广东省潮汕地区食管鳞状细胞癌中p53蛋白的表达

采用ＣＭ－１和ＤＯ－７一抗的免疫组化法，检测了广东省潮汕地区５５例食管鳞状细胞癌中的ｐ５３蛋白 表达的现象。ｐ５３蛋白阳性细胞不但可在呈浸润生长的肿瘤细胞中见到，而且在已具有浸润癌巢ｐ５３蛋白表达的肿瘤癌旁浸润前病变如上皮高度增生，异型增生和原位癌中可出现。     

大肠肿瘤中抑凋亡基因bcl－2和p53蛋白产物的表达和意义

用免疫组化方法观察４５例大肠腺瘤和６１例大肠腺癌中ｂｃ１－２和ｐ５３蛋白的表达。结果显示：正常大肠粘膜中ｂｃ１－２和ｐ５３均未见表达，而大肠腺瘤及大肠腺癌阳性率均较正常组织明显增加（Ｐ＜０．０１）．大肠腺瘤中ｂｃ１－２表达位于不典型增生较重区域。大肠腺瘤ｐ５３表达随腺瘤直径增加而增加，其中≥２０ｍｍ组阳性率显著高于＜１０ｍｍ组（ｐ＜０．０５）。ｐ５３蛋白阳性率也随不典型增生程度增加而增高。ｐ５３表达与大肠腺癌分化程度及Ｄｕｋｅｓ分期有关。大肠腺瘤中ｂｃ１－２和ｐ５３蛋白的表达呈负相关。结果表明，ｂｃ１－２蛋白表达对大肠腺瘤的增殖有一定意义，ｐ５３在大肠腺瘤癌变和大肠腺癌进展中起重要作用。     

p53蛋白表达与上颌窦鳞状细胞癌发生的关系

用免疫组化技术对８例上颌窦正常粘膜、１６例乳头状瘤及癌变、５０例鳞状细胞癌及１４例癌旁粘膜（包括癌旁正常粘膜、单纯增生及不典型增生）ｐ５３蛋白的表达进行了检测。结果显示，上颌窦癌ｐ５３阳性率为６２％（３１／５０）。ｐ５３表达与患者性别、年龄及肿瘤分化程度无关。上颌窦正常粘膜、单纯增生及乳头状瘤ｐ５３表达阴性。不典型增生ｐ５３表达为４／６，其表达与相应鳞癌存在一致性，不典型增生ｐ５３阳性者，相应癌组织亦为阳性，反之亦然。４例乳头状瘤癌变中，３例ｐ５３阳性。研究结果表明，ｐ５３表达是人上颌窦癌发生的早期事件，而与其恶性程度无关，ｐ５３表达发生于不典型增生或乳头状瘤癌变阶段，显示ｐ５３基因突变与上颌窦癌的发生密切相关。研究结果还表明，检测ｐ５３蛋白有助于上颌窦良，恶性病变的鉴别及早期发现上颌窦癌。     

肺肿瘤形成中的癌基因和抗癌基因

人类肿瘤的形成与很多因素有关，随着科学技术的发展和实验手段的提高，人们对肿瘤病因的认识逐渐深入，目前已达分子水平。分子遗传学的研究显示，肿瘤的形成是一个多步骤的过程，经历了支气管粘膜上皮增生、过度增生、鳞状化增生、不典型增生、原位癌、早期浸润癌、浸润癌等阶段。在这些阶段中，发生了一系列遗传学改变，包括细胞水平的染色体片段的缺失和重排，分子水平的基因突变和表达的改变，这些遗传学改变常引起原癌基因的激活和抑癌基因的失活，最终导致肺癌的发生。国内外很多研究表明，肺癌形成中有其特异性的染色体变异和癌基因及抑癌基因的突变。如肺癌中常见１３ｑ和１７ｐ缺失，这两个区域又恰好是抑癌基因Ｒｂ的ｐ５３的定位区。肺癌中还有３ｐ、５ｑ、１３ｑ、１０ｑ、４ｑ等染色体畸变。与之相关的癌基因目前发现的有ｒａｓ、ｍｙｃ、ｃ－ｅｒｂＢ－１、ｃ－ｅｒｂＢ－２、ｃ－ｍｙｂ、ｃ－ｆｏｓ、ｃ－ｊｕｎ、ｃ－ｒａｆ－１等。抑癌基因主要有ｐ５３、Ｒｂ、ｎｍ２３、ＡＰＨ、ＭＣＣ、ＡＰＣ等。最近又发现两种与肿瘤浸润转移有关的基因Ｔｉａｍ－１和ＫＡＩ－１，还有位于９ｑ２１上的一种抑癌基因ｐ１６。     

肺癌中MTS1/p16和p53基因产物的表达与细胞增殖的关系

目的：研究肺癌中ＭＴＳ１／ｐ１６和ｐ５３基因产物的表达与细胞增殖的关系。方法：应用Ｓ－Ｐ免疫组织化学方法研究６２例肺癌组织中ｐ１６蛋白和ｐ５３蛋白的表达情况，并进行增殖细胞核抗原（ＰＣＮＡ）检测，计算细胞增殖指数（ｐｒｏｌｉｆｅｒａｔｉｏｎｉｎｄｅｘ，ＰＩ）。结果：６２例肺癌组织中ｐ１６蛋白和ｐ５３蛋白阳性率分别为５８．１％和５９．７％。腺癌ｐ１６蛋白的阳性率明显高于小细胞癌（Ｐ＜０．０５）；淋巴结转移阳性组ｐ１６蛋白的表达显著低于阴性组（Ｐ＜０．０５）；ＰＩ分级为Ⅱ级的ｐ１６蛋白表达显著高于Ⅳ级（Ｐ＜０．０５）。不同组织类型肺癌中ｐ５３蛋白的表达未见明显差异，淋巴结转移阳性组ｐ５３蛋白的表达高于阴性组（Ｐ＜０．０１）；不同ＰＩ分级中ｐ５３蛋白的表达，Ⅳ级明显高于Ⅰ级（Ｐ＜０．０５）和Ⅱ级（Ｐ＜０．０５），Ⅲ级明显高于Ⅰ级（Ｐ＜０．０５）和Ⅱ级（Ｐ＜０．０１）。ｐ１６蛋白低表达和ｐ５３蛋白过表达之间未见明显相关。结论：ｐ１６蛋白低表达和ｐ５３蛋白过表达均有促进肺癌细胞增殖的作用，ｐ１６蛋白的表达与肺癌的细胞分化有关，ｐ５３蛋白过表达对肺癌细胞的转移起重要作用。抑癌基因ｐ５３对ＭＴＳ１／ｐ１６基因无明显调控作     

H-ras、c-erbB-2、p53蛋白在乳腺增生病中的表达及意义

我们通过检测Ｈｒａｓ、ｃｅｒｂＢ２、ｐ５３蛋白在乳腺增生病和乳腺癌中的表达，探讨不同增生程度的乳腺增生病与乳腺癌的关系。一、材料与方法１．标本：取自成都军区昆明总医院病理科１９９０～１９９６年的存档蜡块，包括导管上皮轻度增生、导管上皮中重度增生、导管上皮不典型增生和导管癌各６０例，共２４０例。标本均经４％甲醛固定，逐级乙醇脱水和常规石蜡包埋。２．乳腺增生病的分级标准：参照Ｐａｇｅ分类［１，２］、李维华和纪小龙［３］提出的标准将导管增生分为轻度增生、中重度增生和不典型增生３级。轻度增生指导管…     

喉癌和不典型增生上皮细胞的P53蛋白表达与S期组分

应用免疫组织化学方法和流式细胞技术对喉癌和不典型增生上皮的Ｐ５３蛋白表达和细胞周期中Ｓ期组分进行了研究。结果显示Ｐ５３蛋白在轻度中度、重度不典型增生上皮的表达率分别为４０．００％，６０．００％，７１．４３％和７３．６８％，声带息肉，不典型增生上皮和喉癌的Ｓ期组分分别为３４．５１％、５０．１８％和６２．２０％。其中有８例在２种指标中均为阳性，有两例均为阴性。结果表明Ｐ５３蛋白免疫组化和Ｓ期组分的测定     

鼻咽癌,宫颈癌和肺癌中p53基因突变和表达的对比研究

目的对比研究ｐ５３在鼻咽癌、宫颈癌、肺癌中的表达及其与ｐ５３基因突变的关系，为进一步研究ｐ５３在癌变过程中的作用奠定基础。方法应用免疫组化结合ＰＣＲ－ＳＳＣＰ银染技术检测２４例鼻咽癌、９例宫颈癌、１０例肺癌中ｐ５３基因的表达与突变。结果２３／２４例鼻咽癌、６／９例宫颈癌、９／１０例肺癌有ｐ５３的过表达，在两种发病与ＤＮＡ致瘤病毒有关的鼻咽癌、宫颈癌中未能检测到ｐ５３基因突变；而发病与化学致癌物有关的肺癌中有５／１０例检测到有ｐ５３基因突变，其中１例发生在外显子８，４例发生在外显子５。这５例均有ｐ５３的过表达。结论（１）鼻咽癌、宫颈癌、肺癌中存在着ｐ５３蛋白的过表达。（２）肺癌中ｐ５３的过表达大部分与ｐ５３基因突变有关，而鼻咽癌与宫颈癌中ｐ５３过表达与ｐ５３基因突变关系不大，其与致瘤性ＤＮＡ病毒的作用是否有关有待进一步研究。     

人肺癌组织中p53,Rb基因突变研究

为探讨肺癌发生的分子遗传学机理，采用聚合酶链反应及聚合酶链反应－单链构象多态性技术，对４１例人肺癌组织中ｐ５３基因外显子５～８及Ｒｂ基因外显子１４～１６、２２～２３进行了突变分析。结果显示：ｐ５３基因突变１６例（１６／４１，３９％），分布于外显子５～７；Ｒｂ基因异常４例（４／４１，９．８％），其中外显子１４～１６区域部分缺失和外显子２２～２３区域突变各２例；在９例小细胞肺癌标本中，７例发生ｐ５３及Ｒ５基因的突变，其中１例存在ｐ５３基因两个外显子突变，另１例同时存在ｐ５３及Ｒｂ基因的突变。对部分ｐ５３基因突变标本序列分析，均在１个或３个密码子上存在导致ｐ５３蛋白异常的单碱基置换或插入突变。以上结果表明：肺癌、特别是小细胞肺癌的发生可能与ｐ５３及Ｒｂ基因的突变有关。     

肺癌和肺癌前病变组织中维甲酸受体的表达

目的： 探讨维甲酸受体（RAR）在肺癌癌前病变（支气管黏膜上皮不典型增生）和肺癌病变中的表达。方法: 在40例正常肺组织、41例支气管黏膜上皮不典型增生组织及143例肺癌患者手术标本中,用免疫组化的方法检测RAR的表达,分析RAR基因在肺癌变不同阶段间的变化。结果: 80.0%（32/40）的正常肺组织中可检测到RAR基因的蛋白表达;68.3%（28/41）的支气管黏膜上皮不典型增生组织和57.6%（83/143）的肺癌组织中可检出RAR（P<0.05）;且随着肺组织癌变过程的变化,RAR基因蛋白的表达呈逐渐降低的趋势（P<0.01）。吸烟者RAR蛋白检出率低于非吸烟者。结论: RAR表达下降与肺组织癌变过程密切相关,吸烟暴露可导致RAR基因表达降低,检测RAR基因表达下降可能为肺癌的早期临床诊断提供科学依据。     

p53 gene mutations are common in uterine serous carcinoma and occur early in their pathogenesis.

Uterine serous carcinoma (USC) is an uncommon but aggressive type of endometrial cancer associated with rapid progression of disease and a poor prognosis. Both USC and its recently described putative precursor, endometrial intraepithelial carcinoma (EIC), demonstrate strong p53 overexpression by immunohistochemistry, suggesting alteration of the p53 gene in their pathogenesis. In the present study, we evaluated 21 USCs and 9 EICs for mutations in the p53 gene using direct sequence analysis and found that 90% of USCs and 78% of EICs contain mutations. Significantly, mutations were found in 3 cases of EIC without associated invasive carcinoma and identical mutations were detected in cases with synchronous USC and EIC. Strong p53 immunoreactivity was seen in the majority of USCs and EICs and correlated with p53 gene mutation, although lack of reactivity did not always indicate the absence of a gene mutation. Loss of heterozygosity of chromosome 17p was observed in 100% of USCs and in 43% of EICs, demonstrating that loss of the wild-type p53 allele occurs early in the development of serous carcinoma. Overall, our results reveal that p53 mutations are very common in USC and EIC. The presence of p53 gene mutations in EIC further suggests that p53 alteration plays an important role early in the pathogenesis of serous carcinoma, possibly accounting for its aggressive biological behavior.     

p53 gene alteration in atypical epithelial lesions and carcinoma in patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is well known to be associated with lung cancer. Several atypical epithelial lesions are frequently observed in the fibrotic area in IPF patients, and they have been suspected to be related to lung carcinogenesis. Several studies have suggested that p53 protein accumulation and mutation occur in the early pathogenesis of squamous cell carcinoma of the lung, suggesting some abnormality of the p53 tumor-suppressor gene in interstitial lung diseases. To examine the cause of the high frequency of lung cancer in IPF, we examined the p53 changes in atypical epithelial lesions and carcinoma in patients with IPF by immunohistochemistry and mutational analysis. We examined 19 lung cancer patients with IPF who underwent surgical resection for lung cancer in our institute. Paraffin-embedded tissues were treated by microwave and stained with an anti-p53 antibody (RSP53) by the avidin-biotin-peroxidase complex method. Mutations in exons 5 through 8 of the p53 gene were also examined by polymerase chain reaction mediated single-strand conformation polymorphism (polymerase chain reaction-single-strand conformation polymorphism) analysis and DNA sequencing. p53 protein was immunohistochemically detected in 13 (62%) of 21 squamous cell carcinomas, 3 (60%) of 5 squamous metaplasia with atypia, 16 (54%) of 30 squamous metaplasia, and 1 (4%) of 26 other hyperplastic lesions. p53 mutation was detected in 12 (57%) of 21 squamous cell carcinomas, 2 (40%) of 5 squamous metaplasia with atypia, 7 (23%) of 30 squamous metaplasia, and 0 (0%) of 26 other hyperplastic lesions. In conclusion, there are frequent p53 gene alterations in squamous metaplasia, which is distributed in the peripheral zone of the fibrotic area in patients with IPF. The present findings might provide a clue to the molecular mechanisms underlying the high incidence of lung cancer, especially peripheral-type squamous cell carcinoma in IPF patients, and suggest that p53 gene alterations play an important role in the early stages of lung carcinogenesis in patients with IPF.     

Frequent expression of p53 protein without mutation in the atypical epithelium of human bronchus.

We investigated the correlation between p53 protein levels and mutations in the p53 gene of atypical bronchial epithelium (ABE). Protein levels were analyzed by immunohistochemistry, whereas mutations were assessed by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and direct sequencing. A total of 78 formalin-fixed, paraffin-embedded bronchial biopsy specimens that had been diagnosed to be ABE were retrieved from the archives and examined. p53 protein was expressed in 44 of the 78 (56%) specimens overall. However, when pathologically classified, 38% of hyperplasias, 58% of metaplasias, and 73% of dysplasias were positive, indicating that an increased frequency of p53 expression correlated with the severity of ABE (P = 0.042). Among the 44 specimens that expressed p53 protein, 40 (91%) did not reveal mutations by PCR-SSCP. In the four specimens with abnormal PCR-SSCP bands, p53 gene mutation was identified by direct sequencing and revealed the same point mutation at codon 248 (CGG-to-CTG transversion) of exon 7 in all four specimens. These four specimens were dysplasias derived from patients with lung cancer. p53 protein expression in ABE was associated with the wild-type gene in most cases; therefore, wild-type p53 protein expressed in ABE might have a protective function from lung carcinogenesis, and mutation of p53 gene may be a late event in the sequential steps of lung carcinogenesis.     

亚硝基诱发大鼠胃腺癌发生发展过程中p53,ras基因表达和突?…

目的 探讨Ｐ５３、ｒａｓ基因在亚硝基胍诱发大鼠胃腺癌发生发展过程中的作用。方法 用免疫组织化学、聚合酶链反应－单链构象多态怀分析（ＰＣＲ－ＳＳＣＰ）技术对大鼠正常腺胃粘膜、癌前病变和胃腺癌组织中ｐ５３、ｒａｓ基因的表达和突变进行检测。结果Ｐ５３蛋白在癌组织 的了性表达率为５０％,正常和癌前病缲为阴性;ｒａｓ蛋白癌前病变组织中阳性率为４４％,在癌组织中为２３％。癌组织中ｐ５３基因突变率为４５％,非     

用显微切割技术定点检测诱发大鼠肺癌发生发展各阶段p53、K-ras基因突变与表达

目的　探讨p5 3、K ras基因突变、蛋白表达在 3 甲基胆蒽 (3 methylcholanthrene ,MCA)和二乙基亚硝胺 (diethylinitrosamine ,DEN)诱发大鼠肺鳞癌发生演进中的作用 ,及其突变与蛋白表达的关系。方法　将大鼠诱发肺癌石蜡标本连续切片 ,切片用于HE染色确定肺癌发生发展的病变阶段 ,及免疫组织化学 (SP法 )检测各阶段p5 3、K ras蛋白表达 ,并用于显微切割 ,定点对位分别切割由正常支气管黏膜上皮细胞演变成癌细胞 ,癌浸润、转移各阶段病灶的主质 ,提取DNA ,用聚合酶链反应 单链构象多态性 (PCR SSCP)检测各阶段p5 3、K ras基因的突变。结果　 30例正常支气管黏膜上皮未检测到p5 3、K ras基因突变及其蛋白表达。在 32例支气管黏膜增生和鳞状化生、2 1例不典型增生、12例原位癌、4 3例浸润癌及 17例转移癌组织中 ,p5 3基因突变率分别为 3 1% ,2 8 6 % ,30 0 % ,5 1 2 % ,5 2 9% ;p5 3蛋白阳性表达率分别为 0 ,4 2 9% ,5 0 0 % ,6 0 5 % ,6 4 7% ;不典型增生阶段与增生、鳞状化生阶段相比 ,p5 3基因突变率及蛋白表达率增高 ,差异均有显著性意义 (P <0 0 2 5 ,P <0 0 0 5 ) ,p5 3基因突变及蛋白阳性表达高度相关 (P <0 0 0 5 ,Pearson′sR =0 5 996 )。K ras基因突变率分别为 0 ,4 8% ,8 3% ,9 3     

Aberrant expression of p53 gene product in malignant melanoma.

According to the current concept of carcinogenesis, the alterations of p53 tumor suppressor gene have been the most frequently detected in both human cancer cell lines and cancer tissues freshly isolated. This study was conducted to investigate the p53 gene alteration in malignant melanoma. Nineteen tumor tissues were obtained from 19 patients with malignant melanoma and examined for the expression of p53 protein by immunohistochemical staining with mouse monoclonal anti-p53 antibody, NCL-p53-DO-7. Twelve out of 19 cases (63%) showed positive reactions for p53 protein: 26, 21 and 16% of which had low, intermediate and high reactivity, respectively. p53 alteration more frequently expressed in female (10/12) than male patients (2/7) with malignant melanoma (p < 0.05). The incidence of expression of p53 protein was compared according to the stages and the sites of tissue obtained. The positive rate for p53 protein was not significantly different between the stages. The positive rates for p53 protein were five out of five (100%), one out of two (50%) and six out of twelve (50%) in tissues obtained from the metastatic, lymph node, and primary sites, respectively. The difference in the positive rates, however, is not statistically significant. These results suggest that p53 gene is a frequent target for mutation in the development of malignant melanoma.     

Molecular abnormalities of p53, MDM2, and H-ras in synovial sarcoma.

Forty-nine cases of synovial sarcoma were evaluated for mutation of the p53 gene, amplification of the MDM2 gene and mutation of the H-ras gene, and for the relation of these factors to overall survival and clinicopathologic parameters. All investigations were carried out on formalin-fixed paraffin-embedded materials. Furthermore, we evaluated the expression of p53 protein, MDM2, and p21(WAF1/CIP1) immunohistochemically in these cases, together with an assessment of proliferative activities using monoclonal antibody MIB-1. Nine of the 49 cases (18.4%) had p53 gene alteration detected by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing. Eleven cases (24%) showed nuclear accumulation of p53 protein in more than 10% of the tumor cells. Among them, only three cases contained gene mutations. There was no correlation between p53 nuclear accumulation and p53 gene alteration. MDM2 gene amplification, as shown by differential PCR, was observed in 19 out of 47 cases (40%). Nineteen out of 49 cases (38.8%) showed immunoreactivity for MDM2. MDM2 gene amplification and the expression of MDM2 protein showed a significant positive relationship (P = 0.0004). Moreover, MDM2 immunoreaction was significantly correlated with nuclear accumulation of p53 protein (P = 0.023). Positive immunoreaction for p21(WAF1/CIP1) was observed in 21 out of 48 cases (43.8%). p21(WAF1/CIP1) expression was correlated with p53 protein expression. H-ras gene mutations were seen in only three cases (6.1%). All mutations were in codon 12 (one GGC-to-AGC [Gly-to-Ser] mutation and two GGC-to-GAC [Gly-to-Ap] mutations). The gene alteration of p53, MDM2, and H-ras did not affect the patients' prognosis. Although the cases with positive immunoreaction for p53 tended to have a worse prognosis, the difference was not statistically significant (P = 0.13). No correlation was observed between MIB-1 LI and the immunohistochemical expression of p53, MDM2, and p21(WAF1/CIP1) or the mutation status of p53 and H-ras. On the other hand, high MIB-1 LI (more than 10) significantly correlated with poor prognosis (P < 0.0001). Our results suggest that p53 gene mutation does not appear to be a major prognostic factor and H-ras mutations are infrequent in synovial sarcoma.     

Expression of p21 WAF1/CIP1 protein in colorectal cancers: study of its relation to p53 mutation and Ki67 antigen expression

Mutations of the p53 gene are the most common genetic alteration in malignant human tumors. A cyclin-dependent kinase inhibitor, p21WAF1/CIP1, is thought to be an important mediator of p53-induced cell cycle arrest. Although numerous studies have reported p53 expression and mutation in colorectal cancer few of them have correlated p53 expression with that of its downstream effector p21 and with the proliferation index as measured by expression of the Ki67 nuclear antigen. We studied p53, p21 and Ki67 expression by immunohistochemistry and molecular biology in 35 colorectal carcinomas. We compared these findings with each other and with clinical factors. Sixty three percent of tumors expressed p53 whereas seventy one percent expressed p21WAF1/CIP1. In adenocarcinomas, p21 staining was heterogeneous: p21-reactive cells were seen in the most differentiated areas. There was no correlation between p21WAF1/CIP1 and p53 expression, p53 mutation, Ki67 expression or clinical factors such as sex or location of the tumor. On the other hand, there was a statistical relationship between p21 expression and survival: our results indicated an association between high p21 expression and lower stages p21WAF1/CIP1 appears to be induced independently of p53 in these tumors and may be associated with differentiation rather than proliferation.     

APC, K-ras codon 12 mutations and p53 gene expression in carcinoma and adenoma of the gall-bladder suggest two genetic pathways in gall-bladder carcinogenesis

Currant histopathoiogical evidence suggests that gall-bladder cancer has two main morphological pathways for Its development: de novo ( ab Inltlo ) origin and adenoma-carcinoma sequence. In order to investigate the genetic difference between them, APC mutations were examined by RNase protection analysis, K-ras mutations by nested porymerase chain reaction-restriction fragment length polymorphism analysis, and p53 gene overexpression by immunoriisto-chemical analysis in both tumors and benign lesions of the gall-bladder. Overexpression of the p53 gene was detected in 105 of 164 (64%) de novo carcinomas regardless of size and depth of Invasion, but not in 16 tumors of carcinoma-ln-pyloric-gland-type adenoma, or In 51 adenomas (47 pyloric gland-type and 4 Intestinal-type). K-ras codon 12 mutation was detected in 4 of 40 (10%) de novo carcinomas, ail four being associated with p53 gene overexpression, but not in 12 tumors of carcinoma in adenoma or 16 adenomas (14 pyloric gland-type and 2 intestinal-type). APC mutation was not found in 16 de novo carcinomas or the one pyloric gland-type adenoma examined. These results suggest that there are two distinct genetic pathways in gall-bladder carcinogenesis; that is, de novo carcinoma develops from a predominant p53 alteration with low K-ras mutation, and carclnoma-in-pylortc-gland-type adenoma develops from p53 -, K-ras -, and APC -gene-unrelated, as yet unknown, alteration.