD8 T cells. We demonstrated that DUSP2 expression in human CD8 T cells is transient, peaking at two-hours post-T cell stimulation with rapid waning thereafter. Downregulation of DUSP2, via siRNA transfection, increased cytotoxicity of human CD8 T cells activated for 48-hours prior to inclusion in a four-hour tumor killing co-culture assay, despite the return of DUSP2 expression to baseline levels at this time-point. Additionally, DUSP2 overexpression, via mRNA transfection, decreased release of effector molecules (Granzyme B and Interferon-gamma) from human CD8 T cells at 24-hours post-activation when compared to control mRNA. When we utilized RNA-sequencing to analyze and compare differentially expressed genes between responders and non-responders of ICI therapy, we found DUSP2 expression in CX3CR1 positive CD8 T cells to be consistently decreased in responders but not in non-responders. Since CX3CR1 positive CD8 T cells identify a highly resilient and cytotoxic subset in response to ICI therapy, we hypothesize that DUSP2 negatively regulates ICI-responsive effector CD8 T cells. Thus, uncovering the role of DUSP2 in CD8 T cells may provide a target to improve the efficacy of ICIs in patients with limited therapeutic alternatives. Mayo Clinic Department of Immunology Pilot Award Mayo Clinic Center for Individualized Medicine

Abstract Though anti-PD-1/PD-L1 immune checkpoint blockade therapy has demonstrated robust anti-tumor immunity in human patients with advanced cancers such as melanoma, only a fraction of patients achieved such durable anti-tumor responses. Among all the non-responders, some patients responded initially but developed resistance to the therapy in the later treatments. This reflects an urgent need to understand the role of PD-1 signals in CD8+ T cells at priming and at effector stages during tumor development for the benefit of immunotherapy. The controversy of rapid tumor progression in PD-1 conditional knockout in regulatory T cells and tumor regression in PD-1 targeted deletion in regulatory T cells indicated germline PD-1 knockout mice might not be sophisticated enough to study the role of PD-1 in CD8+ T cells during tumor development. Thus, we produced a CD8 T cell-specific PD-1 knockout mouse model (CD8 E8ICre-PD-1fl/fl, also called CD8-PD-1 cKO mice) in which PD-1 is deleted in CD8+ T cells in the peripheral tissues. When CD8-PD-1 cKO mice were challenged with B16-OVA mouse melanoma cells, we found a delayed tumor growth and prolonged survival of CD8-PD-1 cKO compared to control mice. However, after 20 days of tumor challenge, the delayed tumor growth rebound in CD8-PD-1 cKO mice. In an OVA antigen immunization model, we found increased effector cells and tissue resident cells in the spleen of CD8-PD-1 cKO mice compared with control mice on day 7 after immunization. Taken together, our results suggest that PD-1 may restrain the expansion of effector T cells that contribute to the early phase of tumor control, but the augmented effector T cell response may cause an attrition of memory T cell pool that will be needed for long term protection. R01CA256927

Abstract Imatinib mesylate (IM) has revolutionized the treatment of gastrointestinal stromal tumor (GIST). However, most patients inevitably acquire IM resistance. Second- and third-line treatments exhibit modest clinical benefits with a median time to disease progression of 4 to 6 months, highlighting the urgency for novel therapeutic approaches. Here, we report that the expression of BCL6, a known oncogenic driver and transcriptional repressor, was significantly induced in GIST cells following IM treatment. Elevated BCL6 levels suppressed apoptosis and contributed to IM resistance. Mechanistically, BCL6 recruited SIRT1 to the TP53 promoter to modulate histone acetylation and transcriptionally repress TP53 expression. The reduction in p53 subsequently attenuated cell apoptosis and promoted tolerance of GIST cells to IM. Concordantly, treatment of GIST cells showing high BCL6 expression with a BCL6 inhibitor, BI-3802, conferred IM sensitivity. Furthermore, BI-3802 showed striking synergy with IM in IM-responsive and IM-resistant GIST cells in vitro and in vivo. Thus, these findings reveal a role for BCL6 in IM resistance and suggest that a combination of BCL6 inhibitors and IM could be a potentially effective treatment for GIST. Significance: BCL6 drives resistance to imatinib by inhibiting p53-mediated apoptosis and can be targeted in combination with imatinib to synergistically suppress tumor growth, providing a therapeutic strategy for treating gastrointestinal stromal tumor.

Abstract Background Cyclin D has been shown to play an essential role in acute lymphoblastic leukemia (ALL) initiation and progression, providing rationale for targeting the CDK4/6‐cyclin D complex that regulates cell cycle progression. Procedure The Children's Oncology Group AINV18P1 phase 1 trial evaluated the CDK4/6 inhibitor, palbociclib, in combination with standard four‐drug re‐induction chemotherapy in children and young adults with relapsed/refractory B‐ and T‐cell lymphoblastic leukemia (ALL) and lymphoma. Palbociclib (50 mg/m 2 /dose) was administered orally once daily for 21 consecutive days, first as a single agent (Days 1–3) and subsequently combined with re‐induction chemotherapy. This two‐part study was designed to determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D), followed by an expansion pharmacokinetic cohort. Results Twelve heavily pretreated patients enrolled, all of whom were evaluable for toxicity. One dose‐limiting hematologic toxicity (DLT) occurred at the starting dose of 50 mg/m 2 /dose orally for 21 days. No additional DLTs were observed in the dose determination or pharmacokinetic expansion cohorts, and overall rates of grade 3/4 nonhematologic toxicities were comparable to those observed with the chemotherapy platform alone. Five complete responses were observed, two among four patients with T‐ALL and three among seven patients with B‐ALL. Pharmacokinetic studies showed similar profiles with both liquid and capsule formulations of palbociclib. Conclusions Palbociclib in combination with re‐induction chemotherapy was well tolerated with a RP2D of 50 mg/m 2 /day for 21 days. Complete responses were observed among heavily pretreated patients.

Abstract Despite recent methodology advancements in clinical natural language processing (NLP), the adoption of clinical NLP models within the translational research community remains hindered by process heterogeneity and human factor variations. Concurrently, these factors also dramatically increase the difficulty in developing NLP models in multi-site settings, which is necessary for algorithm robustness and generalizability. Here, we reported on our experience developing an NLP solution for Coronavirus Disease 2019 (COVID-19) signs and symptom extraction in an open NLP framework from a subset of sites participating in the National COVID Cohort (N3C). We then empirically highlight the benefits of multi-site data for both symbolic and statistical methods, as well as highlight the need for federated annotation and evaluation to resolve several pitfalls encountered in the course of these efforts.