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Introduction MYD88 L265P mutations (MUT) are found in 90% of patients with Waldenstrom macroglobulinemia (WM) and lead to the activation of pro-survival signaling. Limited literature has demonstrated that patients with MYD88 wild-type (WT) WM have poorer responses to conventional therapies and a higher likelihood of histological transformation. Increasingly the role of non-coding genomics in lymphomagenesis has demonstrated importance and currently, the epigenetic landscape of MYD88 WT WM remains underexplored. This study utilized a multi-omics approach to assess DNA methylation in MYD88 WT WM compared to MYD88 MUT patients and controls. Methods Bone marrow samples were prospectively collected from 20 subjects [3 MYD88 WT/CXCR4 WT WM, 12 MYD88 MUT WM and 5 normal controls] and positive selection for CD19+ and/or CD138+ B cells was performed. The analysis focused on the comparison between MYD88 WT WM to normal controls and MYD88-WT to MUT WM. Genome-wide DNA methylation analysis was performed, and CpG methylation ratios were segmented into 200-bp regions. Differentially methylated regions (DMRs) were determined using a Q-value <0.05 and absolute methylation difference ≥10%. Next, RNA-seq data was interrogated and differentially expressed genes (log2fold change>0.5 or <-0.5, false discovery rate<0.05) with corresponding promoter hypo- or hypermethylation were assessed. Ingenuity Pathway Analysis was used to determine potentially epigenetically regulated pathways using a corrected p<0.05. Results Comparing MYD88 WT WM samples to controls, a statistically greater number of hypermethylated DMRs were observed across genomic regions (hyper- vs hypomethylated): promoter (29129 vs 6300), 3'untranslated region (UTR) (2068 vs 1551), 5'UTR (10169 vs 735), intron (30344 vs 24511), exon (2224 vs 6855), CpG island (30372 vs 1240) and CpG shore (14477 vs 5872) (all p<0.001). Next, mRNA analysis demonstrated that 214 genes were differentially up-/downregulated with concordant promoter hypo-/hypermethylation. On the assessment of promoter-methylation-based pathways, multiple pathways involved in cell signaling, proliferation, gene regulation, cytokine and immune signaling and metabolism were found to be deregulated. Most pathways were found to be negatively enriched in MYD88 WT patients compared to controls including several members of interleukin (IL) signaling (IL-2, IL-3, IL,7) and regulators of cell proliferation and signaling including mTOR, PI3K/AKT, and p53. Positive enrichment of the PTEN, FGFR3 and ERK/MAPK pathways were observed. Underlying multiple pathways was promoter hypermethylation with corresponding mRNA downregulation of AKT3 and PIK3CA. Furthermore, promoter hypomethylation with transcript upregulation of PTEN was observed. Next, in comparison of MYD88 WT to MUT samples, a statistically greater number of hypermethylated DMRs were observed across genomic regions (hyper- vs hypomethylated): promoter (24829 vs 2544), 3'UTR (3534 vs 378), 5'UTR (6111 vs 605), intron (52292 vs 5360), exon (21066 vs 2187), CpG island (18334 vs 1551) and CpG shore (16351 vs 1957) (all p<0.001). Assessing mRNA expression, 354 genes were up-/downregulated with concordant promoter hypo-/hypermethylation. Pathway analysis revealed positive enrichment of the mitotic G1 phase to G1/S transition pathway with upregulation of the CDK4, LYN, and PSMA6 transcripts in MYD88 WT compared to MUT samples. Further, negative enrichment of cell signaling regulators including the RHO GTPase family, IL pathways (IL-4, IL-8, IL-13, IL-17) and integrin signaling were found in MYD88 WT compared to MUT samples. Underlying multiple pathways were downregulation with promoter hypermethylation of BCL2, an important regulator of apoptosis. Conclusion Genome-wide methylation analysis of MYD88 WT WM revealed a unique DNA methylation profile, with potential epigenetic alteration of key oncogenic transcripts and pathways. The negative enrichment of cell signaling and cytokine/inflammatory pathways in MYD88 WT samples compared to controls and MYD88 MUT WM suggests an epigenetic mechanism for immune evasion and potentially underlies the reduced response to conventional WM-directed therapies. Overall, this study reveals that MYD88 WT WM is epigenetically distinct from MYD88 MUT WM and regulators such as methylation may underlie the unique clinical and phenotypic properties observed.

Abstract Waldenstrom macroglobulinemia (WM) is a lymphoplasmacytic lymphoma associated with an increase in serum immunoglobulin M (IgM) monoclonal protein. The underlying causes of the disease are still not fully understood, but cells in the bone marrow (BM) microenvironment (TME) are anticipated to play a significant role in disease progression. We previously reported that myeloid-derived suppressor cells (MDSCs), a phenotypically heterogeneous group of cells known for their ability to suppress immune responses, are expanded in the bone marrow of patients with WM and that the expansion of MDSCs is particularly notable for increased numbers of polymorphonuclear MDSCs (PMN-MDSCs). In the present study, we evaluated the ability of WM cells to attract MDSCs to the tumor microenvironment (TME). We also evaluated whether PMN-MDSCs suppress the cytotoxic function of T-cells and thereby promote the survival of WM cells. Our study confirms the expansion of MDSCs, notably PMN-MDSCs in BM of WM patients as compared to normals. We also found that PMN-MDSCs expressing CD66b was a predominant population in WM patients. To determine the phenotype and transcriptome of both PMN-MDSCs and monocytic MDSCs (M-MDSCS) in WM, we compared BM aspirates from WM patients to normal bone marrow (NBM) and performed cellular indexing of transcriptomes and epitopes by sequencing (CITEseq) analysis (WM=3, NBM=2) followed by qPCR validation. We found that MDSCs in WM express significantly higher levels of HIF1-α overall (p<0.02) compared to NBM. Furthermore, PMN-MDSCs are more abundant in WM bone marrow than M-MDSCs and express significantly higher levels of CXCR4 (p<0.001), myeloperoxidase (MPO; p<0.0003), and azuricidin (AZU1; p<0.0004), suggesting an activated phenotype. To determine whether WM cells attract MDSCs to the BM, we performed a transwell assay and showed that WM cells (BCWM.1 cell line) attracted MDSCs, particularly PMN-MDSCs, to a greater extent than M-MDSCs (n=3; p<0.001). We next evaluated whether MDSCs prevent cytotoxic T-cells from lysing WM cells. In-vitro co-culture studies with BCWM.1 cells and T-cytotoxic cells (B:CD8-T) showed that T-cytotoxic cells were able to lyse the WM cell line by 46.4% , (median range 46.4 - 47.8%, p=0.001). However, when BCWM.1 cells and T-cytotoxic cells were co-cultured in the presence of MDSCs (i.e B:CD8-T:M), the specific lysis of BCWM.1 cells was reduced by 12.6% respectively (range 2.2- 12.6%, p<0.001). Furthermore, we observed that B:CD8-T:M co-culture significantly reduced CD8-T cell activation as compared to activated T cells alone (17.9%) (median range 17.8- 18%, p<0.001). In contrast, no affect was observed with B: CD8-T coculture by 91% (range 90.6- 91.4% p<.001) confirming that MDSCs suppress CD8-T cell activation. Our study therefore provides evidence that MDSCs in WM have an activated phenotype and that PMN-MDSCs in WM are attracted by malignant cells in the bone marrow. Furthermore, we find that the increased presence of PMN-MDSCs in the WM bone marrow environment suppresses T-cell lysis of malignant cells and may thereby contribute to disease progression. These findings provide a strong rationale for targeting MDSCs as a potential therapeutic strategy in WM.

ABSTRACT Background Immune exclusion inhibits anti-tumor immunity and response to immunotherapy, but its mechanisms remain poorly defined. In triple-negative breast cancer (TNBC), an aggressive and generally immune-rich subtype, an immune-cold microenvironment predicts poor prognosis due to a limited response to chemotherapy and immune checkpoint inhibitors. This study aimed to identify mechanisms regulating immune infiltration in TNBC. Methods We performed spatial transcriptomic analysis comparing immune-enriched versus immune-cold treatment-naïve TNBCs. Functional analyses, including loss-of-function and reconstitution experiments, were conducted to investigate the role of Trophoblast Cell-Surface Antigen 2 (TROP2), a key target of anti-cancer Antibody Drug Conjugates (ADCs), in promoting TNBC progression. A humanized TROP2 syngeneic TNBC model was used to assess the effects of TROP2-targeting in combination with anti-PD1 therapy. Additionally, data from patients treated with immune checkpoint blockade were used to test hypotheses from the preclinical findings. Results We reveal that TROP2 controls barrier-mediated immune exclusion in TNBC through Claudin 7 association and tight junction regulation. TROP2 expression is inversely correlated with T cell infiltration and predicts poor outcomes in TNBC. We demonstrate that TROP2 is sufficient to drive tumor progression in vivo in a CD8 T cell-dependent manner, while its loss deregulates expression and localization of multiple tight junction proteins, enabling T cell infiltration. We show that TROP2 targeting via hRS7, the antibody component of the ADC Sacituzumab govitecan (SG), enhances the anti-PD1 response and improves T cell accessibility and effector function. Correspondingly, TROP2 expression is highly associated with lack of response to anti-PD1 therapy in human breast cancer. Conclusions This study defines a new mechanism of barrier-mediated immune exclusion in cancer controlled by TROP2-dependent tight junctions. This mechanism drives tumor progression but can be targeted via TROP2-directed therapy to activate anti-tumor immunity and enhance immunotherapy response. What is already known on this topic Barrier-mediated immune exclusion is emerging as a mechanism of immune evasion in cancer such as triple-negative breast cancer (TNBC), but its molecular underpinnings remain poorly understood. TROP2 is a surface glycoprotein targeted by antibody-drug conjugates such as Sacituzumab govitecan, yet its functional role beyond drug delivery has been unclear. What this study adds This study identifies TROP2 as a key regulator of tight junction-mediated immune exclusion in TNBC, independent of its intracellular signaling function. TROP2 promotes an immune-cold tumor microenvironment by enforcing mechanical barriers that limit T cell infiltration. How this study might affect research, practice or policy These findings establish TROP2 as a functional driver of immune evasion and provide a mechanistic rationale for combining TROP2-targeting therapies with immune checkpoint inhibitors to overcome resistance in immune-excluded tumors.

Introduction: Bispecific antibodies (BsAbs) epcoritamab (Epco) and glofitamab (Glofit) are approved in the US for the treatment of patients (pts) with relapsed or refractory (r/r) large B-cell lymphomas (LBCLs) based upon high response rates and manageable toxicity profiles reported in pivotal trials. However, data on outcomes for pts treated with BsAbs outside of clinical trials are limited. As such, we sought to investigate the safety and efficacy of Epco and Glofit for the treatment of pts with r/r LBCLs in real-world settings. Methods: We performed a retrospective study including 19 US centers evaluating pts with r/r LBCLs receiving commercially available Epco or Glofit between 2023 and 2024. Baseline characteristics and outcomes were extracted from medical records. Efficacy outcomes included overall response rate (ORR), complete and partial response (CR, PR) rates, progression-free survival (PFS) and overall survival (OS), analyzed using the Kaplan Meier method. Key safety outcomes included cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Results: As of May 31, 2024, a total of 209 pts with r/r LBCLs were treated with Epco (n=139) or Glofit (n=70). Median age at BsAb start was 67 years (interquartile range [IQR] 58-76). Most were male (62.2%), and the most common histology was diffuse large B-cell lymphoma (n=155 [74.2%]). Of pts with molecular testing (n=168), 34 (20.2%) had double/triple hit lymphoma. Most (78.9%) had Ann Arbor stage III-IV disease and 91% had an ECOG performance status of ≤1. Median number of lines of treatment before BsAb was 3 (range 1-12). Of pts with known response assessment following front-line therapy, 54 (32.1%) had primary refractory disease, defined as failure to achieve PR or CR to front-line therapy. Most pts (n=126 [60.3%]) received prior chimeric antigen receptor (CAR) T-cell therapy and 21 (10.0%) received prior autologous hematopoietic progenitor cell transplant. CD20 expression was assessed in 106 biopsies (50.7%) prior to BsAb and was positive in 90 (84.9%). A total of 172 pts were response-evaluable. The best ORR was 50.6%, with CR in 41 (23.8%), PR in 46 (26.7%), stable disease in 10 (5.8%), and progressive disease in 75 (43.6%). The ORR and CR rates were 49% and 23% for Epco, and 53% and 25% for Glofit, respectively. With a median follow-up of 5 months, the median PFS was 2.7 months (95% CI 2.0, 3.9) and median OS was 7.2 months (95% CI 6.1, not reached [NR]). The estimated PFS and OS at six months were 35% (95% CI 29%-43%) and 59% (95% CI 52%-67%), respectively. For pts who relapsed after CAR T-cell therapy, the median PFS and OS were 2.5 (95% CI 1.8, 4.7) and 7.8 (95% CI 6.2, NR) months, respectively. Pts without detectable CD20 expression via immunohistochemistry or flow cytometry prior to BsAb therapy had significantly inferior outcomes (PFS 1.1 vs 3.4 months, P < 0.001 and OS 1.3 vs 13 months, P < 0.001) compared to pts with detectable CD20. CRS of any grade occurred in 82 pts (39.2%), corresponding to 51.1% and 28.6% of pts treated with Epco and Glofit, respectively. Grade ≥3 CRS occurred in 9 (4.3%), all of whom received Epco. CRS was treated with tocilizumab in 43 pts (53.1%). ICANS occurred in 24 (11.5%) pts; any grade ICANS occurred in 13.7% and 7.1% of pts treated with Epco and Glofit, respectively. Grade ≥3 ICANS occurred in 6 pts (2.9%), 5 of which received Epco (3.6%). Of those experiencing ICANS, 66.0% were treated with corticosteroids. Two pts had deaths attributed to ICANS and one to CRS. A total of 122 (58.4%) pts discontinued therapy after a median of 3 cycles (range 0-48), 91 (74.6%) due to disease progression and 31 (25.4%) due to adverse events (15 due to infection, 3 due to ICANS, 2 due to CRS, and 11 for other reasons). Conclusions: In this heavily pretreated group with adverse disease features and high rate of CAR T exposure, the response rates, PFS, and OS following BsAb therapy were lower compared to pivotal trials. Poor outcomes were observed for those without detectable CD20. Rates of ICANS and grade ≥3 CRS were higher than reported in pivotal trials, as was the rate of discontinuation of therapy due to adverse events. Future efforts to improve outcomes may include intensified dose escalation protocols to more rapidly achieve disease control, combination therapies, and characterization of clinicopathologic factors that predict response to BsAb to identify subgroups of pts who can derive maximal benefit from these agents.

Abstract Background and Aims Artificial intelligence (AI) algorithms in 12-lead electrocardiogram (ECG) provides promising age prediction methods. This study investigated whether the discrepancy between ECG-derived AI-predicted age (AI-ECG age) and chronological age, termed electrocardiographic aging (ECG aging), is associated with atrial fibrillation (AF) risk. Methods An AI-ECG age prediction model was developed using a large-scale dataset (1 533 042 ECGs from 689 639 participants) and validated with six independent and multi-national datasets (737 133 ECGs from 330 794 participants). The AI-ECG age gap was calculated across two South Korean cohorts [mean (standard deviation) follow-up: 4.1 (4.3) years for 111 483 participants and 6.1 (3.8) years for 37 517 participants], one UK cohort [3.0 (1.6) years; 40 973 participants], and one US cohort [12.9 (8.6) years; 90 639 participants]. Participants were classified into two groups: normal group (age gap < 7 years) and ECG-aged group (age gap ≥ 7 years). The predictive capability of ECG aging for new- and early-onset AF risk was assessed. Results The mean AI-ECG ages were 51.9 (16.2), 47.4 (12.5), 68.4 (7.8), and 56.7 (14.6) years with age gaps of .0 (6.8), −.1 (6.0), 4.7 (8.7), and −1.4 (8.9) years in the two South Korean, UK, and US cohorts, respectively. In the ECG-aged group, increased risks of new-onset AF were observed with hazard ratios (95% confidence intervals) of 2.50 (2.24–2.78), 1.89 (1.46–2.43), 1.90 (1.55–2.33), and 1.76 (1.67–1.86) in the two South Korean, UK, and US cohorts, respectively. For early-onset AF, odds ratios were 2.89 (2.47–3.37), 1.94 (1.39–2.70), 1.58 (1.06–2.35), and 1.79 (1.62–1.97) in these cohorts compared with the normal group. Conclusions The AI-derived ECG aging was associated with the risk of new- and early-onset AF, suggesting its potential utility to identify individuals for AF prevention across diverse populations.

Our polyvinyl alcohol feedstock was prepared through carbic anhydride functionalization, UV curing during direct ink writing, and base treatment. The scaffold exhibited mechanical properties similar to pelvic floor tissue.

Importance Typical cysteine-altering NOTCH3 ( NOTCH3 cys ) variants are highly prevalent (approximately 1 in 300 individuals) and are associated with a broad spectrum of small vessel disease (SVD), ranging from early-onset stroke and dementia (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy [CADASIL]) to nonpenetrance. A staging system that captures the full NOTCH3 -SVD severity spectrum is needed and currently lacking. Objective To design a simple disease severity staging system that captures the broad clinicoradiological NOTCH3 -SVD severity spectrum. Design, Setting, and Participants A cohort study was performed in which the NOTCH3 -SVD severity staging system was developed using a discovery cohort (2019-2020) and validated in independent international CADASIL cohorts (1999-2023) and the UK Biobank. Clinical and imaging data were collected from participants originating from 23 international CADASIL cohorts and from the UK Biobank. Eligibility criteria were presence of a NOTCH3 cys variant, availability of brain magnetic resonance imaging, and modified Rankin Scale score. The discovery cohort consisted of 195 NOTCH3 cys -positive cases from families with CADASIL; the validation set included 1713 NOTCH3 cys -positive cases from 15 countries. The UK Biobank cohort consisted of 101 NOTCH3 cys -positive individuals. Data from 2-year (2019-2023) and 18-year (1999-2017) follow-up studies were also analyzed. Data analysis was performed from July 2023 to August 2024. Main Outcomes and Measures Percentage of cases following the sequence of events of the NOTCH3 -SVD stages, and the association between the stages and ischemic stroke, intracerebral hemorrhage, global cognition, processing speed, brain volume, brain microstructural damage, and serum neurofilament light chain (NfL) level. Results The NOTCH3 -SVD staging system encompasses 9 disease stages or substages, ranging from stage 0 (premanifest stage) to stage 4B (end stage). Of all 1908 cases, which included 195 in the discovery cohort (mean [SD] age, 52.4 [12.2] years) and 1713 in the validation cohorts (mean [SD] age, 53.1 [13.0] years), 1789 (94%) followed the sequence of events defined by the NOTCH3 -SVD staging system. The NOTCH3 -SVD stages were associated with neuroimaging outcomes in the NOTCH3 cys -positive cases in the CADASIL cohorts and in the UK Biobank and with cognitive outcomes and serum NfL level in cases from the CADASIL cohorts. The NOTCH3 -SVD staging system captured disease progression and was associated with 18-year survival. Conclusions and Relevance The NOTCH3 -SVD staging system captures the full disease spectrum, from asymptomatic individuals with a NOTCH3 cys variant to patients with end-stage disease. The NOTCH3 -SVD staging system is a simple but effective tool for uniform disease staging in the clinic and in research.

Abstract Systematic multiomic analysis has revealed ancestry-dependent molecular alterations, but their impact on the efficacy of anticancer treatment is yet largely unknown. In this study, we analyzed clinical trials from ClinicalTrials.gov and found that only 8,779/102,721 (8.5%) oncology clinical trials posted information on enrollment by race/ethnicity. The underrepresentation of non-White populations suggests that it remains challenging to determine differences in the efficacy of antitumor treatments among different racial groups. Through a comprehensive analysis of clinically actionable genes, imputed drug responses, and immune features, we identified potential differences in treatment responses to targeted therapy, chemotherapy, and immunotherapy between different ancestral populations. Further analysis of multiple independent cohorts confirmed some of our key findings. Such potential ancestral effects are also identified in response to emerging new treatments like chimeric antigen receptor T-cell therapy and proteolysis-targeting chimeras. These findings are made publicly available in a comprehensive web portal, Ancestral Differences of Efficacy in Cancers (https://hanlaboratory.com/ADEC), to facilitate their further investigation. Significance: Our study charts a global landscape of ancestry-associated differences in therapeutic efficacy, highlighting the importance of considering ancestry in anticancer therapies.

Abstract Rationale Idiopathic pulmonary fibrosis (IPF) is a complex and heterogeneous disease. Given this, we reasoned that differences in genetic profiles may be associated with unique clinical and radiologic features. Computational image analysis, sometimes referred to as radiomics, provides objective, quantitative assessments of radiologic features in subjects with pulmonary fibrosis. Objectives To determine if the genetic risk profile of patients with IPF identifies unique computational imaging phenotypes. Methods Participants with IPF were included in this study if they had genotype data and computed tomography (CT) scans of the chest available for computational image analysis. The extent of lung fibrosis and the likelihood of a usual interstitial pneumonia (UIP) pattern were scored automatically using two separate, previously validated deep learning techniques for CT analysis. UIP pattern was also classified visually by radiologists according to established criteria. Results Among 329 participants with IPF, MUC5B and ZKSCAN1 were independently associated with the deep learning–based UIP score. None of the common variants were associated with fibrosis extent by computational imaging. We did not find an association between MUC5B or ZKSCAN1 and visually assessed UIP pattern. Conclusions Select genetic variants are associated with computer-based classification of UIP on CT in this IPF cohort. Analysis of radiologic features using deep learning may enhance our ability to identify important genotype–phenotype associations in fibrotic lung diseases.