The expression of AIP-related molecules in elucidation of cellular pathways in pituitary adenomas.

Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene predispose to the development of pituitary adenomas. Here, we characterized AIP mutation positive (AIPmut+) and AIP mutation negative (AIPmut-) pituitary adenomas by immunohistochemistry. The expressions of the AIP-related proteins aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT), cyclin-dependent kinase inhibitor 1B encoding p27(Kip1), and hypoxia-inducible factor 1-alpha were examined in 14 AIPmut+ and 53 AIPmut- pituitary adenomas to detect possible expression differences. In addition, the expression of CD34, an endothelial and hematopoietic stem cell marker, was analyzed. We found ARNT to be less frequently expressed in AIPmut+ pituitary adenomas (P = 0.001), suggesting that AIP regulates the ARNT levels. AIP small interfering RNA-treated HeLa, HEK293, or Aip-null mouse embryonic fibroblast cells did not show lowered expression of ARNT. Instead, in the pituitary adenoma cell line GH3, Aip silencing caused a partial reduction of Arnt and a clear increase in cell proliferation. We also observed a trend for increased expression of nuclear AHR in AIPmut+ samples, although the difference was not statistically significant (P = 0.06). The expressions of p27(Kip1), hypoxia-inducible factor 1-alpha, or CD34 did not differ between tumor types. The present study shows that the expression of ARNT protein is significantly reduced in AIPmut+ tumors. We suggest that the down-regulation of ARNT may be connected to an imbalance in AHR/ARNT complex formation arising from aberrant cAMP signaling.

Aryl hydrocarbon receptor interacting protein (AIP) gene mutation analysis in children and adolescents with sporadic pituitary adenomas.

OBJECTIVE: Pituitary adenomas occur rarely in childhood and adolescence. Pituitary adenoma predisposition (PAP) has been recently associated with germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene. The aim of the study was to examine the proportion of germline AIP mutations in apparently sporadic paediatric pituitary adenomas. DESIGN: Genomic DNA was analysed for mutations in the AIP gene, by PCR amplification and direct sequencing. PATIENTS: A population-based cohort consisting of 36 apparently sporadic paediatric pituitary adenoma patients, referred to two medical centres in Italy, was included in the study. Patients were either less than 18 years at diagnosis, or showed clinical evidence of adenoma development before the age of 18 years. RESULTS: A heterozygous in-frame deletion Y248del (c.742_744delTAC) was identified in one GH-secreting adenoma patient. Loss of heterozygosity (LOH) analysis of tumour DNA revealed the loss of the wild-type allele. First degree relatives carrying the mutation were clinically unaffected. CONCLUSIONS: While mutations were absent in non-GH-secreting adenoma patients, germline AIP mutations can be found in children and adolescents with GH-secreting tumours, even in the absence of family history. The present study reports the AIP mutation analysis results on patients of a single ethnic origin. Clearly, further studies are needed to improve our knowledge on the role of AIP in paediatric pituitary adenomas.

Germline CDKN1B/p27Kip1 mutation in multiple endocrine neoplasia.

CONTEXT: Germline mutations in the MEN1 gene predispose to multiple endocrine neoplasia type 1 (MEN1) syndrome, but in up to 20-25% of clinical MEN1 cases, no MEN1 mutations can be found. Recently, a germline mutation in the CDKN1B gene, encoding p27(Kip1), was reported in one suspected MEN1 family with two acromegalic patients. OBJECTIVE: Our objective was to evaluate the role of CDKN1B/p27(Kip1) in human tumor predisposition in patients clinically suspected of MEN1 but testing negative for MEN1 germline mutation as well as in familial and sporadic acromegaly/pituitary adenoma patients. DESIGN: Genomic DNA was analyzed for germline mutations in the CDKN1B/p27(Kip1) gene by PCR amplification and direct sequencing. SETTING: The study was conducted at nonprofit academic research and medical centers. PATIENTS: Thirty-six Dutch and one German suspected MEN1 patient, who previously tested negative for germline MEN1 gene mutations, were analyzed. In addition, 19 familial and 50 sporadic acromegaly/pituitary adenoma patients from Europe and the United States were included in the study. MAIN OUTCOME MEASURES: We analyzed germline CDKN1B/p27(Kip1) mutations in individuals with pituitary adenoma and MEN1-like features. RESULTS: A heterozygous 19-bp duplication (c.59_77dup19) leading to a truncated protein product was identified in one Dutch patient with suspected MEN1 phenotype, pituitary adenoma, carcinoid tumor, and hyperparathyroidism (one of 36, 2.8%). No mutations were detected in either familial or sporadic acromegaly/pituitary adenoma patients. CONCLUSIONS: Our results support the previous finding that germline CDKN1B/p27(Kip1) mutations predispose to a human MEN1-like condition. However, such mutations appear uncommon in suspected MEN1 cases and rare or nonexistent in familial or sporadic acromegaly/pituitary adenoma patients.

Molecular diagnosis of pituitary adenoma predisposition caused by aryl hydrocarbon receptor-interacting protein gene mutations.

Pituitary adenomas are common neoplasms of the anterior pituitary gland. Germ-line mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene cause pituitary adenoma predisposition (PAP), a recent discovery based on genetic studies in Northern Finland. In this population, a founder mutation explained a significant proportion of all acromegaly cases. Typically, PAP patients were of a young age at diagnosis but did not display a strong family history of pituitary adenomas. To evaluate the role of AIP in pituitary adenoma susceptibility in other populations and to gain insight into patient selection for molecular screening of the condition, we investigated the possible contribution of AIP mutations in pituitary tumorigenesis in patients from Europe and the United States. A total of 460 patients were investigated by AIP sequencing: young acromegaly patients, unselected acromegaly patients, unselected pituitary adenoma patients, and endocrine neoplasia-predisposition patients who were negative for MEN1 mutations. Nine AIP mutations were identified. Because many of the patients displayed no family history of pituitary adenomas, detection of the condition appears challenging. Feasibility of AIP immunohistochemistry (IHC) as a prescreening tool was tested in 50 adenomas: 12 AIP mutation-positive versus 38 mutation-negative pituitary tumors. AIP IHC staining levels proved to be a useful predictor of AIP status, with 75% sensitivity and 95% specificity for germ-line mutations. AIP contributes to PAP in all studied populations. AIP IHC, followed by genetic counseling and possible AIP mutation analysis in IHC-negative cases, a procedure similar to the diagnostics of the Lynch syndrome, appears feasible in identification of PAP.

Pituitary adenoma predisposition caused by germline mutations in the AIP gene.

Pituitary adenomas are common in the general population, and understanding their molecular basis is of great interest. Combining chip-based technologies with genealogy data, we identified germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene in individuals with pituitary adenoma predisposition (PAP). AIP acts in cytoplasmic retention of the latent form of the aryl hydrocarbon receptor and also has other functions. In a population-based series from Northern Finland, two AIP mutations account for 16% of all patients diagnosed with pituitary adenomas secreting growth hormone and for 40% of the subset of patients who were diagnosed when they were younger than 35 years of age. Typically, PAP patients do not display a strong family history of pituitary adenoma; thus, AIP is an example of a low-penetrance tumor susceptibility gene.

Thyroid hormone receptor beta1 in normal colon and colorectal cancer-association with differentiation, polypoid growth type and K-ras mutations.

The precursors for colorectal cancer include polypoid (conventional), flat and serrated adenomas. Polypoid growth in polypoid adenomas and serrated adenomas is associated with K-ras mutations. The regulation of polypoid or nonpolypoid growth is not well known, but could be related to trophic stimuli, such as thyroid hormones. Hence, we investigated the expression pattern of thyroid hormone receptor TRbeta1 in colorectal mucosa and in colorectal tumours and its relationship to tumour growth type. One hundred fourteen colorectal carcinoma specimens were evaluated for TRbeta1. Normal mucosa, adjacent adenomatous component (N = 46) and lymph node metastases (N = 28) were analysed when present, and the results were confirmed by Western blot analysis in selected cases. Nuclear TRbeta1 was almost always present in normal epithelium (96%), but less frequent in adenomas (83%) and in cancer (68%; p < 0.001 and p < 0.001, respectively). TRbeta1 was associated with polypoid growth, presence of K-ras mutations and also with a higher WHO histological grade and advanced Dukes' stage. Cytoplasmic expression of TRbeta1 was observed in nonneoplastic and neoplastic epithelium. In Western blot analysis, a 58 kDa band corresponding to TRbeta1 was expressed in normal mucosa and in colorectal cancer specimens with positive immunohistochemistry. Association of TRbeta1 expression with growth pattern and the presence of K-ras mutations suggest that abnormalities in thyroid hormone signalling involving TRbeta1 play a role in the development of some types of colorectal adenocarcinomas.

Inflammation and prognosis in colorectal cancer.

AIM OF THE STUDY: Previous work has indicated that quantification of inflammatory cell reaction is of prognostic value in colorectal cancer. We evaluated the prognostic significance of inflammatory cell reaction patterns in colorectal cancer and developed a grading method which could be used in the routine assessment of tumours. METHODS: The intensity of overall inflammatory cell reaction, numbers of neutrophilic and eosinophilic granulocytes, lymphoid cells and macrophages in both the central region and the invasive margin were estimated in 386 colorectal cancer patients. Prognostic significance was analysed by uni- and multivariate analysis. RESULTS: Our method for classification of inflammatory reaction was reliable. High-grade inflammation at the invasive margin in Dukes' stage A and B cancers (pT1-2N0 and pT3N0, respectively) was associated with better 5-year-survival (87.6%) than low-grade inflammation (47.0%). CONCLUSIONS: Inflammatory cell response at the invasive border is a relevant prognostic indicator and could be easily incorporated into the routine evaluation of histopathological specimens.

Morphology and microsatellite instability in sporadic serrated and non-serrated colorectal cancer.

Colorectal serrated adenocarcinoma originates from serrated adenoma, but definite histological criteria have not yet been established. It presents with frequent DNA microsatellite instability (MSI), but the frequency of low-level (MSI-L) and high-level MSI (MSI-H) and the expression of mismatch-repair (MMR) enzymes in serrated adenocarcinoma are not known. To address these questions, morphological criteria for serrated cancers were established, their validity was tested, and MSI analysis was performed with NIH consensus markers and MMR enzyme immunohistochemistry for hMLH1, hMSH2, and hMSH6 in 35 serrated and 75 non-serrated colorectal carcinomas. Serrated carcinomas frequently showed a serrated, mucinous or trabecular growth pattern; abundant eosinophilic cytoplasm; chromatin condensation; preserved polarity; and the absence of necrosis. With these features, it was possible to distinguish them from non-serrated cancers, with the mean kappa score for five observers being 0.509. MSI analysis was successful in 31 serrated and 73 non-serrated carcinomas. 54.8% of serrated carcinomas were microsatellite-stable (MSS), 29.0% presented with MSI-L, and 16.1% presented with MSI-H, whereas 78.1% of non-serrated carcinomas were MSS, 13.7% were MSI-L, and 8.2% were MSI-H. MSI-L was more common in serrated cancers (p=0.035) and it was associated with patchy immunohistochemical staining (33.3%) of MLH1. MSI-H did not differ between serrated and non-serrated cancers (p=0.14). These results suggest that the biological background of serrated carcinomas differs from sporadic non-serrated colorectal cancer, but is not directly related to MSI.