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Transfusion Transmitted Diseases Department, National Institute of Immunohaematology, 13th Floor, New Multi-storeyed Bldg, KEM Hospital Campus, Parel, Mumbai 400 012, Maharashtra, India
Transfusion Transmitted Diseases Department, National Institute of Immunohaematology, 13th Floor, New Multi-storeyed Bldg, KEM Hospital Campus, Parel, Mumbai 400 012, Maharashtra, India
Transfusion Transmitted Diseases Department, National Institute of Immunohaematology, 13th Floor, New Multi-storeyed Bldg, KEM Hospital Campus, Parel, Mumbai 400 012, Maharashtra, India
Department of Gastroenterology, King Edward Memorial Hospital and Seth Gordhandas Sunderdas Medical College, Acharya Donde Marg, Parel East, Parel, Mumbai, Maharashtra 400012, India
Address for correspondence: Dr. Aruna Shankarkumar Ph.D., Department of Transfusion Transmitted Disease, National Institute of Immunohaematology (ICMR), 13th Floor, New Multi-storeyed Bldg, KEM Hospital Campus, Parel, Mumbai 400012, Maharashtra, India. Tel.: +91 022 2411161 (Ext: 708); fax: +91 22 24138521.
Transfusion Transmitted Diseases Department, National Institute of Immunohaematology, 13th Floor, New Multi-storeyed Bldg, KEM Hospital Campus, Parel, Mumbai 400 012, Maharashtra, India
CTCs were characterized using a biomarker panel (+EpCAM, +CK, +AFP, −CD45, +DRAQ) in LC and HCC patients using IFC.
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Surface markers, EpCAM + CK, were the most expressed biomarkers on CTCs in LC vs. HCC followed by AFP, EpCAM and CK.
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The cell area of AFP was significantly higher in the LC group when compared with the HCC group (P value = 0.035).
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CTCs cell area (EpCAM+CK+ AFP) performed well with an AUC of 0.92, in detecting early-stage and AFP-negative HCC and LC cases.
Background
Hepatocellular carcinoma (HCC) is asymptomatic at an early stage which delays its timely diagnosis and treatment. Circulating tumor cells (CTCs), derived from a primary or secondary tumor, may help in the management of HCC. Here, we evaluate and characterize CTCs in liver disease patients.
Methods
In total, 65 patients, categorized into liver cirrhosis (LC) (n = 30) and HCC (n = 35), were enrolled. Using ImagestreamX MkII imaging flow cytometer, CTCs were detected and characterized using biomarker expression of EpCAM, CK, AFP, CD45, and DRAQ5 in LC and HCC patients.
Results
CTCs were detected in 33/35 (94%) HCC patients and in 28/30 (93%) LC patients. In the HCC group, the number of biomarker-positive CTCs was higher in BCLC stage D when compared with others. EpCAM + CK was the most expressed biomarker on CTCs in LC versus HCC (83.3% vs. 77.14%), followed by AFP (80% vs. 65.71%), EpCAM (30% vs. 28.57%), and CK (16.6% vs. 14.28%). The EpCAM cell area was significantly associated (P value = 0.031) with the CTC-positive status. The combination biomarker expression of CTCs cell area (EpCAM, CK, and AFP) performed well with the area under the curve of 0.92, high sensitivity, and specificity in detecting early-stage and AFP-negative HCC as well as in AFP-negative LC cases.
Conclusion
Enumeration and cell area of CTCs may be used as a biomarker for early detection of HCC and guiding treatment.
HCC is asymptomatic at an early stage which significantly delays its timely diagnosis and treatment, therefore, only about 20–30% of the patients are candidates for surgical intervention.
Therefore, early detection is of paramount importance in using a marker having both sensitivity and specificity.
Circulating tumor cells (CTCs) are tumor cells derived from a primary or secondary tumor and entered the circulation. These cells can travel to other sites such as lymph nodes, bone marrow, or other organs and can cause metastasis.
CTCs are not identical clones of each other but represent a heterogeneous population of cells from different tumor foci, with abilities to change their phenotypic and molecular characteristics under selective microenvironmental and therapeutic pressures.
Detection of circulating tumor cells and their implications as a biomarker for diagnosis, prognostication, and therapeutic monitoring in hepatocellular carcinoma.
CTCs have an important role in the prognosis of HCC, as well as gauging the overall survival, postoperative recurrence, and metastasis of HCC. Although first identified in 1869, only the recent advancements in technology have made it possible to detect CTCs which circulate at very low frequencies in the blood.
There are different methods for enrichment and separation of CTCs which are based on either their physical or biological properties. Espejo-Cruz ML et al. provided comprehensive review of liquid biopsy and CTC including liver and HCC specific markers, epithelial–mesenchymal transition (EMT) markers, as well as dynamic changes of CTC counts after HCC therapy.
The biological property-based method uses different antibodies to the surface markers such as epithelial cell adhesion molecule (EpCAM) and the cytokeratin family (CK8, CK18, CK19) commonly expressed on the CTCs.
although, it is not able to identify the tumor cells which undergo EMT and heterogeneous in the expression of EpCAM on some cells becomes a limitation in identifying CTCs in circulation. Other negative enrichment methods exist such as the Cytelligen system, an integrated subtraction enrichment, and immunostaining fluorescence in situ hybridization platform.
Imaging flow cytometry (IFC) offers an advantage over other methods previously used for CTC detection due to the combined features of flow cytometry and high-resolution imaging offered by this technology. It is an open platform enabling the multitarget phenotyping of CTCs independently of epithelial markers and enables the multiparametric morphological evaluation.
The ability to identify cell subpopulations with a low expression (<0.03%) makes it a great platform for the identification and visualization of CTCs at the same time.
The use of high-resolution imaging technology enables identification of the CTCs not only based on the epithelial markers but also the relative size of cells and their apparent morphology. The technique was successful in identifying CTCs from cultured cells as well as patients with other cancer types.
High-resolution imaging for the detection and characterisation of circulating tumour cells from patients with oesophageal, hepatocellular, thyroid and ovarian cancers.
Due to profound heterogeneity between tumor cells within the primary tumors and within sites of metastasis, liquid biopsy using CTCs provides a novel way to obtain a comprehensive understanding of the heterogeneous tumor cells throughout the body.
Detection of circulating tumor cells and their implications as a biomarker for diagnosis, prognostication, and therapeutic monitoring in hepatocellular carcinoma.
Here, we report our findings on the detection and characterization of CTCs using ImagestreamX MkII in liver cirrhosis and HCC patients with the help of biomarker expression of EpCAM, CK, AFP, CD45, and DRAQ5.
Material and methods
Cell Culture
HCC cell line HepG2 was acquired from American Type Culture Collection and grown in Eagle's Minimum Essential Medium supplemented with a fetal bovine serum to a final concentration of 10%. Cells were maintained in the growth phase and passaged when reached 70% confluency. Cells were confirmed to be mycoplasma-free (MycoAlert mycoplasma detection kit; Lonza).
Patient Enrollment
This study was approved by the Institutional Ethics Committee of Topiwala National Medical College (TNMC) & BYL Nair Charitable Hospital, Mumbai, and written informed consent was obtained from each patient. Total 65 patients, above 18 years of age, were recruited from the outpatient department and inpatient department of Gastroenterology in TNMC and BYL Nair Hospital, Mumbai, during the year 2019–2021. Patients were categorized into liver cirrhosis (n = 30) and hepatocellular carcinoma (n = 35) based on the Asian Pacific Association for the Study of the Liver guidelines and clinical–pathological data of patients were obtained from their medical records. HCC cases were diagnosed using imaging techniques (multiphasic CT and/or dynamic contrast-enhanced MRI) and pathological findings (histological and immune-histological) based on the International Consensus recommendations. Cirrhotic patients without any evidence of hepatic or extrahepatic malignancy were included in the study. Patients were excluded if not willing to provide informed consent, incomplete medical records, age below 18 years, or preexisting malignancy at any other site.
Sample Processing
Total 10 mL of blood from each patient was collected in EDTA and plain BD vacutainer tubes and transported in a cold chain (4 °C) to the laboratory where they were immediately processed. The blood sample was centrifuged, and top plasma layer was carefully collected, aliquoted, and stored until further analysis. To the bottom layer, the sample potentially contains CTCs along with granulocytes, lymphocytes, and mononucleocytes, and RBCs; 1× RBC Lysis Buffer (ThermoFisher Scientific, US) was added and incubated at room temperature, protected from light, for 10–15 min. Following the centrifugation at 350g for 5 min, the cell pellet was washed with 1× PBS twice at 4 °C and resuspended in 1 mL of 1× PBS with 2 mM EDTA and 0.5% BSA. After centrifugation, the cell pellet was resuspended in 1× PBS containing 3% FBS and 10 mM EDTA. The sample was incubated at 4 °C for 15 min using FcR blocking reagent (MACS, Miltenyi) to prevent unspecific antigen reactions. The sample was then immunomagnetically depleted of white blood cells (WBCs) using Invitrogen™ MagniSort™ Human CD45 Depletion Kit (Fisher Scientific, US). CD45 depleted cells were fixed in 2–4% formaldehyde in PBS and incubated at room temperature for 30 min. After centrifugation at 4 °C, cells were resuspended Flow Cytometry Staining Buffer (Thermo Fisher Scientific, US) for 30 min at 4 °C. After centrifugation cells were resuspended in Flow Cytometry Staining Buffer and stained with different immunofluorescent antibodies including EpCAM (Cell Signaling TECHNOLOGY, US), AFP (eBioscience,US), Pan-CK (Cell Signaling TECHNOLOGY, US), CD45 (eBioscience,US). After staining, cells were washed and re-suspended in PBS followed by nuclei staining with DRAQ5 (Cell Signaling TECHNOLOGY, US). Cells were acquired using the ImageStream®X Mark II flow cytometer. IFC 488 nm Blue laser (100 mW) and 642 nm Red laser (20 mW) with 40× magnification was used for detection of fluorescence dye and Side scatter (SSC) images were produced from a dedicated 785 nm laser with 2 mW of power. INSPIRE acquisition software was used for acquiring data. CTCs were identified based on brightfield morphology, size, antigen expression, nuclear signal, and the absence of CD45 expression. Objects which did not meet these criteria but showed a persistent cell morphology and lack of CD45 marker expression were also identified. Cell area was calculated by creating a custom brightfield mask based on the standard brightfield mask eroded by 3 pixels to allow a closer fit to the brightfield image. A new area feature of this mask was created and area values were calculated in um2.
Biomarker Expression in HCC Cell Line
Optimal conditions for antibodies were standardized in HepG2 cell line using the Amnis ImageStream®X Mark II flow cytometer. Fluorophores on the antibodies were selected based on excitation lasers and emission spectra in the respective fluorescence channel.
Data Analysis
Analysis of all raw data files of each processed sample was done using the IDEAS® software platform. A series of mask/feature combinations of the analysis algorithm were utilized for the cellular morphology assay using IFC. Further, focused images of single cells were initially identified from the brightfield images by using the manufacturer's recommended analysis algorithm, the gradient root means square (RMS) feature. The Gradient RMS feature measured the sharpness of an image by detecting large changes of pixel values in the image and was useful for the selection of focused images. The gradient RMS feature was computed using the mean gradient of a normalized pixel for changes in intensity levels. Artifacts and background noise were reduced by using the mask of the IDEAS 6.2 software called adaptive erode 80% mask. Cell images with calibration beads, doublets or more were excluded.
Statistical Analysis
Statistical analyses were carried out using SPSS, version 26 (SPSS Inc. Chicago, USA) and GraphPad Prism Software, version 6 (GraphPad Prism Software Inc, California, USA). Bivariate associations were Pearson's or Spearman's rho correlations for parametric or non-parametric data respectively. Differences between groups of continuous variables were evaluated by t-test (parametric data) or Mann–Whitney U (non-parametric data) tests. A P-value of <0.05 was considered significant.
Results
Patients' Characteristics
The demographics, clinical and laboratory parameters of HCC and LC patients are summarized Supplementary Table 1. The mean age of the study participants was 51.45 ± 12.26 years with male preponderance (n = 46, 70.8%). The most common etiology of HCC was HBV (28.6%) followed by HCV (11.4%) and alcoholic liver disease (2.8%). AFP levels were significantly increased in HCC patients (7095 ± 19,742 ng/mL) when compared to LC (8.1 ± 13.0 ng/mL) (P < 0.05). Clinical parameters of HCC patients are shown in Supplementary Table 2.
Characterization and Expression of CTCs in HCC and LC
The ImageStream Mark II instrument was capable of discriminating CTCs from WBCs based on objective characterization of the size of the high-resolution brightfield images. The surface area of the brightfield images as shown in Supplementary Figure 1 was used for the selection of CTCs. CTCs were identified based on any one of the positive biomarker expressions (CK, EpCAM, AFP) in combination with positive nuclear stain (DRAQ5) and negative CD45 expression. CTCs were detected in 33/35 (94%) HCC patients and in 28/30 (93%) LC patients. The number of CTCs in the HCC group was variable with CTC counts ranging from 1 to 170 cells per 10 mL of blood and 9 (25.7%) patients had marker expression negative CTCs counts ranging from 1 to 10 cells/10 mL blood based on cell morphology. There was no statistical significant difference observed in different etiology of HCC and a status of CTCs. IN HBV, HCV, Alcohol and NASH etiology, a total number of CTCs were [median (range)] 17.5 (7–37), 9.5 (1–15), 4, 89 (17–161), respectively. In HCC, the number of biomarker-positive CTCs were higher in BCLC stage D (37, 14–161; median, range) as compared to stage A (14, 6–170), Stage B (10, 6–37) and Stage C (13, 4–25). No corelations were observed between groups. In the LC group, the number of CTCs count was between 1 and 143 cells per 10 mL of blood and 5 LC patients (16.66%) had marker expression negative CTCs counts ranging from 1 to 10 cells/10 mL blood based on cell morphology. Two HCC patients had high CTC counts of 170 and 161 per 10 mL. The epithelial markers like EpCAM and CK were included in the biomarker panel for LC and HCC cases whereas AFP was used as an HCC precise biomarker. The expression of different markers such as AFP, EpCAM, and CK was highly varied between cases and in the same patients, as shown in the representative image as shown in Figure 1(a–c). EpCAM + CK was the most expressed biomarker on CTCs in LC vs. HCC (83.3% vs. 77.14%), followed by AFP (80% vs. 65.71%), EpCAM (30% vs. 28.57%), and CK (16.6% vs. 14.28%) as shown in Table 1.
Figure 1Differential expression of CTCs in patients with HCC. CTCs observed in LC and HCC patient samples displayed differential expression of individual positive biomarkers as well as in combinations. (a) EpCAM-positive and CK-positive CTCs (b) AFP-positive CTCs (c) Positive CTCs for different combinations of EpCAM and CK markers including doublet and triplet CTCs. Abbreviations: CK, cytokeratin; CTC, circulating tumor cells; EpCAM, epithelial cell adhesion molecule; HCC, hepatocellular carcinoma; LC, liver cirrhosis.
The cell surface area of CTCs was larger than WBCs as shown in Figure 2. The mean surface area of WBCs was 165.57 ± 15.60 μm2, while the HepG2 cell line had 267.02 ± 93.82 μm2 cell surface area. The mean surface area of CK-positive CTCs was larger (478.61 ± 251.53 μm2) when compared with AFP (358.67 ± 192.70 μm2) and EpCAM (287.58 ± 68.97 μm2)-positive CTCs. The cell area of AFP was positively correlated with the cell area of CK-positive CTCs (Spearman's rho: 0.66, P = 0.038) and EpCAM + CK-positive CTCs (Spearman's rho: 0.553, P < 0.001). The cell area of AFP was significantly higher in the LC group when compared with the HCC group (P = 0.035). Regarding the biomarker positivity of CTCs detected, correlations were also explored. CK expressing CTCs were negatively correlated with EpCAM expressing CTCs (Spearman's rho: −0.268, P = 0.31). In contrast, AFP-positive CTCs were strongly and positively correlated with CK-positive CTCs (Spearman's rho: 0.479, P < 0.001) and EpCAM-positive CTCs (Spearman's rho: 0.551, P < 0.001). No significant correlations were found between CTC markers in AFP+ve versus AFP−ve status in HCC and LC patients as shown in the Supplementary Table 3. However, it was found that AFP >20 ng/mL group had increased cell area of biomarker-positive CTCs than AFP <20 ng/mL group in both HCC and LC.
Figure 2Cell surface area of white blood cell compared to CTCs in LC and HCC patients. Abbreviations: CTC, circulating tumor cells; HCC, hepatocellular carcinoma; LC, liver cirrhosis.
Diverse Observations in CTCs Biomarker Identification for LC and HCC Cases
There were a series of other distinct images that were identified by the Imaging flow cytometer capabilities. In some patients, EpCAM and CK-positive cells appeared to have CD45-positive cells attached to them (Figure 3a). In a few patient samples, CTCs were in aggregates (Figure 3b) as previously reported. It can be a tumor-assisted immune response as previously suggested by ogle et al.
The receiver operating characteristic curve was plotted to evaluate the sensitivity, specificity, and area under the curve (AUC) as shown in Figure 4 and Table 2. The EpCAM, CK, and AFP cell area and CTC positive status were compared to generate AUC (0.67, 0.59, and 0.47, respectively) and for EpCAM and CK cell area combination the AUC was 0.57. The EpCAM cell area was significantly associated (P value = 0.031) with the CTC positive status. When the combined marker (EpCAM, CK, EpCAM + CK, and AFP) cell area and CTC positive status were analyzed, it was significantly associated with the AUC of 0.92 (P-value <0.001) and a specificity and sensitivity of 100% and 87.5%, respectively. Poisson regression was run to predict the combined marker (EpCAM, CK, EpCAM + CK, and AFP) cell area and total number of CTCs.
Figure 4The diagnostic capabilities of the ImageStream instrument for detecting circulating tumor cells (CTCs) in HCC and LC patients. (A) The receiver operating characteristic curve of individual markers and with EpCAM + CK cell area. (B) Combined CTC markers cell area. Abbreviations: CK, cytokeratin; CTC, circulating tumor cells; EpCAM, epithelial cell adhesion molecule; HCC, hepatocellular carcinoma; LC, liver cirrhosis.
The present study used imaging flow cytometry technology which allows detection of expression of multiple biomarkers on each cell and produces high-resolution images to identify and characterize CTCs for the early detection of HCC. First, HepG2 cell line-based model was used to standardize the workflow including enrichment of CTCs, immunomagnetic depletion of CD45-positive WBCs as well as generation of antibody panel. Expression of EpCAM, CK, AFP, and CD45 was detected by immunofluorescence. Nuclei identification was done using DRAQ5 and brightfield images of cells were collected. Next, the optimized workflow was used to enumerate and characterize CTCs in the blood of liver cirrhosis and HCC patients using a biomarker panel. This method is reproducible and allows the isolation of CTCs from cancer types including ovarian, esophageal, and thyroid.
High-resolution imaging for the detection and characterisation of circulating tumour cells from patients with oesophageal, hepatocellular, thyroid and ovarian cancers.
In the present study, high EpCAM-positive CTCs were detected in BCLC stage D (>9 CTCs) when compared with other BCLC stages (<2 CTCs) using an imaging flow cytometer. Schulze K et al. demonstrated the frequent presence of EpCAM-positive CTC in 58% of patients with intermediate or advanced HCC (BCLC stage C) using the CellSearch™ system, which is in accordance with this study.
Presence of EpCAM-positive circulating tumor cells as biomarker for systemic disease strongly correlates to survival in patients with hepatocellular carcinoma: EpCAM-Positive CTCs and Hepatocellular Carcinoma.
Among 20 different CKs; 8, 18, and 19 are the most commonly expressed in epithelial cells and their expression is retained in malignant cells, making them a choice candidate for the recognition of tumor cells.
along with raised AFP levels can be correlated with the diagnosis of HCC. This study found a positive correlation of CK-positive CTCs with AFP-positive CTCs (Spearman's rho: 0.479, P < 0.001).
Even though serum AFP lacks HCC diagnostic sensitivity and specificity, it remains the only clinically useful serum biomarker despite its limitations in monitoring HCC progression and stratification for therapy as well as Asian Pacific guidelines for the management of HCC still recommend its use for diagnosis of HCC.
AFP levels were significantly increased in HCC patients compared with LC patients (P < 0.001) as shown in Supplementary Table 1. It is well worth noting that 18/35 (51.44%) HCC patients showed a normal level of serum AFP, conventional serum AFP cutoff <200 ng/mL, at the time of diagnosis. Interestingly, hepatocyte-specific AFP biomarker expression on CTCs was found in 65.71% (23/35) HCC cases and in 80% (24/30) LC cases where all LC cases had serum AFP levels less than the conventional cutoff 200 ng/mL. The present study also reports a positive correlation of AFP-positive CTCs with CK-positive CTCs (Spearman's rho: 0.479, P < 0.001) as well as with EpCAM-positive CTCs (Spearman's rho: 0.551, P < 0.001). Kelley R et al. reported that CTCs ≥1/7.5 mL was associated with alpha-fetoprotein ≥400 ng/mL (P = 0.008). Similarly, in this study average, 30/10 mL CTCs were found in HCC cases with >200 ng/mL AFP levels and status of AFP levels was significantly associated with the expression of AFP on CTCs (P = 0.035).
A significant statistical difference (P = 0.036) was found between the number of CTCs and Child–Turcotte–Pugh. A positive correlation was found between the number of nodules in HCC cases and CK-positive CTCs (P = 0.037) as well as AFP cell area (P = 0.047).
Furthermore, gating based on the measurements of the brightfield area showed that CTCs were larger than CD45 WBCs. The mean surface area of WBCs in LC and HCC was 165.57 ± 15.60 μm2, while HepG2 cell line had 267.02 ± 93.82 μm2 cell surface area. The mean surface area of CK-positive CTCs was larger (478.61 ± 251.53 μm2) when compared with AFP (358.67 ± 192.70 μm2) and EpCAM (287.58 ± 68.97 μm2)-positive CTCs, with no overlap with the WBC cell population. The cell area of AFP was significantly higher in the LC group when compared with the HCC group (P = 0.035). Similar findings were reported by Ogle et al., the surface area of WBCs of volunteers and the patient cohort was mean 88.3 ± 0.20 μm2 and HepG2 had 219.5 ± 54.1 μm2. The average area of biomarker-positive CTCs (mean area of 362.2 ± 55.5 l μm2) was comparable to that of HCC cell line cells which are in accordance with present study findings.
The diagnostic capabilities of the ImageStream instrument for detecting CTCs based on cell area in HCC and LC patients were also explored. Our study demonstrates that the combination of expression of biomarker cell area of CTCs (EpCAM, CK, EpCAM + CK, and AFP) achieved better performance with the AUC of 0.92 (95% CI, 0.79–1.06), specificity 100%, and sensitivity 87.5% distinguishing between HCC and LC. Cheng Y et al. reported the AUC values for total CTCs, AFP, and a combined model (combined use of total CTCs and AFP) was 0.774 (95% CI, 0.704–0.834), 0.669 (95% CI, 0.587–0.750), and 0.821 (95% CI, 0.756–0.886).
Guo W et al. demonstrated that a multimarker CTC detection panel (EpCAM, CD90, CD133, and CK19) with high sensitivity and specificity, capable of differentiating patients with HCC from healthy control and patients with chronic hepatitis B, liver cirrhosis, and benign hepatic lesion. Thus, a combination of markers on CTCs may be very useful for the early diagnosis of HCC instead of a single marker. Several studies have identified the use of liquid biopsy as a marker for early detection and prognosis of HCC. Qi et al. found the significance of CTCs undergoing EMT in patients with hepatocellular carcinoma using CanPatrol CTC-enrichment technique. They reported CTC count ≥16 and mesenchymal–CTC (M-CTC) percentage ≥2% prior to resection were significantly associated with early recurrence, multi-intrahepatic recurrence, and lung metastasis.
Court CM et al. developed a HCC multimarker antibody-based CTC capture assay on the NanoVelcro surface that allowed the identification of HCC-CTCs as well as a distinct subpopulation of vimentin(+)-CTCs, thus, highlighted the potential of mesenchymal CTCs in a prospective study of 80 patients.
Genome-wide mapping of 5-hydroxymethylcytosines in circulating cell-free DNA as a non-invasive approach for early detection of hepatocellular carcinoma.
CtDNA represents tumor-derived fragmented DNA in the bloodstream of cancer patients with a constitution that varies substantially from <0.01% to >60% of alleles in circulation.
Clinical applications of liquid biopsy as prognostic and predictive biomarkers in hepatocellular carcinoma: circulating tumor cells and circulating tumor DNA.
Wu et al. reviewed genetic (RAS, TERT, TP53, and so on) and epigenetic changes (methylation on DBX2, THY1, TGR5 and so on) in the cfDNA that originates from cancer cells (ctDNA) suggested that profiling of molecular changes in ctDNA/cfDNA has the huge potential to detect tumor cells and may be able to guide targeted therapy in HCC.
Li et al. reviewed hypermethylated genes, such as DBX2, TGR5, MT1M, MT1G, and INK4A, in cfDNA from HCC patients that were identified as biomarkers for vascular invasion in HCC.
Clinical applications of liquid biopsy as prognostic and predictive biomarkers in hepatocellular carcinoma: circulating tumor cells and circulating tumor DNA.
The major hurdle for the development of a CTC detection assay is the unfamiliarity of the existence of tumor cells in a blood sample and their number. Another limitation of the present study is pertaining to detection which is specific to markers for epithelial expression. Few patients had cells that were nucleated and cellular in morphology, negative for all the epithelial markers, but of a size resembling a CTC rather than other blood cell types. Additionally, these cells were CD45 negative, unrelated in both size and morphology from the white cells, with higher DNA content. CTCs traveling in clusters and Immune interactions between single CTCs and WBCs were other unusual observations and needs further understanding. CTCs may form aggregates with fibroblasts and other cells to form CTC clusters, which possess a significantly higher metastatic potential and increased ability to survive compared to individual CTCs.
Detection of circulating tumor cells and their implications as a biomarker for diagnosis, prognostication, and therapeutic monitoring in hepatocellular carcinoma.
The major limitation of this study includes follow-up of patients after their enrollment into the study; therefore, it was not possible to study biomarker expression on CTCs before versus after treatment as well as couldn't perform predictive or prognostic biomarker expression on CTCs in LC and HCC cases.
In conclusion, by using Amnis ImageStream®X Mark II flow cytometer, we identified, characterized, and detected circulating tumor cells (CTCs) using a biomarker panel (+EpCAM, +CK, +AFP, −CD45, +DRAQ) in LC and HCC patients. Imaging flow cytometry technology doesn't only enable the enumeration of CTCs in the blood sample but also utilizes cell area for the characterization of CTCs from WBCs. This biomarker panel performed well in detecting early-stage and AFP-negative HCC as well as in AFP negative LC cases. The combination of expression of biomarker cell area of CTCs (EpCAM, CK, and AFP) performed well with the AUC of 0.92, high sensitivity, and specificity in detecting early-stage and AFP-negative HCC as well as in AFP-negative LC cases.
Consent to participate
Informed consent was obtained from all individual participants included in the study.
Consent to publish
Consent for publication was obtained from all authors.
Ethics approval statement
This study was approved by the Institutional Ethics Committee of Topiwala National Medical College (TNMC) & BYL Nair Charitable Hospital, Mumbai, and, the study protocol confirmed to the ethical guidelines of the 1975 Declaration of Helsinki. Written informed consent was obtained from all individual participants included in the study.
Credit authorship contribution statement
Debnath Partha: Methodology, Investigation, Writing – Original draft.
Detection of circulating tumor cells and their implications as a biomarker for diagnosis, prognostication, and therapeutic monitoring in hepatocellular carcinoma.
High-resolution imaging for the detection and characterisation of circulating tumour cells from patients with oesophageal, hepatocellular, thyroid and ovarian cancers.
Presence of EpCAM-positive circulating tumor cells as biomarker for systemic disease strongly correlates to survival in patients with hepatocellular carcinoma: EpCAM-Positive CTCs and Hepatocellular Carcinoma.
Genome-wide mapping of 5-hydroxymethylcytosines in circulating cell-free DNA as a non-invasive approach for early detection of hepatocellular carcinoma.
Clinical applications of liquid biopsy as prognostic and predictive biomarkers in hepatocellular carcinoma: circulating tumor cells and circulating tumor DNA.
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