Loss of extracellular E-cadherin in the normal mucosa of duodenum and colon of patients with familial adenomatous polyposis

The duodenum is the main site for (pre-) malignant extracolonic manifestations in patients with familial adenomatous polyposis (FAP). Changes in the E-cadherin/beta-catenin complex play a pivotal role in the development of malignancies. Loss of E-cadherin has been described in association with loss of SMAD4. To elucidate the pathways leading to the development of duodenal adenomas in patients with FAP, the distributions of E-cadherin, SMAD4, and beta-catenin were analyzed. Furthermore, differences between the duodenum and colon were evaluated. Normal FAP duodenum (n = 13) and FAP duodenal adenomas (n = 50; total, 21 patients) were compared with non-FAP duodenal adenomas (n = 7) and normal non-FAP duodenum (n = 15) by immunohistochemical staining for extracellular and intracellular E-cadherin, beta-catenin, and SMAD4. Colonic biopsies of 10 patients with FAP were also studied, as well as non-FAP colonic adenomas (n = 26) and non-FAP normal colon (n = 12). Compared with the intracellular component of E-cadherin that was present in all cases, a significant loss of extracellular E-cadherin was observed in both duodenal and colonic adenomas and normal tissue of patients with FAP. Nuclear localization of beta-catenin was more often observed in duodenal FAP adenomas compared with non-FAP adenomas. Loss of nuclear SMAD4 was seen in the duodenum and, to a higher degree, in the colon of patients with FAP, as well as non-FAP patients. The loss of extracellular E-cadherin in the normal duodenal and colonic mucosa of patients with FAP might play a role in the high susceptibility of these tissues for (pre-) malignant transformation.     

Cell proliferation and ultrastructural changes of the duodenal mucosa of patients affected by familial adenomatous polyposis

Patients affected by familial adenomatous polyposis (FAP) are at risk of developing duodenal neoplasia. Our objective was to detect early abnormalities of the epithelial cell proliferation and ultrastructure of apparently normal duodenal mucosa of FAP patients. Biopsy specimens were taken from the duodenal mucosa. Cell proliferation was studied by immunohistochemistry with proliferating cell nuclear antigen (PCNA), and ultrastructure, by transmission electron microscopy. We found that the PCNA labeling index for duodenal mucosa of patients with FAP was higher in comparison to the case of hospital controls without cancer risk (P = 0.019). Moreover, ultrastructural changes related to an impairment of cell adhesion function were found in all biopsies of FAP patients but not in the duodenal mucosa of the controls. We conclude that alterations of cell proliferation kinetics and epithelial adherens junction structures were phenotypic characteristics of histologically normal duodenal mucosa of FAP patients. These abnormalities may be considered as intermediate biomarkers of neoplasia and potential surrogate endpoints in chemoprevention studies.     

Molecular analysis of the APC gene in 205 families: extended genotype-phenotype correlations in FAP and evidence for the role of APC amino acid changes in colorectal cancer predisposition

BACKGROUND/AIMS—The development of colorectal cancer and a variable range of extracolonic manifestations in familial adenomatous polyposis (FAP) is the result of the dominant inheritance of adenomatous polyposis coli (APC) gene mutations. In this study, direct mutation analysis of the APC gene was performed to determine genotype-phenotype correlations for nine extracolonic manifestations and to investigate the incidence of APC mutations in non-FAP colorectal cancer.
METHODS—The APC gene was analysed in 190 unrelated FAP and 15 non-FAP colorectal cancer patients using denaturing gradient gel electrophoresis, the protein truncation test, and direct sequencing.
RESULTS—Chain terminating signals were only identified in patients belonging to the FAP group (105 patients). Amino acid changes were identified in four patients, three of whom belonged to the non-FAP group of colorectal cancer patients. Genotype-phenotype correlations identified significant differences in the nature of certain extracolonic manifestations in FAP patients belonging to three mutation subgroups.
CONCLUSIONS—Extended genotypephenotype correlations made in this study may have the potential to determine the most appropriate surveillance and prophylactic treatment regimens for those patients with mutations associated with life threatening conditions. This study also provided evidence for the pathological nature of amino acid changes in APC associated with both FAP and non-FAP colorectal cancer patients.


Keywords: familial adenomatous polyposis; genotype-phenotype; familial colorectal cancer     

Proliferation and apoptosis in proliferative lesions of the colon and rectum

 Classically, neoplasia has been considered to be primarily a disturbance in the regulation of proliferation, but it is now clear that programmed cell death is dysregulated as well as proliferation. The genes that are implicated in the regulation of these processes, such as p53, c-myc and bcl-2, are often also altered in neoplasms. We have studied proliferation and programmed cell death in hyperplastic polyps, adenomas, carcinomas in adenomas and adenocarcinomas of the colorectum, using the MIB-1 antibody which recognizes the Ki-67 proliferation related antigen, and an in situ nick-end labelling procedure for histochemical labelling of proliferating and apoptotic cells. In addition, immunohistochemistry was used to study the expression of the p53, c-myc and bcl-2 proteins. The material studied consisted of 12 samples of normal mucosa, 8 hyperplastic polyps, 39 adenomas with different degrees of dysplasia and including 3 that carried a carcinoma, and 10 adenocarcinomas, all formalin fixed and paraffin embedded. The Ki-67 index indicated that proliferation increased progressively in hyperplasia, through different degrees of dysplasia in adenoma, to reach the highest level (Ki-67 index of 50%) in adenocarcinoma. Apoptosis also increased in hyperplastic polyps and in adenomas, but decreased significantly in adenocarcinomas. p53 Labelling was seen in 77% of the carcinomas but in only 3% of the adenomas. Expression of c-myc increased in adenomas and carcinomas. Furthermore, a shift from predominantly nuclear to predominantly cytoplasmic expression was seen in progressive neoplasms. Expression of bcl-2 was increased in an occasional hyperplastic polyp, but was increased markedly in almost all adenomas. Strikingly, in the adenomas with a carcinoma, the carcinoma showed weaker bcl-2 expression than the adenoma. In 20% of the carcinomas some bcl-2 staining was seen but this was less extensive than in the adenomas. Our findings indicate that in the progression from adenoma to carcinoma both increased proliferation and decreased apoptosis occur. This is paralleled by an increased expression of p53 and an increased and predominantly cytoplasmic expression of c-myc, but a decreased expression of bcl-2. This decreased bcl-2 expression does not lead to an increase in apoptotic activity. Received: 16 January 1997 / Accepted: 10 March 1997     

Germline mutations in APC and MUTYH are responsible for the majority of families with attenuated familial adenomatous polyposis

A small fraction of families with familial adenomatous polyposis (FAP) display an attenuated form of FAP (AFAP). We aimed to assess the presence of germline mutations in the MUTYH and adenomatous polyposis coli (APC) genes in AFAP families and to compare the clinical features between the two causative genes. Families with clinical AFAP were selected from the Dutch Polyposis Registry according to the following criteria: (a) at least two patients with 10-99 adenomas diagnosed at age >30 years or (b) one patient with 10-99 adenomas at age >30 years and a first-degree relative with colorectal cancer (CRC) with a few adenomas, and, applying for both criteria, no family members with more than 100 polyps before the age of 30 years. All probands were screened for germline mutations in the APC and MUTYH genes. Twenty-five of 315 Dutch families with FAP (8%) met our criteria for AFAP. These families included 146 patients with adenomas and/or CRC. Germline APC mutations were identified in nine families and biallelic MUTYH mutations in another nine families. CRC was identified at a mean age of 54 years (range 24-83 years) in families with APC and at 50 years (range 39-70 years) in families with MUTYH (p = 0.29). APC and biallelic MUTYH mutations are responsible for the majority of AFAP families. Based on our results and those reported in the literature, we recommend colonoscopy once every 2 years in AFAP families, starting surveillance from the late teens in APC mutation carriers and from age 20-25 years in biallelic MUTYH mutation carriers.     

Immunohistochemical analysis of biliary tract lesions.

The distinction among inflammatory, benign, and malignant lesions of the biliary tract can at times be difficult. Several methods have been used, including immunohistochemistry (IHC), with variable success. We evaluated a panel of IHC stains to determine their utility in discriminating between bile duct lesions. Formalin-fixed, paraffin-embedded 4-microm sections from 12 inflammatory lesions, 10 bile duct adenomas, and 13 bile duct carcinomas were immunostained using a modified avidin-biotin-complex technique after epitope enhancement using antibodies for p53, Ki-67, and bcl-2. For p53 and bcl-2, greater than 1% of cells staining positive was interpreted as positive. The proliferation index was calculated by determining the number of Ki-67-positive cells in a 1000 cell count. In the inflammatory group, 0 of 12 reacted with anti-p53, 2 of 12 were positive with anti-bcl-2, and the proliferation index with was 22.9% +/- 3.9%. Two of 10 bile duct adenomas showed reactivity with anti-bcl-2, and none were decorated with anti-p53 or Ki-67. In the carcinoma group, 6 of 13 were positive with anti-p53, 9 of 12 were positive with anti-bcl-2, and the proliferation index was 35.3% +/- 5.5%. The proliferation rates differed significantly between groups (P < 0.05). The presence of bcl-2 and p53 immunoreactivity coupled with a high proliferative rate in a biliary tract lesion suggests a malignant process. A panel using these antibodies may be useful in difficult cases.     

Familial adenomatous polyposis associated with multiple adrenal adenomas in a patient with a rare 3' APC mutation

Familial adenomatous polyposis (FAP) is characterised by hundreds of colorectal adenomas. Endocrine neoplasms have occasionally been reported, as have gastric polyps, which are usually hamartomatous in the fundus of the stomach and adenomatous in the antrum. A 57 year old man with colorectal, gastric, and periampullary adenomatous polyposis, in association with three bilateral adrenocortical adenomas, is presented. Mutation screening showed a 5960delA germline mutation in the adenomatous polyposis coli (APC) gene predicted to lead to a premature stop codon. This mutation was found in three of the four children of the patient. Western blot analysis of a lymphoblastoid cell line derived from the patient failed to detect any truncated APC polypeptide. This rare 3' mutation is responsible for an unusually complex and late onset phenotype of FAP.


Keywords: familial adenomatous polyposis; APC mutation; adrenal adenoma     

Different expression of Bcl-2 protein in gastric adenomas and carcinomas

In order to cast light on the possible role of bcl-2 protein (Bcl-2) expression In gastric tumorigenesis, 33 cases of gastric adenomas and carcinomas originating from the same stomachs were immunohistocnemically investigated for Bcl-2 protein (Bcl-2) expression, accumulation of p53 protein and cell proliferation as determined by the KI-67 labeling index (LI). Bcl-2 expression was detected in 24/33 (72.7%) adenomas and in 6/33 (18.2%) carcinomas, the difference being statistically significant (P=0.0O01). Only 4 of 33 (12.1%) cases exhibited expression in both adenoma and carcinoma lesions in the same stomachs. ImmunoreactMty was decreased in areas of cellular and structural atypia in adenoma lesions ( P <0.008), and appeared to be positively linked to the tumor progression and the degree of differentatlon in carcinomas, although It did not reach statistical significance. Accumulation of p53 protein was rare In the adenomas but was found in 15/33 (45.5%) of carcinoma lesions, with a significant dissociation from Bcl-2 immunoreactivity. No apparent relation between Ki-67 U and either adenoma grading or carcinoma typing was noted, although average KI-67 LI of the highest labeling areas in carcinomas was statistically higher than in adenomas ( P =0.0001). These results indicate that the regulation of Bcl-2 expression may differ between gastric adenomas and carcinomas, may be correlated with tumor dlfferentiathre features. In addition, p53 accumulation may play an Important role in the onset of malignancy.     

结直肠腺瘤及癌变组织中增殖基因和细胞凋亡调控基因的表达

目的 了解结直肠腺瘤癌变转化过程中细胞增殖与凋亡相关基因表达在癌变过程中的作用,并寻求判断腺瘤癌变的分子生物学指标。方法 采用免疫组织化学ＳＰ法检测６１份结直肠腺瘤癌变标本中ｐ５３、ｂｃ１－２、Ｆａｓ、ｂａｘ在腺瘤癌变区及腺瘤轻、重度异型增生区的表达;以Ｋｉ－６７指数和ＴＵＮＥＬ法计量细胞增殖率及凋亡率。结果 ６１份结直肠腺瘤癌变标本中ｐ５３蛋白在腺瘤癌变区、重度异型增生区及轻度异型增生区的表达分别为６７．２％、５７．４％和１９．７％;ｂｃ１－２蛋白的表达分别为７７．１％、８０．３％和４９．２％;腺瘤癌变区、重度异型增生区及轻度异型增生区的平均ｋｉ－６７指数分别为３８．４％、３６．５％和１６．１％;平均细胞凋亡指数分别为２．２％、１．９％及０．５％。ｐ５３蛋白、ｂｃ１－２蛋白的表达和Ｋｉ－６７指数、细胞凋亡指数     

Comparative Analysis of Cells with Combined Apoptosis and Proliferation Markers in Thyroid Tissue Specimens from Patients with Cancer,Adenoma, and Autoimmune Diseases

Combined phenotypes of cells with membrane and intracellular expression of apoptosis and proliferation regulation markers (p53, bcl-2, CD95, CD95L, Ki-67) were studied by flow cytometry of cell suspension from thyroid tissue specimens from patients with autoimmune diseases, adenoma, and thyroid cancer. The incidence of cell groups with phenotypes p53/Ki-67, p53/CD95, bcl-2/Ki-67, bcl-2/CD95, CD95/Ki-67, p53/CD95L, CD95/CD95L, and bcl-2/CD95L was evaluated and the density of receptor distribution on/in each cell group are presented. Patients with autoimmune diseases had high incidence of cells with phenotypes p53/Ki-67, p53/CD95, bcl-2/Ki-67, bcl-2/CD95, CD95/Ki-67, p53/CD95L, CD95/CD95L, and bcl-2/CD95L; cells with the bcl-2/CD95 phenotype were the most incident. Patients with thyroid adenoma had high levels of cells with p53/CD95L phenotype, while patients with thyroid cancer had significantly lower levels of p53 expression in the p53/CD95L cell group. The density of CD95L receptors on CD95/CD95L-positive cells was 4-7-fold higher in patients with thyroid tumors; the density of CD95L receptors on CD95/CD95L cells was maximum in thyroid adenoma and minimum in thyroid cancer. These data indicate differences in the expression of apoptosis and proliferation markers in thyroid adenoma, cancer, and autoimmune diseases. Analysis of the expression of these markers in the above diseases can be useful for differential diagnosis.     

High-grade pancreatic intraepithelial neoplasia in a patient with familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is caused by mutation of the adenomatous polyposis coli (APC) gene and is characterized by multiple colorectal adenomas and tumors of other organs and sites. A 58-year-old woman with FAP syndrome and previous total colectomy presented for routine follow-up examination. Abdominal ultrasound and subsequent endoscopic evaluation revealed ampullary and duodenal polyps, as well as inhomogeneity of the pancreatic head. A pancreaticoduodenectomy confirmed multiple duodenal adenomas. In addition, high-grade pancreatic intraepithelial neoplasia (PanIN-3) was found in the smaller pancreatic ducts. Pancreatic precancerous lesions have only rarely been described in FAP, including 2 pancreatic duct adenomas and 2 intraductal papillary mucinous neoplasms. A review of the world English literature revealed no reports of PanIN-3 in association with FAP. Further studies are required to determine if patients with FAP are at increased risk for pancreatic premalignant lesions.     

Proliferation in Adrenocortical Tumors: Correlation with Clinical Outcome and p53 Status

There appears to be a relationship between mitotic activity and malignant behavior in adrenocortical tumors, and carcinomas with a high mitotic index may have a poorer prognosis. This has been investigated further by quantifying and comparing the Ki-67 index using antibody MIB-1 in a series of 14 adrenocortical adenomas and 40 carcinomas. The levels have been correlated with survival and disease-free survival in carcinomas and with evidence of abnormal p53 expression as detected by immunohistochemistry. Nevertheless, many carcinomas have a low level of proliferation that may reflect varying abnormalities within the regulation of both cell division and apoptosis. Expression of bcl-2 protein, an inhibitor of apoptosis has therefore also been examined. The Ki-67 index in carcinomas was significantly higher than in adenomas, but below 4% there was overlap. There was no significant difference in survival between carcinomas with MIB-1 index <3% and those greater, but the lower group had significantly longer disease-free survival (p=0.02). There was no significant difference between p53 immunopositive and p53 immunonegative carcinomas. No tumor showed immunopositivity for bcl-2 protein. It is concluded that MIB-1 index may contribute additional prognostic information in adrenocortical tumors. Inhibition of apoptosis by bcl-2 does not appear to play a role in tumor growth.     

Oncogenic KRAS is not necessary for Wnt signalling activation in APC‐associated FAP adenomas

Recent studies have suggested that APC loss alone may be insufficient to promote aberrant Wnt/β‐catenin signalling. Our aim was to comprehensively characterize Wnt signalling components in a set of APC‐associated familial adenomatous polyposis (FAP) tumours. Sixty adenomas from six FAP patients with known pathogenic APC mutations were included. Somatic APC and KRAS mutations, β‐catenin immunostaining, and qRT‐PCR of APC, MYC, AXIN2 and SFRP1 were analysed. Array‐comparative genomic hybridization (aCGH) was also assessed in 26 FAP adenomas and 24 paired adenoma–carcinoma samples. A somatic APC alteration was present in 15 adenomas (LOH in 11 and four point mutations). KRAS mutations were detected in 10% of the cases. APC mRNA was overexpressed in adenomas. MYC and AXIN2 were also overexpressed, with significant intra‐case heterogeneity. Increased cytoplasmic and/or nuclear β‐catenin staining was seen in 94% and 80% of the adenomas. β‐Catenin nuclear staining was strongly associated with MYC levels (p value 0.03) but not with KRAS mutations. Copy number aberrations were rare. However, the recurrent chromosome changes observed more frequently contained Wnt pathway genes (p value 0.012). Based on β‐catenin staining and Wnt pathway target genes alterations the Wnt pathway appears to be constitutively activated in all APC‐FAP tumours, with alterations occurring both upstream and downstream of APC. Wnt aberrations are present at both the DNA and the RNA level. Somatic profiling of APC‐FAP tumours provides new insights into the role of APC in tumourigenesis. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Parathyroid neoplasms: clinical,histopathological, and tissue microarray-based molecular analysis

We studied 45 patients with typical and 8 with atypical parathyroid adenomas as well as 20 with parathyroid carcinomas. Clinical, pathological, and molecular analyses were conducted on all adenomas. Clinical data were analyzed for 20, histopathologic slides for 16, and tissue specimens for 8 patients with carcinoma. Molecular expression profiles were investigated by immunohistochemistry (IHC) for Ki-67, p53, mdm2, p21, Bcl-2, cyclin D1, and p27 on paraffin-embedded tissues arrayed on tissue microarrays. Trabecular growth and vascular, capsular, and soft-tissue invasion were characteristic of parathyroid carcinomas but not of typical adenomas. No adenomas recurred. Seventy-four percent of carcinomas recurred, most in the neck. Seventy-nine percent of patients with such illness died of disease after an indolent, multiply recurrent course responsive to repeated resections; the 5-year survival rate was 50%. High Ki-67 proliferative index was seen in 2% of adenomas and 25% of carcinomas, whereas p27 expression was present in 80% of adenomas and 18% of carcinomas. The molecular phenotype, p27(+)Bcl-2(+)Ki-67(-)mdm2(+), was observed in 76%, 29%, and 0% of typical and atypical adenomas and carcinomas, respectively. The complexity of molecular phenotypes increased with tumor aggressiveness. Parathyroid carcinoma is an aggressive disease with a propensity for multiple recurrences. It is characterized by capsular, vascular, and soft-tissue invasion. Recurrence portends poor outcome. Molecular markers, Ki-67 and p27, may distinguish parathyroid carcinoma from adenoma. The molecular phenotype, p27(+)Bcl-2(+)Ki-67(-)mdm2(+), appears to be unique to nonmalignant parathyroid tumors, and multimarker phenotypes are more complex in carcinomas.     

Apoptosis regulation differs between ulcerative colitis-associated and sporadic colonic tumors. Association with survivin and bcl-2

To clarify kinetics in ulcerative colitis (UC)-associated lesions, cell proliferation, apoptosis, and expression of apoptosis-inhibitory proteins were studied. Ki-67 labeling and survivin and bcl-2 expression were examined immunohistochemically in 22 low-grade dysplasias (LGDs), 25 high-grade dysplasias (HGDs), and 13 adenocarcinomas associated with UC, and for comparison in 21 sporadic adenomas with LGD, 22 sporadic adenomas with HGD, and 21 invasive adenocarcinomas. Apoptosis was studied with nick-end labeling and immunohistochemical analysis of single-stranded DNA. In UC-associated LGDs, Ki-67--positive cells were more frequent in the lower than the upper half of the crypt, related to bcl-2 expression, while in sporadic adenomas such cells were more common in the upper half. No difference in apoptosis was found between UC-associated LGDs and sporadic adenomas with LGD or between UC-associated HGDs and sporadic adenomas with HGD. However, UC-associated carcinomas exhibited a lower apoptotic count than their sporadic invasive counterparts. This seemed related to higher survivin expression without a significant difference between the 2 types of invasive lesions regarding bcl-2 levels. Apoptosis is less frequent in UC-associated than in sporadic invasive colon carcinomas, this being linked to elevated survivin expression. The control of apoptosis may be different in the 2 types of tumorigenesis.     

Carcinoma of the ampulla of Vater associated with or without adenoma: a clinicopathologic analysis of 198 cases with reference to p53 and Ki-67 immunohistochemical expressions.

Adenomatous areas are found frequently within or in the vicinity of carcinoma of the ampulla of Vater. This makes definite diagnosis difficult in the preoperative examination. The adenoma-carcinoma development hypothesis is generally accepted for colorectal tumors. Recently, a genetic alteration model during colorectal tumor development has attracted much attention, leading to various studies. We studied clinicopathologic features, prognostic factors, and the alteration of the p53 tumor suppressor gene using p53 immunohistochemical staining in pure adenomas, pure carcinomas, and carcinomas with adenomatous areas. A proliferative activity of the tumors using Ki-67 was also evaluated. Nine cases of pure adenoma and 198 cases of carcinoma of the ampulla of Vater were selected for this study. Among the 198 cases of thecarcinoma, 83 cases (42%) had adenomatous areas. Positivity of p53 immunohistochemical staining was 0% in pure adenomas, 36% in the adenomatous areas of carcinomas with adenomatous areas and 62% in the carcinomatous areas of carcinomas with adenomatous areas, and 56% in pure carcinoma. Accumulation of p53 protein and the Ki-67 labeling index revealed no significant difference in prognosis. The clinicopathological factors examined were as follows: degree of invasion of the surrounding tissue, such as duodenal wall; pancreatic parenchyma; the presence or absence of lymphatic permeation; venous invasion; perineural invasion; the presence of regional lymph node metastasis; and TNM stage. Each of the clinicopathological factors showed a significant difference. Multivariate analysis revealed strong predictors for a worse prognosis: presence of lymphatic permeation, invasion of the pancreas, and perineural invasion. In conclusion, our results are consistent with the adenoma--carcinoma development hypothesis. It would seem that the molecular events leading to p53 accumulation in neoplasms of the ampulla of Vater occur relatively late during the oncogenetic process. Moreover, we think it may be useful to refer to the p53 overexpression in the diagnosis of ampullary tumors.     

Immunohistochemical Analysis of the Cell Cycle-Associated Antigens Ki-67 and Retinoblastoma Protein in Parathyroid Carcinomas and Adenomas

The morphologic distinction between parathyroid carcinoma and adenoma can be a difficult diagnostic problem. We analyzed nuclear immunoreactivity for the cell cycle-associated antigen Ki-67 with monoclonal antibody (MAb) MIB-1 and for retinoblastoma (RB) protein with two polyclonal antisera in 24 parathyroid carcinomas and 35 adenomas, which were formalin fixed and paraffin embedded to determine if these antibodies could assist in distinguishing between carcinomas and adenomas. In addition, 10 cases of parathyroid hyperplasia and 5 cases of normal parathyroids were examined as control tissues. The Ki-67 labeling index was significantly higher in parathyroid carcinomas compared to adenomas (7.1 ± 1.0% vs 2.4 ± 0.2%,p<0.001). No patient with a parathyroid adenoma, parathyroid hyperplasia, or normal parathyroid gland had a Ki-67 labeling index >5.3%. Analysis of the primary tumors from patients with recurrent carcinomas and from those with nonrecurrent carcinomas showed a higher mean Ki-67 labeling index (7.8 ± 1.5% vs 5.2 ± 1.1%), in the former group, although these differences were not statistically significant. The RB protein immunoreactivity was not useful in distinguishing between parathyroid carcinomas and adenomas in paraffin-tissue sections. These results indicate that nuclear immunoreactivity for the cell cycle-associated antigen Ki-67 may be another useful method to assist in distinguishing parathyroid carcinomas from adenomas.     

Immunoreactivity of p53, Ki-67, and Bcl-2 in oncocytic adenomas and carcinomas of the thyroid gland.

To analyze relevant factors of neoplastic transformation in oncocytic neoplasms of the thyroid, expression of p53, Ki-67, and bcl-2 has been studied in oncocytic carcinomas (n = 17) and compared with results obtained in oncocytic adenomas (n = 20). P53 protein accumulation was found immunohistochemically in 75% of the oncocytic adenomas (15 of 20) and 88% of the oncocytic carcinomas (15 of 17). Eight of 17 of the carcinomas (47%), but only 3 of the 20 adenomas (15%), showed nuclear p53 accumulation in more than 10% of the cells, mostly in a focal pattern. Ki-67 expression also differed significantly between adenomas and carcinomas. The median of Ki-67-positive cells was 12/10 high-power fields (HPF) for adenomas and 76/10 HPF for carcinomas (P < .001). Furthermore, metastatic carcinomas had a significantly higher Ki-67 positivity than nonmetastasized carcinomas (164/10 HPF v 42/10 HPF, P < .05). Bcl-2 immunohistochemistry showed a constantly positive reaction in normal thyroid tissue. In contrast, bcl-2 protein was not detected in most of the adenomas (70%) and carcinomas (76%). In conclusion, p53 protein and Ki-67 is more prevalent in oncocytic carcinomas than in oncocytic adenomas of the thyroid, indicating that these factors may be involved in the progression of oncocytic neoplasms in the thyroid. In contrast, loss of bcl-2 appears to be an early event in the formation of oncocytic neoplasms of the thyroid. Its importance for malignant transformation is, however, unclear.     

p53 and Ki-67 immunoreactivity and nuclear morphometry of 'carcinoma-in-adenoma' and adenoma of the gall-bladder

Twenty Intramucosal tumors of ‘carclnoma-ln-adenoma’ and 43 adenomas (39 pylorlc gland type, 4 Intestinal type) of the gall-bladder were studied to establish more precise hlsto-loglcal criteria of carcinoma or adenoma in cases of ‘carcinoma in pylorlc gland type adenoma’, to compare carcinoma in adenoma with pure, that is, without adenomatous components, carcinoma, and to confirm the benign nature of spindle cell foci in the adenomas. Ki-67 and p53 immunostaining and nuclear morphometry were used. Eight pure intramucosal cancers were used as controls. The formalin-fixed, paraffin-embedded sections were stained with p53 and Ki-67 antibodies. Spindle cell foci were observed only in the adenoma area of the pylorlc gland type, with a frequency of 23% in 39 adenomas, and of 45% in 20 tumors of carclnoma-Jn-adenoma. Ki-67 staining was negative in 129 of 130 spin-die cell foci examined, regardless of their size, and positive in only one focus (550 μm in size, Ki-67 index 0.2%). All of the spindle cell foci were negative for p53 stain. The Ki-67 positive index was 36.6 ±5.6% in the 8 pure carcinomas, and 12.5 ±1.9% in the carcinoma area of 16 tumors with carci-noma-in-adenoma, while it was 7.9± 1.7% and in the adenoma areas of 16 tumors with carcinoma-in-adenoma and 4.9 ± 0.5% in the 32 pure pyloric gland adenomas. The p53-protein overexpression was found in seven of eight pure intramucosal cancers, and in one of 16 cancer components of carcinoma-in-adenoma. However, it was not found in any of 16 adenoma components of carcinoma-in-adenoma, and 35 adenomas. Cells of the cancer tissue of carcinoma-in-adenoma showed a significantly larger nuclear area and a larger nuclear minor axis than those of the pyloric gland type adenomas, as well as other architectural and cytologic abnormalities differing from the features of adenomas. These results suggest that clustered spindle cells do not indicate a malignant transformation of adenoma cells and that carcinomas in carcinoma-in-adenoma are different from pyloric gland type adenomas in terms of morphology and proliferative activity. Moreover, the results of the present study indicate that carcinomas in carcinoma-in-adenoma are lower in malignancy than pure carcinomas, and that their genetic abnormality may differ from that of pure carcinomas.     

Apoptosis in human colorectal tumours: ultrastructure and quantitative studies on tissue localization and association with bak expression

 Apoptotic cell death in human tumours has been demonstrated by electron and light microscopy. In adenomas, fragmented and apoptotic nuclei and signs of phagocytosis have been observed close to the basement membrane. In carcinomas the characteristic structures were apoptotic bodies with small fragments of chromatin. DNA fragmentation was shown by in situ end-labelling. Quantitative assessment of apoptosis and proliferation revealed a high apoptotic index (AI) in all types of adenoma (tubular: 1.77±0.35%, tubulovillous: 2.38± 0.41%; villous: 3.3±0.39%) as well as loss of compartmentalization of proliferating and dying cells. In carcinomas a shift towards proliferation was evident, as shown by lower AIs than in adenomas (0.9±0.68% and 1.1±0.12% for moderately and poorly differentiated tumours), higher Ki67 indices (38.32±2.23% and 57± 3.89%, respectively) and higher mitosis (0.9±0.56% and 1.21±0.17%, respectively). However, apoptosis was observed in all tumours and is available as a target for therapeutic intervention. Expression of the apoptosis related proteins bcl-2 and bak also reflected loss of compartmentalization. While bcl-2 did not show a consistent relationship to AI in tumour specimens, bak was positively correlated with apoptosis in 4 of 8 adenomas and 4 of 7 carcinomas, suggesting a role for this protein in the induction of apoptosis in a subset of tumours. Received: 22 July 1997 / Accepted: 27 October 1997