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Background and Aims: In patients with Rat sarcoma proto-oncogene (RAS) wild-type metastatic colorectal cancer (mCRC), anti-epidermal growth factor receptor (EGFR) antibodies have been established in first- and further therapy lines. Due to limited treatment options upon disease progression, anti-EGFR re-exposure is increasingly employed in real-world oncology. The aim of this study was to assess clinical implementation and utility of anti-EGFR retreatment strategies in real-world mCRC patients. Methods: In this monocentric retrospective study, we included 524 patients with CRC and identified patients who received an anti-EGFR-based treatment as well as anti-EGFR rechallenge (progression on first-line anti-EGFR therapy) or reintroduction (discontinuation due to intolerance/toxicity/other). Results: In total, 143 patients received an anti-EGFR-based first- or second-line treatment, showing a similar overall survival (OS) compared to the non-anti-EGFR treatment group (38.3 vs. 39.6 months, p = 0.88). Thirty-three patients met the inclusion criteria for anti-EGFR re-exposure and were either assigned to rechallenge (n = 21) or reintroduction (n = 12) subgroups. The median FU after re-exposure was 45.8 months. Cetuximab and Panitumumab were used in 21 and 12 patients, respectively, and the main chemotherapy at re-exposure was FOLFIRI in 39.4%. Anti-EGFR re-exposure was associated with a distinct trend towards a better outcome (median OS 56.0 vs. 35.4 months, p = 0.06). In a subgroup comparison, reintroduction was associated with a higher OS and PFS in trend compared to the rechallenge (mOS 66 vs. 52.4, n.s., mPFS 7.33 vs. 3.68 months, n.s.). Conclusions: This retrospective study provides real-world evidence underscoring that anti-EGFR re-exposure strategies might benefit patients independently of the reason for prior discontinuation.
The Q80K polymorphism in the NS3-4A protease of the hepatitis C virus is associated with treatment failure of direct-acting antiviral agents. This polymorphism is highly prevalent in genotype 1a infections and stably transmitted between hosts. Here, we investigated the underlying molecular mechanisms of evolutionarily conserved coevolving amino acids in NS3-Q80K and revealed potential implications of epistatic interactions in immune escape and variants persistence. Using purified protein, we characterized the impact of epistatic amino acid substitutions on the physicochemical properties and peptide cleavage kinetics of the NS3-Q80K protease. We found that Q80K destabilized the protease protein fold (p < 0.0001). Although NS3-Q80K showed reduced peptide substrate turnover (p < 0.0002), replicative fitness in an H77S.3 cell culture model of infection was not significantly inferior to the WT virus. Epistatic substitutions at residues 91 and 174 in NS3-Q80K stabilized the protein fold (p < 0.0001) and leveraged the WT protease stability. However, changes in protease stability inversely correlated with enzymatic activity. In infectious cell culture, these secondary substitutions were not associated with a gain of replicative fitness in NS3-Q80K variants. Using molecular dynamics, we observed that the total number of residue contacts in NS3-Q80K mutants correlated with protein folding stability. Changes in the number of contacts reflected the compensatory effect on protein folding instability by epistatic substitutions. In summary, epistatic substitutions in NS3-Q80K contribute to viral fitness by mechanisms not directly related to RNA replication. By compensating for protein-folding instability, epistatic interactions likely protect NS3-Q80K variants from immune cell recognition.
Inhibitors against the NS3-4A protease of hepatitis C virus (HCV) have proven to be useful drugs in the treatment of HCV infection. Although variants have been identified with mutations that confer resistance to these inhibitors, the mutations do not restore replicative fitness and no secondary mutations that rescue fitness have been found. To gain insight into the molecular mechanisms underlying the lack of fitness compensation, we screened known resistance mutations in infectious HCV cell culture with different genomic backgrounds. We observed that the Q41R mutation of NS3-4A efficiently rescues the replicative fitness in cell culture for virus variants containing mutations at NS3-Asp168. To understand how the Q41R mutation rescues activity, we performed protease activity assays complemented by molecular dynamics simulations, which showed that protease-peptide interactions far outside the targeted peptide cleavage sites mediate substrate recognition by NS3-4A and support protease cleavage kinetics. These interactions shed new light on the mechanisms by which NS3-4A cleaves its substrates, viral polyproteins and a prime cellular antiviral adaptor protein, the mitochondrial antiviral signaling protein MAVS. Peptide binding is mediated by an extended hydrogen-bond network in NS3-4A that was effectively optimized for protease-MAVS binding in Asp168 variants with rescued replicative fitness from NS3-Q41R. In the protease harboring NS3-Q41R, the N-terminal cleavage products of MAVS retained high affinity to the active site, rendering the protease susceptible for potential product inhibition. Our findings reveal delicately balanced protease-peptide interactions in viral replication and immune escape that likely restrict the protease adaptive capability and narrow the virus evolutionary space.
Gene therapy has garnered increasing interest over recent decades. Several therapies employing gene transfer mechanisms have been developed, and, of these, adeno-associated virus (AAV) vectors have demonstrated viability for use with in vivo gene therapy. Several AAV-based therapeutics have received regulatory approval in the last few years including those for retinal disease, spinal muscular atrophy or aromatic L-amino acid decarboxylase deficiency. Lately, with the introduction of novel liver-directed AAV vector-based therapeutics for the treatment of haemophilia A and B, gene therapy has attracted significant attention in the hepatology community, with the liver increasingly recognised as a target for gene therapy. However, the introduction of foreign DNA into hepatocytes is associated with a risk of hepatic reactions, with raised ALT (alanine aminotransferase) and AST (aspartate aminotransferase) being – so far – the most commonly reported side effects. The complete mechanisms underlying the ALT flairs remain to be determined and the long-term risks associated with these new treatments is not yet known. The liver community is increasingly being asked to support liver-directed gene therapy to mitigate potential liver associated harm. In this review, we focus on AAV vector-based gene therapy, shedding light on this promising technique and its remarkable success in haemophilia, with a special focus on hepatic complications and their management in daily clinical practice.
Cyclic AMP analogs containing hydrophobic modification of C(8) at the adenine ring such as 8-(4-chlorophenylthio)-cAMP (8-pCPT-cAMP) and 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-methyl-cAMP) can penetrate membranes due to their high lipophilicity and directly activate intracellular cAMP effectors. Therefore, these cAMP analogs have been used in numerous studies, assuming that their effects reflect the consequences of direct activation of cAMP effectors. The present study provides evidence that 8-pCPT-modified cAMP analogs and their corresponding putative hydrolysis products (8-(4-chlorophenylthio)-adenosine (8-pCPT-ado) and 8-(4-chlorophenylthio)-2'-O-methyl-adenosine (8-pCPT-2'-O-methyl-ado)) inhibit the equilibrative nucleoside transporter 1 (ENT1). In PC12 cells, in which nucleoside transport strongly depended on ENT1, 8-pCPT-ado, 8-pCPT-2'-O-methyl-ado, and, to a smaller extent, 8-pCPT-2'-O-methyl-cAMP caused an increase of protein kinase A substrate motif phosphorylation and anti-apoptotic effect by an A(2A) adenosine receptor (A(2A)R)-dependent mechanism. In contrast, the effects of 8-pCPT-cAMP were mainly A(2A)R-independent. In HEK 293 showing little endogenous ENT1-dependent nucleoside transport, transfection of ENT1 conferred A(2A)R-dependent increase in protein kinase A substrate motif phosphorylation. Together, the data of the present study indicate that inhibition of ENT1 and activation of adenosine receptors have to be considered when interpreting the effects of 8-pCPT-substituted cAMP/adenosine analogs.
MutLα is essential for human DNA mismatch repair (MMR). It harbors a latent endonuclease, is responsible for recruitment of process associated proteins and is relevant for strand discrimination. Recently, we demonstrated that the MMR function of MutLα is regulated by phosphorylation of MLH1 at serine (S) 477. In the current study, we focused on S87 located in the ATPase domain of MLH1 and on S446, S456 and S477 located in its linker region. We analysed the phosphorylation-dependent impact of these amino acids on DNA binding, MMR ability and thermal stability of MutLα. We were able to demonstrate that phosphorylation at S87 of MLH1 inhibits DNA binding of MutLα. In addition, we detected that its MMR function seems to be regulated predominantly via phosphorylation of serines in the linker domain, which are also partially involved in the regulation of DNA binding. Furthermore, we found that the thermal stability of MutLα decreased in relation to its phosphorylation status implying that complete phosphorylation might lead to instability and degradation of MLH1. In summary, we showed here, for the first time, a phosphorylation-dependent regulation of DNA binding of MutLα and hypothesized that this might significantly impact its functional regulation during MMR in vivo.
Several microRNAs (miRNAs) are associated with the molecular pathogenesis of hepatocellular carcinoma (HCC). However, previous studies analyzing the dysregulation of miRNAs in HCC show heterogeneous results. We hypothesized that part of this heterogeneity might be attributable to variations of miRNA expression deriving from the HCC capsule or the fibrotic septa within the peritumoral tissue used as controls. Tissue from surgically resected hepatitis C–associated HCC from six well-matched patients was microdissected using laser microdissection and pressure catapulting technique. Four distinct histologic compartments were isolated: tumor parenchyma (TP), fibrous capsule of the tumor (TC), tumor-adjacent liver parenchyma (LP), and cirrhotic septa of the tumor-adjacent liver (LC). MiRNA expression profiling analysis of 1105 mature miRNAs and precursors was performed using miRNA microarray. Principal component analysis and consecutive pairwise supervised comparisons demonstrated distinct patterns of expressed miRNAs not only for TP versus LP (e.g., intratumoral down-regulation of miR-214, miR-199a, miR-146a, and miR-125a; P< .05) but also for TC versus LC (including down-regulation within TC of miR-126, miR-99a/100, miR-26a, and miR-125b; P< .05). The tumor capsule therefore demonstrates a tumor-like phenotype with down-regulation of well-known tumor-suppressive miRNAs. Variations of co-analyzed fibrotic tissue within the tumor or in controls may have profound influence on miRNA expression analyses in HCC. Several miRNAs, which are proposed to be HCC specific, may indeed be rather associated to the tumor capsule. As miRNAs evolve to be important biomarkers in liver tumors, the presented data have important translational implications on diagnostics and treatment in patients with HCC.
Cholecystokinin (CCK) and related peptides are potent growth factors in the gastrointestinal tract and may be important for human cancer. CCK exerts its growth modulatory effects through G(q)-coupled receptors (CCK(A) and CCK(B)) and activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). In the present study, we investigated the different mechanisms participating in CCK-induced activation of ERK1/2 in pancreatic AR42J cells expressing both CCK(A) and CCK(B). CCK activated ERK1/2 and Raf-1 to a similar extent as epidermal growth factor (EGF). Inhibition of EGF receptor (EGFR) tyrosine kinase or expression of dominant-negative Ras reduced CCK-induced ERK1/2 activation, indicating participation of the EGFR and Ras in CCK-induced ERK1/2 activation. However, compared with EGF, CCK caused only small increases in tyrosine phosphorylation of the EGFR and Shc, Shc-Grb2 complex formation, and Ras activation. Signal amplification between Ras and Raf in a CCK-induced ERK cascade appears to be mediated by activation of protein kinase Cepsilon (PKCepsilon), because 1) down-modulation of phorbol ester-sensitive PKCs inhibited CCK-induced activation of Ras, Raf, and ERK1/2 without influencing Shc-Grb2 complex formation; 2) PKCepsilon, but not PKCalpha or PKCdelta, was detectable in Raf-1 immunoprecipitates, although CCK activated all three PKC isoenzymes. In addition, the present study provides evidence that the Src family tyrosine kinase Yes is activated by CCK and mediates CCK-induced tyrosine phosphorylation of Shc. Furthermore, we show that CCK-induced activation of the EGFR and Yes is achieved through the CCK(B) receptor. Together, our data show that different signals emanating from the CCK receptors mediate ERK1/2 activation; activation of Yes and the EGFR mediate Shc-Grb2 recruitment, and activation of PKC, most likely PKCepsilon, augments CCK-stimulated ERK1/2 activation at the Ras/Raf level.
In PC12 cells, a well studied model for neuronal differentiation, an elevation in the intracellular cAMP level increases cell survival, stimulates neurite outgrowth, and causes activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). Here we show that an increase in the intracellular cAMP concentration induces tyrosine phosphorylation of two receptor tyrosine kinases, i.e. the epidermal growth factor (EGF) receptor and the high affinity receptor for nerve growth factor (NGF), also termed Trk(A). cAMP-induced tyrosine phosphorylation of the EGF receptor is rapid and correlates with ERK1/2 activation. It occurs also in Panc-1, but not in human mesangial cells. cAMP-induced tyrosine phosphorylation of the NGF receptor is slower and correlates with Akt activation. Inhibition of EGF receptor tyrosine phosphorylation, but not of the NGF receptor, reduces cAMP-induced neurite outgrowth. Expression of dominant-negative Akt does not abolish cAMP-induced survival in serum-free media, but increases cAMP-induced ERK1/2 activation and neurite outgrowth. Together, our results demonstrate that cAMP induces dual signaling in PC12 cells: transactivation of the EGF receptor triggering the ERK1/2 pathway and neurite outgrowth; and transactivation of the NGF receptor promoting Akt activation and thereby modulating ERK1/2 activation and neurite outgrowth.
Erratum in: Correction: Cyclic AMP induces transactivation of the receptors for epidermal growth factor and nerve growth factor, thereby modulating activation of MAP kinase, Akt, and neurite outgrowth in PC12 cells. Journal of biological chemistry 2020 Oct 23;295(43):14792. doi: 10.1074/jbc.AAC120.016177.
Erratum for: Cyclic AMP induces transactivation of the receptors for epidermal growth factor and nerve growth factor, thereby modulating activation of MAP kinase, Akt, and neurite outgrowth in PC12 cells.Journal of biological chemistry, 2002 Nov 15;277(46):43623-30. doi: 10.1074/jbc.M203926200. Epub 2002 Sep 5.
Low platelet count predicts reduced survival in potentially resectable hepatocellular carcinoma
(2022)
The prognostic role of platelet count in hepatocellular carcinoma (HCC) remains unclear, and in fact both thrombocytopenia and thrombocytosis are reported as predictors of unfavourable outcomes. This study aimed to clarify the prognostic value of preoperative platelet count in potentially resectable HCC. We retrospectively reviewed 128 patients who underwent hepatic resection for HCC at a tertiary academic centre (2007–2019). Patient data were modelled by regression analysis, and platelet count was treated as a continuous variable. 89 patients had BCLC 0/A tumours and 39 had BCLC B tumours. Platelet count was higher in patients with larger tumours and lower in patients with higher MELD scores, advanced fibrosis, and portal hypertension (p < 0.001 for all listed variables). After adjusting for BCLC stage and tumour diameter, low platelet count associated with reduced overall survival (hazard ratio 1.25 per 50/nL decrease in platelet count, 95% confidence interval (CI) 1.02–1.53, p = 0.034) and increased perioperative mortality (odds ratio 1.96 per 50/nL decrease in platelet count, 95% CI 1.19–3.53, p = 0.014). Overall, low platelet count correlates with increased liver disease severity, inferior survival, and excess perioperative mortality in resectable HCC. These insights might be applied in clinical practice to better select patients for resection.
Intrahepatic cholangiocarcinoma (iCCA) is the most frequent subtype of cholangiocarcinoma (CCA), and the incidence has globally increased in recent years. In contrast to surgically treated iCCA, data on the impact of fibrosis on survival in patients undergoing palliative chemotherapy are missing. We retrospectively analyzed the cases of 70 patients diagnosed with iCCA between 2007 and 2020 in our tertiary hospital. Histopathological assessment of fibrosis was performed by an expert hepatobiliary pathologist. Additionally, the fibrosis-4 score (FIB-4) was calculated as a non-invasive surrogate marker for liver fibrosis. For overall survival (OS) and progression-free survival (PFS), Kaplan–Meier curves and Cox-regression analyses were performed. Subgroup analyses revealed a median OS of 21 months (95% CI = 16.7–25.2 months) and 16 months (95% CI = 7.6–24.4 months) for low and high fibrosis, respectively (p = 0.152). In non-cirrhotic patients, the median OS was 21.8 months (95% CI = 17.1–26.4 months), compared with 9.5 months (95% CI = 4.6–14.3 months) in cirrhotic patients (p = 0.007). In conclusion, patients with iCCA and cirrhosis receiving palliative chemotherapy have decreased OS rates, while fibrosis has no significant impact on OS or PFS. These patients should not be prevented from state-of-the-art first-line chemotherapy.
There is ample epidemiologic evidence for an association of chronic hepatitis C virus (HCV) infection with B-cell non-Hodgkin lymphoma (B-NHL). B-NHL subtypes most frequently associated with HCV are marginal zone lymphoma and diffuse large B-cell lymphoma. The most convincing evidence for a causal relationship between HCV infection and lymphoma development is the observation of B-NHL regression after HCV eradication by antiviral therapy (AVT). In fact, for indolent HCV-associated B-NHL, first-line AVT instead of standard immune-chemotherapy might be considered. Molecular mechanisms of HCV-NHL development are still poorly understood. Three general theories have emerged to understand the HCV-induced lymphomagenesis: (1) continuous external stimulation of lymphocyte receptors by viral antigens and consecutive proliferation; (2) HCV replication in B cells with oncogenic effect mediated by intracellular viral proteins; (3) permanent B-cell damage, e.g., mutation of tumor suppressor genes, caused by a transiently intracellular virus (“hit and run” theory). This review systematically summarizes the data on epidemiology, interventional studies, and molecular mechanisms of HCV-associated B-NHL.
The nuclear factor kappa beta (NFκB) signaling pathway plays an important role in liver homeostasis and cancer development. Tax1-binding protein 1 (Tax1BP1) is a regulator of the NFκB signaling pathway, but its role in the liver and hepatocellular carcinoma (HCC) is presently unknown. Here we investigated the role of Tax1BP1 in liver cells and murine models of HCC and liver fibrosis. We applied the diethylnitrosamine (DEN) model of experimental hepatocarcinogenesis in Tax1BP1+/+ and Tax1BP1−/− mice. The amount and subsets of non-parenchymal liver cells in in Tax1BP1+/+ and Tax1BP1−/− mice were determined and activation of NFκB and stress induced signaling pathways were assessed. Differential expression of mRNA and miRNA was determined. Tax1BP1−/− mice showed increased numbers of inflammatory cells in the liver. Furthermore, a sustained activation of the NFκB signaling pathway was found in hepatocytes as well as increased transcription of proinflammatory cytokines in isolated Kupffer cells from Tax1BP1−/− mice. Several differentially expressed mRNAs and miRNAs in livers of Tax1BP1−/− mice were found, which are regulators of inflammation or are involved in cancer development or progression. Furthermore, Tax1BP1−/− mice developed more HCCs than their Tax1BP1+/+ littermates. We conclude that Tax1BP1 protects from liver cancer development by limiting proinflammatory signaling.
Standard monitoring of heart rate, blood pressure and arterial oxygen saturation during endoscopy is recommended by current guidelines on procedural sedation. A number of studies indicated a reduction of hypoxic (art. oxygenation < 90% for > 15 s) and severe hypoxic events (art. oxygenation < 85%) by additional use of capnography. Therefore, U.S. and the European guidelines comment that additional capnography monitoring can be considered in long or deep sedation. Integrated Pulmonary Index® (IPI) is an algorithm-based monitoring parameter that combines oxygenation measured by pulse oximetry (art. oxygenation, heart rate) and ventilation measured by capnography (respiratory rate, apnea > 10 s, partial pressure of end-tidal carbon dioxide [PetCO2]). The aim of this paper was to analyze the value of IPI as parameter to monitor the respiratory status in patients receiving propofol sedation during PEG-procedure. Patients reporting for PEG-placement under sedation were randomized 1:1 in either standard monitoring group (SM) or capnography monitoring group including IPI (IM). Heart rate, blood pressure and arterial oxygen saturation were monitored in SM. In IM additional monitoring was performed measuring PetCO2, respiratory rate and IPI. Capnography and IPI values were recorded for all patients but were only visible to the endoscopic team for the IM-group. IPI values range between 1 and 10 (10 = normal; 8–9 = within normal range; 7 = close to normal range, requires attention; 5–6 = requires attention and may require intervention; 3–4 = requires intervention; 1–2 requires immediate intervention). Results on capnography versus standard monitoring of the same study population was published previously. A total of 147 patients (74 in SM and 73 in IM) were included in the present study. Hypoxic events occurred in 62 patients (42%) and severe hypoxic events in 44 patients (29%), respectively. Baseline characteristics were equally distributed in both groups. IPI = 1, IPI < 7 as well as the parameters PetCO2 = 0 mmHg and apnea > 10 s had a high sensitivity for hypoxic and severe hypoxic events, respectively (IPI = 1: 81%/81% [hypoxic/severe hypoxic event], IPI < 7: 82%/88%, PetCO2: 69%/68%, apnea > 10 s: 84%/84%). All four parameters had a low specificity for both hypoxic and severe hypoxic events (IPI = 1: 13%/12%, IPI < 7: 7%/7%, PetCO2: 29%/27%, apnea > 10 s: 7%/7%). In multivariate analysis, only SM and PetCO2 = 0 mmHg were independent risk factors for hypoxia. IPI (IPI = 1 and IPI < 7) as well as the individual parameters PetCO2 = 0 mmHg and apnea > 10 s allow a fast and convenient conclusion on patients’ respiratory status in a morbid patient population. Sensitivity is good for most parameters, but specificity is poor. In conclusion, IPI can be a useful metric to assess respiratory status during propofol-sedation in PEG-placement. However, IPI was not superior to PetCO2 and apnea > 10 s.
Introduction. Balapiravir (R1626, RG1626) is the prodrug of a nucleoside analogue inhibitor of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (R1479, RG1479). This phase 2, double-blind international trial evaluated the optimal treatment regimen of balapiravir plus peginterferon alfa-2a (40KD)/ribavirin.
Material and methods. Treatment-naive genotype 1 patients (N = 516) were randomized to one of seven treatment groups in which they received balapiravir 500, 1,000, or 1,500 mg twice daily, peginterferon alfa2a (40KD) 180 or 90 Mg/week and ribavirin 1,000/1,200 mg/day or peginterferon alfa-2a (40KD)/ribavirin. The planned treatment duration with balapiravir was reduced from 24 to 12 weeks due to safety concerns.
Results. The percentage of patients with undetectable HCV RNA was consistently higher in all balapiravir groups from week 2 to 12. However, high rates of dose modifications and discontinuations of one/all study drugs compromised the efficacy assessment and resulted in similar sustained virological response rates in the balapiravir groups (range 32-50%) and the peginterferon alfa-2a (40KD)/ribavirin group (43%). Balapiravir was discontinued for safety reasons in 28-36% of patients (most often for lymphopenia) and the percentage of patients with serious adverse events (especially hematological, infection, ocular events) was dose related. Serious hematological adverse events (particularly neutropenia, lymphopenia) were more common in balapiravir recipients. Two deaths in the balapiravir/peginterferon alfa-2a/ribavirin combination groups were considered possibly related to study medication.
Conclusion. Further development of balapiravir for the treatment of chronic hepatitis C has been halted because of the unacceptable benefit to risk ratio revealed in this study (www.ClinicalTrials.gov NCT 00517439).