Validation of Protein Biomarker Candidates for Diagnosis of HBV induced HCC

Hepatocellular carcinoma is a major contributor to the global cancer burden. It affects millions of people in Pakistan on a yearly basis. Furthermore, HCC is linked to viral infections Hepatitis B and C, which account for roughly 87 percent of HCC cases in Pakistan. HCC is identified using imaging techniques such as MRI, Ultrasound, and histology, which have radiation hazards and frequently need expensive healthcare systems that are less available in most of the developing countries. Novel HCC biomarkers are being developed as part of a large research project aimed at detecting the disease early. These include the creation of biomarkers based on HCC patients' transcriptome and proteomic profiles. Circulating proteins, which are easily detected in body fluids, including blood serum, may thus provide an opportunity for the development of HCC biomarkers. Blood-based serum biomarkers must be developed for easy, non-invasive, and early detection of HCC. In conjunction with imaging techniques, alpha-fetoprotein (AFP) has been used to detect HCC, although it has little clinical usefulness. Also, the reported AFP negative results make its utility meager. Multiple circulating proteins have been studied as biomarker possibilities for HCC diagnosis in recent years. In this study, Blood serum was used to validate three novel protein biomarker candidates to detect HBV induced HCC that had previously been predicted using a bioinformatics methodology. Proteins named C6, C8A and C8B were measured in the serum of 22 HCC patients infected with HBV in Pakistani population and compared to AFP levels using quantitative ELISA. C8A possesses considerable biomarker potential, with 95.45 percent specificity and 77.27% sensitivity with 0.933 Area Under the Curve (AUC), whereas C6 and C8B showed poor biomarker potential. Hence, C8A demonstrated great promise as a circulating blood-based protein biomarker for HBV induced HCC diagnosis. View Article DOI: 10.47856/ijaast.2022.v09i03.00

Biomarkers and overall survival in patients with advanced hepatocellular carcinoma treated with TGF-βRI inhibitor galunisertib.

BackgroundTransforming growth factor beta (TGF-β) signalling is involved in the development of hepatocellular carcinoma (HCC). We followed changes in biomarkers during treatment of patients with HCC with the TGF-βRI/ALK5 inhibitor galunisertib.MethodsThis phase 2 study (NCT01246986) enrolled second-line patients with advanced HCC into one of two cohorts of baseline serum alpha-fetoprotein (AFP): Part A (AFP ≥1.5x ULN) or Part B (AFP <1.5x ULN). Baseline and postbaseline levels of AFP, TGF-β1, E-cadherin, selected miRNAs, and other plasma proteins were monitored.ResultsThe study enrolled 149 patients (Part A, 109; Part B, 40). Median OS was 7.3 months in Part A and 16.8 months in Part B. Baseline AFP, TGF-β1, E-cadherin, and an additional 16 plasma proteins (such as M-CSF, IL-6, ErbB3, ANG-2, neuropilin-1, MIP-3 alpha, KIM-1, uPA, IL-8, TIMP-1, ICAM-1, Apo A-1, CA-125, osteopontin, tetranectin, and IGFBP-1) were found to correlate with OS. In addition, a range of miRs were found to be associated with OS. In AFP responders (21% of patients in Part A with decrease of >20% from baseline) versus non-responders, median OS was 21.5 months versus 6.8 months (p = 0.0015). In TGF-β1 responders (51% of all patients) versus non-responders, median OS was 11.2 months versus 5.3 months (p = 0.0036).ConclusionsConsistent with previous findings, both baseline levels and changes from baseline of circulating AFP and TGF-β1 function as prognostic indicators of survival. Future trials are needed to confirm and extend these results

Perfusion computed tomography of the liver

Background: Perfusion CT (P-CT) is a relatively new imaging technique that permits the visual and quantitative assessment of the micro- and macrocirculation of a target organ and focal lesions. P-CT has shown promising results in the evaluation of hyper-vascular tumors such as hepatocellular carcinoma (HCC). HCC is the sixth most common cancer globally and it has a poor prognosis when discovered at a late tumor stage. Any improvement in HCC detection would be directly beneficial for patient care. This thesis aims to investigate the strengths and limitations of whole liver P-CT and to evaluate if PCT can improve the detection of hyper-vascular liver lesions in patients with chronic liver disease. Methods: Study I: Twenty-four patients, who underwent dynamic P-CT for detection of HCC were retrospectively divided into three groups: (1) without portal-venous hypertension (PVH) (n = 8), (2) with PVH (n = 8), (3) with PVH and thrombosis (n = 8). Time to peak splenic- and peak renal enhancement (PSE resp. PRE), as well as arterial liver perfusion (ALP), portal- venous liver perfusion (PLP) and hepatic perfusion-index (HPI) of the liver and HCC derived from PSE- versus PRE- based modelling were compared between the groups. Study II: Group A (n=15) and Group B (n= 38) underwent P-CT using 50 ml contrast medium (CM). Group B underwent an additional standard multiphasic liver CT using 120ml (70-143 ml). Triple-arterial CT image sets were reconstructed from P-CT by fusing three dedicated arterial time points. Triple-arterial CT and single-arterial CT were compared by two readers (R1, R2), who assessed subjective image quality (IQ) and HCC detection rate. A third reader assessed objective IQ.Study III: Fifty study participants (Group A) were scanned with P-CT, a high CM volume protocol and bolus-tracking technique to depict ten arterial phases. Time attenuation curves were created for hyper-vascular liver lesions, liver parenchyma and hepatic vascular structures. 16 participants of Group A with lesions were further analyzed and radiation dose-neutral quadruple arterial phase image sets were created (Group A1). Group A1 was then compared to a Control Group (Group B) consisting of 16 consecutive patients undergoing standard single arterial phase scans. Lesion depiction and quantitative IQ were compared. Results: Study I: Time to PSE was significantly delayed in PVH groups 2 and 3 (P = 0.02), whereas PRE was similar in groups 1, 2 and 3 (P > 0.05). In group 1, liver and HCC perfusion parameters were similar for PSE- and PRE-based modelling (all P > 0.05), while significant differences were seen for PLP and HPI (liver only) in group 2 and ALP in group 3 (all P < 0.05). Study II: The mean CTDIvol of triple-arterial CT and single-arterial CT was equivalent (P=0.73). Triple-arterial CT showed lower image noise and better contrast-to-noise-ratio (P<0.001, P=0.032, respectively), but no significant difference in lesion-to-liver-contrast-ratio (P=0.31). Subjective IQ was good for both protocols. The detection rate of the 65 HCC lesions was 82%/83% (R1/R2) at triple-arterial CT and 80%/77% (R1/R2) at single-arterial CT (P=0.4). Study III: Both Group A1 and B had 33 hyper-enhancing liver lesions each. The mean CTDIvol of quadruple-arterial CT and single-arterial CT was equivalent (P=0.16). The mean time to reach peak lesion-to-liver contrast (LLC) was 20.1s (±4.2s) with a range of 12.5s to 29.1s. Quadruple arterial CT performed significantly better than the Control Group in regards to LLC (P= .009), CNR (P= .002), Image Noise (P<0.001) and hepatic artery enhancement(P<0.001). Conclusions: Study I: PSE is significantly delayed in patients with portal venous hypertension, which results in a miscalculation of P-CT parameters. Maximum-slope based P-CT could be improved by replacing the spleen with the kidney as the reference organ. The difference between time to PSE and time to PRE might serve as a non-invasive biomarker of portal venous hypertension. Study II: Radiation dose-equivalent triple arterial phase imaging is feasible and showed superior image quality and similar HCC detection rate as standard single arterial phase CT despite a substantially smaller CM volume. Study III: The optimal scan delay at single arterial phase CT for depiction of hyper-vascular liver lesions occurs at 20 s, when using a high iodine dose CM protocol and bolus-tracking. Fused quadruple arterial phase CT significantly increases lesion depiction, quantitative IQ and hepatic artery enhancement as compared to standard single arterial phase CT, without elevating the total radiation dose

Programmed cell death 6 interacting protein (PDCD6IP) and Rabenosyn-5 (ZFYVE20) are potential urinary biomarkers for upper gastrointestinal cancer

PURPOSE: Cancer of the upper digestive tract (uGI) is a major contributor to cancer-related death worldwide. Due to a rise in occurrence, together with poor survival rates and a lack of diagnostic or prognostic clinical assays, there is a clear need to establish molecular biomarkers. EXPERIMENTAL DESIGN: Initial assessment was performed on urine samples from 60 control and 60 uGI cancer patients using MS to establish a peak pattern or fingerprint model, which was validated by a further set of 59 samples. RESULTS: We detected 86 cluster peaks by MS above frequency and detection thresholds. Statistical testing and model building resulted in a peak profiling model of five relevant peaks with 88% overall sensitivity and 91% specificity, and overall correctness of 90%. High-resolution MS of 40 samples in the 2-10 kDa range resulted in 646 identified proteins, and pattern matching identified four of the five model peaks within significant parameters, namely programmed cell death 6 interacting protein (PDCD6IP/Alix/AIP1), Rabenosyn-5 (ZFYVE20), protein S100A8, and protein S100A9, of which the first two were validated by Western blotting. CONCLUSIONS AND CLINICAL RELEVANCE: We demonstrate that MS analysis of human urine can identify lead biomarker candidates in uGI cancers, which makes this technique potentially useful in defining and consolidating biomarker patterns for uGI cancer screening

Exploring circulating biomarkers in patients with hepatocellular cancer

PhD ThesisHepatocellular cancer (HCC) is the sixth commonest cancer worldwide and the second leading cause of cancer mortality. The majority of patients present with advanced disease, where cure is not an option and palliative therapies are limited. What is more, biomarkers to stratify available therapies effectively are lacking. Tissue is not routinely obtained as diagnosis of HCC is largely reliant on imaging, as this is sensitive and specific in the majority of patients with cirrhosis and liver biopsy carries small but recognised risks - including tumour seeding and bleeding. Serum AFP lacks HCC diagnostic sensitivity and specific, but remains the only clinically useful serum biomarker – employed despite its limitations in surveillance programmes as well as to monitor HCC progression and to stratify patients for therapy. This highlights the need for alternative biomarkers for HCC management. Sampling patient blood is a quick, non-invasive and inexpensive method and may be regarded as a ‘liquid biopsy’ if it could deliver clinically relevant molecular information about the tumour or its microenvironment. Several liquid biopsy methods have been explored: circulating tumour cell (CTC) detection and characterisation; circulating tumour DNA (ctDNA) KRAS mutation detection; circulating immune cell counts and gene expression signatures of peripheral blood mononucleocytes (PBMCs). CTCs can be detected in patients with cancer and have the potential to provide diagnostic, prognostic and treatment stratifying information. A method for CTC detection using the Imagestream – an imaging fluorescent activated cells sorter with multi-channel immunofluorescence - was developed and optimised. A CD45-immunomagnetic depletion resulted in ≥95% reduction in WBCs with the maintenance of a recovery rate of 51.3-65.37% of CTCs. The remaining cell suspension was labelled with a panel of fluorescent antibodies to enable cell characterisation prior to running through the Imagestream. Between 1 and 1642 CTCs were detected in 65% of HCC patients. The presence of CTCs indicated a worse median overall survival (OS) in HCC patients (24.5 months vs 12 months, Log-Rank p>0.0001). Expression of biomarkers on CTCs was heterogeneous with CK being the most commonly detected biomarker, followed by DNA- ii PK. In some patients, CTCs observed were negative for all of the biomarkers in the panel but detectable on the basis of size and morphology. Clusters of CTCs and leucocyte interactions with CTCs were also observed. Studies on plasma ctDNA detected KRAS mutation in 2/38 (5%) of patients with HCC. Retrospective patient blood data analysis demonstrated that circulating neutrophils were the key circulating immune cell driving HCC progression. In two patient cohorts – Newcastle (n=583) and Hong Kong (n=585), circulating neutrophil counts were an independent predictor of prognosis. Furthermore, in the combined cohort (n=1168), the neutrophil count, either alone or combined with platelets and lymphocytes, was associated with significant differences in patients survival. In a small pilot cohort, gene expression analysis of PBMCs identified NFAT in the immune response as being the top altered pathway in patients with NAFLD/NASH-HCC. DNA extracts prepared from PBMCs in patients with NAFLD-NASH-HCC were more similar to samples derived from patients with HCV-HCC compared to non-cancerous NAFLD/NASH controls. These pilot data were encouraging, supporting a potential role for future application of liquid biopsy tools in the clinical setting for patients with HCC

The application of molecular profiling and proteomics in the study of liver cancers

MD ThesisBackground: Hepatocellular cancer [1] is increasing worldwide. The majority are detected too late for curative surgery as effective identification of the at risk population and their subsequent surveillance is inadequate. My specific aims were to study and characterise our own patients, evaluating the known and predicted biomarkers for HCC in patients with non-alcoholic fatty liver disease and exploring novel biomarker methodologies for the detection of either cirrhosis or HCC complicating it. Methods: Details of all HCC patients presenting to the Newcastle hepatopancreatobiliary (HPB) multidisciplinary team were recorded in an NHS intranet database, and a subset were consented for tissue collection. Established and candidate biomarkers were assessed in serum by western blotting and/or ELISA assay and the role of microarray analysis of HepG2 cells, and 2D-gel electrophoresis of immunodepleted serum in novel candidate biomarker identification were explored. Results: The number of HCC cases referred has increased dramatically over the last decade, as has the numbers arising in a background of non-alcoholic fatty liver disease (NAFLD). In our cohort, patients with earlier stage cancers detected by either - surveillance or incidentally had a better prognosis, but only 10% were eligible for definitive treatment. While HCC patients treated with liver transplant had an overall 5 year survival was 62.1%, the median survival of the transplant cohort was 137 months. Exploration of serum levels of Glypican 3, Squamous Cell Carcinoma Antigen-1 and follistatin were poor surveillance biomarkers, but the combination of Alpha-fetoprotein and Protrhombin induced by vitamin-K absence was encouraging. Serum proteomic analysis in a small subgroup identified four isoforms of apolipoproteins as well as CD5L as differentially expressed between patients with no cirrhosis, cirrhosis and HCC, although subsequent CD5L ELISA failed to confirm its ability to specifically detect HCC. Discussion: While the incidence of HCC is increasing, the prognosis for those affected remains poor. Biomarkers identifying both the at risk individuals and those with cancer are urgently required

Serum clusterin as a marker for diagnosing hepatocellular carcinoma

Background: Approximately 80% of patients with hepatocellular carcinoma (HCC) areuntreatable because of advanced tumor stages at presentation. Therefore, finding newer markers for screening and diagnosing HCC is of utmost importance. Clusterin (CLU) is a 449 amino acid, heterodimeric glycoprotein with a plausible role in the regeneration, migration, and anti-apoptosis of tumor cells. It has been implicated in many malignancies such as prostate and pancreatic adenocarcinomas, but its role in HCC is not well defined.Objective: We aimed to evaluate the diagnostic performance of serum CLU level in diagnosing HCC on top of hepatitis C virus-related liver cirrhosis, and comparing it to that of alpha fetoprotein (AFP).Methods: Twenty cases of apparently healthy subjects, 27 cases of hepatitis C virus-related liver cirrhosis (CHC cases), and 44 HCC cases on top of hepatitis C virus-related liver cirrhosis were included in this study. Serum CLU concentration was determined using a quantitative sandwich enzyme immunoassay technique.Results: Serum clusterin level showed a significant increase in the HCC group compared to the control group (151.96 ± 32.74 vs. 111.40 ±27.46) and to the CHC group (151.96 ± 32.74 vs. 89.12 ±31.62), while a significant decrease in serum clusterin level was found in the CHC group compared to the control group (89.12± 31.62 vs. 111.40± 27.46). Based on receiver operator characteristic curve analysis, serum AFP still surpassed serum CLU in diagnostic sensitivity (77.3% vs. 70.5%), specificity (100% vs. 90%), and positive and negative predictive values (100% vs. 86.1% and 83.3% vs. 77.6% respectively). The use of a combined parallel approach improved the diagnostic sensitivity (95.5%) and negative predictive value (95.7%) over the single use of AFP.Conclusions: Although the diagnostic performance of serum AFP outperformed that of serum CLU, their combined parallel approach improved the sensitivity which is required in screening high risk populations such as CHC patients. KEYWORDS Clusterin; Alpha fetoprotein; Tumor marker; Hepatitis C virus related liver cirrhosis; Hepatocellular carcinom

Radiomics analysis in gastrointestinal imaging: a narrative review

Background and Objective: To present an overview of radiomics radiological applications in major gastrointestinal oncological non-oncologic diseases, such as colorectal cancer, pancreatic cancer, gastro- oesophageal cancer, gastrointestinal stromal tumor (GIST), hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and non-oncologic diseases, such as liver fibrosis, nonalcoholic steatohepatitis, and inflammatory bowel disease. Methods: A search of PubMed databases was performed for the terms “radiomic”, “radiomics”, “liver”, “small bowel”, “colon”, “GI tract”, and “gastrointestinal imaging” for English articles published between January 2013 and July 2022. A narrative review was undertaken to summarize literature pertaining to application of radiomics in major oncological and non-oncological gastrointestinal diseases. The strengths and limitation of radiomics, as well as advantages and major limitations and providing considerations for future development of radiomics were discussed. Key Content and Findings: Radiomics consists in extracting and analyzing a vast amount of quantitative features from medical datasets, Radiomics refers to the extraction and analysis of large amounts of quantitative features from medical images. The extraction of these data, integrated with clinical data, allows the construction of descriptive and predictive models that can build disease-specific radiomic signatures. Texture analysis has emerged as one of the most important biomarkers able to assess tumor heterogeneity and can provide microscopic image information that cannot be identified with the naked eye by radiologists. Conclusions: Radiomics and texture analysis are currently under active investigation in several institutions worldwide, this approach is being tested in a multitude of anatomical areas and diseases, with the final aim to exploit personalized medicine in diagnosis, treatment planning, and prediction of outcomes. Despite promising initial results, the implementation of radiomics is still hampered by some limitations related to the lack of standardization and validation of image acquisition protocols, feature segmentation, data extraction, processing, and analysi

Vandetanib-eluting radiopaque beads and stereotactic body radiotherapy in the treatment of liver cancers

Background: Current treatment options for unresectable hepatocellular carcinoma (HCC) and colorectal liver metastases (mCRC) include transarterial chemoembolisation (TACE) and stereotactic body radiotherapy (SBRT). The objectives of this project were: 1. To assess a novel drug-eluting bead for TACE / 2. To report on the safety and efficacy of SBRT in HCC / 3. To assess the feasibility of using radiopaque beads as fiducial markers for SBRT / Methods: In Part 1, a first-in-human trial was performed in patients with HCC and mCRC using a novel vandetanib-eluting radiopaque bead, BTG-002814. Primary trial endpoints were safety/tolerability and the concentrations of vandetanib and its major metabolite in plasma and resected tissue. Biomarker studies included blood cytokines and perfusion imaging parameters. In Part 2, the efficacy of SBRT was explored in a retrospective study of 31 patients with HCC tumours ≤5 cm and in a phase II study of 13 patients with larger tumours. In Part 3 the feasibility of using radiopaque beads as fiducial markers for SBRT was investigated. / Results: BTG-002814 was shown to have a satisfactory safety profile in 8 patients. Vandetanib was present in the plasma of all patients 12 days post-TACE, and present in resected liver tissue up to 32 days post-treatment. There were no significant changes in perfusion parameters. Blood biomarker studies showed increases in leptin, osteopontin and sTie2. SBRT offered 1-year local control rates of 94% in small HCCs and 92% in larger tumours. Radiopaque beads were visible on 4D-CT and CBCT images in all 8 cases and matching successfully performed. / Conclusions: The safety profile and pharmacokinetic characteristics for this novel technology are adequate to proceed to a Phase I/II trial. SBRT is an effective local treatment for HCC. The role of radiopaque beads as fiducial markers is feasible and warrants further exploration as a clinical trial of TACE with SBRT