BRAF基因突变情况与结直肠癌发病的相关性及其临床意义分析

探讨结直肠癌患者BRAF基因突变情况及其临床意义。取2014年2月至2017年2月在我院保存的结直肠癌手术标本174例,采用直接测序技术检测BRAF基因突变情况。174例患者标本中,检测到BRAF基因突变8例,突变率仅为4.60%,突变类型主要以Exon15-codon600突变为主(87.50%);低分化患者BRAF基因突变率为20.00%,明显高于中高分化患者(p0.05);BRAF基因突变与患者年龄、性别、肿瘤部位、TNM分期、病理类型、肿瘤直径及淋巴结转移无明显关系(p0.05);术前CA199升高患者BRAF基因突变率为10.17%,明显高于术前CA199正常者(p0.05)。结直肠癌患者BRAF基因突变率较低,其与肿瘤分化程度有关,且与术前CA199有一定关系,值得进一步研究。     

白介素17 与结直肠癌

结直肠癌是世界范围内的高发癌症,其发病机理尚不明确。大量研究数据表明,基因突变、表观遗传学的改变、饮食习惯以及生活方式等均是结直肠癌发生发展的高危因素。目前,普遍认为慢性炎症在肿瘤的发生发展中起重要作用。白介素17主要由T细胞的亚型Th17细胞分泌产生,能够促进肿瘤相关性炎症,使肿瘤细胞逃避免疫监控。已在胃癌、宫颈癌、食管癌、非小细胞肺癌、肝细胞肝癌、卵巢癌、黑色素瘤、淋巴瘤、乳腺癌、前列腺癌、结直肠癌等多种恶性肿瘤中发现白介素17呈高表达。现有研究表明,白介素17与肠炎和结直肠癌的发生发展密切相关。尽管尚存在争议,多数学者认为白介素17在结直肠癌的发生发展中起促进作用。本文将近年来关于IL-17在结直肠癌的发生发展中的作用以及其与结直肠癌的预后的研究成果进行总结。     

K-ras基因突变与结直肠癌临床病理特征的相关性研究

目的:探讨结直肠癌患者K-ras基因突变特点,并分析K-ras基因突变与结肠癌临床病理特征的相关性及其临床意义。方法:收集哈尔滨医科大学附属第一医院收治的500例结直肠癌患者的手术石蜡标本,采用突变扩增阻滞系统技术(amplification refractory mutation system,ARMS)检测K-ras基因第二外显子第12、13密码子的七种热点突变情况,分析其与结肠癌患者临床病理特征的相关性。结果:ARMS法检出K-ras基因突变213例,突变率为42.6%。K-ras基因突变与患者的年龄、性别、肿瘤发生的部位、肿瘤分化程度及有无淋巴结转移均无相关性(P0.05),而与肿瘤的组织学类型具有相关性(P0.05)。结论:(1)结直肠癌患者K-ras基因突变率较高,其中GLY12ASP位点突变率最高,GLY12ARG为稀有突变位点。(2)K-ras基因突变与结肠癌的组织学类型相关,可能用于结直肠癌患者治疗方案的选择。     

基于锁核酸及等位位点特异性扩增技术的KRAS基因突变检测荧光定量PCR方法研究

基于等位位点特异性扩增的原理,设计锁核酸修饰KRAS基因突变特异性扩增引物,结合封阻探针技术,建立检测KRAS基因突变的荧光定量PCR方法。结果发现,锁核酸修饰的引物及探针可显著提高等位位点特异性扩增技术用于复杂样本中的微量基因突变检测的敏感度,该技术检测KRAS基因突变的敏感性可达0.01%~0.1%。进一步用建立的荧光定量PCR方法检测52例结直肠癌患者血浆标本,并用DNA测序法作为对照,同时用健康人血浆标本建立阴性检测结果判读标准,以初步评价该方法应用于循环DNA中KRAS基因突变检测的可行性。结果发现结直肠癌患者KRAS基因突变主要是G12C、G12A和G12R,而且q PCR法的阳性检出率为46.15%,高于DNA测序法(13.46%),阴性结果与DNA测序法的符合率为100%。此外,结直肠癌患者外周血KRAS基因的突变检出率与文献报道组织标本中的突变检出率及常见突变类型基本相符。上述结果说明该方法检测循环肿瘤DNA(circulating tumor DNA,ct DNA)具有较高的可靠性,可以用于肿瘤患者循环血液中KRAS基因突变的检测。     

基因突变在结直肠癌诊疗及预后中的研究进展

结直肠癌是常见的恶性肿瘤之一,其发病率居全球恶性肿瘤发病率的第三位,死亡率呈逐年上升趋势。中国已成为全球结直肠癌每年新发病例数和死亡病例数最多的国家。对结直肠癌基因突变状态的识别以及对结直肠癌发生发展过程进行精确分类,可实现对患者进行个性化精准治疗的目的,而精准治疗的实现有赖于基因测序技术。目前,二代测序技术(Next generation sequencing,NGS)结合基因捕获技术,集中对研究者感兴趣的候选基因或外显子进行平行测序,极大拓展了对肿瘤特征基因的认识,为发展新的治疗手段和治疗策略奠定了基础。整合癌症基因组数据库IntOgen已明确72个结直肠癌驱动突变基因,包括“TP53”、“KRAS”、“PIK3CA”等;癌基因数据库Cancer Gene Census目前收录的结直肠癌突变基因有59个,包括原癌基因“BRAF”、抑癌基因“SMAD4”等;在线人类孟德尔遗传OMIM数据库已收录55个与结直肠癌相关的体细胞突变基因,包括“SRC”、“APC”等。本文通过26篇国内外文献,对结直肠癌基因突变检测的共识基因进行综述,并总结了与结直肠癌患者临床诊断、分型、预后、治疗等临床病理特征相关的突变基因标志物。     

应用结直肠癌基因检测指导术后化疗

为了探讨结直肠癌基因检测在术后化疗中的应用价值,本研究于2015年2月至2017年2月期间,选取在我院行结直肠癌根治术患者102例,根据患者选取的治疗方案,分为观察组(n=47)和对照组(n=55)。其中观察组根据术后肿瘤组织基因检测结果给予化疗方案,对照组直接选用FOLFOX4方案,随访观察两组无进展生存期和总生存期。研究发现,观察组检测到1例Nras基因突变,3例Braf基因突变,19例Kras基因突变,2例Pik3ca基因突变;Kras基因突变与肿瘤部位和分化程度有关(p<0.05);观察组中位无进展生存期为23个月,明显长于对照组的17个月,差异比较具有统计学意义(p<0.05);观察组中位总生存期为31个月,明显长于对照组的25个月,差异比较具有统计学意义(p<0.05);观察组和对照组Ⅲ～Ⅳ级肝肾损害、骨髓抑制和胃肠道反应比例比较差异无统计学意义(p>0.05)。研究表明结直肠癌基因检测指导术后化疗,可以提高患者无进展生存期和总生存期,具有较好的应用价值。     

应用高分辨率熔解曲线分析石蜡标本中KRAS基因突变

目的:KRAS基因在结直肠癌患者突变率为30％～50％,并且KRAS基因状态与抗EGFR抗体疗效的密切相关.常用的直接测序法其检测灵敏度低、容易出现假阴性结果.采用高分辨率溶解曲线分析法检测结直肠癌中KRAS基因突变,探索其应用于临床检测的可行性.方法:本文收集20例结直肠癌患者石蜡包埋组织标本,应用高分辨率熔解曲线方法检测KRAS基因突变(2,3号外显子).将KRAS基因突变型DNA(p.G12D)和野生型DNA梯度混合稀释,突变型DNA浓度分别为40％、20％、10％、2％,进行高分辨率熔解曲线方法进行检测,并将扩增产物进行直接测序,比较两种方法的灵敏度.结果:在20例结直肠癌患者中检出5例2号外显子突变,p.G12D 2例,p.G12V 2例,p.G13D 1例,3号外显子未检出突变.HRM方法检测基因突变的检出限可达2％,直接测序法检测基因突变的检出限为20％.结论:HRM法比直接测序法灵敏度高,能精确检测出结直肠癌石蜡标本中低浓度的KRAS突变,有望应用于临床基因突变检测.     

直结肠癌BUBR1的过度表达与TP53突变及微卫星状态关系提示其过度表达与染色体不稳定表型有关

在有丝分裂过程中BUBR1监视微管与着丝点的结合,是保证染色体均等分离的重要分子机制之一.BUBIB变异家谱研究及其敲除模型的研究表明,BUBR1缺陷与染色体不稳定性及肿瘤的发生直接相关.近来在数种人类肿瘤,对BUBR1蛋白过度表达有所报道.但在直结肠癌,BUBR1的过度表达是否与染色体不稳定性的发生有关目前仍无定论.在人类结直肠癌的遗传不稳定性主要表现为两种类型,染色体不稳定性及微卫星不稳定性,它们提示了两条独立的肿瘤发生路径.一般认为不存在高频度微卫星不稳定性表型的肿瘤通过染色体不稳定途径癌变.P53蛋白通过多种机制对维护遗传稳定性起到重要的作用,TP53基因突变经常与染色体不稳定现象并存.DNA倍体情况也是染色体不稳定研究不可缺少的指标.本研究采用免疫组织化学法检测了一组93例进展期散发结直肠癌BUBR1蛋白的表达情况,直接测序法检测TP53变异.高分辨率荧光标记微卫星不稳定检测技术检测微卫星状态,固相激光扫描细胞仪技术检测DNA倍体情况.我们分析了BUBR1表达与三种反映遗传背景的因子的关系.BUBR1蛋白过度表达在人结直肠癌较为常见.在非高频度微卫星不稳定的结直肠癌,BUBR1蛋白过度表达率明显为高(P＜0.01),在TP53基因突变的病例其过度表达率亦较高(P＜0.05).BUBR1蛋白的过度表达与DNA异倍体无统计学相关,但DNA异倍体病例的BuBRl过度表达有偏高倾向.BuBRl表达情况与常用的临床病理学指标无统计学相关.BuBRl过度表达同微卫星状态及TP53突变的关系明确的提示,在人类散发结直肠癌,BUBR1蛋白过度表达与染色体不稳定状态有关.BUBR1过度表达作为一种常见的分子异常,对于肿瘤的早诊预防提供新的标志物.并可能成为治疗的新靶点.     

结直肠癌患者中IGF2 LOI以及表达的研究

目的:结直肠癌(CRC)在全球范围内位居恶性肿瘤的第3位,是我国最为高发的恶性肿瘤之一.作为表观调控的一种,基因组印记丢失(LOI)被证明在结直肠癌的发生发展中具有重要的作用.本次研究通过检测结直肠癌患者中胰岛样生长因子2(IGF2)基因组印迹丢失,探讨其在结直肠癌发生及其发展的病理过程中的作用.方法:采用聚合酶链式反应—限制性酶切片段多态性(PCR-RFLP)法筛选出47名结直肠癌患者的杂合子携带者,采用RT-PCR及PCR-RFLP法检测杂合子患者中IGF2 LOI.采用荧光定量PCR检测肿瘤患者体内肿瘤组织中IGF2的表达情况.结果:在31例的杂合子中,发现有11例肿瘤组织发生LOI.对患者的年龄、性别、吸烟史、肿瘤等级以及病理分期的比较结果显示,IGF2 LOI的患者与IGF2基因印记正常的患者中未发现存在显著差异无统计学意义(P＞0.05).IGF2 LOI的患者相比较,IGF2印记正常的患者更易出现肿瘤的转移(P＜0.05).IGF2 LOI患者的IGF2表达量明显高于IGF2印记正常的患者(P＜0.05).结论:本次实验表明IGF2 LOI在结直肠癌发生发展中具有重要作用,特别是结直肠癌伴转移的患者常常表现出IGF2 LOI这一现象.IGF2 LOI可以作为一个新的表现遗传学标志,用于临床结直肠癌的早期诊断,以及肿瘤转移的评估和预防.     

结直肠癌中Galectin-3和β-catenin的表达与相关性研究

目的探讨结直肠癌中Galectin-3和β-catenin的表达与临床病理参数之间的关系。方法采用免疫组化En Vision法检测83例结直肠癌组织中galectin-3和β-catenin的表达。结果Galectin-3在结直肠癌中的阳性表达率为81.9%,β-catenin的异常表达率为62.7%。结直肠癌中galectin-3的表达与肿瘤的分化程度、浸润深度、淋巴结转移和病理分期有关(P0.05),而与患者年龄、肿瘤部位、肿瘤大小和脉管侵犯无关(P0.05)。结直肠癌中β-catenin的异常表达与分化程度、淋巴结转移、脉管侵犯和病理分期有关(P0.05),而与患者年龄、肿瘤部位、肿瘤大小和浸润深度无关(P0.05)。结直肠癌中galectin-3的表达与β-catenin异常表达呈正相关(P0.05)。结论Galectin-3的表达可能与结直肠癌的高浸润转移能力有关,其可能是通过β-catenin表达异常而促进肿瘤的浸润扩散。     

基于微流控芯片的温度梯度毛细管电泳检测大肠癌石蜡标本中K-ras基因突变

K-ras基因突变检测可用于大肠癌的早期筛查与诊断,并有利于筛选出抗表皮生长因子受体靶向药物治疗有效的大肠癌患者,以实现肿瘤的个体化治疗．采用以倾斜式热辐射原理建立的微流控温度梯度毛细管电泳(temperature gradient capillary electrophoresis,TGCE)基因突变检测系统,实现了对98例石蜡包埋大肠癌组织中K-ras基因突变的高灵敏度筛查,突变阳性检出率为47.96%,显著高于PCR产物直接测序的23.47%．克隆测序显示该方法至少能检测到2.08%的K-ras基因突变体．K-ras基因突变与临床病理学参数的关系分析显示,直肠癌中K-ras基因突变率明显高于结肠癌(P < 0.05),而与年龄、性别、组织学类型和肿瘤分期等无显著相关性．该检测方法为肿瘤早期诊断和指导临床用药提供了一种灵敏度高、检测速度快、便于大规模筛查的有效手段.     

The identical 5' splice-site acceptor mutation in five attenuated APC families from Newfoundland demonstrates a founder effect

Inherited mutations of the APC gene predispose carriers to multiple adenomatous polyps of the colon and rectum and to colorectal cancer. Mutations located at the extreme 5' end of the APC gene, however, are associated with a less severe disease known as attenuated adenomatous polyposis coli (AAPC). Many individuals with AAPC develop relatively few colorectal polyps but are still at high risk for colorectal cancer. We report here the identification of a 5' APC germline mutation in five separately ascertained AAPC families from Newfoundland, Canada. This disease-causing mutation is a single basepair change (G to A) in the splice-acceptor region of APC intron 3 that creates a mutant RNA without exon 4 of APC. The observation of the same APC mutation in five families from the same geographic area demonstrates a founder effect. Furthermore, the identification of this germline mutation strengthens the correlation between the 5' location of an APC disease-causing mutation and the attenuated polyposis phenotype.     

Role of Smad4 (DPC4) inactivation in human cancer

The tumor suppressor gene Smad4 (DPC4) at chromosome 18q21.1 belongs to the Smad family, which mediates the TGFbeta signaling pathway suppressing epithelial cell growth. This review summarizes the mutational events of the Smad4 gene in human cancer. The Smad4 gene is genetically responsible for familial juvenile polyposis, an autosomal dominant disease characterized by predisposition to gastrointestinal polyps and cancer. In this syndrome, polyps are formed by inactivation of the Smad4 gene through germline mutation and loss of the unaffected wild-type allele. In pancreatic and colorectal cancer, inactivation of the Smad4 gene through homozygous deletion or intragenic mutation occurs frequently in association with malignant progression. However, mutation of this gene is seen only occasionally in the rest of human cancers. The majority of Smad4 gene mutations in human cancer are missense, nonsense, and frameshift mutations at the mad homology 2 region (MH2), which interfere with the homo-oligomer formation of Smad4 protein and the hetero-oligomer formation between Smad4 and Smad2 proteins, resulting in disruption of TGFbeta signaling. Supporting evidence for the above observation was provided by genetically manipulated mice carrying either a heterozygote of the Smad4 gene or a compound heterozygote of the Smad4 and APC genes, which develop either gastrointestinal polyps/cancer mimicking familial juvenile polyposis or progressed colorectal cancer, respectively.     

MUTYH and the mismatch repair system: partners in crime?

Biallelic germline mutations of MUTYH—a gene encoding a base excision repair protein—are associated with an increased susceptibility of colorectal cancer. Whether monoallelic MUTYH mutations also increase cancer risk is not yet clear, although there is some evidence suggesting a slight increase of risk. As the MUTYH protein interacts with the mismatch repair (MMR) system, we hypothesised that the combination of a monoallelic MUTYH mutation with an MMR gene mutation increases cancer risk. We therefore investigated the prevalence of monoallelic MUTYH mutations in carriers of a germline MMR mutation: 40 carriers of a truncating mutation (group I) and 36 of a missense mutation (group II). These patients had been diagnosed with either colorectal or endometrial cancer. We compared their MUTYH mutation frequencies with those observed in a group of 134 Dutch colorectal and endometrial cancer patients without an MMR gene mutation (0.7%) and those reported for Caucasian controls (1.5%). In group I one monoallelic MUTYH mutation was found (2.5%). In group II five monoallelic germline MUTYH mutations were found (14%), four of them in MSH6 missense mutation carriers (20%). Of all patients with an MMR gene mutation, only those with a missense mutation showed a significantly higher frequency of (monoallelic) MUTYH mutations than the Dutch cancer patients without MMR gene mutations (P=0.002) and the published controls (P=0.001). These results warrant further study to test the hypothesis of mutations in MMR genes (in particular MSH6) and MUTYH acting together to increase cancer risk.     

The first mutations in the MYH gene reported in Moroccan colon cancer patients

Background

Biallelic germline mutations in the MYH gene cause MYH-associated polyposis (MAP) disease, an autosomal recessive form of inherited colorectal cancer. People with MAP tend to develop attenuated multiple adenomatous colon polyps during their lifetime and will have an increased risk of colorectal cancer. Contrary to familial adenomatous polyposis, the number of adenomas is often lower in MAP (from 5 to 100), and even some patients have recently been reported with no identified adenomas.There have been many investigations into MAP that have been conducted in many different countries. Currently there is limited data on MAP in Morocco, and it is reasonable to think, that the prevalence of this form of genetic predisposition is as high as other autosomal recessive genetic diseases found in countries with high rates of consanguinity.The aim of this study is to examine the frequency of MYH mutations in colorectal cancer and/or attenuated polyposis in Moroccan patients.

Patients and methods

The study population consisted of 62 patients; 52 with colorectal cancer, three of them had attenuated polyposis (2 to 99 adenomatous polyps). 10 other patients were referred to our department for polyposis without colorectal cancer.We carried out DNA analysis in 62 patients to screen for the three recurrent mutations c.494A > G (p.Tyr165Cys), c.1145 G > A (p.Gly382Asp) and c.1185_1186dup, p.Glu396GlyfsX43, whereas 40 subjects were screened for germline MYH mutations in the whole coding sequence of the MYH gene by direct DNA sequencing. All these 40 patients, except two, had colorectal cancer without polyposis.

Results

Three patients with colorectal cancer and attenuated polyposis carried biallelic mutations in the MUTYH gene one with the c.494 A > G mutation, one with the c.1105delC mutation, one with the c.1145 G > A mutation. One patient with 25 adenomas without colorectal cancer carried the c.1145 G > A mutation at a homozygote state and one patient with 3 polyps was heterozygote for the mutation c.1145 G > A. No biallelic mutations of MYH gene were detected in colorectal cancer patients and in patients with small number (< 5) of polyps without colorectal cancer.

Conclusion

We report the first biallelic MYH mutations in four Moroccan patients with clinical criteria of MAP; three of them had colorectal cancer with attenuated polyposis. No MYH mutations were found in colorectal patients without polyposis.Despite the relatively small sample size of the current study, our findings suggest that the MAP is not a frequent cause of colon cancer in Morocco as we had expected, and the molecular analysis of MYH gene should be restricted to patients displaying the classical phenotype of MAP.     

Mutation of GDP-Mannose-4,6-Dehydratase in Colorectal Cancer Metastasis

Fucosylation is a crucial oligosaccharide modification in cancer. The known function of fucosylation in cancer is to mediate metastasis through selectin ligand-dependent processes. Previously, we found complete loss of fucosylation in the colon cancer cell line HCT116 due to a mutation in the GDP-fucose synthetic enzyme, GDP-mannose-4,6-dehydratase (GMDS). Loss of fucosylation led to escape of cancer cells from tumor immune surveillance followed by tumor progression and metastasis, suggesting a novel function of fucosylation in tumor progression pathway. In the present study, we investigated the frequency of GMDS mutation in a number of clinical colorectal cancer tissue samples: 81 samples of primary colorectal cancer tissue and 39 samples of metastatic lesion including liver and lymph node. Four types of deletion mutation in GMDS were identified in original cancer tissues as well as metastatic lesions. The frequency of GMDS mutation was slightly higher in metastatic lesions (12.8%, 5/39 samples) than in original cancer tissues (8.6%, 7/81 samples). No mutation of the GMDS gene was observed in normal colon tissues surrounding cancer tissues, suggesting that the mutation is somatic rather than in the germline. Immunohistochemical analysis revealed complete loss of fucosylation in three cases of cancer tissue. All three cases had GMDS mutation. In one of three cases, loss of fucosylation was observed in only metastatic lesion, but not its original colon cancer tissue. These data demonstrate involvement of GMDS mutation in the progression of colorectal cancer.     

粪便DNA突变检测在快速筛选大肠癌中的应用

大肠癌是发病率和死亡率都很高的常见疾病, 对高危人群进行大规模的普查筛选, 可以降低大肠癌的发生率和死亡率。粪便DNA突变检测是新近发展的可用作大肠癌普查的技术, 与常规结肠镜检测和大便隐血实验相比, 具有特异性高、灵敏度高和易于被患者接受等优点。文章阐述了粪便DNA突变检测的相关基因、肿瘤特异性DNA提取方法和检测方法等, 并对其在快速筛选大肠癌中的应用进行了展望。大肠癌是发病率和死亡率都很高的常见疾病, 对高危人群进行大规模的普查筛选, 可以降低大肠癌的发生率和死亡率。粪便DNA突变检测是新近发展的可用作大肠癌普查的技术, 与常规结肠镜检测和大便隐血实验相比, 具有特异性高、灵敏度高和易于被患者接受等优点。文章阐述了粪便DNA突变检测的相关基因、肿瘤特异性DNA提取方法和检测方法等, 并对其在快速筛选大肠癌中的应用进行了展望。     

Histological and molecular evaluation of patient-derived colorectal cancer explants

Mouse models have been developed to investigate colorectal cancer etiology and evaluate new anti-cancer therapies. While genetically engineered and carcinogen-induced mouse models have provided important information with regard to the mechanisms underlying the oncogenic process, tumor xenograft models remain the standard for the evaluation of new chemotherapy and targeted drug treatments for clinical use. However, it remains unclear to what extent explanted colorectal tumor tissues retain inherent pathological features over time. In this study, we have generated a panel of 27 patient-derived colorectal cancer explants (PDCCEs) by direct transplantation of human colorectal cancer tissues into NOD-SCID mice. Using this panel, we performed a comparison of histology, gene expression and mutation status between PDCCEs and the original human tissues from which they were derived. Our findings demonstrate that PDCCEs maintain key histological features, basic gene expression patterns and KRAS/BRAF mutation status through multiple passages. Altogether, these findings suggest that PDCCEs maintain similarity to the patient tumor from which they are derived and may have the potential to serve as a reliable preclinical model that can be incorporated into future strategies to optimize individual therapy for patients with colorectal cancer.     

Missense mutations in hMLH1 associated with colorectal cancer

One of the most prevalent hereditary syndromes associated with colorectal cancer is hereditary nonpolyposis colorectal cancer (HNPCC). The inherited gene defects in HNPCC have been shown to reside in DNA mismatch repair genes, mostly hMSH2 or hMLH1. Most HNPCC patients are heterozygous with regard to the relevant mismatch repair gene; they have one normal and one mutated allele, and mismatch repair in normal somatic cells is functional. Cancer predisposition in HNPCC is believed to be associated with the loss of the wild-type allele in somatic cells, resulting in defective DNA mismatch repair. This gives rise to DNA microsatellite instability (MSI), an increased somatic mutation rate, and eventually, to the accumulation of mutations in genes involved in colorectal carcinogenesis. In support of this theory, colorectal tumors in HNPCC patients and in mice deficient for hMSH2 or hMLH1 show MSI. Here, we describe two missense mutations in hMLH1 exon 16 associated with colorectal cancer. Interestingly, the tumors do not show MSI. This raises some potentially important issues. First, even microsatellite-negative colorectal tumors can be associated with germline mutations and these will be missed if an MSI test is used to select patients for mutation screening. Second, the lack of MSI in these cases suggests that the mechanism involved in carcinogenesis could be different from that generally hypothesized.