Cell Clonal Expansion Research Articles

Effect of American genomic ancestry on severe toxicities in children with acute lymphoblastic leukemia in the Amazon region

BackgroundAcute Lymphoblastic Leukemia (ALL) is a neoplasm of the hematopoietic system characterized by a clonal expansion of abnormal lymphocyte precursor cells. ALL is the most common form of cancer in children, but despite advances in treatment, it can still be fatal. Ethnic differences influence survival rates, and genomic ancestry plays an important role, especially in mixed-race populations such as Latin America. This study aims to analyze the influence of genomic ancestry on toxicity in children with ALL in the Amazon region.MethodsThe study included 171 patients (protocol number 119,649/2012—Ethics Committee) with ALL treated at a pediatric treatment center in Belém do Pará, in the Brazilian Amazon. The patients were submitted to the BFM protocol of induction therapy for ALL. Toxicity was assessed based on laboratory tests and adverse events, classified according to the CTC-NCI guide. Genomic ancestry was determined using autosomal informative markers.ResultsThe majority of children (94.74%) developed some type of toxicity during treatment, 87.04% of which were severe. Infectious toxicity was the most common, present in 84.8% of cases, 77.24% of which were severe. Amerindian ancestry showed an association with the risk of severe general toxicity and severe infectious toxicity, with a contribution of 35.0% demonstrating a significant increase in risk. In addition, post-induction refractoriness and relapse were also associated with an increased risk of death.ConclusionThis study highlights the influence of Amerindian genomic ancestry on response to therapy and toxicity in children with ALL in the Amazon region. Understanding these associations can contribute to personalizing treatment and improving clinical outcomes.

The clonal hematopoiesis mutation Jak2V617F aggravates endothelial injury and thrombosis in arteries with erosion-like intimas

BackgroundSuperficial plaque erosion causes many acute coronary syndromes. However, mechanisms of plaque erosion remain poorly understood, and we lack directed therapeutics for thrombotic complication. Human eroded plaques can harbor neutrophil extracellular traps (NETs) that propagate endothelial damage at experimental arterial lesions that recapitulate superficial erosion. Clonal Hematopoiesis of Indeterminate Potential (CHIP) denotes age-related clonal expansion of bone marrow-derived cells harboring somatic mutations in the absence of overt hematological disease. CHIP heightens the risk of cardiovascular disease, with the greatest increase seen in individuals with JAK2V617F. Neutrophils from mice and humans with JAK2V617F undergo NETosis more readily than Jak2WT (wild-type) cells. We hypothesized that JAK2V617F, by increasing propensity to NETosis, exacerbates aspects of superficial erosion. Methods and resultsWe generated Jak2V617F and Jak2WT mice with heterozygous Jak2V617F in myeloid cells. We induced areas of denuded endothelium that recapitulate features of superficial erosion and assessed endothelial integrity, cellular composition of the erosion, thrombosis rates, and response to ruxolitinib, a clinically available JAK1/2 inhibitor, in relation to genotype. Following experimental erosion, Jak2V617F mice have greater impairment of endothelial barrier function and increased rates of arterial thrombosis. Neointimas in Jak2V617F mice exhibit increased apoptosis, NETosis, and platelet recruitment. Jak2V617F mice treated with ruxolitinib show increased endothelial continuity and reduced apoptosis in the neointima comparable to levels in Jak2WT. ConclusionsThese observations provide new mechanistic insight into the pathophysiology of superficial erosion, the heightened risk for myocardial infarction in JAK2V617F CHIP, and point the way to personalized therapeutics based on CHIP status.

Asymmetric stem cell division maintains the genetic heterogeneity of tissue cells.

Within a given tissue, the stem cell niche provides the microenvironment for stem cells suitable for their self-renewal. Conceptually, the niche space constrains the size of a stem-cell pool, as the cells sharing the niche compete for its space. It has been suggested that either neutral- or non-neutral-competition of stem cells changes the clone dynamics of stem cells. Theoretically, if the rate of asymmetric division is high, the stem cell competition is limited, thus suppressing clonal expansion. However, the effects of asymmetric division on clone dynamics have never been experimentally tested. Here, using the Drosophila germline stem cell (GSC) system, as a simple model of the in-vivo niche, we examine the effect of division modes (asymmetric or symmetric) on clonal dynamics by combining experimental approaches with mathematical modeling. Our experimental data and computational model both suggest that the rate of asymmetric division is proportional to the time a stem cell clone takes to expand. Taken together, our data suggests that asymmetric division is essential for maintaining the genetic variation of stem cells and thus serves as a critical mechanism for safeguarding fertility over the animal age or preventing multiple disorders caused by the clonal expansion of stem cells.

Genetic and epigenetic alterations in precursor lesions of endometrial endometrioid carcinoma.

The hyperplasia-carcinoma sequence is a stepwise tumourigenic programme towards endometrial cancer in which normal endometrial epithelium becomes neoplastic through non-atypical endometrial hyperplasia (NAEH) and atypical endometrial hyperplasia (AEH), under the influence of unopposed oestrogen. NAEH and AEH are known to exhibit polyclonal and monoclonal cell growth, respectively; yet, aside from focal PTEN protein loss, the genetic and epigenetic alterations that occur during the cellular transition remain largely unknown. We sought to explore the potential molecular mechanisms that promote the NAEH-AEH transition and identify molecular markers that could help to differentiate between these two states. We conducted target-panel sequencing on the coding exons of 596 genes, including 96 endometrial cancer driver genes, and DNA methylome microarrays for 48 NAEH and 44 AEH lesions that were separately collected via macro- or micro-dissection from the endometrial tissues of 30 cases. Sequencing analyses revealed acquisition of the PTEN mutation and the clonal expansion of tumour cells in AEH samples. Further, across the transition, alterations to the DNA methylome were characterised by hypermethylation of promoter/enhancer regions and CpG islands, as well as hypo- and hyper-methylation of DNA-binding regions for transcription factors relevant to endometrial cell differentiation and/or tumourigenesis, including FOXA2, SOX17, and HAND2. The identified DNA methylation signature distinguishing NAEH and AEH lesions was reproducible in a validation cohort with modest discriminative capability. These findings not only support the concept that the transition from NAEH to AEH is an essential step within neoplastic cell transformation of endometrial epithelium but also provide deep insight into the molecular mechanism of the tumourigenic programme. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Identification of Clonal Hematopoiesis Driver Mutations through In Silico Saturation Mutagenesis.

Clonal hematopoiesis (CH) is a phenomenon of clonal expansion of hematopoietic stem cells driven by somatic mutations affecting certain genes. Recently, CH has been linked to the development of hematologic malignancies, cardiovascular diseases, and other conditions. Although the most frequently mutated CH driver genes have been identified, a systematic landscape of the mutations capable of initiating this phenomenon is still lacking. In this study, we trained machine learning models for 12 of the most recurrent CH genes to identify their driver mutations. These models outperform expert-curated rules based on prior knowledge of the function of these genes. Moreover, their application to identify CH driver mutations across almost half a million donors of the UK Biobank reproduces known associations between CH driver mutations and age, and the prevalence of several diseases and conditions. We thus propose that these models support the accurate identification of CH across healthy individuals. Significance: We developed and validated gene-specific machine learning models to identify CH driver mutations, showing their advantage with respect to expert-curated rules. These models can support the identification and clinical interpretation of CH mutations in newly sequenced individuals. See related commentary by Arends and Jaiswal, p. 1581.

LINC00606 promotes glioblastoma progression through sponge miR-486-3p and interaction with ATP11B

BackgroundLncRNAs regulate tumorigenesis and development in a variety of cancers. We substantiate for the first time that LINC00606 is considerably expressed in glioblastoma (GBM) patient specimens and is linked with adverse prognosis. This suggests that LINC00606 may have the potential to regulate glioma genesis and progression, and that the biological functions and molecular mechanisms of LINC00606 in GBM remain largely unknown.MethodsThe expression of LINC00606 and ATP11B in glioma and normal brain tissues was evaluated by qPCR, and the biological functions of the LINC00606/miR-486-3p/TCF12/ATP11B axis in GBM were verified through a series of in vitro and in vivo experiments. The molecular mechanism of LINC00606 was elucidated by immunoblotting, FISH, RNA pulldown, CHIP-qPCR, and a dual-luciferase reporter assay.ResultsWe demonstrated that LINC00606 promotes glioma cell proliferation, clonal expansion and migration, while reducing apoptosis levels. Mechanistically, on the one hand, LINC00606 can sponge miR-486-3p; the target gene TCF12 of miR-486-3p affects the transcriptional initiation of LINC00606, PTEN and KLLN. On the other hand, it can also regulate the PI3K/AKT signaling pathway to mediate glioma cell proliferation, migration and apoptosis by binding to ATP11B protein.ConclusionsOverall, the LINC00606/miR-486-3p/TCF12/ATP11B axis is involved in the regulation of GBM progression and plays a role in tumor regulation at transcriptional and post-transcriptional levels primarily through LINC00606 sponging miR-486-3p and targeted binding to ATP11B. Therefore, our research on the regulatory network LINC00606 could be a novel therapeutic strategy for the treatment of GBM.Graphical LINC00606 is highly expressed in GBM patients with carcinogenic function and correlated with poor prognosis. LINC00606 regulates glioblastoma progression by sponging miR-486-3p and interacting with ATP11B.

Abstract PO4-13-07: Association of clonal hematopoiesis (CH) with clinical outcomes among survivors of breast cancer (BC)

Abstract Background: Clonal hematopoiesis (CH) refers to the identification of clonal expansion of hematopoietic cells due to somatic mutations in leukemia-associated genes without evidence of hematologic anomalies. CH, particularly with variant allele frequency (VAF) ≥ 10%, has been associated with adverse outcomes, including reduced survival in advanced malignancies and higher risk of cardiovascular events. The aim of this study was to evaluate the biological and prognostic relevance of CH in survivors of breast cancer (BC). Methods: CANTO (NCT01993498) is a prospective, multicenter cohort enrolling patients diagnosed with stage I-III BC. CANTO collects clinical, tumor and treatment-related characteristics at diagnosis (dx). Follow-up visits are scheduled at 1, 2, 4 and 6 years after dx and data on disease status, type of recurrence, and survival status are collected. We assessed CH among pts ≥ 40 years, without previous solid or hematological cancer, with blood samples available at dx and year 4 or experiencing death or disease recurrence before year 4. CH was determined using Next-Generation Sequencing with unique molecular identifiers (HaloPlexHS, Agilent Technologies) for 17 genes (including DNMT3A, TET2, ASXL1, PPM1D, ATM, JAK2 and TP53) on blood samples obtained at dx. Outcomes of interest included invasive disease-free survival (iDFS), distant disease-free survival (DDFS) and overall survival (OS) defined according to STEEP criteria. Kaplan-Meier estimator, log-rank test and multivariate Cox models assessed prognostic role of CH. Results: In the cohort with available CH data (N=1219) mean age (SD) was 57.5 (10.1) years, 62% of pts were postmenopausal, 40.9% were current or former smokers, 46.2% had stage I BC, 77.7% had HR+/HER2- BC, 53.5% received chemotherapy and 80.8% hormonal therapy. CH was detected with a VAF ≥ 1% in 306 pts (25.1%) including 207 pts (67.6%) with DNMT3A mutation, ≥ 2% in 186 (15.3%) and ≥ 10% in 45 (3.7%) pts. Pts with CH ≥ 10% were more frequently older (65.3 vs 57.2 years, p< .0001) and with a previous history of cardiovascular disease (53.3% vs 37.3%, p< 0.02). At a median (IQR) follow-up of 7.0 (6.2-7.8) years 246 iDFS, 161 DDFS and 118 OS events were observed. Only 8 cases of new hematological malignancies were observed. Pts harboring CH with a VAF ≥ 10% had worse OS (log-rank p=0.04) and CH with a VAF ≥ 10% was associated with higher risk of death (HR= 2.4, 95%CI 1.04-5.6); presence of CH with lower VAF was not associated with worse OS. Table displays complete results for all tested associations. Conclusions: CH at dx was identified in 25% of pts with stage I-III BC aged ≥ 40 years in the CANTO cohort, including 4% with a VAF ≥ 10%. In a large, prospective cohort of survivors of BC, we confirmed known associations of CH with older age and cardiovascular history. We observed few secondary hematological malignancies. CH with a VAF ≥ 10% was significantly associated with higher risk of death while adjusting for other relevant prognostic factors. Furthermore, a trend towards higher risk of DDFS events was observed among pts harboring CH with a VAF ≥ 10%. Table. Citation Format: Davide Soldato, Antonin Della Noce, Antonio Di Meglio, Christophe Marzac, Barbara Pistilli, Anne-Laure Martin, Sibille Everhard, Marie Breckler, Sandrine Boyault, Marina Rousseau, Olivier Trédan, Charles Coutant, Paul Cottu, Olivier Rigal, Christelle Levy, Christelle Jouannaud, Baptiste Sauterey, Nathalie Droin, Bastien Job, Aurélie Bertaut, Fabrice André, Ines Vaz Luis, Jean-Baptiste Micol. Association of clonal hematopoiesis (CH) with clinical outcomes among survivors of breast cancer (BC) [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 PO4-13-07.

Recent advances on anti-HIV chimeric antigen receptor-T-cell treatment to provide sustained HIV remission.

Successful sustained remission of HIV infection has been achieved after CCR5Δ32/Δ32 allogeneic hematopoietic stem cell transplantation for treatment of leukemia in a small cohort of people living with HIV (PLWH). This breakthrough demonstrated that the goal of curing HIV was achievable. However, the high morbidity and mortality associated with bone marrow transplantation limits the routine application of this approach and provides a strong rationale for pursuing alternative strategies for sustained long-term antiretroviral therapy (ART)-free HIV remission. Notably, long-term immune-mediated control of HIV replication observed in elite controllers and posttreatment controllers suggests that potent HIV-specific immune responses could provide sustained ART-free remission in PLWH. The capacity of chimeric antigen receptor (CAR)-T cells engineered to target malignant cells to induce remission and cure in cancer patients made this an attractive approach to provide PLWH with a potent HIV-specific immune response. Here, we review the recent advances in the design and application of anti-HIV CAR-T-cell therapy to provide a functional HIV cure. HIV reservoirs are established days after infection and persist through clonal expansion of infected cells. The continuous interaction between latently infected cells and the immune system shapes the landscape of HIV latency and likely contributes to ART-free viral control in elite controllers. CAR-T cells can exhibit superior antiviral activity as compared with native HIV-specific T cells, particularly because they can be engineered to have multiple HIV specificities, resistance to HIV infection, dual costimulatory signaling, immune checkpoint inhibitors, stem cell derivation, CMV TCR coexpression, and tissue homing ligands. These modifications can significantly improve the capacities of anti-HIV CAR-T cells to prevent viral escape, resist HIV infection, and enhance cytotoxicity, persistence, and tissue penetration. Collectively, these novel modifications of anti-HIV CAR-T cell design have increased their capacity to control HIV infection. Anti-HIV CAR-T cells can be engineered to provide potent and sustained in-vitro and in-vivo antiviral function. The combination of anti-HIV CAR-T cells with other immunotherapeutics may contribute to long-term HIV remission in PLWH.

B and T cell repertoire and gene expression differences in measles infection and vaccination revealed by longitudinal single-cell RNA/VDJ sequencing

Abstract While wild-type measles virus (WT MeV) was demonstrated to induce immune amnesia, it paradoxically still elicited high titers of measles-specific antibodies longitudinally; and much higher than the live-attenuated measles vaccine (LAMV). In order to further understand the longitudinal B and T repertoires, clonotype evolution and gene expression difference in WT MeV and LAMV, we conducted longitudinal single-cell RNA/VDJ sequencing of peripheral blood mononuclear cells (PBMC) from WT MeV and LAMV-infected rhesus macaques (RMs) (n = 3 in each WT MeV and LAMV groups, with 4 timepoints at D0, D11, D42/56, D168/176). Firstly, B cell subtypes (e.g. naïve, memory, activated and antibody-secreting B cells) were successfully annotated in PBMC based on key gene markers and immunogenetic information. Furthermore, preliminary analysis of 1 WT MeV and 1 LAMV revealed interesting differences in the B cell repertoire, including a more diverse usage of IGHV genes, greater isotype switching from IgD/M to IgGs, higher level of somatic hypermutation and later B cell clonal expansion peak in LAMV than WT-MeV infected RMs longitudinally. Interestingly, there was greater T cell clonotype expansion in the WT MeV than LAMV RMs. Our study highlights the differential humoral and adaptive immune responses between LAMV and WT MeV infections, uncovering important immunogenetic understanding in natural infection and vaccination.

Understanding the Role of Connexins in Hepatocellular Carcinoma: Molecular and Prognostic Implications.

Connexins, a family of tetraspan membrane proteins forming intercellular channels localized in gap junctions, play a pivotal role at the different stages of tumor progression presenting both pro- and anti-tumorigenic effects. Considering the potential role of connexins as tumor suppressors through multiple channel-independent mechanisms, their loss of expression may be associated with tumorigenic activity, while it is hypothesized that connexins favor the clonal expansion of tumor cells and promote cell migration, invasion, and proliferation, affecting metastasis and chemoresistance in some cases. Hepatocellular carcinoma (HCC), characterized by unfavorable prognosis and limited responsiveness to current therapeutic strategies, has been linked to gap junction proteins as tumorigenic factors with prognostic value. Notably, several members of connexins have emerged as promising markers for assessing the progression and aggressiveness of HCC, as well as the chemosensitivity and radiosensitivity of hepatocellular tumor cells. Our review sheds light on the multifaceted role of connexins in HCC pathogenesis, offering valuable insights on recent advances in determining their prognostic and therapeutic potential.

HPV oncogenes expressed from only one of multiple integrated HPV DNA copies drive clonal cell expansion in cervical cancer.

Persistent oncogenic HPV infections lead to viral DNA integration into the human genome and the development of cervical, anogenital, and oropharyngeal cancers. The expression of the viral E6 and E7 oncogenes plays a key role in cell transformation and tumorigenesis. However, how E6 and E7 could be expressed from the integrated viral DNA which often lacks a viral polyadenylation signal in the cancer cells remains unknown. By analyzing the integrated HPV DNA sites and expressed HPV RNAs in cervical cancer tissues and cell lines, we show that HPV oncogenes are expressed from only one of multiple chromosomal HPV DNA integrated copies. A host polyadenylation signal downstream of the integrated viral DNA is used for polyadenylation and stabilization of the virus-host chimeric RNAs, making the oncogenic transcripts targetable by siRNAs. This observation provides further understanding of the tumorigenic mechanism of HPV integration and suggests possible therapeutic strategies for the development of precision medicine for HPV cancers.

Identification of hub genes and potential molecular mechanisms related to drug sensitivity in acute myeloid leukemia based on machine learning.

Background: Acute myeloid leukemia (AML) is the most common form of leukemia among adults and is characterized by uncontrolled proliferation and clonal expansion of hematopoietic cells. There has been a significant improvement in the treatment of younger patients, however, prognosis in the elderly AML patients remains poor. Methods: We used computational methods and machine learning (ML) techniques to identify and explore the differential high-risk genes (DHRGs) in AML. The DHRGs were explored through multiple in silico approaches including genomic and functional analysis, survival analysis, immune infiltration, miRNA co-expression and stemness features analyses to reveal their prognostic importance in AML. Furthermore, using different ML algorithms, prognostic models were constructed and validated using the DHRGs. At the end molecular docking studies were performed to identify potential drug candidates targeting the selected DHRGs. Results: We identified a total of 80 DHRGs by comparing the differentially expressed genes derived between AML patients and normal controls and high-risk AML genes identified by Cox regression. Genetic and epigenetic alteration analyses of the DHRGs revealed a significant association of their copy number variations and methylation status with overall survival (OS) of AML patients. Out of the 137 models constructed using different ML algorithms, the combination of Ridge and plsRcox maintained the highest mean C-index and was used to build the final model. When AML patients were classified into low- and high-risk groups based on DHRGs, the low-risk group had significantly longer OS in the AML training and validation cohorts. Furthermore, immune infiltration, miRNA coexpression, stemness feature and hallmark pathway analyses revealed significant differences in the prognosis of the low- and high-risk AML groups. Drug sensitivity and molecular docking studies revealed top 5 drugs, including carboplatin and austocystin-D that may significantly affect the DHRGs in AML. Conclusion: The findings from the current study identified a set of high-risk genes that may be used as prognostic and therapeutic markers for AML patients. In addition, significant use of the ML algorithms in constructing and validating the prognostic models in AML was demonstrated. Although our study used extensive bioinformatics and machine learning methods to identify the hub genes in AML, their experimental validations using knock-out/-in methods would strengthen our findings.

Telomere dysfunction alters intestinal stem cell dynamics to promote cancer

Telomere dynamics are linked to aging hallmarks, and age-associated telomere loss fuels the development of epithelial cancers. In Apc-mutant mice, the onset of DNA damage associated with telomere dysfunction has been shown to accelerate adenoma initiation via unknown mechanisms. Here, we observed that Apc-mutant mice engineered to experience telomere dysfunction show accelerated adenoma formation resulting from augmented cell competition and clonal expansion. Mechanistically, telomere dysfunction induces the repression of EZH2, resulting in the derepression of Wnt antagonists, which causes the differentiation of adjacent stem cells and a relative growth advantage to Apc-deficient telomere dysfunctional cells. Correspondingly, in this mouse model, GSK3β inhibition countered the actions of Wnt antagonists on intestinal stem cells, resulting in impaired adenoma formation of telomere dysfunctional Apc-mutant cells. Thus, telomere dysfunction contributes to cancer initiation through altered stem cell dynamics, identifying an interception strategy for human APC-mutant cancers with shortened telomeres.

Immune features revealed by single-cell RNA and single-cell TCR/BCR sequencing in patients with rheumatoid arthritis receiving COVID-19 booster vaccination.

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, have profoundly affected human health. Booster COVID-19 vaccines have demonstrated significant efficacy in reducing infection and severe cases. However, the effects of booster COVID-19 vaccines on key immune cell subsets and their responses in rheumatoid arthritis (RA) are not well understood. By using single-cell RNA sequencing (scRNA-seq) combined with scTCR/BCR-seq analysis, a total of 8 major and 27 minor cell clusters were identified from paired peripheral blood mononuclear cells (PBMCs) which were collected 1 week before and 4 weeks after booster vaccination in stable RA patients. Booster vaccination only had limited impact on the composition and proportions of PBMCs cell clusters. CD8+ cytotoxic T cells (CD8+T_CTL) showed a trend toward an increase after vaccination, while naive B cells and conventional dendritic cells (cDCs) showed a trend toward a decrease. Transcriptomic changes were observed after booster vaccination, primarily involving T/B cell receptor signaling pathways, phagosome, antigen processing and presenting, and viral myocarditis pathways. Interferon (IFN) and pro-inflammatory response gene sets were slightly upregulated across most major cell subpopulations in COVID-19 booster-vaccinated RA individuals. Plasma neutralizing antibody titers significantly increased after booster COVID-19 vaccination (p = 0.037). Single-cell TCR/BCR analysis revealed increased B cell clone expansion and repertoire diversity postvaccination, with no consistent alterations in T cells. Several clonotypes of BCRs and TCRs were identified to be significantly over-represented after vaccination, such as IGHV3-15 and TRBV28. Our study provided a comprehensive single-cell atlas of the peripheral immune response and TCR/BCR immune repertoire profiles to inactivated SARS-CoV-2 booster vaccination in RA patients, which helps us to understand vaccine-induced immune responses better.

Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1/2 trial

Programmed cell death protein 1 (PD-1) inhibitors have modest efficacy as a monotherapy in hepatocellular carcinoma (HCC). A personalized therapeutic cancer vaccine (PTCV) may enhance responses to PD-1 inhibitors through the induction of tumor-specific immunity. We present results from a single-arm, open-label, phase 1/2 study of a DNA plasmid PTCV (GNOS-PV02) encoding up to 40 neoantigens coadministered with plasmid-encoded interleukin-12 plus pembrolizumab in patients with advanced HCC previously treated with a multityrosine kinase inhibitor. Safety and immunogenicity were assessed as primary endpoints, and treatment efficacy and feasibility were evaluated as secondary endpoints. The most common treatment-related adverse events were injection-site reactions, observed in 15 of 36 (41.6%) patients. No dose-limiting toxicities or treatment-related grade ≥3 events were observed. The objective response rate (modified intention-to-treat) per Response Evaluation Criteria in Solid Tumors 1.1 was 30.6% (11 of 36 patients), with 8.3% (3 of 36) of patients achieving a complete response. Clinical responses were associated with the number of neoantigens encoded in the vaccine. Neoantigen-specific T cell responses were confirmed in 19 of 22 (86.4%) evaluable patients by enzyme-linked immunosorbent spot assays. Multiparametric cellular profiling revealed active, proliferative and cytolytic vaccine-specific CD4+ and CD8+ effector T cells. T cell receptor β-chain (TCRβ) bulk sequencing results demonstrated vaccination-enriched T cell clone expansion and tumor infiltration. Single-cell analysis revealed posttreatment T cell clonal expansion of cytotoxic T cell phenotypes. TCR complementarity-determining region cloning of expanded T cell clones in the tumors following vaccination confirmed reactivity against vaccine-encoded neoantigens. Our results support the PTCV’s mechanism of action based on the induction of antitumor T cells and show that a PTCV plus pembrolizumab has clinical activity in advanced HCC. ClinicalTrials.gov identifier: NCT04251117.

Characterization of Th17 tissue-resident memory cells in non-inflamed intestinal tissue of Crohn's disease patients

Crohn's disease (CD) is a chronic inflammatory disorder affecting the bowel wall. Tissue-resident memory T (Trm) cells are implicated in CD, yet their characteristics remain unclear. We aimed to investigate the transcriptional profiles and functional characteristics of Trm cells in the small bowel of CD and their interactions with immune cells. Seven patients with CD and four with ulcerative colitis as controls were included. Single-cell RNA sequencing and paired T cell receptor sequencing assessed T cell subsets and transcriptional signatures in lamina propria (LP) and submucosa/muscularis propria-enriched fractions (SM/MP) from small bowel tissue samples. We detected 58,123 T cells grouped into 16 populations, including the CD4+ Trm cells with a Th17 signature and CD8+ Trm clusters. In CD, CD4+ Trm cells with a Th17 signature, termed Th17 Trm, showed significantly increased proportions within both the LP and SM/MP areas. The Th17 Trm cluster demonstrated heightened expression of tissue-residency marker genes (ITGAE, ITGA1, and CXCR6) along with elevated levels of IL17A, IL22, CCR6, and CCL20. The clonal expansion of Th17 Trm cells in CD was accompanied by enhanced transmural dynamic potential, as indicated by significantly higher migration scores. CD-prominent Th17 Trm cells displayed an increased interferon gamma (IFNγ)-related signature possibly linked with STAT1 activation, inducing chemokines (i.e., CXCL10, CXCL8, and CXCL9) in myeloid cells. Our findings underscored the elevated Th17 Trm cells throughout the small bowel in CD, contributing to disease pathogenesis through IFNγ induction and subsequent chemokine production in myeloid cells.

Abstract 3627: Volrustomig a novel PD-1/CTLA-4 bispecific antibody leads to robust increase in peripheral and intra-tumoral pharmacodynamic biomarkers in solid tumors from FTiH study

Abstract Background: Volrustomig (MEDI5752) is designed to fully inhibit PD-1 while preferentially inhibiting CTLA-4 on activated PD-1+ T cells. In a FTiH trial, volrustomig demonstrated meaningful clinical activity in patients with solid tumors (including 1L RCC and NSCLC), acceptable safety profile, sustained PD-1 receptor occupancy, and T cell proliferation at levels greater than seen with clinically tolerable doses of CTLA-4 inhibitors with co-administration of anti-PD(L)-1. We sought to further characterize volrustomig pharmacodynamic effect in the periphery and tumor to demonstrate its biological activity and inform dose optimization. Methods: Pre- and on-treatment blood samples from 86 patients enrolled in dose exploration were collected. Peripheral T cell profiling was assayed by flow cytometry, RNAseq, TCRSeq and serum proteomics. Paired tumor biopsies (N=17), pre- and at week 6 on-treatment, were analyzed for T cell infiltration and function using RNAseq and computational image analysis of immune cell marker density detected by histochemistry and multiplex immune fluorescence assays. Results: Volrustomig achieved dose-dependent peripheral pharmacodynamic changes of biomarkers specific to CTLA-4 blockade at ≥ 225mg. At C1D8 with doses ≥ 500mg, increases in CD4 T cell proliferation (ki67), activation (ICOS), and memory T cells (1516%, 321% and 88%, respectively) were seen, each greater than observed with historical controls of patients treated with tremelimumab 3mg/kg (T3) in combination with durvalumab. Similarly, CD8 proliferation and activation were also higher than T3 at C1D8 with 300% and 161% increase respectively. Additional CD4 and CD8 T cell proliferation and activation was observed at C2D8 supporting the benefit of repeat dosing. Higher peripheral T cell clonal expansion at doses ≥ 500mg was seen and associated with PFS and OS benefit. In patients with paired biopsies, volrustomig achieved robust T cell mediated anti-tumor activity including increases in T cell infiltration (CD8+, 2.3 fold, p=0.04), proliferation (CD8+ Ki67+, 2.1 fold, p=0.04) and, T cell cytotoxicity and activation (GrB+, 2.2 fold, p=0.01; T cell effector and IFNg gene signatures, > 2-fold, p< 0.01). Additionally, volrustomig increased CD8 proliferation in tumor compared to historical control durvalumab alone (>2-fold increase in 70% vs 50% patients). Conclusion: Voltrustomig demonstrated robust peripheral and intra-tumoral T cell activation and proliferation, broadening the pool of antigen experienced T cells, at levels greater than seen with dual checkpoint blockade currently used in the clinic. Citation Format: Ikbel Achour, Michael Kuziora, Florian Song, Steven Eck, Lina Meinecke, Jorge Blando, Jennifer Pearson, Megha Saraiya, Christian Eisen, Felix Segerer, Markus Schick, Nicolas Triltsch, Michael Surace, Gisele Silva Boos, Harald Hessel, Mai Bui, Katrin Pratsch, Alma Andoni, Shelby Denise Gainer, Dan Freeman, Deepa Subramaniam, Ben Tran. Volrustomig a novel PD-1/CTLA-4 bispecific antibody leads to robust increase in peripheral and intra-tumoral pharmacodynamic biomarkers in solid tumors from FTiH study [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 3627.

Abstract 6645: Temporal immune profiling reveals systemic anti tumor immunity in triple negative breast cancer(TNBC) patients treated with immunotherapy and radiation

Abstract Combining pembrolizumab (anti-PD1) with radiation therapy (RT) has shown to enhance response rates in TNBC. We hypothesize that this improved efficacy is not just localized to the tumor bed, but also involves a broader systemic immune response, although the precise mechanisms are yet to be fully understood. Therefore, in this study, we aimed to investigate T cell clonal dynamics in Tumor and peripheral blood mononuclear cells (PBMCs) in 9 patients (6 responders(R) and 3 non-responders (NR)) from 3 different timepoints (before therapy (Baseline), after anti-PD1 alone and after anti-PD1+RT). CD45+ positive stained cells were sorted for subsequent single-cell RNA and T-cell receptor (TCR) sequencing analysis.Through single-cell analysis, we identified increased proportions of exhausted CD8+ and Treg cells from baseline to both treatment points, whereas exhaustedCD4+ T cells increased in proportions after PD1 alone in Responders. We aimed to then understand the origin of these cells, and how they might contribute to systemic immunity. Hence, by TCR overlap between Tumor and PBMC’s we detected 590 overlapped clones in CD8 and 1000 overlapped clones in CD4 T cells. Neo-TCR score was higher in these overlapped clones, suggesting tumor reactive clones can be identified in PBMC’s. Compared to exhausted CD4, exhausted CD8 T cell and Treg had higher overlapped clones between Tumor and PBMC. We also found that exhausted CD8+T cells clonal expansion after treatment was higher in pre-existing clones, compared to new clones in Responders. These pre-existing clones consisted of overlapped clones, suggesting tumor reactivity to be present in PBMC’s before treatment. Further investigation suggested that exhausted CD8+ cells also showed increased transition to TEMRA (in PBMC) and a tissue-resident memory CD8 T cell subset, under combination therapy. This pattern was not seen in exhausted CD4+Tcells or Treg cells, indicating that CD8+ T cell subsets in the blood are crucial in driving systemic anti-tumor immunity. Overall, this study has identified the systemic immune response triggered by the combination of anti-PD1 and radiation therapy highlighting its potential to inform and refine future therapeutic strategies in TNBC treatment. Citation Format: Vaishnavi Devarakonda, Jolene Viramontes, Satchel Stevens, Tahir Dar, Emily Ko, Elvin Canesco, Kaiden Anderson, Jlenia Guarnerio, Simon Knott, Stephen L. Shiao. Temporal immune profiling reveals systemic anti tumor immunity in triple negative breast cancer(TNBC) patients treated with immunotherapy and radiation [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 6645.

Abstract 791: Evaluation of BCR spike-in controls using DriverMap adaptive immune receptor (AIR) profiling technology

Abstract The DriverMap™ Adaptive Immune Receptor (AIR) repertoire profiling assay employs multiplex RT-PCR for comprehensive profiling of CDR3 or full-length receptor profiling of all variable heavy and light chains of T-cell receptors (TCR) and B-cell receptors (BCR) from either RNA or DNA. In this study, initiated by a U.S. Food and Drug Administration (FDA) consortium to compare performance of different BCR repertoire profiling technologies, we tested spike-ins in peripheral blood mononuclear cell (PBMC) RNA and DNA control mixtures prepared from nine different B-cell lines. We measured the sensitivity, linearity and accuracy of AIR RNA and DNA BCR profiling for the IGH, IGK and IGL chains of the spike-in controls. Our comprehensive analysis, encompassing CDR3 profiling and full-length receptor profiling (CDR1, CDR2, and CDR3) sequencing, revealed a 100X increase in sensitivity of detection of spike-in controls in RNA compared to the DNA assay. The high sensitivity of the full-length DriverMap AIR RNA assay allowed the detection of controls from all nine cell lines, with 8 out of 9 cell lines detected in the DNA assay, which demonstrates the robustness of the DriverMap AIR profiling technology. This study highlights the remarkable sensitivity of the AIR RNA assay in quantifying transcripts. This approach enables the detection of low-frequency BCR clonotypes, which is particularly advantageous when working with samples containing low numbers of B cells. Furthermore, our AIR DNA assay in combination with spike-in controls offers a quantitative tool for minimal residual disease (MRD) applications, providing accurate insight into cell numbers, which then facilitates tracking clonal expansion of immune cells. Citation Format: Alex Chenchik, Tianbing Liu, Mikhail Makhanov, Dongfang Hu, Khadija Ghias, Paul Diehl. Evaluation of BCR spike-in controls using DriverMap adaptive immune receptor (AIR) profiling technology [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 791.

Abstract 258: An organoid model for investigating Lynch syndrome tumorigenesis

Abstract Lynch syndrome (LS) is the most common inherited predisposition for developing colon cancer and is caused by germline variants in mismatch repair (MMR) genes. Still, the mechanism of tumorigenesis in these patients is unknown. An LS patient inherits one defective copy of an MMR gene. At some point in their lifetime, the remaining wild-type allele is lost in a cell rendering it entirely MMR deficient. MMR deficiency increases mutational rate and the chance of acquiring mutations in an oncogene or tumor suppressor that drive tumor initiation. However, loss of MMR in cells also leads to increased resistance to certain forms of DNA damage. Thus, our question is whether this phenotype imparts a selective advantage in colonic cells that may lead to clonal expansion of MMR-deficient cells as a first step towards tumorigenesis in LS patients. To test this, we are using stem cell-derived human colonic organoids and CRISPR gene editing to examine competition between MMR-deficient (MMRd) and MMR-proficient (MMRp) colonic cells over time. We have initially observed that MMRd stem cells are much more efficient at generating colonic organoids that grow larger and survive longer, consistent with our hypothesis of a selective advantage. To mimic the spontaneous loss of heterozygosity that occurs in LS patients’ colonic crypts, we are forming organoids from differentially labeled MMRd and MMRp stem cells. Live fluorescent imaging of individual organoids is used to quantify changes in cell population over time. Preliminary data indicate that MMRd cells can outgrow MMRp cells resulting in a shift in the ratio of green to red cells over a two-week time period while challenged with a DNA-damaging agent. These results suggest that loss of MMR function imparts an immediate selective advantage in colonic cells in response to DNA damage. Citation Format: Caroline Guild, Christopher Heinen, Samantha Nadeau. An organoid model for investigating Lynch syndrome tumorigenesis [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 258.