OBJECTIVES/GOALS: We use a tissue engineered, biomimetic, 3D model to study the pathogenesis of breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) by comparing the effect of silicone implant shell on proliferation of patient-derived BIA-ALCL to its precursor T cells within the breast microenvironment. METHODS/STUDY POPULATION: Patient-derived breast tissue was processed for component adipocytes, ductal organoids, and stromal vascular fraction. These were suspended within 50 µl of 0.3% type I collagen matrix to which was added 200,000 cells/mL of either patient-derived BIA-ALCL cells or T progenitor cells. These were then plated into 6mm wells. As a control, both BIA-ALCL cells and T progenitor cells were suspended within type I collagen alone at the same seeding density without breast components. Before plating, wells were lined circumferentially with either textured, smooth, or no implant shell. These were 1cm by 2cm pieces dissected from the whole implant. Wells were imaged using confocal microscopy over 8 days. RESULTS/ANTICIPATED RESULTS: Unstimulated T progenitor cell count showed no significant increase in any of the conditions tested. The change in cell count over 8 days was 3.85% in each condition (p = 0.3352). A Tukey’s multiple comparison test comparing each condition revealed no significant increase in cell count over 8 days for all six conditions. Notably, our previous studies have shown proliferation of BIA-ALCL cells to be significantly more robust in the biomimetic platform compared to collagen-only groups, regardless of implant shell type (p < 0.01). BIA-ALCL cells grew nearly 30% faster in textured and smooth shell biomimetic groups compared to biomimetic wells lacking implant shell. DISCUSSION/SIGNIFICANCE OF IMPACT: Towards elucidating BIA-ALCL’s etiopathology, we show that silicone implant shell has a significant effect on proliferation of BIA-ALCL cells, but not their precursor T cells. If breast implant silicone shell is not a sufficient stimulus for T cell proliferation, co-stimulatory factors are required.

ABSTRACT IMPACT: Evaluate the accuracy of applying a predictive algorithm using clinical measures only in persons with stroke in the US. OBJECTIVES/GOALS: PREP2 is an algorithm, that predicts UL functional capacity at 3 months post stroke from measures taken within the first week.(1, 2) Despite its accuracy and ease of use, challenges arise of applying PREP2 in the US. The objective of this study was to evaluate the accuracy of PREP2 using only clinical measures in persons with stroke in the US. METHODS/STUDY POPULATION: Individuals with first-ever stroke were recruited from a local hospital and followed longitudinally, as part of an ongoing observational cohort. Variables captured within two weeks of stroke and entered into the algorithm were: age, SAFE score(1-3) and NIH Stroke Scale(4) total score. The algorithm classifies individuals into one of four expected categories: excellent, good, limited, or poor. The dependent variable was the predicted category of UL functional capacity as defined by ranges of the 3-month Action Research Arm Test score.(5) Accuracy, specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) of the algorithm, were calculated using a 4x4 contingency table. Other statistics analyzed include demographic characteristics and a weighted kappa for the algorithm. RESULTS/ANTICIPATED RESULTS: Data from 49 individuals were analyzed (57% male, 88% ischemic stroke, age = 65±8.56 years). Expected categorization matched observed categorization in 29/49 subjects, with the overall accuracy of the algorithm of 59% (95% CI = 0.44-0.73). The sensitivity of the algorithm was low except for the excellent category (0.95). Specificity was moderate to high for good (0.81), limited (0.98), and poor (0.95) categories. PPV was low for all categories and NPV was high for all categories except the good category. Additional results including weighted kappa and inaccuracy of predictions to be presented. DISCUSSION/SIGNIFICANCE OF FINDINGS: PREP2 algorithm, with clinical measures only, is better than chance (chance = 25% for each of the 4 categories) alone at predicting a category of UL capacity at 3 months post stroke. PREP2 is a simple tool that facilitates evaluation of eventual UL outcome from measures routinely captured after a stroke within most healthcare settings in the US.

OBJECTIVES/GOALS: Traumatic interpersonal experiences are associated with higher rates of chronic pain, increased pain severity and poorer functioning. The objective of this ongoing project is to obtain prevalence rates for various forms of interpersonal trauma among individuals with chronic pain, and to explore the potential mediating effect of heightened sensory and social sensitivity on the experience of pain. METHODS/STUDY POPULATION: Patients at Michigan Medicine between the ages of 18 and 65 complete an online survey. Patients are being recruited through a tertiary-care, outpatient pain clinic, as well as through an online health research portal. We aim to recruit 700 participants; we currently have 59.6% of our goal (n = 417). Participants also have the option to be included in a registry from which we can recruit for future studies. Approximately 85% of our participants have agreed to be in the registry. RESULTS/ANTICIPATED RESULTS: Preliminary data show that, of the 263 (63.4%) participants for whom data on chronic pain is available, 167 (63.5%) report chronic or persistent pain over the previous 3 months. Of these, 54% reported some form of childhood abuse or neglect. Approximately 41% reported four or more adverse childhood experiences. Additionally, of the 122 participants (73%) who were in a current romantic relationship, 20% reported some form of physical violence victimization from their romantic partner. We anticipate that interpersonal trauma will be associated with poorer perceptions of social relationships, higher sensory sensitivity, and higher perceived stress. DISCUSSION/SIGNIFICANCE OF IMPACT: The PASE Project parent study will be used to better understand prevalence rates for various forms of interpersonal trauma in our chronic pain population. Future analyses and studies will explore alternative pathways linking interpersonal trauma to the experience of pain through sensory and social sensitivity, which will inform interventions aimed at reducing pain among patients with a history of trauma.

OBJECTIVES/GOALS: Interleukin (IL)-6 is produced in excess in Systemic Sclerosis (SSc). Likewise, microarray analysis of Insulin-like Growth Factor (IGF)-II-treated NL fibroblasts revealed increased expression of the basic helix-loop-helix transcription factor, BHLHB2. Our goal is to delineate the role of BHLHB2 in the fibrotic response to IGF-II and IL-6. METHODS/STUDY POPULATION: Primary lung fibroblasts were cultured from human lung tissues at 37oC and 5% CO2. Cell cultures were stimulated with IL-6. Gene expression was measured using quantitative PCR (qPCR). IGF-II mRNA expression levels after IL-6 stimulation were compared with those of the housekeeping gene PPIB (Peptidylprolyl Isomerase B). Western blot was performed on nuclear and chromatin-bound subcellular fractions from treated lung fibroblasts. BHLHB2 protein levels were assayed in response to IGF-II in comparison to PBS as vehicle control. RESULTS/ANTICIPATED RESULTS: Results: Our results show that IL-6 increases IGF-II levels in fibroblasts. In turn, IGF-II increases BHLHB2 nuclear localization. We further show that IL-6 increases BHLHB2 levels and its nuclear localization in lung fibroblasts. Our findings are novel since the role of the transcription factor BHLHB2 in the IL-6 induced/ IGF-II-mediated fibrotic response in SSc lung disease remains unexplored. DISCUSSION/SIGNIFICANCE: Our findings may provide a rationale for combination therapy to block IL-6 and IGF-II function concomitantly and thus halt the progression of SSc pulmonary fibrosis (PF). Our findings may have wide implications for lung fibrosis associated with various diseases, since SSc-PF, is characterized by the activation of common fibrotic pathways.

OBJECTIVES/GOALS: Susceptible mucocutaneous membranes of the eye and nasal cavity are easily infected by viruses leading to pink eye or respiratory infections whose direct cost has been estimated as $16 billion annually in the United States. We have developed a novel and effective barrier that will be agnostic to variants enveloped viruses like coronaviruses. METHODS/STUDY POPULATION: We evaluated the efficacy of hydroxypropyl cyclodextrin barrier in preventing respiratory coronavirus infections using 25 humanized angiotensin converting enzyme-2 receptor (hACE-2) mice under a BSL3 laboratory setting. We have shown the barrier is safe and efficacious in preventing coronavirus infections in in vitro respiratory cell lines. We instilled 10 uL aliquot of the barrier into the nostril of the mouse 30 minutes before exposing them to a 10uL titer containing 10,000 plaque forming units of the SARS-CoV-2 delta variant. The control mice received the SARS-CoV-2 infection but not the barrier. The mice were observed for 5 days after which they were sacrificed. We analyzed the lungs and nasal palates for viral load using reverse transcription-polymerase chain reaction. RESULTS/ANTICIPATED RESULTS: We observed our barrier to be effective in preventing SARS-CoV-2 delta variant infection in hACE2 mice models. The lungs and nasal secretions of treated mice were less infectious with lower viral load than the control mice. The lungs of treated mice showed decrease in IFN gene expression and many cytokines and chemokines that regulate virally induced inflammatory responses such as IL-1b, IL-8, CXCL9, CXCL10, and the CCLs. We observed the plasma Angiotensin I and Angiotensin II decreased with barrier treatment, correlating with the viral load observed in the lungs. These peptides may be useful biomarkers for monitoring viral load within the lungs of virally infected individuals. DISCUSSION/SIGNIFICANCE: This supports the barrier’s efficacy to reduce transmission and prevent infections of SARS-CoV-2. This easy to use barrier can augment the mucocutaneous layers of the eye and nasal cavity. Our agnostic barrier will reduce the economic and public health burden of seasonal respiratory and eye viral infections and their related deaths amongst the public.

ABSTRACT IMPACT: This study assesses patient and volumetric risk factors for distant recurrence within 6 months of completion of curative chemoradiation with brachytherapy in locally advanced cervical cancer. OBJECTIVES/GOALS: Initial tumor volume and tumor shrinkage velocity are prognostic of cure and survival after curative chemoradiation (CRT) for cervical cancer. We explored whether local tumor volumetric changes influence time to distant recurrences outside the radiation field. METHODS/STUDY POPULATION: We performed a retrospective cohort study of patients with FIGO Stage IB-IVA cervical cancer treated with curative CRT and brachytherapy at a tertiary academic center with minimum 3 months follow up and standard post-treatment FDG-PET. Patients received 6 weekly fractions of brachytherapy interdigitated with external beam radiation and cisplatin. Tumor volumes were assessed by MRI at brachytherapy planning. Patients who developed distant metastasis were classified as earliest (3-6 months), early (6-24 months) or late (>24 months) following completion of CRT. Absolute and percent decrease in tumor volume for each fraction were calculated with respect to first brachytherapy volume. Fisher’s exact and Mann Whitney-U tests were used for comparison of categorical and continuous variables. RESULTS/ANTICIPATED RESULTS: 143 of 574 (25%) patients developed distant metastasis. Distribution of age, histology, FIGO 2018 stage, primary tumor SUVmax, treatment length, and pre/post treatment squamous cell carcinoma antigen levels were not associated in each group. Para-aortic lymph metastases were more common in patients with earliest distant recurrence (33% earliest, 26% early, 12% late, p=0.03). Median initial tumor volume in the earliest (n=24), early (n =29) and late (n=9) groups was 57, 28 and 40 mL, respectively (p=0.08); 57 (earliest) vs 30mL (early+late groups), p=0.04. Average mid treatment (fraction 4) and end of treatment (fraction 6) percent shrinkage was 80 (earliest) vs 73 (early+late), p=0.84 and 94 vs 92, p=0.95, respectively. Neither absolute nor percent tumor shrinkage differed between early vs. late groups. DISCUSSION/SIGNIFICANCE OF FINDINGS: Tumor volumetric changes during definitive chemoradiation were not associated with the timing of developing distant metastasis, which is linked to presence of lymph node metastasis and tumor volume at diagnosis.

OBJECTIVES/SPECIFIC AIMS: Chlamydia trachomatis (CT) infection can lead to reproductive morbidity in women. Animal models suggest that protection against CT is mediated through the cytokine interferon-gamma (IFN-γ), produced by CD4+ T-cells, which clears CT through intracellular tryptophan depletion. In humans, correlates of protection remain to be elucidated, which hinders chlamydia vaccine development. Natural clearance of CT infection (e.g., clearance before antibiotics) may be an immunological correlate of protection, evidenced by (1) CT clearance without antibiotics; and (2) a 4-fold reduced risk of CT reinfection within 6 months. We have identified women with and without natural clearance of CT infection. By comparing these two groups of women, the role of IFN-γ-mediated natural clearance of CT infection will be investigated. METHODS/STUDY POPULATION: Through collaboration with a cohort study of CT-infected women, we have access to stored specimens from women who naturally cleared CT or had persisting CT infection. Using peripheral blood mononuclear cell (PBMC), we will assess whether natural clearance of CT infection is associated with IFN-γ-producing CD4+ T-cells by stimulating PBMC ex vivo with CT antigens using intracellular cytokine staining. We will also use cervicovaginal lavage (CVL) and untargeted High-Performance Liquid Chromatography-Mass Spectrometry to assess for tryptophan-dependent and -independent metabolic pathways associated with natural clearance of CT infection. RESULTS/ANTICIPATED RESULTS:: To date, IFN-γ has been measured in 10 women who did not clear CT infection, demonstrating that <20% of these women produced significant levels of IFN-γ. Women who naturally cleared CT have yet to be studied. Untargeted HPLC-MS has been performed on 6 women (3 who cleared matched to 3 with persisting CT infection). To date, 11 pathways that are significantly associated with natural clearance have been identified. DISCUSSION/SIGNIFICANCE OF IMPACT: The outcome of natural clearance of CT infection is distinct from women with persisting chlamydia. These studies may inform whether IFN-γ, produced by CD4+ T-cells, or tryptophan-dependent or -independent metabolic pathways are associated with natural clearance, which may advance chlamydia vaccine development.

OBJECTIVES/GOALS: Congenital cytomegalovirus (cCMV) remains to be the leading infectious cause of fetal anomalies. The role of fetal natural killer (NK) cells during cCMV remains largely unknown. The objective of this study is to define the transcriptomes of fetal NK cells exposed to human cytomegalovirus (HCMV) infection during gestation. METHODS/STUDY POPULATION: Four sets of umbilical cord blood and matching umbilical cord tissues were collected from two HCMV seropositive (HCMV+) and two HCMV seronegative (HCMV-) fetuses that did not experience any complications during gestation. These samples were provided by the Medical College of Wisconsin Tissue Bank and were processed within 24 hours following live birth. CD7+ CD3e-CD14-CD19-CD20- fetal NK cells were isolated, using the BD FACSAria sorter. Following cell sorting, single-cell RNA sequencing (scRNA-seq) was performed, and cDNA libraries were constructed and sequenced via NextSeq 550. Cell Ranger was then used to algin the cDNA reads and the Seurat R package was used to analyze the transcriptional data. Cells were filtered and clustered based on the number of uniquely expressed genes. RESULTS/ANTICIPATED RESULTS: Four sets of umbilical cord blood and matching umbilical cord tissues were collected from two HCMV+ and two HCMV- fetuses. We were able to successfully sort and capture fetal NK cells and perform scRNA-seq on these samples. Following unbiased clustering, we observed and characterized five distinct fetal NK cell subsets in the umbilical cord blood and four fetal NK cell subsets in the corresponding umbilical cord tissue. Our findings revealed that HCMV+ fetal NK cells primarily consisted of mature NK cell subsets, while HCMV- fetal NK cells constituted the majority of the immature subsets. Importantly, we identified a unique subset of NKG2CHi fetal NK cells that were significantly elevated in the HCMV+ fetuses. Finally, we defined a group of transcription factors involved in the formation of antiviral fetal NK. DISCUSSION/SIGNIFICANCE: Here, we demonstrate that HCMV infection can induce the formation of distinct NK cell subsets and drive their unique transcriptional profiles. These findings have the potential to guide the development of an innovative NK cell immunotherapy that could help prevent fetuses from developing symptomatic cCMV.

ABSTRACT IMPACT: Urine tumor DNA non-invasively detects minimal residual disease and infers tumor mutational burden in locally advanced bladder cancer prior to radical cystectomy, which may potentially enable the selection of patients for bladder-sparing treatment or facilitate personalized adjuvant immunotherapy. OBJECTIVES/GOALS: Standard-of-care treatment for muscle-invasive bladder cancer (MIBC) is radical cystectomy. The inability to assess minimal residual disease (MRD) non-invasively limits our ability to offer bladder-sparing treatment. We sought to develop a liquid biopsy solution via urine tumor DNA (utDNA) analysis. METHODS/STUDY POPULATION: We applied uCAPP-Seq, a targeted sequencing method for detecting utDNA, to urine cell-free DNA samples acquired on the day of radical cystectomy from 42 patients with bladder cancer. utDNA variant-calling was performed non-invasively without prior tumor mutational knowledge. The overall utDNA level for each patient was represented by the non-silent mutation with the highest variant allele fraction after removing germline variants. Urine was similarly analyzed from 15 healthy adults. Tumor mutational burden (TMB) was inferred from the number of non-silent mutations detected in urine cell-free DNA by applying a linear relationship derived from TCGA whole exome sequencing of 409 MIBC tumors. RESULTS/ANTICIPATED RESULTS: utDNA levels were significantly higher in patients with residual disease detected in their surgical pathology compared to those who achieved a pathologic complete response (P = 0.002). Using an optimal utDNA threshold to define MRD detection, positive utDNA MRD significantly predicted the absence of pathologic complete response with a sensitivity of 81% and specificity of 81%. Positive utDNA MRD also portended significantly worse progression-free survival (HR = 7.4; P = 0.03) compared to negative utDNA MRD. Furthermore, we applied a linear relationship (Pearson r = 0.84; P < 0.0001) to identify patients with high inferred TMB who may have been candidates for early immune checkpoint blockade. DISCUSSION/SIGNIFICANCE OF FINDINGS: utDNA MRD analysis prior to surgery correlated significantly with pathologic response and progression-free survival, which may help select patients for bladder-sparing treatment. utDNA can also non-invasively infer TMB, which could facilitate personalized adjuvant therapy for patients in the future.

OBJECTIVES/GOALS: Cell-cell (CC) and cell-matrix interactions (CM) are known to affect drug sensitivity of cancer cells, but are not effectively recapitulated using 2D platforms. This research aims to determine how cell and matrix interactions confer drug resistivity in 3 distinct culturing models: 2D (no CM/limited CC), 3D spheroids (CC) and 3D fibronectin (both). METHODS/STUDY POPULATION: We examined four breast cancer cell types. The cells were derived from a nonmetastatic primary tumor (HMLE-E2) or overt bone-metastasis (BM). Transglutaminase 2 (TGM2), a matrix crosslinking protein, is overexpressed in metastatic bone tumors and may play a key role in matrix-conferred drug resistivity. In a gain-of-function model, TGM2 was upregulated in HMLE-E2 cells and compared to shTGM2 knockdown BM cells. Growth rates were analyzed using metabolic activity over 8 days, and drug sensitivity to Neratinib (0-1000 nM) was analyzed via cell titer. To account for the different transport properties of the 3 distinct culture environments, we developed a mathematical model for each condition, allowing us to normalize the drug sensitivity results across models to effectively compare true biological resistivity. RESULTS/ANTICIPATED RESULTS: We observed that increased cellular levels of TGM2 significantly increase the growth rate and drug resistivity of cells on fibronectin matrices. Interestingly, in 2D cultures, TGM2 expression was correlated with higher Neratinib resistivity but did not affect growth rates. In spheroid models without a significant matrix component, that rely solely on cell-cell junctions, high levels of TGM2 were correlated with lower survival rates. Lower levels of TGM2 are correlated with a more epithelial phenotype, and using our mathematical model we have identified significant transport differences between high and low TGM2 spheroids. We theorize that the low TGM2 spheroids have denser packing, which lowers the rate of diffusion and, thus reduces the effective concentration of the drug to the majority of the cells. DISCUSSION/SIGNIFICANCE OF IMPACT: Our studies indicate that the cellular response to drugs can be altered by changes in both transport properties of the tissue and the CM interactions. By systematically investigating the effects of CC interactions and CM interactions, we can use mathematical models to delineate physical means of drug resistivity from a biologically driven resistance.

OBJECTIVES/GOALS: Ocular graft versus host disease (oGVHD) affects ~50% of individuals after an allogeneic hematopoietic stem cell transplant for treating blood cancers. OGVHD results in severe dry eye disease and decreased vision. Quantifying specific cytokine changes in tears may reveal biomarkers and future treatment targets for patients with oGVHD. METHODS/STUDY POPULATION: The goal of this study is to determine if cytokines can be measured in tears with the Isoplexis platform. This pilot validation study evaluates the tears of a patient with oGVHD utilizing the Isoplexis platform. The Isoplexis has specific advantages for tear samples including high-throughput analysis, and small sample requirements, but has yet to be validated in tears. A sample from normal and oGVHD patient tears were collected for comparison. Samples were analyzed on two separate backgrounds–standard Bovine Serum Albumin (BSA) background and artificial tears (ATs). The negative control was ATs and positive control was a concentrated cytokine solution. Analysis of 22 cytokines was performed. RESULTS/ANTICIPATED RESULTS: Analysis of 22 cytokines was performed. As expected, the cytokine levels of the ATs alone were below the limit of detection (LOD). The oGVHD patient tears showed elevated TNF-alpha, TNF-beta, perforin, MIP-1a, MIP-1β, MCP-1, IL2, IL4, IL5, IL-7A, IL9, IL-13, IL-15, IFN-γ, granzyme B, and GM-CSF with ATS background, but no cytokines above the LOD in the BSA background plate. The control tears had elevated IP-10. The elevated cytokines for the oGVHD patient corresponded to symptom severity and clinical findings. DISCUSSION/SIGNIFICANCE: These results suggest that using ATs as the background with the Isoplexis platform improves the sensitivity to detect tear cytokines. Findings of elevated IL-7A and GM-CSF in tears parallels literature findings for oGVHD. Further evaluation of samples will continue to validate the Isoplexis multiplex assay for tear cytokine analyses.

Youth are an understudied population requiring additional safeguards when participating in research. Their input is necessary to facilitate participation and interest in studies. To address this, Mayo Clinic established one of the first pediatric advisory boards (PAB) comprised of 18 diverse youth aged 11–17. The PAB members participated in quarterly meetings (in person and then by video conference with the advent of COVID-19) where they provided feedback to researchers on recruitment strategies, study materials, and procedures. The PAB meetings fostered bidirectional conversations with researchers on several health research topics, including mental health. Youth advisory boards can promote engagement in pediatric research.

OBJECTIVES/GOALS: There is increasing evidence that cannabidiol (CBD) has promising potential to treat PTSD. However, more research is warranted to fully understand the benefits of CBD for PTSD. This poster will describe the design and methodology of one of the first ever pilot RCTs examining CBD (vs. placebo) combined with prolonged exposure therapy for PTSD. METHODS/STUDY POPULATION: This study is an early Phase II double-blind, pilot RCT. Participants are 24 individuals 18-65 years old who meet DSM-5 criteria for PTSD on the CAPS-5 and were recruited from local hospitals and the community. Individuals complete a standardized baseline assessment with an independent evaluator to assess study eligibility. Participants who meet study inclusion are randomized to 18 days of CBD 250mg (BID) or placebo delivered in combination with 10-sessions Prolonged Exposure (PE) psychotherapy over 2 weeks. Individuals begin medication 3 days prior to beginning PE to ensure steady state. Participants complete self-report and biomarker outcomes at select timepoints during study participation, and are also asked to complete a 1-month follow-up assessment following treatment. RESULTS/ANTICIPATED RESULTS: This aims of this study are to: 1) examine the safety, feasibility, and PTSD symptom reductions associated with the combined intervention; 2) evaluate biomarkers associated with the endocannabinoid system and stress response; 3) determine the association between changes in biomarkers and PTSD symptoms following treatment. It is expected that CBD+PE will be safe and feasible, and that there will be a detectable signal of CBD vs. placebo in the reduction of PTSD symptoms. It is also anticipated that CBD will have higher levels of endocannabinoids and lower stress response levels compared to placebo. Lastly, we expect that greater changes in biomarkers will be associated with lower levels of PTSD severity following treatment. DISCUSSION/SIGNIFICANCE: Although there is growing interest in cannabinoids for psychiatric conditions, such as PTSD, controlled trials are limited and have yet to examine the proposed intervention for PTSD. If successful, this study will enhance the feasibility of a larger, adequately powered RCT to address immediate and long-term improvements for PTSD treatments.

OBJECTIVES/SPECIFIC AIMS: The first aim of the study is to evaluate the accuracy of serum biomarkers of acute GVHD measured after four weeks of corticosteroid therapy to predict 6 month NRM. The second aim of this study is to compare the accuracy of the biomarker algorithm to that of clinical response to corticosteroids after four weeks. The third aim of the study is to develop a novel regression model that uses weekly biomarker measurements over the first month of corticosteroid therapy to predict 6 month NRM. METHODS/STUDY POPULATION:. Patients who received HCT at one of 22 IRB-approved centers and provided blood samples to the Mount Sinai Acute GVHD International Consortium (MAGIC) biorepository and developed GVHD between January 2008 to May 2018 are included in this study. Patients were divided by time into a training set (Jan 2008-Dec 2015, n=233) for model development and a validation set (Jan 2015-May 2018, n=357) to evaluate the predictive performance of the model. The later time of the validation set was chosen deliberately to model contemporaneous GVHD treatment practices. The size of each group was designed so that there would be roughly equal numbers of deaths in both groups. RESULTS/ANTICIPATED RESULTS:. Serum concentrations of GVHD biomarkers after one month of corticosteroid therapy were measured in the validation set, and the predicted probability of NRM ($\hat{\rm p}$) was computed according to the previously published algorithm: $\log[-\log(1 - \hat{\rm p})]=-11.263 + 1.844({\rm logST}2)+ 0.577({\rm logREG}3\alpha)$. The performance of the biomarker algorithm was evaluated by creating receiver operating characteristic (ROC) curves and calculating the area under the curve (AUC) in the validation set. The AUC of the biomarker algorithm was a significantly better predictor of 6 month NRM than clinical response to treatment after four weeks of corticosteroids (0.84 vs. 0.64, p<0.001), which is a clinically relevant improvement in accuracy. To evaluate serial biomarker monitoring, serum biomarker concentrations will be measured weekly at five time points from treatment initiation to one month after corticosteroid therapy. We will use these values in the training set to develop a regression model for 6 month NRM that accounts for repeated biomarker measurements. The performance of this model will be tested in the validation set and the accuracy of the serial biomarker measurements will be compared to the accuracy of measuring biomarkers at the single time point after four weeks of corticosteroid therapy. An AUC improvement of 0.05 would be considered clinically significant. DISCUSSION/SIGNIFICANCE OF IMPACT: Clinical response to treatment after four weeks has been the standard endpoint in GVHD interventional trials for decades. If biomarkers measured at the same time more accurately predict long term mortality, this study would provide the basis for a novel endpoint in GVHD trials and enable more accurate determination of effect size of experimental interventions. An accurate biomarker algorithm will prove useful in guiding immunosuppressive treatment decisions for patients with GVHD. Patients identified by the algorithm as low-risk may benefit from reduced-dose corticosteroid therapy, potentially reducing lethal opportunistic infections. Patients identified as high-risk will be candidates for more intensive immunosuppression or investigational therapies. This precision medicine approach tailors therapy to the individual patient’s biology.

OBJECTIVES/GOALS: Real-world studies have been gaining momentum in providing evidence of treatment effectiveness and hold great potential for facilitating the drug regulatory process. The U.S. Food and Drug Administration (FDA) has recognized this by providing a framework for using real-world data (RWD) to generate real-world evidence (RWE). The objective of this study is to assess the current level of RWE implementation in clinical studies. METHODS/STUDY POPULATION: Using keywords relevant to RWE, we reviewed studies on drugs, biologics, and medical devices published on PubMed in 2018. Information regarding the therapeutic area of focus, intervention type, study design, primary outcome, and data source was recorded. Further analyses of the three main therapeutic areas of study (oncology, cardiology, and infectious diseases) were performed to determine how RWE was being utilized. In addition, a broad “real-world” search was performed on Clinicaltrials.gov, from which we extracted relevant observational and Phase I, II, II/III, III, III/IV, and IV studies. A supplemental PubMed search was used to evaluate published studies in order to identify which field these trials were concentrated in and the outcome of interest.” RESULTS/ANTICIPATED RESULTS: After application of “real-world” search terms to PubMed, 995 hits were generated and of these, 311 studies were excluded. More than half of the studies were observational and retrospective in nature (64%) with 70% examining drug/biologic outcomes. RWE data sources were largely dominated by medical records and claims data. The primary uses of RWE across oncology, cardiology, and infectious diseases included supporting drug product effectiveness, assessing safety, and evaluating treatment patterns. Of the 207 RWE studies identified on ClinicalTrials.gov, 66 were cancer randomized controlled trials (RCTs), a majority of which were used for post-marketing safety evaluations. Further research will be conducted to determine the precise role of RWE in all studies (e.g. historical comparator, label expansion). DISCUSSION/SIGNIFICANCE OF IMPACT: By examining the use of RWE in regulatory decision making, we can inform stakeholders of the extent to which robust RWE studies complement evidence generated by RCTs. Thought to reflect a product’s performance in a broader and more diverse population, RWE can provide greater insight to clinical trial conduct and ultimately transform patient outcomes.

OBJECTIVES/GOALS: Numerous diseases, including AL amyloidosis, are due to expression of aberrant antibodies. Significant effort has gone into plasma cell toxic therapies with varying degrees of success, but no therapies preventing antibody synthesis have been developed. The goal of this study is to assess BiP targeting to prevent antibody secretion in plasma cells. METHODS/STUDY POPULATION: Using 4 multiple myeloma cell lines (KMS11, RPMI8226, ANBL-6, U266), we knocked down BiP expression with RnaseH dependent siRNA or subA toxin, a bacterial toxin that specifically cleaves BiP, and measured changes in unfolded protein and intracellular light chains by flow cytometry during drug induced ER stress created by the intracellular calcium depleting agent thapsigargin. BiP is the master regulator of the unfolded protein response (UPR), an ER stress pathway important for protein folding. BiP is also an ER resident protein folding chaperone important for proper antibody folding. We hypothesized that BiP downregulation will lead to decreased folded antibody in the cell, increased unfolded antibody and constitutive activation of the UPR. RESULTS/ANTICIPATED RESULTS: 1 to 4 hours after treatment with thapsigargin plus siRNA against BiP, levels of BiP are significantly decreased. The levels of intracellular light chains decrease, and the level of unfolded protein within the cells increases dramatically. Interestingly, in alignment with the UPR literature, 24 hours post treatment, these levels have normalized again in surviving cells. SubA treatment increased BiP expression by 4 hours, contrary to our hypothesis, and minimally increased unfolded proteins and minimally decreased intracellular light chains. We expect that further functional testing of antibody secretion by ELIspot assays will show decreased secretion of antibody with BiP siRNA treatment. Combination therapies with other UPR stressing agents may act synergistically to affect antibody production. DISCUSSION/SIGNIFICANCE: BiP knockdown reduces antibodies and boosts unfolded proteins. SubA toxin ineffectiveness likely stems from increased BiP due to feedback loops. Combining anti-BiP treatments with UPR stressing drugs like bortezomib may halt antibody synthesis and induce cell death. These findings support BiP as a viable drug target for antibody-related diseases.