TGF-β Signature Research Articles

TGF-β signaling-related signature for predicting prognosis and therapeutic response in lower-grade glioma.

Low-grade glioma (LGG) is a tumor that includes World Health Organization (WHO) grade II and III glioma, the treatment of which consistently results in relapse and drug resistance. Transforming growth factor-beta (TGF-β) is a multifunctional cytokine that regulates various cellular processes, which is found to be abnormal in tumors and promotes glioma development and progression. In this study, we aimed to systematically evaluate the importance of the genes associated with TGF-β in LGG and discover the role of these genes in the prognosis and treatment response of LGG. We used the "Bioconductor Limma" and "consensusClusterplus" R packages to screen differential and prognostic TGF-β-related genes. The R package "GSVA" was used to estimate the infiltration of immune cells and metabolism signature. The drug sensitivity for each TGF-β subtype was assessed by the R package "pRRophetic". The Genomic Identification of Significant Targets in Cancer (GISTIC) algorithm was used to assess the copy number variation (CNV). The onco-print tool of the "complexheatmap package" was employed to visualize the somatic mutation and copy number alteration (CNA) among TGF clusters. We reported three subtypes (A, B, and C) of LGG according to the classification of TGF-β-related genes, where subtype A showed the best prognosis. Subtype B was highly enriched in immune cells. Somatic variations were observed to be diverse in all of the three TGF-β subtypes. Furthermore, another three genes (SHA, AC062021.1, and SNCG) related to TGF-β were identified, which can be a superior predictor of prognosis with a risk score. LGG can be divided into three subtypes based on TGF-β signaling-related genes with distinct immune infiltration, metabolism, somatic variations, and prognosis.

Abstract PO2-13-05: Deciphering pregnancy-associated breast cancer: distinctive molecular profile and clinical implications from GEICAM/2017-07 EMBARCAM study

Abstract Background: Pregnancy-Associated Breast Cancer (PABC) is defined as BC diagnosed during pregnancy, breastfeeding, or within the first year postpartum. This challenging entity has a distinct biology and worst prognosis and presents an increased risk for metastasis and death compared to non-PABC patients. Identification of molecular pathways that define this malignancy is crucial to explain its biological characteristics and potential association with pregnancy as well as serve to identify new biomarkers with preventive and clinical implications. This study assesses the gene expression profile of a PABC cohort from the Registry Study of Pregnancy and Breast Cancer (GEICAM/2017-07 EMBARCAM study, NCT04603820) to identify distinct molecular signatures and altered pathways for this entity. Methods: This was a multicenter, observational and ambispective age-matched cohort of PABC patients (n=46) and non-PABC patients (n=49). We analyzed the expression of 776 genes involved in 23 key breast cancer pathways, using the nCounter® Breast Cancer 360™ Panel (NanoString Technologies) on BC tumor samples, including molecular subtypes by PAM50 gene signature. Associations between individual differential genes and epidemiological and clinical features of these patients were explored. Normalized differential expression values were log2-transformed for statistical analysis in nSolver Software (NanoString Technologies). P-values were adjusted within each gene/signature and on the grouping variable level difference t-test using the Benjamini and Yekutieli False Discovery Rate (FDR) adjustment. Results: PABC patients’ clinical subtypes were distributed as 43,5% HR-positive/HER2-negative, 17,4% HER2-positive and 39,1% triple negative BC. These patients reported higher family history of BC/ovarian cancer compared to non-PABC (52.17% vs 26.53%; p=0.0124). Differential gene expression showed distinct molecular profiles in PABC and non-PABC patients: significantly genes enrichment involved in cell proliferation and p53 signature for PABC patients and higher expression of apoptosis, TGF-β, and PTEN signatures for non-PABC (adj p< 0.05). Furthermore, CDK4 expression signature (adj p=0.124) and DNA damage repair signatures Homologous Recombination Deficiency (HDR) (adj p=0.083) and BRCAness (adj p=0.220) were also upregulated in PABC tumors. The most significantly upregulated genes in PABC patients (CCNA2, DEPDC1, FAM83D, CDC20, CDKN3, TRIP13, TTK, MAD2L1, KIF2C, and UBE2C) (adj p< 0.05) were involved in cell cycle regulation and DNA damage repair. PABC basal-like tumors were found to be significantly more prevalent than non-PABC, as per the PAM50 classification (45.7% vs 20.4%; p=0.020). Interestingly, basal-like PABC showed higher expression of ESR1 than basal-like non-PABC (logFC=1.486; p=0.022). In contrast, luminal A PABC tumors exhibited lower ESR1 expression vs non-PABC (logFC=-1.5; p=0.021). Additionally, HER2-enriched PABC tumors displayed lower B7-H3/CD276 expression compared to HER2-enriched non-PABC (adj p=0.031). Conclusions: Our study shows that PABC is potentially a clinical and molecular different entity with predominance of the basal-like subtype. Moreover, our results suggest the activation of oncogenic pathways related to cell proliferation, DNA damage repair and p53 mutations which may lead to a clinically more aggressive phenotype for PABC patients. Likewise, the enrichment of BRCAness and HRD signatures found in PABC patients in our study may suggest an increased genetic instability due to a breakdown in the DNA damage repair. These findings may be clinically relevant and translate to new treatment options in PABC patients, who may benefit from therapies targeting DNA repair or cell-cycle checkpoints, such as PARP inhibitors. Citation Format: Juan de la Haba-Rodríguez, Regina Peña, Marina Pollan, Yolanda Jerez Gilarranz, Jose Ponce, Antonio Fernández Aramburu, Blanca Cantos, Ana Santaballa Bertrán, Elena Galve, María Eva Pérez, Susana de la Cruz, María Helena López-Ceballos, Yolanda Fernández, Fernando Moreno, Sonia González, Manuel Ruíz - Borrego, Isabel Blancas, José L Alonso-Romero, Álvaro Jiménez-Arranz, Raúl Rincón, Silvia Guil, Alejandra Díaz-Chacón, Rosalía Caballero, Begoña Bermejo, Angel Guerrero. Deciphering pregnancy-associated breast cancer: distinctive molecular profile and clinical implications from GEICAM/2017-07 EMBARCAM study [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 PO2-13-05.

Abstract 6400: Tislelizumab combined with XELOX as a neoadjuvant therapy for locally advanced gastric and gastroesophageal junction adenocarcinomas: A prospective, phase II trial

Abstract Background: Combing anti-PD-(L)1 antibody with chemotherapy in neoadjuvant therapy of gastric/gastroesophageal junction (G/GEJ) adenocarcinomas is a promising strategy, but clinical data are remain insufficient. Whether immunotherapy biomarkers in previous studies (TMB, MSI, PD-L1, etc.) have equivalent predictive value for G/GEJ remains to be verified. In this phase II clinical trial of tislelizumab in combination with chemotherapy, potential therapy-related biomarkers in tumor samples, and changes in the tumor microenvironment during treatment were analyzed, and a predictive model for efficacy was developed. Methods: This was a prospective, single-center, single-arm, phase II trial. Patients with G/GEJ adenocarcinomas (stage cT3-4aN+M0) were treated with PD-1 inhibitor tislelizumab in combination with oxaliplatin plus capecitabine (XELOX) as neoadjuvant treatment for 2 or 3 cycles. The primary endpoint was the major pathological response (MPR). The secondary endpoints included pathological complete response (pCR) rate, R0 resection rate, and safety. Tumor samples underwent RNA-sequencing (Amoy Diagnostics Co Ltd, Xiamen, China), to identify potential biomarkers, and a predictive model was developed to explore factors influencing tumor response and survival. Results: 28 patients were enrolled; mean age was 59.5 years (SD = 8.2), 19 (67.9%) patients were male; 14 (50.0%) patients with primary tumor location in the stomach, and 14 (50.0%) patients with primary tumor location in the GEJ. All patients were EB virus negative. Except one patient with PMS2 loss, 27 patients were pMMR. Nine (32.1%) patients achieved MPR after treatment, with four (14.3%) of them achieved pCR. The R0 resection rate was 96.4%, with 14 (50.0%) patients achieved objective response. No serious adverse events were reported. The most common grade 2 TRAEs during neoadjuvant treatment were anemia (5 of 28, 17.86%) and nausea (3 of 28, 10.71%). Analysis of differentially expressed genes revealed enrichment of the antigen processing and presentation pathway in the MPR group, while the TGF-β signaling pathway was enriched in the non-MPR group. Notably, the differential immune microenvironment analysis also uncovered significant differences in the TGF-β signature between the MPR and non-MPR groups. Additionally, a therapeutic response prediction model (HT score) was developed with an AUC of 0.98 (0.932-1) in the training set, and its performance was validated in publicly available datasets. Conclusion: Tislelizumab combined with XELOX holds promise as a neoadjuvant therapy for resectable G/GEJ adenocarcinomas, especially for EBV- and pMMR population, offering a well-tolerated safety profile. Moreover, the HT score has the potential to predict treatment efficacy in this regimen. Trial registration numbers: NCT05507658 and NCT05508399. Citation Format: Haikun Zhou, Tao Han, Lei Tuo, Qingchuan Yang, Ruiqi Gao, Pengfei Yu, Jiangpeng Wei, Changbin Zhu, Jin Wang, Peng Cheng, Xiang Kang, Sihui Pi, Hui Liu, Xiaoxi Pan, Xiaohua Li, Gang Ji. Tislelizumab combined with XELOX as a neoadjuvant therapy for locally advanced gastric and gastroesophageal junction adenocarcinomas: A prospective, phase II trial [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 6400.

Abstract 1187: Sensitization to chemo-immunotherapy by targeting TGF-β in preclinical triple negative breast cancer models

Abstract Metastatic triple-negative breast cancer (mTNBC) is refractory to anti-PD-(L)1 immunotherapy. In order to sensitize it, preclinical data show the value of adding chemotherapy treatment for its ability to induce immunogenic cancer cell death (Kroemer et al, 2013). This would enable the recruitment of cytotoxic T-CD8+ lymphocytes (CTL) and thus initiate an antitumor immune response to sensitize tumors to immunotherapy. Nevertheless, the results of clinical trials using chemo-immunotherapy combinations are divergent (IMpassion 130 and 131). The IMpassion 130 trial demonstrated the efficacy of nab-paclitaxel and atezolizumab (anti-PD-L1) in PD-L1+ patients, but unfortunately, IMpassion 131 with paclitaxel did not replicate these results. The choice of chemotherapy to combine with immunotherapy appears to be crucial for inducing an antitumor immune response. Optimizing chemo-immunotherapy combinations is a major challenge in the management of TNBC. The initial aim of this project was to evaluate the immune effects of an experimental chemotherapy doublet, based on two drugs used in mTNBC: cisplatin (CDDP) and eribulin. The idea was to find an alternative to the use of taxanes in combination with anti-PD-(L)1.The effect of the two molecules, alone or in combination, was evaluated on immunogenic death stigmata, modulation of effector or immunosuppressive immune populations and stromal populations, and sensitization to anti-PD-L1 immunotherapy in the immunologically "cold" model: 4T1. The in vivo studies has been realized by generation of orthotopic 4T1 tumors, the analysis of the immune infiltration was performed by RT-qPCR, IHC and flow cytometry. In vitro, our results indicate that CDDP, and more intensely in combination with eribulin, significantly induces the various immunogenic death stigmas. In vivo, the therapeutic effect of chemotherapies is limited, with a superior effect of CDDP and the combination. Nevertheless, CTL recruitment and functionality induced by CDDP are significantly enhanced by eribulin. Despite these data pointing to a potential therapeutic synergy with anti PD-L1 immunotherapy, chemo-immunotherapy treatment fails to induce a superior treatment effect. The significant increase in a TGF-β signature induced by the combination in parallel with CTL recruitment explains this resistance. The results show that inhibition of TGF-β, combined with chemo-immunotherapy in the 4T1 model, leads to a reduction in tumor immunosuppression and intratumoral fibrosis, as well as an increase in CTL infiltration and cytotoxicity, enabling tumor sensitization. Overall, this project highlights the benefits of using an experimental doublet of CDDP and eribulin-based chemotherapies. This project demonstrates that management of chemo-induced immunosuppression mechanisms is necessary to overcome tumor resistance to immunotherapy. Citation Format: Laura Kalfeist, Fanny Ledys, Stacy Petit, Samia Kada-Mohammed, Mickael Rialland, François Ghiringhelli, Emeric Limagne, Sylvain Ladoire. Sensitization to chemo-immunotherapy by targeting TGF-β in preclinical triple negative breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1187.

Phenotypic and Molecular Landscape of Bone Marrow CD8+ T Cells in Myeloid Neoplasms Predicts Response to Treatment with Hypomethylating Agents

Background and aims Myeloid malignancies encompass a diverse group of conditions, including myelodysplastic neoplasms (MDS) and chronic myelomonocytic leukemia (CMML). There is evidence suggesting the role of bone marrow (BM) environment and immune cells in the development and progression of MDS and CMML. Azacytidine (AZA) remains the backbone therapy for higher-risk MDS and non-proliferative CMML. To date, molecular predictors of response to AZA are not yet well-defined. Herein, we sought to investigate the immune landscape of BM samples from patients with lower-risk MDS (LR-MDS; i.e., IPSS-R <3.5), CMML, higher-risk MDS (HR-MDS; i.e., IPSS-R ≥3.5), and acute myeloid leukemia (AML). Methods Samples were collected before treatment initiation. Mass cytometry (CyTOF) was performed on isolated BM mononuclear cells from 48 patients, using the Maxpar® Direct™ Immune Profiling Assay. Flow cytometry was performed on a secondary cohort of 56 patients. scRNA-sequencing was performed on sorted BM CD8+ T cells from patients with HR-MDS (n=4) and AML (n=5), using a 10x Genomics pipeline. Mann-Whitney U test and the Kruskal-Wallis test were used as appropriate. Kaplan-Meier analysis was utilized for survival analysis, and the log-rank test was employed to assess the significance. The level of significance was established at P < 0.05. Results The immunophenotypic analysis using CyTOF demonstrated increased expression of CXCR3 on cytotoxic CD8+ T-cells in CMML and AML samples compared to samples from patients with MDS. Increased frequency of the CD57 +CXCR3 +CD8 + T-cell subset was further observed in patients with HR-MDS and AML that were non-responders to AZA, compared to responders, as observed by both CyTOF and flow cytometry. After determining the optimal cut-off for the frequency of CD8 +CD57 +CXCR3 + T cells at 29%, we observed that HR-MDS and AML patients (Figure 1A) with a frequency of CD57 +CXCR3 +CD8 + of <29% had better overall survival (OS).Multivariate analysisshowed that a frequency of CD57 +CXCR3 +CD8 + of >29% was independently associated with decreased survival in patients with TET2 mutations (HR=2.603; 1.275-5.317). scRNA-seq analysis of isolated BM CD8 + T cells was further performed to characterize the molecular signature that favors response to AZA, which resulted in the identification of several cell clusters based on gene expression. The cluster characterized by the expression of genes associated with cytotoxicity showed significant differences between responders and non-responders. Specifically, within the cytotoxic cluster, genes associated with IFN-related pathways were upregulated in cells from responders to AZA. In contrast, genes upregulated in non-responders displayed an enrichment for the TGF-β signaling pathway, which is known to be associated with the suppression of the killing capacity of T cells (Figure 1B). The aforementioned molecular signatures were further associated with a higher cytotoxic molecular signature in the cytotoxic cell cluster from the group of responders. Conclusion In this study, we focused on BM CD8 + T cells in samples from patients with myeloid neoplasias and performed deep phenotypic analysis using CyTOF coupled with scRNA, in order to characterize the BM immune environment. We demonstrate for the first time that BM CD8 + T cells show different phenotypic and transcriptomic characteristics in responders to AZA. Specifically, we show that the frequency of the effector CD57 +CXCR3 +CD8 + subset is decreased in responders before treatment initiation, and this is associated with better survival. Despite the decreased proportion of this cytotoxic cell population in responders, there are specific signatures associated with effector T cell functionality that can explain the improved outcomes in these patients. These findings underscore the significance of the crosstalk between leukemic and immune cells in the progression of the disease and the effect of treatment. Figure legend A. Increased (%) CD57 +CXCR3 + correlates significantly with worse survival of HR-MDS and AML patients. Kaplan Meier curves for Overall Survival in HR-MDS and AML patients respectively. B. Gene signature derived from the scRNAseq analysis. Interferon-stimulated gene (ISG) signature is increased in the cytotoxic cell cluster in responders and and TGF-β signature in non-responders.

Phase I/Ib, open-label, multicenter, dose-escalation study of the anti-TGF-β monoclonal antibody, NIS793, in combination with spartalizumab in adult patients with advanced tumors

BackgroundNIS793 is a human IgG2 monoclonal antibody that binds to transforming growth factor beta (TGF-β). This first-in-human study investigated NIS793 plus spartalizumab treatment in patients with advanced solid tumors.MethodsPatients received...

Methanolic Extracts of D. viscosa Specifically Affect the Cytoskeleton and Exert an Antiproliferative Effect on Human Colorectal Cancer Cell Lines, According to Their Proliferation Rate.

Numerous studies have reported the pharmacological effects exhibited by Dittrichia viscosa, (D. viscosa) including antioxidant, cytotoxic, antiproliferative, and anticancer properties. In our research, our primary objective was to validate a prescreening methodology aimed at identifying the fraction that demonstrates the most potent antiproliferative and anticancer effects. Specifically, we investigated the impact of various extract fractions on the cytoskeleton using a screening method involving transgenic plants. Tumors are inherently heterogeneous, and the components of the cytoskeleton, particularly tubulin, are considered a strategic target for antitumor agents. To take heterogeneity into account, we used different lines of colorectal cancer, specifically one of the most common cancers regardless of gender. In patients with metastasis, the effectiveness of chemotherapy has been limited by severe side effects and by the development of resistance. Additional therapies and antiproliferative molecules are therefore needed. In our study, we used colon-like cell lines characterized by the expression of gastrointestinal differentiation markers (such as the HT-29 cell line) and undifferentiated cell lines showing the positive regulation of epithelial-mesenchymal transition and TGFβ signatures (such as the DLD-1, SW480, and SW620 cell lines). We showed that all three of the D. viscosa extract fractions have an antiproliferative effect but the pre-screening on transgenic plants anticipated that the methanolic fraction may be the most promising, targeting the cytoskeleton specifically and possibly resulting in fewer side effects. Here, we show that the preliminary use of screening in transgenic plants expressing subcellular markers can significantly reduce costs and focus the advanced characterization only on the most promising therapeutic molecules.

Malignant Peripheral Nerve Sheath Tumors Activate Distinct Immunosuppressive Pathways Following Radiotherapy and are Associated with Immune Depletion In Vivo

Patients with neurofibromatosis type I, caused by NF1 loss, develop benign plexiform neurofibromas (pNF) in their peripheral nervous system (PNS). Malignant transformation of pNFs into malignant peripheral nerve sheath tumors (MPNSTs) occurs following CDKN2A/B and SUZ12 loss, a process associated with radiotherapy (RT). However, the molecular mechanisms underlying RT responses by different PNS cell types remain unclear. We hypothesized normal peripheral nerve cells, pNFs, and MPNSTs harbor distinct RT responses. Patient derived NF1 WT immortalized peripheral nerve cells (iPNs), NF1 mutant pNF cells, and NF1/CDKN2AB/SUZ12 mutant MPNST cells were used to study RT responses in vitro. CRISPRi was used to assess the functional effects of candidate gene repression. In vitro viability was measured by cell counts. Transcriptomic signatures were measured by bulk RNA-sequencing and integrated with single-cell RNA sequencing (scRNA-seq) data from patient-derived pNF and MPNST resection specimens. Radiation dose response curves revealed pNF cells (IC50 0.61 Gy) were more radiosensitive than MPNST cells (4.15 Gy). WT iPNs, NF1 deficient iPNs, and pNFs cells displayed no difference in cell viability (p = 0.67; t-test) following initiation of 2 Gy x 5 fractions, while MPNST cells were significantly more viable (p = 0.02; t-test). Principal component analysis of bulk RNA-sequencing data at 5 or 14 days following 2 Gy x 5 fractions revealed cell line of origin accounted for the greatest inter-sample variation (64.9% variance), with additional components separating samples based on radiation presence and timing. Using the most variable genes in PCA space to identify markers of RT response, iPNs and pNFs upregulated pro-apoptotic pathways (BAD, DAPK3) at 5 days post-radiation while MPNST cells alone upregulated pro-survival growth factor signaling). At 14 days post radiation, MPNST cells uniquely upregulated TGFβ signaling and interferon response circuits. Incorporation of scRNA-seq data revealed enrichment of growth factor signaling and TGFβ signatures in MPNSTs compared to pNF. Moreover, MPNST harbored significantly fewer immune cells than pNFs (p = 0.008, t-test), suggesting cell-autonomous signaling and crosstalk with the microenvironment are both critical to MPNST radioresistance. Our data indicate additional genetic hits beyond NF1 loss may be required for RT-associated malignant transformation of pNFs and radioresistance in MPNSTs. Analysis of transcriptomic responses to RT suggests that upregulated growth factor signaling and TGFβ-associated immunosuppression are distinct features of MPNST. Future work will focus on CRISPRi screens to unbiasedly nominate functional modifiers of RT response in NF1/CDKN2AB deficient tumors, which may be broadly useful in cancer.

Gene Expressions and High Lymphocyte Count May Predict Durable Clinical Benefits in Patients with Advanced Non-Small-Cell Lung Cancer Treated with Immune Checkpoint Inhibitors.

Not all patients with advanced non-small cell lung cancer (NSCLC) benefit from immune checkpoint inhibitors (ICIs). Therefore, we aimed to assess the predictive potential of gene expression profiling (GEP), peripheral immune cell counts, and clinical characteristics. The primary endpoint of this prospective, observational study was a durable clinical benefit (DCB) defined as progression-free survival >6 months. In a subgroup with histological biopsies of sufficient quality (n = 25), GEP was performed using the nCounter® PanCancer IO 360 panel. DCB was observed in 49% of 123 included patients. High absolute lymphocyte count (ALC) and absence of liver metastases were associated with DCB (OR = 1.95, p = 0.038 and OR = 0.36, p = 0.046, respectively). GEP showed clustering of differentially expressed genes according to DCB, and a strong association between PD-L1 assessed by GEP (CD274) and immunohistochemistry (IHC) was observed (p = 0.00013). The TGF-β, dendritic cell, and myeloid signature scores were higher for patients without DCB, whereas the JAK/STAT loss signature scores were higher for patients with DCB (unadjusted p-values < 0.05). ALC above 1.01 × 109/L and absence of liver metastases were significantly associated with DCB in ICI-treated patients with NSCLC. GEP was only feasible in 20% of the patients. GEP-derived signatures may be associated with clinical outcomes, and PD-L1 could be assessed by GEP rather than IHC.

Identifying a distinct fibrosis subset of NAFLD via molecular profiling and the involvement of profibrotic macrophages

BackgroundThere are emerging studies suggesting that non-alcoholic fatty liver disease (NAFLD) is a heterogeneous disease with multiple etiologies and molecular phenotypes. Fibrosis is the key process in NAFLD progression. In this study, we aimed to explore molecular phenotypes of NAFLD with a particular focus on the fibrosis phenotype and also aimed to explore the changes of macrophage subsets in the fibrosis subset of NAFLD.MethodsTo assess the transcriptomic alterations of key factors in NAFLD and fibrosis progression, we included 14 different transcriptomic datasets of liver tissues. In addition, two single-cell RNA sequencing (scRNA-seq) datasets were included to construct transcriptomic signatures that could represent specific cells. To explore the molecular subsets of fibrosis in NAFLD based on the transcriptomic features, we used a high-quality RNA-sequencing (RNA-seq) dataset of liver tissues from patients with NAFLD. Non-negative matrix factorization (NMF) was used to analyze the molecular subsets of NAFLD based on the gene set variation analysis (GSVA) enrichment scores of key molecule features in liver tissues.ResultsThe key transcriptomic signatures on NAFLD including non-alcoholic steatohepatitis (NASH) signature, fibrosis signature, non-alcoholic fatty liver (NAFL) signature, liver aging signature and TGF-β signature were constructed by liver transcriptome datasets. We analyzed two liver scRNA-seq datasets and constructed cell type-specific transcriptomic signatures based on the genes that were highly expressed in each cell subset. We analyzed the molecular subsets of NAFLD by NMF and categorized four main subsets of NAFLD. Cluster 4 subset is mainly characterized by liver fibrosis. Patients with Cluster 4 subset have more advanced liver fibrosis than patients with other subsets, or may have a high risk of liver fibrosis progression. Furthermore, we identified two key monocyte-macrophage subsets which were both significantly correlated with the progression of liver fibrosis in NAFLD patients.ConclusionOur study revealed the molecular subtypes of NAFLD by integrating key information from transcriptomic expression profiling and liver microenvironment, and identified a novel and distinct fibrosis subset of NAFLD. The fibrosis subset is significantly correlated with the profibrotic macrophages and M2 macrophage subset. These two liver macrophage subsets may be important players in the progression of liver fibrosis of NAFLD patients.

TGF-β based risk model to predict the prognosis and immune features in glioblastoma.

Transforming growth factor-β (TGF-β) is a multifunctional cytokine with an important role in tissue development and tumorigenesis. TGF-β can inhibit the function of many immune cells, prevent T cells from penetrating into the tumor center, so that the tumor cells escape from immune surveillance and lead to low sensitivity to immunotherapy. However, its potential roles in predicting clinical prognosis and tumor microenvironment (TME) immune features need to be deeply investigated in glioblastoma (GBM). The TCGA-GBM dataset was obtained from the Cancer Genome Atlas, and the validation dataset was downloaded from Gene Expression Omnibus. Firstly, differentially expressed TGF-β genes (DEGs) were screened between GBM and normal samples. Then, univariate and multivariate Cox analyses were used to identify prognostic genes and develop the TGF-β risk model. Subsequently, the roles of TGF-β risk score in predicting clinical prognosis and immune characteristics were investigated. The TGF-β risk score signature with an independent prognostic value was successfully developed. The TGF-β risk score was positively correlated with the infiltration levels of tumor-infiltrating immune cells, and the activities of anticancer immunity steps. In addition, the TGF-β risk score was positively related to the expression of immune checkpoints. Besides, the high score indicated higher sensitivity to immune checkpoint inhibitors. We first developed and validated a TGF-β risk signature that could predict the clinical prognosis and TME immune features for GBM. In addition, the TGF-β signature could guide a more personalized therapeutic approach for GBM.

Relationship between tumor microenvironment (TME)-based histomic TGFβ signature (TGFBs), stromal fibroblast recruitment, and exclusion of immune cells as immunotherapy resistance mechanisms.

2585 Background: TGF-beta activates fibroblasts within the TME and may be associated with poor response to immunotherapy.However, objective and reliable assessment of the TGFBs by gene expression profiling (GEP) is challenging. Using H&amp;E whole-slide images (WSI) from The Cancer Genome Atlas (TCGA), we developed an artificial intelligence (AI) model to predict a normalized TGFBs, applying it to real-world datasets (RWDs) of advanced solid tumor patients treated with immunotherapy. Methods: The TGFBs was defined using the mean GEP values of the 'Hallmark TGF-beta signaling' gene set. H&amp;E WSIs of 23 carcinoma types from TCGA (n=6,945) were used to discover the association of the TGFBs with various cell types, including fibroblasts and tumor-infiltrating lymphocytes (TILs) detected by an AI model, Lunit SCOPE IO (AI-1). Another custom AI model (AI-2) to predict TGFBs was developed by integrating self-supervised and supervised features and other semantic content extracted by AI-1 with multilayered perceptron classifiers. TGFBs prediction scores from the AI-2 were applied to 5 different RWDs containing 1,792 immunotherapy-treated patients (&gt; 16 primary tumor types). The immune-excluded phenotype (IEP) was defined by high stromal TIL and low intratumoral TIL density. Results: In the TCGA dataset, TGFBs and fibroblast density detected by AI-1 were closely correlated (R=0.19, p&lt;0.001) compared to other cell types, with both highest in pancreatic adenocarcinoma (PAC)(median [interquartile range, IQR] 0.73 [0.66-0.80], 1306 [985-1588]/mm2, respectively) and CMS4 colorectal cancer (0.61 [0.54-0.67], 868 [582-1128]/mm2) compared to other cancers (0.57 [0.47-0.66], 577 [295-890]/mm2). The AI-2 predicted TGFBs with area-under-the-curve of 0.781 on cross-validation within the TCGA dataset. The TGFBs of immunotherapy-treated RWDs were inferred from their corresponding H&amp;E WSI, with scores comparable to those from the TCGA (0.42 [0.16-0.60] vs 0.58 [0.48-0.67]), with PAC having the highest TGFBs (0.65 [0.45-0.77]) in the RWD as well. Interestingly, TGFBs inferred scores and fibroblast density were significantly correlated (R=0.7, p&lt;0.001) in the RWD and also significantly correlated with the IEP (0.52 [0.30-0.66]) compared to other immune phenotypes (0.42 [0.16-0.60], p&lt;0.001). In the RWD, patients with inferred TGFBs in the upper 75% quartile had a poor clinical response to immunotherapy (objective response rate: 15.1% vs 20.7%, median progression-free survival 3.0 m vs 4.0 m, hazard ratio 1.21, 95% confidence interval 1.07-1.36, p=0.002). Conclusions: TGFBs is closely correlated with fibroblast density within the TME and may confer resistance to immunotherapy. Unique morphologic features of the TGFBs can be captured by AI, enabling scalable application to various datasets for clinical and translational research.

The Immature Morphology of the Megakaryocytes Present in the Bone Marrow of Patients with Myelofibrosis Reflects Changes in the Frequency of Functionally Distinctive Subpopulations

We and others have demonstrated that the immature megakaryocytes (MK) observed in the bone marrow (BM) from patients with myelofibrosis (MF) play a causative role in the establishment of this disease [reviewed in 1]. By leveraging scRNA-seq and other methods, recent studies have identified the existence of at least three megakaryocyte subpopulations, each one exerting different functions [2-4]: the most prevalent is that of platelet producing MK, the second is niche MK (defined here as hematopoietic stem cell (HSC)-supporting for clarity) and the third is classified as immune MK. In addition, the human fetus contains a fourth subpopulation, also defined niche MK, characterized by high expression of extracellular matrix genes, such as COL1A1 and COL3A1, and by a TGF-β signature, which is responsible for shaping the extracellular matrix of the organs during development. Platelet producing MK fall into the high ploidy class, express genes related to thrombopoiesis and are characterized by the CD42b/ARNTL markers. HSC-supporting MKs are mostly >=8N with high expression of IGF1and PF4, which regulate the growth of HSCs, and are characterized by the CD42b/MYLK4 markers. Immune MK are low ploidy with signatures of inflammatory response and myeloid leukocyte activation genes and are characterized as CD41/LSP1 cells. Lastly, niche MK, which are absent in adult BM, are characterized as CD42b/Col I and/or Col III cells. We hypothesize that the immature morphology of the MK from the BM of MF patients flags changes in the relative frequencies of the different MK subpopulations and induction of the formation of niche MK, originally identified in the fetus. To test this hypothesis, we performed extensive confocal microscopy studies with MK-specific markers on the BM from three patients with MF (and three patients with essential thrombocythemia, ET, or with other malignancies but no fibrosis, non-MF, as control) and of the BM from Gata1low mice before (young) and after (old) onset of MF, using age matched wild type (WT) littermates as control (three mice/group). First we confirmed that the BM from MF patients contains greater numbers of MK than that from ET and non-MF patients and that these MF MK have an immature morphology and contain lower levels of GATA1 compared to controls (Fig. 1). We then demonstrated that MF MK are engaged in greater numbers in neutrophils emperipolesis associated with Netosis (Fig. 1), two events predicted to be triggered by the high bioavailability of interleukin-8 in the BM microenvironment of MF patients. Third, we observed that platelet MK (CD42b+ARNTL+) are present in the BM from MF, ET and non-MF patients and from that of WT and Gata1low mice of all ages. By contrast, HSC-supporting MK (CD42b+MYLK4+) are observed in the BM from ET but not in that from MF. They are also observed in the BM from WT mice (both young and old) and from young Gata1low mice, but not in that from old Gata1low mice. Immune MK (CD42b+LSP1+) are instead observed in the BM from ET and non-MF patients but not in MF patients and in the BM from young and old WT mice and in that from young Gata1low mice but not in that from old Gata1low mice. Lastly, great numbers of the CD42b MK in the BM from MF patients, but not in that from ET and non-MF, contain Col I and III (Fig. 1). These cells are also present in the BM from old Gata1low mice but not in their age-matched WT littermates (data not shown) and may represent niche MK the differentiation of which is reactivated by the high TGF-β bioavailability in the BM from MF. In conclusion, these results confirm the hypothesis that changes in frequency of MK subpopulations, rather than in MK maturity state per se, plays a causative role in the development of MF.

Unraveling tumor microenvironment heterogeneity in malignant pleural mesothelioma identifies biologically distinct immune subtypes enabling prognosis determination.

BackgroundMalignant pleural mesothelioma (MPM) is a rare and intractable disease exhibiting a remarkable intratumoral heterogeneity and dismal prognosis. Although immunotherapy has reshaped the therapeutic strategies for MPM, patients react with discrepant responsiveness.MethodsHerein, we recruited 333 MPM patients from 5 various cohorts and developed an in-silico classification system using unsupervised Non-negative Matrix Factorization and Nearest Template Prediction algorithms. The genomic alterations, immune signatures, and patient outcomes were systemically analyzed across the external TCGA-MESO samples. Machine learning-based integrated methodology was applied to identify a gene classifier for clinical application.ResultsThe gene expression profiling-based classification algorithm identified immune-related subtypes for MPMs. In comparison with the non-immune subtype, we validated the existence of abundant immunocytes in the immune subtype. Immune-suppressed MPMs were enriched with stroma fraction, myeloid components, and immunosuppressive tumor-associated macrophages (TAMs) as well exhibited increased TGF-β signature that informs worse clinical outcomes and reduced efficacy of anti-PD-1 treatment. The immune-activated MPMs harbored the highest lymphocyte infiltration, growing TCR and BCR diversity, and presented the pan-cancer immune phenotype of IFN-γ dominant, which confers these tumors with better drug response when undergoing immune checkpoint inhibitor (ICI) treatment. Genetically, BAP1 mutation was most commonly found in patients of immune-activated MPMs and was associated with a favorable outcome in a subtype-specific pattern. Finally, a robust 12-gene classifier was generated to classify MPMs with high accuracy, holding promise value in predicting patient survival.ConclusionsWe demonstrate that the novel classification system can be exploited to guide the identification of diverse immune subtypes, providing critical biological insights into the mechanisms driving tumor heterogeneity and responsible for cancer-related patient prognoses.

Immune microenvironment characteristics in multiple myeloma progression from transcriptome profiling

Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells in the bone marrow (BM). Despite the significant advances in treatment, relapsed and refractory MM has not yet been completely cured due to the immune dysfunction in the tumor microenvironment (TME). In this study, we analyzed the transcriptome data from patients with newly diagnosed (ND) and relapsed/refractory (R/R) MM to characterize differences in the TME and further decipher the mechanism of tumor progression in MM. We observed highly expressed cancer testis antigens and immune suppressive cell infiltration, such as Th2 and M2 cells, are associated with MM progression. Furthermore, the TGF-β signature contributes to the worse outcome of patients with R/R MM. Moreover, patients with ND MM could be classified into immune-low and immune-high phenotypes. Immune-high patients with higher IFN-g signatures are associated with MHC-II–mediated CD4+ T-cell response through CIITA stimulation. The baseline TME status could potentially inform new therapeutic choices for the ND MM who are ineligible for autologous stem cell transplantation and may help predict the response to CAR-T for patients with R/R MM. Our study demonstrates how integrating tumor transcriptome and clinical information to characterize MM immune microenvironment and elucidate potential mechanisms of tumor progression and immune evasion, which will provide insights into MM treatment selection.

HOXB9 mediates resistance to chemotherapy and patient outcomes through the TGFβ pathway in pancreatic cancer.

Background: Although HOXB9 induces tumor proliferation and chemoresistance in several cancer cells, little is known in pancreatic ductal adenocarcinoma (PDAC). In the present study, increased expression of HOXB9 in PDAC was associated with the induction of angiogenic factors and poor overall survival through the TGFβ pathway. Taken together, these results suggested that HOXB9 expression in PDAC could be a surrogate marker in clinical treatment.Methods: In vitro, angiogenic factors, TGFβ signature, Epithelial Mesenchymal Transition (EMT) marker, and chemoresistance were examined in PDAC cell lines by HOXB9 knockdown system. And the reverse effect was confirmed by using TGFβ1 recombinant. Furthermore, in clinical specimens, the correlation between HOXB9 expression and TGFβ signature was analyzed, and the relationship with clinical outcomes were investigated.Results: HOXB9 expression regulated the expression of TGFβ1 signature, angiogenic factors, and EMT markers in vitro, and TGFβ1 recombinant made the reverse effect of these results. And HOXB9 expression regulated the resistance to chemotherapy (Gemcitabine and nab-Paclitaxel) and stem cell population. Moreover, increased HOXB9 expression was significantly associated with poor disease-free survival the prognosis for overall survival. And, a significant positive correlation was observed between HOXB9 expression and several TGFβ signatures in clinical specimens.Conclusions: In conclusion, HOXB9 expression could mediate angiogenesis, EMT, and cancer stemness through the TGFβ pathway, thereby resulting in chemoresistance and poor overall outcomes in patients with pancreatic cancer. Our results suggested that HOXB9 may clinically serve as a novel surrogate biomarker.

The dual role of transforming growth factor-beta signatures in human B viral multistep hepatocarcinogenesis: early and late responsive genes.

Transforming growth factor-beta (TGF-β) has a dichotomous role, functioning as a tumor suppressor and tumor promoter. TGF-β signatures, explored in mouse hepatocytes, have been reported to predict the clinical outcomes of hepatocellular carcinoma (HCC) patients; HCCs exhibiting early TGF-β signatures showed a better prognosis than those with late TGF-β signatures. The expression status of early and late TGF-β signatures remains unclear in defined lesions of human B-viral multistep hepatocarcinogenesis. The expression of TGF-β signatures, early and late responsive signatures of TGF-β were investigated and analyzed for their correlation in cirrhosis, low-grade dysplastic nodules (DNs), high-grade DNs, early HCCs and progressed HCCs (pHCCs) by real-time PCR and immunohistochemistry. The expression levels of TGF-β signaling genes (TGFB1, TGFBR1, TGFBR2 and SMAD4) gradually increased with the progression of hepatocarcinogenesis, peaking in pHCCs. The expression of early responsive genes of TGF-β (GADD45B, FBP1, CYP1A2 and CYP3A4) gradually decreased, and that of the late TGF-β signatures (TWIST and SNAI1) significantly increased according to the progression of multistep hepatocarcinogenesis. Furthermore, mRNA levels of TWIST and SNAI1 were well correlated with those of stemness markers, with upregulation of TGF-β signaling, whereas FBP1 expression was inversely correlated with that of stemness markers. The enrichment of the late responsive signatures of TGF-β with induction of stemness is considered to be involved in the progression of the late stage of multistep hepatocarcinogenesis, whereas the early responsive signatures of TGF-β are suggested to have tumor-suppressive roles in precancerous lesions of the early stage of multistep hepatocarcinogenesis.

Molecular classification of hormone-sensitive and castration-resistant prostate cancer, using nonnegative matrix factorization molecular subtyping of primary and metastatic specimens.

BackgroundDespite the rapidly evolving therapeutic landscape, immunotherapy has demonstrated limited activity in prostate cancer. A greater understanding of the molecular landscape, particularly the expression of immune‐related pathways, will inform future immunotherapeutic strategies. Consensus nonnegative matrix factorization (cNMF) is a novel model of molecular classification analyzing gene expression data, focusing on biological interpretation of metagenes and selecting meaningful clusters.ObjectiveWe aimed to identify molecular subtypes of prostate cancer using cNMF and correlate these with existing biomarkers to inform future immunotherapeutic strategies.MethodsA cohort of archival tumor specimens from hormone‐sensitive and castration‐resistant disease was studied. Whole transcriptomic profiles were generated using TruSeq RNA Access technology and subjected to cNMF. Comprehensive genomic profiling was performed with the FoundationOne assay. NMF subtypes were characterized by gene expression pathways, genomic alterations and correlated with clinical data, then applied to The Cancer Genome Atlas data set.ResultsWe studied 164 specimens, including 52 castration‐resistant and 13 paired primary/metastatic specimens. cNMF identified four distinct subtypes. NMF1 (19%) is enriched for immune‐related and stromal‐related pathways with transforming growth factor β (TGFβ) signature. NMF2 (36%) is associated with FOXO‐mediated transcription signature and AKT signaling, NMF3 (26%) is enriched for ribosomal RNA processing, while NMF4 (19%) is enriched for cell cycle and DNA‐repair pathways. The most common gene alterations included TMPRSS22 (42%), TP53 (23%), and DNA‐repair genes (19%), occurring across all subtypes. NMF4 is significantly enriched for MYC and Wnt‐signaling gene alterations. TMB, CD8 density, and PD‐L1 expression were low overall. NMF1 and NMF4 were NMF2 was associated with superior overall survival.ConclusionsUsing cNMF, we identified four molecularly distinct subtypes which may inform treatment selection. NMF1 demonstrates the most inflammatory signature with asuppressive TGFβ signature, suggesting potential benefit with immunotherapy combination strategies targeting TGFβ and PD‐(L)1. Prospective studies are required to evaluate the use of this novel model to molecularly stratify patients for optimal treatment selection.

Remodeling the tumor microenvironment via blockade of LAIR-1 and TGF-β signaling enables PD-L1-mediated tumor eradication.

Collagens in the extracellular matrix (ECM) provide a physical barrier to tumor immune infiltration, while also acting as a ligand for immune inhibitory receptors. Transforming growth factor-β (TGF-β) is a key contributor to shaping the ECM by stimulating the production and remodeling of collagens. TGF-β activation signatures and collagen-rich environments have both been associated with T cell exclusion and lack of responses to immunotherapy. Here, we describe the effect of targeting collagens that signal through the inhibitory leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) in combination with blockade of TGF-β and programmed cell death ligand 1 (PD-L1). This approach remodeled the tumor collagenous matrix, enhanced tumor infiltration and activation of CD8+ T cells, and repolarized suppressive macrophage populations, resulting in high cure rates and long-term tumor-specific protection across murine models of colon and mammary carcinoma. The results highlight the advantage of direct targeting of ECM components in combination with immune checkpoint blockade therapy.

SMAD4 TGF-β-independent function preconditions naive CD8+ T cells to prevent severe chronic intestinal inflammation.

SMAD4, a mediator of TGF-β signaling, plays an important role in T cells to prevent inflammatory bowel disease (IBD). However, the precise mechanisms underlying this control remain elusive. Using both genetic and epigenetic approaches, we revealed an unexpected mechanism by which SMAD4 prevents naive CD8+ T cells from becoming pathogenic for the gut. Prior to the engagement of the TGF-β receptor, SMAD4 restrains the epigenetic, transcriptional, and functional landscape of the TGF-β signature in naive CD8+ T cells. Mechanistically, prior to TGF-β signaling, SMAD4 binds to promoters and enhancers of several TGF-β target genes, and by regulating histone deacetylation, suppresses their expression. Consequently, regardless of a TGF-β signal, SMAD4 limits the expression of TGF-β negative feedback loop genes, such as Smad7 and Ski, and likely conditions CD8+ T cells for the immunoregulatory effects of TGF-β. In addition, SMAD4 ablation conferred naive CD8+ T cells with both a superior survival capacity, by enhancing their response to IL-7, as well as an enhanced capacity to be retained within the intestinal epithelium, by promoting the expression of Itgae, which encodes the integrin CD103. Accumulation, epithelial retention, and escape from TGF-β control elicited chronic microbiota-driven CD8+ T cell activation in the gut. Hence, in a TGF-β–independent manner, SMAD4 imprints a program that preconditions naive CD8+ T cell fate, preventing IBD.