肉瘤样肝细胞癌中MET基因扩增与预后的相关性分析

背景与目的：肉瘤样肝细胞癌（sarcomatoid hepatocellular carcinoma,SHC）是一种罕见且高度恶性的肝脏肿瘤。MET基因异常具有肿瘤预后预测价值,以MET为靶点的抑制剂已成为晚期肿瘤治疗的重要方向,然而SHC组织中MET基因扩增状态尚不明确。探讨SHC中MET基因扩增与临床病理学因素的相关性及其预后预测价值。方法：收集2008年1月—2016年12月于复旦大学附属中山医院经病理学检查确诊的22例SHC患者资料。采用荧光原位杂交（fluorescence in situ hybridization,FISH）法检测上述患者的MET基因扩增情况,并结合临床病理学资料进行统计学分析。采用Kaplan-Meier模型分析总生存期（overall survival,OS）及无病生存期（disease-free survival,DFS）,采用log-rank检验比较生存曲线,采用多因素COX回归模型分析SHC中独立的预后因素。结果：22例SHC患者中,MET基因扩增5例（22.7%）。MET基因扩增存在异质性,主要位于分化差的梭形细胞区域（4例）。MET基因扩增的SHC患者中位OS明显短于MET阴性患者（6.8个月 vs 24.0个月,P=0.001）。SHC中具有完整肿瘤包膜的患者中位OS明显长于包膜不完整的患者（41.3个月 vs 8.5个月,P=0.001）。单灶肿瘤及中国肝癌分期（China Liver Cancer Staging,CNLC）Ⅰ期患者的中位DFS较多灶肿瘤及Ⅱ+Ⅲ+Ⅳ期患者明显延长（10.4个月 vs 3.8个月,12.4个月 vs 3.8个月,P=0.027和0.017）。MET基因扩增是SHC独立的预后因子。结论：22.7%（5/22）的SHC中存在MET基因扩增。MET基因扩增的SHC患者预后更差,是其预后的独立危险因素。该研究结果为该罕见肿瘤的治疗提供了新策略和临床依据。     

MET gene amplification or EGFR mutation activate MET in lung cancers untreated with EGFR tyrosine kinase inhibitors

We analyzed MET protein and copy number in NSCLC with or without EGFR mutations untreated with EGFR tyrosine kinase inhibitors (TKIs). MET copy number was examined in 28 NSCLC and 4 human bronchial epithelial cell lines (HBEC) and 100 primary tumors using quantitative real‐time PCR. Positive results were confirmed by array comparative genomic hybridization and fluorescence in‐situ hybridization. Total and phospho‐MET protein expression was determined in 24 NSCLC and 2 HBEC cell lines using Western blot. EGFR mutations were examined for exon 19 deletions, T790M, and L858R. Knockdown of EGFR with siRNA was performed to examine the relation between EGFR and MET activation. High‐level MET amplification was observed in 3 of 28 NSCLC cell lines and in 2 of 100 primary lung tumors that had not been treated with EGFR‐TKIs. MET protein was highly expressed and phosphorylated in all the 3 cell lines with high MET amplification. In contrast, 6 NSCLC cell lines showed phospho‐MET among 21 NSCLC cell lines without MET amplification (p = 0.042). Furthermore, those 6 cell lines harboring phospho‐MET expression without MET amplification were all EGFR mutant (p = 0.0039). siRNA‐mediated knockdown of EGFR abolished phospho‐MET expression in examined 3 EGFR mutant cell lines of which MET gene copy number was not amplified. By contrast, phospho‐MET expression in 2 cell lines with amplified MET gene was not down‐regulated by knockdown of EGFR. Our results indicated that MET amplification was present in untreated NSCLC and EGFR mutation or MET amplification activated MET protein in NSCLC. © 2008 Wiley‐Liss, Inc.     

Dynamin2 downregulation delays EGFR endocytic trafficking and promotes EGFR signaling and invasion in hepatocellular carcinoma

Dysregulation of endocytosis was viewed as an emerging feature of cancer development and progression. A large GTPase dynamin2 plays a significant role in receptor tyrosine kinases (RTKs) endocytosis. The study was designed to investigate its roles in hepatocellular carcinoma (HCC) metastasis and its underlying mechanism. Dynamin2 expression in cancer tissues from HCC patients was assessed by immunohistochemistry and its prognostic significance for the patients was conducted using univariate and multivariate analysis. Its role in tumor invasion and metastasis was evaluated in vitro by gene silence using siRNA-mediated approach and the small molecule inhibitor of Dynasore. EGFR expression in HCC cell lines and EGFR downstream signaling ERK1/2 was evaluated by Western-blot and immunofluorescence analyses after Dynamin2 inhibition. Our data indicated that low expression of dynamin2 was well correlated with invasion characteristics and shorter overall survival. HCC cell migration, colony formation and invasion were significantly increased after the inhibition of dynamin2 in HCC cells. Internalization of EGFR was markedly reduced when dynamin2 was knock down or inhibition. In addition, we observed that dynamin2 regulated EGF mediated EGFR downstream Ras/ERK1/2 signaling and p-ERK1/2 accumulation in nucleus. The results demonstrate a possible mechanism of dynamin involved EGFR endocytosis and modulation of metastasis in HCC. Dynamin2 inhibits the invasion and metastasis of HCC cells by the promotion of EGFR endocytosis and downregulation of ERK1/2 phosphorylation.     

Low EGFR/MET ratio is associated with resistance to EGFR inhibitors in non-small cell lung cancer

Purpose

Although activating mutations in the epidermal growth factor receptor (EGFR) gene are predictive markers for response to EGFR inhibitors, 30–40% of EGFR-mutant non-small cell lung cancer (NSCLC) patients are de novo non-responders. Hence, we sought to explore additional biomarkers of response.

Methods

We conducted a prospective pilot study to characterize the expression and/or activation of key receptor tyrosine kinases (RTKs) in stage IIIB-IV NSCLC tumors. A total of 37 patients were enrolled and 34 underwent EGFR inhibitor treatment.

Results

As expected, patients bearing activating EGFR mutations showed increased progression free survival (PFS) compared to patients with wild-type EGFR status (9.3 vs 1.4 months, p = 0.0629). Analysis of baseline tumor RTK profiles revealed that, regardless of EGFR mutation status, higher levels of EGFR relative to MET correlated with longer PFS. At multiple EGFR/MET ratio cut-offs, including 1, 2 and 3, median PFS according to below vs. above cut-offs were 0.4 vs. 6.1 (p = 0.0001), 0.5 vs. 9.3 (p = 0.0006) and 1.0 vs. 11.2 months (p = 0.0008), respectively.

Conclusion

The EGFR/MET ratio measured in tumors at baseline may help identify NSCLC patients most likely to benefit from prolonged PFS when treated with EGFR inhibitors.     

Tivantinib (ARQ197) in hepatocellular carcinoma

Here we review the development of tivantinib, a selective oral inhibitor of c-MET. The initially identified dose and schedule for clinical use was 360 mg twice daily. Biological considerations and early results suggested its activity against hepatocellular carcinoma after progression on sorafenib. The results of randomized Phase II study in this setting have already been reported; while in the overall population, the risk of progression was reduced by 36% (HR: 0.64; 90% CI: 0.43–0.94; p = 0.04), in the pre-defined MET-high population median overall survival (7.2 vs 3.8 months; p = 0.01), median time to progression (2.7 vs 1.4 months; p = 0.03) as well as disease control rate (50 vs 20%), were increased by tivantinib. During study conduction, tivantinib dose was amended to 240 mg twice daily, due to a high incidence of neutropenia, without losing clinical efficacy. Presently, a global Phase III trial is being conducted.     

STMN1 upregulation mediates hepatocellular carcinoma and hepatic stellate cell crosstalk to aggravate cancer by triggering the MET pathway

STMN1 has been regarded as an oncogene and its upregulation is closely associated with malignant behavior and poor prognosis in multiple cancers. However, the detailed functions and underlying mechanisms of STMN1 are still largely unknown in hepatocellular carcinoma (HCC) development. Herein, we analyzed STMN1 expression and the related clinical significance in HCC by using well‐established Protein Atlas, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) cancer databases. Analysis indicated that STMN1 was highly expressed in HCC and closely associated with vascular invasion, higher histological grade, advanced clinical grade and shorter survival time in HCC patients. Overexpressing and silencing STMN1 in HCC cell lines showed that STMN1 could regulate cell proliferation, migration, drug resistance, cancer stem cell properties in vitro as well as tumor growth in vivo. Further experiments showed that STMN1 mediated intricate crosstalk between HCC and hepatic stellate cells (HSC) by triggering the hepatocyte growth factor (HGF)/MET signal pathway. When HSC were cocultured with HCC cells, HSC secreted more HGF to stimulate the expression of STMN1 in HCC cells. Mutually, STMN1 upregulation in HCC cells facilitated HSC activation to acquire cancer‐associated fibroblast (CAF) features. The MET inhibitor crizotinib significantly blocked this crosstalk and slowed tumor growth in vivo. In conclusion, our findings shed new insight on STMN1 function, and suggest that STMN1 may be used as a potential marker to identify patients who may benefit from MET inhibitor treatment.     

Insulin receptor tyrosine kinase substrate activates EGFR/ERK signalling pathway and promotes cell proliferation of hepatocellular carcinoma

Insulin receptor tyrosine kinase substrate (IRTKS) is closely associated with actin remodelling and membrane protrusion, but its role in the pathogenesis of malignant tumours, including hepatocellular carcinoma (HCC), is still unknown. In this study, we showed that IRTKS was frequently upregulated in HCC samples, and its expression level was significantly associated with tumour size. Enforced expression of IRTKS in human HCC cell lines significantly promoted their proliferation and colony formation in vitro, and their capacity to develop tumour xenografts in vivo, whereas knockdown of IRTKS resulted in the opposite effects. Furthermore, the bromodeoxyuridine (BrdU) incorporation analyses and propidium iodide staining indicated that IRTKS can promote the entry into S phase of cell cycle progression. Significantly, IRTKS can interact with epidermal growth factor receptor (EGFR), results in the phosphorylation of extracellular signal-regulated kinase (ERK). By contrast, inhibition of ERK activation can attenuate the effects of IRTKS overexpression on cellular proliferation. Taken together, these data demonstrate that IRTKS promotes the proliferation of HCC cells by enhancing EGFR–ERK signalling pathway.     

Heat shock proteins in hepatocellular carcinoma: Molecular mechanism and therapeutic potential

Heat shock proteins (HSPs) are highly conserved proteins, which are expressed at low levels under normal conditions, but significantly induced in response to cellular stresses. As molecular chaperones, HSPs play crucial roles in protein homeostasis, apoptosis, invasion and cellular signaling transduction. The induction of HSPs is an important part of heat shock response, which could help cancer cells to adapt to stress conditions. Because of the constant stress condition in tumor microenvironment, HSPs overexpression is widely reported in many human cancers. In light of the significance of HSPs for cancer cells to survive and obtain invasive phenotype under stress condition, HSPs are often associated with poor prognosis and treatment resistance in many types of human cancers. It has been described that upregulation of HSPs may serve as diagnostic and prognostic markers in hepatocellular carcinoma (HCC). Targeting HSPs with specific inhibitor alone or in combination with chemotherapy regimens holds promise for the improvement of outcomes for HCC patients. In this review, we summarize the expression profiles, functions and molecular mechanisms of HSPs (HSP27, HSP70 and HSP90) as well as a HSP‐like protein (clusterin) in HCC. In addition, we address progression and challenges in targeting these HSPs as novel therapeutic strategies in HCC.     

MicroRNA-107: a novel promoter of tumor progression that targets the CPEB3/EGFR axis in human hepatocellular carcinoma

MicroRNAs (miRNAs) are dysregulated in many types of malignancies, including human hepatocellular carcinoma (HCC). MiR-107 has been implicated in several types of cancer regulation; however, relatively little is known about miR-107 in human HCC. In the present study, we showed that the overexpression of miR-107 accelerates the tumor progression of HCC in vitro and in vivo through its new target gene, CPEB3. Furthermore, our results demonstrated that CPEB3 is a newly discovered tumor suppressor that acts via the EGFR pathway. Therefore, our study demonstrates that the newly discovered miR-107/CPEB3/EGFR axis plays an important role in HCC progression and might represent a new potential therapeutic target for HCC treatment.     

Preliminary Study of Oxidative Stress in Human Hepatocellular Carcinoma and Adjacent Normal Liver Tissues

OBJECTIVE The antioxidative system in human hepatocellular carcinoma was investigated. METHODS The activities of cytosolic catalase (CAT), superoxide dismu-tase, glutathione peroxidase (GSH-Px), glutathione S-tranferase and levels of reduced glutathione, total protein thiols and malondialdehyde were assayed in 10 cases of hepatocellular carcinoma and adjacent normal liver. RESULTS Hepatoma tissues showed higher activities of CAT, GSH -Px and lower content of total antioxidative capacity compared to adjacent normal liver tissue (P<0.05). CONCLUSION These findings suggest that the antioxidative defense-related enzymes and antioxidants are largely regulated in hepatoma cells. However, the mechanism which is not clear requires further investigation.     

Percutaneous thermal ablation of medium and large hepatocellular carcinoma

BACKGROUND:

Radiofrequency ablation (RFA) and microwave ablation (MWA) were found to be effective in treating hepatocellular carcinoma (HCC) smaller than 3 cm; however, to the authors' knowledge, the usefulness of thermal ablation in treating larger HCC, especially those >5 cm, has not been well documented. The present study evaluated the therapeutic efficacy of percutaneous thermal ablation with curative intention for HCC measuring between 3.0 cm and 7.0 cm.

METHODS:

Percutaneous RFA or MWA were used to treat 109 HCC patients with at least 1 tumor measuring between 3.0 cm and 7.0 cm. Fifty?eight patients received thermal ablation as the first treatment, and the remaining 51 were treated for posthepatectomy recurrent HCC. A total of 89 patients had a main tumor measuring 3.0 cm to 5.0 cm, and 20 patients had main tumors measuring 5.0 cm to 7.0 cm. Local therapeutic efficacy, long‐term outcome, and prognostic factors were analyzed.

RESULTS:

There were no treatment‐related deaths, and the major complication rate was 9.2%. Complete ablation rate was 92.6%. Local recurrence (LR) occurred in 22% patients, with a median time to LR of 4.6 months. Distant recurrences developed in 53.2% patients. The 1‐year, 3‐year, and 5‐year survival rates were 75.8%, 30.9%, and 15.4%, respectively. Univariate analysis indicated that incomplete tumor ablation, posthepatectomy recurrence, and preablation α‐fetoprotein (AFP) ≥200 ng/mL were 3 unfavorable prognostic factors for long‐term survival (P = .000, .015, and .008, respectively). Cox regression analysis confirmed that incomplete tumor ablation, recurrent tumors, and preablation AFP ≥200 ng/mL were independent unfavorable prognostic factors, with an exp(B) of 4.158 (P = .001), 1.568 (P = .082), and 1.593 (P = .082), respectively.

CONCLUSIONS:

Percutaneous thermal ablation was effective and safe in treating HCC between 3 cm and 7 cm, with acceptable local tumor control and long‐term outcomes. Completeness of ablation, previous history of treatment, and preablation AFP level were significant prognostic factors. Cancer 2009. © 2009 American Cancer Society.     

HER2‐positive gastric cancer with concomitant MET and/or EGFR overexpression: A distinct subset of patients for dual inhibition therapy

Growth factor receptors, often carrying tyrosine kinase activities in their cytoplasmic domains, are overexpressed in many cancers. Coactivation of receptor tyrosine kinases (RTKs) plays a critical role in tumor response to targeted therapeutics. We examined concomitant overexpression of EGFR and MET in patients with HER2⁺ and HER2^? gastric cancers (GCs). Tissue microarray samples obtained from 1,589 GC patients who received R0 gastrectomy with extensive node dissection and adjuvant chemoradiationtherapy were analyzed by immunohistochemistry and fluorescence in situ hybridization. HER2⁺ was observed in 169 patients (11%). Out of 169 HER2⁺ patients, 15 (9%) were EGFR⁺ and MET⁺, 29 (17%) were EGFR⁺, 37 (22%) were MET⁺ and the remaining 88 patients (52%) were HER2⁺ only, without concomitant EGFR or MET overexpression. Greater number of overexpressed RTKs correlated with younger age (p < 0.001), larger tumor size (p = 0.027), intestinal histology (p < 0.001) and shorter overall survival (p = 0.002). The mean overall survival was 113 months for HER2^?/EGFR^?/MET^? and 63 months for HER2⁺/EGFR⁺/MET⁺ subgroups. Patients with HER2⁺/EGFR⁺/MET⁺ GCs had a substantial risk of death with a hazard ratio of 3.01 (95% CI: 1.54–5.90), compared with HER2^?/EGFR^?/MET^? GC patients. Using patient‐derived tumor cell models isolated from pericardial effusion of HER2⁺ and MET⁺ GC cases, we demonstrated that the combination of HER2‐inhibitor (lapatinib) and MET‐inhibitor offered a more profound inhibition in the ERK/AKT pathway and cell proliferation than lapatinib alone. Co‐overexpression of RTKs was demonstrated in small subsets of GC associated with aggressive behavior and in these cases, combination therapy may be considered as potential treatment options.     

Survival and intra-hepatic recurrences after laparoscopic radiofrequency of hepatocellular carcinoma in patients with liver cirrhosis

BACKGROUND: The optimal treatment for hepatocellular carcinoma (HCC) is surgical resection. However, only a small percentage of patients are operative candidates. Percutaneous radiofrequency interstitial thermal ablation proved to be effective, too. Our objective was to assess a novel operative combination of laparoscopic ultrasound (LUS) with laparoscopic radiofrequency (LRF) in the treatment of HCC not amenable to liver resection. METHODS: One hundred and four patients with HCC in liver cirrhosis were submitted to laparoscopic LRF. A LRF was indicated in patients not amenable to liver resection that had at least one of the following criteria: (a) severe impairment of the coagulation tests; (b) large tumors (but <5 cm) or multiple lesions requiring repeated punctures; (c) superficial lesions adjacent to visceral structures; (d) deep-sited lesions with a very difficult or impossible percutaneous approach; (e) short-term recurrence of HCC following percutaneous loco-regional therapies. RESULTS: The LRF procedure was completed in 102 out of 104 patients (98% feasibility rate). LUS identified 26 new malignant lesions (25%) undetected by pre-operative imaging. There was no operative mortality. Seventy-six patients had no complication (73%). At 1-month computed tomography (CT) evaluation, a complete response with a 100% necrosis was achieved in 88 out of 101 patients (87%). During the follow-up (mean follow-up: 22.5 +/- 15.9 months), 55 patients (54%) developed new malignant nodules (42% of these recurrences were localized in the same segment of the HCC treated). CONCLUSIONS: LRF of HCC proved to be a safe and effective technique at least in the short and mid-term: in fact it permits to treat lesions not treatable with the per cutaneous approach, to detect 25% of new HCC nodules and it has a low morbidity rate.     

肝细胞癌中Ｓｕｒｖｉｖｉｎ、ＥＧＦＲ和ＶＥＧＦ的表达及临床意义

目的　研究Ｓｕｒｖｉｖｉｎ、ＥＧＦＲ和ＶＥＧＦ在肝细胞癌中的表达情况,为肝癌预后判断和靶向治疗提供理论依据。方法　应用免疫组织化学ＰＶ６０００法检测８１例人肝细胞癌组织、癌旁组织Ｓｕｒｖｉｖｉｎ、ＥＧＦＲ和ＶＥＧＦ的表达并分析其与相关临床病理特征的关系。结果　肝细胞癌组织与癌旁组织Ｓｕｒｖｉｖｉｎ的阳性率无显著差别;肝细胞癌组织ＥＧＦＲ、ＶＥＧＦ的阳性率分别为４９.４％、６３.０％,显著高于癌旁组织的阳性率２１.０％、４４.４％（Ｐ＜０.０５）。Ｓｕｒｖｉｖｉｎ的表达与肿瘤的ＴＮＭ分期和肿瘤直径有关（Ｐ＜０.０５）;ＥＧＦＲ的表达与病理分化和术后复发转移有关（Ｐ＜０.０５）;ＶＥＧＦ的表达与ＴＮＭ分期、术前局部转移及术后复发转移有关（Ｐ＜０.０５）。ＥＧＦＲ与ＶＥＧＦ表达呈正相关（ｒ＝０.３０７,Ｐ＝０.００７）。ＴＮＭ分期、Ｓｕｒｖｉｖｉｎ、ＥＧＦＲ是肝细胞癌的独立预后因子（Ｐ＜０.０５）。结论　肝细胞癌组织Ｓｕｒｖｉｖｉｎ、ＥＧＦＲ和ＶＥＧＦ表达水平较高,其中Ｓｕｒｖｉｖｉｎ、ＥＧＦＲ可以作为肝细胞癌的预后因子。ＥＧＦＲ与ＶＥＧＦ表达正相关,对研发多靶点药物或联合使用多种靶向药物有一定指导意义。     

Thermal ablation of hepatocellular carcinoma in patients with abnormal coagulation function

Objective: To evaluate the safety of thermal ablation for hepatocellular carcinoma (HCC) in patients with abnormal coagulation function.

Methods: Fifty-seven HCC tumours in 50 patients were treated with thermal ablation. All patients had a meted platelet count?<50?×?10⁹/L or international normalised ratio (INR)?≥?1.7. Gastroscopy before ablation, platelet concentrate or fresh frozen plasma transfusion during ablation and contrast enhanced ultrasoundgraphy (CEUS)-guided ablation to cease needle tract bleeding were performed to reduce haemorrhage. The incidences of haemorrhage and other major complications were recorded and patients were followed up to observe the local tumour progression (LTP), intrahepatic distant recurrence (IDR), overall survival (OS) and recurrence-free survival (RFS) rates.

Results: Two incidences of needle tract bleeding and one needle tract bleeding together with bleeding at the suture of the spleen fossa were found. Three needle tract bleeding events were detected by CEUS and ceased after CEUS-guided complementary ablation. CEUS failed to detect bleeding at the suture of the spleen fossa. Therefore, a laparotomy was conducted for haemostasis. No other major complications were found after ablation. The median follow-up periods were 18.7?±?12.0?months (range 1?～?42?months) and 1 LTP and 15 IDRs occurred. The 1-, 2- and 3-year OS rates were 84.8%, 82.7% and 82.7%, and RFS rates were 67.9%, 64.0% and 64.0%, respectively.

Conclusion: With gastroscopy before ablation, platelet concentrate or fresh frozen plasma transfusion during ablation and CEUS-guided ablation to cease needle tract bleeding, thermal ablation is a safe treatment for HCC in patients with abnormal coagulation function.