A processed HLA-A*24:02 pseudogene found in the peripheral blood of a father and his son

We encountered a case that exhibited a discrepancy in human leukocyte antigen-A (HLA-A) type determined by sequence-based typing (SBT) and sequence-specific primer (SSP) molecular typing. The child of this case was identified as A* 02:01 homozygote and A* 02, A* 24, respectively. The HLA-A type of his father was A* 02:01, 26:01, but low-resolution SSP also showed unexpected amplification with A* 24 primers as with the child. Serologic typing of the child and the father was A2/blank and A2/A26, respectively. Sequencing analysis of the A* 24 variant in the child and the father showed a complete deletion of all introns of the A* 24:02 allele. Though rare, this type of processed pseudogene variant can be one of the causes of discrepancies between high- and low-resolution HLA typing.     

Biomarkers of laryngeal squamous cell carcinoma: a review

Laryngeal carcinoma is the second common malignancy of the upper aerodigestive tract after lung cancer; in most cases is a squamous cell carcinoma, whose risk factors include tobacco smoking and alcohol consumption. Despite therapeutic progress, the five-year overall survival rate for this malignancy has remained nearly 50% and many patients already present metastasis at the time of diagnosis.To date, there are no tools that predict the evolution of laryngeal carcinoma: in this light, during the last years, many studies were planned with the aim to investigate the role played by different biomarkers expressed by larynx cancer, which can help make an early diagnosis, predict disease evolution and direct therapeutic choice. This review aims to summarize these markers and correlating them with disease evolution.     

FHIT在喉癌中的研究进展

［摘要］ 脆性组氨酸三联体基因（fragile histidine triad， FHIT）是位于3p14.2上的抑癌基因，它可以通过水解Ap3A、改变线粒体跨膜电位及增强微管蛋白聚合来诱导细胞凋亡。FHIT的改变包括纯合性缺失、微卫星不稳定(MSI)、杂合性缺失（LOH）及启动子甲基化在喉鳞状细胞癌中有非常重要的作用。FHIT基因可能成为喉鳞状细胞癌的基因治疗靶点     

Molecular biology of laryngeal squamous cell carcinoma

Some of the mechanisms involved in neoplastic transformation and progression of laryngeal squamous cell carcinoma (LSCC) are discussed. Although tumor suppressor inactivation of p53 and p16 is common in these tumors (about 50% each), oncogenic activation is less well characterized. Cyclin D1 and epidermal growth factor receptor amplification have been reported in one-third and one-quarter of LSCCs, respectively, both related to advanced stages, whereas c-myc could be amplified in 13% of cases although without associated overexpression. The role of ras in LSCC is, at most, exceptional, and the role of human papillomavirus infection in these neoplasms could have been largely overestimated. The AIS (amplified in squamous carcinoma) gene has been recently proposed as the main oncogenic target in head and neck squamous carcinomas and is a promising line of investigation. This, along with the link that exists between p53 and INK4 suppressor pathways through ARF and MDM-2, and the role of the universal cdk inhibitors (the Cip/Kip family) in these neoplasms deserve further investigation. Not forgotten are the mechanisms leading to cell immortalization and invasive capabilities acquisition, some of which are also briefly described.     

Characterization of homozygous deletions in laryngeal squamous cell carcinoma cell lines

The majority of classical tumor suppressor genes, such as CDKN2A or RB1, were identified by delineation of biallelic losses called homozygous deletions. To systematically identify homozygous deletions in laryngeal squamous cell carcinoma and to unravel novel putative tumor suppressor genes we screened three laryngeal squamous cell carcinoma cell lines (LSCC) using array comparative genomic hybridization (array-CGH). Out of 31 candidate regions for homozygous deletions identified by array-CGH, 5 were verified further by PCR. Among others, these homozygous deletions affected the tumor suppressor gene CDKN2A and the apoptosis-inducing STK17A gene. To assess the frequency of the identified deletions we investigated the affected sites in 9 additional LSCC cell lines. In 5 of the 9 cell lines the CDKN2A gene was homozygously lost. Thus, CDKN2A was homozygously deleted in 7 of the 12 cell lines. No other recurrent homozygous deletions were found. Homozygous deletions was a frequent mechanism of CDKN2A inactivation. Moreover, we identified several other genes, including the putative tumor suppressor gene STK17A, which may be inactivated by homozygous deletions and thus are potentially implicated in laryngeal squamous cell carcinoma development.     

Biochemical changes of extracellular proteoglycans in squamous cell laryngeal carcinoma

Larynx is a complicated organ with peculiar properties, having a noticeable impact in vocal and respiratory physiology. In squamous cell laryngeal carcinoma, the extracellular matrix components underwent significant modifications concerning their fine chemical structure. Degradation of aggrecan is observed, whereas versican and decorin amounts are increased. The expression of aggrecan is almost totally ceased in later cancer stages, whereas decorin is expressed in normal and cancerous samples. But its expression is increased in cancer, being related to cancer stage. However, the expression of versican seems to be characteristic of the tumor, since none or traces expression is observed in normal samples. Chondroitin/dermatan sulfate is the major glycosaminoglycan, but its sulfation shows a shift from C6 position of galactosamine in normal samples to C4 in malignancy. Dermatan sulfate represents minor amounts in normal samples but increases in proportion up to one-fourth of total sulfated glycosaminoglycans in malignancy. In addition, an increase in the amounts of hyaluronan is also observed in malignant samples. Accumulated data demonstrate that tumor progression is closely related to the alteration of the expression and biochemical composition of specific extracellular constituents that describes the mild aggressive phenotype of squamous cell laryngeal carcinoma.     

Annexin A1 subcellular expression in laryngeal squamous cell carcinoma

Aims: Annexin A1 (ANXA1) is a soluble cytoplasmic protein, moving to membranes when calcium levels are elevated. ANXA1 has also been shown to move to the nucleus or outside the cells, depending on tyrosine‐kinase signalling, thus interfering in cytoskeletal organization and cell differentiation, mostly in inflammatory and neoplastic processes. The aim was to investigate subcellular patterns of immunohistochemical expression of ANXA1 in neoplastic and non‐neoplastic samples from patients with laryngeal squamous cell carcinomas (LSCC), to elucidate the role of ANXA1 in laryngeal carcinogenesis. Methods and results: Serial analysis of gene expression experiments detected reduced expression of ANXA1 gene in LSCC compared with the corresponding non‐neoplastic margins. Quantitative polymerase chain reaction confirmed ANXA1 low expression in 15 LSCC and eight matched normal samples. Thus, we investigated subcellular patterns of immunohistochemical expression of ANXA1 in 241 paraffin‐embedded samples from 95 patients with LSCC. The results showed ANXA1 down‐regulation in dysplastic, tumourous and metastatic lesions and provided evidence for the progressive migration of ANXA1 from the nucleus towards the membrane during laryngeal tumorigenesis. Conclusions: ANXA1 dysregulation was observed early in laryngeal carcinogenesis, in intra‐epithelial neoplasms; it was not found related to prognostic parameters, such as nodal metastases.     

p53 Overexpression in laryngeal squamous cell carcinoma and dysplasia

Aim—To investigate the expression of p53 protein in invasive squamous cell carcinoma (SCC) of the larynx and dysplasia in relation to histological grade and tobacco smoking.     

人乳头瘤病毒阴性的喉鳞癌细胞系的建立

目的 建立1株人乳头状瘤病毒（HPV）阴性的喉鳞状细胞癌细胞系,为体外研究喉癌提供理想的实验模型。方法 以逆转录聚合酶链反应（RT-PCR）证实为HPV阴性的高分化喉鳞癌手术切除标本接种于裸鼠皮下,取连续传代2次的裸鼠皮下移植瘤进行体外原代培养。通过光镜、电镜、生长曲线、细胞周期时相、软琼脂克隆实验、异种移植成瘤实验、角蛋白、癌胚抗原及HPV检测,对其生物学特性进行初步分析。结果 经裸鼠过渡所建立的高分化喉鳞癌细胞系（Lscc-02）目前已传至86代,细胞生长增殖稳定。该细胞系呈单层形式生长,群体倍增时间为39．1h。透射电镜下见胞质内典型的张力原纤维,细胞问以桥粒方式连接。染色体为人类核型,呈亚三倍体,众数分布在63～72。该细胞系具有恶性肿瘤细胞生长特征：软琼脂中形成克隆,裸鼠接种成瘤且形态结构、分化程度与原发瘤相似。免疫组织化学显示高分子量细胞角蛋白及癌胚抗原阳性,PCR显示HPV阴性。结论 建立Lscc-02细胞系为研究无HPV感染喉癌的发生、发展规律及HPV与喉癌演进的关系提供了有价值的体外模型。     

喉鳞状细胞癌患者人乳头瘤病毒感染的检测分析

目的 了解喉鳞状细胞癌患者人乳头瘤病毒感染的情况.方法 对收集到的64例临床诊断喉癌病例的石蜡组织标本应用Luminex及PCR的方法对HPV感染进行基因检测,应用免疫组织化学的方法对HPV16/18E6蛋白进行检测.结果 在64例临床诊断的喉癌病例中,通过Luminex及PCR的方法发现7例病例具有HPV的感染;通过免疫组织化学的方法发现18例病例具有HPV16/18的感染,基因及蛋白检测的总阳性率达到39.1％.结论 此研究发现在喉癌患者中较高的HPV感染率间接说明HPV感染对喉癌发生的重要性,为阐明喉癌的发病机制奠定基础.     

HLA-B蛋白在喉癌中的表达及意义

目的研究人白细胞抗原HLA-B蛋白在喉癌组织中的表达及意义。方法应用免疫组化方法检测HLA-B蛋白在50例喉癌组织及癌旁组织的表达,应用Western Blot方法检测HLA-B蛋白在6对喉癌及癌旁组织中的表达。结果 HLA-B蛋白在癌旁正常组织中100%表达,而在喉癌组织中表达水平明显下调,有淋巴结转移组下调明显低于无淋巴结转移组。结论 HLA-B蛋白在喉癌组织中表达下调,可能与颈淋巴结转移相关。     

Spondin-2 is a novel diagnostic biomarker for laryngeal squamous cell carcinoma

Spondin-2, belongs to the SOX (SRY-related HMG box) gene family, plays a vital role in the development of malignancy, however, the role of Spondin-2 in laryngeal squamous cell carcinoma (LSCC) remains unknown. The aim of this study is to investigate the prognostic significance of and probable mechanism of Spondin-2 in LSCC. qRT-PCR, western blotting assays and IHC analysis demonstrated that Spondin-2 was significantly increased in LSCC tissues compared with adjacent non-tumorous tissues. In addition, high levels of Spondin-2 was associated with clinical stage, lymph node metastasis and pathology grade of LSCC patients (P ＜0.05). Kaplan-Meier analysis showed that patients with high expression of Spondin-2 had a lower overall survival rate (P＜0.05) than that with low expression of Spondin-2. Moreover, spondin-2 silencing inhibited the proliferation of LSCC cells through inhibiting the activation of PI3K/AKT signaling. In conclusion, spondin-2 might be a novel therapeutic target and prognostic biomarker for LSCC patients.     

Association between human papillomavirus infection and laryngeal squamous cell carcinoma

The aim of this study was to compare the prevalence of human papillomavirus (HPV) infection in laryngeal squamous cell carcinoma using two methods: PCR‐DNA enzyme immunoassay (PCR/DEIA) and immunohistochemistry (IHC) for detection of HPV in specimens of laryngeal squamous cell carcinoma and to correlate the presence of HPV with the epidemiological and clinicopathological features of recurrence and survival. HPV DNA was amplified from 93 paraffin‐embedded laryngeal squamous cell carcinoma tissue specimens by the short PCR fragment (SPF 10) primer set using PCR/DNA method. HPV detection using monoclonal anti‐human papilloma virus antibodies Clone K1H8 for IHC reaction was performed on 130 specimens. HPV was identified in 35.5% of patients with laryngeal squamous cell carcinoma using PCR/DEIA and 27.7% using IHC. There was no statistically significant association between the presence of HPV and the epidemiological and clinicopathological features and recurrence. There was no statistically significant association between the presence of HPV and overall survival nor disease specific survival. Statistically significant correlation between HPV detection using PCR/DEIA technique and IHC technique was found. The presence of HPV infection in 27.7% and 38.9% of the patients suggests a possible role in the etiology of laryngeal squamous cell carcinoma. The SPF₁₀ PCR/DEIA technique is the most accurate method for detection of HPV in laryngeal squamous cell carcinoma. J. Med. Virol. 82:1017–1023, 2010. © 2010 Wiley‐Liss, Inc.     

喉鳞状细胞癌中MTA2的表达及预后分析

目的探讨转移相关蛋白2(metastasis-associated protein 2,MTA2)在喉鳞状细胞癌组织中的表达,分析其与临床病理特征和患者预后的关系。方法采用免疫组化SP法检测120例喉石蜡包埋组织中MTA2蛋白的表达,采用Western blot法检测喉新鲜组织中MTA2蛋白的表达量,利用Kaplan-Meier法绘制总生存期曲线,Log-rank方法分析两组生存曲线差异,Cox比例风险回归模型筛选喉鳞状细胞癌患者的预后影响因素。结果MTA2在正常喉组织、高级别鳞状上皮内病变和喉鳞状细胞癌组织中的阳性率分别为6.67%、43.33%和73.33%,差异有统计学意义(χ^2=36.11,P<0.001),MTA2表达与喉鳞状细胞癌组织学分级、临床分期及淋巴结转移显著相关(P均<0.05);在正常喉组织、高级别鳞状上皮内病变及喉鳞状细胞癌组织中MTA2的相对表达量分别是0.98±0.07、1.30±0.07和1.46±0.11,差异有统计学意义(F=121.194,P<0.001);Kaplan-Meier生存分析提示MTA2表达与喉鳞状细胞癌患者的生存期显著相关(P<0.05);多因素回归分析表明MTA2蛋白表达、临床分期及淋巴结转移是喉鳞状细胞癌患者有效的独立预后指标(P均<0.05)。结论MTA2过表达在喉鳞状细胞癌发生、进展过程中发挥重要作用,可作为喉鳞状细胞癌独立的预后因素,MTA2有望成为指导喉鳞状细胞癌治疗和判断患者预后的新分子学标志物。     

PDCD4在喉癌中的表达及意义

目的研究PDCD4在喉癌组织中的表达及意义。方法应用免疫组化方法检测PDCD4蛋白在喉癌及癌旁组织中的表达。应用Real-time PCR方法检测PDCD4 mRNA在30例喉癌组织及癌旁正常组织中的表达,并与临床资料进行统计学分析。结果与癌旁组织相比,PDCD4蛋白在喉癌组织中表达水平明显降低,PDCD4 mRNA在喉癌组织中表达水平明显下调(0.05,t=4.714),与分期、分化及淋巴结转移明显相关,但与年龄、性别及分型不相关。结论 PDCD4在喉癌组织低表达,与分期、分化及淋巴结转移相关。     

Expression of paxillin in laryngeal squamous cell carcinoma and its prognostic value

Paxillin (PXN) gene has been reported to act as an oncogene in many malignancies and play important roles in the development of human carcinomas. However, the relationship between the expression of PXN and clinicopathological characteristics in human laryngeal carcinoma remains unclear. This study aimed to examine the expression of PXN, and to evaluate the clinical significance of its expression in human laryngeal squamous cell carcinoma (LSCC). Real-time quantitative PCR (qRT-PCR), Western blotting and immunohistochemistry were performed to analyze the expression of PXN in LSCC tissues and corresponding paracancerous normal tissues. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognosis of patients with LSCC. The expression of PXN was significantly higher in LSCC than in matched paracancerous normal tissues. Immunohistochemical analysis was performed in human LSCC samples and the data were correlated with clinicopathologic features. Levels of PXN in LSCC were related to histopathological grade (P = 0.016), Lymph node metastasis (P = 0.029) and TNM stage (P < 0.001). Kaplan-Meier analysis revealed that survival curves of the overall survival of patients with high PXN expression was significantly worse than that of low PXN expression (P = 0.035). Cox regression analysis revealed that PXN expression level was an independent prognostic factor of the overall survival rate of patients with LSCC (P = 0.002). These findings suggest that PXN expression has potential use as a novel biomarker of LSCC patients and may serve as an independent predictive factor for prognosis of LSCC patients.     

Immunohistochemical analysis of pro- and active-caspase 3 in laryngeal squamous cell carcinoma

Active caspase 3 is considered to be the main executioner caspase in apoptotic process. The mechanisms of apoptosis in laryngeal squamous cell carcinoma (LSCC) have been investigated by examining the expression profiles of pro-caspase 3 and active-caspase 3. The correlation between the two forms of caspase 3 and the p53 status was also determined. LSCCs (n=65) were studied using immunohistochemistry with antibodies to pro-caspase 3, active-caspase 3 and p53. The expression of pro-caspase 3 was absent or weak in 16 (24.6%), moderate in 21 (32.3%) and strong in 28 (43.1%) cases. Survival curves for different levels of pro-caspase 3 differed, but the differences were not statistically significant. An apoptotic index (AI) was determined by quantifying the active-caspase 3-positive cells. The AI ranged from 0.2% to 9.4% and did not differ among the different levels of pro-caspase 3 expression. Even in cases in which the expression of pro-caspase 3 was considered negative, caspase 3-positive apoptotic cells were found. The AIs were significantly higher in supraglottic tumours compared with glottic counterparts (P=0.008) and were higher in poorly differentiated tumours compared with well-differentiated and moderately differentiated LSCC (P=0.06). No correlation between AI and p53 expression was found, although pro-caspase 3 expression trended to be higher in the p53-positive group of LSCC. Our results suggest that the expression of pro-caspase 3, a key executioner caspase in apoptosis, is downregulated in a proportion of LSCC, but this is not associated with decreased apoptotic activity, measured by active-caspase 3 labelling.