United States

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.

AMA Research Challenge 2023

Driving CARs Forward: An OX40 Tale of Immunotherapy

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The role of Lcn2 in Triple Negative Breast Cancer Organoids

The kappa-opioid receptor (KOR) has been a target of recent interest in the treatment of alcohol use disorder, due to its role in negatively reinforced ethanol drinking, whereby aversive stimuli can promote ethanol intake. The KOR is densely expressed in the nucleus accumbens (NAc) on terminals of afferent neurons from the ventral tegmental area (VTA), where their activation has been found to suppress dopamine release such as is observed during alcohol withdrawal. We have previously found that KOR agonism differs along the rostro-caudal axis of the NAc shell, and that there may be a sex-related difference in baseline levels of ethanol intake. In the present study, we sought to determine if KOR stimulation in the rostral NAc shell could reverse effects of dopamine afferent activation on ethanol drinking. In Experiments 1 and 2, we used Long-Evans rats, as well as female transgenic tyrosine hydroxylase (TH)-Cre rats on a Long-Evans background in Experiment 3, which have a TH promoter directing expression of Cre recombinase in dopamine-containing cells, and we allowed them to drink under an intermittent access model, which establishes binge-like ethanol drinking. After the establishment of stable drinking, we bilaterally infused an excitatory designer receptors exclusively activated by designer drugs (DREADD) virus in the VTA of Experiment 2 groups, and a Cre-dependent excitatory DREADD in the VTA of the Experiment 3 group. After time for transduction, we injected into the NAc shell (1) the DREADD agonist, clozapine-N-oxide (CNO) (Experiments 2-3), (2) the KOR agonist, U50,488H (Experiments 1-3), (3) CNO + U50,488H (Experiments 2-3), and (4) saline vehicle (Experiments 1-3), counterbalanced in a Latin-Square design, and measured subsequent ethanol drinking. Surprisingly, we found that both U50,488H and CNO, independently and combined, enhanced ethanol drinking in high-level drinkers, and that these effects were not additive. Furthermore, the opposite trend was seen in low-level drinkers, suggesting a phenotype-dependent effect. These results suggest that both the DREADD activation and the KOR agonist microinjection have a similar net effect on dopamine release into the NAc shell, and that these effects depended on the phenotype of the drinker. The findings support the use of KOR antagonism as a therapy for alcohol use disorder, despite its potential unconventional effects on the dopamine system, and the overall use of individualized medication that considers an individual’s sex and phenotype.

Effects of Dopamine and Kappa Opioid Receptor Activity in the Rostral Nucleus Accumbens Shell on Ethanol Drinking in Rats

Background

Numerous studies have explored COVID-19 in adults and pediatric patients with chronic illnesses, but limited research has focused on previously healthy children. This study reports results from a COVID-19 screening program implemented at two pediatric clinics in central California, focusing on the clinical presentation of COVID-19 in this population.

Methods

Patients seeking care at the clinics completed a screening questionnaire to identify symptoms consistent with COVID-19. Temperature and oxygen saturation were measured, and nasal swabs were collected for antigen testing. Chief complaints, final diagnoses, and test results were recorded. The severity of COVID-19 illness was categorized using the CDC's severity index.
1) Asymptomatic/ pre-symptomatic - patient reported no symptoms and vital signs were normal.
2) Mild disease - respiratory or GI symptoms were present with or without fever.
3) Moderate disease; evidence of lower respiratory tract involvement was present.
4) Severe disease; oxygen saturation was < 94%.
5) Critical disease; the patient was transferred to the ER for further treatment.

Results

Between 11/2020 and 12/2022, 11,649 COVID-19 antigen screening tests were performed. Patients ranged in age from 0.1 to 17.0 years (mean 8.7, SD 4.5). 47.7% of patients identified as female, 50.5% as male, and 1.8% as other. In total, 1,560 pts. (13.4%) tested positive. Among these, 665 (43%) were asymptomatic/pre-symptomatic, 560 (36%) had mild disease, 318 (20%) had moderate disease, and 17 (1%) had severe disease. No critical cases or transfers to the emergency room were reported. A higher body mass index (BMI) was associated with increased severity of illness. 4,446 patients reported no symptoms at the time of screening. Among these, 85% tested negative, and 15% tested positive. Demographics were similar between these 2 groups. 7,203 patients report symptoms consistent with COVID-19. Among these, 87.6% tested negative and were presumed to have an infection other than COVID-19, while 12.4% tested positive. Median severity of illness did not differ significantly between the 2 groups.

Conclusion

The screening program effectively detected pediatric COVID-19 cases, including asymptomatic patients. In healthy children, COVID-19 was mild, with severity similar to other viral infections. Higher BMI is linked to greater illness severity. These findings enhance understanding of acute COVID-19 in outpatient pediatric populations, emphasizing the need for proper screening to ensure safe delivery of care.

Clinical Manifestations of COVID-19 in Previously Healthy Pediatric Patients Presenting to a Primary Care Office.

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: 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. 


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

Background
Obstructive sleep apnea (OSA) is a breathing disorder characterized by recurrent relaxation of the tongue and soft palate during sleep. The genioglossus (GG) muscle, the largest extrinsic tongue muscle, maintains upper airway patency. Obesity-induced OSA decreases muscular tone in the GG, causing airway obstruction. Other muscles impacted by obesity, such as the diaphragm (DIA) and the sternocleidomastoid, may contribute to OSA complications. Leptin partakes in central regulation of upper airway patency and diaphragmatic control. Leptin-
deficient obese (ob/ob) mice exhibit pharyngeal collapsibility and provide an effective model of OSA effects. By using ob/ob mice, we study the impact of obesity on GG, DIA, and sternomastoid (ST) muscles with a focus on the neuromuscular junction (NMJ), the best indicator of muscle function. 

Methods
GG, ST, and DIA muscles were extracted from 16 and 20-week wild-type (WT) and ob/ob male and female mice. Some samples were fixed and incubated with Alexa594TM alpha-bungarotoxin to visualize nicotinic acetylcholine receptors (nAChRs), and Alexa488TM Cholera toxin to label lipid rafts. They were imaged with a NikonTM inverted fluorescence microscope and a Confocal Microscope. Other GG and ST samples were “snap frozen,” sectioned in a cryostat, and stained
with H&E and NADH.

Results
GG muscles of ob/ob male mice showed a significant decrease in postsynaptic nAChRs and lipid rafts density compared to age-matched controls. In contrast to WT, GG of ob/ob male mice displayed multiply-innervated NMJs, nerve sprouting, and multiple Schwann cells engulfing nerves. Furthermore, the GG of ob/ob males exhibited central nuclei, intermuscular fat deposition, and hypertrophy. ST muscles in 16-week male WT and ob/ob mice displayed no differences in nAChR density, but nAChR density decreased in 20-week ob/ob male mice. The DIA showed higher nAChR density in ob/ob mice compared to controls. Female WT and ob/ob
mice showed insignificant changes in nAChR density in GG, ST, and Dia. 

Conclusion
The reduction in postsynaptic nAChRs and lipid rafts densities, and presynaptic remodeling of NMJs of GG observed in male ob/ob mice suggest synaptic disassembly. NMJ dismantlement, not observed in female ob/ob mice, may suggest female sex hormones contribute to synapse preservation through a neuroprotective pathway. Central nuclei in the GG indicate hypertrophic regeneration. Increased nAChR density in the male ob/ob DIA suggests a compensatory mechanism. Therefore, obesity-induced OSA-associated pathology exclusive to male GG, ST, and DIA affects the synaptic strength of these muscles. The observed sexual dimorphism and the possibility of reversing these observed changes should be further explored.

Effects of Obesity on the Neuromuscular Junction of Genioglossus Muscle and Other Associated Muscles of Respiration

Background: Research shows that increased severity of congenital heart disease (CHD) is associated with increased frequency of neurological deficits in children with structural cardiac abnormalities. Young children with more complex CHDs who require cardiopulmonary bypass (CPB) have a significantly higher incidence of developmental abnormalities and neurological deficits, including academic difficulties and behavioral abnormalities. Microglia, the resident immune cells in the central nervous system, play a key role in neural immune function. Mesenchymal stromal cells (MSCs) are multipotent, non-hematopoietic cells that possess immunomodulatory and regenerative properties and regulate microglia activation. It is hypothesized that intra-arterial MSC delivery through CPB in a survival porcine model is neuroprotective by modulating systemic and brain-specific inflammatory responses.

Methods: To mimic the effects in young CHD patients, two-week old female piglets were randomly assigned to one of three treatment groups: control, CPB, and CPB+MSC. The CPB+MSC group consisted of a CPB surgery followed by MSC administration before weaning. Microglia undergo morphological changes from a naïve, ramified to an amoeboid shape under activated condition. To analyze the effects of MSCs on microglia activation, changes in the length of microglial processes and number of branch points from the nuclei were assessed.

Results: Four weeks post-surgery, analysis using Imaris software showed that CPB reduced both the length of processes and number of branch points compared to control in three different cortex regions. CPB+MSC revealed significantly longer processes (p=0.0281) and more branch points (p=0.0232) than CPB alone, indicating that morphological changes in microglia were normalized after MSC delivery.

Conclusion: MSCs regulate microglial activation and participate in the phenotypic switch from a pro-inflammatory state to a repair-permissive state. In contrast to their ramified morphology under normal conditions, activated microglia undergo structural remodeling and adopt an amoeboid morphology with highly retracted processes, a hallmark of brain inflammation, as shown in the CPB group. Microglia routinely migrate toward sites of neuronal degeneration induced by excitotoxic injury. In MSC-treated animals, there are significantly fewer amoeboid microglia with an associated increase in the length of processes and number of branch points to mitigate the effects of CPB, indicating attenuated activation of the microglia. MSC delivery during CPB is highly effective and shows translational potential to minimize CPB-induced systemic inflammation and microglial activation in children with CHD.

Effect of mesenchymal stromal cell delivery through cardiopulmonary bypass in a piglet model

Background:
Since the release of the COVID-19 vaccines, vaccine safety and hesitancy have been significant topics of contention. While studies have described COVID-19 vaccine receptiveness in the general population, its uptake in inflammatory bowel disease (IBD) patients is not well-characterized. This study aimed to investigate COVID-19 immunization rates and barriers to immunization in IBD patients.

Methods:
A retrospective study investigated 545 adult patients with Crohn’s disease (CD) or ulcerative colitis (UC) seen at an IBD subspecialty clinic at a university-based tertiary care center between January 1, 2020 and October 31, 2021. For each patient, data collected includes: demographics, past medical history, use of immunosuppressive medications, and COVID-19 and influenza immunization status. COVID-19 immunization rates in the general U.S. population were obtained from data published by the Center for Disease Control and Prevention (CDC).

Results:
As of October 31, 2021, 87.7% of IBD patients had received at least one dose of the COVID-19 vaccine, 84.4% had received the full vaccine series, and 21.8% had received the booster and/or additional dose. COVID-19 immunization rates in IBD patients were significantly higher than in the general population (p < 0.001). No significant difference was found in COVID-19 immunization rates when stratified by IBD type (CD vs UC), sex, or concurrent immunosuppressive therapy. Patients who received the COVID-19 booster/additional dose were older (mean age = 54.0 ± 17.9) than those who did not (mean age = 50.0 ± 16.5; p = 0.02). Asthmatic patients had a significantly lower rate of receiving one dose (79.5% vs 89.2%, p = 0.02) or the full series (74.7% vs 86.1%, p = 0.01) of the COVID-19 vaccine compared to non-asthmatics. Tobacco users had a significantly lower rate of receiving the full COVID-19 vaccine series (73.9% vs 85.9%, p = 0.02). Patients previously immunized for influenza had a significantly higher rate of receiving the COVID-19 booster/additional dose than those who had not (31.5% vs 17.6%, p < 0.001). History of hypertension, hyperlipidemia, heart failure, diabetes, malignancy, ischemic heart disease, pulmonary circulation disorder, or COPD did not significantly affect COVID-19 immunization rates.

Conclusion:
Our study reveals that IBD patients are more receptive to the COVID-19 vaccine compared to the general U.S. population. Immunosuppressive therapy did not affect IBD patients’ uptake of the COVID-19 vaccine, although younger age, asthma, and tobacco use were associated with decreased uptake. Particular attention is required when counseling IBD patients to identify barriers to vaccination and increase COVID-19 immunization rates.

Uptake of COVID-19 Immunization in the Inflammatory Bowel Disease Population

Background
Acute appendicitis is one of the most common surgical emergencies in the world.
Appendectomies, especially if delayed, can be associated with postoperative complications in up to 16% of the patients. Low serum albumin often indicates malnutrition or systemic inflammatory response. Past studies have demonstrated that a low albumin level, defined as less than 3.5 mg/dl, is associated with greater risks of adverse outcomes following various types of surgery, such as orthopedic or urogenital surgeries. However, limited research has specifically investigated the relationship between serum albumin levels and appendectomy outcomes. This study aimed to investigate the independent association between preoperative serum albumin levels and the likelihood of postoperative complications in adult patients undergoing appendectomy for appendicitis.

Methods
A retrospective cohort study was conducted using the National Surgical Quality Improvement Program (NSQIP) database, including adult appendectomy patients with preoperative serum albumin data and postoperative appendicitis diagnosis. Exclusion criteria included ventilator dependency, ascites, disseminated cancer, open wounds, steroid or immunosuppressant use, bleeding disorders, preoperative transfusions, moribund status, or concurrent procedures. Baseline characteristics of the patients were analyzed to identify potential confounding factors. The strength of the association between low albumin levels and postoperative complications was assessed. A multivariate logistic regression model was constructed to control for potential confounding factors.

Results
A total of 28,051 patients were included in our statistical analysis. Among them, 1,312 (4.68%) had low levels of albumin (< 3.5 g/dl), and 2,022 (7.56%) suffered from postoperative complications. This study revealed that patients with low albumin levels had significantly higher odds of postoperative complications (OR: 3.14, 95% CI: 2.70-3.64). Even after adjusting for potential confounders, including age, sex, BMI, diabetes, dyspnea, COPD, hypertension, ASA classification, sepsis status, and surgery type, patients with low albumin levels still had significantly higher odds of postoperative complications (OR: 2.22, 95% CI: 1.88-2.62). Furthermore, older age, male sex, higher ASA classification, and nonlaparoscopic appendectomy were also independently associated with greater odds of postoperative complications (all p < 0.05).

Conclusion
This study highlights a significant association between low preoperative albumin levels and an increased likelihood of post-appendectomy complications, even after accounting for other known risk factors. Low albumin levels can serve as an important indicator of nutritional status, liver or kidney disease, or ongoing inflammatory processes. Physicians can use this prognostic tool to predict adverse postoperative complications and potentially implement proactive measures to prevent or mitigate these complications, leading to improved patient outcomes.

Association Between Preoperative Serum Albumin and Risk of Complications After Appendectomy

Spalt-Like Transcription Factor 4 (SALL4), a member of the SALL family, is a regulator of embryonic stem cell development that plays a crucial role in cell renewal and proliferation. SALL4 expression is normally repressed in most adult organs but is reactivated in a multitude of solid and blood cancers and upon treatment with hypomethylating agents (HMA). There is outstanding clinical need for the development and validation of a less invasive SALL4 expression assay via peripheral blood, which can be used for monitoring patients on HMA treatment and SALL4-targeting therapy such as molecular glue degraders.

Using the Simoa (Single Molecule Array) platform, a bead-based digital enzyme-linked immunosorbent assay, we cross-tested 5 commercial SALL4 antibodies and 13 antibody pair combinations, using the best antibody pair to test the matrix effects of human plasma, buffy coats, and bone marrow aspirates. Quantitative assay development further included creating recombinant protein standards, antibody pair screening and validation, and pre-analytical optimization. Upon completion of these steps, a retrospective cohort analysis via patient peripheral blood samples is currently being conducted to validate assay accuracy and precision. Additionally, we are aiming to conduct prospective studies to explore the role of the SALL4 protein biomarker in risk stratification and treatment monitoring.

Thus far, for quantitative assay development, we have successfully expressed and purified full-length SALL4 which will be used to calibrate the standard curve and determine the assay’s limit of quantification. We also optimized multiple pre-analytic steps, including our cell lysis protocol, isolation of buffy coats using density gradient separation, and sample collection method comparison. The best antibody pair can detect both SALL4A and SALL4B isoforms, with stronger signal detected with the SALL4A isoform. For the retrospective cohort validation, we have obtained samples for analysis for liver cancer, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) with plans to expand to breast cancer and lung cancer.

In conclusion, we have developed an ultrasensitive SALL4 protein expression diagnostic assay for use in monitoring patient treatment response and for guiding targeted therapy by analyzing peripheral blood for both solid and liquid tumors. Future steps include validating the diagnostic assay for specificity and sensitivity in a retrospective cohort analysis and conducting prospective studies to explore the role of the SALL4 protein biomarker for risk stratification, monitoring on HMA treatment, and guiding targeted therapy.

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Next from AMA Research Challenge 2023

The role of Lcn2 in Triple Negative Breast Cancer Organoids