The role of molecular analyses in the diagnosis and treatment of non-small-cell lung carcinomas

Non-small-cell lung cancer (NSCLC) subtyping has recently been a key factor in determining patient management with novel drugs. In addition, the identification of distinct oncogenic driver mutations frequently associated with NSCLC histotype and coupled to the clinical responses to targeted therapies have revolutionized the impact of histologic type and molecular biomarkers in lung cancer. Several molecular alterations involving different genes (EGFR, KRAS, ALK, BRAF, and HER2) seem to have a remarkable predilection for adenocarcinoma and specific inhibitors of EGFR and ALK are now available for patients with adenocarcinoma harboring the relevant gene alterations. The efficacy of histology-based and molecular-targeted therapies had a deep impact in (1) re-defining classification of lung cancer (particularly adenocarcinomas) and (2) routine clinical practice of pathologists involved in optimization of handling of tissue samples in order to guarantee NSCLC subtyping with the help of immunohistochemistry and adequately preserve tumor cells for molecular analysis. In agreement with the modern multidisciplinary approach to lung cancer, we reviewed here the diagnostic and predictive value of molecular biomarkers according to the clinical, pathologic, and molecular biologist viewpoints.     

抗鼠科肉瘤病毒癌基因同源物B1突变与甲状腺乳头状癌淋巴结转移的关系研究进展

近年来随着检查设备敏感性的提高及人们生活、饮食环境的改变,甲状腺癌(TC)的发病率直线上升,10年内增加了近5倍,不同病理类型甲状腺癌发病机制、临床表现、生物学行为、治疗方法和预后有明显不同。甲状腺乳头状癌(PTC)任何年龄均可发病,约占甲状腺癌的90%,疾病进展缓慢,预后良好,但易出现淋巴结转移。是否伴有淋巴结转移对于PTC的预后转归有着重要影响,抗鼠科肉瘤病毒癌基因同源物B1(BRAF)基因突变在PTC的淋巴结转移中发挥着至关重要的作用,深入了解BRAF基因突变在PTC的具体作用及机制,有望对PTC提供新的诊疗思路。     

中国人非小细胞肺癌EGFR和K-RAS基因突变情况的研究

目的研究中国人非小细胞肺癌表皮生长因子受体(epidermal growth factor receptor,EGFR)基因和K-RAS基因突变情况。方法通过PCR扩增和基因测序的方法检测了101例中国人非小细胞肺癌(non-small cell lung cancers,NSCLCs)EGFR第18、19和21外显子及K-RAS密码子12、13的突变情况,并观察分析了其突变与肺癌临床特征及吉非替尼(gefitinib,商品名:易瑞沙/Iressa)药物治疗肺癌的疗效间的关系。结果共检测到26例EGFR突变(25.7%),3例K-RAS突变(2.9%),并发现腺癌患者、非吸烟患者和女性患者EGFR突变率较高(分别为44.2%、65.7%和48.3%)。10例服用吉非替尼有效的患者9例伴有EGFR突变。结论中国人肺癌的EGFR突变率高于西方人,吉非替尼疗效与EGFR突变有关。     

An immunohistochemical and molecular genetic analysis of KIT and PDGFRA in small cell lung carcinoma in Japanese

KIT and PDGFRA in small cell lung carcinoma (SCLC) have been rarely examined in Japanese. The author investigated protein expression of KIT and PDGFRA in 54 Japanese cases of small cell lung carcinoma by immunohistochemistry, and gene mutations of KIT and PDGFRA in 20 Japanese cases of small cell lung carcinoma by the PCR-direct sequencing method. The molecular genetic analysis showed no mutations of KIT (exons 9, 11, 13, and 17) and PDGFRA (exons 12 and 18) genes in all 20 cases. KIT protein expression was recognized in all cases (100%). Membranous KIT expression was strong in 35 cases, moderate in 7 cases and weak in 12 cases. PDGFRA protein expression was noted in 35 cases (65%); the membranous expression was strong in 2 cases, moderate in 16 cases, and weak in 17 cases. The overall median survival was 13 months. There was no significant difference in the survival between KIT strongly positive cases (median, 12 months) and KIT moderately or weakly positive cases (median, 11 months). Likewise, there was no significant difference in the survival between PDGFRA-positive cases (median, 11 months) and PDGFRA-negative cases (median, 12 months). The protein expressions of KIT and PDGFRA did not correlate with gender, smoking, and disease stage. These findings suggest, in Japanese population, that mutations of KIT and PDGFRA were absent in small cell lung carcinoma of Japan, that KIT protein expression is present in 100%, that PDGFRA expression is present in 65%, and that KIT and PDGFRA protein expressions do not correlate with survival, gender, smoking, and disease stage.     

非小细胞肺癌EGFR突变检测技术的研究进展

针对表皮生长因子受体(epidermal growth factor receptor,EGFR)突变基因的检测是肺癌靶向治疗的重要基石.目前临床中主要针对肺癌组织、脱落细胞以及液态活检标本开展检测工作,此外尿液标本近期也展现出良好的检测效率.针对上述标本,临床中开展多种检测技术,如以特定靶点为目的的技术,包括数字PCR、楔形探针扩增阻滞突变系统等;以筛查为目的的检测技术,包括二代测序(next generation sequencing,NGS)、Sanger测序等,这些技术在特定的标本类型和检测目的中具有独特的优势.一些新兴的技术,如拉曼光谱,也在临床检测中展现出良好的应用前景.     

非小细胞肺癌组织表皮生长因子受体基因第19外显子突变研究

目的 研究非小细胞肺癌的表皮生长因子受体(epidermal growth factor receptor,EGFR)基因第19外显子突变情况.方法 提取72例手术切除肺癌基冈组DNA,用巢式PCR扩增EGFR基因第19外显子并测序,分析EGFR基因第19外显子突变及其与I临床特征的相关性.结果 检测到13例肿瘤组织存在EGFR基因第19外显子多核苷酸的缺失突变,共有4种突变类型,突变率为18.1%,所检测的突变均为杂合型;EGFR基因第19外显子突变与肺腺癌、女性患者与不吸烟者相关.结论 EGFR基因第19外显子存在多核苷酸的框架缺失突变,以女性、腺癌和不吸烟患者为主.     

非小细胞肺癌表皮生长因子受体突变的检测及临床病理意义

目的检测非小细胞肺癌患者体细胞表皮生长因子受体（EGFR）基因第19和21外显子突变情况并探讨其临床病理联系。方法用酚．氯仿法抽提66例NSCLC患者手术标本的基因组DNA,采用PCR技术扩增EGFR基因第19和21号外显子,从正反两个方向对扩增片段进行DNA测序和分析,并寻找EGFR突变与患者115床病理特征之间的联系。结果66例NSCLC患者中有11例（16．7％）存在EGFR杂合性体细胞突变,其中7例为第19外显子缺失突变,4例为第21外显子替代突变。女性患者突变率（9／34,26,5％）高于男性患者突变率（2／32,6．3％）,腺癌患者突变率（10／43,23,3％）高于鳞癌（0／13）和腺鳞癌患者（1／10）,吸烟者与非吸烟者突变率之间差异无统计学意义。伴细支气管肺泡癌成分的腺癌患者EGFR突变率（6／11）,高于无细支气管肺泡癌成分的腺癌患者（4／32,12．5％）。结论EGFR突变率以伴细支气管肺泡癌成分的腺癌和女性较高,更有利于适宜靶向治疗患者的临床筛选。     

Clonality analysis of different histological components in combined small cell and non-small cell carcinoma of the lung

Combined small cell and non-small cell carcinoma is relatively rare in the lung. Examination of the clonal relationship of different components in this type of tumor may give a clue to the rarity. We retrieved 6 such tumors; all 6 had small cell carcinoma and adenocarcinoma components, and 3 had an additional squamous cell carcinoma component. We examined the point mutations in the p53 gene and allelic loss (ie, the loss of heterozygosity [LOH] pattern) of chromosome 3p in each component. p53 mutations were detected in the small cell carcinoma component of 5 tumors and in the non-small cell carcinoma components of 2 tumors. In 1 case, the squamous cell carcinoma component had a p53 mutation locus identical to that in the small cell carcinoma component, but in the other case, the adenocarcinoma component had a different mutation than that in the small cell carcinoma component. Chromosome 3p LOH loci in the squamous cell carcinoma component were present in the small cell carcinoma component in all 3 cases, but some LOH loci were not identical in the small cell carcinoma and adenocarcinoma components in 3 cases. These results suggest that the small cell and squamous cell carcinoma components of combined small cell lung carcinomas have an intimate clonal relationship. On the other hand, the adenocarcinoma component often may be derived from a separate clone or, more likely, undergo a progressive process separate from the squamous cell-small cell carcinoma beginning in a very early stage, that is, before the appearance of p53 and chromosome 3p abnormalities. This tumorigenesis process may explain the relative rarity of combined small cell and non-small cell carcinoma, which occurs primarily in the peripheral lung, an infrequent site of squamous cell carcinoma.     

Role of GSTP1 Ile105Val and XRCC1 Arg194Trp,Arg280His and Arg399Gln gene polymorphisms in the clinical outcome of advanced non-small cell lung cancer

We conducted a study to investigate the association between the clinical outcome and GSTP1 Ile105Val and XRCC1 Arg194Trp, Arg280His and Arg399Gln gene polymorphisms in advanced NSCLC patients with cisplatin-based chemotherapy. Between January 2010 and December 2012, a total of 206 patients with advanced NSCLC were histopathologically confirmed were included into analysis. By logistic regression analysis, individuals carrying the AG and GG genotypes of GSTP1 Ile105Val were associated with better response to chemotherapy when compared with the AA genotype, and the adjusted Ors (95% CI) were 2.06 (1.10-3.86) and 4.89 (1.52-18.33), respectively. The TT genotype of XRCC1 Arg194Trp was correlated with better response to chemotherapy compared to the CC genotype, and the adjusted OR (95% CI) was 3.23 (1.20-9.30). By Cox Hazard Proportional Model, the GG genotype of GSTP1 Ile105Val and the TT genotype of XRCC1 Arg194Trp were found to be associated with lower risk of death from all causes when compared with the wide-type genotype, and the adjusted HRs (95% CI) were 0.05 (0.01-0.18) and 0.20 (0.07-0.62), respectively. Moreover, individuals carrying both the G/A+G/G genotype of GSTP1 Ile105Val and the G/A+A/A of XRCC1 Arg194Trp were associated with heavy greater CR+PR response to chemotherapy (OR=2.98, 95% CI=1.39-6.42), and also correlated with longer overall survival of advanced NSCLC (HR=0.19, 95% CI=0.05-0.61). In conclusion, we found that the GSTP1 Ile105Val and XRCC1 Arg194Trp were associated with better response to chemotherapy and longer survival of advanced NSCLC, compared to the wide-type genotype.     

Small cell carcinoma of the urinary bladder

Primary small cell carcinoma of the urinary bladder is very rare; only several studies have been reported in the English literature. A 62-year-old woman was admitted to our hospital because of hematuria and dysuria. Bladder endoscopy revealed a large polypoid tumor at the bladder base. Transurethral bladder tumorectomy (TUR-BT) was performed. Many TUR-BT specimens were obtained. Histologically, the bladder tumor was pure small cell carcinoma. Immunohistochemically, the tumor cells were positive for cytokeratin (CK) AE1/3, CK CAM5.2, CK8, CK18, neurone-specific enolase, chromogranin, NCAM (CD56), synaptophysin, Ki-67 (labeling=100%), p53, KIT (CD117), and platelet-derived growth factor receptor-α (PDGFRA). The tumor cells were negative for CK5/6, CK 34BE12, CK7, CK14, CK19, CK20, p63, CD45, and TTF-1. A molecular genetic analysis using PCR-direct sequencing showed no mutations of KIT (exons 9, 11, 13 and 17) and PDGFRA (exons 12 and 18) genes. No metastases were found by various imaging techniques. The patient is now treated by cisplatin-based chemotherapy.     

Primary small cell carcinoma of the maxillary sinus: a case report with immunohistochemical and molecular genetic study involving KIT and PDGFRA

Primary small cell carcinoma of the nose and paranasal sinuses is very rare; only a few reports are present in the English literature. The author herein reports a very rare case of primary small cell carcinoma of the maxillary sinus with an emphasis on immunohistochemistry and on KIT and PDGFRA. A 64-year-old man was admitted to our hospital because of left nasal obstruction. Endoscopy revealed three nasal polyps, and imaging modalities revealed an infiltrative tumor (45 x 45 mm) in the left maxillary sinus with invasion into nasal cavity. Multiple biopsies are taken from the nasal lesions. Histologically, the tumor consists of proliferation of malignant small epithelioid cells with hyperchromatic nuclei, fine chromatin, scant cytoplasm, molded nuclei, and absent nucleoli. Immunohistochemically, the malignant cells were positive for cytokeratin (CK) 18, synaptophysin, CD56, p53, Ki-67 (labeling=95%), bcl-2, KIT, and PDGFRA. However, they were negative for pancytokeratins, high molecular weight CK, CK5/6, CK7, CK 14, CK 19, CK20, vimentin, neuron-specific enolase, chromogranin, CD15, CD45, S100 protein, CEA, CA19-9, glial fibrillary acidic protein, neurofilaments, neuroblastoma, CD99, surfactant apoprotein A, melanosome, and TTF-1. The pathologic diagnosis was small cell carcinoma. A molecular genetic analysis using PCR-direct sequencing was performed using paraffin sections, and it showed no mutations of KIT (exons 9, 11, 13, and 17) and PDGFRA (exons 12 and 18) genes. Imaging modalities including CT, MRI and PET did not reveal any tumors, including the lung, other than the maxillary sinus tumor. The present case is the first of small cell carcinoma of the maxillary sinus with a comprehensive immunohistochemical examination and a gene analysis of KIT and PDGFRA.     

Renal cell carcinoma risk is associated with the interactions of APOE,VHL and MTHFR gene polymorphisms

Objective: The study was designed to explore the association of renal cell carcinoma (RCC) with VHL (rs779805), MTHFR (rs1801133) and APOE (rs8106822 and rs405509) polymorphisms, investigate the interactions among the single nucleotide polymorphisms (SNPs), and explore roles of the interactions in the pathogenesis of RCC in Chinese Han population. Methods: 81 RCC patients and 80 healthy controls were included in the study. Polymerase chain reaction (PCR) and direct sequencing methods were used in the analysis on the genotypes of APOE, VHL and MTHFR gene polymorphisms. Multifactor dimensionality reduction (MDR) method was adopted to conduct gene-gene interaction analysis. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were utilized to evaluate the correlation between gene-gene interactions and RCC risk. Results: Significant correlations were found between RCC risk and 3 SNPs (rs8106822, rs779805 and rs1801133). Genotype AA and allele A of APOE rs8106822 were significantly associated with RCC susceptibility (OR=2.65, 95% CI=1.05-6.69). Meanwhile, we found that the frequencies of genotype GG and allele G were much higher in case group, compared with controls (P<0.05 for both) and they appeared to be risk factors for RCC (OR=2.90, 95% CI=1.22-6.87; OR=1.78, 95% CI=1.14-2.27). While, allele T of MTHFR rs1801133 could decrease the risk of RCC (OR=0.62, 95% CI=0.40-0.97). MDR analysis showed that gene-gene interactions among APOE, VHL and MTHFR SNPs were closely related with RCC susceptibility. Conclusion: APOE, VHL and MTHFR gene polymorphisms were related to the risk of RCC. The interactions among APOE, VHL and MTHFR genes could increase the risk of RCC.     

乳腺癌易感基因1的表达与乳腺癌亚型关系的实验研究

目的 探讨乳腺癌易基因1（BRCA1）的表达与乳腺癌分子亚型的关系.方法 运用免疫组化法检测乳腺癌组织中BRCA1的表达与雌二醇受体（ER）和人表皮生长因子受体-2（Her-2）的关系,分析BRCA1的表达在乳腺癌各亚型中的意义.结果 ER阳性和阴性时,BRCA1的阳性率分别为40.2％和60.0％,表明BRCA1表达与ER有关（P＜0.05）,与Her-2无关（P＞0.05）.在乳腺癌各分子亚型中,Luminal B型和Her-2过表达型的BRCA1阳性率（66.7％和70.0％）与Basal-like型的BRCA1阳性率（75.0％）差别无统计学意义（P＞0.05）;Luminal A型的BRCA1阳性率（23.7％）低于Basal-like型的BRCA1阳性率（75.0％）,差别具有统计学意义（P＜0.05）.结论 BRCA1在Luminal B型、Her-2过表达型和Basal-like型乳腺癌中表达较高,在Luminal A型乳腺癌中表达不高,提示BRCA1可成为治疗乳腺癌的重要靶点.     

Time course and side-by-side analysis of mesodermal,pre-myogenic,myogenic and differentiated cell markers in the chicken model for skeletal muscle formation

The chicken is a well-established model for amniote (including human) skeletal muscle formation because the developmental anatomy of chicken skeletal muscle matches that of mammals. The accessibility of the chicken in the egg as well as the sequencing of its genome and novel molecular techniques have raised the profile of this model. Over the years, a number of regulatory and marker genes have been identified that are suited to monitor the progress of skeletal myogenesis both in wildtype and in experimental embryos. However, in the various studies, differing markers at different stages of development have been used. Moreover, contradictory results on the hierarchy of regulatory factors are now emerging, and clearly, factors need to be able to cooperate. Thus, a reference paper describing in detail and side-by-side the time course of marker gene expression during avian myogenesis is needed. We comparatively analysed onset and expression patterns of the key markers for the chicken immature paraxial mesoderm, for muscle-competent cells, for cells committed to myogenesis and for cells entering terminal differentiation. We performed this analysis from stages when the first paraxial mesoderm is being laid down to the stage when mesoderm formation comes to a conclusion. Our data show that, although the sequence of marker gene expression is the same at the various stages of development, the timing of the expression onset is quite different. Moreover, marker gene expression in myogenic cells being deployed from the dorsomedial and ventrolateral lips of the dermomyotome is different from those being deployed from the rostrocaudal lips, suggesting different molecular programs. Furthermore, expression of Myosin Heavy Chain genes is overlapping but different along the length of a myotube. Finally, Mef2c is the most likely partner of Mrf proteins, and, in contrast to the mouse and more alike frog and zebrafish fish, chicken Mrf4 is co-expressed with MyoG as cells enter terminal differentiation.     

Caveolin-1 promotes an invasive phenotype and predicts poor prognosis in large cell lung carcinoma

Purpose

This study investigated the relationships of caveolin-1 expression with clinical pathologic parameters and the prognosis of patients with large cell lung carcinoma. This study also explored the roles of caveolin-1 in cell invasiveness, matrix metalloproteinase (MMP) expression, and non-small cell lung carcinoma activity in vitro.

Methods

A total of 120 tissue samples were immunohistochemically analyzed for caveolin-1 expression. Cell invasion ability was measured by a Transwell invasion assay. Protein expression was assessed by Western blotting. MMP activity was detected by gelatin zymography.

Results

Caveolin-1 was expressed in 54 of 120 (45.0%) cases of large cell lung carcinoma. Caveolin-1 expression was significantly correlated with node status (N0 vs. N1, N2, and N3; P = 0.005) and advanced pTNM stage (Stages I and II vs. Stage III, P < 0.001). Patients with caveolin-1-positive expression had a poorer prognosis than did those with caveolin-1-negative expression (P < 0.001). The knockdown of caveolin-1 in H460 and 95D cells reduced the invasive ability of the cells and the expression of phosphorylated epidermal growth factor receptor (EGFR), phospho-extracellular signal-regulated kinases 1 and 2, MMP2, and MMP9; the protein level and activity of MMP2 and MMP9 were also decreased by the inhibition of EGFR activity in H460 and 95D cells.

Conclusions

The expression of caveolin-1 was positively correlated with an advanced pathologic stage. Thus, caveolin-1 could act as a predictor of a poor prognosis in patients with large cell lung carcinoma. In addition, the downregulation of caveolin-1 reduced both the invasive ability of tumor cells and the protein and activity levels of MMP2 and MMP9 in vitro, suggesting the involvement of EGFR/MMP signaling in this process.