Clinical Significance of Molecular Expression Profiles of Hürthle Cell Tumors of the Thyroid Gland Analyzed via Tissue Microarrays

Hürthle cell tumors are rare thyroid neoplasms for which disease biology is poorly understood and diagnosis of carcinoma can be challenging. The aim of the study was to characterize molecular expression profiles of Hürthle cell tumors and to determine the clinical significance of identified phenotypes. Paraffin-embedded tissue cores of normal thyroid (n = 18), and histopathologically well-defined Hürthle cell adenomas (n = 27), Hürthle cell tumors of unknown malignant behavior (n = 7), and minimally (n = 14) and widely (n = 21) invasive Hürthle cell carcinomas were arrayed in triplicate on tissue microarrays. Expression profiles of p53, mdm-2, p21, Bcl-2, cyclin D1, and Ki-67 were detected by immunohistochemistry and correlated with clinicopathological data and patient outcome using standard statistical methodology. Median follow-up time was 8 years. High Ki-67 proliferative index was evident only in the clinically aggressive widely invasive Hürthle cell carcinomas and was associated with significantly reduced relapse-free (P = 0.001) and disease-specific (P < 0.001) survival. The molecular phenotype of Hürthle cell tumors, independent of histopathological subtype diagnosis, was characterized by p53(-), mdm-2(+), p21(+/-), cyclin D1(-), and Bcl-2(+/-). Normal thyroid tissue demonstrated a p53(-), mdm-2(-), p21(-), cyclin D1(-), and Bcl-2(+) phenotype. The Bcl-2(+) phenotype was associated with improved relapse-free survival (P = 0.04) and disease-specific survival (P = 0.01) in widely invasive carcinomas and the Ki-67(+)/Bcl-2(-) phenotype was associated with the diagnosis of widely invasive Hürthle cell carcinoma (P < 0.001). This study demonstrates that tissue microarray-based profiling allows identification of molecular markers that are associated with patient prognosis. High Ki-67 proliferative index was associated with adverse outcome in Hürthle cell neoplasms. Together with down-regulation of Bcl-2, high Ki-67 proliferative index may be useful for diagnosing widely invasive Hürthle cell carcinomas. Molecular alterations in the p53 pathway play a role in Hürthle cell tumorigenesis, but other unidentified molecular changes seem to be required to induce the malignant phenotype.     

Adrenocortical adenoma and carcinoma: histopathological and molecular comparative analysis.

We compared histomorphological features and molecular expression profiles of adrenocortical adenomas (ACAd) and carcinomas (ACCa). A critical histopathological review (mean, 11 slides per patient) was conducted of 37 ACAd and 67 ACCa. Paraffin-embedded tissue cores of ACAd (n = 33) and ACCa (n = 38) were arrayed in triplicate on tissue microarrays. Expression profiles of p53, mdm-2, p21, Bcl-2, cyclin D1, p27, and Ki-67 were investigated by immunohistochemistry and correlated with histopathology and patient outcome using standard statistical methodology. Median follow-up period was 5 years. Tumor necrosis, atypical mitoses, and >1 mitosis per 50 high-power fields were factors that were highly specific for ACCa (P <.001). Number (0 to 4) of unfavorable markers [Ki-67 (+), p21 (+), p27 (+), mdm-2(-)] expressed was significantly associated with mitotic activity and morphologic index (i.e., number of adverse morphologic features) and highly predictive of malignancy (P <.001). Ki-67 overexpression occurred in 0 ACAd and 36% ACCa (P <.001) and was significantly associated with mitotic rate and unfavorable morphologic index (P <.001). Tumor necrosis, atypical mitoses, >5 mitoses per 50 high-power fields, sinusoidal invasion, histologic index of >5, and presence of more than two unfavorable molecular markers were associated significantly with metastasis in ACCa. Well-established histopathologic criteria and Ki-67 can specifically distinguish ACCAd from ACCa. Tumor cell proliferation (Ki-67) correlates with mitotic activity and morphologic index. Tumor morphology is a better predictor of metastatic risk in ACCa than current immunohistochemistry-detected cell cycle regulatory and proliferation-associated proteins.     

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.     

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.     

Expression of mdm2 and p53 in epithelial neoplasms of the colorectum.

AIMS: To evaluate the respective roles of mdm2 (murine double minute 2) and p53 in the development of colorectal carcinoma. METHODS: Formalin fixed, paraffin wax embedded tissues from 72 sporadic adenomas and 55 carcinomas were investigated by means of immunohistochemistry for mdm2 and p53. RESULTS: mdm2 was expressed weakly in 17 of 72 (23.6%) adenomas and in 14 of 55 (25.4%) carcinomas. p53 was expressed in 19 of 72 (26.4%) adenomas and in 23 of 55 (41.8%) carcinomas. Four adenomas and five carcinomas showed positive staining for both proteins. Overexpression of p53 in adenomas was associated with moderate and severe dysplasia but not with tumour size. No associations were found between the expression of mdm2 and either the degree of dysplasia or tumour size. In carcinomas, neither the expression of p53 nor mdm2 correlated with Dukes's stage, metastasis, or differentiation. No associations were found between the expression of p53 and mdm2 in either adenomas or carcinomas. CONCLUSIONS: Although mdm2 has been reported to be an oncogene, it does not appear to play a major role in the development of colorectal carcinoma.     

Aberrant p27kip1 expression in endocrine and other tumors.

The p27kip1 (p27) gene encodes an inhibitor of cyclin-dependent kinase activity. The expression of p27 protein in normal and neoplastic tissues was investigated by immunoblotting and immunohistochemistry. Immunoblotting studies detected a 27-kd protein band that was decreased in neoplastic pituitary tissues compared with normal pituitary. Immunostaining of 177 tissues showed abundant expression of p27 protein in normal tissues with decreased numbers of immunoreactive cells in adenomas and carcinomas in both endocrine and nonendocrine tissues. p27 expression was inversely related to the proliferation marker Ki-67 antigen detected with monoclonal antibody MIB-1. Parathyroid adenomas and hyperplasias had similar Ki-67 labeling indices; however, hyperplasias had threefold more p27-positive cells than parathyroid adenomas, suggesting that p27 immunostaining may be useful in distinguishing between these two conditions. These results indicate that there is widespread aberrant p27 expression in hyperplastic tissues and in benign and malignant neoplasms compared with normal tissues. Immunohistochemical analysis of p27 along with Ki-67 may be used to assess the biological behavior of various neoplasms, to classify hyperplastic and neoplastic tissues, and to study cell cycle regulation during tumor progression.     

Parafibromin and APC as Screening Markers for Malignant Potential in Atypical Parathyroid Adenomas

The identification of parathyroid carcinomas is based upon histopathological criteria in which an invasive growth pattern or distant metastasis is demonstrated. A dilemma arises when tumours present with atypical histopathological features but lack direct evidence of malignancy. Recently, reduced expression or loss of the tumour suppressor proteins parafibromin and adenomatous polyposis coli (APC) has been associated with parathyroid malignancy. We report results from APC and parafibromin expression analyses by immunohistochemistry and Western blot in five cases of atypical adenoma, a single case of carcinoma and 54 adenomas without atypical features. Complete loss of APC immunoreactivity and reduced expression of parafibromin was evident in two of the atypical adenomas and in the parathyroid carcinoma. By contrast, all adenomas displayed APC expression, including two cases with hyperparathyroidism 2 gene (HRPT2) mutations and loss of parafibromin expression. We conclude that loss of APC is a frequent molecular event in atypical adenomas and carcinomas, but not in adenomas. Following verification in an independent material, APC could become a valuable tool when assessing parathyroid tumours in the clinical setting. Furthermore, the molecular resemblance of atypical adenomas with carcinoma concerning parafibromin and APC expression indicates that atypical adenomas should be subjects to watchful follow-up.     

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.     

Galectin-3 expression in parathyroid carcinoma: immunohistochemical study of 26 cases

The diagnosis of parathyroid carcinoma (PC) is difficult and based on morphological features that are not totally reliable. Several molecular markers proved useful in the evaluation of PC, but their sensitivity, specificity, or both are rather low. With the aim of identifying a marker of malignancy in parathyroid tumors, we tested the expression of galectin-3 (Gal-3), a lectin expressed in several malignant tumors, including follicular carcinomas (but not adenomas) of the thyroid. Twenty-six PCs and 30 control parathyroid adenomas (PAs) were collected. The PCs had been diagnosed based on capsular/vascular invasion (26/26 cases), extraparathyroid infiltration (16), local recurrence (9), and distant metastases (6). All cases were immunohistochemically tested for Gal-3 and for other markers claimed to be useful in the differential diagnosis of parathyroid neoplasms, namely, Ki67, p27, and bcl2. Gal-3 was expressed by 24 of the PC (92.3%), but only 1 PA (3.3%) (P < .001). All metastasizing PCs were Gal-3-positive. As expected, the Ki67 proliferative index was higher in PCs (mean, 6.7%) than in PAs (1.9%); p27 was down-regulated in 61.5% of PCs and only 33.3% of PAs, whereas bcl2 was strongly positive in most PAs and in 38.5% of PCs. In a suspected PC, the association of Gal-3 with Ki67 (using a cutoff of 6% for the proliferative activity) appeared the best marker combination (sensitivity 96.2%, specificity 90%), and the profile Gal-3-positive/Ki67 >6% was unique to PCs. We conclude that Gal-3 immunostaining is a valuable tool to support a diagnosis of PC in highly proliferating (Ki67 >6%) tumors affecting a single parathyroid gland.     

Expression of c-erbB2, p53, Bcl-2, Bax,c-myc and Ki-67 in apocrine metaplasia and apocrine change within sclerosing adenosis of the breast

Molecular evidence has recently suggested a number of different pathways leading to the development of ductal carcinoma of the breast. The links between atypical ductal hyperplasia and low-grade ductal carcinoma in situ and lobular neoplasia and lobular carcinoma are well known pathologically, but high-grade in situ and invasive carcinomas appear to have a different biological oncogenetic pathway. Morphologically there is a similarity between apocrine cells and some cases of high-grade ductal carcinoma. In order to investigate this possibility a number of different biological markers known to occur in high-grade breast carcinomas were assessed in both apocrine metaplasia (APM) and a putative premalignant lesion called apocrine change within sclerosing adenosis (AA). In 64 cases of APM and 18 cases of AA we examined for expression of c-erbB2, p53, Bcl-2, Bax, c-myc and Ki-67 proteins using immunocytochemistry. c-erbB2 expression was seen in 55.6% of AA cases and in 10.9% of APM cases. p53 expression was detected in 27.8% of AA cases but only 1.6% of APM cases. All cases of AA and APM were negative for the anti-apoptotic protein Bcl-2, but all the APM and 33.3% of AA cases showed cytoplasmic positivity for Bax, a pro-apoptotic protein. All the cases of AA and APM were positive for c-myc oncoprotein, however, the mean percentage of nuclear positivity was 50% in AA and 37% in cases of APM cases. The mean percentage positivity for Ki-67, a proliferation associated antigen, was 3.6% in AA and 1.3% in APM. The results indicate that a subset of breast lesions containing APM epithelium show abnormal oncoprotein and apoptosis-related protein expression and have a higher proliferation rate.     

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.     

Differential expression of the calcium sensing receptor and combined loss of chromosomes 1q and 11q in parathyroid carcinoma

Malignant transformation of parathyroid tumours is rare. Nevertheless, this small subset of malignant tumours often creates diagnostic and therapeutic problems. In this work, the morphological characteristics of 26 primary parathyroid carcinomas and seven metastases have been studied. Furthermore, immunohistochemical expression profiles for the calcium sensing receptor (CASR), cyclin D1 (CCND1), and Ki-67 were determined for parathyroid carcinomas and compared with adenomas and hyperplasias using a tissue microarray. Loss of heterozygosity (LOH) of the chromosome 1q region containing the HRPT2 gene and chromosome 11q (MEN1) was determined in the carcinomas. In contrast to the adenomas and hyperplasias, 31% of carcinomas demonstrated down-regulation of CASR. A significant correlation was found between CASR expression and the Ki-67 proliferation index. Chromosome 1q and chromosome 11q LOH were found in 12 of 22 (55%) and 11 of 22 (50%) carcinomas tested, respectively. Combined 1q and 11q LOH was seen in 8 of 22 (36%) carcinomas, in contrast to the low percentage of LOH reported in both regions in adenomas. In conclusion, this study demonstrates that combined 1q and 11q LOH in parathyroid tumours is suggestive of malignant behaviour. Strong down-regulation of the CASR protein is seen in a proportion of parathyroid carcinomas with a high proliferation index.     

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

Twenty lntramucosal tumors of ‘carclnomaln-adenoma’ and 43 ademas (39 pylorlc gland type, 4 Intestinal type) of the gall-bladder were studied to establish more precise histo-logical criteria of carcinoma or adenoma In cases of ‘carcinoma In pyforic gland type adenoma’, to compare carcinoma In adenoma with pure, that Is, without adenomatous components, carcinoma, and to confirm the benign nature of spin-dle cell fd in the adenomas. Ki-67 and p53 immunostaining and nuclear morphomety were used. Eight pure intramucessl cancers were used as controls. The formalin-fixed, paraffln+mbedded sections were stained with p53 and Ki-67 antibodies. Splndle cell foci were observed only In the adenoma area of the pyloric gland type, wlth a frequency of 23% In 39 adenomas, and of 45% in 20 tumors of carclnoma-lrradenoma. Ki-67 staining was negative in 129 of 130 spin-die cell foci examlned, regardless of their size, and positive in only one focus (550 pm 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 cancer areas of 16 tumors with carcinoma-in-adenoma, while it was 7.9 ± 1.7% 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 over-expression was found in seven of eight pure intramucosal cancers, and in one of 16 cancer components of carclnoma-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 indmte a malignant transformation of adenoma cells and that carcinomas in carcinoma-in-adenoma are dtfferent from pylorlc gland type adenomas In terms of morphology and proliferative activity. Moreover, the results of the present study indicate that carcinomas In carcinoma-ln-adenoma are lower In malignancy than pure carcinomas, and that their genetic abnormaltty may differ from that of pure carcinomas.     

Morphogenesis of nonpolypoid colorectal adenomas and early carcinomas assessed by cell proliferation and apoptosis

Nonpolypoid neoplasms, as well as ordinary polypoid tumours, are occasionally found in the colorectum. To clarify whether cell kinetic status affects the macroscopic morphology of colorectal neoplasms, we investigated proliferative indices (PI), apoptotic indices (AI), and the expression of apoptosis-related gene products. We examined 110 colorectal neoplasms comprised of 36 polypoid, 38 flat elevated and 36 depressed tumours. According to WHO’s criteria these tumours consisted of 61 adenomas with low grade dysplasia (LGD), 30 adenomas with high grade dysplasia (HGD) and 19 carcinomas with submucosal invasion. Apoptotic cells were detected by TUNEL staining. Proliferating cells and apoptosis-related gene products were assessed by immunohistochemistry for Ki-67, p53, Bcl-2, and Bax antigens. AI were closely associated with macroscopic morphology in adenomas but not in carcinomas. PI were relatively constant among the three macroscopic types in adenomas and carcinomas. Median AI values of polypoid, flat elevated and depressed tumours were 1.8%, 2.1% and 4.6% for adenomas with LGD, 0.8%, 2.4% and 6.2% for adenomas with HGD and 2.9%, 4.0% and 3.6% for carcinomas, respectively. Overall PI were significantly higher in carcinomas than in adenomas with LGD, whereas AI were not different. Although the incidence of expression was significantly higher in carcinomas for p53 and in adenomas for Bcl-2 than the others, the expression of apoptosis-related gene products (p53, Bcl-2 and Bax) was similar among polypoid, flat elevated and depressed tumours. Macroscopic morphology of colorectal adenomas is determined by the apoptosis not by proliferation, and high apoptosis found in depressed adenomas implies their low net growth. Received: 1 July 1999 / Accepted: 17 January 2000     

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.     

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.     

Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors.

A total of 111 pulmonary neuroendocrine tumors comprising 13 typical carcinoids, five atypical carcinoids, 44 large-cell neuroendocrine carcinomas and 49 small-cell carcinomas were immunohistochemically studied for dysregulated cyclin B1 expression and disruption of the Rb/p16/cyclin D1 pathway (Rb pathway), and the results were correlated with tumor proliferation activity and clinical outcome. Overexpression of cyclins B1 and D1, respectively, was detected in no and 15% typical carcinoids, 20 and 20% atypical carcinoids, 84 and 32% large-cell neuroendocrine carcinomas, 84 and 10% small-cell carcinomas. Loss of Rb and p16 expression, respectively, was observed in no and 14% typical carcinoids, no and 40% atypical carcinoids, 49 and 18% large-cell neuroendocrine carcinomas, 84 and 8% small-cell carcinomas. In summary, 29% typical carcinoids, 20% atypical carcinoids, 78% large-cell neuroendocrine carcinomas and 93% small-cell carcinomas had Rb pathway aberrations. Rb pathway aberration was mostly attributed to Rb loss in small-cell carcinomas, while p16 loss and/or cyclin D1 overexpression besides Rb loss also played an important role in large-cell neuroendocrine carcinomas, while cyclin D1 overexpression was the only cause of Rb pathway aberration in carcinoid tumors. Thus, both cyclin B1-associated G2/M arrest and Rb-mediated G1 arrest are consistently compromised in high-grade large-cell neuroendocrine carcinoma and small-cell carcinoma, but are generally intact or occasionally altered in carcinoid tumor; the mechanisms involved in Rb pathway aberration among the tumor categories are different, reflecting a genetic divergence among the individual tumor categories. Cyclin B1 expression closely correlated with the Ki-67 labeling index either in the individual tumor categories or overall tumors (P < 0.0001, r = 0.742), suggesting that cyclin B1 is one of the key factors regulating cell proliferation in pulmonary neuroendocrine tumors. Neither cyclins B1 and D1, Rb, p16, nor Ki-67 correlated with patient survival in individual tumor categories, suggesting that the prognostic significance of these factors is tumor-type specific.     

Biomarkers of Parathyroid Carcinoma

The diagnosis of parathyroid carcinoma can be challenging, and adjuvant therapies such as radiotherapy and chemotherapy are not particularly beneficial in the management of this disease, creating a challenge when dealing with unresectable recurrent and metastatic malignancy. We investigated the expression profile of biomarkers that represent potential markers of malignancy or targets for novel therapies in this disease. We constructed a tissue microarray of parathyroid carcinomas from 10 patients as well as parathyroid adenomas from 25 patients and stained the slides for 34 proteins involved in angiogenesis (platelet-derived growth factor receptor (PDGFR)-??, PDGFR-??, vascular endothelial growth factor receptor-2 (VEGFR-2), and epidermal growth factor receptor (EGFR)), inflammation (cyclooxygenase (COX)-1 and COX-2), cell adhesion (matrix metalloproteinase (MMP)-1, CD9, and keratin 7), cell cycle (Cdc2p34, cyclin D1, retinoblastoma (Rb), p27, p21, parafibromin, Bmi-1, 14-3-3??, and p53), and apoptosis (Bcl-2a, Mcl-1, Bcl-xL, and glutathione-S-transferase-isoenzyme ?? (Gst-??)) along with some markers of the sonic hedgehog (Smo, SHH, Gli-1, Gli-2, Gli-3, and patched), mTOR (AKT, mammalian target of rapamycin (mTOR), and Forkhead box O (FoxO)-1), and WNT (Wisp-1, Wisp-2, and ??-catenin) signal transduction pathways. Protein expression was determined using computerized image analysis software (Spectrum Plus?, Aperio). Bcl-2a, parafibromin, Rb, and p27 were significantly decreased to variable degrees in all parathyroid carcinomas. COX-1/2, CD9, MMP-1, FoxO-1, VEGFR-2, PDGFR-??/??, Gst-??, Gli-1, Gli-2, Gli-3, and patched were expressed in the majority of benign and malignant tumor cells. These results indicate that the use of a panel that includes Bcl-2a, parafibromin, Rb, and p27 may be helpful in the assessment of atypical parathyroid neoplasms. Although the majority of other markers studied are also expressed in both benign and malignant parathyroid neoplasms, we have identified several potentially important target proteins related to angiogenesis and cell proliferation along with COX-1/2, Gst-?? and members of sonic hedgehog pathway that may be therapeutic targets in parathyroid carcinoma. While these results are preliminary, a successful outcome of a clinical trial directed against these novel targets would provide much needed systemic adjuvant treatment for patients with metastatic parathyroid carcinoma.