Molecular Markers of Response to Anti-PD1 Therapy in Advanced Hepatocellular Carcinoma.

BACKGROUND & AIMS: Single-agent anti-PD1 checkpoint inhibitors convey outstanding clinical benefits in a small fraction (∼20%) of patients with advanced hepatocellular carcinoma (aHCC) but the molecular mechanisms determining response are unknown. To fill this gap, we herein analyze the molecular and immune traits of aHCC in patients treated with anti-PD1. METHODS: Overall, 111 tumor samples from patients with aHCC were obtained from 13 centers before systemic therapies. We performed molecular analysis and immune deconvolution using whole-genome expression data (n = 83), mutational analysis (n = 72), and histologic evaluation with an endpoint of objective response. RESULTS: Among 83 patients with transcriptomic data, 28 were treated in frontline, whereas 55 patients were treated after tyrosine kinase inhibitors (TKI) either in second or third line. Responders treated in frontline showed upregulated interferon-γ signaling and major histocompatibility complex II-related antigen presentation. We generated an 11-gene signature (IFNAP), capturing these molecular features, which predicts response and survival in patients treated with anti-PD1 in frontline. The signature was validated in a separate cohort of aHCC and >240 patients with other solid cancer types where it also predicted response and survival. Of note, the same signature was unable to predict response in archival tissue of patients treated with frontline TKIs, highlighting the need for fresh biopsies before immunotherapy. CONCLUSION: Interferon signaling and major histocompatibility complex-related genes are key molecular features of HCCs responding to anti-PD1. A novel 11-gene signature predicts response in frontline aHCC, but not in patients pretreated with TKIs. These results must be confirmed in prospective studies and highlights the need for biopsies before immunotherapy to identify biomarkers of response.

Mapping the hepatic immune landscape identifies monocytic macrophages as key drivers of steatohepatitis and cholangiopathy progression.

BACKGROUND AND AIMS: The progression of chronic liver diseases towards liver cirrhosis is accompanied by drastic tissue changes. This study combines elaborate transcriptomic and histological methods aiming at spatially resolving the hepatic immune microenvironment in NAFLD (including NASH, primary sclerosing cholangitis, primary biliary cholangitis, and severe alcoholic hepatitis). APPROACH AND RESULTS: Human liver samples were subjected to RNA-sequencing (n=225) and imaging cytometry (n=99) across 3 independent patient cohorts. Liver samples from alcoholic hepatitis and primary biliary cholangitis patients were used for comparison. Myeloid populations were further characterized in corresponding mouse models. Imaging, clinical, and phenotypical data were combined for multidimensional analysis. NAFLD/NASH and primary sclerosing cholangitis disease stages were associated with loss of parenchymal areas, increased ductular cell accumulation, and infiltration of immune cells. NASH patients predominantly exhibited myeloid cell accumulation, whereas primary sclerosing cholangitis patients additionally had pronounced lymphoid cell responses. Correlating to disease stage, both etiologies displayed intense IBA1 + CD16 low CD163 low macrophage aggregation in nonparenchymal areas, with a distinct spatial proximity to ductular cells. Mouse models revealed that disease-associated IBA1 + hepatic macrophages originated from bone marrow-derived monocytes. Using an unbiased, machine learning-based algorithm, IBA1 in combination with hepatocyte and ductular cell immunostaining-predicted advanced cirrhosis in human NASH, primary sclerosing cholangitis, and alcoholic hepatitis. CONCLUSIONS: Loss of hepatocytes and increased ductular reaction are tightly associated with monocyte-derived macrophage accumulation and represent the most prominent common immunological feature revealing the progression of NAFLD, primary sclerosing cholangitis, primary biliary cholangitis, and alcoholic hepatitis, suggesting IBA1 + CD163 low macrophages are key pathogenic drivers of human liver disease progression across diverse etiologies.

Models of Gastroenteropancreatic Neuroendocrine Neoplasms: Current Status and Future Directions.

Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) are a rare, heterogeneous group of tumors that originate from the endocrine system of the gastrointestinal tract and pancreas. GEP-NENs are subdivided according to their differentiation into well-differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinomas (NECs). Since GEP-NENs represent rare diseases, only limited data from large prospective, randomized clinical trials are available, and recommendations for treatment of GEP-NEN are in part based on data from retrospective analyses or case series. In this context, tractable disease models that reflect the situation in humans and that allow to recapitulate the different clinical aspects and disease stages of GEP-NET or GEP-NEC are urgently needed. In this review, we highlight available data on mouse models for GEP-NEN. We discuss how these models reflect tumor biology of human disease and whether these models could serve as a tool for understanding the pathogenesis of GEP-NEN and for disease modeling and pharmacosensitivity assays, facilitating prediction of treatment response in patients. In addition, open issues applicable for future developments will be discussed.

The Role of Microbiota in Primary Sclerosing Cholangitis and Related Biliary Malignancies.

Primary sclerosing cholangitis (PSC) is an immune-related cholangiopathy characterized by biliary inflammation, cholestasis, and multifocal bile duct strictures. It is associated with high rates of progression to end-stage liver disease as well as a significant risk of cholangiocarcinoma (CCA), gallbladder cancer, and colorectal carcinoma. Currently, no effective medical treatment with an impact on the overall survival is available, and liver transplantation is the only curative treatment option. Emerging evidence indicates that gut microbiota is associated with disease pathogenesis. Several studies analyzing fecal and mucosal samples demonstrate a distinct gut microbiome in individuals with PSC compared to healthy controls and individuals with inflammatory bowel disease (IBD) without PSC. Experimental mouse and observational human data suggest that a diverse set of microbial functions may be relevant, including microbial metabolites and bacterial processing of pharmacological agents, bile acids, or dietary compounds, altogether driving the intrahepatic inflammation. Despite critical progress in this field over the past years, further functional characterization of the role of the microbiota in PSC and related malignancies is needed. In this review, we discuss the available data on the role of the gut microbiome and elucidate important insights into underlying pathogenic mechanisms and possible microbe-altering interventions.

From Liver Cirrhosis to Cancer: The Role of Micro-RNAs in Hepatocarcinogenesis.

In almost all cases, hepatocellular carcinoma (HCC) develops as the endpoint of a sequence that starts with chronic liver injury, progresses to liver cirrhosis, and finally, over years and decades, results in liver cancer. Recently, the role of non-coding RNA such as microRNA (miRNA) has been demonstrated in the context of chronic liver diseases and HCC. Moreover, data from a phase II trial suggested a potential role of microRNAs as therapeutics in hepatitis-C-virus infection, representing a significant risk factor for development of liver cirrhosis and HCC. Despite progress in the clinical management of chronic liver diseases, pharmacological treatment options for patients with liver cirrhosis and/or advanced HCC are still limited. With their potential to regulate whole networks of genes, miRNA might be used as novel therapeutics in these patients but could also serve as biomarkers for improved patient stratification. In this review, we discuss available data on the role of miRNA in the transition from liver cirrhosis to HCC. We highlight opportunities for clinical translation and discuss open issues applicable to future developments.

The Role of miRNA in the Pathophysiology of Neuroendocrine Tumors.

Neuroendocrine tumors (NETs) represent a tumor group that is both rare and heterogeneous. Prognosis is largely determined by the tumor grading and the site of the primary tumor and metastases. Despite intensive research efforts, only modest advances in diagnostic and therapeutic approaches have been achieved in recent years. For patients with non-respectable tumor stages, prognosis is poor. In this context, the development of novel diagnostic tools for early detection of NETs and prediction of tumor response to therapy as well as estimation of the overall prognosis would greatly improve the clinical management of NETs. However, identification of novel diagnostic molecules is hampered by an inadequate understanding of the pathophysiology of neuroendocrine malignancies. It has recently been demonstrated that microRNA (miRNA), a family of small RNA molecules with an established role in the pathophysiology of quite different cancer entities, may also play a role as a biomarker. Here, we summarize the available knowledge on the role of miRNAs in the development of NET and highlight their potential use as serum-based biomarkers in the context of this disease. We discuss important challenges currently preventing their use in clinical routine and give an outlook on future directions of miRNA research in NET.

In Vivo Models for Cholangiocarcinoma-What Can We Learn for Human Disease?

Cholangiocarcinoma (CCA) comprises a heterogeneous group of primary liver tumors. They emerge from different hepatic (progenitor) cell populations, typically via sporadic mutations. Chronic biliary inflammation, as seen in primary sclerosing cholangitis (PSC), may trigger CCA development. Although several efforts were made in the last decade to better understand the complex processes of biliary carcinogenesis, it was only recently that new therapeutic advances have been achieved. Animal models are a crucial bridge between in vitro findings on molecular or genetic alterations, pathophysiological understanding, and new therapeutic strategies for the clinic. Nevertheless, it is inherently difficult to recapitulate simultaneously the stromal microenvironment (e.g., immune-competent cells, cholestasis, inflammation, PSC-like changes, fibrosis) and the tumor biology (e.g., mutational burden, local growth, and metastatic spread) in an animal model, so that it would reflect the full clinical reality of CCA. In this review, we highlight available data on animal models for CCA. We discuss if and how these models reflect human disease and whether they can serve as a tool for understanding the pathogenesis, or for predicting a treatment response in patients. In addition, open issues for future developments will be discussed.

Microbial Players in Primary Sclerosing Cholangitis: Current Evidence and Concepts.

Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease with progressive biliary inflammation, destruction of the biliary tract, and fibrosis, resulting in liver cirrhosis and end-stage liver disease. To date, liver transplantation is the only definitive treatment option for PSC. The precise etiology of PSC remains elusive, but it is widely accepted to involve a complex interplay between genetic predisposition, immunologic dysfunction, and environmental influence. In recent years, the gut-liver axis has emerged as a crucial pathway contributing to the pathogenesis of PSC, with particular focus on the role of gut microbiota. However, the role of the fungal microbiome or mycobiome has been overlooked for years, resulting in a lack of comprehensive studies on its involvement in PSC. In this review, we clarify the present clinical and mechanistic data and concepts concerning the gut bacterial and fungal microbiota in the context of PSC. This review sheds light on the role of specific microbes and elucidates the dynamics of bacterial and fungal populations. Moreover, we discuss the latest insights into microbe-altering therapeutic approaches involving the gut-liver axis and bile acid metabolism.

Biliary microbial patterns in primary sclerosing cholangitis are linked to poorer transplant-free survival.

BACKGROUND: Factors that determine individual disease course of patients with primary sclerosing cholangitis (PSC) are poorly understood. Although an association between gut microbes and disease outcome has been suggested, little is known about the role of microbes in the biliary tract. METHODS: We analyzed microbial cultures from bile specimens obtained during routine endoscopic retrograde cholangiopancreatography (ERCP) and intraoperatively before liver transplantation in 114 patients with PSC in our tertiary academic center. The presence of bacterial and fungal species was correlated with clinical characteristics and outcome data. RESULTS: A total of 87 patients (76%) had positive bile culture results. The presence of concomitant inflammatory bowel disease (IBD) was associated with positive bile culture results in multivariate analysis (OR, 4.707; 95% CI, 1.688-13.128; p=0.003). Enterococcus spp. in the bile was associated with a more frequent occurrence of liver transplantation and/or death (OR, 2.778; 95% CI, 1.147-6.728; p=0.021) and recurrent (≥3) cholangitis episodes (OR, 2.839; 95% CI, 1.037-7.768; p=0.037). Biliary candidiasis was linked to a higher frequency of recurrent (≥3) cholangitis episodes (OR, 5.677; 95% CI, 1.940-16.616; p=0.001). Proton pump inhibitor intake conferred a clinical feature associated with biliary candidiasis in multivariate analysis (OR, 3.559; 95% CI, 1.275-9.937; p=0.016). CONCLUSIONS: Our data indicate that in patients with PSC, presence of Enterococcus spp. and Candida spp. in bile is associated with an adverse outcome. Concomitant IBD is linked to presence of microbes in bile, and proton pump inhibitor intake is a feature associated with biliary candidiasis in patients with PSC.

Soluble Urokinase Plasminogen Activator Receptor Levels Are Associated with Severity of Fibrosis in Patients with Primary Sclerosing Cholangitis.

The soluble urokinase-type plasminogen activator receptor (suPAR) has evolved as a useful biomarker for different entities of chronic liver disease. However, its role in patients with primary sclerosing cholangitis (PSC) is obscure. We analyzed plasma levels of suPAR in 84 patients with PSC and compared them to 68 patients with inflammatory bowel disease (IBD) without PSC and to 40 healthy controls. Results are correlated with clinical records. suPAR concentrations were elevated in patients with PSC compared to patients with IBD only and to healthy controls (p < 0.001). Elevated suPAR levels were associated with the presence of liver cirrhosis (p < 0.001) and signs of portal hypertension (p < 0.001). suPAR revealed a high accuracy for the discrimination of the presence of liver cirrhosis comparable to previously validated noninvasive fibrosis markers (area under the curve (AUC) 0.802 (95%CI: 0.702−0.902)). Further, we demonstrated that suPAR levels may indicate the presence of acute cholangitis episodes (p < 0.001). Finally, despite the high proportion of PSC patients with IBD, presence of IBD and its disease activity did not influence circulating suPAR levels. suPAR represents a previously unrecognized biomarker for diagnosis and liver cirrhosis detection in patients with PSC. However, it does not appear to be confounded by intestinal inflammation in the context of IBD.

PD-L1 - inhibitors in neuroendocrine neoplasia: Results from a real-life study.

ABSTRACT: Immune check-point inhibitors (ICIs) have changed our view on how to treat cancer. Despite their approval in treatment of many different cancers, efficacy of immune check-point inhibitors (ICI) in neuroendocrine neoplasia is limited and poorly understood. Established treatment options of neuroendocrine tumors (NET) and neuroendocrine carcinomas (NECs) are based on surgery, tumor-targeted medical treatments, Peptide Receptor Radionuclide Therapy (PRRT), and locoregional therapies. However, in many patients these treatments lose efficacy over time, and novel therapies are urgently needed. We report on 8 patients diagnosed with neuroendocrine neoplasms (NEN) that were treated with ICI (pembrolizumab, avelumab, nivolumab plus ipilimumab) as salvage therapy. In this cohort, we observed tumor response with partial remission in 3 patients and stable disease in 1 patient. Four patients showed progressive disease. Of note, responses were observed both in PD-L1 positive and PD-L1 negative patients. Here, we discuss clinical courses of these patients in the context of available literature to highlight limitations and drawbacks currently preventing the use of ICI in routine management of patients with NEN.

A rare case of a patient with a high grade neuroendocrine tumor developing neutropenic sepsis after receiving PRRT combined with Capecitabine or Temozolomide: A case report.

Neuroendocrine tumors (NET) are rare and demonstrate variable clinical behavior depending on the degree of tumor differentiation. Patients with poorly differentiated tumors (NET G3) have a poor prognosis. Systemic treatment with cytotoxic chemotherapy is considered to be the treatment of choice. In patients that are refractory or intolerant to first-line therapy, experts recommend peptide receptor radionuclide therapy (PRRT) in tumors that express somatostatin receptors. Recently, combinations of PRRT and chemotherapy were tested in patients with NET. Available data have reported promising tumor control rates and an excellent toxicity profile in cases where PRRT had been administered with capecitabine/temozolomide, even when administered as salvage therapy. The current study reported an exceptional case of advanced NET G3 with severe toxicity upon receiving PRRT in combination with capecitabine/temozolomide as third line therapy. The patient developed a life-threatening neutropenic fever, fungal pneumonia and necrotizing mastitis 23 days after the first cycle of therapy was administered. However, the treatment led to a significant reduction in tumor size. A total of 5 months after treatment initiation, the patient was alive and in excellent clinical condition with sustained tumor response. In summary, the current study presented a rare case of high grade NET exhibiting an almost complete response to PRRT in combination capecitabine/temozolomide, despite facing unexpected severe toxicity.

Lessons From Immune Checkpoint Inhibitor Trials in Hepatocellular Carcinoma.

The implementation of immune checkpoint inhibitors (ICI) into the clinical management of different malignancies has largely changed our understanding of cancer treatment. After having proven efficacy in different tumor entities such as malignant melanoma and lung cancer, ICI were intensively tested in the setting of hepatocellular carcinoma (HCC). Here they could achieve higher and more durable response rates compared to tyrosine-kinase inhibitors (TKI), that were sole standard of care for the last decade. Most recently, ICI treatment was approved in a first line setting of HCC, for cases not suitable for curative strategies. However, only a subset of patients benefits from ICI therapy, while others experience rapid tumor progression, worsening of liver function and poor prognosis. Efforts are being made to find immune characteristics that predict tumor responsiveness to ICI, but no reliable biomarker could be identified so far. Nevertheless, data convincingly demonstrate that combination therapies (such as dual inhibition of PD-L1 and VEGF) are more effective than the application of single agents. In this review, we will briefly recapitulate the current algorithms for systemic treatment, discuss available results from checkpoint inhibitor trials and give an outlook on future directions of immunotherapy in HCC.

Atezolizumab and bevacizumab-induced encephalitis in advanced hepatocellular carcinoma: Case report and literature review.

INTRODUCTION: On the basis of the results of the IMBRAVE-150 trial, the combination of atezolizumab, a programmed cell death ligand 1 (PD-L1) antibody, as well as bevacizumab, a vascular endothelial growth factor (VEGF) antibody, represents a promising novel first-line therapy in patients with advanced hepatocellular carcinoma (HCC). Despite favorable safety data, serious adverse events have been described. However, central nervous system complications such as encephalitis have rarely been reported. We present the case of a 70-year-old woman with hepatitis C virus (HCV)-related liver cirrhosis and advanced HCC who developed severe encephalitis after only one cycle of atezolizumab/bevacizumab. PATIENT CONCERNS: Ten days after administration, the patient presented with confusion, somnolence, and emesis. Within a few days, the patient's condition deteriorated, and mechanical ventilation became necessary. DIAGNOSIS: Cerebrospinal fluid (CSF) analysis showed increased cell count and elevated protein values. Further work-up revealed no signs of an infectious, paraneoplastic, or other autoimmune cause. INTERVENTION: Suspecting an atezolizumab/bevacizumab-related encephalitis, we initiated a high-dose steroid pulse therapy as well as repeated plasmapheresis, which resulted in clinical improvement and remission of CSF abnormalities. OUTCOME: Despite successful weaning and transfer to a rehabilitation ward, the patient died of progressive liver cancer 76 days after initial treatment with atezolizumab/bevacizumab, showing no response. CONCLUSION: This case illustrates that rapid immunosuppressive treatment with prednisolone can result in remission even of severe encephalitis. We discuss this case in the context of available literature and previously reported cases of atezolizumab-induced encephalitis in different tumor entities, highlighting the diagnostic challenges in oncologic patients treated with immune checkpoint-inhibitors.

Low Serum Levels of Soluble Receptor Activator of Nuclear Factor κ B Ligand (sRANKL) Are Associated with Metabolic Dysregulation and Predict Long-Term Mortality in Critically Ill Patients.

Soluble receptor activator of nuclear factor κ B ligand (sRANKL) is a member of the tumor necrosis factor receptor superfamily, and therefore, involved in various inflammatory processes. The role of sRANKL in the course of bone remodeling via activation of osteoclasts as well as chronic disease progression has been described extensively. However, the potential functional importance of sRANKL in critically ill or septic patients remained unknown. Therefore, we measured sRANKL serum concentrations in 303 critically ill patients, including 203 patients with sepsis and 100 with non-sepsis critical illness. Results were compared to 99 healthy controls. Strikingly, in critically ill patients sRANKL serum levels were significantly decreased at intensive care unit (ICU) admission (p = 0.011) without differences between sepsis and non-sepsis patients. Inline, sRANKL was correlated with markers of metabolic dysregulation, such as pre-existing diabetes and various adipokines (e.g., adiponectin, leptin receptor). Importantly, overall mortality of critically ill patients in a three-year follow-up was significantly associated with decreased sRANKL serum concentrations at ICU admission (p = 0.038). Therefore, our study suggests sRANKL as a biomarker in critically ill patients which is associated with poor prognosis and overall survival beyond ICU stay.

Systemic treatment of hepatocellular carcinoma: from sorafenib to combination therapies.

For almost a decade, systemic therapy of advanced hepatocellular carcinoma (HCC) was limited to the tyrosine kinase inhibitor (TKI) sorafenib. Different agents including checkpoint inhibitors, TKIs and anti-VEGFR antibodies demonstrated efficacy in treatment. For the first time, the combination of atezolizumab and bevacizumab, a first-line treatment that is superior to the current standard was identified, potentially changing the way we treat HCC. In this review, we summarize current data on systemic treatment of patients with advanced HCC, focusing on combination therapies comprising immune checkpoint inhibitors, TKIs and locoregional therapies. We elucidate findings from recent trials and discuss such challenges as the lack of predictive biomarkers for identification of subgroups that will benefit from novel treatment strategies.

A case report of an excellent response to interferon- α in a patient with functional metastasized neuroendocrine tumor refractory to other treatments.

INTRODUCTION: Interferon alpha (IFNα) has been used for a long time in patients with functionally active neuroendocrine tumors (NET). However, due to the unfavorable toxicity profile of interferon, the perceived limited efficacy as well as the development of novel substances, IFNα is only used sparingly in the treatment of NET to date. PATIENTS CONCERNS AND DIAGNOSIS: We describe the case of a 63-year-old male patient with highly differentiated, functional NET of the ileum and synchronous liver metastasis. INTERVENTIONS: After failure of classical therapies including dose-intensified somatostatin analog treatment and palliative primary tumor resection, a therapy with pegylated IFNα2a (135 µg/wk) was initiated. Following this treatment, the patient fully recovered from signs of hypersecretion and demonstrated an impressive tumor response. OUTCOMES: Thirty months after initiating IFNα, the patient is still free of clinical symptoms and shows a sustained tumor response. Notably, no relevant side effects were observed. CONCLUSION: Our case report supports the use of IFNα in patients with functional NET refractory to classical treatments.

Primary Neuroendocrine Neoplasms of the Breast: Case Series and Literature Review.

Primary neuroendocrine carcinoma of the breast (NECB) as defined by the World Health Organization (WHO) in 2012 is a rare, but possibly under-diagnosed entity. It is heterogeneous as it entails a wide spectrum of diseases comprising both well-differentiated neuroendocrine tumors of the breast as well as highly aggressive small cell carcinomas. Retrospective screening of hospital charts of 612 patients (2008-2019) from our specialized outpatient unit for neuroendocrine neoplasia revealed five patients diagnosed with NECB. Given the low prevalence of these malignancies, correct diagnosis remains a challenge that requires an interdisciplinary approach. Specifically, NECB may be misclassified as carcinoma of the breast with neuroendocrine differentiation, carcinomas of the breast of no special type/invasive ductal carcinoma, or a metastasis to the breast. Therefore, this study presents multifaceted characteristics as well as the clinical course of these patients and discusses the five cases from our institution in the context of available literature.

Soluble Urokinase Plasminogen Activator Receptor (suPAR) Concentrations are Elevated in Patients with Neuroendocrine Malignancies.

Neuroendocrine neoplasia (NEN) comprises heterogeneous tumors that are challenging to diagnose and, especially in cases of poorly differentiated (G3) NEN, are associated with very limited survival. Novel biomarkers allowing an early diagnosis as well as an optimal selection of suitable treatment options are urgently needed to improve the outcome of these patients. Recently, alterations of soluble urokinase-type plasminogen activator receptor (suPAR) serum levels were described in various types of cancers. However, the role of circulating suPAR as a biomarker in patients with NEN is unknown. In this study, we measured suPAR serum levels in a large and well-characterized cohort of 187 patients with NEN (neuroendocrine carcinomas (NEC) n = 30; neuroendocrine tumors (NET), n = 157) as well as 44 healthy controls. suPAR concentrations were significantly elevated in patients compared to controls. However, suPAR concentrations were independent of tumor-related factors such as the proliferation activity according to Ki-67, tumor grading, TNM (TNM classification of malignant tumors) stage, somatostatin receptor expression or clinical features such as functional or nonfunctional disease and the presence of tumor relapse. Interestingly, suPAR concentrations in NET patients were similar when compared to those measured in NEC patients. In contrast to previous results from other malignancies, in our analysis suPAR levels were not a significant predictor of overall survival. In conclusion, our data suggests that suPAR serum concentrations are elevated in NEN patients but do not allow prediction of outcome.