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The heterogeneity underlying cancer organoid phenotypes is not yet well understood. Here, the authors develop an imaging analysis assay for high throughput phenotypic screening of colorectal organoids that allows to define specific morphological changes that occur following different drug treatments.

Summary: Background: The overall survival of patients with advanced and refractory oesophageal squamous cell carcinoma, mostly aged 65 years and older, is poor. Treatment with PD-1 antibodies showed improved progression-free survival and overall survival. We assessed the safety and efficacy of combined nivolumab and ipilimumab therapy in this population. Methods: This multicentre, open-label, phase 2 trial done in 32 sites in Germany included patients aged 65 years and older with oesophageal squamous cell carcinoma and disease progression or recurrence following first-line therapy. Patients were treated with nivolumab (240 mg fixed dose once every 2 weeks, intravenously) in the safety run-in phase and continued with nivolumab and ipilimumab (nivolumab 240 mg fixed dose once every 2 weeks and ipilimumab 1 mg/kg once every 6 weeks, intravenously). The primary endpoint was overall survival, which was compared with a historical cohort receiving standard chemotherapy in the intention-to-treat population. This study is registered with ClinicalTrials.gov, NCT03416244. Findings: Between March 2, 2018, and Aug 20, 2020, we screened 75 patients with advanced oesophageal squamous cell carcinoma. We enrolled 66 patients (50 [76%] men and 16 [24%] women; median age 70·5 years [IQR 67·0–76·0]), 44 (67%) of whom received combined nivolumab and ipilimumab therapy and 22 (33%) received nivolumab alone. Median overall survival time at the prespecified data cutoff was 7·2 months (95% CI 5·7–12·4) and significantly higher than in a historical cohort receiving standard chemotherapy (p=0·0063). The most common treatment-related adverse events were fatigue (12 [29%] of 42), nausea (11 [26%]), and diarrhoea (ten [24%]). Grade 3–5 treatment-related adverse events occurred in 13 (20%) of 66 patients. Treatment-related death occurred in one patient with bronchiolitis obliterans while on nivolumab and ipilimumab treatment. Interpretation: Patients aged at least 65 years, with advanced oesophageal squamous cell carcinoma might benefit from combined nivolumab and ipilimumab therapy in second-line treatment. Funding: Bristol Myers Squibb.

Monitoring the mutational patterns of solid tumors during cancer therapy is a major challenge in oncology. Analysis of mutations in cell‐free (cf) DNA offers a noninvasive approach to detect mutations that may be prognostic for disease survival or predictive for primary or secondary drug resistance. A main challenge for the application of cfDNA as a diagnostic tool is the diverse mutational landscape of cancer. Here, we developed a flexible end‐to‐end experimental and bioinformatic workflow to analyze mutations in cfDNA using custom amplicon sequencing. Our approach relies on open‐software tools to select primers suitable for multiplex PCR using minimal cfDNA as input. In addition, we developed a robust linear model to identify specific genetic alterations from sequencing data of cfDNA. We used our workflow to design a custom amplicon panel suitable for detection of hotspot mutations relevant for colorectal cancer and analyzed mutations in serial cfDNA samples from a pilot cohort of 34 patients with advanced colorectal cancer. Using our method, we could detect recurrent and patient‐specific mutational patterns in the majority of patients. Furthermore, we show that dynamic changes of mutant allele frequencies in cfDNA correlate well with disease progression. Finally, we demonstrate that sequencing of cfDNA can reveal mechanisms of resistance to anti‐Epidermal Growth Factor Receptor(EGFR) antibody treatment. Thus, our approach offers a simple and highly customizable method to explore genetic alterations in cfDNA.

Abstract Background Advanced esophageal squamous cell cancer (ESCC) is frequently diagnosed in elderly patients. The impact of 2nd line chemotherapy is poorly defined. Recent data demonstrated effectiveness of checkpoint inhibitors in different squamous cell carcinomas. Therefore, we assess combined nivolumab/ipilimumab as 2nd line therapy in elderly ESCC patients. Methods RAMONA is a multicenter open-label phase II trial. The primary objective is to demonstrate a significant survival benefit of nivolumab/ipilimumab in advanced ESCC compared to historical data of standard chemotherapy. Primary endpoint is therefore overall survival (OS). Major secondary objective is the evaluation of tolerability. Time to QoL deterioration will thus be determined as key secondary endpoint. Further secondary endpoints are tumor response, PFS and safety. We aim to recruit a total of n = 75 subjects that have to be > 65 years old. Eligibility is determined by the geriatric status (G8 screening and Deficit Accumulation Frailty Index (DAFI)). A safety assessment will be performed after a 3 cycle run-in phase of nivolumab (240 mg Q2W) to justify escalation for eligible patients to combined nivolumab (240 mg Q2W) and ipilimumab (1 mg/kg Q6W), while the other patients will remain on nivolumab only. RAMONA also includes translational research sub-studies to identify predictive biomarkers, including PD-1 and PD-L1 evaluation at different time points, establishment of organoid cultures and microbiome analyses for response prediction. Discussion The RAMONA trial aims to implement checkpoint inhibitors for elderly patients with advanced ESCC as second line therapy. Novel biomarkers for checkpoint-inhibitor response are analyzed in extensive translational sub-studies. Trial registration EudraCT Number: 2017–002056-86; NCT03416244, registered: 31.1.2018.

Only a small subset of colorectal cancer (CRC) patients benefits from immunotherapies, comprising blocking antibodies (Abs) against checkpoint receptor “programmed-cell-death-1” (PD1) and its ligand (PD-L1), because most cases lack the required mutational burden and neo-antigen load caused by microsatellite instability (MSI) and/or an inflamed, immune cell-infiltrated PD-L1+ tumor microenvironment. Peroxisome proliferator-activated-receptor-gamma (PPARγ), a metabolic transcription factor stimulated by anti-diabetic drugs, has been previously implicated in pre/clinical responses to immunotherapy. We therefore raised the hypothesis that PPARγ induces PD-L1 on microsatellite stable (MSS) tumor cells to enhance Ab-target engagement and responsiveness to PD-L1 blockage. We found that PPARγ-agonists upregulate PD-L1 mRNA/protein expression in human gastrointestinal cancer cell lines and MSS+ patient-derived tumor organoids (PDOs). Mechanistically, PPARγ bound to and activated DNA-motifs similar to cognate PPARγ-responsive-elements (PPREs) in the proximal −2 kb promoter of the human PD-L1 gene. PPARγ-agonist reduced proliferation and viability of tumor cells in co-cultures with PD-L1 blocking Ab and lymphokine-activated killer cells (LAK) derived from the peripheral blood of CRC patients or healthy donors. Thus, metabolic modifiers improved the antitumoral response of immune checkpoint Ab, proposing novel therapeutic strategies for CRC.

Abstract Background In the group of elderly patients (≥70 years) with metastatic pancreatic ductal adenocarcinoma (mPDAC), it is not known who benefits from intensive 1st line nab-paclitaxel/gemcitabine (nab-p/gem) combination chemotherapy or who would rather suffer from increased toxicity. We aim to determine whether treatment individualization by comprehensive geriatric assessments (CGAs) improves functional outcome of the patients. Methods/Design GrantPax is a multicenter, open label phase 4 interventional trial. We use a CGA to stratify elderly patients into three parallel treatment groups (n = 45 per arm): 1) GOGO (nab-p/gem), 2) SLOWGO (gem mono) or 3) FRAIL (best supportive care). After the 1st cycle of chemotherapy (or 4 weeks in FRAIL group) another CGA and safety assessment is performed. CGA-stratified patients may not decline in their CGA performance in response to the first cycle of chemotherapy (primary objective), measured as a loss of 5 points or less in Barthels activities of daily living. Based on the second CGA, patients are re-assigned to their definite treatment arm and undergo further CGAs to monitor the course of treatment. Secondary endpoints include CGA scores during the course of therapy (CGA1–4), response rates, safety and survival rates. Discussion GrantPax is the first trial implementing a CGA-driven treatment to personalize therapy for elderly patients with pancreatic cancer. This may lead to standardization of therapy decisions for elderly patients and may optimize standard of care for this increasing group of patients. Trial registration NCT02812992, registered 24.06.2016.

Deregulation of the insulin‐like growth factor (IGF) axis and dysbalance of components of the IGF system as potential therapeutic targets have been described in different tumor types. IGF2 is a major embryonic growth factor and an important activator of IGF signaling. It is regulated by imprinting in a development‐ and tissue‐dependent manner and has been implicated in a broad range of malignancies including prostate cancer (PCa). Loss of imprinting (LOI) usually results in bi‐allelic gene expression and increased levels of IGF2. However, the regulatory mechanisms and the pathophysiological impact of altered IGF2 expression in PCa remain elusive. Here, we show that in contrast to many other tumors, IGF2 mRNA and protein levels were decreased in 80% of PCa in comparison with non‐neoplastic adjacent prostate and were independent of LOI status. Instead, IGF2 expression in both tumors and adjacent prostate depended on preferential usage of the IGF2 promoters P3 and P4. Decreased IGF2 expression in tumors was strongly related to hypermethylation of these two promoters. Methylation of the A region in promoter P4 correlated specifically with IGF2 expression in the 20% of PCa where IGF2 was higher in tumors than in adjacent prostate. We conclude that IGF2 is downregulated in most PCa and may be particularly relevant during early stages of tumor development or during chemotherapy and androgen deprivation. PCa differs from other tumors in that IGF2 expression is mainly regulated through methylation of promoter‐specific and not by imprinting. Targeting of promoter‐specific regions may have relevance for the adjuvant treatment of PCa.

Docking protein-1 (DOK1) is a tumor suppressor frequently lost in malignant cells, however, it retains the ability to control activities of immune receptors in adjacent stroma cells of the tumor microenvironment. We therefore hypothesized that addressing DOK1 may be useful for cancer immunotherapy. DOK1 mRNA and DOK1 protein expression were downregulated in tumor cells of gastric cancer patients (n = 249). Conversely, its expression was up-regulated in cases positive for Epstein Barr Virus (EBV+) together with genes related to macrophage biology and targets of clinical immunotherapy such as programmed-cell-death-ligand-1 (PD-L1). Notably, high DOK1 positivity in stroma cells conferred poor prognosis in patients and correlated with high levels of inducible nitric oxide synthase in CD68+ tumor-associated macrophages. In macrophages derived from human monocytic leukemia cell lines, DOK1 (i) was inducible by agonists of the anti-diabetic transcription factor peroxisome proliferator-activated receptor-gamma (PPARγ), (ii) increased polarization towards an inflammatory phenotype, (iii) augmented nuclear factor-κB-dependent transcription of pro-inflammatory cytokines and (iv) reduced PD-L1 expression. These properties empowered DOK1+ macrophages to decrease the viability of human gastric cancer cells in contact-dependent co-cultures. DOK1 also reduced PD-L1 expression in human primary blood monocytes. Our data propose that the drugability of DOK1 may be exploited to reprogram myeloid cells and enforce the innate immune response against EBV+ human gastric cancer.

Full-length (FL) docking protein-1 (DOK1) is an adapter protein which inhibits growth factor and immune response pathways in normal tissues, but is frequently lost in human cancers. Small DOK1 variants remain in cells of solid tumors and leukemias, albeit, their functions are elusive. To assess the so far unknown role of DOK1 in colorectal cancer (CRC), we generated DOK1 mutants which mimic the domain structure and subcellular distribution of DOK1 protein variants in leukemia patients. We found that cytoplasmic DOK1 activated peroxisome-proliferator-activated-receptor-gamma (PPARγ) resulting in inhibition of the c-FOS promoter and cell proliferation, whereas nuclear DOK1 was inactive. PPARγ-agonist increased expression of endogenous DOK1 and interaction with PPARγ. Forward translation of this cell-based signaling model predicted compartmentalization of DOK1 in patients. In a large series of CRC patients, loss of DOK1 protein was associated with poor prognosis at early tumor stages (*p = 0.001; n = 1492). In tumors with cytoplasmic expression of DOK1, survival was improved, whereas nuclear localization of DOK1 correlated with poor outcome, indicating that compartmentalization of DOK1 is critical for CRC progression. Thus, DOK1 was identified as a prognostic factor for non-metastatic CRC, and, via its drugability by PPARγ-agonist, may constitute a potential target for future cancer treatments.

To identify novel hubs for cancer immunotherapy, we generated C57BL/6J mice with concomitant deletion of the drugable transcription factor PPARγ and transgenic overexpression of the mutant KRASG12V oncogene in enterocytes. Animals developed epithelial hyperplasia, transmural inflammation and serrated adenomas in the small intestine with infiltration of CD3+ FOXP3+ T-cells and macrophages into the lamina propria of the non-malignant mucosa. Within serrated polyps, CD3+ CD8+ T-cells and phosphorylated ERK1/2 were reduced and the senescence marker P21 and macrophage counts up-regulated, indicative of an immunosuppressive tissue microenvironment. Treatment of mutant KRASG12V mice with the PPARγ-agonist rosiglitazone augmented M1 macrophage numbers, reduced IL4 expression and diminished polyp load in mice. Rosiglitazone also promoted M1 polarisation of human THP1-derived macrophages and decreased Il4 mRNA in isolated murine lymphocytes. Thus, inhibition of the oncogenic driver mutant RAS by PPARγ in epithelial and immune cell compartments may be a future target for the prevention or treatment of human malignancies associated with intestinal inflammation.