Apocynin,an NADPH oxidase inhibitor,suppresses progression of prostate cancer via Rac1 dephosphorylation

Recently, considerable evidence has been generated that oxidative stress contributes to the etiology and pathogenesis of prostate cancer. The present study focused on the effects of apocynin, an inhibitor of the NADPH oxidase which generates intracellular superoxide, on a rat androgen-independent prostate cancer cell line (PLS10) in vitro and in vivo. Apocynin significantly inhibited cell proliferation of PLS10 cells via G1 arrest of the cell cycle in vitro. Surprisingly, it did not affect reactive oxygen species (ROS) but inhibited phosphorylation of Rac1, one component of the NADPH oxidase complex. A Rac1 inhibitor, NSC23766, also inhibited cell proliferation, and both apocynin and NSC23766 reduced phosphorylation of Rac1 and NF-κB, as well as cyclin D1. Furthermore, in a xenograft model of prostate cancer with PLS10, apocynin suppressed tumor growth and metastasis in a dose dependent manner in vivo, with reduction of cell proliferation and vessel number in the tumors. Expression and secretion of vascular endothelial growth factor (VEGF) were reduced by apocynin treatment in vivo and in vitro, respectively. In conclusion, despite no apparent direct relationship with oxidative stress, apocynin inhibited growth of androgen-independent prostate cancer in vitro and in vivo. Apocynin thus warrants further attention as a potential anti-tumor drug.     

Rac1信号通路在小鼠可卡因情景线索记忆过程中的作用

目的:探讨Rac1信号通路在小鼠可卡因情景线索记忆过程中的作用。方法:将成年雄性C57BL/6J小鼠随机分为对照组(control)、可卡因配对的条件性位置偏爱组(CPP)和Rac1抑制剂NSC23766处理组(NSC),利用CPP实验检测可卡因是否可诱导小鼠产生CPP,利用立体定位技术注射AAV-GFP病毒于小鼠海马CA1区并做免疫荧光染色观察海马CA1区神经元的形态;利用Western Blot及GST-pull down方式检测蛋白表达及活性变化;采用Rac1抑制剂NSC23766阻断信号通路探讨其对小鼠可卡因诱导的CPP的影响。结果:可卡因可以诱导小鼠形成CPP,并导致Rac1 GTPase活性增强及其下游信号分子cofilin磷酸化活性升高,并最终导致海马CA1区锥体神经元发生神经可塑性变化。结论:Rac1信号通路通过影响小鼠海马CA1区锥体神经元发生结构可塑性,从而参与调控可卡因情景线索记忆。     

Expression of the EphA1 protein is associated with Fuhrman nuclear grade in clear cell renal cell carcinomas

Aberrant expression of receptor tyrosine kinase EphA1 in malignant tissues has been reported. However, the expression profile of EphA1 in renal cell carcinoma (RCC) and its association with clinicopathological parameters remain unknown. The aim of this study was to determine the cancerous value of the EphA1 protein expression in patients with renal cell carcinomas. This study included 144 patients with clear cell RCC (ccRCC), 18 patients with chromophobe RCC and 6 patients with papillary RCC. The EphA1 protein was detected in RCC tissue samples by an immunohistochemical staining with a specific polycolonal antibody. The correlation of the expression of the EphA1 protein with clinicopathological parameters was evaluated. High level of the expression of EphA1 was observed in all normal renal tubes. The EphA1 protein was negatively or weakly expressed in 93 out of 144 ccRCC (64.6%) and positively expressed in 51 out of 144 ccRCC (35.4%). The high level expression of the EphA1 protein was significantly associated with younger patients (P<0.001), sex (P=0.016) and lower nuclear grade (P<0.001). No significant relation between the expression of EphA1 and tumor diameter was found (P=0.316). Positive expression of EphA1 was observed in all samples of chromophobe RCC and papillary RCC. Our data indicated that the EphA1 protein may be a new marker for the prognosis of ccRCC.     

抑制Rac1通过降低磷酸化p38MAPK提高1型糖尿病小鼠的心脏功能

目的:探讨抑制Rac1对1型糖尿病小鼠心肌细胞肥大、心脏功能的影响及其作用机制。方法:50只8周龄雄性C57小鼠随机分为对照组(control,n=10)、Rac1抑制剂NSC23766对照组(NSC,n=10)、1型糖尿病组(STZ,n=15)及NSC治疗组(STZ+NSC,n=15)。小鼠腹腔注射链脲佐菌素(STZ)建立1型糖尿病动物模型,血糖升高后给予小鼠腹腔注射NSC23766,实验于8周末结束,记录实验小鼠生存率、测量小鼠体重及左室重量并计算左室重量指数,运用超声心动图检测小鼠心脏功能,心肌组织进行HE染色结合图像分析软件定量心肌细胞大小,运用实时定量RT-PCR技术检测心肌组织心房利钠肽(ANP)、脑利尿肽(BNP)、β-肌球蛋白重链(β-MHC)mRNA表达,以Westernblotting定量心肌组织磷酸化p38丝裂素活化蛋白激酶(pho-p38MAPK)表达。结果:抑制Rac1后:(1)糖尿病小鼠生存率提高、糖尿病小鼠左室重量指数降低(P0.01)、心脏左室射血分数(EF)增加、左室短轴缩短率增加(FS)(P0.01);(2)心肌细胞大小明显降低(P0.01);(3)心肌肥大相关基因ANP、BNP、β-MHC表达明显降低(P0.01);(4)心肌组织pho-p38MAPK表达明显降低(P0.01)。结论:抑制Rac1活性显著改善1型糖尿病小鼠心脏功能、降低心肌细胞肥大,其机制可能与明显降低心肌组织磷酸化p38MAPK密切相关。     

肺炎衣原体感染通过PI3K激活Rac1而诱导血管平滑肌细胞迁移

 目的:研究Rac1活化在肺炎衣原体（C.pn）感染诱导血管平滑肌细胞（VSMCs）迁移中的作用及磷脂酰肌醇3-激酶（PI3K）对其活化的影响。方法:谷胱甘肽巯基转移酶（GST）-p21活化激酶1 p21结合结构域（PBD）即GST-PBD重组质粒转化感受态细菌,诱导融合蛋白表达并纯化;GST-pull down实验检测C.pn感染VSMCs后Rac1活性的变化;PI3K特异性抑制剂LY294002 （25 μmol/L）预处理VSMCs,GST-pull down实验检测Rac1活性的变化;Rac1特异性抑制剂NSC23766（50 μmol/L）预处理VSMCs,wound-healing 实验和Transwell 实验观察VSMCs迁移能力的变化。结果:重组质粒转化感受态细菌后,经诱导表达和纯化,得到足量有效的GST-PBD融合蛋白;GST-pull down实验结果显示,C.pn感染VSMCs后Rac1活性增强且显著高于正常对照组（P<0.05）;LY294002预处理VSMC后,C.pn感染诱导的Rac1活性明显下降（P<0.05）;细胞迁移实验结果显示,NSC23766预处理的 C.pn感染组细胞迁移能力明显低于单纯感染组（P<0.05）。结论: C.pn感染可能通过PI3K激活Rac1,从而诱导VSMCs迁移。     

Clinical Implications of HSC70 Expression in Clear Cell Renal Cell Carcinoma

Purpose: The role of heat shock protein 70 (HSC70) in the progression of clear cell renal cell carcinoma (ccRCC) is unclear. This study explored the effect of the HSC70 on the survival of ccRCC patients.Methods: Immunohistochemical analysis was performed to determine HSC70 expression in samples obtained from 121 ccRCC patients with at least 5 years of follow-up. We also analyzed the association between HSC70 expression and clinicopathological characteristics. Furthermore, the association of overall survival (OS) with HSC70 expression was analyzed using Kaplan-Meier curves. Finally, we used the Oncomine and CCLE databases to determine the effects of HSC70 mRNA expression on ccRCC.Results: HSC70 expression was associated with distant metastasis and death of ccRCC patients. HSC70 was expressed in the nucleus and/or cytoplasm of ccRCC cells. The incidence of distant organ metastasis and death was higher in patients with HSC70 expression than in those without it. Survival analysis revealed that patients with HSC70 expression had significantly shorter OS. Oncomine analyses also showed that the HSC70 mRNA was significantly upregulated in ccRCC tissues.Conclusions: HSC70 expression was related to adverse prognosis, and patients with HSC70 expression had a worse prognosis than those without HSC70 expression. HSC70 may thus serve as a potential therapeutic target for ccRCC.     

Decreased expression of EphA5 is associated with Fuhrman nuclear grade and pathological tumour stage in ccRCC

The incidence of renal cell carcinoma is increasing all over the world. The molecular mechanisms for tumorigenesis, progression and prognosis are still unknown. The erythropoietin‐producing hepatoma amplified sequence (Eph) receptors have been reported to be expressed aberrantly in many types of human cancers and in particular EphA5 may play a role in certain human cancers. In this study, a set of clear cell renal cell carcinoma (ccRCC) tissues were subjected to immunohistochemistry. The relationship between EphA5 protein expression and clinicopathological parameters was statistically analysed. Our data show that EphA5 protein was negatively (0) or weakly (1+) expressed in 48 of 78 (61.5%), moderately (2+) expressed in 15 of 78 (19.2%) and strongly (3+) expressed in 15 of 78 (19.2%) tumour samples of ccRCC. Decreased expression of EphA5 was detected more often in females than in males (P = 0.017, r_s = ?0.267). Expression of EphA5 was related negatively to Fuhrman grade (P = 0.013, r_s = ?0.279) and pathological tumour stage pT (P = 0.003, r_s = ?0.334). No relation between the expression of EphA5 and age of patients was found (P = 0.107, r_s = 0.184). Fuhrman grade and pT stage are the most important factors used in prognosis of ccRCC. Hence this study may provide a new and useful prognostic marker in the clinical practice of ccRCC.     

Low expression of miR-99b promotes progression of clear cell renal cell carcinoma by up-regulating IGF1R/Akt/mTOR signaling

Objective: Dysfunctions of microRNAs have been implicated in the progression of clear cell renal cell carcinoma (ccRCC). Here, we investigated the roles of miR-99b and miR-99b* in ccRCC development. Methods: The expression levels of miR-99b and miR-99b* in tumor and tumor-adjacent tissues from ccRCC patients were quantified by quantitative Real-Time PCR (qRT-PCR). MicroRNA mimics and inhibitors were employed to evaluate the functions of miR-99b and miR-99b*. The effects of miR-99b on the proliferation and migration of ccRCC cells were analyzed by MTT and wound-healing assays, respectively. The effect of miR-99b on the expression of its target gene IGF1R and mTOR was determined by western blotting and qRT-PCR. Results: The abundances of miR-99b and miR-99b* were lower in ccRCC tissues than in the tumor-adjacent tissues from patients. Similarly, the expression of these two microRNAs was higher in the normal kidney HK-2 cells than in the ccRCC cell lines. Moreover, miR-99b and miR-99b* inhibited the proliferation and migration of ccRCC cells. MiR-99b was found to down-regulate IGF1R and mTOR expression, likely through targeting their mRNAs to induce degradation. Consistently, the mRNA levels of IGF1R and mTOR were higher in ccRCC tissues than in the tumor-adjacent tissues, and Akt, a downstream factor of IGF1R, was highly activated correspondingly in ccRCC tissues. Conclusion: The low expression of miR-99b and miR-99b* contributes to ccRCC development and miR-99b acts as an onco-suppressor by suppressing IGF1R and mTOR expression to down-regulate IGF1R/AKT/mTOR signaling.     

透明细胞肾细胞癌组织中DNMT3B异构体表达及其整体甲基化的分析

目的检测透明细胞肾细胞癌组织中DNA甲基转移酶3B(DNA methyltransferase 3B,DNMT3B)异构体表达及其整体甲基化的变化,探讨这些变化与透明细胞肾细胞癌的关系。方法选取15例透明细胞肾细胞癌及其癌旁组织,应用Real-time PCR技术检测DNMT1、DNMT3A、DNMT3B与DNMT3B六种主要异构体的mRNA表达,应用Western blot法检验DNMT3B4蛋白表达;应用联合亚硫酸氢钠的限制性内切酶分析法(COBRA)分析重复序列Alu和LINE-1的甲基化水平。结果透明细胞肾细胞癌组织中DNMT3B4 mRNA和蛋白表达水平比癌旁组织高;重复序列Alu和LINE-1的甲基化水平在透明细胞肾细胞癌组织中比癌旁组织低。结论 DNMT3B4表达增加可能与整体甲基化降低及透明细胞肾细胞癌的发生有密切关系。     

High expression of long non-coding RNA SPRY4-IT1 predicts poor prognosis of clear cell renal cell carcinoma

Introduction: Long non-coding RNAs (lncRNAs) play a key role in cellular processes, such as cell growth, apoptosis, and carcinogenesis. lncRNAs SPRY4-IT1 has recently been identified to be involved in tumorigenesis of several cancers such as non-small cell lung cancer and esophageal squamous cell carcinoma. However, the role of SPRY4-IT1 in clear cell renal cell carcinoma (ccRCC) remains unclear. Methods: The expression of SPRY4-IT1 was examined in ccRCC patients and renal cancer cell lines by using quantitative real-time PCR (qRT-PCR). The relationship between SPRY4-IT1 level and clinicopathological parameters of ccRCC was analyzed with the Kaplan-Meier method and Cox proportional hazards model. Small interfering RNA (siRNA) was used to suppress SPRY4-IT1 expression in renal cancer cell line 786-O. In vitro assays were performed to further explore its role in renal cancer progressio. Results: The relative level of SPRY4-IT1 was significantly higher in ccRCC tissues compared to the adjacent normal renal tissues. And higher expression of SPRY4-IT1 was found in renal cancer cell lines compared with the normal human proximal tubule epithelial cell line HK-2. The ccRCC patients with higher SPRY4-IT1 expression had an advanced clinical stage and poorer prognosis than those with lower SPRY4-IT1 expression. Multivariate analyses by Cox’s proportional hazard model revealed that expression of SPRY4-IT1 was an independent prognostic factor in ccRCC. In vitro assays, our results indicated that knockdown of SPRY4-IT1 reduced renal cancer cell proliferation, migration, and invasion. Conclusions: Our data suggested that lncRNA SPRY4-IT1 might be considered as a potential prognostic indicator and a potential target for therapeutic intervention in RC.     

The post-operative pathological prognostic parameters of clear cell renal cell carcinoma in pT1a cases

There has been a recent increase in the number of small clear cell renal cell carcinoma (ccRCC) cases, particularly in pT1a cases. The prognostic parameters for small ccRCC, however, are not well described. Herein, we assess the pathological parameters of pT1a patients. Various clinicopathological parameters were analyzed in 293 patients with pT1a ccRCC without pre-operative metastasis to predict the disease-free survival rate (DFS) and the cancer-specific survival rate (CSS). Clinicopathological parameters included age, tumor location, Fuhrman grade, lymph-vascular invasion (LVI), tumor necrosis, and growth pattern (expansive or infiltrative). In the univariate analysis, Fuhrman grade (grade 1 + 2 vs. 3 + 4), LVI, growth pattern, and tumor necrosis were parameters associated with a worse prognosis (P < 0.0001) in both the DFS and CSS. In the multivariate analysis, Fuhrman grade (P = 0.0048), growth pattern (P = 0.0275), and tumor necrosis (P = 0.0188) were statistically significant in the DFS. Fuhrman grade (P = 0.0189) and growth pattern (P = 0.0016) were also statistically significant in the CSS. Fuhrman grade, tumor necrosis, and growth pattern were independent prognostic parameters in pT1a ccRCC. Growth pattern, a previously unrecognized parameter for prognosis, can be considered a new prognostic parameter in ccRCC.     

Downregulated Rac1 promotes apoptosis and inhibits the clearance of apoptotic cells in airway epithelial cells,which may be associated with airway hyper‐responsiveness in asthma

The accumulation of airway apoptotic cells may be an important factor causing airway hyper‐responsiveness (AHR). Whether the apoptotic cells can be promptly removed is related to the occurrence and course of asthma. In recent years, studies have shown that Rac1 is involved in many cellular biological activities including the formation and elimination of apoptotic cells. In this study, based on the analysis of airway local cells and related factors in asthmatic mice, we evaluated the expression of Rac1 in airway epithelial cells or phagocytes and analysed its relationship with the incidence of apoptosis or scavenging of apoptotic cells. Our data showed that the expression level of Rac1 in asthmatic mice decreased significantly, while the expression of IL‐33 increased obviously. The airway epithelial cell line was stimulated by curcumin at 50 μmol/L for 24‐48 hours; more than 50% of the cells were apoptotic, and of which, about 20% were late apoptosis. Rac1 inhibitor (NSC23766) can enhance the apoptosis effect. In addition, the ability of phagocytosis and migration in the epithelial cells or macrophages was increased following the application of Rac1 inhibitors or specific siRNA in a dose‐dependent manner, and the expression level of IL‐33 was simultaneously increased after blocking Rac1. It is suggested that the down regulation of Rac1 in asthma may contribute to the apoptosis of airway epithelial cells and affect the clearance of apoptotic cells, which will lead to the aggregation of the apoptotic cells in the respiratory tract and participate in AHR.     

ERK activation of p21 activated kinase-1 (Pak1) is critical for medulloblastoma cell migration

We previously identified that overexpression of the platelet-derived growth factor receptor (PDGFR) is associated with metastatic medulloblastoma (MB) and showed that PDGF treatment increases ERK activity and promotes MB cell migration. In this study, we investigated whether ERK regulates Rac1/Pak1 signaling and is critically linked to MB cell migration. Herein we demonstrate that PDGF-BB treatment of MB cells induces concomitant activation of PDGFRβ, MEK1/ERK, Rac1 and Pak1, but suppresses Rho activity, which together significantly promotes cell migration. Conversely, cells transfected with either PDGFRβ or Pak1 siRNA or treated with an inhibitor of Rac1 (NSC23766) or N-myristoyltransferase-1 (Tris-dipalladium) are unable to activate Rac1 or Pak1 in response to PDGF, and consequently, are unable to undergo PDGF-mediated cell migration. Furthermore, we also demonstrate that either chemical inhibition of MEK/ERK (U0126) or stable downregulation of PDGFRβ by shRNA similarly results in the loss of PDGF-induced ERK phosphorylation and abolishes Rac1/Pak1 activation and cell migration in response to PDGF. However, specific depletion of Pak1 by siRNA has no effect on PDGF-induced ERK phosphorylation, indicating that in MB cells ERK signaling is Pak1-independent, but PDGF-induced migration is dependent on ERK-mediated activation of Pak1. Finally, using tissue microarrays, we detect phosphorylated Pak1 in 53% of medulloblastomas and show that immunopositivity is associated with unfavorable outcome. We conclude that Rac1/Pak1 signaling is critical to MB cell migration and is functionally dependent on PDGFRβ/ERK activity.     

DRAM1 plays a tumor suppressor role in clear cell renal cell carcinoma through modulating Akt signaling

BackgroundClear cell renal carcinoma (ccRCC) is one of the most common malignant tumors worldwide. DNA damage-regulated autophagy modulator1 (DRAM1) plays an important roles in apoptosis and tumor progression. However, the role of DRAM1 in ccRCC is still unknown. In our study, we aimed to investigate the effect of DRAM1 in the progression of ccRCC.MethodsThe expression and prognostic information of DRAM1 in ccRCC were obtained by immunohistochemistry staining and bioinformatics database. Cell proliferation, migration, invasion were detected by CCK-8 assay, wound-healing and transwell assays, and the cell apoptosis was examined by tunel assay and flow cytometry analysis. Western blot was used to detect the expression of DRAM1, Bax, Bcl2, Akt, p53,E-cadherin, N-cadherin of ccRCC cells.ResultsDecreased expression of DRAM1 was found in ccRCC tissues, which predicted a shorter survival rate in ccRCC patient. We confirmed that DRAM1 inhibited the proliferation, migration, invasion and epithelial mesenchymal transformation (EMT), while enhanced the apoptosis of ccRCC cells. In addition, the results of inhibition of Akt signaling were consistent with the above. We further proved that DRAM1 over-expression decreased the phosphorylation of Akt signaling, and overexpression of DRAM1 could reverse oncogenic function induced by the over-activating of Akt in ccRCC cells.Conclusionoverexpression of DRAM1 plays a tumor suppressive role in ccRCC through inactivation of Akt and highlights the potential role of DRAM1 as a prognostic biomarker in ccRCC.     

Characteristics and prognostic value of potential dependency genes in clear cell renal cell carcinoma based on a large-scale CRISPR-Cas9 and RNAi screening database DepMap

Background: Large-scale loss-of-function screening database such as Cancer Dependency Map (Depmap) provide abundant resources. Investigation of these potential dependency genes from human cancer cell lines in the real-world patients cohort would evaluate their prognostic value thus facilitate their clinical application and guide drug development.Methods: A few genes were selected from top clear cell renal cell carcinoma (ccRCC) lineage preferential dependency candidates from Depmap. Their characteristic including expression levels both in normal and tumor tissues and correlations with methylation or copy number, genetic alterations, functional enrichment, immune-associated interactions, prognostic value were evaluated in KIRC cohort from TCGA, GTEx, and multiple other open databases and platforms.Results: 16 genes were collected from 106 ccRCC preferential candidates and further analyzed including B4GALT4, BCL2L1, CDH2, COPG1, CRB3, FERMT2, GET4, GPX4, HNF1B, ITGAV, MDM2, NFE2L2, PAX8, RUVBL1, TFRC, and TNFSF10. The normalized gene effect scores of these genes varied from different ccRCC cell lines and principal component analysis (PCA) showed their tissue specificity expression profiles. Genetic alteration rates of them were low to moderate (0.7%-13%) in KIRC cohort. CDH2, MDM2, TNFSF10 showed a statistically significant higher level in tumors than normal tissues while PAX8 and FERMT2 were significantly downregulated. Moderate positive or negative correlations were observed in several genes between their expression and relative gene copy number or methylation levels, respectively. Based on the multivariable COX regression model adjusted by critical clinical variables revealed the expression of GET4 (p=0.002, HR=1.023 95%CI 1.009-1.038) and CRB3 (p<0.001, HR=0.969 95%CI 0.960-0.980) were independent predictive factors for overall survival in KIRC cohort.Conclusions: A dependency gene validated in cell lines didn''t directly represent its role in corresponding patients with same histological type and their prognostic value might be determined by multiple factors including dependency driven types, genetic alteration rates and expression levels. GET4 and CRB3 were the independent prognostic factors for ccRCC patients. CRB3 seemed like a potential broad tumor suppressor gene while GET4 might be a ccRCC preferential dependency gene with a ligandable structure.     

Synchronous clear cell renal cell carcinoma and clonal plasmacytoid cell infiltration: A case report and literature review

Recent studies of tumor microenvironments have revealed that clonal B cells reacting to tumor-derived antigens play an important role in anti-tumor immunity. We report a case of a 72-year-old Japanese man with a complaint of fever for 1 month. Computed tomography revealed a 48 mm mass in his right kidney. The patient underwent a right nephrectomy and histology revealed clear cell renal cell carcinoma (ccRCC) of Fuhrman Grade 4 with rhabdoid morphology. Focally, marked plasmacytoid cell infiltration was detected in the carcinoma. These plasmacytoid cells were immunohistochemically positive for immunoglobulin (Ig) G, and kappa light chain restriction was confirmed using mRNA in situ hybridization. Programmed death-ligand 1 (PD-L1) immunostaining and RNA in situ hybridization of transforming growth factor beta (TGF-β) revealed that both PD-L1 and TGF-β were highly expressed in the area with clonal plasmacytoid cell infiltration. The patient developed bone metastasis 3 months after surgery, and plasmacytoma was not detected during the observation period. We identified a potential link between an immunosuppressive microenvironment and clonal B cell proliferation. The latter posed a differential diagnosis problem between reactive and neoplastic clonal B cell proliferation vis-à-vis a plasmacytoma complicating carcinoma.     

Epidermal growth factor receptor: a novel biomarker for aggressive head and neck cutaneous squamous cell carcinoma

There is currently no prognostic tool that reliably predicts the risk of metastasis in cutaneous squamous cell carcinoma, most of which occur in the head and neck region. Epidermal growth factor receptor has received much interest in recent years with the advent of epidermal growth factor receptor-targeted molecular therapy in clinical oncology. We investigate the role of epidermal growth factor receptor as a biomarker for head and neck cutaneous squamous cell carcinoma. Using immunohistochemistry and fluorescence in situ hybridization, we assessed the epidermal growth factor receptor protein expression and gene copy in 3 groups of head and neck cutaneous squamous cell carcinoma: primary lesions not associated with metastasis (P), primary lesions associated with subsequent metastasis (PM), and metastatic nodal disease (M). Epidermal growth factor receptor overexpression was detected in 36% and 79% of P and PM cases, respectively. Epidermal growth factor receptor overexpression was significantly associated with PM (P = .03) and was found to be an independent prognostic factor for metastasis on multivariate analysis (P = .05). However, epidermal growth factor receptor overexpression was only maintained in 47% of cases in the M group. None of the 27 cases that overexpressed the epidermal growth factor receptor protein showed gene amplification: the results were uninterpretable in 2, and polysomy and balanced disomy were detected in 5 and 20 cases, respectively. These observations may have important prognostic and therapeutic implications for head and neck cutaneous squamous cell carcinoma.