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.     

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.     

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.     

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.     

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.     

p53 expression patterns in colorectal adenomas and early carcinomas: A special reference to depressed adenoma and non-polypoid carcinoma

The purpose of the present study was to investigate the role of p53 in tumor progression of colorectal adenomas and early carcinomas, while especially focusing on flat tumors (depressed adenomas and non-polypoid carcinomas). Paraffin sections of 61 pure adenomas (33 polypoid, 28 depressed), 26 carcinomas in polypoid adenoma (CIA) and 63 pure carcinomas (36 polypoid, 27 non-polypoid) were examined for immunostaining using p53 monoclonal antibody (PAb 1801). All of the carcinomas were restricted to the mucosa. The number and distribution of the p53 positive tumor cells was evaluated, and then compared with tumor growth patterns and histologlcal features. The incidence of p53 expression in carcinomas (58% in CIA and 51% in pure carcinomas) was significantly higher than that in polypoid adenoma (27% in CIA and 21% in pure adenomas). However, the same incidence In depressed adenomas (51%) was significantly higher than In polypoid adenomas. No correlation in carcinomas was observed between p53 expression and cllnlco-pathologic data except for age. The distribution of p53 positive cells was different between adenomas and carcinomas. There tended to be fewer p53 positive cells in adenomas, even in depressed ones, than in carcinomas and they also tended to be confined to the superficial areas in adenomas, while they were diffusely distributed in carcinomas. Interestingly, the p53 positive cells were more frequently present in the deep mucosal areas than in the superficial areas of some non-polypoid carcinomas. In conclusion, the following hypotheses are suggested: (i) the increase of p53 expression from adenoma to carcinoma supports the hypothesis of an adenoma-carcinoma sequence in a polypoid tumor; (ii) the unique p53 expression in non-polypoid carcinoma suggests the existence of another type of carcinogenesis; and (iii) depressed adenomas are thus considered to have a high potential risk of carcinoma.     

Usefulness of endoscopic biopsy using immunostaining of p53 and Ki-67 in tumors of the ampulla of Vater

We investigated the diagnostic and prognostic value of p53 expression and proliferative activity, as indicated by the Ki-67, in endoscopic biopsy specimens. Specimens were immunologically stained with p53 and MIB-1 (Ki-67), and the MIB-1/Ki-67 labeling index (LI) was calculated. Classification of adenomas was based on findings of H&E-stained preparations into those with low- or high-grade atypia. Well-differentiated tubular and papillary adenocarcinomas were classified as carcinomas with low- or high-grade atypia. There were significant differences among the control and adenoma patients in MIB-1/Ki-67 LI (P < 0.05). No significant difference was identified between adenomas with high grade atypia and carcinomas with low grade atypia. The p53 expression was negative in all adenomas, but it was positive in 68.2% of carcinomas. The current study demonstrated that p53 protein expression in endoscopic biopsy specimens was of preoperative diagnostic value for carcinoma of the ampulla of Vater. The p53 protein positive tumors had a relatively higher malignant potential than p53 protein negative ones. The MIB-1/Ki-67 LI was useful in differentiating non-tumorous lesions from adenomas and adenomas with low- or high-grade atypia. The MIB-1/Ki-67 LI had a prognostic value because clinicopathological factors of carcinoma of ampulla of Vater correlated with MIB-1/Ki-67 LI.     

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.     

Analysis of mucin, p53 protein and Ki-67 expressions in gastric differentiated-type intramucosal neoplastic lesions obtained from endoscopic mucosal resection samples: a proposal for a new classification of intramucosal neoplastic lesions based on nuclear atypia

There are differing views between Western and Japanese pathologists on the use of histological criteria to classify gastrointestinal tumors. It is therefore a priority to create a new histological classification of the stomach in order to resolve the confusion. Expression patterns were examined of mucin (MUC2, CD10, MUC5AC, pyloric gland-type mucin), p53 protein, and Ki-67 in tumor cells according to the following new classification system for differentiated-type intramucosal neoplastic lesions of the stomach, based on nuclear atypia: borderline neoplasia (adenoma (including dysplasia), indefinite tumor of adenoma or low-grade cancer, and low-grade cancer) and definite carcinoma (intermediate cancer, and high-grade cancer). The resulting grades were: adenoma, 23; indefinite tumor for adenoma or low-grade cancer, 6; low-grade cancer, 28; intermediate cancer, 48; high-grade cancer, 20. While the frequency of intestinal-type borderline neoplasias was higher than that of definite carcinomas, the mixed-type of definite carcinomas occurred with higher frequency than borderline neoplasias. The p53 protein overexpression and the Ki-67-positive rate increased with an increase in the grade assigned according to the new classification. The correlated expression levels of p53 protein, Ki-67, and various mucins, support the conclusion that this classification of intramucosal neoplastic lesions is useful for obtaining a consensus diagnosis of gastric intramucosal neoplasia between pathologists and gastrointestinal clinicians.     

Clinical significance of Fhit expression in development of colorectal carcinoma of various macroscopic types

It is unclear how expression of the FHIT (fragile histidine triad) gene by the colorectal neoplasm correlates with histogenesis and progression of the disease. We studied the association between expression of Fhit protein and development of colorectal carcinoma (CRC). We also examined relations between Fhit protein expression, macroscopic type, Ki-67 labeling index (LI), and p53 overexpression in carcinoma in situ. We examined 27 colorectal adenomas, 82 carcinomas in situ and 21 invasive CRCs resected endoscopically or surgically. The carcinomas in situ comprised three macroscopic types: polypoid (n=27), superficial (flat elevated, n=27; depressed, n=10) and granulonodular laterally spreading tumor (G-LST, n=23). Fhit, Ki-67, and p53 overexpression were examined immunohistochemically. Levels of Fhit protein were lower in invasive CRC than in adenoma and carcinoma in situ (p<0.01). In carcinoma in situ, reduced Fhit expression was observed in 7 of 22 (31.8%) polypoid types, 13 of 27 (48.1%) superficial flat elevated types, 8 of 10 (80%) superficial depressed types and 7 of 23 (30.4%) G-LST. Frequencies of reduced Fhit expression were significantly higher in the polypoid type and G-LST lesions than in the depressed type (p<0.05). Reduced expression of Fhit protein was related significantly to Ki-67 LI and p53 overexpression in carcinoma in situ (p<0.01). The present findings suggest that reduced expression of Fhit protein is related to development of colorectal neoplasm. Polypoid CRC and G-LST appear to differ from superficial depressed CRC in terms of Fhit expression.     

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     

Genetic Changes in Chromosomes 1p and 17p in Thyroid Cancer Progression

Little is known about the genetic alterations that occur during the progression of thyroid neoplasms. To understand better the biology of thyroid tumors, we investigated several genetic loci in benign and malignant thyroid neoplasms. Forty-one thyroid tumors (6 adenomas, 16 papillary, 14 follicular, and 5 anaplastic carcinomas) were studied. Normal and tumor cells were microdissected from paraffin-embedded tissues. DNA was used for polymerase chain reaction-based loss of heterozygosity (LOH) analysis with the following markers: D1S243 (1p35–36), D1S165 (1p36) and D1S162 (1p32), TP53 (17p13), and INT-2 (11q13). Immunohistochemistry for Ki-67 was performed. The Ki-67 labeling index (LI) was the percentage of positive tumor cells. LOH at 1p was seen in 2 of 5 (40%) informative cases of anaplastic carcinoma (2 of 2 at D1S162 and 1 of 2 at D1S165) and in 2 of 11 (18%) informative cases of follicular carcinoma (2 of 7 at D1S243, 2 of 7 at D1S165, and 1 of 6 at D1S162). One anaplastic (20%) and two follicular carcinomas (14%) had LOH in at least two of the 1p loci analyzed. None of the adenomas and papillary carcinomas had LOH at these loci. LOH at 17p and 11q13 were infrequent. Ki-67 LI was 1.4, 7, 16, and 65% in adenomas, papillary, follicular, and anaplastic carcinomas, respectively. Allelic loss at 1p may occur in aggressive types of thyroid carcinoma and may be a marker of poor prognosis. LOH at 1p may represent a late genetic event in thyroid carcinogenesis. LOH at 17p and 11q13 (MEN gene locus) is uncommon in thyroid neoplasms.     

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.     

Adenomas of the gallbladder. Morphologic features, expression of gastric and intestinal mucins, and incidence of high-grade dysplasia/carcinoma in situ and invasive carcinoma

We report 201 gallbladder adenomas from 91 patients most of whom were adult females. Fifty-three (58%) patients had gallstones. In 83 (91%) patients the adenomas were single. One gallbladder had 102 adenomas. Histologically, 165 (82%) of 201 adenomas were classified as pyloric, 28 (14%) as intestinal, 5 (2.4%) as foveolar, and 3 (1.4%) as biliary. Two patients had intestinal-type adenomas coexisting with biliary papillomatosis. Twenty-eight percent of pyloric gland adenomas contained squamoid morules. Two pyloric gland adenomas were composed predominantly of columnar oxyphil cells. High-grade dysplasia/carcinoma in situ was identified in 44 (27%) of 165 pyloric gland adenomas and low-grade dysplasia in 25 (15%) of 165. However, only 2 (1%) invasive adenocarcinomas, both of intestinal type, arose in pyloric gland adenomas. Both patients survived more than 5 years. Intestinal-type adenomas were classified as tubular, papillary, and tubulopapillary. High-grade dysplasia/carcinoma in situ was recognized in 13 (46%) of 28 intestinal adenomas. However, only 1 (3.5%) invasive adenocarcinoma with biliary phenotype arose in an intestinal-type adenoma. Foveolar adenomas showed low-grade dysplasia, and biliary adenomas were composed of columnar cells similar to the normal biliary cells of the gallbladder. None of these tumors progressed to adenocarcinoma. MUC5AC and MUC6 labeled 44 (95%) of 46 pyloric gland adenomas, whereas CDX2 was positive in 14 (78%) of 18 intestinal adenomas and MUC2 in 6 (33%) of 18. MUC5AC and MUC6 labeled 2 foveolar adenomas, and 2 biliary adenomas expressed only CK7. The immunophenotype of gallbladder adenomas justifies their classification into pyloric, intestinal, foveolar, and biliary. Our results indicate that adenomas of the gallbladder play a minor role in the pathway of gallbladder carcinogenesis.     

Neoplasia of the ampulla of Vater. Ki-ras and p53 mutations.

Eleven tumors of the ampulla of Vater (5 stage IV and 2 stage II adenocarcinomas, 1 stage II papillary carcinoma, 1 neuroendocrine carcinoma, and 2 adenomas, one with foci of carcinoma) were examined for Ki-ras and p53 gene mutations by single-strand conformation polymorphism analysis and direct sequencing of polymerase chain reaction-amplified DNA fragments. Ki-ras mutations were found in one adenocarcinoma and in the adenoma with foci of carcinoma, both involving mainly the intraduodenal bile duct component of the ampulla. Seven cases showed p53 gene mutations: four advanced-stage adenocarcinomas, the papillary carcinoma, the neuroendocrine carcinoma, and the adenoma with foci of carcinoma. Nuclear accumulation of p53 protein was immunohistochemically detected in the morphologically high-grade areas of the five cancers harboring a p53 gene missense point mutation. The adenomas, the two frame shift-mutated cancers, and the adenomatous and low-grade cancer areas of mutated carcinomas were immunohistochemically negative. Our data suggest that in ampullary neoplasia 1) p53 mutations are common abnormalities associated with the transformation of adenomas and low-grade cancers into morphologically high-grade carcinomas, and 2) Ki-ras mutations are relatively less frequent and might be restricted to tumors originating from the bile duct component of the ampulla.     

Evaluation of malignant grade of salivary gland tumors: Studies by cytofluorometric nuclear DNA analysis, histochemistry for nucleolar organizer regions and immunohistochemistry for p53

Investigations were carried out in 109 surgically resected salivary gland tumors, 46 adenomas and 63 carcinomas for nuclear DNA content by cytofluorometry, nucleolar organizer regions (AgNOR) by histochemistry and nuclear p53 accumulation by immunohistochemistry. The mean nuclear DNA content and the incidence of aneuploidy as well as the mean number of the AgNOR in carcinomas were significantly higher than those in adenomas. In the DNA histogram pattern, the aneuploidy frequently appeared in high grade carcinoma groups (adenocarcinoma, salivary duct carcinoma, carcinoma in pleomorphic adenoma and oncocytic carcinoma). Immunohistochemical nuclear p53 accumulation was observed in 15 tumors, of which ten showed DNA aneuploidy. In high grade carcinomas, DNA aneuploidy was closely related to p53 immunoreactivity. This study revealed that combined cytofluorometric nuclear DNA analysis, histochemical AgNOR count and immunoreactivity for p53 might be quite useful for evaluating the malignant grade of salivary gland tumors.     

Expressions of cell-cycle regulatory gene products in conventional gastric adenomas: possible immunohistochemical markers of malignant transformation

In 54 lesions of gastric adenomas consisting of 31 low-grade adenomas (LGAs) and 23 high-grade adenomas (HGAs), 28 intramucosal carcinomas (IMCs), and 23 carcinomas invading the submucosa (SMCs), the expression of cell-cycle regulatory gene products (p53, p21/waf1, p27/kip1, and Ki-67) was studied using immunohistochemical techniques. Several lesions were also analyzed by the fluorescence in situ hybridization method. The overexpression of p53 was found in no LGAs and in 9% of HGAs, whereas a considerable number of cases showed an overexpression in IMCs (39%) and SMCs (43%). A reduced expression of p21/waf1 was present in only 4% of HGAs. Superficial eccentric positivity was present in all LGAs and 74% of HGAs, whereas it was present in 46% of IMCs and 4% of SMCs. P53-positive and p21/waf1-negative lesions, which were supposed to have a mutated p53 gene, were observed in no LGAs, in 4% of HGAs, in 11% of IMCs, and in 26% of SMCs. The expression of cyclin E was more frequently present in carcinomas than in adenomas. However, no high expression of cyclin E was observed among the adenomas. A reduced expression of p27/kip1 was encountered more frequently in carcinoma than adenoma. By a semiquantitative evaluation comparing adenoma and carcinoma in the same stomach, the increased degrees of both p21/waf1 and cyclin E were highlighted. A chromosome gain was detected among 7% of the adenomas and 85% of the carcinomas. In conclusion, the expressions of p53, p21/waf1, p27/kip1, and cyclin E were considered to be of great value for estimating the dysplastic progression of gastric adenomas. Especially, various aspects of protein expression, including its distribution and semiquantitative evaluation of positive cells, and a combined analysis with several proteins, may thus be useful as possible markers of dysplastic evolution in gastric adenomas.     

Assessment of Mitotic Activity in Pituitary Adenomas and Carcinomas

Assessment of mitotic activity represents one of the oldest and most routinely used histopathologic methods of evaluating the biological aggressiveness of human tumors. In the case of pituitary tumors, however, the relevance of this approach as a means of gaging tumor behavior remains ill-defined. In this article, the relationship between the mitotic index and biological aggressiveness of pituitary tumors was evaluated in a series of 54 pituitary adenomas and 6 primary pituitary carcinomas. All tumors were fully classified by immunohistochemistry and electron microscopy; adenomas were further stratified on the basis of their invasion status, the latter being defined as gross, operatively, or radiologically apparent infiltration of dura or bone. Mitotic figures were present in 11 tumors, 10 being either invasive adenomas or pituitary carcinomas. A significant association between the presence of mitotic figures and tumor behavior was noted, as evidenced by progressive increments in the proportion of cases expressing mitotic figures in the categories of noninvasive adenoma, invasive adenoma, and pituitary carcinoma (3.9, 21.4, and 66.7%, respectively; Fisher’s exact test, two-tailed,p<0.001). The mitotic index, however, appeared to be a less informative parameter, being extremely low in all cases (mean=0.016%±0.005 [±SEM]). Although the mean mitotic index in pituitary carcinomas (0.09%±0.035) was significantly higher than the mean mitotic index of either noninvasive adenomas (0.002%±0.002) or invasive adenomas (0.013%±0.005), no practical threshold value capable of distinguishing these three groups was evident. Comparison of the mitotic index with Ki-67 derived growth fractions in these tumors revealed a significant but weak linear correlation (r=0.41,p<0.01). These data suggest that when, mitotic figures are present, they do provide some indication of the behavior and invasive potential of pituitary tumors. For routine diagnostic purposes, however, the discriminating power of this parameter is somewhat limited, being superseded by alternative and more informative methods of growth fraction determination such as that provided by the Ki-67 immunolabeling.     

Usefulness of proliferative activity, DNA ploidy pattern and p53 products as diagnostic adjuncts in colorectal adenomas and intramucosal carcinomas

Although numerous studies have assessed the biologic parameters of tumors, measurement of these parameters has had, to date, little impact on histologic diagnosis. Furthermore, analysis of a single parameter is insufficient to evaluate tumor malignant potential. In the present study, cell proliferation, DNA ploidy and p53 product were analyzed to objectify the tumor malignant potential in colorectal adenomas and intramucosal carcinomas. Sixty-one adenomas and 49 intramucosal carcinomas were studied using immunohistochemical analysis of Ki-67 and p53, silver-staining nucleolar organizer region (AgNOR) stain and DNA ploidy in fresh samples. Intramucosal carcinoma exhibited a greater Ki-67-positive rate and AgNOR count than the adenomas, although these parameters varied widely among samples. The incidence of aneuploidy and p53 over-expression in colorectal intramucosal carcinomas was significantly higher than in colorectal adenomas. These results indicate that DNA aneuploidy and p53 accumulation are the most reliable parameters for distinguishing benign and malignant lesions.     

Intraductal tubular carcinoma, intestinal type, of the pancreas

Presented herein is an unusual case of intraductal tubular carcinoma, intestinal type, of the pancreas. This tumor was characterized by intraductal adenoma with a few malignant foci, and also by entire involvement of the main pancreatic duct and no involvement of its branches. A 67-year-old man was admitted to hospital because of abdominal pain. On endoscopy and endoscopic retrograde cholangiopancreatography, irregular pancreatic duct was seen. No mucus secretion was observed on endoscopy. Because a biopsy showed tubular atypical cells, pancreato-duodenectomy was performed. Grossly, the entire main pancreatic duct had intraductal tumor, sparing its branches. No intraductal mucus was noted. Microscopically, the entire main pancreatic duct had proliferation of tubular adenomatous tumor without secretory mucins. Goblet cells were present in some areas. No pyloric type tubules were recognized. Malignant transformation was present in a few areas. No invasive features were recognized. On mucin histochemistry the tumor cell cytoplasm contained a little or no neutral and acidic mucus, and no secretory mucins were recognized. Immunohistochemically, the tumor cells were positive for cytokeratins (CK), CK 8, 9, 18, 19 and 20, epithelial membrane antigen, CDX2, carbohydrate antigen 19-9, and Ki-67 (labeling 30%), MUC2, MUC5AC and MUC6, and CD10. The tumor cells were negative for C-erbB2, MUC1, trypsin, pancreatic amylase and pancreatic lipase. The tumor cells were negative for p53 protein, but the malignant foci were positive for p53 protein and had high Ki-67 antigen (labeling 60%). The patient was free of disease 4 years after the operation. In summary, presented here is an extremely rare case of intraductal tubular carcinoma, intestinal type, showing focal malignant foci.