Time Points Research Articles

Abstract 3858: Peripheral CD38+ effector memory (EM) T cells are dynamic biomarkers of response to neoadjuvant immune checkpoint blockade (NeoICB) in patients (pts) with resectable dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS)

Abstract Introduction: NeoICB is a promising approach for pts with DDLPS and UPS. However, biomarkers to identify which pts benefit from this treatment and understanding the mechanisms of response and resistance to ICB remains a challenge. Methods: We conducted a randomized phase 2 trial of neoICB (anti-PD1 +/- CTLA4) in pts with resectable DDLPS (n=17) and UPS (n=10). Pts with UPS received concurrent neoICB and radiation. The primary endpoint of the trial was pathological response; 3 DDLPS and 9 UPS pts were responders (R) and 14 DDLPS and 1 UPS pts were non-responders (NR). Peripheral blood mononuclear cells (PBMCs) were collected at baseline, prior to cycle 2 of neoICB (C2D1), and at the time of surgery (SURG). We performed mass cytometry (CyTOF) to identify immune cells associated with response and their dynamic changes with neoICB. We designed a custom 41-parameter panel using CD45 live cell barcoding with 31 markers for immune profiling, of which 23 were selected to detect key cell lineage and 8 to detect signaling markers. Batch correction and high dimensional data analyses were performed using CyCombine and CyTOF workflow R packages. Longitudinal comparisons between immune cell populations were conducted using Mann-Whitney U Test. Results: Of 79 available PBMC samples from 27 pts, 63 passed quality control after thawing (≥30% viability) and data clean-up (at least 10,000 events). Data was down-sampled to 5000 events for analysis. Of 19 immune cell types identified, monocytes were the most common followed by CD4+ and CD8+ T cells. There was an early increase in CD38+ effector/effector memory (EM) T cells at C2D1 in pts with DDLPS (CD8+ p=0.005, CD4+ p<0.001) and a similar increase observed in pts with UPS (CD8+ C2D1 p=NS, SURG p=0.034; CD4+ C2D1 p=0.024, SURG p=0.06) and these cells had higher Ki67 expression than CD38- T cells (p<0.005). At baseline, R pts with DDLPS had higher fractions of CD38+ EM CD8+ T cells than NR pts (p=0.044). Analysis in the UPS cohort was limited by the low number of NR pts. Higher expression of TIGIT was observed on CD38+ EM CD8+ T cells compared to CD38- counterparts at all time points. Conclusion: Peripheral CD38+ EM CD8+ T cells are associated with response to neoICB in pts with DDLPS. Longitudinal peripheral immune profiling reveals an increase in CD38+ EM CD8+ and CD4+ T cells with neoICB in pts with DDLPS and UPS. These cells are proliferative and express higher levels of TIGIT, which could be a future target for therapeutic exploration. Importantly, changes in the peripheral immune populations may precede changes in the tumor microenvironment (TME) as preliminary analyses of the TME did not identify early (C2D1) changes. TME imaging mass cytometry analyses are currently underway to associate peripheral and intratumor changes in immune populations. Citation Format: Elise F. Nassif, Hsinyi Lu, Michael Stanford, Karen Millerchip, Diana Romero, Jared Malke, Brenna Matejka, Jeffrey E. Gershenwald, Susmita Kumari, Duncan H. Mak, Angelique J. Lin, Jared K. Burks, Wei-Lien Wang, Alexander J. Lazar, Neeta Somaiah, Cara Haymaker, Christina L. Roland, Emily Z. Keung. Peripheral CD38+ effector memory (EM) T cells are dynamic biomarkers of response to neoadjuvant immune checkpoint blockade (NeoICB) in patients (pts) with resectable dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3858.

Abstract 347: Tumor necrosis factor related apoptosis inducing ligand (TRAIL)-induced cytokine production in TNBC promotes neutrophil chemotaxis and immune suppression

Abstract Background: TRAIL induces apoptosis in many preclinical cancer models including breast cancers and has been extensively studied as a potential cancer therapeutic. However, its efficacy in clinical trials is limited, suggesting an unknown modulatory mechanism responsible for lack of TRAIL activity in vivo. Here, we describe that TRAIL treatment elicits transcriptional changes in TNBC cells that alter the immune milieu. Method: We performed RNAseq of MDA-MB-231 cells treated with TRAIL for different time points, followed by validation with RT-PCR in various TNBC cells. RNAi, silencing key differentially regulated genes, along with RT-PCR, ELISA and CHIP assays, were used to validate RNAseq findings. Elucidation of the functional relevance of the outcome was supported both in vitro and in vivo by chemotaxis assay, cytotoxicity assay, RNAseq analysis of donor isolated neutrophils and intravital microscopy, CODEX analysis in TNBC xenografts from mice treated with the TRAIL respectively. Results: TRAIL treatment of the TNBC significantly induced expression of several cytokines, such as CXCLs 1, 2, 3, 8,11 and IL6, both in vitro and in vivo which are known to affect neutrophil function. Mechanistically, induction of these cytokines was predominantly mediated by death receptor 5 and caspase-8 protein, but not caspase-8 enzymatic activity. GSEA of the RNAseq data indicated that NFKB pathway was significantly enriched. Concordantly, we confirmed that both canonical NFKB1 and non-canonical NFKB2 pathways were activated by TRAIL in vitro and in vivo. However, the induction of the cytokine mRNAs was primarily dependent on the NFKB2 pathway. Neutrophils isolated from healthy human donors incubated with supernatants from TNBC cells in vitro indicated that TRAIL-induced CXCLs and IL6 significantly increased neutrophil chemotaxis. Additionally, CODEX analysis as well as intravital imaging confirmed that TRAIL treatment increases the number of neutrophils in the tumor. Preincubation of neutrophils with supernatants from TRAIL-treated TNBC significantly inhibited their cytotoxic effect against TNBCs. Further, transcriptome analysis of neutrophils incubated with either TRAIL or supernatant of TRAIL treated TNBC revealed significant enrichment of expression of inflammatory cytokine genes, immune modulating and immune checkpoint genes like PDL1. Functional studies with these neutrophils confirmed their suppressive effect on T cell function as well as the effect of TRAIL on decreased neutrophil apoptosis. Conclusion: Collectively, our study suggests the novel role of TRAIL-induced NFKB2-dependent cytokine production promoting neutrophil chemotaxis and immune suppression. This study implies that alterations in the innate immune system may modulate the effects of TRAIL on TNBC tumors. Citation Format: Manjari Kundu, Yoshimi Endo Greer, Lisa Ann Ridnour, David A Wink, Yeap S. Ng, Roberto Weigert, Stan Lipkowitz. Tumor necrosis factor related apoptosis inducing ligand (TRAIL)-induced cytokine production in TNBC promotes neutrophil chemotaxis and immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 347.

Abstract 3897: Isolation and molecular characterization of exosomes from glioblastoma patients using a microfluidic device after ultrasound-based opening of the blood-brain barrier

Abstract Glioblastoma (GBM) is a highly aggressive tumor of the brain. Current techniques of detection like magnetic resonance imaging and tissue biopsy are inaccurate or highly invasive, requiring alternatives like liquid biopsy. Exosomes are nanosized vesicles that contain cargo, reflective of their cellular origin and have been used for early diagnosis of cancer. However, in GBM, the blood-brain barrier (BBB) leads to a paucity of pathological information in blood. This study investigates the effect of opening the BBB on circulating exosomes and their potential for early detection of GBM using a microfluidic platform (GlioExoChip). In 20 patients enrolled in a clinical trial NCT03744026, a pulsed ultrasound emitter- Sonocloud-9 was implanted into their brains to open the BBB. We show that GlioExoChip isolates GBM-specific exosomes with a high capture efficiency using phosphatidylserine and Annexin-V chemistry. Further, the amount of circulating EVs increases twofold after opening the BBB. Finally, the correlation between tumor growth rate and the exosome concentrations is evaluated longitudinally. Methods: GlioExoChip is a microfluidic platform with a polydimethylsiloxane top and a glass bottom, functionalized with neutravidin-biotinylated annexin-V. Experiments are performed with GBM cell-line derived exosomes, followed by patient plasma. Isolated exosomes are characterized using Nanoparticle tracking analysis (NTA), scanning electron microscopy, Bicinchoninic acid (BCA) assay and western blot. Statistical analyses are performed using Student’s t-test and Pearson correlation. Results: The microfluidic device GlioExoChip has a high capture efficiency of exosomes derived from GBM cell lines, i.e., 93.03±3.31% and 96.77±0.72% for GBM6 and U87, respectively. NTA and BCA showed that 15 times more exosomes were captured from patient plasma when compared with healthy. Further, western blot proved the presence of a glial protein and Flotillin-1 in exosomes isolated from patients. Exosome concentrations varied with different time points after sonication and had an average twofold increase from the BBB opening. In our longitudinal study with 8 patients, an association between the volumetric enhancement of the tumor and exosome concentrations after sonication shows that the average Pearson correlation among short progression-free survival (PFS) patients was 0.73 (avg. p=0.39), while no correlation was seen among long PFS patients (avg. r value=0.10; avg. p-value=0.54). Conclusions: A microfluidic approach was used to investigate the role of circulating exosomes using phosphatidylserine-annexin V chemistry in GBM. BBB opening via sonication leads to an increase in the average GBM-specific exosome concentrations in plasma. Thus, exosomes are promising biomarkers for early detection in GBM. Citation Format: Abha Kumari, Mark Youngblood, Andrew Gould, Yoon-Tae Kang, Li Chen, Karl Habashy, Chris Amidei, Rachel Ward, Cristal Gomez, Guillaume Bouchoux, Michael Canney, Roger Stupp, Adam M. Sonabend, Sunitha Nagrath. Isolation and molecular characterization of exosomes from glioblastoma patients using a microfluidic device after ultrasound-based opening of the blood-brain barrier [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3897.

Abstract 119: A non-surgical method for developing a pre-clinical orthotopic mouse model for colorectal cancer

Abstract Recapitulating physiologically relevant tumor microenvironment is important yet a significant challenge in modeling colorectal cancer (CRC) for basic and translational studies. Among the different methods, using the cecal-wall injection technique is the most common way to create animal models of colorectal cancer (CRC). However, this approach requires complex surgery, and its unnatural placement creates a significant obstacle for developing treatments. Here, we report a non-surgical procedure for establishing orthotopic CRC mouse models, reflecting the appropriate tumor microenvironment to better model the disease development and facilitate translational studies including drug identification and validation. In this procedure, anesthesia is induced in overnight fasted study immunocompetent or immunodeficient mice while placed on a thermostat-controlled heating blanket. A specialized catheter is inserted and distended into the mouse colon to gently block the colon and ensure the localization for tumor development. Hereafter, the colonic epithelium is treated with proteases and mechanical abrasion to cause mild disruption. Following the local gut inflammation, the targeted colon region is incubated with tumor cells, leading to the adhesion of tumor cells to the colonic epithelium. Based on the animal background and the cell line used, the tumor developed within 3-4 weeks. The stages of tumor development at different time points were successfully confirmed using hematoxylin and eosin stain. Tumor microenvironment data revealed a significant increase in the immune cell infiltration specifically CD4+ T-cells and tumor-associated macrophages in the non-surgical procedure as compared to the cecal-wall injection. Furthermore, a significant increase in the PD-1 expression of the CD8+ T-cells in the cecal-wall injection as compared to the non-surgical procedure, suggests that the precise location of the tumor site may exert a substantial influence on pre-clinical therapeutic outcomes of immune-checkpoint inhibitors, thereby potentially hampering the clinical efficacy of drug treatments. Additional adaptations of this procedure may include: i) tumor development in different mouse strains, ii) local delivery of adeno-Cre in transgenic mice for CRC, and iii) delivering tumor organoids and patient-derived xenografts in immuno-deficient mice. Overall, our non-surgical procedure overcomes the technical difficulties of surgery and offers several advantages like ease of handling, cost-effectiveness, pain-free with nearly no procedure-associated mortality and adaptability to several strains. In conclusion, this procedure provides novel and promising alternative to conventional surgical orthotopic CRC mouse models for therapeutic interventions. Citation Format: Naresh Singh, Samantha Sharma, Kevin Van Der Jeught, Zhuolong Zhou, Xinna Zhang, Xiongbin Lu. A non-surgical method for developing a pre-clinical orthotopic mouse model for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 119.

Abstract 4788: Noninvasive longitudinal monitoring of residual disease in chemotherapy-treated colorectal cancer patients

Abstract Noninvasive monitoring of cancer shows great promise in assessing therapy response and improving patient outcomes. Recently, various groups have developed methods that detect circulating tumor DNA (ctDNA) in the plasma to measure minimal or molecular residual disease (MRD). Aberrant DNA methylation patterns are a hallmark of cancers, and robust signals can be detected by sensitive ctDNA assays. Here, we present a methylation-based approach for longitudinal monitoring of tumor burden that is tumor-naive, i.e., with no reliance on prior characterization of tumor molecular characteristics. We assess the effectiveness of our approach in a cohort of colorectal cancer (CRC) patients receiving chemotherapy with or without additional targeted agents. Longitudinal blood samples were collected from patients (n = 57) while on therapy, which averaged 4.9 months, for a total of 239 sample collection time points (median per patient = 5). Based on RECIST status, 16 patients were classified as complete responders (CR) at the end of treatment. We generated plasma cell free DNA-derived libraries for methylation sequencing targeting regions relevant to CRC. Next, we computed a disease burden score for each sample and related these scores to clinical response (CR vs. non-CR). We trained a classifier on an independent cohort of CRC and healthy donor plasma cfDNA samples, and used this classifier to check for residual disease at the end of treatment for each of our 57 patients. The classifier detected residual disease in only 3/16 CRs (19%) but 35/41 non-CRs (85%). Two of the non-CR patients were initially assessed as CRs at earlier time points. In both cases, our method successfully detected residual disease at or prior to the CR assessment and consistently showed residual disease in all follow up samples. This suggests improved sensitivity relative to RECIST and highlights the potential of using noninvasive blood tests for continuous monitoring of CRC patients receiving therapy. Citation Format: Alison D. Tang, Rebecca Gupte, Victoria Cheung, Tao Qing, Austin Cauwels, Emily Leff, Kimberly Walter, Ehsan Tabari, Alex Lovejoy, Jimmy C. Lin. Noninvasive longitudinal monitoring of residual disease in chemotherapy-treated colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4788.

Abstract 2047: Scale up and scale down approaches for screening of 3D patient-derived cell models

Abstract 3D cell culture is becoming more prevalent as scientists work to better model human physiology using in vitro systems. However, differences in handling 3D cell cultures compared to traditional 2D cell lines have limited adoption of 3D techniques in compound and cell therapy screening experiments. To facilitate use of patient-derived 3D tumoroid models, we developed protocols for both scale up and scale down of these models in Gibco™ OncoPro™ Tumoroid Culture Medium. OncoPro Tumoroid Culture Medium is a serum-free, conditioned medium-free culture medium designed to promote the growth of patient-derived 3D cancer models (tumoroids, cancer organoids). OncoPro medium is compatible with a suspension culture approach for easier handling compared to traditional embedded culture formats. We studied the growth of tumoroids in different flask sizes during suspension culture to optimize scale up for downstream assays. Tumoroid growth rates and morphologies were consistent across non-tissue culture treated flask sizes, and viable cell yield increased with surface area during scale up. In some cases, as many as 100 × 106 dissociated tumoroid cells can be recovered from a single Nunc™ TripleFlask™ after a week of growth. After scaling up, tumoroids were dissociated and seeded in multiwell plates to test multiple screening conditions (compound identity, compound concentration) in parallel. Dissociated cells were seeded in 96-well plates using both manual and automated liquid handling (Tecan Fluent 780). Due to the tendency of 3D cell models to aggregate and fall out of solution, initial seeding in a 96-well flat bottom plate resulted in high well-to-well variability. Optimization of cell seeding protocols led to standard deviations in tumoroid metabolic activity, number, and size that were comparable to manual seeding, with a coefficient of variation of less than 3% between wells for multiple tumoroid lines when automation was implemented. Generally, variability in cell seeding was lower when using the optimized, automated cell seeding protocol compared to manual methods. After seeding, cells were grown for three days to form tumoroids prior to the addition of compounds. At this time point, there was low well-to-well variability in tumoroid metabolic activity (CV<2%). Compounds and reagents to analyze cell health could also be added using automated liquid handling prior to analysis on plate readers or high-content imaging platforms. Citation Format: Colin Paul, Anthony Chatman, Amber Bullock, Xiaoyu Jenny Yang, Garrett Wong, Brittany Balhouse, Chris Yankaskas, Erik Willems, Matt Dallas, David Kuninger. Scale up and scale down approaches for screening of 3D patient-derived cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2047.

Abstract 2053: Spatiotemporal tumor immune modulation by localized delivery of cancer therapeutics using an implantable microdevice

Abstract Mechanism of action studies are imperative to translating oncology therapeutics into the clinic informing testable hypotheses and potential combination partners. This often involves large and time-consuming mouse studies to test a small number of compounds in an attempt to show statistically significant benefit. Likewise, testing multiple compounds and combination partners in these systems is limited by time, cost, and a desire to reduce the number of animals used in biological research. To overcome these limitations, we utilized an implantable microdevice (IMD) developed by KiburMed to measure intra-tumoral drug responses and to differentiate the mechanism of action of multiple cytokine agents in parallel in the MC38 mouse model. After sizable tumor growth in mice and implantation of the IMD device with 9 different therapeutic candidates, MC38 mice were taken down at different time points (24 hour, 4 days,7 days) to assess response. Local tumor response in the study was measured by cyclical immunofluorescence for deep cellular response phenotyping with a panel of 32 markers for comprehensive immune cell phenotyping. Among the candidate murine surrogate compounds in the device, SAR445877 (anti-PD-1-IL-15 mutein) was tested as a monotherapy, as a combination partner with anti-PDL1 and compared against anti-PD1 monotherapy. With further validation of this novel platform, results from this work are aligned with previous studies (1) showing a murine surrogate for SAR445877 in combination with anti-PDL1 increases immune cell populations more than either single agent alone. These analyses show CD3, CD4, and CD8 were significantly increased in SAR’877 murine surrogate + anti-PDL1 compared to SAR’877 alone or anti-PD1 alone. In addition, T cells could infiltrate at a distance away from treatment release region, well into tumor core. Other significant changes in immune cell markers include increases in CD45+, CD11b, CD11c, CD20 and CD27. In conclusion, we have shown that multiple oncology therapeutics can be screened simultaneously in a small number of mice using an implantable microdevice and data obtained with this technology is consistent with that obtained from traditional mouse tumor experiments. (1) Preclinical characterization of SAR445877, an anti-PD-1 antibody-IL-15 mutein fusion protein with robust anti-tumor efficacy as monotherapy and in combination with PD-L1 blockade. Marie Bernardo; Yu-an Zhang; Dan Lu; Stella Martomo; Fatima Menas; Chen Zhu; Raymond Perez; Jeegar Patel; Donald Shaffer; Xiangming Li, Cancer Res (2023) 83 (7_Supplement): 2972. *This work was supported and funded by Sanofi. Citation Format: Xiangming Li, Julien Tessier, Joon Sang Lee, Seth Garren, Virna Cortez-Retamozo, Meenu Sharma, Angela Jankowski, Erik Zarazinski, Ann Fiore, Oliver Jonas, Colin Brenan, Donald Shaffer, Angela Hadjipanayis. Spatiotemporal tumor immune modulation by localized delivery of cancer therapeutics using an implantable microdevice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2053.

Exploring the ageing methylome in the model insect, Nasonia vitripennis

BackgroundThe ageing process is a multifaceted phenomenon marked by the gradual deterioration of cellular and organismal functions, accompanied by an elevated susceptibility to diseases. The intricate interplay between genetic and environmental factors complicates research, particularly in complex mammalian models. In this context, simple invertebrate organisms have been pivotal, but the current models lack detectable DNA methylation limiting the exploration of this critical epigenetic ageing mechanism.This study introduces Nasonia vitripennis, the jewel wasp, as an innovative invertebrate model for investigating the epigenetics of ageing. Leveraging its advantages as a model organism and possessing a functional DNA methylation system, Nasonia emerges as a valuable addition to ageing research.ResultsWhole-genome bisulfite sequencing unveiled dynamic alterations in DNA methylation, with differentially methylated CpGs between distinct time points in both male and female wasps. These changes were associated with numerous genes, enriching for functions related to telomere maintenance, histone methylation, and mRNA catabolic processes. Additionally, other CpGs were found to be variably methylated at each timepoint. Sex-specific effects on epigenetic entropy were observed, indicating differential patterns in the loss of epigenetic stability over time. Constructing an epigenetic clock containing 19 CpGs revealed a robust correlation between epigenetic age and chronological age.ConclusionsNasonia vitripennis emerges as a promising model for investigating the epigenetics of ageing, shedding light on the intricate dynamics of DNA methylation and their implications for age-related processes. This research not only expands the repertoire of ageing models but also opens avenues for deeper exploration of epigenetic mechanisms in the context of ageing.

Abstract 6942: Single cell resolution of intrapatient drug resistance mechanisms in metastatic melanoma

Abstract Intrapatient heterogeneity among different metastatic sites contributes to immunotherapy (ICi) and targeted therapy resistance. Determining the evolution of metastatic melanoma lesions that develop within the same patient could help elucidate adaptive mechanisms that drive tumor progression during therapy. To assess how metastatic melanoma persists through immunotherapy and targeted inhibitor treatments, we utilized samples from a single patient who had received anti-CTLA4, BRAFi/MEKi, and BRAFi/MEKi+CDK4/6i treatments during the LOGIC2 clinical trial. Eight short-term cultures were established from different metastatic site biopsies at chronological time points throughout the patient’s treatment and subsequent post-mortem resection of the bone, ovary, and brain metastatic sites. While the initial treatment naïve metastatic tumor in the breast was BRAFV600E mutant, NRASQ61 mutations were acquired after treatment with CDK4/6i in the bone and ovary tumors. Tumors that persist through treatment and colonize distant organs adapt to and rely on organ-specific microenvironments. To assess treatment- and organ-specific adaptations, single-cell RNA sequencing was performed on seven of the short-term cultures to identify subpopulations that persist through each treatment. We analyzed samples and subpopulations of each culture to determine differences in their transcriptional cell states. The regulon transcriptional network of each sample and subpopulation was evaluated to determine site- and treatment-specific transcriptional regulation differences. In addition, we identified the treatment naïve culture is split into two major subpopulations by therapy resistance features which are further made up of six distinct subpopulations with heterogeneous regulatory networks. We determined the longitudinal evolution of subpopulations in response to treatment and organ site to evaluate how the pressure of therapies and the microenvironment influence metastatic progression. We further evaluated site-specific resistance mechanisms and detected increased HER3 expression in the melanoma brain metastasis (MBM) sample. Treatment with Neuregulin 1, a ligand for the HER3 receptor that is abundant in brain tissue, partially rescued the growth of MBM cells and restored MAPK and AKT-mTOR signaling in the presence of BRAFi/MEKi. Targeting the NRG1-HER3 signaling axis could have therapeutic potential in melanoma patients with active brain metastases. Additionally, we evaluated treatment-specific resistance mechanisms and identified high Brevican (BCAN) expression in an early emerging subpopulation that arises after ICi in this patient and in others. BCAN is implicated in both ICi- and cross-resistance. We will functionally investigate whether targeting BCAN sensitizes cells to ICi. Future studies aim to functionally examine novel site- and treatment-specific cross patient targets. Citation Format: Haley P. Wilson, Glenn L. Mersky, Jelan I. Haj, Jessica Teh, Phil F. Cheng, Signe Caksa, Casey D. Stefanski, Vivian Chua, Claudia Capparelli, Mitch P. Levesque, Reinhard Dummer, Timothy J. Purwin, Andrew E. Aplin. Single cell resolution of intrapatient drug resistance mechanisms in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6942.

Abstract 265: Reconstructing the early steps of breast tumor initiation

Abstract Breast cancer screening programs, detect not only the early stages of the disease but also various preneoplastic lesions which are very difficult to predict if and when they may develop into cancer. This uncertainty can lead to over-diagnosis, over-treatment and stress for millions of screened women. As consequence, discovering reliable biomarkers of this evolution could make it possible to monitor these patients appropriately and propose an effective prevention strategy. To meet this challenge, we urgently need to better understand the mechanisms that regulate human mammary homeostasis during early steps of tumor initiation. The hierarchy and the composition of human mammary epithelium, composed of stem, myoepithelial, luminal progenitors and mature luminal cells, are complex. This complexity raises the question of whether a given mammary epithelial cell is equally likely to be the cell-of-origin of breast cancer. Interestingly, increasing evidences show that disruption of lineage integrity can be a precursor of breast tumor initiation, and the basal and the luminal lineages respond differently to a given oncogenic stress and may each follow their own rule towards malignant transformation. These results strongly suggest that each lineage may have its own pliancy to be transformed by a given oncogene, hijacking the inter-tumoral heterogeneity observed in breast cancers. The objectives of this study are to develop a model to reconstruct the early steps of breast tumor initiation and understand the interplay between the initiating oncogenic event and the cell-of-origin. To answer these questions, we used a human mammary epithelial cell lines (HME) that maintains a cellular hierarchy with mammary stem cells (MaSC) and luminal cells (LC). Upon oncogenic expression of HRASG12V or c-Myc, we observed different modifications of cell homeostasis, with altered proportions of the lineages populations in the cell line. Depending on the oncogenic stress induced, we also demonstrated a modification in the morphology and the cellular composition of HME organoids, as well as in the capacity to form tumors. HRASG12V induces increase of MaSC compartment, formation of more branched organoids and generate aggressive basal-like tumors. Conversely, c-MYC leads to an almost loss of MaSC and formation of more proliferative unbranched organoid and generate tumors with limited growth. To address more precisely how cell-of-origin and lineage plasticity influence malignant transformation, we are developing for the first time a lineage tracing model in human mammary epithelium with HME. Moreover, a single cell RNA-seq was performed at different early time points during HME cellular differentiation in homeostasis and upon oncogenic stress to decipher the molecular mechanisms that underpin the very earliest steps of breast tumor initiation. Citation Format: Anaïs Grandon, Shuheng Lin, Martin Castagné, Julien Wicinski, Olivier Rosnet, Christophe Ginestier, Emmanuelle Charafe-Jauffret. Reconstructing the early steps of breast tumor initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 265.

Neurocognitive effects a combined polyphenolic-rich herbal extract in healthy middle-aged adults – a randomised, double-blind, placebo-controlled study

ABSTRACT Objective: This study assessed whether polyphenolic rich supplement containing Bacopa monnieri (BM: 300 mg), Panax quinquefolius ginseng (PQ: 100 mg) and whole coffee fruit extract (WCFE: 100 mg) could enhance cognitive performance, affect and cerebral-cortical activation over 28-days of intervention. Method: A randomised, double-blind, placebo-controlled, between-group study of 52 healthy adults between 35 and 65 years (M = 50.20, SD = 9.37) was conducted. Measures of cognition, affect and brain activity were measured at three time points: baseline, 28 days post intervention and 14 days post washout. At each time point, haemodynamic response in the prefrontal cortex (PFC) was measured using functional near-infrared spectroscopy (fNIRS), and serum brain-derived neurotrophic factor (BDNF). Results: The polyphenolic-rich supplement reliably improved positive affect and delayed recall compared to placebo following 28 days of supplementation. For the brain, those in the active condition showed greater PFC activation on performance of the 2-back tasks post supplementation compared to placebo (p < .05, d = 0.6). Discussion: This is the first report of a 28-day supplement intervention and 2-week follow-up study to assess changes in affect, cognition, cerebral haemodynamic response and BDNF in healthy middle-aged adults. The potential synergistic effects of polyphenolic compounds on neurocognitive function in middle-aged adults through emotional-cognitive processing and cognitive reserve are important for promoting brain and cognitive health.

Exploring the molecular regulation of vernalization-induced flowering synchrony in Arabidopsis.

Many plant populations exhibit synchronous flowering, which can be advantageous in plant reproduction. However, molecular mechanisms underlying flowering synchrony remain poorly understood. We studied the role of known vernalization-response and flower-promoting pathways in facilitating synchronized flowering in Arabidopsis thaliana. Using the vernalization-responsive Col-FRI genotype, we experimentally varied germination dates and daylength among individuals to test flowering synchrony in field and controlled environments. We assessed the activity of flowering regulation pathways by measuring gene expression across leaves produced at different time points during development and through a mutant analysis. We observed flowering synchrony across germination cohorts in both environments and discovered a previously unknown process where flower-promoting and repressing signals are differentially regulated between leaves that developed under different environmental conditions. We hypothesized this mechanism may underlie synchronization. However, our experiments demonstrated that signals originating from sources other than leaves must also play a pivotal role in synchronizing flowering time, especially in germination cohorts with prolonged growth before vernalization. Our results suggest flowering synchrony is promoted by a plant-wide integration of flowering signals across leaves and among organs. To summarize our findings, we propose a new conceptual model of vernalization-induced flowering synchrony and provide suggestions for future research in this field.

Longitudinal effects of prenatal alcohol exposure on visual neurodevelopment over infancy.

Prenatal alcohol exposure (PAE) affects neurodevelopment in over 59 million individuals globally. Prior studies using dichotomous categorization of alcohol use and comorbid substance exposures provide limited knowledge of how prenatal alcohol specifically impacts early human neurodevelopment. In this longitudinal cohort study from Cape Town, South Africa, PAE is measured continuously-characterizing timing, dose, and drinking patterns (i.e., binge drinking). High-density electroencephalography (EEG) during a visual-evoked potential (VEP) task was collected from infants aged 8 to 52 weeks with prenatal exposure exclusively to alcohol and matched on sociodemographic factors to infants with no substance exposure in utero. First trimester alcohol exposure related to altered timing of the P1 VEP component over the first 6 months postnatally, and first trimester binge drinking exposure altered timing of the P1 VEP components such that increased exposure was associated with longer VEP latencies while increasing age was related to shorter VEP latencies (n = 108). These results suggest alcohol exposure in the first trimester may alter visual neurodevelopmental timing in early infancy. Exploratory individual-difference analysis across infants with and without PAE tested the relation between VEP latencies and myelination for a subsample of infants with usable magnetic resonance imaging (MRI) T1w and T2w scans collected at the same time point as EEG (n = 47). Decreased MRI T1w/T2w ratios (an indicator of myelin) in the primary visual cortex (n = 47) were linked to longer P1 VEP latencies. Results from these two sets of analyses suggest that prenatal alcohol and postnatal myelination may both separately impact VEP latency over infancy. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Longitudinal Associations Between Optimism and Objective Measures of Physical Functioning in Women.

Identifying factors contributing to sustained physical functioning is critical for the health and well-being of the aging population, especially as physical functioning may precede and predict subsequent health outcomes. Prior work suggests optimism may protect health, but less is known about the association between optimism and objective physical functioning measures as individuals age. To evaluate the longitudinal association between optimism and 3 physical functioning measures. This was a prospective cohort study using data from the Women's Health Initiative (WHI) with participants recruited from 1993 to 1998 and followed up over 6 years. Data analysis was conducted from January 2022 to July 2022. Participants included postmenopausal women older than 65 years recruited from 40 clinical centers in the US. Optimism was assessed at baseline using the Life Orientation Test-Revised. Physical functioning was measured at 4 time points across 6 years by study staff evaluating performance in grip strength, timed walk, and chair stands. The final analytic sample included 5930 women (mean [SD] age, 70 [4] years). Linear mixed-effects models controlling for demographics, depression, health status, and health behaviors showed that higher optimism was associated with higher grip strength (β = 0.36; 95% CI, 0.21-0.50) and number of chair stands (β = 0.05; 95% CI, 0.01-0.10) but not timed walk at baseline. Higher optimism was also associated with slower rates of decline in timed walk (β = -0.09; 95% CI, -0.13 to -0.04) and number of chair stands (β = 0.01; 95% CI, 0-0.03) but not grip strength over time. Cox proportional hazards models showed that higher optimism was associated with lower hazards of reaching clinically defined thresholds of impairment for all 3 outcomes over 6 years of follow-up. For example, in fully adjusted models, for a 1-SD increase in optimism, hazard ratios for reaching impairment thresholds were 0.86 (95% CI, 0.80-0.92) for grip strength, 0.94 (95% CI, 0.88-1.01) for timed walk, and 0.91 (95% CI, 0.85-0.98) for chair stands. In this cohort study of postmenopausal women, at baseline, higher optimism was associated with higher grip strength and number of chair stands but not with the time it took to walk 6 m. Higher optimism at baseline was also associated with maintaining healthier functioning on 2 of the 3 performance measures over time, including less decline in walking speed and in number of chair stands women could perform over 6 years of follow-up. Given experimental studies suggesting that optimism is modifiable, it may be a promising target for interventions to slow age-related declines in physical functioning. Future work should explore associations of optimism with maintenance of physical functioning in diverse populations.

Humoral and cellular immunity to SARS-CoV-2 following vaccination with non-mRNA vaccines in adolescent/young adults with cancer: A prospective cohort study

BackgroundData on SARS-CoV-2 vaccine responsiveness in adolescent/young adult (AYA) cancer patients are sparse. The present study assessed humoral and cellular immune responses post-vaccination in this population. MethodsIn this prospective study, patients aged 12–30 years undergoing cancer therapy (“on therapy”) and survivors (“off therapy”) were recruited. Anti-receptor binding domain (RBD) protein IgG levels were measured at baseline, four weeks post-first vaccine dose (T1), and six weeks post-second dose (T2). Cellular immunity was assessed using activation-induced markers and intracellular cytokine staining in a patient subset. The primary outcome was to quantify humoral responses in both cohorts at T2 compared to baseline. Clinical predictors of log antibody titres at T2 were identified. ResultsBetween April-December 2022, 118 patients were recruited of median age 15.4 years. Among them, 77 (65.2 %) were in the “on therapy” group, and 77 (65.2 %) had received the BBV152 vaccine. At baseline, 108 (91.5 %) patients were seropositive for anti-RBD antibody. The log anti-RBD titre rose from baseline to T2 (p-value = 0.001) in the whole cohort; this rise was significant from baseline-T1 (p-value < 0.001), but not from T1 to T2 (p-value = 0.842). A similar pattern was seen in the “on therapy” cohort. BECOV-2 vaccine was independently associated with higher log anti-RBD titres than BBV152 (regression coefficient: 0.41; 95 % CI: 0.10–0.73; p = 0.011). Cellular immune responses were similar in the “on-” and “off therapy” groups at the three time points. ConclusionAmong AYA cancer patients, a single non-mRNA vaccine dose confers robust hybrid humoral immunity with limited benefit from a second dose.

The Female Thumb Carpometacarpal Joint: Motion and Force Changes Due to Arthritis and Surgical Intervention.

Thumb carpometacarpal (CMC) osteoarthritis (OA) has been one of the most common locations of hand OA. CMC OA disproportionately occurs in females over males. In severe cases, surgical intervention may be needed. However, to determine the effects of surgical treatment, normative, pre-, and postsurgery function must be understood. The goals of this work were to compare the thumb motion and force abilities of older healthy (OH) females without CMC OA to those of females with CMC OA and who received ligament reconstruction with tendon interposition (LRTI) surgery at time points presurgery, 3- and 6-months postsurgery. On average, CMC OA participants 3- and 6-months postsurgery showed 35.6% and 32.9% less overall metacarpal motion compared to presurgery, 31.9% and 29.1% less than OH, and exhibited altered motion. Metacarpal flexion/extension and abduction/adduction ranges were 51.9 deg and 43.4 deg for OH, 52.9 deg and 40.3 deg presurgery, 39.9 deg and 33.5 deg at 3-months, and 42.6 deg and 32.7 deg at 6-months postsurgery. On average, participants had increased force generation at 6-months postsurgery compared to presurgery, and 20% of participants returned to the level of OH females. These data sets highlight changes in thumb metacarpal movement and thumb force generation due to disease and surgical intervention. This work has the ability to support both surgeons and patients through improved outcome assessments as well as additional data to inform the decision process on intervention.

Lack of clinical utility of serum macrophage migration inhibitory factor (MIF) for monitoring therapy response and estimating prognosis in advanced lung cancer.

Lung cancer is a major burden to global health and is still among the most frequent and most lethal malignant diseases. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in a variety of processes including tumorigenesis, formation of a tumor microenvironment and metastasis. It is therefore a potential prognostic biomarker in malignant diseases. In this study, we investigated the applicability of MIF in serum samples as a biomarker in lung cancer. In a retrospective approach, we analyzed the sera of 79 patients with non-small-cell lung cancer (NSCLC) and 14 patients with small-cell lung cancer (SCLC) before the start of chemotherapy, as well as before the second and third chemotherapy cycle, respectively. Serum MIF levels were measured using a sandwich immunoassay with a sulfo-tag-labelled detection antibody, while pro-gastrin releasing peptide (proGRP) levels were determined with an enzyme-linked immunosorbent assay. No difference in serum MIF levels between responders and non-responders to chemotherapy was observed at all time points, while proGRP levels were significantly lower in responders before the second chemotherapy cycle (p = 0.012). No differences in biomarker levels depending on the histopathological classification of NSCLC patients was found. Moreover, in ROC curve analyses MIF was not able to distinguish between responders and non-responders to therapy. proGRP could differentiate between responders and non-responders before the second chemotherapy cycle (p = 0.015) with sensitivities of 43% at 90% and 95% specificity, respectively. Likewise, proGRP yielded significantly longer survival times of patients with low proGRP concentrations before the second chemotherapy cycle (p = 0.015) in Kaplan-Meier analyses, yet MIF showed no significant differences in survival times at all time points. Comparison with the biomarkers CEA and CYFRA 21-1 in the same cohort showed that these established biomarkers clearly performed superior to MIF and proGRP. From the present results, there is no indication that serum MIF may serve as a biomarker in prognosis and monitoring of response to therapy in lung cancer. Limitations of this study include its retrospective design, the inclusion of a larger NSCLC and a smaller SCLC subgroup, the classical chemotherapeutic treatment, the use of a non-diagnostic immunoassay (RUO-test) for MIF measurement and the lack of a validation cohort. Strengths of the study are its highly standardized procedures concerning sample collection, preanalytic treatment, measurements and quality control of the laboratory assays.

Network analysis of maternal parenting practices and adolescent mental health problems: a longitudinal study

BackgroundAn extensive literature has shown a strong connection between maternal parenting practices and adolescent mental health problems. However, it has been difficult for previous research to map a dynamic concurrent and prospective relationships within and between types of parenting practices and adolescent mental health problems. The present study addressed these issues using a network analysis approach and a longitudinal design.MethodsThis study involved 591 Chinese adolescents (249 males; mean age at T1 = 13.53) and their mothers (mean age at T1 = 39.71) at two time points (T1 and T2) with eighteen months apart. Mothers reported their parenting practices including warmth, monitoring, inductive reasoning, hostility, and harshness, while adolescents reported their mental health problems including anxiety, depression, aggression, and conduct problems. Network analysis was conducted for contemporaneous networks at T1 and T2 and temporal networks from T1 to T2.ResultsThe contemporaneous networks revealed the negative association between monitoring and conduct problems served as the main pathway through which parenting practices and adolescent mental health mutually influenced each other, and further, warmth was the most influential parenting practice on adolescent mental health. The temporal network revealed that maternal hostility exerted the most influence on adolescent mental health problems, whereas adolescents’ depression was most influenced by maternal parenting practices. Moreover, maternal hostility was most predicted by maternal harshness.ConclusionsThis study presents a novel perspective to gain a better understanding of the dynamics between and within maternal parenting practices and adolescent mental health problems. Findings highlight maternal harshness and warmth as potential prevention and intervention targets for adolescent mental health problems.

Prognostic value of tumor markers ProGRP, NSE and CYFRA 21-1 in patients with small cell lung cancer and chemotherapy-induced remission.

Despite successful response to first line therapy, patients with small-cell lung cancer (SCLC) often suffer from early relapses and disease progression. To investigate the relevance of serum tumor markers for estimation of prognosis at several time points during the course of disease. In a prospective, single-center study, serial assessments of progastrin-releasing peptide (ProGRP), neuron-specific enolase (NSE), cytokeratin-19 fragments (CYFRA 21-1) and carcino-embryogenic antigen (CEA) were performed during and after chemotherapy in 232 SCLC patients, and correlated with therapy response and overall survival (OS). ProGRP, NSE and CYFRA 21-1 levels decreased quickly after the first chemotherapy cycle and correlated well with the radiological response. Either as single markers or in combination they provided valuable prognostic information regarding OS at all timepoints investigated: prior to first-line therapy, after two treatment cycles in patients with successful response to first-line therapy, and prior to the start of second-line therapy. Furthermore, they were useful for continuous monitoring during and after therapy and often indicated progressive disease several months ahead of radiological changes. The results indicate the great potential of ProGRP, NSE and CYFRA 21-1 for estimating prognosis and monitoring of SCLC patients throughout the course of the disease.

Clinical Efficacy and Patient Reported Impacts of Roflumilast Foam 0.3% in Seborrheic Dermatitis: An Analysis of STRATUM Data for Patients Unresponsive or Intolerant to Topical Corticosteroids

Objective. Roflumilast foam 0.3% has demonstrated efficacy and tolerability in a Phase 3 clinical trial of moderate-to-severe seborrheic dermatitis (SD) patients (STRATUM). The aim of this subgroup analysis is to assess the efficacy and patient-reported quality of life (QOL) effects of roflumilast vs vehicle in patients with an inadequate response, intolerance, or contraindication to topical corticosteroids (TCS).&#x0D; Methods: Patients 9 years or older with at least moderate seborrheic dermatitis (Investigator Global Assessment [IGA] ≥ 3) were randomized 2:1 to roflumilast or vehicle for 8 weeks. Patients reporting a history of inadequate response, intolerance, or contraindication to topical corticosteroids (TCS) were included in this subgroup analysis. The primary efficacy endpoint was IGA success (Clear or Almost Clear with at least a 2-grade improvement) at Week 8. QOL was assessed using the Dermatology Life Quality Index (DLQI) for patients aged ≥17 years. QOL endpoints included percentage change from baseline in DLQI score, achievement of a minimal important difference (MID; defined as at least a 4-point reduction in DLQI score), and patients achieving a DLQI score of 0 or 1 by treatment group at weeks 2, 4, and 8. Differences in change from baseline DLQI scores were assessed using the Kruskal-Wallis rank sum test. The Cochran–Mantel–Haenzel test was used to assess differences in the proportion of patients achieving binary endpoints between treatments.&#x0D; Results: 189 patients (129 roflumilast, 60 vehicle) were included in the subgroup analysis. At Week 8, 78.8% of roflumilast patients achieved IGA success vs. 48.3% of vehicle patients (odds ratio [OR] 3.45; 95% confidence interval [CI]: 1.62, 7.36; p&lt;0.001). At each time point, percentage change from baseline in DLQI score was significantly larger for roflumilast-treated patients relative to vehicle (p≤0.001). Treatment with roflumilast significantly increased the odds of achieving an MID in DLQI across all time points (OR: 6.97; 95% confidence interval [CI]: 3.97, 12.24; p&lt;0.001) and achieving a DLQI score of 0 or 1 (OR: 2.46; 95% CI: 1.58, 3.81; p&lt;0.001).&#x0D; Conclusions: Roflumilast foam 0.3% provided significant and meaningful efficacy with improved QOL in patients with moderate-to-severe SD who had a prior history of an inadequate response, intolerance, or contraindication to TCS. Clinical improvement aligned with patient-reported outcomes at all key assessment periods.