Clearance of Hepatitis C Virus following Immune Checkpoint Inhibitor Therapy for Hepatocellular Carcinoma: Case Report.

Introduction: Patients with advanced hepatocellular carcinoma (HCC) have limited treatment options in the context of decompensated cirrhosis. HCC occurs in patients with hepatitis C virus (HCV) infection and cirrhosis at 1-4% per year. Direct-acting antiviral (DAA) efficacy is decreased in the presence of HCC. We present a case where immunotherapy may have resulted in HCV clearance, when DAA therapy had been ineffective. We hypothesise that immune checkpoint inhibitors targeting the PD-1/PD-L1 pathway can reverse T-cell exhaustion and aid in the clearance of chronic HCV. Case Presentation: This case study describes a male in his 40 s identified by a re-engagement initiative for HCV, who had been unaware of his diagnosis. On further investigation he was found to have compensated for liver cirrhosis and HCC. He was treated with HCV DAA therapy (sofosbuvir/velpatasvir) and then systemic immunotherapy for HCC with atezolizumab and bevacizumab, in an attempt to downstage the disease. Hepatitis C therapy did not achieve sustained virological response, with viral relapse after the end of treatment. This, combined with ongoing alcohol use, resulted in hepatic decompensation and cessation of immunotherapy after the fifth cycle. The HCV RNA subsequently became undetectable without further DAA re-treatment. Conclusion: To our knowledge, this is the first case of HCV clearance after DAA relapse and the timing of this event after immunotherapy suggests a causal link. We hypothesise that this may be due to the reversal of antiviral T-cell exhaustion. This would therefore support further investigation into other chronic viral infections that create tumour associated with immunosuppressive microenvironments.

A Systematic Review of Surgical Management Strategies in the Treatment of Peritoneal Carcinomatosis of Neuroendocrine Origin.

Cytoreductive surgery (CRS) represents the cornerstone of surgical management for peritoneal carcinomatosis (PC) and involves peritonectomy procedures aimed at complete peritoneal tumour resection. Frequently, CRS is combined with hyperthermic intraperitoneal chemotherapy (HIPEC). The combination of CRS + HIPEC is now considered the standard of care in patients with colorectal and ovarian PC. However, the role of this multi-modality treatment approach in patients with PC of neuroendocrine tumour origin (NET-PC) is less well understood. This systematic review provides a summary of available evidence on management strategies for patients with NET-PC. A systematic literature search was performed using Ovid Medline, EMBASE and Cochrane Library databases to identify studies reporting outcomes for patients with NET-PC undergoing surgical treatment. Eligible studies were assessed for methodological quality and design and evaluated for a method of surgical treatment, method of HIPEC delivery, oncological outcomes, and treatment-related morbidity. Eight studies, including a total of 1240 patients with NET-PC, met predefined inclusion criteria and have been included in this review. In three of the included studies, CRS alone was performed for patients with NET-PC, while five studies reported outcomes with combined treatment using CRS plus HIPEC. All studies were performed at tertiary peritoneal malignancy centres. Only one study directly compared outcomes in patients with NET-PC undergoing CRS plus HIPEC compared with CRS in isolation, with no significant difference in overall survival reported. Carefully selected patients with NET-PC may benefit from aggressive surgical treatment in the form of CRS +/- HIPEC. These procedures are best undertaken at centres with expertise in the management of both neuroendocrine tumours and peritoneal malignancy, as both are conditions that require tertiary-level care. The additional benefit of the HIPEC component in this group of patients remains unclear and warrants further investigation in clinical trials. Overall, the quality of data on this subject is restricted by the low number of studies and the variability in treatment methods employed. A multi-national data registry for patients with NET-PC may offer the opportunity to better define treatment algorithms. Translational research efforts in parallel should focus on developing a better biological understanding of NET-PC, with a view to identifying more effective intraperitoneal cytocidal agents.

Strain-Dependent Restriction of Human Cytomegalovirus by Zinc Finger Antiviral Proteins.

Cellular antiviral factors that recognize viral nucleic acid can inhibit virus replication. These include the zinc finger antiviral protein (ZAP), which recognizes high CpG dinucleotide content in viral RNA. Here, we investigated the ability of ZAP to inhibit the replication of human cytomegalovirus (HCMV). Depletion of ZAP or its cofactor KHNYN increased the titer of the high-passage HCMV strain AD169 but had little effect on the titer of the low-passage strain Merlin. We found no obvious difference in expression of several viral proteins between AD169 and Merlin in ZAP knockdown cells, but observed a larger increase in infectious virus in AD169 compared to Merlin in the absence of ZAP, suggesting that ZAP inhibited events late in AD169 replication. In addition, there was no clear difference in the CpG abundance of AD169 and Merlin RNAs, indicating that genomic content of the two virus strains was unlikely to be responsible for differences in their sensitivity to ZAP. Instead, we observed less ZAP expression in Merlin-infected cells late in replication compared to AD169-infected cells, which may be related to different abilities of the two virus strains to regulate interferon signaling. Therefore, there are strain-dependent differences in the sensitivity of HCMV to ZAP, and the ability of low-passage HCMV strain Merlin to evade inhibition by ZAP is likely related to its ability to regulate interferon signaling, not the CpG content of RNAs produced from its genome. IMPORTANCE Determining the function of cellular antiviral factors can inform our understanding of virus replication. The zinc finger antiviral protein (ZAP) can inhibit the replication of diverse viruses. Here, we examined ZAP interaction with the DNA virus human cytomegalovirus (HCMV). We found HCMV strain-dependent differences in the ability of ZAP to influence HCMV replication, which may be related to the interaction of HCMV strains with the type I interferon system. These observations affect our current understanding of how ZAP restricts HCMV and how HCMV interacts with the type I interferon system.

Premalignant Conditions of Bone.

Development of malignancy is a multifactorial process, and there are multitude of conditions of bone that may predispose patients to malignancy. Etiologies of malignancy include benign osseous conditions, genetic predisposition, and extrinsic conditions. New-onset pain or growth in a previously stable lesion is that should concern for malignant change and should prompt a diagnostic workup for malignancy.

Minimal impact of ZAP on lentiviral vector production and transduction efficiency.

The antiviral protein ZAP binds CpG dinucleotides in viral RNA to inhibit replication. This has likely led to the CpG suppression observed in many RNA viruses, including retroviruses. Sequences added to retroviral vector genomes, such as internal promoters, transgenes, or regulatory elements, substantially increase CpG abundance. Because these CpGs could allow retroviral vector RNA to be targeted by ZAP, we analyzed whether it restricts vector production, transduction efficiency, and transgene expression. Surprisingly, even though CpG-high HIV-1 was efficiently inhibited by ZAP in HEK293T cells, depleting ZAP did not substantially increase lentiviral vector titer using several packaging and genome plasmids. ZAP overexpression also did not inhibit lentiviral vector titer. In addition, decreasing CpG abundance in a lentiviral vector genome did not increase its titer, and a gammaretroviral vector derived from murine leukemia virus was not substantially restricted by ZAP. Overall, we show that the increased CpG abundance in retroviral vectors relative to the wild-type retroviruses they are derived from does not intrinsically sensitize them to ZAP. Further understanding of how ZAP specifically targets transcripts to inhibit their expression may allow the development of CpG sequence contexts that efficiently recruit or evade this antiviral system.

Rapid Response to Lorlatinib in a Patient With TFG-ROS1 Fusion Positive Inflammatory Myofibroblastic Tumor of the Chest Wall Metastatic to the Brain and Refractory to First and Second Generation ROS1 Inhibitors.

Most inflammatory myofibroblastic tumors (IMTs) harbor ALK fusions but oncogene fusions involving ROS1, RET, NTRK, and PDGFR also occur. The recognition that most IMTs harbor receptor tyrosine kinase fusions has provided a rationale for the use of tyrosine kinase inhibitors to target these oncogenic drivers in advanced IMTs. Crizotinib has been effective in ALK and ROS1-positive IMTs but resistance eventually develops. Here we report the successful use of lorlatinib in a patient with heavily pretreated ROS1-positive IMT of the chest wall with acquired crizotinib-resistance and metastasis to the brain.

CpG Dinucleotides Inhibit HIV-1 Replication through Zinc Finger Antiviral Protein (ZAP)-Dependent and -Independent Mechanisms.

CpG dinucleotides are suppressed in the genomes of many vertebrate RNA viruses, including HIV-1. The cellular antiviral protein ZAP (zinc finger antiviral protein) binds CpGs and inhibits HIV-1 replication when CpGs are introduced into the viral genome. However, it is not known if ZAP-mediated restriction is the only mechanism driving CpG suppression. To determine how CpG dinucleotides affect HIV-1 replication, we increased their abundance in multiple regions of the viral genome and analyzed the effect on RNA expression, protein abundance, and infectious-virus production. We found that the antiviral effect of CpGs was not correlated with their abundance. Interestingly, CpGs inserted into some regions of the genome sensitize the virus to ZAP antiviral activity more efficiently than insertions into other regions, and this sensitivity can be modulated by interferon treatment or ZAP overexpression. Furthermore, the sensitivity of the virus to endogenous ZAP was correlated with its sensitivity to the ZAP cofactor KHNYN. Finally, we show that CpGs in some contexts can also inhibit HIV-1 replication by ZAP-independent mechanisms, and one of these is the activation of a cryptic splice site at the expense of a canonical splice site. Overall, we show that the location and sequence context of the CpG in the viral genome determines its antiviral activity.IMPORTANCE Some RNA virus genomes are suppressed in the nucleotide combination of a cytosine followed by a guanosine (CpG), indicating that they are detrimental to the virus. The antiviral protein ZAP binds viral RNA containing CpGs and prevents the virus from multiplying. However, it remains unknown how the number and position of CpGs in viral genomes affect restriction by ZAP and whether CpGs have other antiviral mechanisms. Importantly, manipulating the CpG content in viral genomes could help create new vaccines. HIV-1 shows marked CpG suppression, and by introducing CpGs into its genome, we show that ZAP efficiently targets a specific region of the viral genome, that the number of CpGs does not predict the magnitude of antiviral activity, and that CpGs can inhibit HIV-1 gene expression through a ZAP-independent mechanism. Overall, the position of CpGs in the HIV-1 genome determines the magnitude and mechanism through which they inhibit the virus.

KHNYN is essential for the zinc finger antiviral protein (ZAP) to restrict HIV-1 containing clustered CpG dinucleotides.

CpG dinucleotides are suppressed in most vertebrate RNA viruses, including HIV-1, and introducing CpGs into RNA virus genomes inhibits their replication. The zinc finger antiviral protein (ZAP) binds regions of viral RNA containing CpGs and targets them for degradation. ZAP does not have enzymatic activity and recruits other cellular proteins to inhibit viral replication. We found that KHNYN, a protein with no previously known function, interacts with ZAP. KHNYN overexpression selectively inhibits HIV-1 containing clustered CpG dinucleotides and this requires ZAP and its cofactor TRIM25. KHNYN requires both its KH-like domain and NYN endonuclease domain for antiviral activity. Crucially, depletion of KHNYN eliminated the deleterious effect of CpG dinucleotides on HIV-1 RNA abundance and infectious virus production and also enhanced the production of murine leukemia virus. Overall, we have identified KHNYN as a novel cofactor for ZAP to target CpG-containing retroviral RNA for degradation.

Integration of a Personalized Molecular Targeted Therapy into the Multimodal Treatment of Refractory Childhood Embryonal Tumor with Multilayered Rosettes (ETMR).

Embryonal tumor with multilayered rosettes (ETMR) are rare pediatric brain tumors with increased malignant potential. Despite the advances in multimodal treatment schemes the overall 5-year event free survival rates for ETMR are not favorable. Further, therapeutic regimes are limited to a case by case basis due to the limited amount of literature and guidelines available for treating childhood ETMR. We report one patient with refractory ETMR who was successfully treated by implementing a molecular profiling approach which identified the tyrosine kinase inhibitor dasatinib as a viable therapy. Our results suggest that utilizing this precision medicine approach might prove useful in treating patients with refractory ETMR.

CNS Langerhans cell histiocytosis: Common hematopoietic origin for LCH-associated neurodegeneration and mass lesions.

BACKGROUND: Central nervous system Langerhans cell histiocytosis (CNS-LCH) brain involvement may include mass lesions and/or a neurodegenerative disease (LCH-ND) of unknown etiology. The goal of this study was to define the mechanisms of pathogenesis that drive CNS-LCH. METHODS: Cerebrospinal fluid (CSF) biomarkers including CSF proteins and extracellular BRAFV600E DNA were analyzed in CSF from patients with CNS-LCH lesions compared with patients with brain tumors and other neurodegenerative conditions. Additionally, the presence of BRAFV600E was tested in peripheral mononuclear blood cells (PBMCs) as well as brain biopsies from LCH-ND patients, and the response to BRAF-V600E inhibitor was evaluated in 4 patients with progressive disease. RESULTS: Osteopontin was the only consistently elevated CSF protein in patients with CNS-LCH compared with patients with other brain pathologies. BRAFV600E DNA was detected in CSF of only 2/20 (10%) cases, both with LCH-ND and active lesions outside the CNS. However, BRAFV600E+ PBMCs were detected with significantly higher frequency at all stages of therapy in LCH patients who developed LCH-ND. Brain biopsies of patients with LCH-ND demonstrated diffuse perivascular infiltration by BRAFV600E+ cells with monocyte phenotype (CD14+ CD33+ CD163+ P2RY12- ) and associated osteopontin expression. Three of 4 patients with LCH-ND treated with BRAF-V600E inhibitor experienced significant clinical and radiologic improvement. CONCLUSION: In LCH-ND patients, BRAFV600E+ cells in PBMCs and infiltrating myeloid/monocytic cells in the brain is consistent with LCH-ND as an active demyelinating process arising from a mutated hematopoietic precursor from which LCH lesion CD207+ cells are also derived. Therapy directed against myeloid precursors with activated MAPK signaling may be effective for LCH-ND. Cancer 2018;124:2607-20. © 2018 American Cancer Society.

Resistance of Transmitted Founder HIV-1 to IFITM-Mediated Restriction.

Interferon-induced transmembrane proteins (IFITMs) restrict the entry of diverse enveloped viruses through incompletely understood mechanisms. While IFITMs are reported to inhibit HIV-1, their in vivo relevance is unclear. We show that IFITM sensitivity of HIV-1 strains is determined by the co-receptor usage of the viral envelope glycoproteins as well as IFITM subcellular localization within the target cell. Importantly, we find that transmitted founder HIV-1, which establishes de novo infections, is uniquely resistant to the antiviral activity of IFITMs. However, viral sensitivity to IFITMs, particularly IFITM2 and IFITM3, increases over the first 6 months of infection, primarily as a result of neutralizing antibody escape mutations. Additionally, the ability to evade IFITM restriction contributes to the different interferon sensitivities of transmitted founder and chronic viruses. Together, these data indicate that IFITMs constitute an important barrier to HIV-1 transmission and that escape from adaptive immune responses exposes the virus to antiviral restriction.

Signal transducer and activator of transcription 5b (Stat5b) serine 193 is a novel cytokine-induced phospho-regulatory site that is constitutively activated in primary hematopoietic malignancies.

Signal transducer and activator of transcription 5b (Stat5b) is a critical node in the signaling network downstream of external (cytokines or growth factors) or internal (oncogenic tyrosine kinases) stimuli. Maximum transcriptional activation of Stat5b requires both tyrosine and serine phosphorylation. Although the mechanisms governing tyrosine phosphorylation and activation of Stat5b have been extensively studied, the role of serine phosphorylation remains to be fully elucidated. Using mass spectrometry and phospho-specific antibodies, we identified Ser-193 as a novel site of cytokine-induced phosphorylation within human Stat5b. Stat5b Ser(P)-193 was detected in activated primary human peripheral blood mononuclear cells or lymphoid cell lines in response to several γ common (γc) cytokines, including interleukin (IL)-2, IL-7, IL-9, and IL-15. Kinetic and spatial analysis indicated that Stat5b Ser-193 phosphorylation was rapid and transient and occurred in the cytoplasmic compartment of the cell prior to Stat5b translocation to the nucleus. Moreover, inducible Stat5b Ser-193 phosphorylation was sensitive to inhibitors of mammalian target of rapamycin (mTOR), whereas inhibition of protein phosphatase 2A (PP2A) induced phosphorylation of Ser-193. Reconstitution assays in HEK293 cells in conjunction with site-directed mutagenesis, EMSA, and reporter assays indicated that Ser(P)-193 is required for maximal Stat5b transcriptional activity. Indeed, Stat5b Ser-193 was found constitutively phosphorylated in several lymphoid tumor cell lines as well as primary leukemia and lymphoma patient tumor cells. Taken together, IL-2 family cytokines tightly control Stat5b Ser-193 phosphorylation through a rapamycin-sensitive mechanism. Furthermore, constitutive Ser-193 phosphorylation is associated with Stat5b proto-oncogenic activity and therefore may serve as a novel therapeutic target for treating hematopoietic malignancies.

Fetal, infant, and child mortality in a context of alcohol use.

Alcohol is an important element in the causal chain of risk for fetal, infant, and childhood mortality. Mortality risk is influenced by interactions of alcohol with other environmental and genetic factors and temporal periods of susceptibility. In this paper we discuss four time periods (preconceptual, gestational, infancy, and childhood) where alcohol use may create a context of risk. Alcohol use in one period increases the risk of alcohol use in subsequent periods. Gestational alcohol use can influence risk of mortality from abuse of mother/fetus, can cause other adverse outcomes, and can result in fetal alcohol spectrum disorders (FASDs). In infancy alcohol use can increase risk from impairment of arousal in adults who use alcohol. Infants with gestational exposure can be behaviorally difficult with sleep disturbance, irritability, and colic. This can increase the risk of harm or death, especially during periods of caretakers' alcohol use. Caretakers' alcohol use and smoking are strongly correlated. The all-cause mortality rate in people diagnosed with fetal alcohol syndrome (FAS) is over 5%. All-cause mortality in siblings of diagnosed cases of FAS is increased 530% compared to siblings of matched controls. We recommend that a context of alcohol use be considered as a marker for multifactorial risk in all fetal, infant, and child deaths. A schema to collect data on alcohol use is provided to increase awareness of alcohol use as an environmental risk marker for mortality.

Serotonergic brainstem abnormalities in Northern Plains Indians with the sudden infant death syndrome.

The rate of the sudden infant death syndrome (SIDS) among American Indian infants in the Northern Plains is almost 6 times higher than in U.S. white infants. In a study of infant mortality among Northern Plains Indians, we tested the hypothesis that receptor binding abnormalities to the neurotransmitter serotonin (5-HT) in SIDS cases, compared with autopsied controls, occur in regions of the medulla oblongata that contain 5-HT neurons and that are critical for the regulation of cardiorespiration and central chemosensitivity during sleep, i.e. the medullary 5-HT system. Tritiated-lysergic acid diethylamide binding to 5-HT(1A-D) and 5-HT2 receptors was measured in 19 brainstem nuclei in 23 SIDS and 6 control infants using tissue receptor autoradiography. Binding in the arcuate nucleus, a part of the medullary 5-HT system along the ventral surface, in the SIDS infants (mean age-adjusted binding 7.1 +/- 0.8 fmol/mg tissue, n = 23) was significantly lower than in controls (mean age-adjusted binding 13.1 +/- 1.6 fmol/mg tissue, n = 5) (p = 0.003). Binding also demonstrated significant diagnosis x age interactions (p < 0.04) in 4 other nuclei that are components of the 5-HT system. These data suggest that medullary 5-HT dysfunction can lead to sleep-related, sudden death in affected SIDS infants, and confirm the same binding abnormalities reported by us in a larger dataset of non-American Indian SIDS and control infants. This study also links 5-HT abnormalities in the arcuate nucleus with exposure to adverse prenatal exposures, i.e. cigarette smoking (p = 0.011) and alcohol (p = 0.075), during the periconceptional period or throughout pregnancy. Prenatal exposure to cigarette smoke and/or alcohol may contribute to abnormal fetal medullary 5-HT development in SIDS infants.

Risk factors for sudden infant death syndrome among northern plains Indians.

CONTEXT: Sudden infant death syndrome (SIDS) is a leading cause of postneonatal mortality among American Indians, a group whose infant death rate is consistently above the US national average. OBJECTIVE: To determine prenatal and postnatal risk factors for SIDS among American Indians. DESIGN, SETTING, AND PARTICIPANTS: Population-based case-control study of 33 SIDS infants and 66 matched living controls among American Indians in South Dakota, North Dakota, Nebraska, and Iowa enrolled from December 1992 to November 1996 and investigated using standardized parental interview, medical record abstraction, autopsy protocol, and infant death review. MAIN OUTCOME MEASURES: Association of SIDS with maternal socioeconomic and behavioral factors, health care utilization, and infant care practices. RESULTS: The proportions of case and control infants who were usually placed prone to sleep (15.2% and 13.6%, respectively), who shared a bed with parents (59.4% and 55.4%), or whose mothers smoked during pregnancy (69.7% and 54.6%) were similar. However, mothers of 72.7% of case infants and 45.5% of control infants engaged in binge drinking during pregnancy. Conditional logistic regression revealed significant associations between SIDS and 2 or more layers of clothing on the infant (adjusted odds ratio [aOR], 6.2; 95% confidence interval [CI], 1.4-26.5), any visits by a public health nurse (aOR, 0.2; 95% CI, 0.1-0.8), periconceptional maternal alcohol use (aOR, 6.2; 95% CI, 1.6-23.3), and maternal first-trimester binge drinking (aOR, 8.2; 95% CI, 1.9-35.3). CONCLUSIONS: Public health nurse visits, maternal alcohol use during the periconceptional period and first trimester, and layers of clothing are important risk factors for SIDS among Northern Plains Indians. Strengthening public health nurse visiting programs and programs to reduce alcohol consumption among women of childbearing age could potentially reduce the high rate of SIDS.