Article

IGFBP1 in epithelial circulating tumor cells as a potential response marker to selective internal radiation therapy in hepatocellular carcinoma

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Abstract

Background: Local ablative techniques such as selective internal radiation therapy (SIRT) have become the mainstay of treating hepatocellular carcinoma (HCC) in the bridging-to-transplant and palliative setting. We recently demonstrated that epithelial circulating tumor cells (CTCs) correlate to an unfavorable outcome. We wanted to scrutinize whether molecular markers detected in this specific CTC subgroup may also have clinical implications. Materials & methods: Mononuclear cells and CTCs were isolated from peripheral blood samples using density gradient centrifugation followed by depletion of hematopoietic and enrichment of epithelial (EpCAM(+)) cells employing immunomagnetic beads. The mRNA expression of candidate markers was correlated with response to SIRT in 25 patients using quantitative real-time reverse-transcription PCR. Results: IGFBP1 mRNA expression levels were significantly correlated with time to progression in a Kaplan-Meier log rank test (p = 0.04; 0 vs 4 months) and receiver operating characteristic analysis demonstrated a potential use to predict patients with shortened time to progression (area under the curve: 0.8; 95% CI: 0.44-0.98; p = 0.03). Conclusion: The EpCAM fraction of CTCs may be useful to detect novel molecular markers to individualize treatment decision in patients with HCC.

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... We recently developed a CTC detection method based on multi-parameter immunofluorescence microscopy (MPIM) that includes epithelial markers such as CK or EpCAM and cells with mesenchymal and stem cell-like characteristics. We were able to identify an individual composition of CTC subtypes as profiles that associate to therapeutic success in hepatocellular carcinoma, nonsmall cell lung carcinoma and head and neck squamous carcinoma [9][10][11][12][13]. In this study, we have addressed the question whether different types of CTC are identifiable in the peripheral blood of patients with mRCC and, if so, whether their distribution may serve as a predictor of treatment response or outcome. ...
... Duplicates of 20 ml citrated peripheral venous blood were drawn from mRCC patients after response assessment (2 cycles of therapy) and processed within 24 h after collection. Blood sample preparation was done as described previously [9][10][11][12]. Briefly, 20 ml of blood were diluted with 10 ml PBS and carefully layered into a Leucosep tube containing 16 ml Ficoll-Paque (GE-Healthcare) below a porous barrier. ...
... For gene expression analyses epithelial CTC were sequentially enriched by depletion of hematopoietic cells and subsequent positive selection using anti-EpCAM immunomagnetic beads as described above [12]. Total RNA was extracted from recovered EpCAM-positive tumor cells using MagAttract RNA Cell Mini M48 Kits (Qiagen, Hilden, Germany) and King Fisher mL magnetic particle processor (Thermo Fisher, Waltham, MA) according to the manufacturer's instructions. ...
Article
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Purpose: Due to their minimal-invasive yet potentially current character circulating tumor cells (CTC) might be useful as a "liquid biopsy" in solid tumors. However, successful application in metastatic renal cell carcinoma (mRCC) has been very limited so far. High plasticity and heterogeneity of CTC morphology challenges currently available enrichment and detection techniques with EpCAM as the usual surface marker being underrepresented in mRCC. We recently described a method that enables us to identify and characterize non-hematopoietic cells in the peripheral blood stream with varying characteristics and define CTC subgroups that distinctly associate to clinical parameters. With this pilot study we wanted to scrutinize feasibility of this approach and its potential usage in clinical studies. Experimental design: Peripheral blood was drawn from 14 consecutive mRCC patients at the West German Cancer Center and CTC profiles were analyzed by Multi-Parameter Immunofluorescence Microscopy (MPIM). Additionally angiogenesis-related genes were measured by quantitative RT-PCR analysis. Results: We detected CTC with epithelial, mesenchymal, stem cell-like or mixed-cell characteristics at different time-points during anti-angiogenic therapy. The presence and quantity of N-cadherin-positive or CD133-positive CTC was associated with inferior PFS. There was an inverse correlation between high expression of HIF1A, VEGFA, VEGFR and FGFR and the presence of N-cadherin-positive and CD133-positive CTC. Conclusions: Patients with mRCC exhibit distinct CTC profiles that may implicate differences in therapeutic outcome. Prospective evaluation of phenotypic and genetic CTC profiling as prognostic and predictive biomarker in mRCC is warranted.
... Metformin, an activator of AMPK, a central metabolic regulator, was found to increase IGFBP1 expression, thereby inhibiting endometrial cancer cell proliferation [20]. The role of IGFBP1 in HCC has been reported, demonstrating that IGFBP1 inhibited the invasion and metastasis of HCC cells, and this could be considered as an important marker for the prognosis of HCC [21,22]. Nevertheless, the insight true role of IGFBP1 in cancer cell biology, especially in growth and progression of HCC, still remains controversial. ...
... However, the expression and function of IGFBP1 in HCC development remains controversial, and little is known about its true role including diagnostic and prognostic values in HCC. In fact, paradoxical data have been reported in terms of the serum level and expression of IGFBP1 in patients with HCC [21,22]. One study showed that that IGFBP1 may function as a tumor suppressor gene by blunting the IGF axis [21]. ...
Article
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Ursolic acid (UA), a natural pentacyclic triterpenoid, exerts anti-tumor effects in various cancer types including hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying this remain largely unknown. Cell viability and cell cycle were examined by MTT and Flow cytometry assays. Western blot analysis was performed to measure the phosphorylation and protein expression of p38 MAPK, insulin-like growth factor (IGF) binding protein 1 (IGFBP1) and forkhead box O3A (FOXO3a). Quantitative real-time PCR (qRT-PCR) was used to examine the mRNA levels of IGFBP1 gene. Small interfering RNAs (siRNAs) method was used to knockdown IGFBP1 gene. Exogenous expressions of IGFBP1 and FOXO3a were carried out by transient transfection assays. IGFBP1 promoter activity was measured by Secrete-Pair™ Dual Luminescence Assay Kit. In vivo nude mice xenograft model and bioluminescent imaging system were used to confirm the findings in vitro. We showed that UA stimulated phosphorylation of p38 MAPK. In addition, UA increased the protein, mRNA levels, and promoter activity of IGFBP1, which was abrogated by the specific inhibitor of p38 MAPK (SB203580). Intriguingly, we showed that UA increased the expression of FOXO3a and that overexpressed FOXO3a enhanced phosphorylation of p38 MAPK, all of which were not observed in cells silencing of endogenous IGFBP1 gene. Moreover, exogenous expressed IGFBP1 strengthened UA-induced phosphorylation of p38 MAPK and FOXO3a protein expression, and more importantly, restored the effect of UA-inhibited growth in cells silencing of endogenous IGFBP1 gene. Consistent with these, UA suppressed tumor growth and increased phosphorylation of p38 MAPK, protein expressions of IGFBP1 and FOXO3a in vivo. Collectively, our results show that UA inhibits growth of HCC cells through p38 MAPK-mediated induction of IGFBP1 and FOXO3a expression. The interactions between IGFBP1 and FOXO3a, and feedback regulatory loop of p38 MAPK by IGFBP1 and FOXO3a resulting in reciprocal pathways, contribute to the overall effects of UA. This in vitro and in vivo study corroborates a potential novel mechanism by which UA controls HCC growth and implies that the rational targeting IGFBP1 and FOXO3a can be potential for the therapeutic strategy against HCC.
... cant association of IGFBP-1 mRNA expression with shortened time to progression (ROC: 0.8; 95% CI: 0.44-0.98; P=0.03)[71].The function of IGFBP-1 that inhibits the invasion and metastasis of HCC cells via downregulation of matrix metalloproteinase 9 (MMP-9) expression has been proven. Furthermore, in the human liver non-tumor cell line HL-7702, HCC cell lines HuH-7, HepG2, SMMC-7721 and MHCC97-H, the expression of IGFBP-1 was slowly decreased with gradually increased invasion of the above cell lines[70]. ...
Article
Insulin-like growth factor binding protein-1 (IGFBP-1) belongs to the insulin-like growth factor (IGF) system, which plays an indispensable role in normal growth and development, and in the pathophysiology of various tumors. IGFBP-1 has been shown to be associated with the risk of various tumors, and has a vital function in regulating tumor behaviors such as proliferation, migration, invasion and adhesion through different molecular mechanisms. The biological actions of IGFBP-1 in cancer are found to be related to its phosphorylation state, and the IGF-dependent and -independent mechanisms. In this review, we provided an overview of IGFBP-1 in normal physiology, and its aberrantly expression and the underlying molecular mechanisms in a range of common tumors, as well as discussed the potential clinical implications of IGFBP-1 as diagnostic or prognostic biomarkers in cancer.
... CTC showed a positive correlation with Barcelona-Clinic Liver Cancer (BCLC) stage, suggesting that positive CTCs may predict the prognosis of patients with HCC. Meanwhile, Nel et al. [7] revealed that insulin-like growth factor binding protein (IGFBP)-1 mRNA level was correlated with the time to progression (TTP) by detecting CTCs in HCC patients undergone selective internal radiotherapy (SIRT), supporting that CTCs can be a new molecular marker for individualized treatment of HCC. In addition, Chen et al. [8] carried out a retrospective analysis in terms of the level and classification of CTCs in 195 HCC patients. ...
Article
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Background This research was to develop a special method for enriching Circulating tumor cells (CTCs) of Hepatocellular carcinoma (HCC) by Glypican-3 immunoliposomes (GPC3-IML), and to analyze the correlation between the CTCs count and tumor malignancy, as well as to investigate the mutation characteristics of CTC-derived NGS. Results In this study characterization of physical parameters was performed with the preparation of GPC3-IML. CTCs in peripheral blood of HCC patients were further separated and identified. Immunofluorescence was used to identify CTCs for further counting. By this means, the correlation between CTCs count and clinicopathological features was analyzed, and the genetic mutation characteristics of NGS derived from CTCs were investigated and compared with that of tissue NGS. Results showed that compared with EpCAM and vimentin, GPC-3 had a stronger CTCs separation ability. There was a correlation between "positive" count of CTCs (≥ 5 PV-CTC per 7.5 ml blood) and BCLC stage (P = 0.055). The result of CTC-NGS was consistent with that of tissue-NGS in 60% cases, revealing that KMT2C was a common highly-frequent mutated gene. Conclusion The combination of immunomagnetic separation of CTCs and anti-tumor marker identification technology can be regarded as a new technology of CTCs detection in peripheral blood of patients with HCC. Trial registration EHBHKY2020-k-024. Registered 17 August 2020—Retrospectively registered
... Insulin-like growth factor-binding protein-1 [IGFBP-1], the most predominant IGFBP which is primarily secreted by the fetal liver, has been shown to involve in cellular proliferation, development, apoptosis, and tumor growth [69][70][71]. Paradoxical data have been reported on the expression of IGFBP1 in HCC [72,73]. However, consistent with the report from Hwang et al. [74], we found elevated serum IGFBP-1 levels in HCC, implying that IGFBP1 may be potential target in the early diagnosis of HCC. ...
Preprint
Background: Hepatocellular carcinoma (HCC) is one of most common cancers with a high mortality rate. HBV/HCV infection is an important risk factor to trigger HCC. Therefore, developing serum biomarkers for early diagnosis is crucial to prolong survival in HCC patients. Methods: An antibody array technology was utilized to detect serum from 20 HBV-related HCC patients, 20 chronic hepatitis B patients and 20 normal population, whose results were further validated by ELISA. Results: Both antibody array and ELISA showed that ten growth factors (SCF R, GDF-15, HGF, FGF-4, IGFBP-1, PIGF, GH, GDNF, BDNF and IGF-1) were significantly differential in HCC patients when compared to the non-HCC population. Among these growth factors, the levels of SCF R, GDF-15, HGF, GH and IGF-1 showed significant correlation with hepatitis B and its severity, indicating that these growth factors may promote HCC progression by an HBV-specific mechanism. A therapy targeting these growth factors in hepatitis B patients may help to prevent the development of HCC. FGF4 and GH were found, for the first time, to be upregulated in HCC, suggesting that these two growth factors may serve as novel serum biomarkers for the early diagnosis of HCC. Conclusion: The combined detection of all the differential growth factors may improve the diagnostic accuracy of HCC.
... A recent study identified a candidate marker, insulin like growth factor binding protein 1 (IGFBP1), in CTCs of 25 HCC patients, and this marker was shown to be correlated with the responsiveness to selective internal radiation therapy, which is a local ablative technique for HCC treatment [39]. In addition, the sensitivity of CTCs to chemotherapeutic drugs was tested by another study. ...
Article
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Hepatocellular carcinoma (HCC) is ranked as the sixth most common cancer around the world. With the emergence of the state-of-the-art modalities lately, such as liver transplantation, image-guided ablation, and chemoembolization, the death rate is still high due to high metastasis rate after therapy. Observation by biannual ultrasonography allows effective diagnosis at an early stage for candidates with no extrahepatic metastasis, but its effectiveness still remains unsatisfactory. Developing a new test with improved effectiveness and specificity is urgently needed for HCC diagnosis, especially for patients after first line therapy. Circulating tumor cells (CTCs) are a small sub-population of tumor cells in human peripheral blood, they release from the primary tumor and invade into the blood circulatory system, thereby residing into the distal tissues and survive. As CTCs have specific and aggressive properties, they can evade from immune defenses, induce gene alterations, and modulate signal transductions. Ultimately, CTCs can manipulate tumor behaviors and patient reactions to anti-tumor treatment. Given the fact that in HCC blood is present around the immediate vicinity of the tumor, which allows thousands of CTCs to release into the blood circulation daily, so CTCs are considered to be the main cause for HCC occurrence, and are also a pivotal factor for HCC prognosis. In this review, we highlight the characteristics and enrichment strategies of CTCs, and focus on the use of CTCs for tumor evaluation and management in patients with HCC.
... There is an abundance of research results involving the correlation between CTCs and AFP. Nel et al. (22) found that AFP mRNA is 75% sensitive and 71% specific by detecting EpCAM + CTC-specific gene expression. Schulze et al. (23) reported that the AFP levels of liver cancer patients with EpCAM + CTC patients were >400 ng/mL. ...
Article
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Background: This study was to determine circulating tumor cells (CTCs) and the expression of CXC chemokine receptor type 4 (CXCR4) in primary hepatocellular carcinoma (HCC) and the relationships with prognosis. Methods: We used an advanced CanPatrolTM CTC-enrichment technique to collect CTCs for isolation and characterization from blood samples. The RNA in situ hybridization (RNA-ISH) method, which is based on branched DNA (bDNA) signal amplification technology, was used to determine the expression of CXCR4 according to epithelial-mesenchymal transition (EMT) markers in 99 patients with primary liver cancer in blood samples pre-operatively. The relationship between the EMT markers and HCC was determined. Results: The positive rates of CTCs and CXCR4 were 89.9% and 58.8%, respectively. CTCs were positively correlated with the Barcelona clinic liver cancer (BCLC) staging, tumor diameter and number, envelope, microsatellite damage, portal vein thrombosis, alpha-fetoprotein (AFP), and hepatitis B DNA, and negatively correlated with Edmondson grade. There were significant differences in the expression of CXCR4 between interstitial CTCs and mixed CTCs. A total of 99 patients underwent CTCs testing prior to surgery. The tumor-free survival time of HCC patients with interstitial CTCs <1 (13.3 months) was significantly longer than patients with interstitial CTCs ≥1 (5.0 months) pre-operatively. Conclusions: CTC-positivity was shown to be associated with HCC and can be used as an independent prognostic factor for HCC. High CXCR4 protein expression was more common in mixed CTCs.
... By using PCR-based method for detection of liverspecific or tumor-associated gene expressions in peripheral blood mononuclear cells, HCC CTCs have been demonstrated to be present in circulating peripheral blood. These gene mRNA markers include AFP, albumin, TERT, Snail, and insulin-like growth factor-binding protein 1 (IGFBP1) [119,128,136,187]. Among them, only AFP is a well-established HCC marker [210]. ...
Chapter
Hepatocellular carcinoma (HCC) is among the most aggressive of cancers, and the prognosis remains poor. Data are accumulating that circulating tumor cells (CTCs) are an active source of recurrence or metastasis in HCC patients. As a novel biomarker for the metastatic disease process, CTC study has become a very active field of research. As for the detection of CTCs in HCC patients, some interesting and encouraging results have currently been obtained although the knowledge about their clinical relevance is lagging behind other major cancers. This chapter describes recent discoveries related to the biology of CTCs, outlines their potential clinical utility as a real-time liquid tumor biopsy, reviews current advances in different techniques or strategies available for their detection and isolation, and introduces progress obtained from clinical studies on detection and molecular characterization of CTCs and circulating cancer stem cells (CCSCs) in HCC patients and current challenges.
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Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related death worldwide. Diagnostic, prognostic, and predictive biomarkers are urgently needed in order to improve patient survival. Indeed, the most widely used biomarkers, such as alpha-fetoprotein (AFP), have limited accuracy as both diagnostic and prognostic tests. Liver biopsy provides an insight on the biology of the tumor, but it is an invasive procedure, not routinely used, and not representative of the whole neoplasia due to the demonstrated intra-tumoral heterogeneity. In recent years, liquid biopsy, defined as the molecular analysis of cancer by-products, released by the tumor in the bloodstream, emerged as an appealing source of new biomarkers. Several studies focused on evaluating extracellular vesicles, circulating tumor cells, cell-free DNA and non-coding RNA as novel reliable biomarkers. In this review, we aimed to provide a comprehensive overview on the most relevant available evidence on novel circulating biomarkers for early diagnosis, prognostic stratification, and therapeutic monitoring. Liquid biopsy seems to be a very promising instrument and, in the near future, some of these new non-invasive tools will probably change the clinical management of HCC patients.
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The cancer stem cell marker, EpCAM, is an important indicator of Wnt/β-catenin signaling activation and a functional component of hepatocellular tumor-initiating cells. A high-throughput screening assay was developed to identify inhibitors of EpCAM-dependent growth of hepatocellular carcinoma (HCC) cells. EpCAM(+) and EpCAM(-) HCC cell lines were assessed for differential sensitivity to a Wnt/β-catenin pathway inhibitor. Libraries comprising 22 668 pure compounds and 107 741 crude or partially purified natural product extracts were tested, and 12 pure compounds and 67 natural product extracts were identified for further study. Three active compounds and the positive control were further characterized in terms of effects on EpCAM expression. Treatment of EpCAM(+) Hep3B cells resulted in loss of EpCAM expression as assessed by flow cytometry. This reduction was incomplete (most cells continued to express EpCAM), but resulted in generation of cell populations expressing lower levels of EpCAM. Sublethal concentrations (~IC50 ) reduced median EpCAM expression to 28% of control after 1 day and 19% of control after 2 days. Reduction in EpCAM expression preceded growth inhibition suggesting that a threshold of EpCAM expression may be required for growth of EpCAM-dependent cells. The identification of compounds with a variety of possible molecular targets suggests a likelihood of multiple mechanisms for modulation of EpCAM-dependent cell growth.
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Circulating tumor cells (CTCs) have long been considered a reflection of tumor aggressiveness. Hematogenous spreading of CTCs from a primary tumor is a crucial step in the metastasis cascade, which leads ultimately to the formation of overt metastases. However, owing to the rarity of CTCs in peripheral blood, detecting these cells requires methods combined with high sensitivity and specificity, which sets tremendous challenges for the implementation of these assays into clinical routine. Generally, CTCs detection methods are composed of the following two steps: enrichment (isolation) process (morphological and immunological techniques) and detection (identification) process (cytometric and nucleic acid techniques), which may or may not be separate from enrichment. Genetic and molecular characterization of CTCs carried out by fluorescent in situ hybridization (FISH), comparative genomic hybridization (CGH), PCR-based techniques, and biomarker immunofluorescent staining extract more information about malignant profile, metastatic potential of CTCs, and the extent to which CTCs are genetically identical to the primary tumor. Recent technical advances made it possible to detect CTCs. The efficacy of circulating tumor cell (CTC) detection among patients with solid malignancy has been investigated, which shows great potential to become a tool for real-time parameter of prognosis and serve as an early marker to assess the therapeutic response in overt cancers. Improvements in detection and characterization of CTCs will hopefully lead to refinement of clinical management of cancer patients. This review addresses the majority of assays that have been published thus far, including the enrichment and detection steps and the markers used in these assays, accompanied by some biological issues of CTC and the results of clinical application harvested.
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To establish a sensitive and specific isolation and enumeration system for circulating tumor cells (CTC) in patients with hepatocellular carcinoma (HCC). HCC cells were bound by biotinylated asialofetuin, a ligand of asialoglycoprotein receptor, and subsequently magnetically labeled by antibiotin antibody-coated magnetic beads, followed by magnetic separation. Isolated HCC cells were identified by immunofluorescence staining using Hep Par 1 antibody. The system was used to detect CTCs in 5 mL blood. Blood samples spiked with Hep3B cells (ranging from 10 to 810 cells) were used to determine recovery and sensitivity. Prevalence of CTCs was examined in samples from HCC patients, healthy volunteers, and patients with benign liver diseases or non-HCC cancers. CTC samples were also analyzed by FISH. The average recovery was 61% or more at each spiking level. No healthy, benign liver disease or non-HCC cancer subjects had CTCs detected. CTCs were identified in 69 of 85 (81%) HCC patients, with an average of 19 ± 24 CTCs per 5 mL. Both the positivity rate and the number of CTCs were significantly correlated with tumor size, portal vein tumor thrombus, differentiation status, and the disease extent as classified by the TNM (tumor-node-metastasis) classification and the Milan criteria. HER-2 gene amplification and TP53 gene deletion were detected in CTCs. Our system provides a new tool allowing for highly sensitive and specific detection and genetic analysis of CTCs in HCC patients. It is likely clinically useful in diagnosis and monitoring of HCC and may have a role in clinical decision making.
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The global burden of cancer continues to increase largely because of the aging and growth of the world population alongside an increasing adoption of cancer-causing behaviors, particularly smoking, in economically developing countries. Based on the GLOBOCAN 2008 estimates, about 12.7 million cancer cases and 7.6 million cancer deaths are estimated to have occurred in 2008; of these, 56% of the cases and 64% of the deaths occurred in the economically developing world. Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females, accounting for 23% of the total cancer cases and 14% of the cancer deaths. Lung cancer is the leading cancer site in males, comprising 17% of the total new cancer cases and 23% of the total cancer deaths. Breast cancer is now also the leading cause of cancer death among females in economically developing countries, a shift from the previous decade during which the most common cause of cancer death was cervical cancer. Further, the mortality burden for lung cancer among females in developing countries is as high as the burden for cervical cancer, with each accounting for 11% of the total female cancer deaths. Although overall cancer incidence rates in the developing world are half those seen in the developed world in both sexes, the overall cancer mortality rates are generally similar. Cancer survival tends to be poorer in developing countries, most likely because of a combination of a late stage at diagnosis and limited access to timely and standard treatment. A substantial proportion of the worldwide burden of cancer could be prevented through the application of existing cancer control knowledge and by implementing programs for tobacco control, vaccination (for liver and cervical cancers), and early detection and treatment, as well as public health campaigns promoting physical activity and a healthier dietary intake. Clinicians, public health professionals, and policy makers can play an active role in accelerating the application of such interventions globally.
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This article reviews the role of selective internal irradiation (SIR) with yttrium-90 ((90)Y) microspheres for hepatocellular carcinoma (HCC). Studies were identified by searching Medline and PubMed databases for articles from 1990 to 2009 using the keywords "selective internal irradiation," "hepatocellular carcinoma," "therapeutic embolization," and "yttrium-90." (90)Y microspheres are a safe and well-tolerated therapy for unresectable HCC (median survival range, 7 -21.6 months). The evidence was limited to cohort studies and comparative studies with historical control. (90)Y microspheres have been reported to downstage unresectable HCC to allow for salvage treatments with curative intent, act as a bridging therapy before liver transplantation, and treat HCC with curative intent for patients who are not surgical candidates because of comorbidities. (90)Y microsphere is recommended as an option of palliative therapy for large or multifocal HCC without major portal vein invasion or extrahepatic spread. It can also be used for recurrent unresectable HCC, as a bridging therapy before liver transplantation, as a tumor downstaging treatment, and as a curative treatment for patients with associated comorbidities who are not candidates for surgery.
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Survivin (SVV) mRNA expression levels in peripheral blood of patients with gastrointestinal malignancies change significantly during the course of treatment. We wanted to scrutinize these findings in patients with esophageal carcinoma and furthermore evaluate whether the detection of mRNA and the change in detecting ability have an association with overall survival. Whole blood was drawn 1 day pre- and 10 days post-operatively from 62 patients with esophageal carcinoma. Tumor cells were enriched from whole blood by density-gradient centrifugation prior to extraction of total cellular RNA and subsequent direct quantitative reverse transcriptase-PCR assays. SVV was detectable in 48 out of 62 patients (77%). Stepwise multivariate Cox linear regression models demonstrated a significant and independent association of measured SVV with overall survival (6.6 exp[b]; 95% CI: 1.97-22.12; p = 0.002). Increased SVV levels after the operation were linked to shorter overall survival (p = 0.04). Preoperative SVV expression levels appear to be associated with overall survival in patients with esophageal cancers. Increasing levels could potentially indicate a higher risk for shorter overall survival and therefore demand adapted treatment modalities.
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We evaluated whether detection of human telomerase reverse transcriptase (hTERT) mRNA after immunomagnetic separation is useful to detect circulating cancer (CC) cells. Experimental Design: Two ml of peripheral blood were collected from 55 cases with hepatocellular carcinoma (HCC), 20 cases with chronic liver diseases devoid of cancer, and 20 healthy volunteers. Then 1500 and 500 micro l were subjected to immunomagnetic separations using Ber-EP4 and anti-CD45 antibodies, harvested and supernatant cells were collected as epithelial and nonleukocyte fractions, respectively. Samples of each fraction were subjected to reverse transcription-PCR detecting beta-actin, interleukin-2 receptor (IL-2r), alpha-fetoprotein, and hTERT mRNAs. The cases were judged to be positive, equivocal, or negative for CC cells when hTERT positivity with IL-2r negativity, hTERT positivity with IL-2r positivity, or hTERT negativity was seen in epithelial and/or nonleukocyte fractions, respectively. The dilution experiments revealed that our system could detect 10(0-1) HeLa cells involved in 2 ml of blood. The Ber-EP4-harvested cells from cases with distant metastasis were positive for immunostaining using Hep Par 1 monoclonal antibody. CC cells were judged to be positive in 29 of 55 (53%) HCC cases. On the contrary, no cases without HCC were determined to be positive. The frequency of positivity was significantly correlated with disease extent of HCC. These results strongly suggest that detection of hTERT mRNA after immunomagnetic separation is a specific and sensitive tool to detect CC cells and that it would provide useful source for further investigation of cancer metastasis.
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The TNM Classification describes the anatomic extent of cancer. TNM's ability to separately classify the individual tumor (T), node (N), and metastasis (M) elements and then group them into stages differs from other cancer staging classifications (e.g., Dukes), which are only concerned with summarized groups. The objectives of the TNM Classification are to aid the clinician in the planning of treatment, give some indication of prognosis, assist in the evaluation of the results of treatment, and facilitate the exchange of information. During the past 50 years, the TNM system has evolved under the influence of advances in diagnosis and treatment. Radiographic imaging (e.g., endoscopic ultrasound for the depth of invasion of esophageal and rectal tumors) has improved the accuracy of the clinical T, N, and M classifications. Advances in treatment have necessitated more detail in some T4 categories. Developments in multimodality therapy have increased the importance of the "y" symbol and the R (residual tumor) classification. New surgical techniques have resulted in the elaboration of the sentinel node (sn) symbol. The use of immunohistochemistry has resulted in the classification of isolated tumor cells and their distinction from micrometastasis. The most important challenge facing users of the TNM Classification is how it should interface with the large number of non-anatomic prognostic factors that are currently in use or under study. As non-anatomic prognostic factors become widely used, the TNM system provides an inviting foundation upon which to build a prognostic classification; however, this carries a risk that the system will be overwhelmed by a variety of prognostic data. An anatomic extent-of-disease classification is needed to aid practitioners in selecting the initial therapeutic approach, stratifying patients for therapeutic studies, evaluating non-anatomic prognostic factors at specific anatomic stages, comparing the weight of non-anatomic factors with extent of disease, and communicating the extent of disease data in a uniform manner. Methods are needed to express the overall prognosis without losing the vital anatomic content of TNM. These methods should be able to integrate multiple prognostic factors, including TNM, while permitting the TNM system to remain intact and distinct. This article discusses examples of such approaches.
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Hepatocellular carcinoma (HCC) is the fifth most common cause of cancer, and its incidence is increasing worldwide because of the dissemination of hepatitis B and C virus infection. Patients with cirrhosis are at the highest risk and should be monitored every 6 months. Surveillance can lead to diagnosis at early stages, when the tumour might be curable by resection, liver transplantation, or percutaneous treatment. In the West and Japan, these treatments can be applied to 30% of patients, and result in 5-year survival rates higher than 50%. Resection is indicated among patients who have one tumour and well-preserved liver function. Liver transplantation benefits patients who have decompensated cirrhosis and one tumour smaller than 5 cm or three nodules smaller than 3 cm, but donor shortage greatly limits its applicability. This difficulty might be overcome by living donation. Most HCC patients are diagnosed at advanced stages and receive palliative treatments, which have been assessed in the setting of 63 randomised controlled trials during the past 25 years. Meta-analysis shows that only chemoembolisation improves survival in well-selected patients with unresectable HCC.
Article
Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors and is the second most common cause of cancer death in China. Therefore, it is very important to detect this disease and the recurrence at its earlier period. Serum tumor markers, as the effective method for detecting hepatocellular carcinoma for a long time, could be divided into 4 categories: oncofetal antigens and glycoprotein antigens; enzymes and isoenzymes; genes; and cytokines. Serum alpha fetoprotein (AFP) is the most widely used tumor marker in detecting patients with hepatocellular carcinoma, and has been proven to have capability of prefiguring the prognosis. However, it has been indicated that AFP-L3 and DCP excel AFP in differentiating hepatocellular carcinoma from nonmalignant hepatopathy and detecting small hepatocellular carcinoma. Some tumor markers, such as human cervical cancer oncogene and human telomerase reverse transcriptase mRNA, have also been indicated to have higher accuracies than AFP. Furthermore, some other tumor markers, such as glypican-3, gamma-glutamyl transferase II, alpha-l-fucosidase, transforming growth factor-beta1, tumor-specific growth factor, have been indicated to be available supplementaries to AFP in the detection. AFP mRNA has been shown to correlate with the metastasis and recurrence of HCC, and it may be the most useful marker to prefigure the prognosis. Some other markers, such as gamma-glutamyl transferase mRNA, vascular endothelial growth factor, and interleukin-8, could also be used as available prognostic indicators, and the simultaneous determination of AFP and these markers may detect the recurrence of HCC at its earlier period.
Article
Increasing the sensitivity and specificity of detecting circulating carcinoma cells of patients with hepatocellular carcinoma (HCC) is very important for monitoring recurrence. To establish a novel method of detecting circulating carcinoma cells. For method development, 3 sets of controls using HCC cell line HepG2 cells were used. (A): Serial dilutions of HepG2 cells were directly used to extract total RNA for nested reverse transcription-polymerase chain reaction (RT-PCR). (B): Five milliliter of healthy blood was spiked with a serial dilution of HepG2 cells and was used for Ficoll density gradient centrifugation to recover cells. The cells were used to extract total RNA for RT-PCR. (C): After cell recovery with the same procedure as B, the cells were sorted sequentially by CD45 and Ber-EP4 immunomagnetic beads and used for RNA extraction and RT-PCR. For clinical samples, 44 patients with HCC and 7 healthy subjects were included. The alpha-fetoprotein mRNA was amplified using nested RT-PCR technique. The spiking experiments using HepG2 cells showed that 10 cells in 5 mL blood could be detected by method C and an excellent dose-response to the number of spiked cells. Whereas, method B lacked any dose-response and would yield high false-positive rates. In clinical samples, the improved method led to a positive detection rate of 52.9%, 76.9%, and 92.9% in Child-Plug class A, B, and C, respectively. There was significant difference between class A and class C (P<0.05). The total positive detection rate was 72.7%. Combining negative and positive immunomagnetic beads with RT-PCR technique may improve the sensitivity and specificity of detecting circulating HCC cells.
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The clinical impact of circulating tumor cell (CTC) detection is controversial, mainly due to drawbacks of molecular approaches applied to this field. We sought to determine if the specific identification and counting of circulating tumor cells by cytomorphologic analysis has clinical usefulness. Peripheral blood (6 mL), treated using isolation by size of epithelial tumor cells, was obtained from 44 patients with primary liver cancer (PLC) and without metastases, 30 patients with chronic active hepatitis, 39 with liver cirrhosis, and 38 healthy individuals, and followed up for a mean period of 1 year. We searched for beta-catenin mutations in 60 single microdissected CTCs. One patient with liver cancer developed extrahepatic metastases during follow-up. CTCs and microemboli were found in 23 of the 44 patients with liver cancer and in none of the patients with chronic active hepatitis, patients with cirrhosis, or healthy subjects. Their presence was significantly associated with tumor diffusion (P =.0001) and portal tumor thrombosis (P =.006). Both the presence (P =.01) and number (P =.02) of CTCs and microemboli were significantly associated with a shorter survival. Beta-catenin mutations were found in 3 of 60 CTCs, arguing against their impact on the initial step of tumor cell invasion. In conclusion, the highly sensitive and specific detection of CTCs and microemboli may have clinical implications for cancer staging and outcome prediction. We also show the feasibility of molecular studies of individual circulating tumor cells, aimed at identifying gene mutations involved in tumor invasion.