Safety and Efficacy of Avelumab in Small Bowel Adenocarcinoma.

INTRODUCTION: Small bowel adenocarcinomas (SBAs) are rare and frequently treated like large intestinal adenocarcinomas. However, SBAs have a very different microenvironment and could respond differently to the same therapies. Our previous data suggested that SBAs might benefit from targeting the PD-1/PD-L1 axis based on PD-L1 staining in almost 50% of SBA tissue samples tested. Thus, we designed a phase 2 study to explore safety and efficacy of avelumab in SBA. PATIENTS AND METHODS: Patients with advanced or metastatic disease were enrolled; ampullary tumors were considered part of the duodenum and allowed. Prior PD-1/PD-L1 inhibition was not allowed. Avelumab (10 mg/kg) was given every 2 weeks, and imaging was performed every 8 weeks. Primary endpoint was response rate. RESULTS: Eight patients (n = 5, small intestine; n = 3, ampullary) were enrolled, with a majority (88%) being male and a median age of 61 years. Of 7 efficacy-evaluable patients, 2 (29%) experienced partial responses; stable disease occurred in 3 additional patients (71%). Median progression-free survival was 3.35 months. Most frequent, related toxicities were anemia, fatigue, and infusion-related reaction (25% each), mostly grade ≤2; grade 3 hypokalemia and hyponatremia occurred in one patient, and another reported grade 4 diabetic ketoacidosis. CONCLUSIONS: Despite the observed benefit, accrual was slower than expected and the study was closed early due to feasibility. A general clinic observation was that patients were receiving immunotherapy off-label as the availability of these agents increased. Off-label availability and disease rarity were likely drivers of insufficient accrual.

Randomized Phase II Study of PARP Inhibitor ABT-888 (Veliparib) with Modified FOLFIRI versus FOLFIRI as Second-line Treatment of Metastatic Pancreatic Cancer: SWOG S1513.

PURPOSE: PARP inhibitors synergize with topoisomerase inhibitors, and veliparib plus modified (m) FOLFIRI (no 5-FU bolus) had preliminary activity in metastatic pancreatic cancers. This study evaluated the safety and efficacy of second-line treatment with veliparib and mFOLFIRI versus FOLFIRI (control) for metastatic pancreatic cancer. PATIENTS AND METHODS: This randomized phase II clinical trial led by the SWOG Cancer Research Network enrolled patients between September 1, 2016 and December 13, 2017. The median follow-up was 9 months (IQR 1-27). BRCA1/2 and homologous recombination DNA damage repair (HR-DDR) genetic defects were tested in blood and tumor biopsies. Patients received veliparib 200 mg twice daily, days 1-7 with mFOLFIRI days 3-5, or FOLFIRI in 14-day cycles. RESULTS: After 123 of planned 143 patients were accrued, an interim futility analysis indicated that the veliparib arm was unlikely to be superior to control, and the study was halted. Median overall survival (OS) was 5.4 versus 6.5 months (HR, 1.23; P = 0.28), and median progression-free survival (PFS) was 2.1 versus 2.9 months (HR, 1.39; P = 0.09) with veliparib versus control. Grade 3/4 toxicities were more common with veliparib (69% vs. 58%, P = 0.23). For cancers with HR-DDR defects versus wild-type, median PFS and OS were 7.3 versus 2.5 months (P = 0.05) and 10.1 versus 5.9 months (P = 0.17), respectively, with FOLFIRI, and 2.0 versus 2.1 months (P = 0.62) and 7.4 versus 5.1 months (P = 0.10), respectively, with veliparib plus mFOLFIRI. CONCLUSIONS: Veliparib plus mFOLFIRI did not improve survival for metastatic pancreatic cancer. FOLFIRI should be further studied in pancreatic cancers with HR-DDR defects.

Interobserver agreement in pathologic evaluation of bile duct biopsies.

Intraductal biopsy is commonly used for preoperative evaluation of the etiology of biliary strictures. Interpretation of intraductal biopsies is frequently challenging. The diagnosis often suffers from interobserver disagreement, which has not been studied in the literature. We sought to assess interobserver concordance in the interpretation of intraductal biopsies. Eighty-five biopsies were retrieved, falling into five diagnostic categories: negative for dysplasia (NED), indefinite for dysplasia (IND), low-grade dysplasia (LGD), high-grade dysplasia (HGD), and carcinoma (CA). Eight gastrointestinal pathologists blindly reviewed all the slides. Agreement among pathologists was analyzed using Fleiss κ and weighted concordance coefficient S∗. A face-to-face consensus/training session was held to discuss the classification criteria, followed by a second round review. The overall interobserver agreement was fair in the first round review (κ = 0.39; S∗ = 0.56) and improved to moderate in the second round review (κ = 0.48; S∗ = 0.69). The agreement before and after consensus meeting was substantial to nearly perfect for CA (κ = 0.65, S∗ = 0.83; and κ = 0.80, S∗ = 0.91), fair for HGD (κ = 0.28, S∗ = 0.69; and κ = 0.40, S∗ = 0.63), and moderate for NED (κ = 0.47, S∗ = 0.50; and κ = 0.47, S∗ = 0.53). Agreement improved from fair to moderate for LGD (κ = 0.36, S∗ = 0.61; and κ = 0.49, S∗ = 0.71) and slight to fair for IND (κ = 0.16, S∗ = 0.51; and κ = 0.33, S∗ = 0.50). Compared with Hollande's fixed specimens, the agreement was higher in almost all diagnostic categories in formalin-fixed biopsies. Overall, interobserver concordance was improved after a consensus/training session. Interobserver reproducibility was high at the end of the diagnostic spectrum (CA) but fair to moderate for other diagnostic categories.

Copper Deficiency in Liver Diseases: A Case Series and Pathophysiological Considerations.

Copper is an indispensable trace element. It serves as a cofactor for enzymes involved in cellular energy metabolism, antioxidant defense, iron transport, and fibrogenesis. Although these processes are central in the pathogenesis of liver disorders, few studies have attributed them to copper deficiency. We herein describe in detail a case series of liver disease patients (n = 12) who presented with signs of copper deficiency based on serum and liver copper measurements. Median age of the group at the time of presentation was 39 (range 18-64 years). Six patients were female. The median serum copper was 46 µg/dL (normal range: 80-155 µg/dL for women and 70-140 µg/dL for men). Seven of the 12 patients had hepatic copper concentration less than 10 µg/g dry weight (normal range: 10-35 µg/g). Most cases presented with acute-on-chronic liver failure (n = 4) and decompensated cirrhosis (n = 5). Only 3 patients had a condition known to be associated with copper deficiency (ileocolonic Crohn's disease following resection n = 1, Roux-en-Y gastric bypass n = 2) before presenting with hepatic dysfunction. Notable clinical features included steatohepatitis, iron overload, malnutrition, and recurrent infections. In 2 of the 3 patients who received copper supplementation, there was an improvement in serum copper, ceruloplasmin, and liver function parameters. Conclusion: Copper deficiency in the serum or liver occurs in a wide range of liver diseases. Given the biological essentiality of copper, the mechanism and clinical significance of this association require systematic study.

Validation of a Congestive Hepatic Fibrosis Scoring System.

Congestive hepatopathy is a complication of right heart failure and chronically elevated right heart pressure. Histologic findings include sinusoidal dilatation, centrilobular hepatocellular plate atrophy, and fibrosis. We performed a validation study of a recently proposed scoring system (0 to 4 scale) for congestive hepatic fibrosis on 38 liver biopsies. Glutamine synthetase immunohistochemistry was also performed, and loss of centrizonal immunoreactivity correlated with increasing fibrosis score (P<0.01). Interobserver concordance for congestive hepatic fibrosis score based on Masson trichrome stain was initially fair (Fleiss κ=0.28, weighted concordance coefficient=0.60) and significantly improved (κ=0.40, weighted concordance coefficient=0.66) following a multiheaded microscope training session and inclusion of glutamine synthetase immunohistochemistry. Average congestive hepatic fibrosis score correlated with significantly higher right atrial pressure, severity of right atrial dilation, presence of right ventricular dilation, elevated serum alanine aminotransferase, platelet counts, prothrombin time, and model for end-stage liver disease score. In conclusion, the congestive hepatic fibrosis scoring system is reproducible among pathologists and correlates with clinical and laboratory markers of congestive hepatopathy.

Mobilization of CD8+ T Cells via CXCR4 Blockade Facilitates PD-1 Checkpoint Therapy in Human Pancreatic Cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDA) is rarely cured, and single-agent immune checkpoint inhibition has not demonstrated clinical benefit despite the presence of large numbers of CD8+ T cells. We hypothesized that tumor-infiltrating CD8+ T cells harbor latent antitumor activity that can be reactivated using combination immunotherapy. EXPERIMENTAL DESIGN: Preserved human PDA specimens were analyzed using multiplex IHC (mIHC) and T-cell receptor (TCR) sequencing. Fresh tumor was treated in organotypic slice culture to test the effects of combination PD-1 and CXCR4 blockade. Slices were analyzed using IHC, flow cytometry, and live fluorescent microscopy to assess tumor kill, in addition to T-cell expansion and mobilization. RESULTS: mIHC demonstrated fewer CD8+ T cells in juxtatumoral stroma containing carcinoma cells than in stroma devoid of them. Using TCR sequencing, we found clonal expansion in each tumor; high-frequency clones had multiple DNA rearrangements coding for the same amino acid binding sequence, which suggests response to common tumor antigens. Treatment of fresh human PDA slices with combination PD-1 and CXCR4 blockade led to increased tumor cell death concomitant with lymphocyte expansion. Live microscopy after combination therapy demonstrated CD8+ T-cell migration into the juxtatumoral compartment and rapid increase in tumor cell apoptosis. CONCLUSIONS: Endogenous tumor-reactive T cells are present within the human PDA tumor microenvironment and can be reactivated by combined blockade of PD-1 and CXCR4. This provides a new basis for the rational selection of combination immunotherapy for PDA.See related commentary by Medina and Miller, p. 3747.

Searching for Bacterial Biofilm in Recurrent Cholangitis in Primary Sclerosing Cholangitis: A Case Presentation and Introduction of an Unexplored Disease Mechanism.

Inflammation and fibrosis of the bile ducts are the defining pathological characteristics of primary sclerosing cholangitis (PSC). A previously unexplored mechanism for recurrent cholangitis, one of PSC's most common presentations, is bacterial colonization of the biliary epithelium in the form of biofilm, which may confer resistance to antibiotics and host phagocytic machinery. The aim of the current study was to assess whether bacteria could be seen on the liver explant and whether they organized in the form of biofilm. An explanted PSC liver from a 60-year-old male who suffered from recurrent cholangitis was formalin-fixed, paraffin-embedded and Gram stained. The specimens were observed under light microscopy. Neither bacteria nor biofilm were detected. We did not detect bacteria or biofilm in the liver explant of a single PSC patient with recurrent cholangitis using standard light microscopy. We suspect this may be in part due to techniques related to tissue preservation and microscopy.

High prevalence of Helicobacter pylori clarithromycin resistance mutations among Seattle patients measured by droplet digital PCR.

BACKGROUND: Treatment of Helicobacter pylori infection is often empiric; however, current guidelines for management of Helicobacter pylori infection advise against the use of standard triple therapy (clarithromycin, amoxicillin, and proton-pump inhibitor) when clarithromycin resistance exceeds 20%. We developed and tested a new culture-free assay to detect clarithromycin resistance-conferring mutations to determine the prevalence of H. pylori clarithromycin resistance in patients from the United States Pacific Northwest. MATERIALS AND METHODS: Droplet digital PCR (ddPCR) was used to detect the H. pylori 23S rRNA gene, and resistance-conferring mutations, in archived, formalin-fixed, paraffin-embedded (FFPE) gastric tissue and to retrospectively determine the prevalence of clarithromycin-resistant H. pylori among 110 patients at an academic medical center in the Northwest United States between 2012 and 2014. RESULTS: Of 102 patients with the H. pylori 23S rRNA gene detected by the ddPCR assay, 45 (44%) had clarithromycin resistance mutations. Thirty-three of the 45 patients with clarithromycin resistance mutations had a mix of wild-type and resistance alleles. Prevalence of clarithromycin resistance mutations differed among racial groups and was highest among Asians, with mutations detected in 14 (67%) of the 21 patient samples. CONCLUSIONS: The prevalence of clarithromycin resistance detected in this region exceeds 20%, indicating that standard triple therapy should not be the first-line antibiotic treatment for H. pylori infection. Culture-free assays for detecting clarithromycin resistance mutations can be performed on archived tissue samples and will aid in informing tailored treatment for effective H. pylori eradication.

Acute Liver Failure Due to Echovirus 9 Associated With Persistent B-Cell Depletion From Rituximab.

We describe a case of fatal acute liver failure due to echovirus 9 in the setting of persistent B-cell depletion and hypogammaglobulinemia 3 years after rituximab therapy. Metagenomic next-generation sequencing further specified the etiologic agent. Early recognition may provide an opportunity for interventions including intravenous immunoglobulin and liver transplantation.

Long-lived pancreatic ductal adenocarcinoma slice cultures enable precise study of the immune microenvironment.

Pancreatic ductal adenocarcinoma (PDA) remains a deadly disease that is rarely cured, despite many recent successes with immunotherapy for other malignancies. As the human disease is heavily infiltrated by effector T cells, we postulated that accurately modeling the PDA immune microenvironment would allow us to study mechanisms of immunosuppression that could be overcome for therapeutic benefit. Using viable precision-cut slices from fresh PDA, we developed an organotypic culture system for this purpose. We confirmed that cultured slices maintain their baseline morphology, surface area, and microenvironment after at least 6 d in culture, and demonstrated slice survival by MTT assay and by immunohistochemistry staining with Ki-67 and cleaved-Caspase-3 antibodies. Immune cells, including T cells (CD3+, CD8+, and FOXP3+) and macrophages (CD68+, CD163+ and HLA-DR+), as well as stromal myofibroblasts (αSMA+) were present throughout the culture period. Global profiling of the PDA proteome before and after 6 d slice culture indicated that the majority of the immunological proteins identified remain stable during the culture process. Cytotoxic effects of drug treatment (staurosporine, STS and cycloheximide, CHX) on PDA slices culture confirmed that this system can be used to assess functional response and cell survival following drug treatment in both a treatment time- and dose-dependent manner. Using multicolor immunofluorescence, we stained live slices for both cancer cells (EpCAM+) and immune cells (CD11b+ and CD8+). Finally, we confirmed that autologous CFSE-labeled splenocytes readily migrate into co-cultured tumor slices. Thus, our present study demonstrates the potential to use tumor slice cultures to study the immune microenvironment of PDA.

Superior Patient and Graft Survival in Adult Liver Transplant with Rabbit Antithymocyte Globulin Induction: Experience with 595 Patients.

OBJECTIVES: The use of induction therapy in liver transplant is debatable. We aimed to compare clinical outcomes of different induction protocols in liver transplant recipients. MATERIALS AND METHODS: This was a retrospective cohort analyses using the University of Washington Transplant Database from January 2005 to May 2012 for adult (≥ 18 y old) primary liver transplant patients. All patients received induction therapy. Maintenance immunosuppressive agents were tacrolimus or tacrolimus-mycophenolate mofetil. Primary endpoints were acute cellular rejection, patient survival, and graft survival. In patients with chronic hepatitis C, the degree of histologic inflammation or fibrosis at 1 year was assessed. Cox proportional hazards models were constructed to evaluate variables associated with both patient and graft survival. RESULTS: We identified 595 patients: 322 patients received rabbit antithymocyte globulin and 273 received interleukin 2 receptor blocker. Acute cellular rejection was higher in patients who received interleukin 2 receptor blocker than in patients who received rabbit antithymocyte globulin (27% vs 18%; P < .03). Both patient survival at 1 year (95% vs 90%), 3 years (92% vs 87%), and 5 years (86% vs 80%) and graft survival at 1 year (93% vs 88%), 3 years (90% vs 86%), and 5 years (83% vs 78%) were superior with rabbit antithymocyte globulin than with the interleukin 2 receptor blocker (P < .002). In patients with hepatitis C virus, type of induction therapy did not have any effect on the timing of hepatitis C virus recurrence. At 1 year after transplant, 33.3% in the rabbit antithymocyte globulin group had grade 3/4 inflammation and 10.2% had stage 3/4 fibrosis, compared with 16.8% and 4.8% in the interleukin 2 receptor blocker group (P ≤ .002 and not significant). Female recipient, Model for End-Stage Liver Disease score, hepatocellular carcinoma, and high preoperative serum creatinine levels were associated with less favorable patient and graft survival. CONCLUSIONS: Rabbit antithymocyte globulin is associated with lower rejection rate and improved patient and graft survival in liver transplant. Type of therapy affects the degree of histologic hepatitis C virus recurrence.

Analysis of ROR1 Protein Expression in Human Cancer and Normal Tissues.

Purpose: This study examines cell surface ROR1 expression in human tumors and normal tissues. ROR1 is considered a promising target for cancer therapy due to putative tumor-specific expression, and multiple groups are developing antibodies and/or chimeric antigen receptor-modified T cells to target ROR1. On-target, off-tumor toxicity is a challenge for most nonmutated tumor antigens; however, prior studies suggest that ROR1 is absent on most normal tissues.Experimental Design: Our studies show that published antibodies lack sensitivity to detect endogenous levels of cell surface ROR1 by immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded tissues. We developed a ROR1-specific monoclonal antibody (mAb) targeting the carboxy-terminus of ROR1 and evaluated its specificity and sensitivity in IHC.Results: The 6D4 mAb is a sensitive and specific reagent to detect cell surface ROR1 by IHC. The data show that ROR1 is homogenously expressed on a subset of ovarian cancer, triple-negative breast cancer, and lung adenocarcinomas. Contrary to previous findings, we found ROR1 is expressed on several normal tissues, including parathyroid; pancreatic islets; and regions of the esophagus, stomach, and duodenum. The 6D4 mAb recognizes rhesus ROR1, and ROR1 expression was similar in human and macaque tissues, suggesting that the macaque is a suitable model to evaluate safety of ROR1-targeted therapies.Conclusions: ROR1 is a promising immunotherapeutic target in many epithelial tumors; however, high cell surface ROR1 expression in multiple normal tissues raises concerns for on-target off-tumor toxicities. Clinical translation of ROR1-targeted therapies warrants careful monitoring of toxicities to normal organs and may require strategies to ensure patient safety. Clin Cancer Res; 23(12); 3061-71. ©2016 AACR.

'Non-classical' HER2 FISH results in breast cancer: a multi-institutional study.

The 2013 CAP/ASCO HER2 Testing Guidelines Update modified HER2 FISH categories such that some cases with 'monosomy', 'co-amplification/polysomy', low-level increased HER2 signals or clustered heterogeneity now are considered amplified or equivocal. This study examines the frequency and clinico-pathologic characteristics of breast cancers with equivocal or 'non-classical' HER2 FISH results. Breast cancers (2001-2014) with HER2 FISH results, HER2 immunohistochemistry, ER, grade, and age from three institutions (Stanford, UCSF, UWMC) were collected. HER2 FISH was interpreted using the updated recommendations. Amplified cases with non-classical results were grouped into the following categories: (1) 'monosomy' (ratio ≥2.0, mean HER2/cell<4.0); (2) 'co-amplified' (ratio<2.0, mean HER2/cell ≥6.0); (3) 'low amplified' (ratio ≥2.0, mean HER2/cell 4.0-5.9). Heterogeneous cases with clustered HER2-positive cells were also included. Of 8068 cases, 5.2% were equivocal and 4.6% had a 'non-classical' HER2 amplified result; 1.4% 'monosomy', 0.8% 'co-amplified', 2.1% 'low amplified', and 0.3% clustered heterogeneity. These cancers had a high frequency of ER positive (80.4%), Nottingham grade 3 (52.1%) results. The highest percentage of grade 3 cancers (66.7%) and positive HER2 immunohistochemistry (31.7%) was in the 'co-amplified' group. The 'monosomy' group had the highest percent grade 1 cancers (13.3%) and was most frequently HER2 immunohistochemistry negative (30.1%). Equivocal cases had very similar characteristics to the 'low-amplified' category. Cases with non-classical HER2 amplification or equivocal results are typically ER positive, higher grade cancers. 'Co-amplified' cases have the highest frequencies of aggressive characteristics and 'monosomy' cases the highest frequencies of lower risk features. With little clinical outcomes data currently available on these non-classical HER2 results, these results support the current classification scheme for HER2 FISH, with case-by-case correlation with additional clinical-pathologic factors when evaluating whether to offer HER2-targeted therapies in these non-classical cases.

CT of Atypical and Uncommon Presentations of Hepatocellular Carcinoma.

OBJECTIVE: The purpose of this article is to familiarize radiologists with uncommon presentations of hepatocellular carcinoma (HCC) with an emphasis on the CT spectrum of atypical appearances. CONCLUSION: HCC is the fifth most common neoplasm worldwide and the second most common cause of cancer-related death. In many cases, HCC can be confidently diagnosed with noninvasive imaging. However, there are numerous unusual appearances of HCC with which the radiologist must be familiar.

Proliferative capacity of vein graft smooth muscle cells and fibroblasts in vitro correlates with graft stenosis.

OBJECTIVE: About a quarter of peripheral vein grafts fail due in part to intimal hyperplasia. The proliferative capacity and response to growth inhibitors of medial smooth muscle cells and adventitial fibroblasts in vitro were studied to test the hypothesis that intrinsic differences in cells of vein grafts are associated with graft failure. METHODS: Cells were grown from explants of the medial and adventitial layers of samples of vein grafts obtained at the time of implantation. Vein graft patency and function were monitored over the first 12 months using ankle pressures and Duplex ultrasound to determine vein graft status. Cells were obtained from veins from 11 patients whose grafts remained patent (non-stenotic) and from seven patients whose grafts developed stenosis. Smooth muscle cells (SMCs) derived from media and fibroblasts derived from adventitia were growth arrested in serum-free medium and then stimulated with 1 muM sphingosine-1-phosphate (S1P), 10 nM thrombin, 10 ng/ml epidermal growth factor (EGF), 10 ng/ml platelet-derived growth factor-BB (PDGF-BB), PDGF-BB plus S1P, or PDGF-BB plus thrombin for determination of incorporation of [(3)H]-thymidine into DNA. Cells receiving PDGF-BB or thrombin were also treated with or without 100 microg/ml heparin, which is a growth inhibitor. Cells receiving thrombin were also treated with or without 150 nM AG1478, an EGF receptor kinase inhibitor. RESULTS: SMCs and fibroblasts from veins of patients that developed stenosis responded more to the growth factors, such as PDGF-BB alone or in combination with thrombin or S1P, than cells from veins of patients that remained patent (P = .012). In addition, while PDGF-BB-mediated proliferation of fibroblasts from grafts that remained patent was inhibited by heparin (P < .03), PDGF-BB-mediated proliferation of fibroblasts from veins that developed stenosis was not (P > .5). CONCLUSION: Inherent differences in the proliferative response of vein graft cells to PDGF-BB and heparin may explain, in part, the variability among patients regarding long term patency of vein grafts.