Patient-derived head and neck cancer organoids allow treatment stratification and serve as a tool for biomarker validation and identification

Background: Organoids are in vitro three-dimensional structures that can be grown from patient tissue. Head and neck cancer (HNC) is a collective term used for multiple tumor types including squamous cell carcinomas and salivary gland adenocarcinomas.Methods: Organoids were established from HNC patient tumor tissue and characterized using immunohistochemistry and DNA sequencing. Organoids were exposed to chemo- and radiotherapy and a panel of targeted agents. Organoid response was correlated with patient clinical response. CRISPR-Cas9-based gene editing of organoids was applied for biomarker validation.Findings: A HNC biobank consisting of 110 models, including 65 tumor models, was generated. Organoids retained DNA alterations found in HNC. Comparison of organoid and patient response to radiotherapy (primary [n = 6] and adjuvant [n = 15]) indicated potential for guiding treatment options in the adjuvant setting. In organoids, the radio-sensitizing potential of cisplatin and carboplatin could be validated. However, cetuximab conveyed radioprotection in most models. HNC-targeted treatments were tested on 31 models, indicating possible novel treatment options with the potential for treatment stratification in the future. Activating PIK3CA mutations did not predict alpelisib response in organoids. Protein arginine methyltransferase 5 (PRMT5) inhibitors were identified as a potential treatment option for cyclin-dependent kinase inhibitor 2A (CDKN2A) null HNC.Conclusions: Organoids hold potential as a diagnostic tool in personalized medicine for HNC. In vitro organoid response to radiotherapy (RT) showed a trend that mimics clinical response, indicating the predictive potential of patient-derived organoids. Moreover, organoids could be used for biomarker discovery and validation.</p

Patient-derived head and neck cancer organoids allow treatment stratification and serve as a tool for biomarker validation and identification

Background: Organoids are in vitro three-dimensional structures that can be grown from patient tissue. Head and neck cancer (HNC) is a collective term used for multiple tumor types including squamous cell carcinomas and salivary gland adenocarcinomas. Methods: Organoids were established from HNC patient tumor tissue and characterized using immunohistochemistry and DNA sequencing. Organoids were exposed to chemo- and radiotherapy and a panel of targeted agents. Organoid response was correlated with patient clinical response. CRISPR-Cas9-based gene editing of organoids was applied for biomarker validation. Findings: A HNC biobank consisting of 110 models, including 65 tumor models, was generated. Organoids retained DNA alterations found in HNC. Comparison of organoid and patient response to radiotherapy (primary [n = 6] and adjuvant [n = 15]) indicated potential for guiding treatment options in the adjuvant setting. In organoids, the radio-sensitizing potential of cisplatin and carboplatin could be validated. However, cetuximab conveyed radioprotection in most models. HNC-targeted treatments were tested on 31 models, indicating possible novel treatment options with the potential for treatment stratification in the future. Activating PIK3CA mutations did not predict alpelisib response in organoids. Protein arginine methyltransferase 5 (PRMT5) inhibitors were identified as a potential treatment option for cyclin-dependent kinase inhibitor 2A (CDKN2A) null HNC. Conclusions: Organoids hold potential as a diagnostic tool in personalized medicine for HNC. In vitro organoid response to radiotherapy (RT) showed a trend that mimics clinical response, indicating the predictive potential of patient-derived organoids. Moreover, organoids could be used for biomarker discovery and validation

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Investigating the innate and adaptive immune response in patients with metastatic colorectal cancer

© 2019 Rosemary Magdalena MillenThe immune response is strongly associated with outcome in CRC (stages I-III). Cytotoxic CD8+ T-cells are the most important subset of immune cells positively associated with outcome, in most solid malignancies and especially CRC. However, in the advanced stage of CRC, this is not always the case. Stage-IV CRC metastasises (mCRC) commonly to the liver, which this thesis addresses. The gold standard for treatment of colorectal liver metastasis (CRLM) is surgical liver resection. Indeed, improvements in surgical techniques have greatly improved the 5-year survival of these patients; however, up to 60% of patients still recur following surgical liver resection. Understanding the progression of mCRC in the context of the immune response is the main focus of this thesis. To investigate the immune response at the primary site of CRC, a unique retrospective cohort of de novo or synchronous mCRC patients (n=109) was explored. Included in this cohort were patients that had microsatellite unstable tumours (MSI) (n=12), which in the metastatic setting have a reduced overall survival (OS) and have been found to respond to checkpoint blockade inhibition (CBI). I analysed the primary tumours of these patients in the context of tumour infiltrating lymphocytes (TILs), immune escape mechanisms and oncogenic potential of these primary tumours, where the patients had synchronous metastatic disease. Despite high frequencies of cytotoxic CD8+ T-cells in some tumours, there was no association with OS, indicating the tumour had surpassed immune control. Expression of PD-L1 >1% on tumour cells was independently correlated with OS in multivariate analysis suggesting that tumour cells have the ability to progress in part at least by evading the immune response. To evaluate the immune response at the metastatic site in the liver, a prospective cohort of CRLM patients was recruited (n=11) to examine the immune context in these tumours. Patients undergoing liver resection were included in the study, and freshly isolated lymphocytes from the tumour; normal liver and peripheral blood were analysed by flow cytometry. These tumours were found to have a reduced infiltration of cytotoxic CD8+ T-cells and increased infiltration of CD4+ T-cells, including T-regulatory cells that are known to suppress immune responses. This immune milieu in these tumours alludes to a reduced cytotoxic and increased immunosuppressive environment. To investigate the functional capacity of these immune cells, a novel immune cytotoxic assay was developed. This assay involved co-culturing patient-derived tumouroids with expanded autologous TILs to assess dynamic interaction of function of these cells. TILs expanded from these tumours were able to kill matched tumouroids, further indicating that when removed from the immunosuppressive TME these cells have functional ability to kill tumouroids. To assess if these TILs respond to CBI, addition of anti-PD-1 antibody was included in the co-culture assays where no improvement in killing was observed. Further investigation of other immune cell subsets in CRLM tumours was undertaken to gain an insight into other immune cell populations that may contribute to the tumour microenvironment (TME). One population of unconventional T-cells abundant in the liver are mucosal-associated invariant T-cells (MAIT cells). These cells play a role in bacterial infections and bridge a gap between innate and adaptive immune responses, and their role in tumour immunity is less defined. These cells are of interest in the context of the TME as they have cytotoxic capability and rapid produce cytokine. When assessing MAIT cell presence in CRLM (n=25) by flow cytometry, MAIT cell frequency was reduced in the tumour compared to surrounding normal liver. The phenotype of MAIT cells in the tissue was phenotypically distinct compared to the periphery, with high expression of PD-1 and CD69, both markers of activation. To assess the potential of MAIT cells, peripheral MAIT cells were isolated from healthy donors and co-cultured with patient-derived tumouroids in an unstimulated and stimulated state. MAIT cells in both states were able to kill patient-derived tumouroids. This is the first documentation of MAIT cell killing with patient-derived material. Despite PD-1 expression on these cells, addition of anti-PD-1 antibody did not enhance this killing. Even though at reduced frequency in the tumour, MAIT cells are activated and may contribute to the tumour microenvironment (TME) in CRLM. Patients with advanced-stage CRC have a reduced survival compared to earlier stage CRC patients. In the context of the immune response, it is evident that these tumours have evaded the immune response to progress to metastasis. The work of this thesis highlights that at the primary tumour site of patients with de novo mCRC, despite high frequencies of cytotoxic CD8+ T-cells, there is an inability at controlling tumour progression. This has likely arisen through immune evasive mechanisms. Therefore there should be a focus on improving the immunogenicity of these tumours to again be recognised by the immune cells. Secondly, at the metastatic site, the TME is immunosuppressed and reinvigorating the function of cytotoxic immune cells present may restore improved immune responses. Importantly, understanding the immune biology of these tumours will provide greater guidance to improve potential immune therapies for these patients into the future

Levels of regulatory B cells do not predict serological responses to hepatitis B vaccine

This study investigated the immunomodulatory influence of IL10 producing B regulatory cells, Bregs (CD19+CD24hiCD38hi) to standard Twinrix® vaccination. We also investigated HBsAg specific T-cell mediated IFN-γ responses to Twinrix® which in theory could provide effective immunity despite low anti-HBs titer. A total of 309 hepatitis B negative health care students and workers completed a standard Twinrix® vaccination schedule (0, 1 and 6 months). Depending on the vaccination response the participants were divided in to non-, low- and high responders according to anti-HBs titer (1000 mIU/mL respectively) two months after completed vaccination schedule. Blood samples from baseline and after vaccination from all non- and low-responders (23 participants) and the same number of high-responders were used for flow cytometric analyses of IL10 producing Bregs and T-cell mediated IFN-γ responses. A decrease in levels of IL10 producing Bregs was observed after vaccination in high responders compared to non- and low-responders. Compiling non-and low-responders against high-responders showed a lower T-cell mediated IFN-γ response at baseline in non-and low-responders when stimulated with Engerix® vaccine. In contrary no positive correlation between IL10 producing Bregs or IFN-γ positive T-cells and anti-HBs titer was observed. Hence this study cannot prove that levels of IL10 producing Bregs or IFN-γ positive T cell affect HBV vaccine response

A practical critique of antifungal treatment guidelines for haemato-oncologists

The management of invasive fungal disease (IFD) in the haemato-oncology setting remains a challenge. This article reviews recent guidelines relating to IFD for their similarities and differences, as well as applying the Appraisal of Guidelines Research and Evaluation (AGREE) criteria. The guidelines’ recommendations on antifungal prophylaxis, empirical and definitive treatment of candidiasis and aspergillosis are summarized; also, minimum standards for diagnosis and follow-up are discussed. This critique of the reviewed guidelines is a practical guide to physicians and commissioners in making local policies for IFD management

Patient-derived micro-organospheres enable clinical precision oncology

Patient-derived xenografts (PDXs) and patient-derived organoids (PDOs) have been shown to model clinical response to cancer therapy. However, it remains challenging to use these models to guide timely clinical decisions for cancer patients. Here, we used droplet emulsion microfluidics with temperature control and dead-volume minimization to rapidly generate thousands of micro-organospheres (MOSs) from low-volume patient tissues, which serve as an ideal patient-derived model for clinical precision oncology. A clinical study of recently diagnosed metastatic colorectal cancer (CRC) patients using an MOS-based precision oncology pipeline reliably assessed tumor drug response within 14 days, a timeline suitable for guiding treatment decisions in the clinic. Furthermore, MOSs capture original stromal cells and allow T cell penetration, providing a clinical assay for testing immuno-oncology (IO) therapies such as PD-1 blockade, bispecific antibodies, and T cell therapies on patient tumors

Rapid tissue prototyping with micro-organospheres

In vitro tissue models hold great promise for modeling diseases and drug responses. Here, we used emulsion microfluidics to form micro-organospheres (MOSs), which are droplet-encapsulated miniature three-dimensional (3D) tissue models that can be established rapidly from patient tissues or cells. MOSs retain key biological features and responses to chemo-, targeted, and radiation therapies compared with organoids. The small size and large surface-to-volume ratio of MOSs enable various applications including quantitative assessment of nutrient dependence, pathogen-host interaction for anti-viral drug screening, and a rapid potency assay for chimeric antigen receptor (CAR)-T therapy. An automated MOS imaging pipeline combined with machine learning overcomes plating variation, distinguishes tumorspheres from stroma, differentiates cytostatic versus cytotoxic drug effects, and captures resistant clones and heterogeneity in drug response. This pipeline is capable of robust assessments of drug response at individual-tumorsphere resolution and provides a rapid and high-throughput therapeutic profiling platform for precision medicine

Uncovering the mode of action of engineered T cells in patient cancer organoids

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a 'super engager' behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies