Nivolumab With or Without Ipilimumab Combined with Stereotactic Body Radiotherapy in patients with Metastatic Biliary Tract Cancer: A Randomized Phase 2 Study.

PURPOSE: Evaluate the clinical benefits of nivolumab with/without ipilimumab combined with stereotactic body radiotherapy (SBRT) in patients with pretreated metastatic biliary tract cancer (mBTC). EXPERIMENTAL DESIGN: The study was a phase 2 randomized trial with Simon's optimal 2-stage design requiring 36 evaluable patients per group after second stage. Sixty-one patients were included from September 2018 to January 2022 and randomized (1:1) to receive SBRT (15 Gy × 1 on day one to a primary or metastatic lesion) and nivolumab (3 mg/kg intravenously on day one and every 2 weeks) with/without ipilimumab (1 mg/kg intravenously on day one and every 6 weeks). Primary endpoint was clinical benefit rate (CBR), defined as the percentage of patients with complete response, partial response or stable disease. Decision to continue accrual into the second stage depended on CBR from first stage. RESULTS: Forty-two patients received SBRT/nivolumab/ipilimumab with a CBR of 31.0% (95% CI, 17.6-47.1). Five patients (11.9%) achieved partial response with median duration of 4.4 months (range, 1.1-21.5). Nineteen patients received SBRT/nivolumab. This group was closed after the initial stage based on a CBR of 10.5% (95% CI, 1.3-33.1). Adverse events were graded with National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0. Grade ≥3 treatment-related adverse events occurred in 13 (31%) and 3 (16%) patients in the SBRT/nivolumab/ipilimumab and SBRT/nivolumab groups, respectively. One patient died from immune-related hepatitis in the SBRT/nivolumab/ipilimumab group. CONCLUSION: Combining SBRT, nivolumab and ipilimumab is well tolerated, feasible, and shows response in a subgroup of patients with mBTC.

Resolution of Acinar Dedifferentiation Regulates Tissue Remodeling in Pancreatic Injury and Cancer Initiation.

BACKGROUND & AIMS: Acinar-to-ductal metaplasia (ADM) is crucial in the development of pancreatic ductal adenocarcinoma. However, our understanding of the induction and resolution of ADM remains limited. We conducted comparative transcriptome analyses to identify conserved mechanisms of ADM in mouse and human. METHODS: We identified Sox4 among the top up-regulated genes. We validated the analysis by RNA in situ hybridization. We performed experiments in mice with acinar-specific deletion of Sox4 (Ptf1a: CreER; Rosa26-LSL-YFPLSL-YFP; Sox4fl/fl) with and without an activating mutation in Kras (KrasLSL-G12D/+). Mice were given caerulein to induce pancreatitis. We performed phenotypic analysis by immunohistochemistry, tissue decellularization, and single-cell RNA sequencing. RESULTS: We demonstrated that Sox4 is reactivated in ADM and pancreatic intraepithelial neoplasias. Contrary to findings in other tissues, Sox4 actually counteracts cellular dedifferentiation and helps maintain tissue homeostasis. Moreover, our investigations unveiled the indispensable role of Sox4 in the specification of mucin-producing cells and tuft-like cells from acinar cells. We identified Sox4-dependent non-cell-autonomous mechanisms regulating the stromal reaction during disease progression. Notably, Sox4-inferred targets are activated upon KRAS inactivation and tumor regression. CONCLUSIONS: Our results indicate that our transcriptome analysis can be used to investigate conserved mechanisms of tissue injury. We demonstrate that Sox4 restrains acinar dedifferentiation and is necessary for the specification of acinar-derived metaplastic cells in pancreatic injury and cancer initiation and is activated upon Kras ablation and tumor regression in mice. By uncovering novel potential strategies to promote tissue homeostasis, our findings offer new avenues for preventing the development of pancreatic ductal adenocarcinoma.

Hepatic granulomas following liver transplantation: A retrospective survey, and analysis of possible microbiological etiology.

BACKGROUND: Liver granulomas have always been a diagnostic challenge for pathologists. They have been described in up to 15% of liver biopsies and can also be seen in liver allograft biopsy specimens, but there is a paucity of information regarding the prevalence and associated etiologic factors of granulomas in liver transplanted patients. The aim of this study is to shed light on the etiology of liver granulomas. METHODS: Liver biopsies from liver transplanted patients, in the period from 01.01.2011 - 01.05.2017, were examined. We registered the histo-morphological characteristics and clinicopathological data of all biopsies and performed next-generation sequencing (NGS) to detect possible pathogens (bacteria, fungi, and parasites) in the biopsies containing granulomas. RESULTS: We reviewed a total of 400 liver biopsies from 217 liver transplant patients. Of these, 131 liver biopsies (32.8%) from 98 patients (45.2%) revealed granulomas. Most were epithelioid granulomas located parenchymal and were detected in 115 (87.7%) biopsies. We also identified 10 cases (7.6%) with both lobular and portal granulomas and six biopsies (4.5%) with portal granulomas alone. In 54 biopsies (41.2%), granulomas were found in biopsies with acute cellular rejection (ACR). Fifty (51%) patients with granulomas underwent liver transplantation for autoimmune-related end-stage liver disease (AILD). The granulomas were found most frequently in the first six months after transplantation, where patients also more often were biopsied. NGS analysis did not reveal any potential infectious agent, and no significant differences were observed in the microbiological diversity (microbiome) between clinical- and granuloma characteristics concerning bacteria, fungi, and parasites. CONCLUSION: Our study confirmed that granulomas are frequently seen in liver allograft biopsy specimens, and most often localized in the parenchyma, occurring in the first post-transplant period in patients with AILD, and often seen simultaneously with episodes of ACR. Neither a specific microbiological etiological agent nor a consistent microbiome was detected in any case.

Prognostic value of margin clearance in total and distal pancreatectomy specimens with pancreatic ductal adenocarcinoma in a Danish population-based nationwide study.

BACKGROUND: The prognostic role of resection margin status following total (TP) and distal (DP) pancreatectomy for pancreatic ductal adenocarcinoma (PDAC) is insufficiently evaluated. In Denmark, pancreatic surgery, including the postoperative pathological examination of the resection specimens, is confined to four centres, all reporting to the Danish Pancreatic Cancer Database (DPCD). In this Danish population-based nationwide study on TP and DP for PDAC from 2015-2019, based on data from DPCD, we evaluated whether there is a prognostically relevant minimum margin clearance definition and whether certain margins hold independent prognostic information. METHODS: Clinical and pathological data were retrieved from DPCD and supplemented by review of pathology reports and re-microscopy, if needed. One of the study pathologists performed all re-microscopy. The prognostic significance of margin status was evaluated by dichotomisation of the TP cohort (n = 101) and the DP cohort (n = 90) into involved and uninvolved groups, using different clearance definitions (0.5 - ≥3.0 mm). RESULTS: Following TP, direct involvement of the superior mesenteric artery (SMA) margin had independent prognostic value. When using a clearance definition of ≥ 0.5 or ≥ 1.5 mm for SMA, median survival for R0 versus R1 was 19 (95% CI 14-26) versus 10 (95% CI 5-20) months (p = 0.010), and 21 (95% CI 15-30) versus 10 (95% CI 8-19) months (p = 0.011), respectively. Overall margin status was not of significant prognostic importance following neither DP nor TP. CONCLUSION: In this Danish population-based nationwide study, SMA margin involvement was a significant isolated prognostic factor following TP, whereas combined assessment of all circumferential margins did not hold statistically significant prognostic information. Following DP, resection margin status did not affect survival.

Prognostic importance of margin clearance in ampullary adenocarcinoma following pancreaticoduodenectomy in a Danish population-based nationwide study.

BACKGROUND: In this Danish nationwide population-based study, we evaluated the prognostically relevant minimum tumour-free margin width following pancreaticoduodenectomy (PD) for ampullary adenocarcinoma (AAC) and evaluated whether certain margins hold independent prognostic information. METHODS: We included 128 patients who underwent PD for AAC from 2015 to 2019. Clinical and pathological data including well-known prognostic factors were retrieved from the Danish Pancreatic Cancer Database. Missing data were obtained by review of pathology reports and re-microscopy of resection specimens. All PD specimens were examined using a standardised pathological protocol including multicolour inking, axial slicing and exact reporting of margin widths. The cohort was dichotomised into involved and uninvolved groups, using different margin clearance definitions (0.5-≥3.0 mm). RESULTS: Following PD for AAC, margin clearance of ≥1 mm was independently associated with improved chance of survival compared with <1 mm (HR: 0.30, 95 % CI: 0.14-0.64 (p = 0.002)). Posterior and anterior margin widths were narrower compared with superior mesenteric artery and vein margins. Posterior margin and anterior surface had isolated prognostic significance in multivariable analysis. CONCLUSION: Following PD for AAC, margin clearance of at least 1 mm is independently associated with improved survival. Our data further indicate that anterior surface and posterior margin hold particular prognostic value.

Validation of a Novel EUS-FNB-Derived Organoid Co-Culture System for Drug Screening in Patients with Pancreatic Cancer.

Cancer-associated fibroblasts (CAFs) have been shown to impact the chemosensitivity of patient-derived tumor organoids (PDTOs). However, the published literature comparing PDTO response to clinical outcome does not include CAFs in the models. Here, a co-culture model was created using PDTOs and CAFs derived from endoscopic ultrasound-guided fine-needle biopsies (EUS-FNBs) for potential use in drug screening applications. Co-cultures were established, and growth was compared to monocultures using image metrics and a commercially available assay. We were able to establish and expand validated malignant PDTOs from 19.2% of adenocarcinomas from EUS-FNBs. CAFs could be established from 25% of the samples. The viability of PDTOs in the mixed cell co-culture could be isolated using image metrics. The addition of CAFs promoted PDTO growth in half of the established co-cultures. These results show that co-cultures can be established from tiny amounts of tissue provided by EUS-FNB. An increased growth of PDTOs was shown in co-cultures, suggesting that the present setup successfully models CAF-PDTO interaction. Furthermore, we demonstrated that standard validation techniques may be insufficient to detect contamination with normal cells in PDTO cultures established from primary tumor core biopsies.

Promoter hypermethylation of SFRP1 as a prognostic and potentially predictive blood-based biomarker in patients with localized pancreatic ductal adenocarcinoma.

Introduction: Current prognostic blood-based biomarkers for pancreatic adenocarcinoma (PDAC) are limited. Recently, promoter hypermethylation of SFRP1 (phSFRP1) has been linked to poor prognosis in patients with gemcitabine-treated stage IV PDAC. This study explores the effects of phSFRP1 in patients with lower stage PDAC. Methods: Based on a bisulfite treatment process, the promoter region of the SFRP1 gene was analyzed with methylation-specific PCR. Kaplan-Meier curves, log-rank tests, and generalized linear regression analysis were used to assess restricted mean survival time survival at 12 and 24 months. Results: The study included 211 patients with stage I-II PDAC. The median overall survival of patients with phSFRP1 was 13.1 months, compared to 19.6 months in patients with unmethylated SFRP1 (umSFRP1). In adjusted analysis, phSFRP1 was associated with a loss of 1.15 months (95%CI -2.11, -0.20) and 2.71 months (95%CI -2.71, -0.45) of life at 12 and 24 months, respectively. There was no significant effect of phSFRP1 on disease-free or progression-free survival. In stage I-II PDAC, patients with phSFRP1 have worse prognoses than patients with umSFRP1. Discussion: Results could indicate that the poor prognosis may be caused by reduced benefit from adjuvant chemotherapy. SFRP1 may help guide the clinician and be a possible target for epigenetically modifying drugs.

Prognostic significance of margin clearance in pancreaticoduodenectomy specimens with pancreatic ductal adenocarcinoma in a Danish population-based nationwide study.

BACKGROUND: In this nationwide population-based cohort study, we investigated the overall minimum margin width that is independently associated with improved survival following pancreaticoduodenectomy (PD) for pancreatic ductal adenocarcinoma (PDAC) and evaluated whether certain margins or surfaces hold independent prognostic significance. METHODS: Data from 367 patients who underwent PD for PDAC in the period 2015-2019 were retrieved from the Danish Pancreatic Cancer Database. Missing data were obtained by review of pathology reports and re-microscopy of resection specimens. Surgical specimens were evaluated using a standardised pathological protocol involving multicolour inking, axial slicing and exact reporting of circumferential margin clearances in 0.5 mm increments. RESULTS: When categorised according to margin widths of <0.5, <1.0, <1.5, <2.0, <2.5 and <3.0 mm, R1 resections were detected in 34%, 57%, 75%, 78%, 86% and 87% of cases, respectively. In multivariable analyses, an overall margin clearance of ≥1.5 mm was associated with improved survival compared with a clearance of <1.5 mm (HR 0.70 95% CI 0.51-0.97 (p = 0.031)). When evaluating the margins separately, no margin had independent prognostic significance. CONCLUSION: Margin clearance of at least 1.5 mm was independently associated with improved survival following PD for PDAC.

Pre-existing TGF-ß-specific T-cell immunity in patients with pancreatic cancer predicts survival after checkpoint inhibitors combined with radiotherapy.

BACKGROUND: Circulating transforming growth factor-ß (TGF-ß)-specific T cells that recognize TGF-ß-expressing immune regulatory cells have been described in patients with cancer. TGF-ß-derived peptide vaccination modulates the tumor microenvironment and has shown clinical effects in animal models of pancreatic cancer (PC). TGF-ß-expressing regulatory cells are especially elevated in PC and may prevent the clinical response to immune checkpoint inhibitors (ICIs). Thus, in the present study we investigated the significance of TGF-ß-specific T-cell immunity in patients with PC treated with ICI combined with radiotherapy in a randomized phase 2 study (CheckPAC). METHODS: Immune responses to a TGF-ß-derived epitope entitled TGF-ß-15 as well as epitopes from Clostridium tetani (tetanus) and influenza were measured in peripheral blood mononuclear cells (PBMCs) with interferon-É£ enzyme-linked immunospot assays. PBMCs were isolated before and after treatment. Correlations between immune response data and clinical data were evaluated with parametric and non-parametric statistical methods. Survival was analyzed with univariate and multivariate Cox-regression. TGF-ß-15 specific T cells were isolated and expanded and examined for recognition of autologous regulatory immune cells by flow cytometry. RESULTS: PBMCs from 32 patients were analyzed for immune responses to the TGF-ß-derived epitope entitled TGF-ß-15. Patients with a strong TGF-ß-specific immune response at treatment initiation had longer progression-free and overall survival, compared with patients with a weak or no TGF-ß-specific immune response. This remained significant in multivariate analysis. Patients with weak and strong TGF-ß-specific responses displayed similar responses towards viral antigens. Furthermore, we show that TGF-ß-specific T cells from a clinical responder specifically reacted to and lysed autologous, regulatory immune cells. Finally, mimicking a TGF-ß-15 vaccination, we showed that repeated stimulations with the TGF-ß-15 epitope in vitro enhanced the immune response to TGF-ß-15. CONCLUSION: A strong TGF-ß-15 specific immune response was associated with clinical benefit and improved survival after ICI/radiotherapy for patients with PC. Importantly, the lack of TGF-ß-specific T cells in some patients was not caused by a general immune dysfunction. TGF-ß-specific T cells recognized regulatory immune cells and could be introduced in vitro in patients without spontaneous responses. Taken together, our data suggest that combining TGF-ß-based vaccination with ICI/radiotherapy will be beneficial for patients with PC.

Phase 2 study of ipilimumab, nivolumab, and tocilizumab combined with stereotactic body radiotherapy in patients with refractory pancreatic cancer (TRIPLE-R).

BACKGROUND: Interleukin-6 blockade and radiation combined with immunotherapy may modulate the tumour microenvironment to overcome immune resistance. We assessed the efficacy of ipilimumab, nivolumab, and tocilizumab combined with stereotactic body radiotherapy (SBRT) in patients with refractory pancreatic cancer (PC). METHODS: Patients with PC who had progressive disease (PD) or intolerance to gemcitabine- or fluorouracil-containing regimens were enrolled in Part A of the two-part, single-centre, phase 2 study (NCT04258150). SBRT with 15 Gy was administered on day one of the first cycle. Ipilimumab was administered (1 mg/kg every 6 weeks) for a maximum of two infusions. Nivolumab (6 mg/kg) and tocilizumab (8 mg/kg) were given every four weeks until the PD or unacceptable toxicity, or for up to one year. The primary end-point was the objective response rate, with a threshold of 15%. RESULTS: Twenty-six patients were enrolled and treated between April 17, 2020, and January 25, 2021. The median follow-up time at the time of data cutoff (February 7, 2022) was 4.9 months (interquartile range 2.1-7.7). No responses were observed. Five patients (19%; 95% confidence intervals [CI], 7-39) achieved a stable disease. The median progression-free survival was 1.6 months (95% CI 1.4-1.7), and the median overall survival was 5.3 months (95% CI 2.3-8.0). Overall, 19 (73%) experienced adverse events related to the treatment including two (8%) with grade 3 or higher events. CONCLUSION: The combination of ipilimumab, nivolumab, tocilizumab, and SBRT in patients with PC did not meet the prespecified criteria for expansion for full accrual.

Prognostic value of circulating proteins in patients undergoing surgery for pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death. Less than 20% of patients are diagnosed with resectable disease. Identifying truly resectable disease is challenging because 20%-40% of the patients subjected to resection are found to have advanced disease during surgery. The aim of our study was to identify panels of circulating proteins that could be used to distinguish patients with unresectable PDAC from patients with resectable PDAC and to identify prognostic signatures for both groups. METHODS: We measured 92 circulating immuno-oncology-related proteins using the proximity extension assay from Olink Proteomics in 273 patients eligible for surgery for PDAC. Two bioinformaticians worked independently of one another on the same data. LASSO and Ridge regression were used in the statistical analyses. RESULTS: One protein index for determining resectability had an AUC value of 0.66. Several indices for prognosis had AUC values between 0.50 and 0.75 and were therefore not better than existing prognostic markers. DISCUSSION: Our study did not reveal any new high-performing protein panels that could be used to identify patients with inoperable PDAC before surgery. The panel of 92 proteins investigated has previously been found to be applicable for diagnostic use in patients with PDAC, but it does not seem to warrant further investigation regarding resectability in the subgroup of patients with PDAC referred to surgery.

Targeted next-generation sequencing of EUS-guided through-the-needle-biopsy sampling from pancreatic cystic lesions.

BACKGROUND AND AIMS: Recent advances have introduced molecular subtyping of pancreatic cystic lesions (PCLs) as a possible amendment to the diagnostic algorithm. The study evaluated the feasibility and diagnostic accuracy of molecular analysis and subtyping of PCLs using the recently introduced EUS-guided through-the-needle-biopsy (TTNB) sampling. METHODS: We prospectively included 101 patients in the study who presented with PCLs >15 mm in the largest cross-section. EUS-guided TTNB samples were obtained by a micro-biopsy forceps introduced through a 19-gauge needle. The TTNB samples were analyzed by next-generation sequencing (NGS) for point mutations in tumor suppressors and oncogenes using a 51-gene customized hotspot panel. Sensitivity and specificity were calculated with the histologic diagnosis as reference. RESULTS: After initial microscopic evaluation of the samples, 91 patients had residual TTNB samples available for NGS. Of these, 49 harbored mutations, most frequently in KRAS and GNAS, reflecting an excess frequency of intraductal papillary mucinous neoplasms (IPMNs) in the study population. A sensitivity and specificity of 83.7% (95% confidence interval [CI], 70.3-92.7) and 81.8% (95% CI, 48.2-97.7), respectively, were demonstrated for the diagnosis of a mucinous cyst and 87.2% (95% CI, 74.2-95.2) and 84.6% (95% CI, 54.5-98.1) for the diagnosis of an IPMN. CONCLUSIONS: Thus, molecular analysis of TTNB samples by NGS has high sensitivity and specificity for diagnosing mucinous cysts and IPMNs. Although the procedure comes with a risk of adverse events of 9.9%, TTNB samples are a robust alternative to cyst fluid for a combined histologic and molecular diagnosis of PCLs. (Clinical trial registration number: NCT03578445.).

Randomized Phase II Study of Nivolumab With or Without Ipilimumab Combined With Stereotactic Body Radiotherapy for Refractory Metastatic Pancreatic Cancer (CheckPAC).

PURPOSE: To evaluate the clinical benefit of nivolumab with or without ipilimumab in combination with stereotactic body radiotherapy (SBRT) in patients with refractory metastatic pancreatic cancer (mPC). METHODS: Between November 2016 and December 2019, patients with refractory mPC were randomly assigned 1:1 to SBRT of 15 Gy with nivolumab or nivolumab/ipilimumab stratified by performance status (ClinicalTrials.gov identifier: NCT02866383). The primary end point was the clinical benefit rate (CBR), defined as the percentage of patients with complete or partial response (PR) or stable disease, according to RECIST 1.1. Simon's 2-stage phase II optimal design was used independently for both arms, with CBR determining expansion to the second stage. Secondary end points included safety, response rate, duration of response, progression-free survival, and overall survival. Exploratory analyses included biomarkers related to the benefits. RESULTS: Eighty-four patients (41 SBRT/nivolumab and 43 SBRT/nivolumab/ipilimumab) received at least one dose of study treatment. CBR was 17.1% (8.0 to 30.6) for patients receiving SBRT/nivolumab and 37.2% (24.0 to 52.1) for SBRT/nivolumab/ipilimumab. PR was observed in one patient receiving SBRT/nivolumab and lasted for 4.6 months. Six patients receiving SBRT/nivolumab/ipilimumab achieved a PR with a median duration of response of 5.4 months (4.2 to not reached). Grade 3 or higher treatment-related adverse events occurred in 10 (24.4%) and 13 (30.2%) patients in the SBRT/nivolumab and SBRT/nivolumab/ipilimumab groups, respectively. Programmed cell death ligand-1 expression by tumor proportion score or combined positivity score of ≥ 1% was not associated with clinical benefits. On-treatment decreased serum interleukin-6, interleukin-8, and C-reactive protein levels were associated with better overall survival. CONCLUSION: Clinically meaningful antitumor activity and favorable safety profiles were demonstrated after treatment with SBRT/nivolumab/ipilimumab in patients with refractory mPC. However, the contribution from SBRT is unknown. Further studies are warranted.

A Randomized Placebo-Controlled Phase 2 Study of Gemcitabine and Capecitabine with or without T-ChOS as Adjuvant Therapy in Patients with Resected Pancreatic Cancer (CHIPAC).

The antitumor activity of chitooligosaccharides has been suggested. This phase 2 trial evaluated the efficacy and safety of T-ChOS™, in addition to adjuvant chemotherapy, in patients after resection of pancreatic ductal adenocarcinoma (PDAC). In this single-center, randomized, double-blind, placebo-controlled trial using patients ≥18 years of age after complete macroscopic resection for PDAC, patients were randomly assigned (1:1) to either a continuous oral T-ChOS group or a placebo group, in combination with gemcitabine (GEM) and oral capecitabine (CAP), for a maximum of six cycles. The primary endpoint was disease-free survival (DFS). Recruitment was stopped prematurely in July 2018, with 21 of planned 180 patients included, due to poor accrual and because modified FOLFIRINOX replaced GEM/CAP for the target population. Nine patients received T-ChOS and twelve received the placebo. The median DFS was 10.8 months (95% CI 5.9-15.7) for the T-ChOS arm and 8.4 months (95% CI 0-21.5) in the placebo arm. Overall, seven patients (78%) in the T-ChOS arm and eight patients (67%) in the placebo arm experienced at least one grade 3-4 treatment-related adverse event, most frequently neutropenia. Altogether, the addition of T-ChOS to chemotherapy in patients after resection of PDAC seems safe. However, the clinical benefit cannot be assessed due to the premature cessation of the trial.

Transient congenital hyperinsulinism and hemolytic disease of a newborn despite rhesus D prophylaxis: a case report.

BACKGROUND: In neonates, rhesus D alloimmunization despite anti-D immunoglobulin prophylaxis is rare and often unexplained. Rhesus D alloimmunization can lead to hemolytic disease of the newborn with anemia and unconjugated hyperbilirubinemia. In past reports, transient congenital hyperinsulinism has been described as a rare complication of rhesus D alloimmunization. Our case report illustrates that rhesus D alloimmunization can result in a pseudosyndrome with severe congenital hyperinsulinism, anemia, and conjugated hyperbilirubinemia, despite correctly administered anti-D immunoglobulin prophylaxis. CASE PRESENTATION: We report of a 36-year-old, Caucasian gravida 1, para 1 mother with A RhD negative blood type who received routine antenatal anti-D immunoglobulin prophylaxis. Her full term newborn boy presented with severe congenital hyperinsulinism, anemia, and conjugated hyperbilirubinemia up to 295 µmol/L (ref. < 9), accounting for 64% of the total bilirubin. Syndromic congenital hyperinsulinism was suspected. Examinations showed a positive direct antiglobulin test, initially interpreted as caused by irregular antibodies; diffuse congenital hyperinsulinism by 18F-DOPA positron emission tomography/computed tomography scan; normal genetic analyses for congenital hyperinsulinism; mildly elevated liver enzymes; delayed, but present bile excretion by Tc99m-hepatobiliary iminodiacetic acid scintigraphy; and cholestasis and mild fibrosis by liver biopsy. The maternal anti-D titer was 1:16,000 day 20 postpartum. Y-chromosome material in the mother's blood could not be identified. This could, however, not exclude late intrapartum fetomaternal hemorrhage as the cause of immunization. No causative genetic findings were deetrmined by trio whole exome sequencing. The child went into clinical remission after 5.5 months. CONCLUSION: Our case demonstrates that rhesus D alloimmunization may present as a pseudosyndrome with transient congenital hyperinsulinism, anemia, and inspissated bile syndrome with conjugated hyperbilirubinaemia, despite anti-D immunoglobulin prophylaxis, possibly due to late fetomaternal hemorrhage.

Clinical assessment of tumor regression grade systems in gastroesophageal adenocarcinoma following neoadjuvant chemotherapy.

BACKGROUND: The standard treatment for gastroesophageal cancer is neoadjuvant chemotherapy, followed by surgery, which has been shown to increase survival compared with surgery alone. Evidence is mounting that characterization of the oncologically induced tumor regression is of prognostic importance. However, no consensus regarding the optimal system for describing tumor regression exists. Thus, this study aims to explore three validated/promising tumor regression systems with a focus on their interobserver reliability and usability. METHODS: We included 100 consecutive patients with gastroesophageal adenocarcinoma who had undergone neoadjuvant oncological treatment followed by surgery. The tumors underwent tumor regression grade (TRG) assessment according to the Standard Mandard-, Modified Mandard-, and Becker systems to assess the interobserver reliability between two consultant pathologists. The interobserver reliability was determined by both Fleiss kappa and weighted kappa metrics. Besides, a semi-quantitative usability questionary was completed and it was expanded with usability comments. RESULTS: The Fleiss kappa interobserver agreement was 0.67 [95% CI, 0.55-0.79], 0.88 [95% CI, 0.73-1.00], and 0.88 [95% CI, 0.73-1.00] for Standard Mandard-, Modified Mandard-, and the Becker systems, respectively. The weighted kappa (linear) was 0.80 [95% CI, 0.72-0.89], 0.91 [95% CI, 0.84-0.98], and 0.91 [95% CI, 0.84-0.98] for the Standard Mandard-, Modified Mandard-, and the Becker systems, respectively. The usability was scored on a scale of 8-24 by both raters. The systems were scored accordingly: 47 (Modified Mandard system), 43 (Becker system), and 37 (Standard Mandard system). CONCLUSION: The Modified Mandard- and Becker systems had excellent interobserver reliability and usability. However, the systems could be improved by a better characterization of the different tiers and tumor morphology.

Molecular heterogeneity of pancreatic intraductal papillary mucinous neoplasms and implications for novel endoscopic tissue sampling strategies.

AIMS: Intraductal papillary mucinous neoplasms (IPMNs) may be precursor lesions of pancreatic cancer. The path towards malignancy is associated with mutations in tumour suppressor-and oncogenes that may serve as biomarkers during diagnostic investigation. A novel micro forceps has made it possible to obtain biopsies from the cyst wall for analysis by next generation sequencing (NGS), providing an opportunity for early detection and intervention. However, the impact of spatial tumour heterogeneity on the representability of the biopsies has not been determined. The primary aim is to characterise the impact of molecular heterogeneity of the luminal cyst wall on tissue sampling strategies with small biopsies. METHODS: We performed NGS and immunohistochemical phenotyping on 18 resected IPMNs with varying degrees of dysplasia and for a subset, concomitant carcinoma, using a commercially available NGS-panel of 51 oncogenes. We simulated endoscopic biopsies by performing punch biopsies (PBs) of the cyst wall from resected specimens. RESULTS: In total, 127 NGS analyses were performed. Concomitant KRAS and GNAS was a common feature of the IPMNs. Mutations in KRAS and GNAS were associated with low-grade dysplasia whereas alterations in TP53, SMAD4, CDKN2A and PIK3CA were associated with high-grade dysplasia and/or carcinoma. The mutational analysis of the PBs from the cyst wall was compared with the whole lesion. No difference was detected between PBs and whole lesions when the cumulated mutational profile in increasing order of randomly performed PBs was compared. CONCLUSIONS: Small IPMN biopsies from the cyst wall are adequate to yield a molecular diagnosis.

Prevalence of HER2 overexpression and amplification in squamous cell carcinoma of the esophagus: A systematic review and meta-analysis.

Accurate data on HER2 positivity in esophageal squamous cell carcinoma patients (ESCC) is lacking. We conducted a systematic review and meta-analysis (Single Incidence Rates; metarate package, R) to examine the prevalence of HER2 in ESCC. Data on in situ hybridization (ISH) and immunohistochemistry (IHC) were extracted to derive pooled prevalence estimates, characteristics of the studies were extracted for subgroup analysis. Eighteen studies with 1505 patients were identified. HER2 gene amplification by ISH were prevalent in 10 % (95 % CI 6.9 %-15 %). Prevalence of HER2 overexpression (IHC3+) and borderline HER2 expression (IHC2+) were 6 % (95 % CI: 3.5 %-8.7 %) and 10 % (95 % CI: 6.0 %-17 %), respectively. An estimated 8.6 % (95 % CI: 5.5 %-13 %) of ESCC were HER2 positive using initial IHC followed by reflex ISH confirmation of borderline HER2 expression. In conclusion: Estimated prevalence of HER 2 positivity in ESCC were 10 % assessed by ISH and 8.6 % assessed by initial IHC followed by ISH.

Histopathological evaluation of resected intraductal papillary mucinous neoplasms reveals distinct patterns of invasion in associated carcinomas.

Invasive intraductal papillary mucinous neoplasms (inv-IPMNs) have a better prognosis than regular pancreatic ductal adenocarcinoma (PDAC), but no association with status of surgical margins and microscopic infiltration patterns has previously been described. The aim of this study is to review patterns of invasion and the predictive value of clinical guidelines in terms of rates of resection of high-grade dysplasia (HGD) and cancer among intraductal papillary mucinous neoplasms (IPMNs). Consecutively, resected IPMNs between 2011 and 2017 were analyzed. Data were obtained from a prospectively maintained database. A total of 132 patients were identified. Out of these, 38 patients with inv-IPMNs, initially identified as solid lesions suspicious of cancer, were compared with a control group of 101 patients with ordinary PDAC. Lower rates of vascular invasion, perineural invasion, lymph node metastasis, advanced T stage, and R1 status were characteristic of the inv-IPMNs in addition to better overall survival (OS) for a low tumor stage. Furthermore, as novel findings, the PDACs presented with resection margin involvement of 3 or more positive margins (31.3% vs. 9.5%, p = 0.044), associated with poor OS. Of the patients presenting as pT3, the inv-IPMN less often invaded more than one extrapancreatic anatomical structure (40.1% vs. 63.9%, p = 0.03). Regarding the predictive value of clinical guidelines, the frequency of resected HGD in IPMNs with high-risk stigmata (n = 54) and IPMNs with worrisome features was 30.7%, and the frequency of invasive carcinoma was 5.7%. In conclusion, we report a low resection rate of high-risk IPMNs and present novel findings describing inv-IPMNs as a less infiltrative phenotype compared with regular PDAC.