Patient-derived organoids (PDOs) have recently emerged as robust preclinical models; however, their potential to predict clinical outcomes in patients has remained unclear. We report on a living ...biobank of PDOs from metastatic, heavily pretreated colorectal and gastroesophageal cancer patients recruited in phase 1/2 clinical trials. Phenotypic and genotypic profiling of PDOs showed a high degree of similarity to the original patient tumors. Molecular profiling of tumor organoids was matched to drug-screening results, suggesting that PDOs could complement existing approaches in defining cancer vulnerabilities and improving treatment responses. We compared responses to anticancer agents ex vivo in organoids and PDO-based orthotopic mouse tumor xenograft models with the responses of the patients in clinical trials. Our data suggest that PDOs can recapitulate patient responses in the clinic and could be implemented in personalized medicine programs.
ATR inhibitors (ATRi) are in early phase clinical trials and have been shown to sensitize to chemotherapy and radiotherapy preclinically. Limited data have been published about the effect of these ...drugs on the tumor microenvironment.
We used an immunocompetent mouse model of HPV-driven malignancies to investigate the ATR inhibitor AZD6738 in combination with fractionated radiation (RT). Gene expression analysis and flow cytometry were performed posttherapy.
Significant radiosensitization to RT by ATRi was observed alongside a marked increase in immune cell infiltration. We identified increased numbers of CD3
and NK cells, but most of this infiltrate was composed of myeloid cells. ATRi plus radiation produced a gene expression signature matching a type I/II IFN response, with upregulation of genes playing a role in nucleic acid sensing. Increased MHC I levels were observed on tumor cells, with transcript-level data indicating increased antigen processing and presentation within the tumor. Significant modulation of cytokine gene expression (particularly CCL2, CCL5, and CXCL10) was found
, with
data indicating CCL3, CCL5, and CXCL10 are produced from tumor cells after ATRi + RT.
We show that DNA damage by ATRi and RT leads to an IFN response through activation of nucleic acid-sensing pathways. This triggers increased antigen presentation and innate immune cell infiltration. Further understanding of the effect of this combination on the immune response may allow modulation of these effects to maximize tumor control through antitumor immunity.
Soft tissue sarcomas (STS) are rare and diverse mesenchymal cancers with limited treatment options. Here we undertake comprehensive proteomic profiling of tumour specimens from 321 STS patients ...representing 11 histological subtypes. Within leiomyosarcomas, we identify three proteomic subtypes with distinct myogenesis and immune features, anatomical site distribution and survival outcomes. Characterisation of undifferentiated pleomorphic sarcomas and dedifferentiated liposarcomas with low infiltrating CD3 + T-lymphocyte levels nominates the complement cascade as a candidate immunotherapeutic target. Comparative analysis of proteomic and transcriptomic profiles highlights the proteomic-specific features for optimal risk stratification in angiosarcomas. Finally, we define functional signatures termed Sarcoma Proteomic Modules which transcend histological subtype classification and show that a vesicle transport protein signature is an independent prognostic factor for distant metastasis. Our study highlights the utility of proteomics for identifying molecular subgroups with implications for risk stratification and therapy selection and provides a rich resource for future sarcoma research.
BackgroundTriple negative breast cancer (TNBC) remains a challenge to treat due to its heterogeneous tumour microenvironment, rapid metastasis, and high recurrence rates.1 TNBCs are currently still ...treated with chemo- and radiotherapy, both of which cause significant side effects.1 Interleukin-12 (IL-12) is a powerful anti-tumour cytokine but its use in the clinic is limited by significant toxicity.2 We bioengineered IL-12 by conjugating it to both a collagen-binding domain (CBD) and a mask. The former delivers the IL-12 payload into the collagenous stroma of solid tumours,3whilst the latter temporarily suppresses IL-12 activity in the bloodstream to suppress toxicity. The mask is linked to CBD-IL12 using a protein linker, that is cleaved by proteases upon infiltrating the tumour stroma. The removal of the mask activates masked CBD-IL12 (M-CBD-IL12) in a tumour-specific manner.MethodsM-CBD-IL12 with a protease-sensitive linker was produced in HEK293F cells and purified using immobilised metal affinity and size exclusion chromatography. Murine tumour lysate and serum were used to assess the cleaving of the linker through western blotting and flow cytometry. M-CBD-IL12 was tested in two murine models of TNBC (EMT6/4T1), each with unique immune landscapes. Plasma was collected for analysis of cytokine markers and tumours were analysed using flow cytometry.ResultsM-CBD-IL12 significantly prolonged the survival of mice with orthotopic EMT6 tumours, which are considered more myeloid cell infiltrated. A complete remission was seen in 4 out of 6 mice treated with M-CBD-IL12, each of which remained tumour-free for 284 days following tumour inoculation. In an intervention setting, the IL-12- and M-CBD-IL12-treated mice had significantly smaller tumours than PBS controls. Mice treated with IL-12 or M-CBD-IL12 had significantly higher levels of intra-tumoural IFN-gamma with significantly fewer T regulatory cells. The CD8+ T-cells expressed significantly less PD-1 following M-CBD-IL12; this suggests M-CBD-IL12 reduces immunosuppressive pathways in the EMT6 tumour microenvironment. Despite similar efficacies, the mice treated with M-CBD-IL12 had significantly less plasma IFN-gamma (toxicity marker) than mice treated with IL-12, suggesting the mask and CBD are suppressing IL-12-related toxicity. The 4T1 model of TNBC is thought to be more T-cell inflamed; M-CBD-IL12 did not prolong survival of mice with 4T1 tumours, suggesting a selective efficacy of M-CBD-IL12 for TNBC.ConclusionsM-CBD-IL12 cures murine EMT6 TNBC with low toxicity and prevents its recurrence long-term. Further studies are required to understand the immunological mechanism behind the observed responses.AcknowledgementsWe would like to thank the Cancer Research UK Convergence Science Centre for funding this research and the Institute of Cancer Research Core Facilities for their support.ReferencesMarra A, Trapani D, Viale G, et al. Practical classification of triple-negative breast cancer: intratumoral heterogeneity, mechanisms of drug resistance, and novel therapies. npj Breast Cancer 2020;6:54.Nguyen KG, Vrabel MR, Mantooth SM, et al. Localized Interleukin-12 for Cancer Immunotherapy. Frontiers in Immunology 2020;11.Mansurov A, Ishihara J, Hosseinchi P, et al. Collagen-binding IL-12 enhances tumour inflammation and drives the complete remission of established immunologically cold mouse tumours. Nature Biomedical Engineering 2020;4:531–543.
Previously, we classified colorectal cancers (CRCs) into five CRCAssigner (CRCA) subtypes with different prognoses and potential treatment responses, later consolidated into four consensus molecular ...subtypes (CMS). Here we demonstrate the analytical development and validation of a custom NanoString nCounter platform-based biomarker assay (NanoCRCA) to stratify CRCs into subtypes. To reduce costs, we switched from the standard nCounter protocol to a custom modified protocol. The assay included a reduced 38-gene panel that was selected using an in-house machine-learning pipeline. We applied NanoCRCA to 413 samples from 355 CRC patients. From the fresh frozen samples (n = 237), a subset had matched microarray/RNAseq profiles (n = 47) or formalin-fixed paraffin-embedded (FFPE) samples (n = 58). We also analyzed a further 118 FFPE samples. We compared the assay results with the CMS classifier, different platforms (microarrays/RNAseq) and gene-set classifiers (38 and the original 786 genes). The standard and modified protocols showed high correlation (> 0.88) for gene expression. Technical replicates were highly correlated (> 0.96). NanoCRCA classified fresh frozen and FFPE samples into all five CRCA subtypes with consistent classification of selected matched fresh frozen/FFPE samples. We demonstrate high and significant subtype concordance across protocols (100%), gene sets (95%), platforms (87%) and with CMS subtypes (75%) when evaluated across multiple datasets. Overall, our NanoCRCA assay with further validation may facilitate prospective validation of CRC subtypes in clinical trials and beyond.
•By comprehensively profiling the immune transcriptome of triple-negative breast cancers (TNBCs) and applying machine learning methods, we identified three immunotypes (1, 2 and 3) with significant ...differences in prognosis and therapy responses.•TNBC is much more common in India than in the West. However, there was no significant difference in immune transcriptome between Indian and Western TNBCs in our study.•Our analysis shows biological, signaling and clinical features of three TNBC immunotypes.•Immunotype-1 gene signature is associated with improved prognosis and treatment responses in a cross-cancer comparison analysis of melanoma patients treated with anti-PDL1 therapy.•Our study identified a potential opportunity to stratify patients for MAGEA3 and anti-PDL1-based therapies, which warrants further validation.
Triple-negative breast cancer (TNBC) is a heterogeneous disease with a significant challenge to effectively manage in the clinic worldwide. Immunotherapy may be beneficial to TNBC patients if responders can be effectively identified. Here we sought to elucidate the immune landscape of TNBCs by stratifying patients into immune-specific subtypes (immunotypes) to decipher the molecular and cellular presentations and signaling events of this heterogeneous disease and associating them with their clinical outcomes and potential treatment options.
We profiled 730 immune genes in 88 retrospective Indian TNBC samples using the NanoString platform, established immunotypes using non-negative matrix factorization-based machine learning approach, and validated them using Western TNBCs (n=422; public datasets). Immunotype-specific gene signatures were associated with clinicopathological features, immune cell types, biological pathways, acute/chronic inflammatory responses, and immunogenic cell death processes. Responses to different immunotherapies associated with TNBC immunotypes were assessed using cross-cancer comparison to melanoma (n=504). Tumor-infiltrating lymphocytes (TILs) and pan-macrophage spatial marker expression were evaluated.
We identified three robust transcriptome-based immunotypes in both Indian and Western TNBCs in similar proportions. Immunotype-1 tumors, mainly representing well-known claudin-low and immunomodulatory subgroups, harbored dense TIL infiltrates and T-helper-1 (Th1) response profiles associated with smaller tumors, pre-menopausal status, and a better prognosis. They displayed a cascade of events, including acute inflammation, damage-associated molecular patterns, T-cell receptor-related and chemokine-specific signaling, antigen presentation, and viral-mimicry pathways. On the other hand, immunotype-2 was enriched for Th2/Th17 responses, CD4+ regulatory cells, basal-like/mesenchymal immunotypes, and an intermediate prognosis. In contrast to the two T-cell enriched immunotypes, immunotype-3 patients expressed innate immune genes/proteins, including those representing myeloid infiltrations (validated by spatial immunohistochemistry), and had poor survival. Remarkably, a cross-cancer comparison analysis revealed the association of immunotype-1 with responses to anti-PD-L1 and MAGEA3 immunotherapies.
Overall, the TNBC immunotypes identified in TNBCs reveal different prognoses, immune infiltrations, signaling, acute/chronic inflammation leading to immunogenic cell death of cancer cells, and potentially distinct responses to immunotherapies. The overlap in immune characteristics in Indian and Western TNBCs suggests similar efficiency of immunotherapy in both populations if strategies to select patients according to immunotypes can be further optimized and implemented.
BackgroundRectal cancers show a highly varied response to neoadjuvant radiotherapy/chemoradiation (RT/CRT) and the impact of the tumor immune microenvironment on this response is poorly understood. ...Current clinical tumor regression grading systems attempt to measure radiotherapy response but are subject to interobserver variation. An unbiased and unique histopathological quantification method (change in tumor cell density (ΔTCD)) may improve classification of RT/CRT response. Furthermore, immune gene expression profiling (GEP) may identify differences in expression levels of genes relevant to different radiotherapy responses: (1) at baseline between poor and good responders, and (2) longitudinally from preradiotherapy to postradiotherapy samples. Overall, this may inform novel therapeutic RT/CRT combination strategies in rectal cancer.MethodsWe generated GEPs for 53 patients from biopsies taken prior to preoperative radiotherapy. TCD was used to assess rectal tumor response to neoadjuvant RT/CRT and ΔTCD was subjected to k-means clustering to classify patients into different response categories. Differential gene expression analysis was performed using statistical analysis of microarrays, pathway enrichment analysis and immune cell type analysis using single sample gene set enrichment analysis. Immunohistochemistry was performed to validate specific results. The results were validated using 220 pretreatment samples from publicly available datasets at metalevel of pathway and survival analyses.ResultsΔTCD scores ranged from 12.4% to −47.7% and stratified patients into three response categories. At baseline, 40 genes were significantly upregulated in poor (n=12) versus good responders (n=21), including myeloid and stromal cell genes. Of several pathways showing significant enrichment at baseline in poor responders, epithelial to mesenchymal transition, coagulation, complement activation and apical junction pathways were validated in external cohorts. Unlike poor responders, good responders showed longitudinal (preradiotherapy vs postradiotherapy samples) upregulation of 198 immune genes, reflecting an increased T-cell-inflamed GEP, type-I interferon and macrophage populations. Longitudinal pathway analysis suggested viral-like pathogen responses occurred in post-treatment resected samples compared with pretreatment biopsies in good responders.ConclusionThis study suggests potentially druggable immune targets in poor responders at baseline and indicates that tumors with a good RT/CRT response reprogrammed from immune “cold” towards an immunologically “hot” phenotype on treatment with radiotherapy.
A comprehensive analysis of the immune landscape of pancreatic neuroendocrine tumours (PanNETs) was performed according to clinicopathological parameters and previously defined molecular subtypes to ...identify potential therapeutic vulnerabilities in this disease.
Differential expression analysis of 600 immune-related genes was performed on 207 PanNET samples, comprising a training cohort (n=72) and two validation cohorts (n=135) from multiple transcriptome profiling platforms. Different immune-related and subtype-related phenotypes, cell types and pathways were investigated using different in silico methods and were further validated using spatial multiplex immunofluorescence.
The study identified an immune signature of 132 genes segregating PanNETs (n=207) according to four previously defined molecular subtypes: metastasis-like primary (MLP)-1 and MLP-2, insulinoma-like and intermediate. The MLP-1 subtype (26%-31% samples across three cohorts) was strongly associated with elevated levels of immune-related genes, poor prognosis and a cascade of tumour evolutionary events: larger hypoxic and necroptotic tumours leading to increased damage-associated molecular patterns (viral mimicry), stimulator of interferon gene pathway, T cell-inflamed genes, immune checkpoint targets, and T cell-mediated and M1 macrophage-mediated immune escape mechanisms. Multiplex spatial profiling validated significantly increased macrophages in the MLP-1 subtype.
This study provides novel data on the immune microenvironment of PanNETs and identifies MLP-1 subtype as an immune-high phenotype featuring a broad and robust activation of immune-related genes. This study, with further refinement, paves the way for future precision immunotherapy studies in PanNETs to potentially select a subset of MLP-1 patients who may be more likely to respond.
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