Drug screening and genome editing in human pancreatic cancer organoids identifies drug-gene interactions and candidates for off-label treatment.

Pancreatic cancer (PDAC) is a highly aggressive malignancy for which the identification of novel therapies is urgently needed. Here, we establish a human PDAC organoid biobank from 31 genetically distinct lines, covering a representative range of tumor subtypes, and demonstrate that these reflect the molecular and phenotypic heterogeneity of primary PDAC tissue. We use CRISPR-Cas9 genome editing and drug screening to characterize drug-gene interactions with ARID1A and BRCA2. We find that missense- but not frameshift mutations in the PDAC driver gene ARID1A are associated with increased sensitivity to the kinase inhibitors dasatinib (p < 0.0001) and VE-821 (p < 0.0001). We conduct an automated drug-repurposing screen with 1,172 FDA-approved compounds, identifying 26 compounds that effectively kill PDAC organoids, including 19 chemotherapy drugs currently approved for other cancer types. We validate the activity of these compounds in vitro and in vivo. The in vivo validated hits include emetine and ouabain, compounds which are approved for non-cancer indications and which perturb the ability of PDAC organoids to respond to hypoxia. Our study provides proof-of-concept for advancing precision oncology and identifying candidates for drug repurposing via genome editing and drug screening in tumor organoid biobanks.

De novo expression of gastrokines in pancreatic precursor lesions impede the development of pancreatic cancer.

Molecular events occurring in stepwise progression from pre-malignant lesions (pancreatic intraepithelial neoplasia; PanIN) to the development of pancreatic ductal adenocarcinoma (PDAC) are poorly understood. Thus, characterization of early PanIN lesions may reveal markers that can help in diagnosing PDAC at an early stage and allow understanding the pathology of the disease. We performed the molecular and histological assessment of patient-derived PanINs, tumor tissues and pancreas from mouse models with PDAC (KC mice that harbor K-RAS mutation in pancreatic tissue), where we noted marked upregulation of gastrokine (GKN) proteins. To further understand the role of gastrokine proteins in PDAC development, GKN-deficient KC mice were developed by intercrossing gastrokine-deficient mice with KC mice. Panc-02 (pancreatic cancer cells of mouse origin) were genetically modified to express GKN1 for further in vitro and in vivo analysis. Our results show that gastrokine proteins were absent in healthy pancreas and invasive cancer, while its expression was prominent in low-grade PanINs. We could detect these proteins in pancreatic juice and serum of KC mice. Furthermore, accelerated PanIN and tumor development were noted in gastrokine deficient KC mice. Loss of gastrokine 1 protein delayed apoptosis during carcinogenesis leading to the development of desmoplastic stroma while loss of gastrokine 2 increased the proliferation rate in precursor lesions. In summary, we identified gastrokine proteins in early pancreatic precursor lesions, where gastrokine proteins delay pancreatic carcinogenesis.

ß6-Integrin Serves as a Potential Serum Marker for Diagnosis and Prognosis of Pancreatic Adenocarcinoma.

INTRODUCTION: Despite enormous efforts during the past decades, pancreatic adenocarcinoma (PAC) remains one of the most deleterious cancer entities. A useful biomarker for early detection or prognosis of PAC does not yet exist. The goal of our study was the characterization of ß6-integrin (ITGB6) as a novel serum tumor marker for refined diagnosis and prognosis of PAC. Serum ITGB6 levels were analyzed in 3 independent PAC cohorts consisting of retrospectively and prospectively collected serum and/or (metastatic) PAC tissue specimens. METHODS: Using 2 independent cohorts, we measured serum ITGB6 concentrations in 10 chronic pancreatitis patients, 10 controls, as well as in 27 (cohort 1) and 24 (cohort 2) patients with PAC, respectively. In these patients, we investigated whether ITGB6 serum levels correlate with known clinical and prognostic markers for PAC and whether they might differ between patients with PAC or benign inflammatory diseases of the pancreas. RESULTS: We found that elevated serum ITGB6 levels (≥0.100 ng/mL) in patients suffering from metastasizing PAC presented an unfavorable prognostic outcome. By correlating the ITGB6 tissue expression in primary and metastatic PAC with clinical parameters, we found that positive ITGB6 expression in the tumor tissue is linked to increased serum ITGB6 levels in nonmetastatic PAC and correlates with carbohydrate antigen 19-9 and clinical outcome. DISCUSSION: Our findings suggest that ITGB6 might serve as a novel serum biomarker for early diagnosis and prognosis of PAC. Given the limited specificity and sensitivity of currently used carbohydrate antigen 19-9-based assays, ITGB6 may have the potential to improve the diagnostic accuracy for PAC.

Pancreatic fistulas following distal pancreatectomy are unrelated to the texture quality of the pancreas.

PURPOSE: The relevance of pancreatic texture for pancreatic fistula (POPF) formation after distal pancreatectomy (DP) remains ill defined. Recent POPF definition adjustments and common subjective pancreatic texture assessment are further drawbacks in the investigation of pancreatic texture as a factor for POPF development after DP. METHODS: The predictive value of pancreatic texture by histologic assessment was investigated for POPF formation after DP, respecting the updated 2016 fistula definition. Histologic evaluation at the resection margin included amount of steatosis, degree of fibrosis, and pancreatic duct size. RESULTS: A total of 102 patients who underwent DP were included. Thirty-six patients developed POPF. There was no difference in histologic variables in patients with and without POPF. In the univariate analysis, none of the three histologic features showed significant correlation with POPF formation. The ROC (receiver operating characteristic) curve demonstrated poor utility for the grade of steatosis 0.481 ± 0.058 (p = 0.75) and grade of fibrosis 0.466 ± 0.058 (p = 0.57) as predictive factors for POPF formation. CONCLUSION: Results indicate that pancreatic texture does not predict POPF formation following DP. This is particularly relevant in the context of the increasing use of robotic and laparoscopic approaches for DPs with limited clinical pancreatic texture assessment by palpation.

Prospective Evaluation of Residual Breast Tissue After Skin- or Nipple-Sparing Mastectomy: Results of the SKINI-Trial.

OBJECTIVE: This study was designed to investigate the presence of residual breast tissue (RBT) after skin-sparing mastectomy (SSM) and nipple-sparing mastectomy (NSM) and to analyse patient- and therapy-related factors associated with RBT. Skin-sparing mastectomy and NSM are increasingly used surgical procedures. Prospective data on the completeness of breast tissue resection is lacking. However, such data are crucial for assessing oncologic safety of risk-reducing and curative mastectomies. METHODS: Between April 2016 and August 2017, 99 SSM and 61 NSM were performed according to the SKINI-trial protocol, under either curative (n = 109) or risk-reducing (n = 51) indication. After breast removal, biopsies from the skin envelope (10 biopsies per SSM, 14 biopsies per NSM) were taken in predefined radial localizations and assessed histologically for the presence of RBT and of residual disease. RESULTS: Residual breast tissue was detected in 82 (51.3%) mastectomies. The median RBT percentage per breast was 7.1%. Of all factors considered, only type of surgery (40.4% for SSM vs. 68.9% for NSM; P < 0.001) and surgeon (P < 0.001) were significantly associated with RBT. None of the remaining factors, e.g., skin flap necrosis, was associated significantly with RBT. Residual disease was detected in three biopsies. CONCLUSIONS: Residual breast tissue is commonly observed after SSM and NSM. In contrast, invasive or in situ carcinomas are rarely found in the skin envelope. Radicality of mastectomy in this trial is not associated with increased incidence of skin flap necrosis. ClinicalTrials.gov Identifier NCT03470909.

Preoperative Evaluation of Pancreatic Fibrosis and Lipomatosis: Correlation of Magnetic Resonance Findings With Histology Using Magnetization Transfer Imaging and Multigradient Echo Magnetic Resonance Imaging.

OBJECTIVES: The purpose of this study was to evaluate the diagnostic performance of magnetization transfer (MT) imaging and multigradient echo magnetic resonance imaging (MRI) to quantify pancreatic fibrosis and lipomatosis in patients before pancreatoduodenectomy for postoperative risk stratification with histopathology as the reference standard. MATERIALS AND METHODS: Twenty-four patients (age, 68 ± 8 years, 16 males) prospectively underwent quantitative MT imaging using a 2-dimensional gradient echo sequence with and without MT prepulse and multigradient echo imaging on a 3 T MRI 1 day before pancreatoduodenectomy due to adenocarcinoma of the pancreatic head region (n = 20), neuroendocrine tumor (n = 3), or intraductal papillary mucinous neoplasm (n = 1). Magnetization transfer ratio (MTR) and proton density fat fraction (PDFF) were measured in pancreatic tail (PT) and at the resection margin (RM). Histopathologically, pancreatic fibrosis was graded as mild, moderate, or severe (F1-F3), lipomatosis was graded as 0% to 10%, 11% to 30%, and greater than 30% fat deposition (L1-L3). In addition, MTR and histopathologic fibrosis was assessed in pancreatic adenocarcinoma. Mann-Whitney U test and Spearman correlation were used. RESULTS: Patients with advanced pancreatic fibrosis (F3) showed a significantly higher MTR compared with the F1 group at the RM and PT (38 ± 4 vs 32.3 ± 1.6, P = 0.018 and 39.7 ± 5.5 vs 31.2 ± 1.7, P = 0.001). Spearman correlation coefficient of MTR and fibrosis grade was r = 0.532 (P = 0.011) and 0.554 (P = 0.008), respectively. Pancreatic parenchyma with advanced fat deposition (L2-L3) showed significantly higher PDFF compared with lipomatosis grade L1 (RM: P = 0.002 and PT: P = 0.001). Proton density fat fraction of pancreatic parenchyma exhibited a moderate and significant correlation with histopathologic lipomatosis grade (RM: r = 0.668 and PT: r = 0.707, P < 0.001). Magnetization transfer ratio was significantly higher in pancreatic adenocarcinoma compared with pancreatic parenchyma (44 ± 5.5 vs 36.0 ± 4.4 and 37.4 ± 5.4, P = 0.004). CONCLUSIONS: Multiparametric MRI of the pancreas including MTR and PDFF maps may provide quantitative and noninvasive information on pancreatic fibrosis and lipomatosis before surgery.

Value of histomorphometric tumour thickness and smoothelin for conventional m-classification in early oesophageal adenocarcinoma.

AIM: To test the validity of tumour thickness measurement in distinguishing between the different infiltration depths, especially when the duplication of muscularis mucosae cannot be demarcated clearly. METHODS: We re-evaluated 100 completely embedded Barrett's adenocarcinomas regarding m-classification, maximum tumour thickness, and muscularis mucosae duplication. For validation, smoothelin staining was performed on a subset of cases. RESULTS: The m1-, m2- and m3-classified adenocarcinomas showed a significant lower tumour thickness compared to the m4- and sm1-classified lesions (P < 0.001). Smoothelin staining determined a clear muscularis mucosae duplication in 64% of the tested samples and enabled the differentiation of the two layers in diffuse and merged splits. CONCLUSION: Tumour thickness in early oesophageal adenocarcinoma significantly correlates with the depth of infiltration and demonstrates its worth as an accurate pT classification in non-polypoid lesions. We created a new algorithm, which combines histomorphology with morphometric analyses. It is noteworthy that it facilitates the assessment of mucosal vs submucosal infiltration depth. The smoothelin staining strengthened our results of the tumour thickness evaluation and can be used in cases of doubt.

Lymph node size as a simple prognostic factor in node negative colon cancer and an alternative thesis to stage migration.

BACKGROUND: Stage migration is an accepted explanation for the association between lymph node (LN) yield and outcome in colon cancer. To investigate whether the alternative thesis of immune response is more likely, we performed a retrospective study. METHODS: We enrolled 239 cases of node negative cancers, which were categorized according to the number of LNs with diameters larger than 5 mm (LN5) into the groups LN5-very low (0 to 1 LN5), LN5-low (2 to 5 LN5), and LN5-high (≥6 LN5). RESULTS: Significant differences were found in pT3/4 cancers with median survival times of 40, 57, and 71 months (P = .022) in the LN5-very low, LN5-low, and LN5-high groups, respectively. Multivariable analysis revealed that LN5 number and infiltration type were independent prognostic factors. CONCLUSIONS: LN size is prognostic in node negative colon cancer. The correct explanation for outcome differences associated with LN harvest is probably the activation status of LNs.

Specific immunohistochemical pattern of carbonic anhydrase IX is helpful for the diagnosis of CNS hemangioblastoma.

Hemangioblastomas are rare capillary-rich tumors predominantly found in the CNS. The histological appearance of these tumors varies across a broad spectrum. Several entities show considerable histomorphological similarities to hemangioblastomas. Therefore, morphological evaluation can be challenging. In this study, we evaluated the diagnostic utility of immunohistochemistry using antibodies against carbonic anhydrase IX and cytokeratin staining. Within our files, we identified 20 hemangioblastomas. A collection of 46 other tumors relevant to the differential diagnosis (12 pilocytic astrocytomas, 11 meningiomas, one pleomorphic xanthoastrocytoma, one angiomatous fibrous histiocytoma, 14 carcinoma metastases and seven gliomas grades II-IX) served as control. The pattern of strong, diffuse expression of carbonic anhydrase IX with membranous accentuation in combination with keratin negativity was considered diagnostic for hemangioblastomas. It was found in 18 out of 20 (90%) hemangioblastomas and in none of the control cases (P < 0.001). This resulted in a sensitivity of 90% and a specificity of 100%. The positive and negative predictive values were 100% and 96%, respectively. Carbonic anhydrase IX with cytokeratin is thus a highly sensitive and specific marker combination for hemangioblastomas. It is therefore very helpful in the diagnosis of these tumors and in their discrimination from other entities.

Integrative analysis of head and neck cancer identifies two biologically distinct HPV and three non-HPV subtypes.

PURPOSE: Current classification of head and neck squamous cell carcinomas (HNSCC) based on anatomic site and stage fails to capture biologic heterogeneity or adequately inform treatment. EXPERIMENTAL DESIGN: Here, we use gene expression-based consensus clustering, copy number profiling, and human papillomavirus (HPV) status on a clinically homogenous cohort of 134 locoregionally advanced HNSCCs with 44% HPV(+) tumors together with additional cohorts, which in total comprise 938 tumors, to identify HNSCC subtypes and discover several subtype-specific, translationally relevant characteristics. RESULTS: We identified five subtypes of HNSCC, including two biologically distinct HPV subtypes. One HPV(+) and one HPV(-) subtype show a prominent immune and mesenchymal phenotype. Prominent tumor infiltration with CD8(+) lymphocytes characterizes this inflamed/mesenchymal subtype, independent of HPV status. Compared with other subtypes, the two HPV subtypes show low expression and no copy number events for EGFR/HER ligands. In contrast, the basal subtype is uniquely characterized by a prominent EGFR/HER signaling phenotype, negative HPV-status, as well as strong hypoxic differentiation not seen in other subtypes. CONCLUSION: Our five-subtype classification provides a comprehensive overview of HPV(+) as well as HPV(-) HNSCC biology with significant translational implications for biomarker development and personalized care for patients with HNSCC.