3D genomic mapping reveals multifocality of human pancreatic precancers.

Pancreatic intraepithelial neoplasias (PanINs) are the most common precursors of pancreatic cancer, but their small size and inaccessibility in humans make them challenging to study1. Critically, the number, dimensions and connectivity of human PanINs remain largely unknown, precluding important insights into early cancer development. Here, we provide a microanatomical survey of human PanINs by analysing 46 large samples of grossly normal human pancreas with a machine-learning pipeline for quantitative 3D histological reconstruction at single-cell resolution. To elucidate genetic relationships between and within PanINs, we developed a workflow in which 3D modelling guides multi-region microdissection and targeted and whole-exome sequencing. From these samples, we calculated a mean burden of 13 PanINs per cm3 and extrapolated that the normal intact adult pancreas harbours hundreds of PanINs, almost all with oncogenic KRAS hotspot mutations. We found that most PanINs originate as independent clones with distinct somatic mutation profiles. Some spatially continuous PanINs were found to contain multiple KRAS mutations; computational and in situ analyses demonstrated that different KRAS mutations localize to distinct cell subpopulations within these neoplasms, indicating their polyclonal origins. The extensive multifocality and genetic heterogeneity of PanINs raises important questions about mechanisms that drive precancer initiation and confer differential progression risk in the human pancreas. This detailed 3D genomic mapping of molecular alterations in human PanINs provides an empirical foundation for early detection and rational interception of pancreatic cancer.

Long-Term Postsurgical Outcomes of Neoadjuvant Chemoradiation (CROSS) Versus Chemotherapy (FLOT) for Multimodal Treatment of Adenocarcinoma of the Esophagus and the Esophagogastric Junction.

BACKGROUND: The question of the ideal neoadjuvant therapy for locally advanced esophagogastric adenocarcinoma has not been answered to date. Multimodal treatment has become a standard treatment for these adenocarcinomas. Currently, perioperative chemotherapy (FLOT) or neoadjuvant chemoradiation (CROSS) is recommended. METHODS: A monocentric retrospective analysis compared long-term survival after CROSS versus FLOT. The study enrolled patients with adenocarcinoma of the esophagus (EAC) or the esophagogastric junction type I or II undergoing oncologic Ivor-Lewis esophagectomy between January 2012 and December 2019. The primary objective was to determine the long-term outcome in terms of overall survival. The secondary objectives were to determine differences regarding the histopathologic categories after neoadjuvant treatment and the histomorphologic regression. RESULTS: The findings showed no survival advantage for one or the other treatment in this highly standardized cohort. All the patients underwent open (CROSS: 9.4% vs. FLOT: 22%), hybrid (CROSS: 82% vs. FLOT: 72%), or minimally invasive (CROSS: 8.9% vs. FLOT: 5.6%) thoracoabdominal esophagectomy. The median post-surgical follow-up period was 57.6 months (95% confidence interval [CI] 23.2-109.7 months), and the median survival was longer for the CROSS patients (54 months) than for the FLOT patients (37.2 months) (p = 0.053). The overall 5-years survival was 47% for the entire cohort (48% for the CROSS and 43% for the FLOT patients). The CROSS patients showed a better pathologic response and fewer advanced tumor stages. CONCLUSION: The improved pathologic response after CROSS cannot be translated into longer overall survival. To date, the choice of which neoadjuvant treatment to use can be made only on the basis of clinical parameters and the patient's performance status.

High NANOG expression correlates with worse patients' survival in esophageal adenocarcinoma.

BACKGROUND: Patients diagnosed with esophageal cancer demonstrate a low overall survival even despite the established multimodal therapy as the current standard of care. Therefore, further biomarkers for patients with high-risk and additional therapy options are needed. NANOG is a transcription factor, which can be found in stem cells and is known to support tumorigenesis. METHODS: Six hundred sixty patients with esophageal adenocarcinoma, who were operated at the University of Cologne with a curative intent, were included. Immunohistochemical stainings for NANOG were performed. The study population was divided into NANOG-positive and -negative subgroups. RESULTS: Positive NANOG expression correlates significantly with worse overall survival (p = 0.002) and could be confirmed as an independent risk factor for worse patient survival in multivariate analysis (HR = 1.40, 95%CI = 1.09-1.80, p = 0.006). This effect could be detected in the subgroup of primarily operated patients, but not in patients after neoadjuvant therapy. CONCLUSIONS: We describe a NANOG-positive subgroup of patients with esophageal cancer, who exhibit worse overall survival in a large patient cohort. This discovery suggests the potential use of NANOG as a biomarker for both intensified therapy and stricter follow-up regimes. Additionally, NANOG-positive stem cell-like cancer cells could be used as a new antitumoral treatment target if validated in mechanistic and clinical studies.

Influence of patient sex on outcomes after pancreatic surgery: multicentre study.

BACKGROUND: Recent findings support the hypothesis of sex-related differences in inflammatory and immunological responses to trauma. The aim of this study was to address sex-specific aspects in patients who underwent pancreatic surgery. METHODS: This retrospective study used data from the German StuDoQ registry. Patients who underwent pancreatic surgery between 2010 and 2020 were stratified according to procedure (pancreatic head resection, distal pancreatectomy (DP), total pancreatectomy (TP)). Each cohort underwent propensity score matching (PSM) with the co-variables BMI, ASA, age, coronary heart disease (CHD), diabetes, hypertension with medication, and histology to level the distribution of co-morbidities between men and women. The main outcomes were morbidity and mortality. RESULTS: The total cohort consisted of 10 224 patients (45.3 per cent women). Men had higher ASA grades, and more often had CHD, diabetes, and hypertension with medication. Women had fewer overall complications (57.3 versus 60.1 per cent; P = 0.005) and a lower mortality rate (3.4 versus 4.9 per cent; P < 0.001). Rates of pancreatic surgery-specific complications, such as clinically relevant postoperative pancreatic fistula (POPF) (grade B/C: 14 versus 17 per cent; P < 0.001), delayed gastric emptying (grade B/C: 7.8 versus 9.2 per cent; P = 0.014), and postpancreatectomy haemorrhage (grade B/C: 7.1 versus 9.0 per cent; P < 0.001), were also lower in women. After PSM, 8358 patients were analysed. In the pancreatic head resection cohort (5318 patients), women had fewer complications (58.6 versus 61.4 per cent; P = 0.044), a lower in-hospital mortality rate (3.6 versus 6.1 per cent; P < 0.001), and less often had clinically relevant POPF (11.6 versus 16.2 per cent; P < 0.001). After DP, the clinically relevant POPF rate was lower in women (22.5 versus 27.3 per cent; P = 0.012). In the TP cohort, men more often developed intra-abdominal abscess requiring drainage (5.0 versus 2.3 per cent; P = 0.050). CONCLUSION: Women had favourable outcomes after pancreatic surgery.

Tumor biology and multidisciplinary strategies of oligometastasis in gastrointestinal cancers.

More than 70% of gastrointestinal (GI) cancers are diagnosed with metastases, leading to poor prognosis. For some cancer patients with limited sites of metastatic tumors, the term oligometastatic disease (OMD) has been coined as opposed to systemic polymetastasis (PMD) disease. Stephan Paget first described an organ-specific pattern of metastasis in 1889, now known as the "seed and soil" theory where distinct cancer types are found to metastasize to different tumor-specific sites. Our understanding of the biology of tumor metastasis and specifically the molecular mechanisms driving their formation are still limited, in particular, as it relates to the genesis of oligometastasis. In the following review, we discuss recent advances in general understanding of this metastatic behavior including the role of specific signaling pathways, various molecular features and biomarkers, as well as the interaction of carcinoma cells with their tissue microenvironments (both primary and metastatic niches). The unique features that underlie OMD provide potential targets for localized therapy. As it relates to clinical practice, OMD is emerging as treatable with surgical resection and/or other local therapy options. Strategies currently being applied in the clinical management of OMD will be discussed including surgical, radiation-based therapy, ablation procedures, and the results of emerging clinical trials involving immunotherapy.

Study protocol of an open-label, single arm phase II trial investigating the efficacy, safety and quality of life of neoadjuvant chemotherapy with liposomal irinotecan combined with Oxaliplatin and 5-fluorouracil/Folinic acid followed by curative surgical resection in patients with hepatic Oligometastatic adenocarcinoma of the pancreas (HOLIPANC).

BACKGROUND: According to current guidelines, treatment of patients with hepatic oligometastasis in pancreatic cancer is not reflected and systemic chemotherapy is recommended in those patients. Retrospective data suggest beneficial outcomes in patients with hepatic oligometastasis, though prospective data from clinical trials addressing this particular patient group is not available. METHODS: In this single arm, phase-2 trial, survival data from patients receiving neoadjuvant chemotherapy followed by R0/R1 resection will be compared to historic data from patients with oligometastatic adenocarcinoma of the pancreas. The clinical trial will focus on a well-defined patient collective with metastatic load limited to the liver as target organ with a maximum of five metastases. The combination of liposomal irinotecan (nal-IRI), oxaliplatin (OX) and 5-fluouracil (5-FU)/folinic acid (FA) (nal-IRI + OX+ 5-FU/FA, NAPOX) was chosen as neoadjuvant chemotherapy; the choice was based on an ongoing clinical study in which NAPOX appeared manageable, with promising anti-tumor activity in first-line treatment of patients with metastatic pancreatic adenocarcinoma. In total 150 patients will be enrolled for this trial with an aim of 55 patients receiving a complete macroscopic synchronous tumor and metastatic resection. DISCUSSION: This is the first clinical study to prospectively evaluate the value of multimodality therapy concepts in oligometastatic pancreatic cancer. TRIAL REGISTRATION NUMBERS: EudraCT 2019-002734-37 ; NCT04617457 .

Proposal for a definition of "Oligometastatic disease in pancreatic cancer".

BACKGROUND: To date, patients with metastasized pancreatic ductal adenocarcinoma (PDAC M1) are regarded as a uniform collective. We hypothesize the existence of oligometastatic disease (OMD): a state of PDAC M1 disease with better tumor biology, limited metastasis, and increased survival. METHODS: Data of 128 PDAC M1 patients treated at the University of Cologne between 2008 and 2018 was reviewed. Interdependence between clinical parameter was calculated using the Mann-Whitney U-Test. Survival curves were generated using the Kaplan-Meier method and analyzed using the log-rank test. RESULTS: Eighty-one (63%) patients had metastases confined to one organ (single organ metastasis, SOG) whereas the remaining 47 (37%) showed multiple metastatic sites (multi-organ metastasis, MOG). Survival analysis revealed a median overall survival (OS) of 12.2 months for SOG vs 4.5 months for MOG (95% CI 5.7-9.8; p < 0.001). We defined limited disease by the presence of ≤4 metastases in liver or lung. Limited disease together with CA 19-9 baseline < 1000 U/ml and response or stable disease after first-line chemotherapy defined OMD. We identified 8 patients with hepatic metastases and 2 with pulmonary metastases matching all OMD criteria. This group of 10 (7.8%) had a median overall survival of 19.4 vs 7.2 months compared to the remaining patients (95% CI 5.7-9.8; p = 0.009). CONCLUSION: We propose a definition of oligometastatic disease in PDAC including anatomical criteria and biological criteria reflecting better tumor biology. The 10 OMD patients (7.8%) survived significantly longer and might even benefit from surgical resection in the future.

Upregulation of the histone γ-H2AX correlates with worse patient survival and basal-like subtype in pancreatic ductal adenocarcinoma.

PURPOSE: Patients with pancreatic ductal adenocarcinoma (PDAC) have yet to experience significant benefits from targeted therapy. Olaparib is currently the only active substance in BRCA-mutated PDACs that successfully influences the DNA repair of carcinoma cells. H2AX belongs to the histone family and is known as a part of the DNA repair system. The inhibition of γ-H2AX could lead to the inhibition of mitotically active tumor cells. Therefore, we aimed to evaluate the predictive value of the γ-H2AX in patients with PDAC. METHODS: All included patients (n = 311) received a pancreatic resection with curative intention in one of our PANCALYZE study centers. Subsequently, they were enrolled in a standardized follow-up protocol. Immunohistochemical stainings for γ-H2AX were conducted on tissue microarrays. RESULTS: Patients exhibiting high levels of γ-H2AX expression experience more frequent R1 resections, indicating advanced tumor stages in this subgroup. Additionally, patients with high γ-H2AX expression demonstrated significantly poorer survival compared to those with low expression (median OS: 15 vs. 25 months, p < 0.001). In multivariate analyses, high γ-H2AX expression could be identified as an independent risk factor for worse patient survival. Moreover, high γ-H2AX expression could be more frequently observed in the more aggressive basal-like subtype. CONCLUSION: γ-H2AX can be characterized as a predictive biomarker for poorer patient survival. Consequently, upcoming clinical trials focused on the efficacy of targeted therapies influencing the DNA repair system and radiotherapy should evaluate γ-H2AX as a potential biomarker for therapy response. Furthermore, γ-H2AX may serve as a viable target for treatment in the future.

Correlation of primary tumor volume and histopathologic response following neoadjuvant treatment of esophageal adenocarcinoma.

INTRODUCTION: In esophageal cancer, histopathologic response following neoadjuvant therapy and transthoracic esophagectomy is a strong predictor of long-term survival. At the present, it is not known whether the initial tumor volume quantified by computed tomography (CT) correlates with the degree of pathologic regression. METHODS: In a retrospective analysis of a consecutive patient cohort with esophageal adenocarcinoma, tumor volume in CT prior to chemoradiotherapy or chemotherapy alone was quantified using manual segmentation. Primary tumor volume was correlated to the histomorphological regression based on vital residual tumor cells (VRTC) (Cologne regression scale, CRS: grade I, >50% VRTC; grade II, 10-50% VRTC; grade III, <10% VRTC and grade IV, complete response without VRTC). RESULTS: A total of 287 patients, 165 with neoadjuvant chemoradiotherapy according to the CROSS protocol and 122 with chemotherapy according to the FLOT regimen, were included. The initial tumor volume for patients following CROSS and FLOT therapy was measured (CROSS: median 24.8 ml, IQR 13.1-41.1 ml, FLOT: 23.4 ml, IQR 10.6-37.3 ml). All patients underwent an Ivor-Lewis esophagectomy. 180 patients (62.7 %) were classified as minor (CRS I/II) and 107 patients (37.3 %) as major or complete responder (CRS III/IV). The median tumor volume was calculated as 24.2 ml (IQR 11.9-40.3 ml). Ordered logistic regression revealed no significant dependence of CRS from tumor volume (OR = 0.99, p-value = 0.99) irrespective of the type of multimodal treatment. CONCLUSION: The initial tumor volume on diagnostic CT does not aid to differentiate between potential histopathological responders and non-responders to neoadjuvant therapy in esophageal cancer patients. The results emphasize the need to establish other biological markers of prediction.

Extent and clinical significance of the therapy-relevant tight junction protein Claudin 18.2 in pancreatic ductal adenocarcinoma - real-world evidence.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis, wherefore targeted therapies have experienced increasing interest. Zolbetuximab is a novel targeted therapy under investigation in patients with PDAC and targets Claudin 18.2 (CLDN18.2), which is a component of tight junctions and is of significance in various solid tumors. As its role in PDAC is not definitively elucidated, this study aims to clarify the significance of CLDN18.2 expression in PDAC in a real-world setting. METHODS: All patients (n = 309) were recruited at one of the PANCALYZE study centers and received pancreatic resection with curative intention. Paraffin samples were analyzed using an antibody against CLDN18.2, which is known to be comparable to the antibody used by the SPOTLIGHT and GLOW studies. RESULTS: 94 PDACs are positive for CLDN18.2 (30.4 %). Positive CLDN 18.2 expression was associated with significantly better cancer differentiation (p < 0.001). Patients with positive CLDN18.2 expression showed significantly better overall survival when compared to patients with negative expression (median OS: 30 versus 18 months, p = 0.003). Additionally, in multivariable analyses, CLDN18.2 expression was identified as an independent factor for better survival in patients with PDAC (HR = 0.686, 95 %CI = 0.492-0.956, p = 0.026). CONCLUSION: Significant improvement in survival could be demonstrated by adding Zolbetuximab to known chemotherapy regimes in patients with gastro-esophageal junction adenocarcinoma with at least 75 % CLDN18.2 positive cancer cells. Our findings demonstrate, that 30.4 % of the included patients with PDAC would potentially be eligible for therapy with Zolbetuximab in a real-world patient cohort. Results of trials targeting Claudin 18.2 are pending in patients with PDAC.

Therapy of Locally Advanced and Oligometastatic Pancreatic Adenocarcinoma.

Pancreatic adenocarcinoma is a lethal disease, and surgical resection remains the only curative treatment option. Unfortunately, upon primary diagnosis, only 15-20% of all patients with pancreatic ductal adenocarcinoma (PDAC) have localized disease that is eligible for operation. The remainder of patients either have borderline resectable or locally advanced disease or present with distant metastasis. In this review, we present a comprehensive overview regarding the current strategies and future directions in the multimodal therapy of locally advanced and oligometastasized pancreatic adenocarcinoma and discuss the benefit of surgery following neoadjuvant therapy in these patients.

Specific Subtypes of Carcinoma-Associated Fibroblasts Are Correlated with Worse Survival in Resectable Pancreatic Ductal Adenocarcinoma.

PURPOSE: The pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancer entities. Effective therapy options are still lacking. The tumor microenvironment possibly bears further treatment possibilities. This study aimed to describe the expression patterns of four established carcinoma-associated fibroblast (CAFs) markers and their correlation in PDAC tissue samples. METHODS: This project included 321 patients with PDAC who underwent surgery with a curative intent in one of the PANCALYZE study centers. Immunohistochemical stainings for FAP, PDGFR, periostin, and SMA were performed. The expression patterns of each marker were divided into low- and high-expressing CAFs and correlated with patients' survival. RESULTS: Tumors showing SMAhigh-, PeriostinhighSMAhigh-, or PeriostinhighSMAlowPDGFRlowFAPhigh-positive CAFs demonstrated significantly worse survival. Additionally, a high expression of SMA in PDAC tissue samples was shown to be an independent risk factor for worse survival. CONCLUSION: This project identified three subgroups of PDAC with different expression patterns of CAF markers which showed significantly worse survival. This could be the base for the further characterization of the fibroblast subgroups in PDAC and contribute to the development of new targeted therapy options against CAFs.

Cytokeratin 6 identifies basal-like subtypes of pancreatic ductal adenocarcinoma with decreased survival.

PURPOSE: Rising incidence of pancreatic ductal adenocarcinoma (PDAC) bind with insufficient therapy options showcases a great medical challenge. Further biomarkers are required to identify patients, who will benefit from more aggressive therapy. METHODS: 320 patients were included by the PANCALYZE study group. Cytokeratin 6 (CK6) immunohistochemical staining as a putative marker for the basal-like subtype of PDAC was performed. The correlation between CK6 expression patterns and survival data, as well as various markers of the (inflammatory) tumor microenvironment, were analyzed. RESULTS: We divided the study population based on the expression pattern of CK6. Patients with a high CK6 tumor expression had a significantly shorter survival (p = 0.013), confirmed in a multivariate cox regression model. CK6-expression is an independent marker for a decreased overall survival (HR = 1.655, 95% CI 1.158-2.365, p = 0.006). In addition, the CK6-positive tumors showed significantly less plasma cell infiltration and more cancer-associated fibroblasts (CAFs) expressing Periostin and SMA. CONCLUSIONS: CK6 could be considered as an independent biomarker for a shorter overall survival. CK6 is a clinically easily accessible biomarker for the identification of the basal-like subtype of PDAC. Therefore, it could be taken into consideration in deciding for the more aggressive therapy regimes. Prospectively, studies addressing the chemosensitive characteristics of this subtype are required.

Tissue clearing and 3D reconstruction of digitized, serially sectioned slides provide novel insights into pancreatic cancer.

Pancreatic cancer is currently the third leading cause of cancer death in the United States. The clinical hallmarks of this disease include abdominal pain that radiates to the back, the presence of a hypoenhancing intrapancreatic lesion on imaging, and widespread liver metastases. Technologies such as tissue clearing and three-dimensional (3D) reconstruction of digitized serially sectioned hematoxylin and eosin-stained slides can be used to visualize large (up to 2- to 3-centimeter cube) tissues at cellular resolution. When applied to human pancreatic cancers, these 3D visualization techniques have provided novel insights into the basis of a number of the clinical characteristics of this disease. Here, we describe the clinical features of pancreatic cancer, review techniques for clearing and the 3D reconstruction of digitized microscope slides, and provide examples that illustrate how 3D visualization of human pancreatic cancer at the microscopic level has revealed features not apparent in 2D microscopy and, in so doing, has closed the gap between bench and bedside. Compared with animal models and 2D microscopy, studies of human tissues in 3D can reveal the difference between what can happen and what does happen in human cancers.

X-linked inhibitor of apoptosis protein is a prognostic marker for a favorable outcome in three identified subsets in resectable adenocarcinoma of the pancreas.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is currently one of the leading causes of cancer death worldwide. Therefore, building further subgroups as well as enabling individual patient therapy and diagnostics are needed. X-linked inhibitor of apoptosis protein (XIAP) is known to modulate apoptotic and inflammatory pathways. Its expression was found to correlate with patients' survival in other tumor entities. This study aims to examine the role of XIAP in patients with PDAC in relation to the inflammatory microenvironment. METHODS: The PANCALYZE multicenter study group included 257 patients with PDAC. Paraffin-embedded tumor samples were stained immunohistochemically for CD3, CD20, CD38, CD56, CD66b, CD117, and CD163 and XIAP. These stainings were further analyzed digitally with QuPath and survival analyses were done. RESULTS: XIAP-positive patients with T-cell, respectively, neutrophil enriched tumors survived significantly longer compared to XIAP-negative patients (CD3: 37.6 vs. 24.6 months, p = 0.028; CD66b: 34.1 vs. 14.9 months, p = 0.027). Additionally, XIAP-positive patients showed better survival in the lymph node-negative population (48.4 vs. 24.2 months, p = 0.019). Regarding the total population, our findings did not show a correlation between XIAP expression and survival. In multivariate cox regression analyzes XIAP proves to be an independent factor for better survival in the identified subgroups (CD3: p = 0.043; CD66b: p = 0.012, N0: p = 0.040). CONCLUSION: We found XIAP-positive subgroups with significantly better survival in patients with PDAC in T-cell-rich, neutrophil-rich, or lymph node-negative cohorts. This could lead to further individualized cancer treatment with less aggressive therapy protocols for XIAP-positive tumors or more intensive follow-up for XIAP-negative tumors.

Artificial intelligence for tumour tissue detection and histological regression grading in oesophageal adenocarcinomas: a retrospective algorithm development and validation study.

BACKGROUND: Oesophageal adenocarcinoma and adenocarcinoma of the oesophagogastric junction are among the most common malignant epithelial tumours. Most patients receive neoadjuvant therapy before complete tumour resection. Histological assessment after resection includes identification of residual tumour tissue and areas of regressive tumour, data which are used to calculate a clinically relevant regression score. We developed an artificial intelligence (AI) algorithm for tumour tissue detection and tumour regression grading in surgical specimens from patients with oesophageal adenocarcinoma or adenocarcinoma of the oesophagogastric junction. METHODS: We used one training cohort and four independent test cohorts to develop, train, and validate a deep learning tool. The material consisted of histological slides from surgically resected specimens from patients with oesophageal adenocarcinoma and adenocarcinoma of the oesophagogastric junction from three pathology institutes (two in Germany, one in Austria) and oesophageal cancer cohort of The Cancer Genome Atlas (TCGA). All slides were from neoadjuvantly treated patients except for those from the TCGA cohort, who were neoadjuvant-therapy naive. Data from training cohort and test cohort cases were extensively manually annotated for 11 tissue classes. A convolutional neural network was trained on the data using a supervised principle. First, the tool was formally validated using manually annotated test datasets. Next, tumour regression grading was assessed in a retrospective cohort of post-neoadjuvant therapy surgical specimens. The grading of the algorithm was compared with that of a group of 12 board-certified pathologists from one department. To further validate the tool, three pathologists processed whole resection cases with and without AI assistance. FINDINGS: Of the four test cohorts, one included 22 manually annotated histological slides (n=20 patients), one included 62 sides (n=15), one included 214 slides (n=69), and the final one included 22 manually annotated histological slides (n=22). In the independent test cohorts the AI tool had high patch-level accuracy for identifying both tumour and regression tissue. When we validated the concordance of the AI tool against analyses by a group of pathologists (n=12), agreement was 63·6% (quadratic kappa 0·749; p<0·0001) at case level. The AI-based regression grading triggered true reclassification of resected tumour slides in seven cases (including six cases who had small tumour regions that were initially missed by pathologists). Use of the AI tool by three pathologists increased interobserver agreement and substantially reduced diagnostic time per case compared with working without AI assistance. INTERPRETATION: Use of our AI tool in the diagnostics of oesophageal adenocarcinoma resection specimens by pathologists increased diagnostic accuracy, interobserver concordance, and significantly reduced assessment time. Prospective validation of the tool is required. FUNDING: North Rhine-Westphalia state, Federal Ministry of Education and Research of Germany, and the Wilhelm Sander Foundation.

Three-dimensional genomic mapping of human pancreatic tissue reveals striking multifocality and genetic heterogeneity in precancerous lesions.

Pancreatic intraepithelial neoplasia (PanIN) is a precursor to pancreatic cancer and represents a critical opportunity for cancer interception. However, the number, size, shape, and connectivity of PanINs in human pancreatic tissue samples are largely unknown. In this study, we quantitatively assessed human PanINs using CODA, a novel machine-learning pipeline for 3D image analysis that generates quantifiable models of large pieces of human pancreas with single-cell resolution. Using a cohort of 38 large slabs of grossly normal human pancreas from surgical resection specimens, we identified striking multifocality of PanINs, with a mean burden of 13 spatially separate PanINs per cm3 of sampled tissue. Extrapolating this burden to the entire pancreas suggested a median of approximately 1000 PanINs in an entire pancreas. In order to better understand the clonal relationships within and between PanINs, we developed a pipeline for CODA-guided multi-region genomic analysis of PanINs, including targeted and whole exome sequencing. Multi-region assessment of 37 PanINs from eight additional human pancreatic tissue slabs revealed that almost all PanINs contained hotspot mutations in the oncogene KRAS, but no gene other than KRAS was altered in more than 20% of the analyzed PanINs. PanINs contained a mean of 13 somatic mutations per region when analyzed by whole exome sequencing. The majority of analyzed PanINs originated from independent clonal events, with distinct somatic mutation profiles between PanINs in the same tissue slab. A subset of the analyzed PanINs contained multiple KRAS mutations, suggesting a polyclonal origin even in PanINs that are contiguous by rigorous 3D assessment. This study leverages a novel 3D genomic mapping approach to describe, for the first time, the spatial and genetic multifocality of human PanINs, providing important insights into the initiation and progression of pancreatic neoplasia.

Fructose-1,6-bisphosphatase 1 (FBP1) is an independent biomarker associated with a favorable prognosis in esophageal adenocarcinoma.

INTRODUCTION: Despite modern multimodal therapeutic regimens, the prognosis of esophageal adenocarcinoma (EAC) is still poor and there is a lack of biological markers estimating the patients' prognosis. Fructose-1,6-biphosphatase (FBP1) is a key enzyme in gluconeogenesis and is associated with tumor initiation in several cancers. Therefore, this study aims to characterize its implication for EAC patients. METHODS AND MATERIALS: A total of 571 EAC patients who underwent multimodal treatment between 1999 and 2017 were analyzed for FBP1 expression using immunohistochemistry. RESULTS: 82.5% of the EACs show FBP1 expression in the tumor albeit with different intensities categorizing specimens accordingly into score 0 (no expression), score 1 (weak expression), score 2 (moderate expression) and score 3 (strong expression) (score 1 = 25.0%, score 2 = 35.9%, score 3 = 21.5%). Intratumoral FBP1 expression was significantly associated with a better prognosis (p = 0.024). This observation was particularly relevant among patients who received primary surgery without neoadjuvant treatment (p = 0.004). In multivariate analysis, elevated FBP1 expression was an independent biomarker associated with a favorable prognosis. DISCUSSION: Despite being associated with a favorable prognosis, the majority of patients with high FBP1 expression also require individualized therapy options to ensure long-term survival. Recently, it has been shown that the presence of the FBP1 protein increases the sensitivity of pancreatic cancer cells to the bromodomain and extraterminal domain (BET) inhibitor JQ1. CONCLUSION: We described for the first time the prognostic and possibly therapeutic relevance of FBP1 in EAC. The efficiency of the BET inhibitor in EAC should be verified in clinical studies and special attention should be paid to the effects of neoadjuvant therapy on FBP1 expression.