Background
Leukemia, lymphoma and myeloma accounted for nearly 10% of new cancer cases and more than 50,000 deaths in 2022. Although multiple treatment modalities exist, Chimeric Antigen Receptor (CAR) T cells have been particularly useful, using genetic modification of a patient’s harvested T cells to better target cancer cells. Current methods of altering antigen specificity and targeting, however, have proven to be inadequate over time, requiring additional genes to boost the immune response. One such gene is OX40, a co-stimulatory cassette member of the Tumor Necrosis Factor Receptor (TNFR) superfamily that is thought to boost T cell migration, proliferation and survival. Given these inherent qualities of OX40, we hypothesized that incorporating OX40 variants into CAR T cells could enhance their duration of response and tumor-targeting capabilities.

Methods
We combined variants of the OX40 gene with our 2nd generation CAR T cells, adding a costimulatory cassette to CD19 CARs. We started with transformations of plasmid cloning vectors containing OX40 genes, using these CD19 CAR/OX40 constructs to transiently transfect retroviral cell lines. The transient virus produced was used to transduce donor human T cells, inducing uptake of the CD19 CAR/OX40 vectors. Once transduced, T cell reactivity and longevity were assessed using ELISA assays, flow cytometry and cell counts.

Results
We successfully used our CD19 CAR/OX40 T cells to transduce various human donor T cells, demonstrating a 1:1 linear relationship between OX40 and CAR T cell expression via flow cytometry. We also found that the constructs produced markedly higher levels of OX40+ expression compared to endogenous levels, particularly in regulatory T cells. When tested against a Burkitt’s lymphoma line, our
constructs showed reactivity above the standard deviation. With regards to longevity, our highest proliferating construct was a truncated version that survived over 60 days in culture.

Conclusion
We were able to produce CD19 CAR/OX40 donor T cells that had increased longevity and enhanced hematologic tumor targeting capabilities over time, likely due to an increased regulatory T cell response. Surprisingly, our truncated construct survived and proliferated the most, suggesting that the missing signaling domain could be key to balancing OX40’s enhanced growth effects versus over-proliferation.
With the endogenous signaling domain in place, OX40 is a promising addition to our CAR repertoire, boosting the in vitro antitumor response while balancing sustained proliferation and longevity. Our next steps will include translation into in vivo mouse models before bringing it to patients’ bedsides as a novel immunotherapy treatment.

Driving CARs Forward: An OX40 Tale of Immunotherapy

**Scoring is only possible in the semifinals hall**

**Abstract:**



Uncovering Patterns between Obstructive Sleep Apnea and Disease Comorbidities: An Exploration of Patient Demographics

Glioblastoma Multiforme (GBM) is one of the most common and aggressive high-grade primary brain tumors that arises from the glial cells of the central nervous system and occurs most often in the frontal and temporal lobes. Despite current treatment options, long-term prognosis of GBM remains low with no significant improvement rates. Chimeric antigen receptor (CAR)-expressing T cells target the B cell marker CD19 and have shown increased efficacy in B cell lymphomas, and lymphoblastic leukemias suggesting benefits in GBM. This study aims to quantify the persistence of CAR-T cells in the context of GBM tumor volume following neurosurgical debulking procedure. A radiology software known as the mint Lesion was utilized to provide Tumor Response Assessment by Criteria (TRAC) reports that consist of volumetric measurements of the tumor lesions (target enhancing lesions, non-target enhancing lesions, and non-target non-enhancing lesions). Fluorescence-activated cell sorting (FACS) analysis takes a sample mixture of cells and sorts them into different populations based on specific biomarkers and characteristics. B7-H3 CAR-T cell persistence was increased in patients with a larger post-surgical tumor volume (3.04% Subject 001 with 2438 mm² volume and 2.38% Subject 003 with 224 mm² tumor volume). However, the number of CAR-T cells per 1 mm² was significantly reduced in patients with a larger post-surgical tumor volume (0.0012 cells/mm² Subject 001 and 0.010 cells/mm² Subject 003). This study shows that although there is an overall increased CAR-T cell persistence for a larger tumor volume, the number of CAR-T cells per 1 mm² is significantly smaller for larger tumor volumes, while illustrating that a larger post-surgical tumor bulk volume is correlated with an increased CAR-T cell persistence within 1-week post-infusion. These findings provide novel information and complement other correlative studies as part of the Phase 1 interventional clinical trial for glioblastoma patients.

Glioblastoma Multiforme Tumor Volume and Persistence of Chimeric Antigen Receptor T Cells Following Neurosurgical Debulking

Background:
The spectrum of vascular access-site complications following cardiac catheterization can vary widely in severity ranging from simple to complex that can contribute to significant morbidity with a high economic burden and healthcare costs.

Aim:
The aim of the case presentation was to describe post-discharge delayed complication (acute limb ischemia) of cardiac catheterization due to noncalcified plaque.

Case Presentation:
A 58-year-old male with a history of coronary artery disease, diabetes mellitus, and hyperlipidemia underwent cardiac catheterization status-post angioplasty to the right coronary artery via right-femoral artery access. The patient was discharged home post-procedure, however, 8 hours later, he developed a sudden-onset cold foot, and worsening numbness, and tingling sensation in his right extremity that prompted him to present to the ER. Physical examination was notable for cold skin in his right leg with diminished dorsalis pedis and popliteal pulses, and a mild loss of “touch sensation” below the level of the knee. The patient was staged “IIa” on the Rutherford Acute Limb Ischemia score. Vitals and labs were within normal limits. CT Angiogram revealed focal high-grade stenosis in the right common femoral artery at the site of a recent arterial puncture from cardiac catheterization. This was likely secondary to nonocclusive, noncalcified plaque that has been disrupted from recent instrumentation. A Heparin drip was initiated and the patient underwent emergent revascularization with a diagnostic angiogram and balloon angioplasty of the right common femoral artery with the placement of a 27mm stent. A completion angiogram showed the resolution of the high-grade stenosis with good flow through the profound and superficial femoral artery. 

Discussion:
The risk of access-site complications following cardiac catheterization, although quite low, is increased with the femoral artery with an incidence estimated to be as high as 17%. Bleeding and hematoma are often reported, however, thrombosis is infrequent with mechanisms involving atherosclerotic plaque rupture or subintimal dissection of the plaque with secondary thrombosis. When the severity of limb ischemia is threatening, immediate anticoagulation followed by surgical revascularization is the most appropriate intervention. Thrombosis at the access site of cardiac catheterization can cause critical limb ischemia, particularly with the femoral artery, thus highlighting the need for careful post-operative follow-up. 


Acute limb ischemia due to iatrogenic plaque rupture in the common femoral artery at the site of cardiac catheterization - A Case Report

Background
Guillotine amputations involve amputating an extremity with the wound left open, and are indicated in the setting of contamination. With lower extremity amputation, surgical options include below-knee amputation (BKA), through-knee amputation or knee disarticulation (KD), and above-knee amputation (AKA). BKAs preserve the knee joint and have been shown to have fewer complications and improved rehabilitation compared to KDs and AKAs. When BKAs are not feasible for a traumatic injury, an AKA is often chosen, due to difficulty or lack of understanding of KD surgery. While few RCTs have compared KDs to AKAs, they may be a viable alternative to AKAs.
Case Presentation
A 28-year-old male presented to the ED after being hit by a truck while on a motorcycle. Physical exam revealed severe injuries to the left lower extremity (LLE), including open tibia and fibula fractures, degloving of the associated soft tissue, obliteration of the anterior compartment of muscles, and a cool foot with no identifiable pulse. Radiographs revealed open, comminuted fractures of the left tibia and fibula. CTA of the LLE revealed abrupt cut-off of contrast at the level of the distal trifurcation of the popliteal artery, proximally in the tibia. 
The patient was urgently taken to the OR, where a guillotine amputation was performed through the knee joint (KD), due to the proximal nature of the fractures and level of vessel tear. KD was chosen over AKA in order to preserve the thigh musculature and therefore maximal function in this young, healthy patient. Three days later, he underwent irrigation and debridement of the LLE with wound closure. The patient was discharged home five days after wound closure, or eight days after admission. Three months out of surgery, the patient has done well with physical therapy and prosthetic usage; he will continue to be followed closely for rehabilitation and recovery. 
Discussion
Factors to be considered in the level of amputation include soft tissue viability for bone coverage, blood vessel viability, and wound contamination. When BKA is not possible, KD can offer an alternative to AKA. KDs present concerns of poor wound healing and stump breakdown, as well as increased risk of revision surgery. However, compared to ambulating on a normal leg, patients undergoing AKAs require 70% more energy expenditure. As compared to AKA, KD preserves the adductor muscles, leading to improved stability and reduced metabolic expenditure with ambulation. Therefore, KDs may result in improved rehabilitation and increased long-term functionality.


A Case of Traumatic Guillotine Knee Disarticulation

Background
Over 500 clinical trials combining radiation (RT) and immune checkpoint blockade (ICB) have been initiated based on preclinical evidence that RT can augment local immunity and improve the efficacy of ICB. However, the majority of clinical trials have found no benefit of combining RT and ICB, raising questions about whether a synergy exists. We examined whether RT and ICB interact to beneficially stimulate the immune response in patients and explored genomic biomarkers of response to combination RT and ICB.

Methods
We analyzed 2,046 patients from 4 cohorts. The COSINR dataset is a randomized clinical trial of 22 patients with non-small cell lung cancer (NSCLC) treated with concurrent or sequential RT and ICB. Paired pre/on-treatment biopsies of an irradiated metastasis underwent exome and RNA-seq. On-treatment biopsies were obtained after RT and prior to ICB (sequential) or after RT and one cycle of ICB (concurrent). The UC cohort contains targeted sequencing of 58 NSCLCs treated with ICB alone, sequential RT and ICB, or concurrent RT and ICB. The SU2C cohort comprises targeted sequencing of 306 ICB-treated NSCLCs from 11 institutions. The pan-cancer MSKCC cohort contains targeted sequencing of 1,660 patients treated with ICB. 

Aneuploidy score (AS) was defined as the fraction of chromosome arms with arm-level copy number alterations. Single-sample gene set enrichment analysis was used to compute Hallmark immune signaling pathway scores. Survival analyses utilized the Kaplan-Meier method and Cox proportional hazards models. 

Results
In the COSINR trial, RT and ICB increased, whereas RT alone decreased, expression of effector T-cell IFN-gamma and adaptive immune signatures (P<0.05). Baseline AS was negatively associated with expression of interferon, interleukin, and inflammatory signaling pathways (P<0.05). High AS was independently associated with poor OS following ICB after controlling for TMB and ICB paradigm in the MSKCC cohort (hazard ratio[95% CI]: 1.44[1.07–1.94], P=0.014) and controlling for TMB in the SU2C cohort (hazard ratio[95% CI]: 3.06[1.58–5.94], P=9.5e-4). In the UC cohort, high (≥median) AS tumors treated with concurrent RT and ICB had superior 1-year OS vs. sequential RT and ICB or ICB alone (76% vs. 38% vs. 31%, P=0.044). RT did not improve OS in low AS patients. Our findings were corroborated in the COSINR cohort: patients whose tumors exhibited high, but not low, AS had improved survival when treated with concurrent vs. sequential RT and ICB (1-year overall survival [OS] 100% vs. 17%, P=0.025).

Conclusions
Our findings distinguish the genomic and transcriptomic effects of RT versus RT and ICB and challenge the prevailing paradigm that local ablative RT alone positively stimulates the immune response. We propose the use of aneuploidy as a biomarker in personalizing immunotherapeutic treatment approaches. Concurrent RT and ICB may augment survival in patients with highly aneuploid, immunologically cold tumors.

Concurrent radiation and immunotherapy augments immunity and survival in aneuploid lung cancer

Background: Rural communities rely heavily on EMS teams to provide high quality care during transport to often distant medical facilities. Compliance with standardized protocols has been shown to increase triage effectiveness on a global scale, and our providers use the EMS Prehospital Treatment Protocol Handbook to guide care. The purpose of this study is to evaluate if the clinical documentation of our EMS providers follows standard protocols. 

Methods: Tri-County regional trauma calls in the Maine Incident Reporting System database from July 1st, 2022 to July 31st, 2022 were reviewed. The logs were compared to the EMS Prehospital Treatment Protocol Handbook. Documentation was analyzed for inclusion of relevant details as outlined by the appropriate protocol. The author of each log was documented to establish trends among personnel.

Results: 121 cases were included in the final analysis. The most common chief concerns included falls (n = 76) and motor vehicle collisions (n = 24). The most commonly used protocol was the Trauma Triage protocol. 28 cases (23.1%) did not include a Glasgow Coma Scale (GCS). Two of the GCS scores were below 8, but neither patient was intubated. Of the 28 cases without GCS documentation, 20 cases involved the chief concern of “fall” (71.4%) and 7 were motor vehicle collisions (25%). Of the 38 unique authors, 13 providers were responsible for the cases without GCS documentation, with two providers being responsible for 11 of the 28 cases.

Conclusion: The Trauma Triage protocol was broadly used to cover injuries ranging from standing height falls to head on motor vehicle collisions. Although it is possible that clinical care was provided appropriately when a GCS was not recorded, it could also indicate that our patients were not triaged effectively in the field. Given that two providers accounted for 39% of documentation issues, targeted education could drastically increase the rate of GCS documentation in our group. GCS could also be programmed as a required field for all trauma calls in order to increase compliance throughout the entire group. Locating more validated trauma protocols based on the mechanism of injury could further define how patients need to be triaged as opposed to using one protocol for all trauma. The next step in this study would be to investigate how the rates of GCS documentation changed after implementation of these proposed strategies. 

References:
Van Rein, E.A.J. et al. “Compliance to prehospital trauma triage protocols worldwide: A systematic review.” Injury. 2018; 49(8): 1373-1380. https://pubmed.ncbi.nlm.nih.gov/30135040/

Documentation of Prehospital Treatment Protocols in Emergency Medical Services Trauma Calls

The role of Lcn2 in Triple Negative Breast Cancer Organoids

Background: SARS-CoV-2 or (CoV2) utilizes the receptor binding domain (RBD) of its surface SPIKE protein to infect human cells that express ACE2 receptor. Infected persons can be asymptomatic or progress to a potentially fatal pneumonia-associated illness. Antibodies that prevent SPIKE/RBD/ACE2 interaction can result from infection or vaccination. However, their quantity, function, and cross-reactive protection requires serological testing. We obtained 50 deidentified serum samples, and were blinded to CoV2 vaccination (Vax+; Vax-) or CoV2 positive test (CoV2+; CoV2-). Serum IgG antibodies to SPIKE and RBD were quantified and efficacy in preventing CoV2 infection in vitro determined. We hypothesized that high titer RBD IgG would be efficient at preventing viral infection and be effective against a later CoV2 variant.

Methods: ELISA was used to quantify SPIKE and RBD antibodies in sera. Neutralizing capability of antibodies was determined by mixing sera with a firefly luciferase lentivirus psuedotyped with Wuhan CoV2 SPIKE. The mixture was applied to ACE2 expressing cells and luciferase activity used to calculate infection rates. Samples were unblinded to determine correlation between in vitro results and CoV2 exposure or vaccination. Cross-protection was determined by repeating neutralization assays with the Omicron BA.2 variant.

Results Thirteen participants reported no CoV2 vaccine or CoV2 positive test (Vax-/CoV2-) yet 7 had anti-SPIKE and -RBD titers and neutralizing function suggesting prior asymptomatic CoV2 infection. The remaining 37 participants reported Vax-/CoV2+ (n=8), Vax+/CoV2- (n=27), or Vax+/CoV2+ (n=2). Titers to RBD were comparatively lower than titers to SPIKE in most patient samples. Average RBD antibody titers were higher for the Vax+/CoV2- group (1:2600) versus Vax-/CoV2+ cohort (1:900). Highest RBD antibody titers were found in Vax+/CoV2+ patients (1:5400). Finally, sera with RBD titer ≥1:2700 and neutralization capacity had equal neutralizing function on Omicron BA.2, a variant with 28 amino acid changes in SPIKE and 16 in the critical RBD region.

Conclusion High-titer RBD sera correlated with greater viral neutralization activity highlighting the functional importance of this domain in infection. High-titer anti-RBD IgG to the initial Wuhan CoV2 virus also provided potent neutralization activity against the BA.2 variant despite 16 amino acid changes in its RBD region. This information could be used in future vaccination efforts and passive immunization, as well as provide a greater level of confidence to healthcare workers, first responders, and other high-risk populations regardless of the CoV2 strain. Serial immune studies in high risk populations could provide indications for booster vaccines.

Functional Serologic Testing and Cross Reactivity of Serum Antibodies to SARS-CoV-2 SPIKE Protein

Human Immunodeficiency Virus 1 (HIV-1) is a retrovirus that primarily infects human CD4+ T cells, a key cell-type in the adaptive immune system. The virus establishes a latent reservoir in the early phases of infection that persists in people living with HIV-1 (PLWH) despite effective antiretroviral treatment (ART). Designing strategies to target the HIV-1 reservoir is critical to achieve progress towards a cure. In eukaryotic and prokaryotic cells, and in viruses, antisense transcripts have been shown to exist as long, non-coding RNAs (lncRNA) or coding RNAs (mRNA). Some viruses, like Human T-cell Leukemia Virus Type 1, encode antisense genes to regulate viral expression and replication. HIV-1 antisense transcripts (AST) have been detected in in vitro and ex vivo studies and have been shown to induce viral latency through methylation of histone 3 lysine 27 by recruiting the polycomb repressive complex 2 (PRC2). Our previous studies demonstrated the presence of HIV-1 AST in vivo at levels of 1-3 copies in a median of 5% infected peripheral blood mononuclear cells (PBMC), suggesting a potential role in inducing viral latency in vivo. To determine if HIV-1 AST expression affects levels of plasma viremia, we designed an assay to measure AST expression in PLWH with high viral loads (non-controllers, viral load 5,000-200,000 copies/mL), with low viral loads (viremic controllers, 50-2,000 copies/mL), and with undetectable viral loads (elite controllers, <40 copies/mL). The assay is a limited-dilution, digital PCR approach that targets HIV-1 AST in donor blood cells using donor-specific primers. First, nucleic acid is extracted and subjected to thorough DNase treatment. The remaining antisense RNA is reverse transcribed using a primer targeting the AST region. Detection is achieved with a fluorescent probe and reported as the number of HIV-1 AST molecules per 100 infected cells. We measured AST levels using ACH-2 cells (a model of HIV-1 latency) as a positive control with an average of 3.18 copies of AST per 100 infected cells, as expected. Negative controls included no reverse transcriptase and no template to ensure complete DNA digestion and no mis-priming nor contamination. Our preliminary data includes 4 donor samples in which HIV-1 AST was detected at 1-53 per 100 infected cells. Future studies will determine if AST functions to control HIV-1 replication and induce viral latency in vivo.

Investigation of HIV-1 Antisense Expression In Vivo

Abnormal calcium deposition is among the sequelae that may follow major burn injury. This may manifest as heterotopic ossification or dystrophic calcification (DC) of scarred skin. While the two processes are distinct, the term heterotopic ossification (HO) is often used by clinicians in reference to both processes. While HO is well-documented, there appears to be less attention devoted to DC, the most common subtype of calcinosis cutis. HO is the abnormal formation of mature lamellar extraskeletal bone in muscle and soft tissues. Risk factors include traumatic injuries and surgeries such as spinal cord injury, hip arthroplasty, and burns with >30% TBSA. It commonly occurs in upper extremities, especially the elbows, and on average takes weeks to months to develop. In contrast, DC is the pathologic calcification of scar itself, and can present in cases of burn survivors as the calcification of burn and skin graft scar, often decades after the original burn injury. It manifests as the build-up of insoluble calcium salts in damaged subcutaneous tissues in the absence of abnormal serum calcium and phosphate levels. Applicability of this research is to increase awareness of burn sequelae; this research was not externally funded. 
We present a case of DC to highlight the distinct differences between DC and HO in hopes of promoting use of the semantically correct terms. A 67 year old female presented to the clinic 65 years after a >40% TBSA burn treated with excision and skin grafting at the time of injury. She had a months-long history of a draining sinus with a~5mm diameter white hard plaque at the base, and a broader contiguous area of hard plaque palpable beneath the surrounding area of skin graft scar. She had been experiencing recurrent bouts of infection and sought definitive treatment. The area was widely excised and ultimately resurfaced with a new split thickness skin graft. Clinical and histopathologic photographs will be presented. The histopathologic findings were consistent with the dystrophic calcification variant of calcinosis cutis. The presentation was consistent with ~10-12 other cases seen by the senior author over a 20 year career, and consistent with other cases reported in the literature. The term "heterotopic ossification" is sometimes used too broadly to describe any pathologic calcium deposition in burn survivors. We highlight the difference between heterotopic ossification and dystrophic calcification out of a belief that as subject matter experts in burns and their sequelae, burn care providers should want to use the most accurate terminology wherever possible.

Revisiting the Taxonomy of Abnormal Calcium Deposits in Burn Survivors: Heterotopic Ossification versus Calcinosis Cutis

Background: Recurrent urinary tract infections (rUTI) are common in postmenopausal women with type 2 diabetes mellitus (T2DM). However, little is known about the composition of the urinary microbiome in this population and how it contributes to rUTI susceptibility. We aimed to identify key differences in urinary microbiome composition and function between women with and without T2DM through shotgun metagenomic sequencing of their urine.

Methods: Following IRB approval, urine samples were collected from consenting postmenopausal women with T2DM across 3 groups: rUTI remission (rUTI history, no active infection), rUTI relapse (active infection), and no history of UTI. Groups were age and estrogen-matched and DNA was purified from 20 mL of urine per patient. Sample concentrations were standardized and 20 pg of spike-in DNA of a rare bacterial genome was added. Shotgun metagenomic sequencing was performed using the Illumina NextSeq2000 platform. Reads were quality controlled and purified of human-mapping sequences using CLC Genomics Workbench. All microbial reads were taxonomically analyzed using MetaPhlan3. For further controls, data for non-diabetic women in the same groups were obtained.

Results: Fifteen TD2M and 15 non-T2DM samples were compared for this study. The mean genomic DNA yield across diabetic samples was 18.5 ng/uL. We observed an average of 75.8% human mapping reads, which were censored from further analysis. The remaining dataset contained an average of 5.1 million microbial reads. In the No UTI group, common urogenital flora microbes dominated across all samples in both T2DM and non-T2DM groups. In the rUTI Remission group, urogenital flora microbes were the most common again in non-diabetics, while common UTI pathogens such as Escherichia coli, Staphylococcus spp. and Pseudomonas aeruginosa were present in diabetic samples. For the rUTI Relapse group, both T2DM and non-T2DM were infected with common UTI pathogens (Escherichia coli, Klebsiella pneumoniae, Anaerococcus vaginalis, Enterococcus faecalis), however, 5/5 diabetic samples showed multi-strain infections while only 2/5 non-diabetic samples did.

Conclusion: We aim to identify differences in urinary microbiomes between women with T2DM with rUTI relapse, rUTI remission, and no history of UTI. By comparing our results with a published study on women without T2DM, we found that our cohort had ~10% more human mapping reads. Our pilot study suggest that T2DM patients may have higher rates of multi-strain infections compared to their non-T2DM counterparts. An expanded study (n = 60) is currently in progress to further understand the differences in urinary microbiomes in women with and without T2DM. 


Underline digital video library

Driving CARs Forward: An OX40 Tale of Immunotherapy

Uncovering Patterns between Obstructive Sleep Apnea and Disease Comorbidities: An Exploration of Patient Demographics

Glioblastoma Multiforme Tumor Volume and Persistence of Chimeric Antigen Receptor T Cells Following Neurosurgical Debulking

Acute limb ischemia due to iatrogenic plaque rupture in the common femoral artery at the site of cardiac catheterization - A Case Report

A Case of Traumatic Guillotine Knee Disarticulation

Concurrent radiation and immunotherapy augments immunity and survival in aneuploid lung cancer

Documentation of Prehospital Treatment Protocols in Emergency Medical Services Trauma Calls

The role of Lcn2 in Triple Negative Breast Cancer Organoids

Functional Serologic Testing and Cross Reactivity of Serum Antibodies to SARS-CoV-2 SPIKE Protein

Investigation of HIV-1 Antisense Expression In Vivo

Revisiting the Taxonomy of Abnormal Calcium Deposits in Burn Survivors: Heterotopic Ossification versus Calcinosis Cutis

Metagenomic Sequencing of the Urinary Microbiome of Postmenopausal Women with Recurrent Urinary Tract Infection and Type 2 Diabetes

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