Nat Comm Research Articles

Abstract PO-022: Distinct intratumoral microbiome signatures enrichment in patients with different Anaplaplastic Large Cell Lymphoma (ALCL) subtypes

Abstract Background: The intratumoral microbiome is a tumor-specific component of the tumor microenvironment (TME), including microorganisms localized inside and around cancer cells and immune cells (Nejman Science2020), which was recently added to the “Hallmarks of Cancer” (Hanahan, Cancer Discovery 2022). Specific intratumoral microbiome signatures (IMS) can be detected using our targeted sequencing platform Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) (Prashkin Nat Comm 2023) data through the implementation of a specific bioinformatic pipeline (Vanderbilt J Mol Diagn 2022, Abate Ann Surg 2022). In lymphoma, where antigen stimulation is known to promote lymphomagenesis, studying the intratumoral microbiome might inform lymphoma biology and potentially new treatment strategies. In anaplastic large cell lymphoma (ALCL), a specific histologic phenotype can be found in a very diverse spectrum of diseases, ranging from smoldering lymphomas such as breast-implant associated (BIA-ALCL) and primary cutaneous (pcALCL) to systemic aggressive lymphomas such as ALK-positive or ALK-negative ALCL. We hypothesized that the different IMS might be present in different ALCL clinical presentations. Methods: We used metagenomic techniques to analyze IMS on MSK-IMPACT-HEME as previously described (Vanderbilt J Mol Diagn 2022) of patients with ALCL in our lymphoma database. All patients signed informed consent. We calculated the odds ratio (OR) of each intra-lymphoma IMS in one ALCL subtype compared to that of the same IMS in the other ALCL subtypes. We used several different analyses to process the data for commensals and contaminants. In this regard, we built a database of potentially pathogenic or commensal microorganisms integrated in the decontam (R package) pipeline dataset. For BIA-ALCL, we compared the IMS results with those of known components of explanted textured implant biofilm. Results: We re-analyzed the MSK-IMPACT-HEME BAM files of 58 patients with ALCL, including 12 BIA-ALCL, 11 pcALCL, and 35 systemic ALK+/- ALCL. After reviewing the data for commensals and contaminants, we could identify more than one potentially lymphoma-specific or site-specific microorganism in pcALCL (n=2) and in BIA-ALCL (n=14) (slow-growing subtypes) compared with systemic ALCL. Fewer differences were noted between pcALCL and BIA-ALCL; however, Achromobacter and Rothia were significantly enriched in BIA-ALCL compared to systemic ALCL and pcALCL (OR 14.9 p=0.001; OR=5, p=0.025) and Micromonospora in pcALCL compared to BIA-ALCL and systemic ALCL (OR=5.5, p=0.05). No specific IMS was found in systemic ALCL, as the only potentially enriched IMS (Delftiae) is likely a contaminant. Conclusions: This analysis of IMS using targeted sequencing data, highlighted some possible associations between intratumoral microorganisms and lymphoma-specific subtypes, especially in indolent subtypes of ALCL (BIA- and pc-) compared to systemic ALCL. Further analyses with more cases and confirmatory techniques might confirm these initial findings. Citation Format: Paola Ghione, Horwitz Steven, Peter Cordeiro, Maria Arcila, Natasha Galasso, Ahmet Dogan, Gilles Salles, Chad Vanderbilt. Distinct intratumoral microbiome signatures enrichment in patients with different Anaplaplastic Large Cell Lymphoma (ALCL) subtypes [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-022.

Ex vivo assessment of cellular mass response for personalized therapy selection of hyperthermic intraperitoneal chemotherapy (HIPEC) agents.

3132 Background: Despite progress in the development of HIPEC protocols for the treatment of peritoneal carcinomatosis, agent selection remains largely empiric with few biomarkers for guiding treatment. Ex vivo assessment of drug response using single-cell mass measurements offers a promising option for therapy selection. Previously, we have shown that mass response testing (MRT) is a malignancy- and drug-agnostic means of assessing drug response (1) and can predict a patient’s clinical response to therapy in a range of solid and hematological malignancies (2). Methods: Tissue specimens were collected from patients with peritoneal carcinomatosis as part of an ancillary study for a HIPEC clinical trial (NCT04847063) to explore the feasibility of performing MRT with these specimen types. Briefly, after overnight shipment of viable tumor tissue, tumor cells were isolated and treated with various therapies ex vivo. Single-cell mass distributions of vehicle and drug-treated populations were compared to determine the functional activity of each agent. Results: MRT was attempted for 60 patients with metastatic disease, 10 of whom had peritoneal carcinomatosis and ultimately received HIPEC. The HIPEC cases included histologies such as peritoneal mesothelioma (n=4), colorectal cancer (n=3), ovarian cancer (n=2), and small bowel adenocarcinoma (n=1). In total, 8 out of 10 peritoneal specimens had a sufficiently high tumor cell count, viability, and purity to perform MRT. These specimens displayed clear and statistically distinguishable mass response patterns of response and non-response to several different agents commonly used for HIPEC treatment such as cisplatin, oxaliplatin, doxorubicin, and mitomycin C. Study design:These feasibility results serve as the foundational data for launching a single-site phase II interventional study at the NCI’s Center for Cancer Research. The study will evaluate the potential benefit of personalized treatment regimens determined by MRT for patients with peritoneal carcinomatosis, particularly mesothelioma, a context where therapeutic options have relative equipoise in clinical efficacy. Tumor biopsies will be obtained via laparoscopy prior to HIPEC and submitted to a CLIA-certified lab for MRT to assess the responses of various clinically acceptable agents. The HIPEC regimen will be selected based on these MRT results returned within two days of sample collection. Enrolled patients will then undergo cytoreduction and HIPEC, and progression-free and overall survival will be monitored. As a single-arm, non-randomized trial, patient responses will be compared with a matched synthetic control to assess the impact of MRT on efficacy of HIPEC regimens. Conclusions: MRT is feasible for peritoneal carcinomatosis tissues, with future studies focusing on its role in guiding HIPEC treatment. 1. Nat Comm Bio,2022. 2. JCO PO, 2024. Clinical trial information: NCT04847063 .

Abstract PO2-15-06: ctDNA-based DNADX in hormone receptor-positive and HER2-negative (HR+/HER2-) advanced breast cancer following endocrine therapy and CDK4/6 inhibition: a correlative analysis from the randomized phase 2 PARSIFAL trial

Abstract ctDNA-based DNADX in hormone receptor-positive and HER2-negative (HR+/HER2-) advanced breast cancer following endocrine therapy and CDK4/6 inhibition: a correlative analysis from the randomized phase 2 PARSIFAL trial Background: DNADX, a novel machine learning-based approach, utilizes DNA from tumor tissue or plasma ctDNA to identify clinically relevant phenotypic tumor features and classify breast cancer into 4 subtypes (Nat Comm 2023). Here, we evaluated DNADX's ability to predict prognosis and treatment benefit in HR+/HER2- advanced breast cancer following endocrine therapy and a CDK4/6 inhibitor. Methods: DNADX was evaluated centrally in available baseline plasma ctDNA samples from PARSIFAL trial (NCT02491983) which randomized 486 patients (pts) with HR+/HER2- advanced breast cancer to receive (1:1 ratio) first line palbociclib with either fulvestrant or letrozole. Shallow whole genome sequencing was performed on ctDNA, and the 4 DNA-based subtypes (Clusters-1, -2, -3, and -4) were identified if the ctDNA tumor fraction (TF)≥3%. The main objective was to evaluate the association of DNADX subtypes with progression-free survival (PFS) and overall survival (OS). Secondary objective was to identify the subgroup of pts who benefit more from each endocrine treatment. Uni- and multi-variable Cox regression models were used after adjusting for TF, menopausal status, ECOG status, de novo metastasis (vs. recurrence), visceral disease and number of metastatic sites. Results: DNADX was evaluated in plasma ctDNA samples from 122 pts (25.1%). Clinical variables and median PFS (27.6 months) were similar as the overall PARSIFAL population. DNADX identified 56.6% pts with TF of < 3%, 14.8% with Cluster-1, 19.7% with Cluster-2, 5.7% with Cluster-3 and 3.3% with Cluster-4. In terms of PFS, pts classified with TF< 3% had a lower risk of progression compared to Cluster-1, Cluster-2, Cluster-3, Cluster-4 subtypes (pairwise PFS hazard ratios [HRs] of 1.88, 2.02, 3.15, and 5.62, respectively, with a global log-rank test of p=0.010). Similar results were obtained after adjusting for other clinical-pathologic variables. A numerical benefit of fulvestrant in comparison with letrozole was observed in pts classified in Cluster-1 and Cluster-4 (HR=0.42, 95% CI 0.14-1.24) in contrast to the other groups (HR=1.21, 95% CI 0.68-2.18), and the interaction test was statistically significant after adjusting for clinical-pathologic variables (anova p-value=0.037). In terms of OS, pts classified with TF< 3% had a lower risk of death compared to Cluster-1, Cluster-2, Cluster-3, and Cluster-4 subtypes (pairwise OS HRs of 1.90, 4.23, 11.13, and 6.80, respectively, with a global log-rank test of p=0.003). In the multivariable analysis, results were consistent after adjusting for clinical-pathologic variables and TF. Conclusions: Liquid biopsy-based DNADX subtypes predict outcomes in pts with HR+/HER2- advanced breast cancer on endocrine therapy and CDK4/6 inhibitors, potentially identifying the most optimal endocrine treatment for each pt. Citation Format: Fara Brasó-Maristany, Javier Cortés, José Manuel Pérez-García, Rosario Vega-León, Laia Paré, Guillermo Villacampa, Judit Matito, Francisco Pardo, Marina Gomez-Rey, Mario Mancino, Elena Martínez-García, Carmen Mora Gallardo, Leonardo Mina, Florence Dalenc, Meritxell Bellet- Ezquerra, Manuel Ruíz - Borrego, Miguel Gil-Gil, Peter Schmid, Charles M. Perou, Joel S. Parker, Patricia Villagrasa, Ana Vivancos, Aleix Prat, Antonio Llombart-Cussac. ctDNA-based DNADX in hormone receptor-positive and HER2-negative (HR+/HER2-) advanced breast cancer following endocrine therapy and CDK4/6 inhibition: a correlative analysis from the randomized phase 2 PARSIFAL trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-15-06.

MITOCHONDRIAL DYSFUNCTION CAUSES CECAL PATCH (CP) INFLAMMATION IN ULCERATIVE COLITIS

Abstract BACKGROUND Up to 17% of distal ulcerative colitis (UC) patients have discontinuous (proximal) inflammation referred to as Cecal Patch (CP), associated with greater abdominal pain, bleeding and diarrhea. Despite these known associations, the pathogenesis of CP lesions is poorly understood. Recent data suggest that reduced mitochondrial (Mito) function predicts unfavorable clinical outcomes in active UC (Haberman et al; Nat Comm 2019). We posit that inflammation and tissue injury induced in UC-associated CP involves Mito dysfunction. METHOD UC patients with/without cecal disease and normal controls were biopsied. Tissue from CP lesions or normal caecum were collected in Allprotect® (AP) and formalin. Total RNA from AP-preserved biopsies were used for cDNA synthesis and RT-qPCR for Mito complexes, cytokines, anti-oxidant and calcineurin (CaN) signaling genes and tissue processed for IHC for Mito complexes. RESULT Transcriptomic analysis showed significant downregulation in genes associated with Mito biogenesis (PGC1α, TFAM, NRF2) and Mito complexes (Ndufa1, Ndufa4, Ndufb2, Ndufb6, mtCO1, mtCO2, Cox5B, Cox6A1, ATP5A1, ATP5B, ATP5E) in CP lesions compared to normal caecum from active patients. Major regulators of Mito biogenesis (PGC1α and TFAM) revealed ~39% and ~24% reductions of mRNA respectively, while NRF2 (anti-oxidant regulator) showed ~62% reduced mRNA in CP lesions compared to normal cecum from distal UC patients. Electron transport chain complex mRNAs showed 31%, 47%, and 48% reduction in complex I (Ndufa1, Ndufa4, Ndufb2, Ndufb6), complex IV (mtCO1, mtCO2, Cox5B, Cox6A1), and complex V (ATP5A1, ATP5B, ATP5E) levels, respectively in CP lesions compared to normal controls. Concomitantly, the IHC staining also showed significant reductions in Ndufb6, MTCO1 and Cox5A protein levels in CP lesions compared to normal cecum sections. The CP-related decreased markers of Mito biogenesis and function correlates with increased proinflammatory cytokine mRNA (IL-6: 2-fold; TGFβ1: 1.6-fold; IL-2: 2.6-fold) and decreased anti-inflammatory (IL10: 3.2-fold) gene expression in CP lesions. Further investigation demonstrated that repressed Mito function in CP lesions is associated with downregulation of anti-oxidant genes (SOD1, SOD2, Prdx3, Gpx4, Trx2) compared to normal cecum. Inflammation-induced Mito dysfunction in CP lesions also stimulates CaN signaling as demonstrated by significant increases in mRNA levels of CaN and downstream regulated genes (Cathepsin-L, Rel-A Glut4, CREB-BP and Igf-1r). CONCLUSION These data represent the first indication that Mito dysfunction occurs in CP associated with distal UC. Furthermore, we speculate that Mito dysfunction-induced CaN signaling promotes regional (CP) inflammatory responses. These important findings indicate that targeting Mito metabolism may be an important therapeutic goal of treating UC patients with CP.

Epigenetic Age Acceleration in Hematological Malignancies: Beyond Chronological Age, Clinical Implications, and Therapeutic Perspectives

Background: Age is important in prognosticating hematological malignancies. Biological based on DNA methylome changes offers new insights into the complex interplay between epigenetics, genetics, environment, and cancer. DNA methylation levels at specific CpG sites evolve in a predictable fashion with aging and can be used to develop biomarkers that estimate the epigenetic age (EA) of a tissue. Such biomarkers are called epigenetic clocks (Fig 1-B). Based on the EA, we can determine Epigenetic Age Acceleration (EAA), a tool to identify patients aging faster biologically than their chronological age. We sought to build a blood-based clock to predict epigenetic age in healthy young and old adults and subsequently assess the degree of EAA in AML patients. Finally, among multiple planned downstream applications, we propose to use EAA as a biomarker for monitoring treatment responses in targeted therapies (Fig 1-A). Methods: We used ClockBase and NCBI's Gene Expression Omnibus (GEO) repositories to collect methylation data based on Illumina's 450k array for healthy adults and patients with AML/MDS. Pre-processing was done using the minifi BioConductor package. We used a cohort of healthy young adults (YA, aged 18-30 ys.) to train our epigenetic clock and calibrate it with three well-studied epigenetic clocks: Horvath clock (HC), Hannum clock (HAC), and PhenoAge (PA). We hypothesized that EA and chronological ages (CA) should approximate each other in younger patients as they do not generally carry comorbidities or have a higher CHIP prevalence which could influence EA. We compared our clock's performance to known epigenetic clocks, and after fine-tuning, all 4 models were validated in a cohort of healthy older adults (OA, aged 70-85 ys.) with a higher risk of CHIP. Then, we tested our clock in an AML patient cohort and illustrated how EAA can predict therapy responses in AML patients treated with IDH1/2 inhibitors (Published methylation data from Wang et al 2021, Nat Comm.) Results: We designed the clock using an Elastic-Net regression scheme. We screened 130,000 patient samples from GEO and 1,813 eligible samples were divided into 3 cohorts: 800 healthy younger adults (aged 18-30 ys.), 800 healthy elderly adults (aged 70-85 ys.), and 213 patients with AML. To train and test our clock, we used the YA cohort (n=800) with a median age of 20 ys. (SD 3.168 ys) and our epigenetic clock predicted a median EA (MEA) of 19.1 ys. (SD 3.4 ys). HC, HAC and PA predicted MEA of 21.6 ys. (SD 3.9 ys.), 19.4 ys. (SD 4.1 ys.) and 21.5 ys. (SD 3.9 ys.). After fine-tuning of weights, we tested our clock in OA cohort (n=800) with a median age of 78 ys. (SD 4.8 ys.). Our clock predicted a MEA of 82.3 ys. (SD 4.9 ys.). HC, HAC and PA predicted a MEA of 83.7 ys. (SD 5.7 ys.), 85.2 ys. (SD 6.0 ys.) and 84.9 ys. (SD of 5.1 ys.) resp. The AML cohort (n=213) had a median age of 80.9 ys. (SD 2.5 ys.). We expected a significantly higher EA due to the mutation burden along with genomic and epigenomic changes induced by the disease, our clock predicted a MEA of 84.7 ys. (SD 3.0 ys.). EAA was calculated from the difference between EA and CA. YA cohort had a median EAA of 0.9 ys. (SD 1.8 ys.) and our clock predicted a median EAA of 1.1 ys. (SD 2.0 ys.). HC, HAC, and PA predicted a median EAA of 1.4 ys. (SD 2.3 ys.), 1.4 ys. (SD 2.9 ys.), and 1.5 ys. (SD 2.4 ys.). In the OA cohort, we expected higher EAA due to age-associated cardiometabolic disease and CHIP; our clock predicted a median EAA of 4.2 (SD 2.1). HC, HAC, and PA predicted a median EAA of 6.0 ys. (SD 2.7), 5.6 ys. (SD 3.0 ys.), and 5.1 ys. (SD 2.2 ys.). For the AML cohort, our clock predicted a median EAA of 8.0 ys. (SD 5.6). We then applied our clock to a cohort of 60 AML patients treated with IDH inhibitors to predict whether a patient cleared the IDH clone after therapy. We discovered that patients who failed to clear the IDH-clone (n=14) had a higher EAA (3.23 vs 7.91 ys., p=0.012) and shorter RFS compared to patients who cleared the IDH-clone (n=7) [RFS 20.6 vs 6.1 mo., p=0.0086]. Conclusion: We built an epigenetic clock that outperforms three standard clocks in predicting epigenetic age and applied it to AML patients demonstrating a significant increase in epigenetic age. We also illustrated how EAA can be used as a biomarker for targeted therapies. Ongoing work at Karmanos Cancer Center is focused on improving the underlying regression model for more sensitive epigenetic age predictions and implementing novel pace-of-aging algorithms for EAA calculations.

Genomic Profiling to Interpret the Outcomes of Early Intervention for High-Risk Smoldering Myeloma

Introduction Early intervention for High-Risk Smoldering Multiple Myeloma (HR-SMM) achieves deeper and more prolonged responses compared to Newly Diagnosed (ND) MM. Various clinical risk models estimate the risk of SMM progression to MM but there is significant discordance between them (Hill et al., JAMA Onc. 2021). It is unclear if beneficial outcomes of interventional studies in HR-SMM are due to treatment of less complex, susceptible disease or inaccuracy in clinical definition of cases entered. Methods To gain greater biologic insight into treatment outcomes, we performed the first whole genome sequencing (WGS) analysis of treated HR-SMM for 27 patients treated with carfilzomib, lenalidomide, and dexamethasone (KRd) and R maintenance (NCT01572480, presented in parallel). We pooled genomic features from 27 patients with HR-SMM treated with Elotuzumab (Elo)R+/-d; (E-PRISM). Genomic features were compared to those of 701 patients with NDMM from CoMMpass (NCT01454297). Results After a median follow-up of 52.8 months, median PFS was not reached with KRd/R. After 8 cycles of KRd, 19 (70.3%) achieved minimal residual disease (MRD) negativity (LOD 10 -5). At data cutoff, June 13, 2023, 14 patients (51.9%) achieved sustained MRD-negativity, 6 patients (22.2%) lost an initial MRD-negative response and 5 patients clinically progressed (18.5%). Overall, there was discordance between risk models: 3 patients (11.1%) were HR by Mayo2008 criteria, 14 (51.9%) by Mayo 20/2/20, 18 (66.7%) by PETHEMA, and 21 (77.8%) by Rajkumar/Landgren/Mateos criteria (Rajkumar et al., Blood. 2015). Eighteen (66.7%) met criteria for 2 or more scores. The estimated 5-year risk of progression ranged 4.8 to 82.1% (Pangea, median 18.6%). We compared the pooled HR-SMM to NDMM from CoMMpass. The frequency of HR translocations was similar (t(4;14), t(14;16), t(14;20); p>0.05). Consistent with the early disease stage of SMM, mutations of NRAS were lower in SMM (p = <0.001) as were events at the MYC locus (8q24; p <0.001) and gains of 1q (p = 0.039). Next, tumor suppressor genes (TSG) were interrogated together with copy number loss at their loci. Consistent with their late onset in tumor evolution, aberrations at key TSG were less common in HR-SMM (p < 0.05): CDKN2C, CYLD, TENT5C, FUBP1, MAX, NCOR1, NF1, NFKBIA, PRDM1, RB1, RPL5,and TRAF3 (p < 0.05). In a genome-corrected comparison, APOBEC (SBS2+SBS13) mutational signatures were diminished in KRd WGS compared to 60 Dara-KRd-treated NDMM (Maura et al., ASH. 2021; 48% vs 87%, p < 0.001) and in E-PRISM vs CoMMpass (15% vs 45%, p = 0.001). We next related genomic features associated with HR-SMM to treatment outcomes. Patients treated with KRd/R had yearly MRD testing and gain1q, MYC dysregulation via loss of MAX, and t(4:14) were all associated with failure to sustain MRD-negativity. Across pooled HR-SMM, inactivation of CYLD, CREBBP, MAX, and HIST1H2BK; t(4;14), APOBEC expression, loss at select GISTIC peaks ( Fig 1A) and chromothripsis all were associated with HR-SMM progression in the face of triplet therapy (p<0.05). Presence of any one or more of these features was associated with progression (p = 0.005; Fig1B). Conversely, no clinical risk score was able to discern those with this molecular high risk. Conclusion In patients treated on 2 parallel clinical trials for HR-SMM, we found a uniform and relative genomic simplicity. Moreover, non-progressors appear genomically similar to patients with non-progressive/stable MGUS and SMM under observation (Oben et al., Nat Comm 2021). However, within clinical HR-SMM, a set of high-risk genomic features portends progression despite intervention. These results suggest that clinical risk scores do not effectively discriminate between genomically indolent and aggressive disease. Though possible that results reflect treatment before the acquisition of key drivers, prior data suggest that many of these high-risk features are not acquired within the 5 years preceding clinical diagnosis of MM (Bolli et al., Nat Comm. 2018; Bustoros et al., J Clin Onc. 2020). Altogether, these results support the use of genomics to contextualize the advantage of early intervention in SMM (i.e., to avoid overtreatment of non-progressors and to better identify cases likely to progress without therapy). Fig1A: Heatmap of High-Risk Features in Patients treated with KRD/R and EPRISM. Fig1B: Kaplan-Meier curve for time to progression by presence of HR features for KRD/R.

Comprehensive Genomic Characterization of Response and Resistance to Daratumumab-Based Quadruplet Induction in Newly Diagnosed Multiple Myeloma Patients

Introduction: Current prognostic scores for newly diagnosed multiple myeloma (NDMM) rely on measures of tumor bulk and limited genomic information. A recent artificial-intelligence-based model suggested that individualized prognostication and prediction of response to specific therapies may be improved by more comprehensive genomic assessment (IRMMa, Maura et al Blood 2022). The 20 significant genomic contributors included APOBEC mutational activity, gain/amp1q, TP53 aberration, and loss of any of CDKN2C, RPL5, FUBP1, RB1, RASA2, 10q23 or 12p13. The highest impact on progression-free (PFS) and overall survival (OS) came from copy number (CN) signature profiles suggesting the structural variant chromothripsis (Maclachlan et al Nat Comm 2021). We have previously demonstrated that CN features consistent with chromothripsis can be detected in targeted sequencing data, potentially increasing clinical application (Maclachlan el at Blood 2020). One limitation of the recently presented IRMMa model was an absence of patients prescribed daratumumab-based quadruplet induction, (dara-quads; daratumumab, lenalidomide, dexamethasone with carfilzomib [DKRd] or bortezomib [DVRd]). Methods: We applied comprehensive genomic assessment to a cohort of 234 NDMM patients from Memorial Sloan Kettering Cancer Center initiated on dara-quad induction prior to May 2023. Genomic data included FISH (n=215), SNP-array (n=127), targeted sequencing (MSK-IMPACT-Heme, n=78) and whole genome sequencing (WGS, n=45). A further 80 samples are undergoing MSK-IMPACT-Heme (n=25) and WGS (n=55) currently. Results: Among 234 patients analyzed, 86 patients received DKRd and 148 DVRd. Overall median follow-up was 1.4 years (y); 4.2y with DKRd (IQR 1.9-4.7, max 5.7) and 1y with DVRd (IQR 0.5-1.4, max 3.4). 25 patients progressed following DKRd and 20 following DVRd, with 9 progressing during induction chemotherapy. ISS stage did not predict for PFS or OS, either in the whole cohort, or in individual regimens. The inclusion of genomic assessment improved prognostication; while R-ISS, high-risk cytogenetics as per R-ISS, and R2-ISS each improved PFS prediction (p<0.01), consideration of every feature assessable by FISH (including del1p, amp1q, t(14;20), MYC-translocations) further increased discrimination (p=0.009). Assessing as per the Master study (Costa JCO 2022), 2+ co-occurring high-risk features predicted for shorter PFS when compared with 1 or 0 risk factors (p=0.002). Within the 9 who progressed during induction, 4 were ISS III, 6 had a risk factor evident via FISH, and 4 had multiple high-risk features, while 4 had no risk factors by standard methods. Using comprehensive genomic assessment (SNP-array, MSK-IMPACT-Heme and WGS) allowed for the assessment of additional genomic features in those with adequate follow up (predominantly DKRd). Del1p22.1 ( RPL5) was strongly predictive of shorter PFS (p<0.0001), as were del22q12.1 ( XBP1, p=0.02) and high APOBEC-mutational activity (p=0.01). Additional features demonstrated a trend to significance, including del1p12 ( TENT5C, p=0.06) and the complex structural variant chromothripsis. Parallel performance of MSK-IMPACT-Heme and WGS on the same samples allowed demonstration that CN signatures detected by targeted sequencing were accurate indications of complex chromothripsis events. The expanded WGS cohort will further define sensitivity and specificity of this detection. Discussion: In the context of highly effective dara-quad induction, ISS stage did not predict for either PFS or OS. Limited cytogenetic assessment improved prognostication, while advanced genomic assessment was highly discriminatory. The current study provides the rationale for more comprehensive genomic assessment in patients receiving dara-quads in order to refine prognostication and consider novel treatment approaches for high-risk patients. We are significantly expanding our cohort of WGS in dara-quad-treated patients, formalizing the accuracy for CN signature detection of chromothripsis from targeted sequencing data, allowing application in routine clinical practice.

MODL-16. GLIOBLASTOMA GROWTH IS SHAPED BY INVASION ROUTE-SPECIFIC FUNCTIONAL SIGNATURES

Abstract One of the defining features of glioblastomas (GBMs) is the capacity for invasive growth along multiple anatomical pathways in the brain. GBM is well-studied on a genetic and molecular level, but clinically relevant and experimentally tractable models of invasive growth are largely lacking. Here, we report an integrated study of patient-matched information, genomic- and molecular profiles with growth in mouse brains to expose treatments and biomarkers associated with glioblastoma invasion and recurrence. In total, 64 patient-derived cell lines (PDCLs) were injected into the striatum of n ≥ 4 mice each. The 45 tumor-forming PDCLs were each scored for 10 distinct growth characteristics (n = 182 mice). The repertoire of phenotypes was highly divergent, and our material included clear cases of perivascular route invasion, white matter route invasion, perineuronal satellitosis, and gliosarcoma. We explored if cellular pathways, monitored by RNA-sequencing, could account for these differences. GSEA highlighted a positive enrichment for highly proliferative proneural tumors characterized by Notch activation, neuronal signaling, and epigenetic gene regulatory programs in the tumor-initiating lines. Transcriptional signatures were also strongly predictive of route-specific invasion. Diffuse invasion was predominantly seen in classical-subtype PDCLs with astrocytic or outer radial glia-like signatures. Proneural PDCLs, in turn, grew as solid tumors with an invasive peripheral region around vasculature, and mesenchymal tumors were more demarcated. To explore the therapeutic implications of our findings, we used our data-driven method (TargetTranslator, Nat Comm 2020) to predict the drug vulnerabilities of different types of invasive glioblastoma. Defined GBM tumors with perivascular invasion are characterized by increased IGFR1, MAPK/ERK, PI3K/AKT/mTOR, and JAK2 signaling. Diffusively growing GBM tumors, on the other hand, depend more on Wnt/β-catenin signaling, neuronal signaling, and active inflammatory response. Using a sphere invasion assay, we confirm that targeting both PI3K- and Wnt signaling selectively reduces glioblastoma invasion, highlighting their therapeutic potential.

Phase 1/2 Study of Pacritinib in Combination with Azacitidine in Chronic Myelomonocytic Leukemia

Introduction and preclinical rationale Treatment options for chronic myelomonocytic leukemia (CMML) are limited. Hypomethylating agents (HMAs) are the only approved therapies but are associated with unsatisfactory response rates and do not impact disease evolution (Merlevede Nat Comm 2016). JAK inhibition can reduce GM-CSF signaling, which contributes to CMML pathobiology; ruxolitinib has been clinically evaluated in CMML demonstrating promising, but insufficient clinical activity. Pacritinib is a JAK inhibitor that has a unique profile which suggests it may be uniquely well suited for the treatment of CMML, including targeting of CSF1R and IRAK1, both of which are upregulated in CMML and related diseases (Obba Autophagy 2015, Rhyasen Cancer Cell 2013). Additionally, pacritinib was recently shown to be a potent inhibitor of ACVR1 (Oh AJH 2023), potentially allowing for increased iron availability and improvement in anemia. Pacritinib has been evaluated preclinically in CMML primary samples and mouse models where it demonstrated significant, albeit heterogenous activity (Yoshimi Blood 2017). To enhance the activity of pacritinib in CMML, it was also evaluated in combination with azacitidine in GM-CSF hypersensitive cell lines and primary CMML cells where profound synergy was noted (Ma ASH 2015). Based on encouraging preclinical data, we hypothesize that the combination of pacritinib with azacitidine will be an effective treatment strategy in CMML patients. Methods In this phase 1/2 trial, we will evaluate the safety and efficacy of pacritinib and azacitidine in newly diagnosed or previously treated CMML. Patients must be JAK inhibitor and HMA naïve (≤1 cycle of HMA allowed). Phase 1 will follow a ‘3+3‘ dose de-escalation design and will enroll 6-12 patients. The primary objective of this phase is to determine the recommended phase 2 dose (RP2D) of pacritinib in combination with a fixed dose of azacitidine. Pacritinib will be administered at 200mg twice daily (with a lower dose of 100mg twice daily being explored in the case of dose limiting toxicity) in combination with azacitidine 75mg/m 2 for 7 days. Phase 2 will utilize a Simon's two stage design to assess the preliminary efficacy of pacritinib in combination with azacitidine at the RP2D. In the first stage, after treating 6 patients at the RP2D, there will be an interim response assessment as part of the phase 2 portion. If at least 4 among these 6 patients show a response, the trial will proceed to a second stage and enroll an additional 12 patients. Patients will be treated for 6 cycles of 28 days each with an interim bone marrow biopsy after cycle 3. The primary endpoint of the phase 1 portion is the recommended RP2D through the assessment of dose limiting toxicities and of the phase 2 portion is the overall response rate (ORR) by the end of cycle 6 as defined by a clinical benefit or greater by myelodysplastic syndrome/myeloproliferative neoplasm International Working Group response criteria (Savona 2015). Secondary endpoints include duration of response, disease free survival, overall survival, changes in MPN-SAF total symptom scores, palpable splenomegaly, and adverse events. Several correlative studies are planned as part of this clinical trial. Modulation of biomarkers of disease activity with treatment including GM-CSF dependent STAT5 phosphorylation, MAPK, and PI3K signaling will be analyzed. We will also evaluate changes in mutational and cytogenetic burden with treatment as well as at baseline to identify predictive biomarkers of response.

Radchip: A Laminin-Lined Red Blood Cell (RBC) Adhesion Device for Functional Characterization and Clinical Evaluation of Sickle RBCs at Steady-State

Sickle cell disease (SCD) is a genetic disorder caused by a point mutation in the beta-globin gene producing an abnormal hemoglobin (HbS) which, under deoxygenation, polymerizes and causes severe distortion of the red blood cell (RBC). RBCs may be dehydrated, poorly deformable and highly adhesive to the endothelium, with abnormal adhesion being of primary interest to this study. Recently, several new disease modifying drugs have been FDA approved, with more in development. These novel agents target specific aspects of SCD pathophysiology, including adhesion. Changes induced by such therapies are not always detectable by common conventional lab instruments. LORRCA (oxygen-gradient ektacytometry) is the gold-standard for deformability measurement; it has been well described both analytically and clinically in SCD. Adhesion and deformability have a dynamic, yet complementary relationship that we explore here. While these may behave in an interdependent manner, each determinant must be considered independently. There is a growing demand for innovative tools to evaluate rheological properties under physiological conditions, particularly microfluidic devices. However, many current devices are limited in their clinical utility due to, choice of ligand, complexity of design, or reliance on endothelial cell culture (typically HUVEC cells) which are difficult to use, vary with passage number, and do not represent adult SCD endothelium. We developed a Red blood cell Adhesion Chip (RAdChip) to model RBC to endothelial adhesion using laminin, a physiologically relevant ligand, to easily, effectively, and reproducibly measure RBC adhesion. Here, we describe our analytic and clinical validation of our device. Peripheral blood samples were collected in EDTA from individuals with SCD and healthy controls under an IRB approved protocol. RBCs were extracted via centrifugation, washed and resuspended in PBS to a 0.5% HCT. Microfluidic devices were fabricated using polydimethylsiloxane (PDMS). A 5ug/ml solution of recombinant laminin was applied to each channel, then blocked with 1% bovine serum albumin (BSA). Resuspended RBCs were introduced to the device through tubing connected to the inlet port. Harvard Apparatus syringe pumps were used to perfuse samples and wash channels at ~1dyne/cm 2. Adhesion was visualized with a Keyence inverted bright-field microscope at 20x (Itasca, IL). Adhered cells were counted within a 1.8mm 2 region of interest (Roi) using the iCLOTS automated cell quantification software (Fay et al, Nat comm accepted 7/2023). We measured adhesion index via RAdChip for common SCD genotypes: HbSS/SB0: (median (x͂) =756; range (r): 154-1889; n=26) HbSC/SB+ : (x͂ =299; r=: 114-731; n=11 ) HbAA: (x͂) =13; r= 5-90; n=7). RBC adhesion values were independent of age (p=0.65) and gender (p=0.63). An increase of 0.1 unit in EImin and EImax decreases RBC adhesion by 214 and 268 cells/mm 2, respectively. A 1mmHg increase in PoS increased adhesion by 19.5 cells/mm 2. sRBC adhesion had a significant positive association with vaso-occlusive events (VOE) (p=0.005), absolute neutrophil count (ANC) (p=0.005), and EImax (p<0.001). Notably, analytical validation of RAdChip produced a mean CV of 0.13 ± 0.94. RAdChip adds a key component of functional RBC evaluation that conventional lab equipment does not measure. We have analytically validated our device, demonstrating an excellent coefficient of variability. 44 patient samples, each examined in triplicate produced a mean CV of 0.13. We have demonstrated clinical validity of our device by establishing correlations with known markers of severity and pain event frequency. RBC adhesion values increase as the number of VOE events increase, reaffirming the belief that adhesion is a principal element in the multistep VOE cascade. In like manner, improvements in deformability are associated with decreased RBC adhesion, strengthening the interdependent nature of VOE molecular events. Recognizing the dynamic nature of SCD, it is necessary to explore sickle erythrocyte adhesion longitudinally to investigate changes before, during, and after VOE occurrences. Continued and increased integration of adhesion measurements with RAdChip and other rheology devices into routine SCD care may be helpful in assessing disease severity, identifying need for additional disease modifying agents, and assist with second line agent selection and monitoring.

MOSAIC: Multi-Omic Spatial Atlas in Cancer, effect on precision oncology.

e15076 Background: Precision oncology aims to first break diagnoses into biologically distinct subtypes, then pursue personalized therapies for each group of patients ( Garraway et al, J Clin Onc, 2013). This strategy is enabled by recent advances in technologies for medical imaging, molecular profiling, and artificial intelligence. Spatially resolved molecular profiling (“spatial omics”) is an emerging technology that harnesses all three of these advances ( Navarro et al, Science, 2016; Rao et al, Nature, 2021). Cancer is driven by localized interactions between tumor cells, immune and non-immune stromal components. Methods: Spatial omics studies have revealed specific cell types and pathways that control those tumor-microenvironment crosstalks ( Hunter et al, Nat Comm, 2021; Alon et al, Science, 2021), involving both innate and adaptive immunity ( Binnewies et al, Nat Med, 2018). Identifying new cancer subtypes based on the spatially localized tumor-host interactions across many patients have the potential to revolutionize immuno-oncology. The advances in utilizing AI-based methods have so far been unattainable due to a small number of patients in spatial omics studies. Results: Here we present the MOSAIC project - a collaborative initiative across industry and top oncology hospitals to build the largest collection of spatial omics data in cancer. We combine comprehensive clinical annotation with new deep profiling methods to both discover cancer subtypes and identify drug targets and biomarkers within them. MOSAIC aims to generate multimodal data for a total of 7,000 patients in seven cancer indications. Data modalities will include spatial and single cell transcriptomics and proteomics, bulk molecular profiling, pathology images, and curated clinical information. This collaboration harnesses the strengths of academia and industry to provide patient samples, generate high-quality data, develop AI-based analytical tools, and compile a resource that will eventually be made widely available for medical research. Conclusions: Here we present the workflow of the MOSAIC study, demonstrate the data processing pipelines and show spatial distributions of transcripts within the tumor microenvironment. Building upon these initial datasets, we discuss how the MOSAIC parties will collaborate to build an unprecedented database for research and discovery of new immuno-oncology therapeutic approaches.

Assessing tumor-infiltrating immune cells and outcomes with cisplatin- versus carboplatin-based treatment in advanced urothelial cancer (UC): Survival analysis of three platinum-treated UC cohorts.

4583 Background: The association between tumor-infiltrating immune cells and the response to platinum-based chemotherapy (cisplatin vs carboplatin) in advanced urothelial cancer is not well characterized. We dissected the immune microenvironment of 3 large UC cohorts to define populations of immune cells associated with survival following carboplatin- or cisplatin-based chemotherapy. Methods: Two advanced UC cohorts – Hospital del Mar (HM; n = 55) and GREEK (n = 62) – treated with platinum-based chemotherapy were profiled using bulk RNA sequencing (RNA-seq) with tumor-infiltrating immune cells inferred using CIBERSORTx. Our findings were validated in a publicly available platinum-treated UC cohort (n = 93; Taber et al. Nat Comm 2020). Additionally, we assessed the impact of tumor-infiltrating immune cells in IMvigor210, an anti-PD-L1 treated cohort. Analysis was adjusted by tumor stage, and treatment regimen (cisplatin, carboplatin, atezolizumab) to determine associations between tumor-immune cell populations and overall survival (OS). Results: In platinum-treated patients, increased lymphoid cell infiltration was associated with improved OS. Specifically, the presence of naive and memory B-cells and CD4+ T cells was associated with prolonged OS (Table). Interestingly, infiltration of memory B-cells was associated with a better prognosis in patients treated with cisplatin (HR = 0.14) and detrimental in patients treated with carboplatin (HR = 249.53; difference p = 0.006). Additionally, macrophages (HR = 3.44; p = 0.027) and resting dendritic cell (HR = 41.47; p = 0.009) infiltration was associated with worse OS in patients treated with platinum therapy alone, but enrichment of pro-inflammatory M1 macrophages was associated with prolonged OS in patients treated with immunotherapy (p < 0.001). Conclusions: Immune cell infiltration is associated with differential outcomes to cisplatin- and carboplatin-based chemotherapy in advanced UC. Our findings suggest better OS in advanced UC treated with cisplatin in tumors enriched with memory B-cells compared to carboplatin treatment. [Table: see text]

Expanded Reference Tissue Methylome for Tissue-of-origin Deconvolution of Cell-free DNA in Plasma from ALS Patients (P11-8.014)

<h3>Objective:</h3> To depict systemic cell death from plasma cell-free DNA (cfDNA) from ALS patients with an expanded reference tissue methylome dataset. <h3>Background:</h3> CfDNA is fragments of DNA released by dying cells with a half-life of minutes to 2 hours in plasma that has been utilized for non-invasive prenatal testing and cancer detection. Cell-type-specific methylation of DNA can serve as a marker for the cell-of-origin of cfDNA. Caggiano et al. developed a machine-learning algorithm for tissue-of-origin deconvolution of cfDNA, CelFiE, and reported that muscle-derived cfDNA is significantly elevated in plasma of ALS patients than controls with 19 reference tissues excluding nervous tissues (Caggiano et al, <i>Nat Comm</i>, 2020). <h3>Design/Methods:</h3> Reference tissue methylome was expanded to 39 tissues with multiple immune cells and brain-derived NeuN positive and negative cells. Plasma cfDNA methylome of ALS patients (n=16) and controls (n=16) was re-analyzed with CelFiE and the expanded reference tissue methylome, using the tissue identification markers (TIMs) deduced by the original 19 reference tissues. Estimated cfDNA origins were visualized along with the clinical variables (sex, ethnicity, region of onset, days from the date of onset, and ALSFRS-R) for further limited statistical testing. <h3>Results:</h3> Expanded reference methylome with the original TIMs replicated the elevated muscle-derived cfDNA in ALS patients. Brain-derived cfDNA did not differ between ALS and control. Natural killer cells and eosinophils-derived cfDNA were significantly decreased in ALS than control. Neutrophil-derived cfDNA was significantly decreased in patients with ALSFRS-R ≤31 compared to patients with ALSFRS-R &gt;31. <h3>Conclusions:</h3> CfDNA can depict systemic cell death, even for tissues that have not been studied in the context of ALS and that are otherwise not detectable (e.g., hematopoietic stem cells). To apply cfDNA to biomarker and pathophysiology research for ALS, further study is needed to improve the resolution of deconvolution algorithms with alike tissues, and cost-effective sequencing methods. <b>Disclosure:</b> Dr. Yamakawa has nothing to disclose. Miss Caggiano has nothing to disclose. The institution of Prof. Zaitlen has received research support from NIH. Dr. Rexach has nothing to disclose.

Abstract P5-02-31: Genetic determinants of response to patritumab deruxtecan (HER3-DXd) in hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer: a correlative analysis from SOLTI TOT-HER3 trial

Abstract Background: Baseline HER3 protein or ERBB3 mRNA levels do not seem to predict efficacy from HER3-DXd in early-stage and advanced HR+/HER2- breast cancer (Prat et al. ESMO Breast 2022; Krop et al. ASCO 2022). Here, we evaluated potential baseline pre-treatment genetic determinants of efficacy to HER3-DXd. Methods: SOLTI TOT-HER3 (NCT04610528) is a window of opportunity, multicenter, pre-operative trial which enrolled, in part A, 77 evaluable patients with untreated HR+/HER2- operable (≥1 cm) breast cancer. Patients received a single dose of HER3-DXd (6.4 mg/kg). The primary objective was to evaluate the CelTIL score variation between pre- and post-treatment (day 21) samples. CelTIL combines % of tumor cellularity and % of tumor-infiltrating lymphocytes into a single score. DNA and RNA were purified from pre-treatment baseline FFPE tumor samples. Gene expression was evaluated using a custom 67-gene panel on the nCounter. NGS-based DNA-seq was performed using the VHIO-300 panel, which estimates tumor mutational burden (TMB), identifies copy-number aberrations (CNAs) across the entire genome and calls mutational status of &amp;gt;300 genes. From CNA data, 150 previously defined DNA-based signatures (Xia et al. Nat Comm 2019) trained to capture RNA- and protein-based phenotypes such as the PAM50-related biology were evaluated. Associations of each variable with efficacy (i.e., CelTIL relative changes, and tumor cellularity relative changes) were adjusted for multiple-testing (false discovery rate [FDR] &amp;lt; 5%). The area under the ROC Curve (AUC) was used to estimate the discrimination performance of each variable. Results: RNA and DNA data were obtained from 45 (58%) patients. Baseline characteristics in this subset of patients were generally similar to the original TOT-HER3 population. Among 228 variables (single mutation status, single gene expression, PAM50 signatures, TMB, and DNA CNA-based signatures), 139 (61%) were found significantly associated (FDR&amp;lt; 5%) with CelTIL changes at day 21. Among them, TP53 mutations (n=7) were found associated with higher CelTIL response compared to TP53 wild type (71% [95% CI=-5.4-17.8] vs. 24% [95% CI=15.9-55.4], FDR=2.1%). In addition, RNA-based genes tracking Basal-related biology (e.g., CCNE1, AUC=0.71) or immune expression (e.g., PDCD1, AUC=0.73, or CD68, AUC=0.62), together with RNA/DNA-based signatures tracking proliferation and/or basal-related biology (e.g., retinoblastoma loss-of-heterozygozity [RB-LOH], AUC=0.76), were associated with high CelTIL response. Conversely, RNA/DNA-based signatures tracking endocrine sensitivity/Luminal A-related biology (e.g., Scorr_IE_Correlation, AUC=0.76) were associated with low/lack of CelTIL response. PIK3CA somatic mutations (n=14, 31% of cases), and TMB (range 2.2-12.7) were not found associated with CelTIL response. Similar overall results were obtained when relative changes in tumor cellularity (instead of CelTIL) was evaluated as the efficacy endpoint. Conclusions: TP53 mutations, immune-related genes, and DNA/RNA-based phenotypic signatures tracking Basal- or Luminal A-related biology such as the DNA-based RB-LOH score or the endocrine sensitivity score (Scorr_IE_Correlation) are associated with CelTIL changes in response to HER3-DXd in HR+/HER2- breast cancer. Further RNA- and DNA-based analyses will be evaluated. Citation Format: Fara Brasó-Maristany, Claudette Falato, Olga Martínez-Sáez, Juan Miguel Cejalvo, Mireia Margelí, Pablo Tolosa, Francisco Javier Salvador Bofill, Josefina Cruz, Blanca González-Farré, Esther Sanfeliu, Míriam Arumí, Guillermo Villacampa, Eva Ciruelos, Martín Espinosa-Bravo, Yann Izarzuzaga, Patricia Galván, Judit Matito, Sonia Pernas, Anu Santhanagopal, Stephen Esker, Parul Patel, Pang-Dian Fan, Juan Manuel Ferrero-Cafiero, Ana Vivancos, Tomás Pascual, Aleix Prat, Mafalda Oliveira. Genetic determinants of response to patritumab deruxtecan (HER3-DXd) in hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer: a correlative analysis from SOLTI TOT-HER3 trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-31.

Towards cloud-enabled fully automated end-to-end single molecule imaging.

Single Molecule Super-resolution imaging has revolutionised understanding biological processes on the sub-100 nm scale. Our group developed Fluorophore Localisation Imaging with Photobleaching (FLImP), a super-resolution imaging method that can achieve <4 nm resolution. In concert with molecular dynamics simulations, FLImP was used to reveal the in-situ atomic resolution structures of ligand-bound EGFR oligomers at the plasma membrane of cells (Needham et al., Nat Comms 2016), and those of ligand-unbound dimers and oligomers on cells (Zanetti-Domingues et al., Nat Comms 2018), shedding new light on the mechanisms of EGFR auto-inhibition, phosphorylation and signalling.

The role of the constriction in defining the molecular sensing trap of aerolysin-based pores.

It has recently been shown that ionic current modulation by analytes visiting the pore formed by wild-type or mutant aerolysin (AeL) toxin is sensitive not only to molecular volume but also to molecular shape (1). One prerequisite of the remarkable sensitivity of this “whole molecule sensing” regime is the prolonged (1-100 ms) trapping of single molecules in the beta-barrel of the pore. At the same time, it is well known that macromolecular analytes are unable to enter this portion of the AeL-pore directly from the trans-side (2) but must enter from cis passinga constriction. We use high-bandwidth (0-100 kHz) recording of blockades induced by short polynucleotides (dA3,dA4,dA5) and oligoarginine peptides (R3,R5,R7) in conjunction with mutations of charged residues defining the trans-ward boundary of the constriction (D222C, R220S) to probe its role in the sensing mechanism. Both mutations induced rectification (D222C cis-ward; R220S trans-ward) and significantly affected the probability of transient deep blocks indicating analyte returns to the constriction after the first visit to the trap, with D222C potentiating and R220S reducing it for nuleotides. However, the initial deep block indicative of constriction passage remained in R220S. In the case of peptides, however, D222C left deep blocks largely unaffected while R220S nearly abolished returns and prolonged total blockage durations. These and other results indicate that the residues at the border between the constriction and the trap do not merely act by their charges but exert important steric influences and that further modifications of these residues may tailor the pore to various analytes. (1) Ouldali et al. 2020. Nat. Biotech. 38:176-181; Ensslen et al. 2022. JACS 144:16060-16068. (2) Baaken et al. 2015. ACS Nano. 9:6443-6449. (3) Iacovache et al. 2016. Nat Comms. 7:12062-8.

OP01 Sequencing-based gene network analysis reveals a profound role for ferroptosis key gene GPX4 in post-operative endoscopic recurrence in Crohn’s disease

Abstract Background An ileocolonic resection with ileocolonic anastomosis is often required in patients with Crohn’s disease (CD). Postoperative recurrence (POR) is common, with host-luminal interactions being implicated. However, these interactions are poorly understood. We aimed to obtain insights in the early mechanisms of POR CD focusing on sequencing-based gene networks in the mucosa. Methods We included 36 CD patients who underwent ileocolonic resection with ileocolonic anastomosis, and collected mucosal biopsies from the neoterminal ileum 6 months after surgery (Table 1). We defined POR by a Rutgeerts score ≥i2b at month 6, and compared this to complete absence of endoscopic activity (i0). All biopsies underwent single-end RNA sequencing (Illumina TruSeq Stranded mRNA). Gene co-expression network clusters were identified using weighted gene co-expression network analysis (R\WGCNA), were tested for correlation with the primary outcome POR and clinical factors, and multiple testing correction was applied (FDR&amp;lt;0.05). Cellular deconvolution was performed using xCell; and top enriched pathways and upstream regulators were analysed using IPA (p&amp;lt;0.05). Results WGCNA analysis identified 25 co-expression clusters, of which five correlated with the postoperative outcome (clusters V, VI, IX, XV, XXV) (Fig.1). All but cluster XXV were upregulated in POR, with a profound immune-related enrichment in cluster V and VI (eg. (a)granulocyte adhesion and diapedesis, and IL17 signalling); microRNA biogenesis (cluster IX) and wound healing and fibrosis signalling (cluster XV). Interestingly, one cluster (XXV) was inversely correlated with POR, and genes within this cluster were mainly involved in autophagy, ferroptosis and oxidative stress response. The main hub gene (r=0.95, FDR=8.17E-19) within this cluster XXV was GPX4 (Glutathione peroxidase 4), a negative regulator of ferroptosis. Moreover, after cellular deconvolution, we found a positive correlation between the epithelial gene enrichment signature and the expression of GPX4 (r=0.52, p=0.0011). Finally, the main upstream regulator of the genes within this cluster XXV was HNF4A, a transcription factor known to regulate ferroptotic response. Conclusion We identified a mucosal transcriptomic signature associated with postoperative CD recurrence. Both autophagy and ferroptosis were found to be key protective pathways for recurrence with GPX4 as central hub. Our results complement recent findings of impaired epithelial GPX4 activity in ileal CD lesions evoking an inflammatory response to dietary lipids (Mayr et al Nat Comm 2020).

Development of a Novel Chromatin Activation Score As a Powerful Independent Prognostic Factor in Chronic Lymphocytic Leukemia

Background & Aim: In a previous study, we identified a signature of 498 regulatory regions with de novo increased H3K27ac levels (a mark of chromatin activation) in patients with chronic lymphocytic leukemia (CLL), which seemed to be present in all cases studied (Beekman et al., Nat Med 2018). In this abstract, we show that such de novo chromatin activation is not stable among CLL patients but heterogeneous, and therefore, we aim at exploring whether such heterogeneity has a biological and clinical impact. Results: The signature of 498 de novo active regulatory regions was related to 434 specific H3K27ac peaks based on chromatin immunoprecipitation sequencing (ChIPseq) data from 104 CLLs. Analyzing the signal variability among patients, we observed a high degree of heterogeneity. An unsupervised clustering revealed two different sets of regions whose mean H3K27ac signal was associated with time to first treatment (TTFT) using a univariate Cox model. In detail, a higher mean H3K27ac intensity was associated with a shorter TTFT in one of the sets (P=0.001 and hazard ratio (HR)=3.5) while, intriguingly, showed a trend towards longer TTFT (P=0.09 HR=0.6) in the other set. These findings suggest that not only disease progression but also disease stability seem to be active processes in CLL involving de novo chromatin changes. Then, we argued that the clinical behavior of CLL patients could be better captured by the balance between the activation levels of these two sets of regions. Indeed, the ratio of both means was statistically more significant (P=1.4x10-9 HR=15.4). Next, we further refined these groups by selecting individual regions associated with shorter (HR>1, 77 peaks) or longer (HR<1, 144 peaks) TTFT (FDR 0.2). We subsequently defined the score SPP as the mean signal in the pro-progressive (PP) group (HR>1), the score SPS as the mean signal in the pro-stable (PS) disease group (HR<1), and SB as their ratio. Ultimately, this led to an improvement of the statistical significance of the SB with TTFT (P=1.4x10-14 HR=8.6). From a biological perspective, we explored the validity of the SB performing ChIPseq for H3K27ac in 10 primary CLLs before and after a co-culture system inducing strong proliferation (Mangolini et al., Nat Comms in press). In these experiments, we observed an increased SB (P<10-3) that was mostly associated with a decreased SPS while maintaining the SPP levels. To further validate the biological implications of our findings, we mined RNAseq data in 64 cases with concurrent ChIPseq experimentation. We identified target genes whose expression levels correlated with H3K27ac levels (43 genes associated with the PP regions and 62 target genes associated with the PS regions). Both lists included genes previously described in CLL (e.g. CTLA4, FMOD or KSR2). Analogously to H3K27ac, we defined three new scores based on expression levels: SPP-exp, SPS-exp and SB-exp. These scores highly correlated with the H3K27ac-based scores (Pearson r between 0.82 to 0.88). Interestingly, cases with higher SB-exp were enriched in TNF-α and NF-κB pathways and in RELB, FOS, JUN, JUNB and EGR1 transcription factors regulons. We also identified 21 genetic driver alterations related to a higher SB-exp including trisomy 12, 11q loss and mutations in IGLV3-21R110, ATM, SF3B1, NOTCH1 and U1. Finally, we evaluated the potential of SB and SB-exp as independent prognostic factors in multivariate models. In the ChIPseq series (n=104), SB was significant independently of the IGHV mutational status, Binet stage and number of genetic driver alterations. Similarly, using three independent CLL series of gene expression data (n=110, 174 and 140), we observed that the SB-exp was a highly significant prognostic factor independent from IGHV status, Binet stage and number of drivers. To favour potential clinical translation, we generated a reduced SB-exp with only 12 genes which maintained the clinical impact. Conclusions: This study revealed an unexpected heterogeneity within de novo active chromatin regions in CLL, and that such heterogeneity has a dual clinical impact on CLL. A balance score between regions related to the two tendencies (i.e. more progressive vs more stable disease) identified groups of patients with different biological features. Finally, using both chromatin and gene expression data as a surrogate, the balance score was a highly significant independent prognostic factor in independent CLL series.

Abstract 15274: Pediatric Valved Conduit From Biological Engineering in the Growing Lamb Model

Introduction: A valved conduit with growth potential for pediatric patients remains an unmet need. Here we present a novel valved conduit with growth potential based on initial data in a growing lamb model. The valve is constructed from three biologically-engineered tubes of acellular collagenous matrix possessing somatic growth potential (Syedain et al., Nat Comms, 2016) using resorbable suture with each tube contributing to create a “leaflet” (Syedain et al, Sci Transl Med, 2021). This tri-tube valve previously demonstrated growth over 52 weeks when implanted interpositionally in the pulmonary artery. Methods: In this study, a fourth tube of the same matrix is placed around the tri-tube valve to create a tubular valved conduit. The inlet segment is cut diagonally to allow for direct attachment to the myocardium, emulating a clinical repair (see figure). Results: The first two lambs to date implanted in this study exhibited normal leaflet function, a pressure gradient less than 5mmHg without regurgitation or dilatation of the conduit at 12-week echocardiography (see figure). During this time the animal’s weight increased from 34±2kg to 48±1kg. The inflow pattern with this myocardial implantation was more uniform than in the previous study that used a pulmonary artery interposition implant of the just the valve (with no conduit). Discussion: Demonstrating integration of the conduit’s matrix to the myocardium is a key step toward a clinical trial. Previous pulmonary interposition implants of the valve, while achieving integration with the adjacent artery prior to dissolution of the Maxon suture (50% loss of tensile strength in ~3 weeks, complete by ~26 weeks), did not establish this clinically relevant point of myocardial integration. Conclusion: The preliminary finding is that the valved conduit exhibits excellent function at 12 weeks in the growing lamb and integration of the conduit’s matrix with the valve annulus-myocardium will likely occur. Funding: R01 HL107572

Abstract 3277: SWI/SNF inactivation vulnerability

Abstract Genetic inactivation in specific SWI/SNF complex members induce sensitivity to the inhibition of KDM6A/UTX and KDM6B/JMJD3 in cancer. Despite the genetic inactivation of the tumor suppressor SMARCA4 is frequently found in cancer, there are no therapies that effectively target SMARCA4-deficient tumors. In our previous work, we reported that unlike the cells with activated MYC oncogene, cells with SMARCA4 inactivation are refractory to the histone deacetylase inhibitor, SAHA, leading to the aberrant accumulation of H3K27me3. In this context, SMARCA4 deficient cells showed an impaired transactivation and significantly reduced levels of the histone H3K27me3 specific demethylases, KDM6A/UTX and KDM6B/JMJD3, and their inhibition compromises cell viability specifically in SMARCA4 mutant cells. Furthermore, the in vivo administration of KDM6s inhibitor (GSK-J4) to mice orthotopically implanted with SMARCA4 mutant lung cancer cell lines or primary tumors of small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), showed a strong anti-tumor effect, highlighting the vulnerability of the SMARCA4 deficient tumors to KDM6s inhibition as a biomarker that could be exploited for treating SMARCA4-mutant cancer patients (Romero OA, Vilarrubi A, et al. SMARCA4 deficient tumors are vulnerable to KDM6A/UTX and KDM6B/JMJD3 blockade. Nat Comm 12, 4319, 2021). Considering our previous observations, and due to the biological role of SMARCA4 as a core component of the SWI/SNF chromatin-remodeling complex, whose members are genetically inactivated in approximately 20% of all human cancers, we wanted to determine the potential response to KDM6s inhibitors in a context of genetic alterations in genes encoding for the other subunits of this remodeling complex. For this propose, we have integrated state of the art technology like genome-wide chromatin modification analysis (ChIP-seq) and transcriptome analysis (RNA-seq), using human cancer cell lines and preclinical models of different cancer types with genetic inactivation at different SWI/SNF-complex members, including mouse models such as orthoxenografts, to design a personalized epigenetic treatment based on the genetic background. Our results showed that, like SMARCA4 deficient cells, specific mutations at some, but not all, SWI/SNF components induce refractoriness to SAHA, aberrant increase of H3K27me3 mark and sensitizes cancer cells to KDM6 inhibitor accompanied with significantly reduced levels of KDM6A and KDM6B expression. These results suggest a strong functional relationship between KDM6A/6B activity and the SWI/SNF-complex, in the control of gene expression and cancer development. These results will be of great value for the stratification of tumors according to their genetic background for tailored treatments, opening the possibility to use SWI/SNF mutations as potential biomarkers for personalized epigenetic-based therapeutics in cancer. Citation Format: Andrea Vilarrubi, Fernando Setien, Eva Pros, Pedro P. Medina, Antonio Gomez, Alberto Villanueva, Octavio A. Romero, Montse Sanchez-Cespedes. SWI/SNF inactivation vulnerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3277.