中国人非小细胞肺癌EGFR和K-ras基因突变与临床病理特征及厄洛替尼治疗效果的关系

目的 研究中国小细胞肺癌(NSCLC)患者表皮生长因与受体(EGFR)和K-ras基因突变情况,探讨其与NSCLC临床病理学特征及厄洛替尼治疗效果的关系.方法 利用PCR扩增和基因测序的方法检测301例中国NSCLC患者EGFR基因第18、19、20和21外显子及K-ras基因第1213密码子的突变情况,并分析其与NSCLC临床病理学特征及厄洛替尼治疗效果的关系.结果 301例患者中,99例(32.9%)有EGFR基因突变,其中第18外显子上发生突变3例,第19外显子上发生突变59例,第20外显子上发生突变2 例,第21外显子上发生突变35例.14例(4.7%)有K-ras基因突变,其中13例位于第12密码子.无同时存在EGFR和K-ras基因突变者.腺癌、无吸烟史和女性患者EGFR基因突变率较高,分别为45.7%、48.4%和49.6%.10例服用厄洛替尼有效的患者中7例携带有EGFR基因突变.结论 中国NSCLC患者EGFR基因突变率显著高于西方人群,而K-ras基因突变率则较西方人群低.联合检测EGFR和K-ras基因的突变情况可以筛选EGFR酪氨酸激酶抑制剂治疗的获益人群,并较好地预测厄洛替尼治疗晚期NSCLC的疗效.     

EGFR mutation testing in NSCLC: Patterns of care and outcomes in Western Australia

Aims:   This study evaluated the EGFR mutation status, administration of gefitinib or erlotinib and outcomes of patients assessed for EGFR mutations since the commencement of testing in Western Australia.
Methods:   A retrospective study identified patients with NSCLC who undergone EGFR mutation testing in the Department of Anatomical Pathology, Royal Perth Hospital, Western Australia from March 2005 until May 2007. Patient characteristics, cancer history, treatment, outcomes and survival were collected from the medical records and pathology reports.
Results:   Tumor samples from 64 patients were sequenced for mutations in exons 18–21 EGFR and, of these, 53 patients with NSCLC were included in the analysis. The mean age at diagnosis was 61 years (range 19–80) and most of the tumor samples tested were from female patients (76%). Overall 36% of patients tested were mutation-positive with 95% of mutations occurring in exons 19 or 21. A total of 63% of mutation-positive and 18% of mutation-negative patients were treated with gefitinib or erlotinib. Of these, 83% of patients whose tumors had an EGFR mutation had a favorable response following treatment, compared to 17% of mutation-negative patients. The duration of treatment was longer in mutation-positive patients (mean 30 weeks vs 9 weeks).
Conclusion:   EGFR mutation testing is not routinely performed in NSCLC in Western Australia. Referral for testing is at the discretion of the treating physician, accounting for the high proportion of women and adenocarcinoma histology. Selection of mutation-positive tumors for treatment with gefitinib or erlotinib is associated with good responses to treatment. This study supports the use of gefitinib or erlotinib in routine clinical practice in patients with NSCLC carrying an EGFR mutation.     

Loss of Trop2 causes ErbB3 activation through a neuregulin-1-dependent mechanism in the mesenchymal subtype of HNSCC

In head and neck squamous cell cancer (HNSCC), four intrinsic subtypes (or groups) have been identified, and each one possesses a unique biology that will require specific treatment strategies. We previously reported that mesenchymal (group 2) tumors exhibit reduced levels of Trop2 expression. In this study, we investigated the functional role of Trop2 in HNSCC and find that loss results in autocrine activation of the EGFR family member ErbB3 via neuregulin-1. Trop2 localizes to both the cell surface and cytosol of HNSCC cells and forms a complex with neuregulin-1, which is predominantly cytosolic. Inactivation of Trop2 increases the concentration of neuregulin-1 at the cell surface where it is cleaved to activate ErbB3. In primary HNSCC, detection of ErbB3 activation was limited to Trop2 negative tumors. An analysis of the Cancer Genome Atlas (TCGA) HNSCC dataset confirms enrichment for ErbB3 activity in mesenchymal tumors. Notably, Trop2 loss triggers sensitivity to anti-ErbB3 antibodies, which results in reduced proliferation and tumorigenic growth of Trop2 negative HNSCC cancer cells. These results uncover a molecular mechanism by which tumor cells control the amount of cell-surface neuregulin-1 available for cleavage and ErbB3 activation. Moreover, we demonstrate that Trop2 is a potential surrogate biomarker to identify tumors with ErbB3 activation and may therefore respond to anti-ErbB3 therapeutics.     

非小细胞肺癌中EGFR和K-ras基因突变检测

目的:探讨不同类型非小细胞肺癌的EGFR和K-ras基因突变情况及其与肺癌相关临床病理特征的关系。方法:用厦门艾德ADxARMS试剂盒进行98例非小细胞肺癌患者肿瘤组织中EGFR（18,19,20,21外显子）基因和K-ras（12,13,61密码子）基因突变的检测。所有患者均未接受过吉非替尼的治疗。结果:98例样本中31例发生了EGFR基因突变,突变率为31.6%（31/98）,其中15例为19外显子缺失,13例为21 L858R外显子点突变,3例为20外显子突变,1例为18外显子突变。其中1例既有19外显子缺失突变,又有20外显子突变。腺癌中EGFR基因突变率较鳞癌、腺鳞癌、大细胞癌高。女性患者EGFR基因突变率较男性高。不吸烟患者EGFR基因突变率较吸烟患者高。低分化腺癌患者EGFR基因突变率较中、高分化患者高。21例发生了K-ras基因突变（21.4%）,其中12、13、61密码子均发现突变。突变率腺癌较鳞癌、腺鳞癌、大细胞癌高,与是否吸烟、患者性别、分化程度均无相关性。结论:非小细胞肺癌患者EGFR基因突变检出率较高,K-ras基因突变率较低,且两者不存在同时突变,EGFR基因突变与肺癌组织学类型、分化程度、性别等相关。K-ras基因突变与组织学类型相关。     

Combined molecular targeted drug therapy for EGFR and HER-2 in head and neck squamous cell carcinoma cell lines

The epidermal growth factor receptor (EGFR) and related family member, HER-2, are often overexpressed simultaneously in patients with a variety of malignant tumors, and the combination may cooperatively promote cancer cell growth and survival. The purpose of this study was to examine antitumor effects of the combination treatment of cetuximab and trastuzumab on head and neck squamous cell carcinoma (HNSCC) using 16 HNSCC cell lines in terms of antiproliferative effect and antibody-dependent cell-mediated-cytotoxicity (ADCC). Previously we have reported the expression levels of EGFR mRNA on 16 HNSCC cell lines. All cell lines expressed mRNA for EGFR, HER-2 and HER-3; 12 cell lines expressed mRNA for HER-4; and 4 cell lines did not express mRNA for HER-4. In in vitro proliferation assay, the combination treatment of cetuximab and trastuzumab significantly lowered cell viability compared to either drug alone. The mRNA expression levels of EGFR and HER-2 were not correlated with the efficacy of the combination treatment of cetuximab and trastuzumab and the expression levels of HER-3 and HER-4 also showed no correlation with the efficacy of the combination treatment. We evaluated the gene status of HER-2 exons 23 and 24 in 16 HNSCC cell lines, but there was no mutation of HER-2 in any of the cell lines. Either drug showed ADCC in the 3 cell lines using peripheral blood mononuclear cells (PBMCs), however, a significant combination effect was not observed. Combined molecular targeted antibody drug therapy for EGFR and HER-2 may be useful in the treatment of HNSCC.     

EGFR,HER-2/neu,cyclin D1, p21 and p53 in correlation to cell proliferation and steroid hormone receptor status in ductal carcinoma in situ of the breast

Abnormalities in G₁/S transition in cell cultures have been attributed to alterations in ErbB (erythroblastic leukaemia viral [v-erb-b] oncogene homologue, avian) signalling, cyclin D1 overexpression or disturbance of the p21^WAF1(p21)-mediated cell cycle arrest induced by p53. To investigate the significance of these mechanisms on an early stage of human breast tumour growth, we studied the expression of EGFR (ErbB1), HER-2/neu (ErbB2), cyclin D1, p21 and p53 as well as oestrogen (ER) and progesterone receptor (PgR) in paraffin sections of 45 ductal carcinoma in situ (DCIS) by immunohistochemistry. Cell proliferation was assessed by immunohistochemical quantification of Ki-67. Five cases with cyclin D1 overexpression were analysed by FISH for CCND1 amplification. Increased proliferative activity was observed in 46% of DCIS. It was correlated with the expression of EGFR and HER-2/neu (p < 0.05), but neither with cyclin D1 and p21 overexpression nor with p53 accumulation. ErbB positive status was associated with p21 overexpression (p < 0.05). In addition we found a correlation between the overexpression of p21 and cyclin D1 restricted to ErbB-positive cases (p = 0.013). ErbB-negative tumours with increased proliferative activity were ER and cyclin D1 positive. No CCND1 amplification was detected in the analysed cases. In conclusion, our data support that EGFR and HER-2/neu play an important role in cell cycle control in DCIS. p21 appears to be a potential mediator of ErbB signalling. We propose that cyclin D1 could be indirectly induced by ErbB signalling through p21. Besides, ER-mediated upregulation of cyclin D1 seems to be a possible mechanism of maintaining cell proliferation in DCIS in case of EGFR- and HER-2/neu-negativity.     

Pharmacological characterization of MP-412 (AV-412), a dual epidermal growth factor receptor and ErbB2 tyrosine kinase inhibitor

Epidermal growth factor receptor (EGFR) and ErbB2 are currently recognized as validated target molecules in cancer treatment strategies. MP-412 (AV-412) is a potent dual inhibitor of EGFR and ErbB2 tyrosine kinases, including the mutant EGFR^L858R,T790M, which is clinically resistant to the EGFR-specific kinase inhibitors erlotinib and gefitinib. In an enzyme assay, MP-412 inhibited the EGFR variants and ErbB2 in the nanomolar range with over 100-fold selectivity compared with other kinases, apart from abl and flt-1, which were both moderately sensitive to the compound. In cells, MP-412 inhibited autophosphorylation of EGFR and ErbB2 with IC₅₀ of 43 and 282 nM, respectively. It also inhibited epidermal growth factor (EGF)-dependent cell proliferation with an IC₅₀ of 100 nM. Moreover, MP-412 abrogated EGFR signaling in the gefitinib-resistant H1975 cell line, which harbors a double mutation of L858R and T790M in EGFR . In animal studies using cancer xenograft models, MP-412 (30 mg/kg) demonstrated complete inhibition of tumor growth of the A431 and BT-474 cell lines, which overexpress EGFR and ErbB2, respectively. MP-412 suppressed autophosphorylation of EGFR and ErbB2 at the dose corresponding to its antitumor efficacy. When various dosing schedules were applied, MP-412 showed significant effects with daily and every-other-day schedules, but not with a once-weekly schedule, suggesting that frequent dosing is preferable for this compound. Furthermore, MP-412 showed a significant antitumor effect on the ErbB2-overexpressing breast cancer KPL-4 cell line, which is resistant to gefitinib. These studies indicate that MP-412 has potential as a therapeutic agent for the treatment of cancers expressing EGFR and ErbB2, especially those resistant to the first generation of small-molecule inhibitors. ( Cancer Sci 2007; 98: 1977–1984)     

非小细胞肺癌EGFR基因突变的临床意义研究

  目的   探讨非小细胞肺癌EGFR基因外显子突变与其临床病理特征的关系。   方法   利用ADx-ARMS?EGFR基因突变检测试剂盒,检测214例未接受过Gefitinib治疗的非小细胞肺癌患者组织中EGFR基因外显子18、19、20和21突变。   结果   非小细胞肺癌组织中EGFR基因总突变率为45.8%（98/214）,外显子18、19、20和21的突变率分别为0.93%（2/214）、22.0%（47/214）、2.3%（5/ 214）和20.6%（44/214）。另有2例19和21外显子双重突变。EGFR基因在肺腺癌组织中的总突变率为50.3%（93/185）明显高于肺鳞状细胞癌17.2%（5/29）（P=0.001）。EGFR基因在女性患者中的突变率57.0%（57/100）高于男性36.0%（41/114）（P=0.002）,EGFR基因在NSCLC淋巴结转移患者中的突变率（66.7%）显著高于无淋巴结转移患者（39.5%）（P < 0.05）,但EGFR基因突变率与肺癌患者的年龄、肿瘤分级和临床分期均无显著性差异（P>0.05）。   结论   中国肺癌尤其是肺腺癌患者存在EGFR基因的较高突变率,EGFR外显子19、21突变结合肺癌的临床病理特征有望成为评估TKI治疗非小细胞肺癌疗效的分子标志。      

Targeting epidermal growth factor receptor and SRC pathways in head and neck cancer

Epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases (RTKs), is highly expressed in head and neck squamous cell carcinoma (HNSCC) where increased EGFR expression levels in tumors are associated with decreased survival. HNSCC patient responses to EGFR-targeted monotherapies in clinical trials, though significant, have been limited. Tumor signaling pathway components that work in cooperation with EGFR or provide compensation for the loss of EGFR-initiated signaling will be ideal targets for therapies to be used in combination with EGFR-targeted agents. Based on the current understanding of molecular signaling pathways and available agents, ErbB family-targeted and Src family-targeted agents represent strategies for further exploration. Here, we discuss agents targeting ErbB and Src family kinases in clinical development, provide an overview of completed and ongoing clinical trials, and outline a molecular rationale for combining ErbB- and Src-targeted therapeutics.     

Epidermal growth factor receptor expression and mutational analysis in synovial sarcomas and malignant peripheral nerve sheath tumors

BACKGROUND: Synovial sarcomas (SnSrcs) and malignant peripheral nerve sheath tumors (MPNSTs) are rare mesenchymal tumors of adolescence and young adulthood. Previous work from our laboratory has demonstrated that SnSrcs express epidermal growth factor receptor (EGFR) and human EGFR (HER)-2/neu. The present study extends that work to examine the expression of EGFR in MPNSTs and the characterization of potential targets of the EGFR tyrosine kinase domain. METHODS: Tissue microarrays containing 48 cases of SnSrc and 32 cases of MPNST were stained for EGFR, EGFRvIII, and activated EGFR (pY1068-EGFR). Tumor DNA was extracted from fresh and formalin-fixed, paraffin-embedded tissue blocks and sequenced for exons 17-21 of EGFR and exon 2 of K-ras and b-raf. RESULTS: Immunohistochemistry (IHC) demonstrated that EGFR is expressed in a majority of SnSrcs and MPNSTs (71% and 62.5%, respectively). EGFRvIII immunoreactivity was negative. IHC was weakly immunopositive for activated EGFR (18.7% and 3.1%, respectively). Sequence analysis of the EGFR genomic DNA did not demonstrate mutations in exons 17-21. No K-ras or b-raf mutations were observed in either tumor type. CONCLUSIONS: Expression of EGFR in SnSrcs and MPNSTs with an intact EGFR/mitogen-activated protein kinase pathway has been hypothesized to contribute to the malignant potential of these tumors. Our study reveals the absence of known activating mutations in EGFR, which suggests that trials of small-molecule inhibitors would be of little clinical benefit. A clinical study of treatment with cetuximab is ongoing and may help elucidate whether blockade of EGFR with antibodies is likely to be more active.     

Overexpression of cell cycle regulatory proteins correlates with advanced tumor stage in head and neck squamous cell carcinomas

Squamous cell carcinoma of the head and neck region (HNSCC) is the sixth most frequent cancer worldwide. In the USA, 30,000 new cases and 8,000 deaths are reported each year. Differences between normal epithelium and cancer cells from the upper aerodigestive tract arise from alterations in expression of specific genes controlling proliferation and immortalization. The protein products of these genes include growth factor receptors, cell cycle regulators, and tumor suppressors which affect a variety of intracellular signaling pathways. To determine how altered expression of these gene products contribute to HNSCC progression, we examined expression of epidermal growth factor receptor (EGFR), cyclins, p16INK4A, c-myc, proliferating cell nuclear antigen (PCNA), and telomerase in archival pathology specimens by immunohistochemistry. A substantial majority of HNSCC tumors showed loss of p16INK4A expression and dramatic overexpression of EGFR. Overexpression of this receptor correlated with increased cyclin A levels and high mitotic index. EGFR, cyclins A, -B1, -E, and c-myc overexpression was significantly increased in stage III and IV tumors compared to early stage cancers. hTERT was expressed in all tumors and primarily in the basal layer cells of dysplastic epithelial lesions. Suprabasal expression of hTERT was found in a significantly higher number of HNSCC cases than in dysplastic lesions. These results indicate that overexpression of cell cycle regulatory proteins correlates with advanced tumor stage in HNSCC.     

表皮生长因子受体在人头颈部鳞状细胞癌中的作用研究进展

头颈部鳞状细胞癌（head and neck squamous cell carcinoma,HNSCC）是常见的恶性肿瘤,恶性程度较高,患者常出现复发和转移。表皮生长因子受体（epidermal growth factor receptor,EGFR）是重要的癌基因,在头颈鳞癌中广泛过表达,且与HNSCC患者的预后呈负相关,是重要的治疗靶点。但HNSCC中的EGFR靶向治疗效果却不如在非小细胞肺癌治疗中那么有效。近几年的研究发现,EGFR促进肿瘤细胞对治疗耐受的机制可能与其过表达、突变、单核苷酸多态性、入核和自噬有关。本文将就这几个方面进行综述,并探讨如何在HNSCC的治疗中更有效地利用EGFR这一靶点,为探索HNSCC的治疗策略提供新的方向。      

Low Prevalence of K-RAS,EGF-R and BRAF Mutations in Sinonasal Adenocarcinomas. Implications for Anti-EGFR Treatments

We have previously shown that a subset of sinonasal intestinal-type adenocarcinomas (ITAC) shows activation of the epidermal growth factor-receptor (EGFR) pathway. In this study we examine the status of the EGFR, KRAS and BRAF genes in a series of sinonasal intestinal (ITAC) and non-intestinal type adenocarcinomas (non-ITAC). Eighteen ITACs and 12 non-ITACs were studied immunohistochemically for EGFR expression. Point mutations were analyzed for EGFR exons 19 and 21, KRAS exon 2 and BRAF exon 15 by direct sequencing. Non-ITACs showed significantly higher expression of EGFR (p?=?0.015). Mutation analysis revealed one ITAC with EGFR and one ITAC with KRAS mutation, while two non-ITACs presented mutation of BRAF. We conclude that a subset of sinonasal adenocarcinomas shows overexpression of EGFR, while activating mutations of the signaling cascade downstream of EGFR are rare, suggesting that these tumors could be good candidates for anti-EGFR therapies.     

Clinical significance of genetic alterations and expression of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas

The significance of epidermal growth factor receptor (EGFR) signaling is well studied in a number of different tumors, but limited data is available with regard to head and neck squamous cell carcinoma (HNSCC). Since anti-EGFR therapies are currently under investigation in these malignancies as well, comprehensive information about the alteration of EGFR in HNSCC is necessary to design the most appropriate therapeutic protocols. We examined retrospectively the gene copy number of EGFR by FISH and the protein expression by immunohistochemistry using different epitope-specific antibodies in paraffin-embedded primary tumors of five different regions, from 71 HNSCC patients who had not been treated with anti-EGFR therapy. In seven cases corresponding lymph node metastases were also available for comparative analyses. We also determined the mutational status of tyrosine kinase (TK) domain (exon 19 and 21) and the extracellular deletion mutation (vIII) of EGFR, the KRAS mutation at codon 12 and the presence of HPV infection. Eight of the 71 cases (11.3%) showed EGFR gene amplification (most of them localized into the hypopharyngeal region) and the increased gene copy number (amplification+polysomy) was 43.7%. Despite pronounced intratumoral heterogeneity of EGFR protein expression being found, the high EGFR expression correlated with poor prognosis. On the other hand, the phosphorylation of EGFR was associated with prolonged survival. No mutations in the TK domain of EGFR were found in any of the HNSCC patients and only two cases were KRAS mutant at codon 12. We detected vIII deletion mutation of EGFR in 21% of the samples, but there was no statistically significant correlation between the presence of vIII mutant form and patient survival. EGFR vIII mutation was, however, associated with increased gene copy number. Fourteen of 71 cases (19.7%) were HPV-positive and the incidence of infection showed a decreasing tendency from the oral cavity towards the larynx. Interestingly, in contrast to previous findings, we could not observe improved survival in HPV-positive patients compared to non-infected patients, most probably due to the fact that the majority of these HNSCC patients were smokers and alcohol consumers. In conclusion, we found that increased EGFR protein levels and gene copy numbers (not gene amplification alone) have prognostic significance in the investigated HNSCC patient population. However, the relatively high incidence of the EGFR-vIII mutant form warrants careful therapeutic decision-making when choosing between different anti-EGFR treatment options.     

Expression of fatty acid synthase, ErbB2 and Ki-67 in head and neck squamous cell carcinoma. A clinicopathological study

Fatty acid synthase (FAS) is a multifunctional enzyme responsible for the synthesis of saturated fatty acids using acetyl-CoA and malonyl-CoA as substrates. Overexpression of FAS has been reported in several human malignancies and suggested as a potential prognostic factor. ErbB2 (Her-2/neu), a transmembrane tyrosine kinase member of the ErbB receptor family, is known to be overexpressed in a variety of tumors and was recently shown to regulate FAS production in breast epithelial cell lines. Herein we analyzed by immunohistochemistry the expression of FAS, ErbB2, and the proliferation marker Ki-67 in 62 head and neck squamous cell carcinoma (HNSCC) samples. Approximately 78% of the cases were positive for FAS or ErbB2 at the cell membrane and 70% of the tumors that showed a high expression of FAS were also strongly positive for ErbB2 (Fisher's exact test, p = 0.01). The immunolabeling for both FAS and ErbB2 was stronger in histologically well-differentiated lesions. Additionally, Ki-67 expression was significantly associated with a poor prognosis (log-rank test, p = 0.03). Taken together, the results presented here suggest that ErbB2 regulates FAS expression in HNSCC and point out Ki-67 as a useful prognostic marker for these tumors.