Karoliina Tuppurainen

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.

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.

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. Copyright

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.

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.

No evidence of somatic aryl hydrocarbon receptor interacting protein mutations in sporadic endocrine neoplasia

Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were recently observed in patients with pituitary adenoma predisposition (PAP). Though AIP mutation-positive individuals with prolactin-, mixed growth hormone/prolactin-, and ACTH-producing pituitary adenomas as well as non-secreting pituitary adenomas have been reported, most mutation-positive patients have had growth hormone-producing adenomas diagnosed at relatively young age. Pituitary adenomas are also component tumors of some familial endocrine neoplasia syndromes such as multiple endocrine neoplasia type 1 (MEN1) and Carney complex (CNC). Genes underlying MEN1 and CNC are rarely mutated in sporadic pituitary adenomas, but more often in other lesions contributing to these two syndromes. Thus far, the occurrence of somatic AIP mutations has not been studied in endocrine tumors other than pituitary adenomas. Here, we have analyzed 32 pituitary adenomas and 79 other tumors of the endocrine system for somatic AIP mutations by direct sequencing. No somatic mutations were identified. However, two out of nine patients with prolactin-producing adenoma were shown to harbor a Finnish founder mutation (Q14X) with a complete loss of the wild-type allele in the tumors. These results are in agreement with previous studies in that prolactin-producing adenomas are component tumors in PAP. The data also support the previous finding that somatic AIP mutations are not common in pituitary adenomas and suggest that such mutations are rare in other endocrine tumors as well.

Aryl hydrocarbon receptor interacting protein mutations seem not to associate with familial non-medullary thyroid cancer

Background: Over 95% of all thyroid malignancies are non-medullary thyroid carcinomas (NMTC). Familial NMTC are more aggressive and mortality is higher as compared with sporadic carcinomas. Known genetic factors do not explain all familial NMTC. Recently, thyroid disorders have been observed in families with germline mutations in aryl hydrocarbon receptor interacting protein (AIP) but, due to frequent occurrence of these conditions in the population, the significance of this co-occurrence is not clear. Aim, subjects and methods: To examine whether AIP is involved in familial NMTC, we performed AIP mutation screening in 93 familial NMTC cases. In addition, the AIP status was studied in one follicular thyroid adenoma patient with a known AIP mutation from an additional cohort. Results: No potentially pathogenic changes were identified, but two likely rare polymorphisms were detected. AIP mutation-positive patient’s follicular thyroid adenoma showed no loss of heterozygosity or lack of immunohistochemical AIP staining. Conclusion: Our study indicates that germline AIP mutations are rare or do not exist in familial NMTC.

Abstract LB-121: Serrated colorectal adenocarcinoma: Specific copy number alterations

The serrated pathway in the colorectal cancer (CRC) development shows characteristic morphological and genetic differences as compared with the traditional Vogelstein9s adenoma-carcinoma sequence. According to current estimations, about 15-20% of colorectal cancers originate from serrated polyps. Activating mutations of BRAF and KRAS genes and microsatellite instability are common features in serrated polyps and cancers. To identify the driver genes behind the serrated colorectal pathway we analyzed gene copy number variations (CNVs) in 19 serrated colorectal carcinomas. DNA was extracted from fresh-frozen tissues according to standard procedures. By using the Illumina HumanCNV-370 platform we analyzed the samples for somatic copy number alterations. Raw-data were normalized using the built-in Illumina BeadStudio normalization method. To segment the LRRs (tumor vs. normal logarithmic intensity ratios) we used CBS (circular binary segmentation) and BAFsegmentation (B allele frequency) and the driver genomic alterations were searched using GISTIC algorithm. To determine genetic changes specific for serrated adenocarcinomas, the recurrent genomic variations detected were compared with those previously reported in colorectal carcinomas. The recurrent genomic alterations in serrated carcinomas overlap substantially with the previously reported alterations seen in colorectal carcinomas; losses in 1p, 4p, 4q, 5q, 8p, 17p, 18p and 18q, and the gains in 7p, 7q, 8q, 13q and 20q. However, regions that are frequently altered specifically in serrated carcinomas were also found. For example, the gains of 1q, 5p, 6p and 6q are not commonly found in primary tumors in colorectal cancer, but only in liver metastases. The frequent losses specific for serrated carcinoma occur in 3p, 6q, 9p, 11q, 16p, 22q, Xp and Xq. The highest deletion peaks observed in the current sample set are those located at 9p21.3. In summary, our whole-genome screening of serrated colorectal adenocarcinomas with SNP arrays revealed several copy number alterations previously reported in colorectal cancers. Moreover, we detected several novel copy number alterations, which were specific for the serrated adenocarcinoma. The upcoming sequence analysis of genes within regions of copy number alterations may allow detection of mutations of genes linked to serrated adenocarcinoma pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-121.