Stratifying esophago-gastric cancer treatment using a patient-derived organoid-based threshold.

BACKGROUND AND AIMS: This study sought to determine the value of patient-derived organoids (PDOs) from esophago-gastric adenocarcinoma (EGC) for response prediction to neoadjuvant chemotherapy (neoCTx). METHODS: Endoscopic biopsies of patients with locally advanced EGC (n = 120) were taken into culture and PDOs expanded. PDOs' response towards the single substances of the FLOT regimen and the combination treatment were correlated to patients' pathological response using tumor regression grading. A classifier based on FLOT response of PDOs was established in an exploratory cohort (n = 13) and subsequently confirmed in an independent validation cohort (n = 13). RESULTS: EGC PDOs reflected patients' diverse responses to single chemotherapeutics and the combination regimen FLOT. In the exploratory cohort, PDOs response to single 5-FU and FLOT combination treatment correlated with the patients' pathological response (5-FU: Kendall's τ = 0.411, P = 0.001; FLOT: Kendall's τ = 0.694, P = 2.541e-08). For FLOT testing, a high diagnostic precision in receiver operating characteristic (ROC) analysis was reached with an AUCROC of 0.994 (CI 0.980 to 1.000). The discriminative ability of PDO-based FLOT testing allowed the definition of a threshold, which classified in an independent validation cohort FLOT responders from non-responders with high sensitivity (90%), specificity (100%) and accuracy (92%). CONCLUSION: In vitro drug testing of EGC PDOs has a high predictive accuracy in classifying patients' histological response to neoadjuvant FLOT treatment. Taking into account the high rate of successful PDO expansion from biopsies, the definition of a threshold that allows treatment stratification paves the way for an interventional trial exploring PDO-guided treatment of EGC patients.

Deep learning trained on lymph node status predicts outcome from gastric cancer histopathology: a retrospective multicentric study.

AIM: Gastric cancer (GC) is a tumour entity with highly variant outcomes. Lymph node metastasis is a prognostically adverse biomarker. We hypothesised that GC primary tissue contains information that is predictive of lymph node status and patient prognosis and that this information can be extracted using deep learning (DL). METHODS: Using three patient cohorts comprising 1146 patients, we trained and validated a DL system to predict lymph node status directly from haematoxylin and eosin-stained GC tissue sections. We investigated the concordance between the DL-based prediction from the primary tumour slides (aiN score) and the histopathological lymph node status (pN). Furthermore, we assessed the prognostic value of the aiN score alone and when combined with the pN status. RESULTS: The aiN score predicted the pN status reaching area under the receiver operating characteristic curves of 0.71 in the training cohort and 0.69 and 0.65 in the two test cohorts. In a multivariate Cox analysis, the aiN score was an independent predictor of patient survival with hazard ratios of 1.5 in the training cohort and of 1.3 and 2.2 in the two test cohorts. A combination of the aiN score and the pN status prognostically stratified patients by survival with p-values <0.05 in logrank tests. CONCLUSION: GC primary tumour tissue contains additional prognostic information that is accessible using the aiN score. In combination with the pN status, this can be used for personalised management of GC patients after prospective validation.

Fast and label-free intraoperative discrimination of malignant pancreatic tissue by attenuated total reflection infrared spectroscopy.

Significance: Pancreatic surgery is a highly demanding and routinely applied procedure for the treatment of several pancreatic lesions. The outcome of patients with malignant entities crucially depends on the margin resection status of the tumor. Frozen section analysis for intraoperative evaluation of tissue is still time consuming and laborious. Aim: We describe the application of fiber-based attenuated total reflection infrared (ATR IR) spectroscopy for label-free discrimination of normal pancreatic, tumorous, and pancreatitis tissue. A pilot study for the intraoperative application was performed. Approach: The method was applied for unprocessed freshly resected tissue samples of 58 patients, and a classification model for differentiating between the distinct tissue classes was established. Results: The developed three-class classification model for tissue spectra allows for the delineation of tumors from normal and pancreatitis tissues using a probability score for class assignment. Subsequently, the method was translated into intraoperative application. Fiber optic ATR IR spectra were obtained from freshly resected pancreatic tissue directly in the operating room. Conclusion: Our study shows the possibility of applying fiber-based ATR IR spectroscopy in combination with a supervised classification model for rapid pancreatic tissue identification with a high potential for transfer into intraoperative surgical diagnostics.

CRISPR/Cas9 Screen in Gastric Cancer Patient-Derived Organoids Reveals KDM1A-NDRG1 Axis as a Targetable Vulnerability.

Viability CRISPR screens have proven indispensable in parsing genome function. However, their application in new, more physiologically relevant culturing systems like patient-derived organoids (PDOs) has been much slower. To probe epigenetic contribution to gastric cancer (GC), the third leading cause of cancer-related deaths worldwide, the first negative selection CRISPR screen in GC PDOs that faithfully preserve primary tumor characteristics is performed. Extensive quality control measurements showing feasibility of CRISPR screens in primary organoid culture are provided. The screen reveals the histone lysine demethylase-1A (KDM1A) to constitute a GC vulnerability. Both genetic and pharmacological inhibition of KDM1A cause organoid growth retardation. Further, it is shown that most of KDM1A cancer-supporting functions center on repression of N-myc downstream regulates gene-1 (NDRG1). De-repression of NDRG1 by KDM1A inhibitors (KDM1Ai) causes inhibition of Wnt signaling and a strong G1 cell cycle arrest. Finally, by profiling 20 GC PDOs, it is shown that NDRG1 upregulation predicts KDM1Ai response with 100% sensitivity and 82% specificity in the tested cohort. Thus, this work pioneers the use of negative selection CRISPR screens in patient-derived organoids, identifies a marker of KDM1Ai response, and accordingly a cohort of patients who may benefit from such therapy.

A Tumor Microenvironment Model of Pancreatic Cancer to Elucidate Responses toward Immunotherapy.

Pancreatic cancer is a devastating malignancy with minimal treatment options. Standard-of-care therapy, including surgery and chemotherapy, is unsatisfactory, and therapies harnessing the immune system have been unsuccessful in clinical trials. Resistance to therapy and disease progression are mediated by the tumor microenvironment, which contains excessive amounts of extracellular matrix and stromal cells, acting as a barrier to drug delivery. There is a lack of preclinical pancreatic cancer models that reconstruct the extracellular, cellular, and biomechanical elements of tumor tissues to assess responses toward immunotherapy. To address this limitation and explore the effects of immunotherapy in combination with chemotherapy, a multicellular 3D cancer model using a star-shaped poly(ethylene glycol)-heparin hydrogel matrix is developed. Human pancreatic cancer cells, cancer-associated fibroblasts, and myeloid cells are grown encapsulated in hydrogels to mimic key components of tumor tissues, and cell responses toward treatment are assessed. Combining the CD11b agonist ADH-503 with anti-PD-1 immunotherapy and chemotherapy leads to a significant reduction in tumor cell viability, proliferation, metabolic activity, immunomodulation, and secretion of immunosuppressive and tumor growth-promoting cytokines.

Robotic Esophagectomy Compared With Open Esophagectomy Reduces Sarcopenia within the First Postoperative Year: A Propensity Score-Matched Analysis.

INTRODUCTION: Sarcopenia is a known risk factor for adverse outcomes after esophageal cancer (EC) surgery. Robot-assisted minimally invasive esophagectomy (RAMIE) offers numerous advantages, including reduced morbidity and mortality. However, no evidence exists to date comparing the development of sarcopenia after RAMIE and open esophagectomy (OE). The objective was to evaluate whether the development of sarcopenia within the first postoperative year after esophagectomy is associated with the surgical approach: RAMIE versus OE. METHODS: A total of 168 patients with EC were analyzed who either underwent total robotic or fully open Ivor Lewis esophagectomy in a propensity score-matched analysis. Sarcopenia was assessed using the skeletal muscle index (cm2/m2) and psoas muscle thickness per height (mm/m) on axial computed tomography scans during the first postoperative year; in total 540 computed tomography scans were evaluated. RESULTS: After 1-to-1 propensity score matching for confounders, 67 patients were allocated to RAMIE and OE groups, respectively. Skeletal muscle index in the OE group was significantly lower compared with the RAMIE group at the third (43.2 ± 7.6 cm2/m2 versus 49.1 ± 6.9 cm2/m2, p = 0.001), sixth (42.7 ± 7.8 cm2/m2 versus 51.5 ± 8.2 cm2/m2, p < 0.001) and ninth (43.0 ± 7.0 cm2/m2 versus 49.9 ± 6.6 cm2/m2, p = 0.015) postoperative month. Similar results were recorded for psoas muscle thickness per height. CONCLUSIONS: To our knowledge, this study is the first to suggest a substantial benefit of RAMIE compared with open esophagectomy in terms of postoperative sarcopenia. These results add further evidence to support the implementation of the robotic approach in multimodal therapy of EC.

Sensitization of Patient-Derived Colorectal Cancer Organoids to Photon and Proton Radiation by Targeting DNA Damage Response Mechanisms.

Pathological complete response (pCR) has been correlated with overall survival in several cancer entities including colorectal cancer. Novel total neoadjuvant treatment (TNT) in rectal cancer has achieved pathological complete response in one-third of the patients. To define better treatment options for nonresponding patients, we used patient-derived organoids (PDOs) as avatars of the patient's tumor to apply both photon- and proton-based irradiation as well as single and combined chemo(radio)therapeutic treatments. While response to photon and proton therapy was similar, PDOs revealed heterogeneous responses to irradiation and different chemotherapeutic drugs. Radiotherapeutic response of the PDOs was significantly correlated with their ability to repair irradiation-induced DNA damage. The classical combination of 5-FU and irradiation could not sensitize radioresistant tumor cells. Ataxia-telangiectasia mutated (ATM) kinase was activated upon radiation, and by inhibition of this central sensor of DNA damage, radioresistant PDOs were resensitized. The study underlined the capability of PDOs to define nonresponders to irradiation and could delineate therapeutic approaches for radioresistant patients.

Combined Systemic Drug Treatment with Proton Therapy: Investigations on Patient-Derived Organoids.

To optimize neoadjuvant radiochemotherapy of pancreatic ductal adenocarcinoma (PDAC), the value of new irradiation modalities such as proton therapy needs to be investigated in relevant preclinical models. We studied individual treatment responses to RCT using patient-derived PDAC organoids (PDO). Four PDO lines were treated with gemcitabine, 5-fluorouracile (5FU), photon and proton irradiation and combined RCT. Therapy response was subsequently measured via viability assays. In addition, treatment-naive PDOs were characterized via whole exome sequencing and tumorigenicity was investigated in NMRI Foxn1nu/nu mice. We found a mutational pattern containing common mutations associated with PDAC within the PDOs. Although we could unravel potential complications of the viability assay for PDOs in radiobiology, distinct synergistic effects of gemcitabine and 5FU with proton irradiation were observed in two PDO lines that may lead to further mechanistical studies. We could demonstrate that PDOs are a powerful tool for translational proton radiation research.

Sensitivity towards HDAC inhibition is associated with RTK/MAPK pathway activation in gastric cancer.

Gastric cancer ranks the fifth most common and third leading cause of cancer-related deaths worldwide. Alterations in the RTK/MAPK, WNT, cell adhesion, TP53, TGFß, NOTCH, and NFκB signaling pathways could be identified as main oncogenic drivers. A combination of altered pathways can be associated with molecular subtypes of gastric cancer. In order to generate model systems to study the impact of different pathway alterations in a defined genetic background, we generated three murine organoid models: a RAS-activated (KrasG12D , Tp53R172H ), a WNT-activated (Apcfl/fl , Tp53R172H ), and a diffuse (Cdh1fl/fl , Apcfl/fl ) model. These organoid models were morphologically and phenotypically diverse, differed in proteome expression signatures and possessed individual drug sensitivities. A differential vulnerability to RTK/MAPK pathway interference based on the different mitogenic drivers and according to the level of dependence on the pathway could be uncovered. Furthermore, an association between RTK/MAPK pathway activity and susceptibility to HDAC inhibition was observed. This finding was further validated in patient-derived organoids from gastric adenocarcinoma, thus identifying a novel treatment approach for RTK/MAPK pathway altered gastric cancer patients.

Efficient Correction of Oncogenic KRAS and TP53 Mutations through CRISPR Base Editing.

KRAS is the most frequently mutated oncogene in human cancer, and its activating mutations represent long-sought therapeutic targets. Programmable nucleases, particularly the CRISPR-Cas9 system, provide an attractive tool for genetically targeting KRAS mutations in cancer cells. Here, we show that cleavage of a panel of KRAS driver mutations suppresses growth in various human cancer cell lines, revealing their dependence on mutant KRAS. However, analysis of the remaining cell population after long-term Cas9 expression unmasked the occurence of oncogenic KRAS escape variants that were resistant to Cas9-cleavage. In contrast, the use of an adenine base editor to correct oncogenic KRAS mutations progressively depleted the targeted cells without the appearance of escape variants and allowed efficient and simultaneous correction of a cancer-associated TP53 mutation. Oncogenic KRAS and TP53 base editing was possible in patient-derived cancer organoids, suggesting that base editor approaches to correct oncogenic mutations could be developed for functional interrogation of vulnerabilities in a personalized manner for future precision oncology applications. SIGNIFICANCE: Repairing KRAS mutations with base editors can be used for providing a better understanding of RAS biology and may lay the foundation for improved treatments for KRAS-mutant cancers.

p57Kip2 imposes the reserve stem cell state of gastric chief cells.

Adult stem cells constantly react to local changes to ensure tissue homeostasis. In the main body of the stomach, chief cells produce digestive enzymes; however, upon injury, they undergo rapid proliferation for prompt tissue regeneration. Here, we identified p57Kip2 (p57) as a molecular switch for the reserve stem cell state of chief cells in mice. During homeostasis, p57 is constantly expressed in chief cells but rapidly diminishes after injury, followed by robust proliferation. Both single-cell RNA sequencing and dox-induced lineage tracing confirmed the sequential loss of p57 and activation of proliferation within the chief cell lineage. In corpus organoids, p57 overexpression induced a long-term reserve stem cell state, accompanied by altered niche requirements and a mature chief cell/secretory phenotype. Following the constitutive expression of p57 in vivo, chief cells showed an impaired injury response. Thus, p57 is a gatekeeper that imposes the reserve stem cell state of chief cells in homeostasis.

Robotic-assisted minimally invasive Ivor Lewis esophagectomy within the prospective multicenter German da Vinci Xi registry trial.

PURPOSE: Robotic-assisted minimally invasive esophagectomy (RAMIE) has become one standard approach for the operative treatment of esophageal tumors at specialized centers. Here, we report the results of a prospective multicenter registry for standardized RAMIE. METHODS: The German da Vinci Xi registry trial included all consecutive patients who underwent RAMIE at five tertiary university centers between Oct 17, 2017, and Jun 5, 2020. RAMIE was performed according to a standard technique using an intrathoracic circular stapled esophagogastrostomy. RESULTS: A total of 220 patients were included. The median age was 64 years. Total minimally invasive RAMIE was accomplished in 85.9%; hybrid resection with robotic-assisted thoracic approach was accomplished in an additional 11.4%. A circular stapler size of ≥28 mm was used in 84%, and the median blood loss and operative time were 200 (IQR: 80-400) ml and 425 (IQR: 335-527) min, respectively. The rate of anastomotic leakage was 13.2% (n=29), whereas the two centers with >70 cases each had rates of 7.0% and 12.0%. Pneumonia occurred in 19.5% of patients, and the 90-day mortality was 3.6%. Cumulative sum analysis of the operative time indicated the end of the learning curve after 22 cases. CONCLUSIONS: High-quality multicenter registry data confirm that RAMIE is a safe procedure and can be reproduced with acceptable leak rates in a multicenter setting. The learning curve is comparably low for experienced robotic surgeons.

Microbiota-dependent activation of the myeloid calcineurin-NFAT pathway inhibits B7H3- and B7H4-dependent anti-tumor immunity in colorectal cancer.

Bacterial sensing by intestinal tumor cells contributes to tumor growth through cell-intrinsic activation of the calcineurin-NFAT axis, but the role of this pathway in other intestinal cells remains unclear. Here, we found that myeloid-specific deletion of calcineurin in mice activated protective CD8+ T cell responses and inhibited colorectal cancer (CRC) growth. Microbial sensing by myeloid cells promoted calcineurin- and NFAT-dependent interleukin 6 (IL-6) release, expression of the co-inhibitory molecules B7H3 and B7H4 by tumor cells, and inhibition of CD8+ T cell-dependent anti-tumor immunity. Accordingly, targeting members of this pathway activated protective CD8+ T cell responses and inhibited primary and metastatic CRC growth. B7H3 and B7H4 were expressed by the majority of human primary CRCs and metastases, which was associated with low numbers of tumor-infiltrating CD8+ T cells and poor survival. Therefore, a microbiota-, calcineurin-, and B7H3/B7H4-dependent pathway controls anti-tumor immunity, revealing additional targets for immune checkpoint inhibition in microsatellite-stable CRC.

Detecting drug resistance in pancreatic cancer organoids guides optimized chemotherapy treatment.

Drug combination therapies for cancer treatment show high efficacy but often induce severe side effects, resulting in dose or cycle number reduction. We investigated the impact of neoadjuvant chemotherapy (neoCTx) adaptions on treatment outcome in 59 patients with pancreatic ductal adenocarcinoma (PDAC). Resections with tumor-free margins were significantly more frequent when full-dose neoCTx was applied. We determined if patient-derived organoids (PDOs) can be used to personalize poly-chemotherapy regimens by pharmacotyping of treatment-naïve and post-neoCTx PDAC PDOs. Five out of ten CTx-naïve PDO lines exhibited a differential response to either the FOLFIRINOX or the Gem/Pac regimen. NeoCTx PDOs showed a poor response to the neoadjuvant regimen that had been administered to the respective patient in 30% of cases. No significant difference in PDO response was noted when comparing modified treatments in which the least effective single drug was removed from the complete regimen. Drug testing of CTx-naïve PDAC PDOs and neoCTx PDOs may be useful to guide neoadjuvant and adjuvant regimen selection, respectively. Personalizing poly-chemotherapy regimens by omitting substances with low efficacy could potentially result in less severe side effects, thereby increasing the fraction of patients receiving a full course of neoadjuvant treatment. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Comparative Analysis of Postoperative Complications after Cytoreductive Surgery and HIPEC in Gastric Cancer.

INTRODUCTION: Patients with advanced gastric cancer (AGC) frequently show peritoneal carcinomatosis (PC). PC reduces life expectancy and quality of life. Cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) has been shown to improve overall survival. Nevertheless, it has been reported that CRS and HIPEC are accompanied by an increase in postoperative complications. The purpose of this study was to investigate the complications associated with CRS and HIPEC and overall and disease-free survival. METHODS: Patients with AGC and PC, who received complete CRS and HIPEC, were included in the HIPEC group (n = 15). Patients with AGC but without PC, who received resection of the primary tumor alone, constituted the control group (n = 43). RESULTS: Patients enrolled in the HIPEC group presented with a median PCI of 7. In comparison with the control group, no differences were found in patient characteristics, risk factors, pathological findings, and operative procedures. Twenty-five percentage of the patients in both groups suffered from serious postoperative complications (CDC ≥3a). Surgical and medical complications, rate of reoperation, and mortality did not differ. Also, the recurrence pattern, median survival, and 1- and 2-year survival rates showed no differences. CONCLUSION: CRS and HIPEC do not lead to an increased postoperative morbidity and mortality in AGC with PC. Albeit the poorer prognosis of patients with PC, survival of both groups was comparable.

Stem Cells, Helicobacter pylori, and Mutational Landscape: Utility of Preclinical Models to Understand Carcinogenesis and to Direct Management of Gastric Cancer.

Several genetic and environmental factors increase gastric cancer (GC) risk, with Helicobacter pylori being the main environmental agent. GC is thought to emerge through a sequence of morphological changes that have been elucidated on the molecular level. New technologies have shed light onto pathways that are altered in GC, involving mutational and epigenetic changes and altered signaling pathways. Using various new model systems and innovative approaches, the relevance of such alterations for the emergence and progression of GC has been validated. Here, we highlight the key strategies and the resulting achievements. A major step is the characterization of epithelial stem cell behavior in the healthy stomach. These data, obtained through new reporter mouse lines and lineage tracing, enabled insights into the processes that control cellular proliferation, self-renewal, and differentiation of gastric stem cells. It has become evident that these cells and pathways are often deregulated in carcinogenesis. Second, insights into how H pylori colonizes gastric glands, directly interacts with stem cells, and alters cellular and genomic integrity, as well as the characterization of tissue responses to infection, provide a comprehensive picture of how this bacterium contributes to gastric carcinogenesis. Third, the development of stem cell- and tissue-specific reporter mice have driven our understanding of the signals and mutations that promote different types of GC and now also enable the study of more advanced, metastasized stages. Finally, organoids from human tissue have allowed insights into gastric carcinogenesis by validating mutational and signaling alterations in human primary cells and opening a route to predicting responses to personalized treatment.

Gastrointestinal cancer organoids-applications in basic and translational cancer research.

Cancer is a major health problem and a leading cause of death worldwide. Early cancer detection and continuous changes in treatment strategies have improved overall patient survival. The recent development of targeted drugs offers new opportunities for personalized cancer treatment. Nevertheless, individualized treatment is accompanied by the need for biomarkers predicting the response of a patient to a certain drug. One of the most promising breakthroughs in recent years that might help to overcome this problem is the organoid technology. Organoid cultures exhibit self-renewal capacity, self-organization, and long-term proliferation, while recapitulating many aspects of their primary tissue. Generated patient-derived organoid (PDO) libraries constitute "living" biobanks, allowing the in-depth analysis of tissue function, development, tumor initiation, and cancer pathobiology. Organoids can be derived from all gastrointestinal tissues, including esophageal, gastric, liver, pancreatic, small intestinal and colorectal tissues, and cancers of these tissues. PDOs are amenable to various techniques, including sequencing analyses, drug screening, targeted therapy testing, tumor microenvironment studies, and genetic engineering capabilities. In this review, we discuss the different applications of gastrointestinal organoids in basic cancer biology and clinical translation.

Tracing oncogene-driven remodelling of the intestinal stem cell niche.

Interactions between tumour cells and the surrounding microenvironment contribute to tumour progression, metastasis and recurrence1-3. Although mosaic analyses in Drosophila have advanced our understanding of such interactions4,5, it has been difficult to engineer parallel approaches in vertebrates. Here we present an oncogene-associated, multicolour reporter mouse model-the Red2Onco system-that allows differential tracing of mutant and wild-type cells in the same tissue. By applying this system to the small intestine, we show that oncogene-expressing mutant crypts alter the cellular organization of neighbouring wild-type crypts, thereby driving accelerated clonal drift. Crypts that express oncogenic KRAS or PI3K secrete BMP ligands that suppress local stem cell activity, while changes in PDGFRloCD81+ stromal cells induced by crypts with oncogenic PI3K alter the WNT signalling environment. Together, these results show how oncogene-driven paracrine remodelling creates a niche environment that is detrimental to the maintenance of wild-type tissue, promoting field transformation dominated by oncogenic clones.

EGFR amplification and outcome in a randomised phase III trial of chemotherapy alone or chemotherapy plus panitumumab for advanced gastro-oesophageal cancers.

OBJECTIVE: Epidermal growth factor receptor (EGFR) inhibition may be effective in biomarker-selected populations of advanced gastro-oesophageal adenocarcinoma (aGEA) patients. Here, we tested the association between outcome and EGFR copy number (CN) in pretreatment tissue and plasma cell-free DNA (cfDNA) of patients enrolled in a randomised first-line phase III clinical trial of chemotherapy or chemotherapy plus the anti-EGFR monoclonal antibody panitumumab in aGEA (NCT00824785). DESIGN: EGFR CN by either fluorescence in situ hybridisation (n=114) or digital-droplet PCR in tissues (n=250) and plasma cfDNAs (n=354) was available for 474 (86%) patients in the intention-to-treat (ITT) population. Tissue and plasma low-pass whole-genome sequencing was used to screen for coamplifications in receptor tyrosine kinases. Interaction between chemotherapy and EGFR inhibitors was modelled in patient-derived organoids (PDOs) from aGEA patients. RESULTS: EGFR amplification in cfDNA correlated with poor survival in the ITT population and similar trends were observed when the analysis was conducted in tissue and plasma by treatment arm. EGFR inhibition in combination with chemotherapy did not correlate with improved survival, even in patients with significant EGFR CN gains. Addition of anti-EGFR inhibitors to the chemotherapy agent epirubicin in PDOs, resulted in a paradoxical increase in viability and accelerated progression through the cell cycle, associated with p21 and cyclin B1 downregulation and cyclin E1 upregulation, selectively in organoids from EGFR-amplified aGEA. CONCLUSION: EGFR CN can be accurately measured in tissue and liquid biopsies and may be used for the selection of aGEA patients. EGFR inhibitors may antagonise the antitumour effect of anthracyclines with important implications for the design of future combinatorial trials.