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Supplementary Figures S4-5 show 4) Total- and phsoporylated-RB1 inmmunohostochemistry in CA- and CS-tumors. 5) PTPR and PTPRK immunohistochemistry in CA- and CS-tumors.

Supplementary Methods text containing details about mutation status validation of established pituitary adenoma predisposing genes and IHC scoring.

Supplementary Figures S1-S3 and Supplementary Tables S1-S3.

Supplementary Figure S6 shows Ki-67- and PTTG1 immunostainings.

Supplementary Tables S1-S10 shows: 1) All the coding region variants (missense, premature stop codon, frameshift) of 21 sequenced somatotropinomas that passed the somatic filtering. 2) Results of correlation tests between genetic instability (GI%) and clinical variables. 3) Differentially expressed transcripts (FDR {less than or equal to} 5%, FC -2 {greater than or equal to} x {greater than or equal to} 2) in CA- and CS-tumors. 4) Significantly enriched canonical pathways carried out from differentially expressed genes (FDR < 5% and |FC|>2; CA- vs CS-tumors). 5) Immunostaining intensities of total- and phosphorylated-RB1 in CA- and CS-tumors. 6) CpG quality statistics. 7) Replication timing and CpG methylation. 8) Differentially methylated regions (DMRs) between Gsp+ versus Gsp- tumors, and nearby genes. 9) Integration of DMRs with matched transcriptomes by expression quantitative trait methylation (eQTM) analysis (at 5% FDR). 10) The complete list of significantly (p< 0.05) enriched cis-eQTM pathways.

Somatic driver mechanisms of pituitary adenoma pathogenesis have remained incompletely characterized; apart from mutations in the stimulatory Gα protein (Gαs encoded by GNAS) causing activated cAMP synthesis, pathogenic variants are rarely found in growth hormone–secreting pituitary tumors (somatotropinomas). The purpose of the current work was to clarify how genetic and epigenetic alterations contribute to the development of somatotropinomas by conducting an integrated copy number alteration, whole-genome and bisulfite sequencing, and transcriptome analysis of 21 tumors. Somatic mutation burden was low, but somatotropinomas formed two subtypes associated with distinct aneuploidy rates and unique transcription profiles. Tumors with recurrent chromosome aneuploidy (CA) were GNAS mutation negative (Gsp−). The chromosome stable (CS) –group contained Gsp+ somatotropinomas and two totally aneuploidy-free Gsp− tumors. Genes related to the mitotic G1–S-checkpoint transition were differentially expressed in CA- and CS-tumors, indicating difference in mitotic progression. Also, pituitary tumor transforming gene 1 (PTTG1), a regulator of sister chromatid segregation, showed abundant expression in CA-tumors. Moreover, somatotropinomas displayed distinct Gsp genotype–specific methylation profiles and expression quantitative methylation (eQTM) analysis revealed that inhibitory Gα (Gαi) signaling is activated in Gsp+ tumors. These findings suggest that aneuploidy through modulated driver pathways may be a causative mechanism for tumorigenesis in Gsp− somatotropinomas, whereas Gsp+ tumors with constitutively activated cAMP synthesis seem to be characterized by DNA methylation activated Gαi signaling.Implications:These findings provide valuable new information about subtype-specific pituitary tumorigenesis and may help to elucidate the mechanisms of aneuploidy also in other tumor types.

The circadian clock regulates daily variations in physiologic processes. CLOCK acts as a regulator in the circadian apparatus controlling the expression of other clock genes, including PER1. Clock genes have been implicated in cancer-related functions; in this work, we investigated CLOCK as a possible target of somatic mutations in microsatellite unstable colorectal cancers. Combining microarray gene expression data and public gene sequence information, we identified CLOCK as 1 of 790 putative novel microsatellite instability (MSI) target genes. A total of 101 MSI colorectal carcinomas (CRC) were sequenced for a coding microsatellite in CLOCK. The effect of restoring CLOCK expression was studied in LS180 cells lacking wild-type CLOCK by stably expressing GST-CLOCK or glutathione S-transferase empty vector and testing the effects of UV-induced apoptosis and radiation by DNA content analysis using flow cytometry. Putative novel CLOCK target genes were searched by using ChIP-seq. CLOCK mutations occurred in 53% of MSI CRCs. Restoring CLOCK expression in cells with biallelic CLOCK inactivation resulted in protection against UV-induced apoptosis and decreased G2-M arrest in response to ionizing radiation. Using ChIP-Seq, novel CLOCK-binding elements were identified near DNA damage genes p21, NBR1, BRCA1, and RAD50. CLOCK is shown to be mutated in cancer, and altered response to DNA damage provides one plausible mechanism of tumorigenesis. Mol Cancer Res; 8(7); 952–60. ©2010 AACR.

Precision medicine carries great potential for management of all tumor types. The aim of this retrospective study was to investigate if the two most common genetically distinct uterine fibroid subclasses, driven by aberrations in MED12 and HMGA2 genes, respectively, influence response to treatment with the progesterone receptor modulator ulipristal acetate. Changes in diameter and mutation status were derived for 101 uterine fibroids surgically removed after ulipristal acetate treatment. A significant difference in treatment response between the two major subclasses was detected. MED12 mutant fibroids had 4.4 times higher odds of shrinking in response to ulipristal acetate treatment as compared to HMGA2 driven fibroids (95% confidence interval 1.37–13.9; P = 0.013), and in a multivariate analysis molecular subclassification was an independent predictive factor. Compatible with this finding, gene expression and DNA methylation analyses revealed subclass specific differences in progesterone receptor signaling. The work provides a proof-of-principle that uterine fibroid treatment response is influenced by molecular subclass and that the genetic subclasses should be taken into account when evaluating current and future uterine fibroid therapies.

Uterine leiomyomas (ULs) are benign smooth muscle tumors that are common in premenopausal women. Somatic alterations in MED12, HMGA2, FH, genes encoding subunits of the SRCAP complex, and genes involved in Cullin 3-RING E3 ligase neddylation are mutually exclusive UL drivers. Established predisposition genes explain only partially the estimated heritability of leiomyomas. Here, we examined loss-of-function variants across 18,899 genes in a cohort of 233,614 White European women, revealing variants in four genes encoding SRCAP complex subunits (YEATS4, ZNHIT1, DMAP1, and ACTL6A) with a significant association to ULs, and YEATS4 and ZNHIT1 strikingly rank first and second, respectively. Positive mutation status was also associated with younger age at diag-nosis and hysterectomy. Moderate-penetrance UL risk was largely attributed to rare non-synonymous mutations affecting the SRCAP complex. To examine this disease phenotype more closely, we set out to identify inherited mutations affecting the SRCAP complex in our in-house sample collection of Finnish individuals with ULs (n 1/4 860). We detected one individual with an ACTL6A splice-site mutation, two individuals with a YEATS4 missense mutation, and four individuals with DMAP1 mutations: one splice-site, one nonsense, and two missense variants. These individuals had large and/or multiple ULs, were often diagnosed at an early age, and many had family history of ULs. When a somatic second hit was found, ACTL6A and DMAP1 were silenced in tumors by somatic mu-tation and YEATS4 by promoter hypermethylation. Decreased H2A.Z staining was observed in the tumors, providing further evidence for the pathogenic nature of the germline mutations. Our results establish inactivation of genes encoding SRCAP complex subunits as a central contributor to moderate-penetrance UL predisposition.

Parathyroid tumors are mostly sporadic but can also occur in familial forms, including different kinds of genetic syndromes with varying phenotypes and penetrance. Recently, somatic mutations of the tumor suppressor gene PRUNE2 were found to be frequent in parathyroid cancer (PC). The germline mutation status of PRUNE2 was investigated in a large cohort of patients with parathyroid tumors from the genetically homogenous Finnish population, 15 of which had PC, 16 atypical parathyroid tumors (APT), and 6 benign parathyroid adenomas (PA). Mutations in previously established hyperparathyroidism-related genes were screened with a targeted gene panel analysis. Nine PRUNE2 germline mutations with a minor allele frequency (MAF) of

Uterine leiomyoma (UL) is a common benign neoplasm which can sometimes be difficult to differentiate from the uterine inflammatory myofibroblastic tumor (IMT) based on morphology alone. IMT is a myofibroblastic/fibroblastic neoplasm which has typically been considered to be rare in the uterus. Its clinical behavior is usually indolent although aggressive variants exist. The majority of IMTs harbor genomic rearrangement of anaplastic lymphoma kinase ( ALK ), while ALK fusion has not been thus far detected in ULs. We analyzed 2263 ULs of which 9 (0.4%) had tyrosine-kinase activation. Seven of the samples were ALK immunopositive: 6 had an ALK fusion gene and 1 overexpressed an ALK transcript skipping exons 2 to 3, Moreover, 1 sample had a RET , and 1 a PDGFRB fusion gene. While no recurrent somatic mutations were found, 1 patient had an ALK germline mutation. Seven tumors showed leiomyoma-like morphology, 1 tumor had slightly loose, and 1 fibrous growth pattern. Six tumors had mild to moderate lymphocyte infiltration, while no immune cell infiltration was detected in 3 cases. None of the tumors showed aggressive behavior. Except for strong ALK positivity (7/9 tumors) the protein expression profile of the tumors was identical to ULs and distinct from other mesenchymal uterine tumors. In gene expression level, these tumors and the known UL subclasses did not separate perfectly. However, vitamin C metabolism and epithelial-mesenchymal transition pathways were uniquely enriched in these lesions. The overall similarity of the analyzed tumors to UL raises the question whether an UL diagnosis would be more proper for a subset of uterine IMTs.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a tumor predisposition syndrome caused by germline fumarate hydratase (FH) mutations and characterized by uterine and cutaneous leiomyomas and renal cell cancer. Currently, there is no generally approved method to differentiate FH-deficient uterine leiomyomas from other leiomyomas. Here, we analyzed 3 antibodies (S-(2-succino)-cysteine [2SC], aldo-keto reductase family 1, member B10 [AKR1B10], and FH) as potential biomarkers. The study consisted of 2 sample series. The first series included 155 formalin-fixed paraffin-embedded uterine leiomyomas, of which 90 were from HLRCC patients and 65 were sporadic. The second series included 1590 unselected fresh frozen leiomyomas. Twenty-seven tumors were from known HLRCC patients, while the FH status for the remaining 1563 tumors has been determined by copy number analysis and Sanger sequencing revealing 45 tumors with monoallelic (n=33) or biallelic (n=12) FH loss. Altogether 197 samples were included in immunohistochemical analyses: all 155 samples from series 1 and 42 available corresponding formalin-fixed paraffin-embedded samples from series 2 (15 tumors with monoallelic and 7 with biallelic FH loss, 20 with no FH deletion). Results show that 2SC performed best with 100% sensitivity and specificity. Scoring was straightforward with unambiguously positive or negative results. AKR1B10 identified most tumors accurately with 100% sensitivity and 99% specificity. FH was 100% specific but showed slightly reduced 91% sensitivity. Both FH and AKR1B10 displayed also intermediate staining intensities. We suggest that when patient's medical history and/or histopathologic tumor characteristics indicate potential FH-deficiency, the tumor's FH status is determined by 2SC staining. When aberrant staining is observed, the patient can be directed to genetic counseling and mutation screening.

Context Parathyroid carcinoma (PC) is extremely rare. Prognosis is poor, with no known evidence-based systemic therapies. We previously reported complete remission in a patient with metastasized parathyroid carcinoma and high tumor MGMT promoter methylation status who was treated with temozolomide. Objective To study MGMT promoter methylation status in an additional set of aggressive parathyroid tumors. Design/Setting The study included 12 patients: 7 with sporadic and 5 with familial primary hyperparathyroidism (two of the latter carried a CDC73 gross deletion). Patient 9 is the previously described patient with PC and high MGMT methylation status. Her daughter (patient 12) had surgery for severe primary hyperparathyroidism due to atypical parathyroid adenoma during pregnancy. Eleven patients thus had PC and one had atypical parathyroid adenoma. MGMT promoter methylation status was determined from DNA extracted from primary (n = 10) or metastatic (n = 2) tumors. A mean methylation level >20% was considered high. Patient 11 had metastatic PC and received temozolomide cycles. Results Only the previously published patient (patient 9) had high tumor MGMT promoter methylation status. This was not a characteristic of the atypical parathyroid adenoma of the daughter (patient 12). Patient 11 (CDC73 intragenic deletion) has disseminated PC, low MGMT promoter methylation, and stable disease on follow-up after temozolomide treatment. Conclusion High MGMT promoter methylation status seems rare in PC. However, as demonstrated in other neuroendocrine tumors, some patients with disseminated PC might benefit from temozolomide. Demonstration of high methylation status could be a predictor of positive response to temozolomide treatment.

Pituitary adenomas (PAs) are intracranial tumors associated with significant morbidity due to hormonal dysregulation, mass effects and have a heavy treatment burden. Growth hormone (GH)-secreting PAs (somatotropinomas) cause acromegaly-gigantism. Genetic forms of somatotropinomas due to germlineAIPmutations (AIPmut+) have an early onset and are aggressive and resistant to treatment with somatostatin analogs (SSAs), including octreotide. The molecular underpinnings of these clinical features remain unclear. We investigated the role of miRNA dysregulation inAIPmut+ vsAIPmut- PA samples by array analysis. miR-34a and miR-145 were highly expressed inAIPmut+ vsAIPmut- somatotropinomas. Ectopic expression ofAIPmut (p.R271W) inAip(-/-)mouse embryonic fibroblasts (MEFs) upregulated miR-34a and miR-145, establishing a causal link betweenAIPmut and miRNA expression. In PA cells (GH3), miR-34a overexpression promoted proliferation, clonogenicity, migration and suppressed apoptosis, whereas miR-145 moderately affected proliferation and apoptosis. Moreover, high miR-34a expression increased intracellular cAMP, a critical mitogenic factor in PAs. Crucially, high miR-34a expression significantly blunted octreotide-mediated GH inhibition and antiproliferative effects. miR-34a directly targetsGnai2encoding G alpha i2, a G protein subunit inhibiting cAMP production. Accordingly, G alpha i2 levels were significantly lower inAIPmut+ vsAIPmut- PA. Taken together, somatotropinomas withAIPmutations overexpress miR-34a, which in turn downregulates G alpha i2 expression, increases cAMP concentration and ultimately promotes cell growth. Upregulation of miR-34a also impairs the hormonal and antiproliferative response of PA cells to octreotide. Thus, miR-34a is a novel downstream target of mutantAIPthat promotes a cellular phenotype mirroring the aggressive clinical features ofAIPmut+ acromegaly.

Somatic driver mechanisms of pituitary adenoma pathogenesis have remained incompletely characterized; apart from mutations in the stimulatory Gα protein (Gαs encoded by GNAS) causing activated cAMP synthesis, pathogenic variants are rarely found in growth hormone–secreting pituitary tumors (somatotropinomas). The purpose of the current work was to clarify how genetic and epigenetic alterations contribute to the development of somatotropinomas by conducting an integrated copy number alteration, whole-genome and bisulfite sequencing, and transcriptome analysis of 21 tumors. Somatic mutation burden was low, but somatotropinomas formed two subtypes associated with distinct aneuploidy rates and unique transcription profiles. Tumors with recurrent chromosome aneuploidy (CA) were GNAS mutation negative (Gsp−). The chromosome stable (CS) –group contained Gsp+ somatotropinomas and two totally aneuploidy-free Gsp− tumors. Genes related to the mitotic G1–S-checkpoint transition were differentially expressed in CA- and CS-tumors, indicating difference in mitotic progression. Also, pituitary tumor transforming gene 1 (PTTG1), a regulator of sister chromatid segregation, showed abundant expression in CA-tumors. Moreover, somatotropinomas displayed distinct Gsp genotype–specific methylation profiles and expression quantitative methylation (eQTM) analysis revealed that inhibitory Gα (Gαi) signaling is activated in Gsp+ tumors. These findings suggest that aneuploidy through modulated driver pathways may be a causative mechanism for tumorigenesis in Gsp− somatotropinomas, whereas Gsp+ tumors with constitutively activated cAMP synthesis seem to be characterized by DNA methylation activated Gαi signaling. Implications: These findings provide valuable new information about subtype-specific pituitary tumorigenesis and may help to elucidate the mechanisms of aneuploidy also in other tumor types.

Clonal hematopoiesis driven by somatic heterozygous TET2 loss is linked to malignant degeneration via consequent aberrant DNA methylation, and possibly to cardiovascular disease via increased cytokine and chemokine expression as reported in mice. Here, we discover a germline TET2 mutation in a lymphoma family. We observe neither unusual predisposition to atherosclerosis nor abnormal pro-inflammatory cytokine or chemokine expression. The latter finding is confirmed in cells from three additional unrelated TET2 germline mutation carriers. The TET2 defect elevates blood DNA methylation levels, especially at active enhancers and cell-type specific regulatory regions with binding sequences of master transcription factors involved in hematopoiesis. The regions display reduced methylation relative to all open chromatin regions in four DNMT3A germline mutation carriers, potentially due to TET2-mediated oxidation. Our findings provide insight into the interplay between epigenetic modulators and transcription factor activity in hematological neoplasia, but do not confirm the putative role of TET2 in atherosclerosis., Somatic heterozygous TET2 loss drives clonal hematopoiesis, which is linked to malignant cell degeneration and potentially cardiovascular disease. Here, the authors investigate the molecular impact of a germline TET2 mutation in a lymphoma family, finding elevated blood DNA methylation levels and no predisposition to atherosclerosis

Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene predispose humans to pituitary adenomas, particularly of the somatotroph lineage. Mice with global heterozygous inactivation of Aip (Aip+/−) also develop pituitary adenomas but differ from AIP-mutated patients by the high penetrance of pituitary disease. The endocrine phenotype of these mice is unknown. The aim of this study was to determine the endocrine phenotype of Aip+/− mice by assessing the somatic growth, ultradian pattern of GH secretion and IGF1 concentrations of longitudinally followed male mice at 3 and 12 months of age. As the early stages of pituitary tumorigenesis are controversial, we also studied the pituitary histology and somatotroph cell proliferation in these mice. Aip+/− mice did not develop gigantism but exhibited a leaner phenotype than wild-type mice. Analysis of GH pulsatility by deconvolution in 12-month-old Aip+/− mice showed a mild increase in total GH secretion, a conserved GH pulsatility pattern, but a normal IGF1 concentration. No pituitary adenomas were detected up to 12 months of age. An increased ex vivo response to GHRH of pituitary explants from 3-month-old Aip+/− mice, together with areas of enlarged acini identified on reticulin staining in the pituitary of some Aip+/− mice, was suggestive of somatotroph hyperplasia. Global heterozygous Aip deficiency in mice is accompanied by subtle increase in GH secretion, which does not result in gigantism. The absence of pituitary adenomas in 12-month-old Aip+/− mice in our experimental conditions demonstrates the important phenotypic variability of this congenic mouse model.

Microsatellite instability (MSI) leads to accumulation of an excessive number of mutations in the genome, mostly small insertions and deletions. MSI colorectal cancers (CRCs), however, also contain more point mutations than microsatellite-stable (MSS) tumors, yet they have not been as comprehensively studied. To identify candidate driver genes affected by point mutations in MSI CRC, we ranked genes based on mutation significance while correcting for replication timing and gene expression utilizing an algorithm, MutSigCV. Somatic point mutation data from the exome kit-targeted area from 24 exome-sequenced sporadic MSI CRCs and respective normals, and 12 whole-genome-sequenced sporadic MSI CRCs and respective normals were utilized. The top 73 genes were validated in 93 additional MSI CRCs. The MutSigCV ranking identified several well-established MSI CRC driver genes and provided additional evidence for previously proposed CRC candidate genes as well as shortlisted genes that have to our knowledge not been linked to CRC before. Two genes, SMARCB1 and STK38L, were also functionally scrutinized, providing evidence of a tumorigenic role, for SMARCB1 mutations in particular. © 2018 The Authors. Published under the terms of the CC BY 4.0 license

Background: Uterine leiomyomas can be classified into molecularly distinct subtypes according to their genetic triggers: MED12 mutations, HMGA2 upregulation, or inactivation of FH. The aim of this study was to identify metabolites and metabolic pathways that are dysregulated in different subtypes of leiomyomas. Methods: We performed global metabolomic profiling of 25 uterine leiomyomas and 17 corresponding myometrium specimens using liquid chromatography-tandem mass spectroscopy. Results: A total of 641 metabolites were detected. All leiomyomas displayed reduced homocarnosine and haeme metabolite levels. We identified a clearly distinct metabolomic profile for leiomyomas of the FH subtype, characterised by metabolic alterations in the tricarboxylic acid cycle and pentose phosphate pathways, and increased levels of multiple lipids and amino acids. Several metabolites were uniquely elevated in leiomyomas of the FH subtype, including N6-succinyladenosine and argininosuccinate, serving as potential biomarkers for FH deficiency. In contrast, leiomyomas of the MED12 subtype displayed reduced levels of vitamin A, multiple membrane lipids and amino acids, and dysregulation of vitamin C metabolism, a finding which was also compatible with gene expression data. Conclusions: The study reveals the metabolomic heterogeneity of leiomyomas and provides the requisite framework for strategies designed to target metabolic alterations promoting the growth of these prevalent tumours.

The somatic landscape of pituitary adenomas is largely unknown. Identification of somatic alterations aims at better understanding of tumor pathology.The objective of the study was a genome-wide characterization of somatic single-nucleotide variants, structural variants, and copy-number aberrations in somatotropinomas.Whole-genome sequencing and single-nucleotide polymorphism array analyses were performed on 12 fresh-frozen somatotropinomas and their corresponding blood samples. All the coding somatic variants were confirmed by Sanger sequencing.Studied tumors were somatotropinomas. Apart from one AIP mutation-positive patient, all cases were mutation negative for the established germline mutations associated with pituitary adenomas.There were no interventions.Somatic variants were identified with an established computational pipeline and filtered against germline data. Somatic copy number alteration analyses were performed using segmentation-based approaches.A genome-wide analysis revealed on average 129 somatic single-nucleotide variants per tumor. Further analysis of coding regions showed on average 2.3 single-nucleotide variants per tumor. The only recurrent somatic events were the oncogenic GNAS mutation (p.Arg201Cys) and shared chromosome losses (chromosomes 1, 6, 13, 14, 15, 16, 18, 22). Analysis of somatic structural variants revealed one tumor with a complex chromosomal rearrangement.Somatotropinomas showed a low number of somatic genetic alterations. Whereas no novel recurrently mutated genes could be identified, the somatic landscape has potential to affect the Ca(2+) and ATP pathways known to be involved in the pituitary tumorigenesis. Further studies, eg, methylome and transcriptome analyses, are needed to investigate possible interplay between the recurrent chromosome losses and epigenetic factors.

The aryl hydrocarbon receptor interacting protein (AIP) is a tumor-suppressor gene underlying the pituitary adenoma predisposition. Thus far, the exact molecular mechanisms by which inactivated AIP exerts its tumor-promoting action have been unclear. To better understand the role of AIP in pituitary tumorigenesis, we performed gene expression microarray analysis to examine changes between Aip wild-type and knockout mouse embryonic fibroblast (MEF) cell lines. Transcriptional analyses implied that Aip deficiency causes a dysfunction in cyclic adenosine monophosphate (cAMP) signaling, as well as impairments in signaling cascades associated with developmental and immune-inflammatory responses. In vitro experiments showed that AIP deficiency increases intracellular cAMP concentrations in both MEF and murine pituitary adenoma cell lines. Based on knockdown of various G protein α subunits, we concluded that AIP deficiency leads to elevated cAMP concentrations through defective Gαi-2 and Gαi-3 proteins that normally inhibit cAMP synthesis. Furthermore, immunostaining of Gαi-2 revealed that AIP deficiency is associated with a clear reduction in Gαi-2 protein expression levels in human and mouse growth hormone (GH)-secreting pituitary adenomas, thus indicating defective Gαi signaling in these tumors. By contrast, all prolactin-secreting tumors showed prominent Gαi-2 protein levels, irrespective of Aip mutation status. We additionally observed reduced expression of phosphorylated extracellular signal-regulated kinases 1/2 and cAMP response element-binding protein levels in mouse and human AIP-deficient somatotropinomas. This study implies for the first time that a failure to inhibit cAMP synthesis through dysfunctional Gαi signaling underlies the development of GH-secreting pituitary adenomas in AIP mutation carriers.

Introduction In sporadic acromegaly, downregulation of AIP protein of the adenomas associates with invasive tumor features and reduced responsiveness to somatostatin analogues. AIP is a regulator of Gai signaling, but it is not known how the biological function of the Gai pathway is controlled. Aim To study GNAS and AIP mutation status, AIP and Gai-2 protein expressions, Ki-67 proliferation indices and clinical parameters in patients having primary surgery because of acromegaly at a single center between years 2000 and 2010. Results Sixty patients (F/M, 31/29), mean age 49 (median 50), mean follow-up 7.7 years (range 0.6–14.0) underwent primary surgery. Four adenoma specimens (6.8%) harbored an AIP and 21 (35.6%) an activating GNAS (Gsp+) mutation. Altogether 13/56 (23%) adenomas had low AIP protein levels, and 14/56 (25%) low Gai-2 staining. In regression modeling, AIP expression associated with Gai-2 (P = 2.33 × 10−9) and lower Ki-67 (P = 0.04). In pairwise comparison, low AIP protein predicted high GH at last follow-up (mean 7.7 years after surgery, q = 0.045). Extent of treatments given for acromegaly associated with higher preoperative GH (P = 7.94 × 10−4), KNOSP (P = 0.003) and preoperative hypopituitarism (P = 0.03) and remission at last follow-up with change in 3-month postoperative IGF1 (P = 2.07 × 10−7). Conclusions We demonstrate, for the first time, that AIP protein expression associates with Gai-2 protein intensities in sporadic somatotropinomas, suggesting a joint regulation on somatostatin signaling. Low AIP level associates with higher proliferative activity and predicts high GH concentrations after long-term follow-up. The AIP mutation rate of 6.8% is fairly high, reflecting the genetic composition of the Finnish population.

OBJECTIVE: Colorectal cancer (CRC) has a substantial heritable component. Common genetic variation has been shown to contribute to CRC risk. A study was conducted in a large multi-population study to assess the feasibility of CRC risk prediction using common genetic variant data combined with other risk factors. A risk prediction model was built and applied to the Scottish population using available data. DESIGN: Nine populations of European descent were studied to develop and validate CRC risk prediction models. Binary logistic regression was used to assess the combined effect of age, gender, family history (FH) and genotypes at 10 susceptibility loci that individually only modestly influence CRC risk. Risk models were generated from case-control data incorporating genotypes alone (n=39,266) and in combination with gender, age and FH (n=11,324). Model discriminatory performance was assessed using 10-fold internal cross-validation and externally using 4187 independent samples. The 10-year absolute risk was estimated by modelling genotype and FH with age- and gender-specific population risks. RESULTS: The median number of risk alleles was greater in cases than controls (10 vs 9, p5% predicted 10-year absolute risk. CONCLUSION: Genotype data provide additional information that complements age, gender and FH as risk factors, but individualised genetic risk prediction is not currently feasible. Nonetheless, the modelling exercise suggests public health potential since it is possible to stratify the population into CRC risk categories, thereby informing targeted prevention and surveillance.

Defects in the mismatch repair system lead to microsatellite instability (MSI), a feature observed in ∼ 15% of all colorectal cancers (CRCs). Microsatellite mutations that drive tumourigenesis, typically inactivation of tumour suppressors, are selected for and are frequently detected in MSI cancers. Here, we evaluated somatic mutations in microsatellite repeats of 790 genes chosen based on reduced expression in MSI CRC and existence of a coding mononucleotide repeat of 6–10 bp in length. All the repeats were initially sequenced in 30 primary MSI CRC samples and whenever frameshift mutations were identified in >20%, additional 70 samples were sequenced. To distinguish driver mutations from passengers, we similarly analyzed the occurrence of frameshift mutations in 121 intronic control repeats and utilized a statistical regression model to determine cut-off mutation frequencies for repeats of all types (A/T and C/G, 6–10 bp). Along with several know target genes, including TGFBR2, ACVR2, and MSH3, six novel candidate driver genes emerged that harbored significantly more mutations than identical control repeats. The mutation frequencies in 100 MSI CRC samples were 51% in G8 of GLYR1, 47% in T9 of ABCC5, 43% in G8 of WDTC1, 33% in A8 of ROCK1, 30% in T8 of OR51E2, and 28% in A8 of TCEB3. Immunohistochemical staining of GLYR1 revealed defective protein expression in tumors carrying biallelic mutations, supporting a loss of function hypothesis. This is a large scale, unbiased effort to identify genes that when mutated are likely to contribute to MSI CRC development. Defects in the mismatch repair system lead to microsatellite instability (MSI), a feature observed in ∼15% of all colorectal cancers (CRCs). Microsatellite mutations that drive tumourigenesis, typically inactivation of tumour suppressors, are selected for and are frequently detected in MSI cancers. Here, we evaluated somatic mutations in microsatellite repeats of 790 genes chosen based on reduced expression in MSI CRC and existence of a coding mononucleotide repeat of 6-10 bp in length. All the repeats were initially sequenced in 30 primary MSI CRC samples and whenever frameshift mutations were identified in >20%, additional 70 samples were sequenced. In order to distinguish driver mutations from passengers we similarly analyzed the occurrence of frameshift mutations in 121 intronic control repeats and utilized a statistical regression model to determine cut-off mutation frequencies for repeats of all types (A/T and C/G, 6-10 bp). Along with several know target genes, including TGFBR2, ACVR2, and MSH3, six novel candidate driver genes emerged that harbored significantly more mutations than identical control repeats. The mutation frequencies in 100 MSI CRC samples were 51% in G8 of GLYR1, 47% in T9 of ABCC5, 43% in G8 of WDTC1, 33% in A8 of ROCK1, 30% in T8 of OR51E2, and 28% in A8 of TCEB3. Immunohistochemical staining of GLYR1 revealed defective protein expression in tumours carrying biallelic mutations, supporting a loss of function hypothesis. This is a large scale, unbiased effort to identify genes that when mutated are likely to contribute to MSI CRC development.

Colorectal serrated adenocarcinoma forms about 15-20% of colorectal carcinomas. We have previously shown that downregulation of PTCH1 is distinctive for this type of colorectal cancer. In several other tumor types, somatic inactivating PTCH1 mutations have been shown to lead to aberrant Hedgehog signaling, but in colorectal cancer the role of PTCH1 mutations has not been thoroughly studied. Here, we have analyzed the mutation status of PTCH1 in a series of 33 colorectal serrated adenocarcinomas by sequencing all 23 coding exons. We detected 11 previously known SNPs and eight new alterations. The latter included five synonymous changes and two previously unknown missense variations, somatic M319V, and germline V1231A. V1231A was also present in population controls and likely represents polymorphism. The somatic M319V variant does not appear to be an attractive candidate for a disease-associated mutation because in silico analyses did not support the pathogenic nature of the change. A somatic, intronic 1-bp deletion was detected in a short poly(T) stretch in two microsatellite unstable tumors. None of the three changes had predicted effect on splicing when analyzed in silico. Our results did not reveal any clearly deleterious inactivating PTCH1 mutations in our collection of colorectal serrated adenocarcinomas. This suggests that other mechanisms are involved in the observed downregulation of the PTCH1 gene. These might include, e.g., constantly active MAPK signaling by KRAS or BRAF mutations or silencing of PTCH1 by hypermethylation, and further studies are needed to reveal these mechanisms.

Frameshift mutations frequently accumulate in microsatellite-unstable colorectal cancers (MSI CRCs) typically leading to downregulation of the target genes due to nonsense-mediated messenger RNA decay. However, frameshift mutations that occur in the 3′ end of the coding regions can escape decay, which has largely been ignored in previous works. In this study, we characterized nonsense-mediated decay-escaping frameshift mutations in MSI CRC in an unbiased, genome wide manner. Combining bioinformatic search with expression profiling, we identified genes that were predicted to escape decay after a deletion in a microsatellite repeat. These repeats, located in 258 genes, were initially sequenced in 30 MSI CRC samples. The mitotic checkpoint kinase TTK was found to harbor decay-escaping heterozygous mutations in exon 22 in 59% (105/179) of MSI CRCs, which is notably more than previously reported. Additional novel deletions were found in exon 5, raising the mutation frequency to 66%. The exon 22 of TTK contains an A 9 –G 4 –A 7 locus, in which the most common mutation was a mononucleotide deletion in the A 9 (c.2560delA). When compared with identical non-coding repeats, TTK was found to be mutated significantly more often than expected without selective advantage. Since TTK inhibition is known to induce override of the mitotic spindle assembly checkpoint (SAC), we challenged mutated cancer cells with the microtubule-stabilizing drug paclitaxel. No evidence of checkpoint weakening was observed. As a conclusion, heterozygous TTK mutations occur at a high frequency in MSI CRCs. Unexpectedly, the plausible selective advantage in tumourigenesis does not appear to be related to SAC.

Pituitary adenomas are common in the general population. Although most of them are sporadic, some occur in a familial setting. In familial pituitary adenoma patients it is common that no germline defects are found after screening of aryl hydrocarbon receptor interacting protein (AIP) and other genes known to underlie the condition, suggesting the existence of yet unknown predisposition genes. Recently, the RET proto-oncogene was found to be a novel in vivo interaction partner of AIP in the pituitary gland. Here, we have screened patients from 16 AIP mutation negative (AIPmut-) pituitary adenoma families for RET germline mutations to assess whether RET could play a role in pituitary adenoma predisposition, similar to AIP. We found five novel germline RET changes: one in RET Exon 4 and the rest in noncoding regions of RET. Two changes, c.1560*G > A and -1285 G > A, were segregated in affected family members. We also analyzed the RET region with enhancer element locator (EEL) to identify RET regulatory elements, and to see whether the changes resided in these. None of the variants mapped to the regions predicted by EEL. Expression of RET was examined in ten AIPmut- and seven AIP mutation positive (AIPmut+) somatotropinomas by immunohistochemistry, with a trend showing reduced expression in the latter (P = 0.05). We conclude that the RET variants are presumably not related to pituitary adenoma predisposition, although reduced RET expression may play a role in AIP-related genesis of somatotropinomas.

Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene have been shown to predispose to pituitary adenoma predisposition, a condition characterized by growth hormone (GH)-secreting pituitary tumors. To study AIP-mediated tumorigenesis, we generated an Aip mouse model. Heterozygous mice developed normally but were prone to pituitary adenomas, in particular to those secreting GH. A complete loss of AIP was detected in these lesions, and full penetrance was reached at the age of 15 months. No excess of any other tumor type was found. Ki-67 analysis indicated that Aip-deficient tumors have higher proliferation rates compared with Aip-proficient tumors, suggesting a more aggressive disease. Similar to human AIP-deficient pituitary adenomas, immunohistochemical studies showed that expression of aryl hydrocarbon receptor nuclear translocator 1 or 2 (ARNT or ARNT2) protein was lost in the mouse tumors, suggesting that mechanisms of AIP-related tumorigenesis involve aberrant ARNT function. The Aip(+/-) mouse appears to be an excellent model for the respective human disease phenotype. This model constitutes a tool to further study AIP-associated pituitary tumorigenesis and may be potentially valuable in efforts to develop therapeutic strategies to treat pituitary adenomas.

Right atrial isomerism (RAI) is a heterotaxy syndrome with disturbances in the left-right axis development, resulting in complex heart malformations and abnormal lateralization of other thoracic and abdominal organs. Although autosomal-recessive inheritance of heterotaxy syndrome is seen in multiple families, underlying gene defects have remained unknown. Here we identify the molecular genetic basis of a kindred with five siblings with RAI. Linkage analysis and positional candidate gene approach showed that the affected children were compound heterozygotes for truncating mutations in the growth/differentiation factor 1 (GDF1) gene. Individuals heterozygous for the mutations were clinically healthy. This finding, supported by the similar phenotype in Gdf1 knockout mouse, provides firm evidence that RAI can occur as a recessively inherited condition, with GDF1 as the culprit gene. The results will shed light on the biological basis of human laterality defects and facilitate molecular diagnosis of RAI.

Contains fulltext : 89001.pdf (Publisher’s version ) (Closed access) Mixed lineage kinase 3 (MLK3) is a serine/threonine kinase, regulating MAPkinase signalling, in which cancer-associated mutations have never been reported. In this study, 174 primary gastrointestinal cancers (48 hereditary and 126 sporadic forms) and 7 colorectal cancer cell lines were screened for MLK3 mutations. MLK3 mutations were significantly associated with MSI phenotype in primary tumours (P = 0.0005), occurring in 21% of the MSI carcinomas. Most MLK3 somatic mutations identified were of the missense type (62.5%) and more than 80% of them affected evolutionarily conserved residues. A predictive 3D model points to the functional relevance of MLK3 missense mutations, which cluster in the kinase domain. Further, the model shows that most of the altered residues in the kinase domain probably affect MLK3 scaffold properties, instead of its kinase activity. MLK3 missense mutations showed transforming capacity in vitro and cells expressing the mutant gene were able to develop locally invasive tumours, when subcutaneously injected in nude mice. Interestingly, in primary tumours, MLK3 mutations occurred in KRAS and/or BRAF wild-type carcinomas, although not being mutually exclusive genetic events. In conclusion, we have demonstrated for the first time the presence of MLK3 mutations in cancer and its association to mismatch repair deficiency. Further, we demonstrated that MLK3 missense mutations found in MSI gastrointestinal carcinomas are functionally relevant.

ObjectiveMutations in the aryl hydrocarbon receptor-interacting protein (AIP) were recently shown to confer a pituitary adenoma predisposition in patients with familial isolated pituitary adenomas (FIPA). We report a large Samoan FIPA kindred from Australia/New Zealand with an R271W mutation that was associated with aggressive pituitary tumors.Design and methodsCase series with germline screening of AIP and haplotype analyses among R271W families.ResultsThis previously unreported kindred consisted of three affected individuals that either presented with or had first symptoms of a pituitary macroadenoma in late childhood or adolescence. The index case, a 15-year-old male with incipient gigantism and his maternal aunt, had somatotropinomas, and the maternal uncle of the index case had a prolactinoma. All tumors were large (15, 40, and 60 mm maximum diameter) and two required transcranial surgery and radiotherapy. All three affected subjects and ten other unaffected relatives were found to be positive for a germline R271W AIP mutation. Comparison of the single nucleotide polymorphism patterns among this family and two previously reported European FIPA families with the same R271W mutation demonstrated no common ancestry.ConclusionsThis kindred exemplifies the aggressive features of pituitary adenomas associated with AIP mutations, while genetic analyses among three R271W FIPA families indicate that R271W represents a mutational hotspot that should be studied further in functional studies.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a tumor predisposition syndrome with cutaneous and uterine leiomyomatosis as well as renal cell cancer (RCC) as its clinical manifestations. HLRCC is caused by heterozygous germline mutations in the fumarate hydratase (fumarase) gene. In this study, we used array comparative genomic hybridization to identify the specific copy number changes characterizing the HLRCC‐associated RCCs. The study material comprised formalin‐fixed paraffin‐embedded renal tumors obtained from Finnish patients with HLRCC. All 11 investigated tumors displayed the papillary type 2 histopathology typical for HLRCC renal tumors. The most frequent copy number changes detected in at least 3/11 (27%) of the tumors were gains in chromosomes 2, 7, and 17, and losses in 13q12.3‐q21.1, 14, 18, and X. These findings provide genetic evidence for a distinct copy number profile in HLRCC renal tumors compared with sporadic RCC tumors of the same histopathological subtype, and delineate chromosomal regions that associate with this very aggressive form of RCC.

The common single-nucleotide polymorphism (SNP) rs3802842 at 11q23.1 has recently been reported to be associated with risk of colorectal cancer (CRC). To examine this association in detail we genotyped rs3802842 in eight independent case-control series comprising a total of 10 638 cases and 10 457 healthy individuals. A significant association between the C allele of rs3802842 and CRC risk was found (per allele OR = 1.17; 95% confidence interval [CI]: 1.12-1.22; P = 1.08 x 10(-12)) with the risk allele more frequent in rectal than colonic disease (P = 0.02). In combination with 8q21, 8q24, 10p14, 11q, 15q13.3 and 18q21 variants, the risk of CRC increases with an increasing numbers of variant alleles for the six loci (OR(per allele) = 1.19; 95% CI: 1.15-1.23; P(trend) = 7.4 x 10(-24)). Using the data from our genome-wide association study of CRC, LD mapping and imputation, we were able to refine the location of the causal locus to a 60 kb region and screened for coding changes. The absence of exonic mutations in any of the transcripts (FLJ45803, LOC120376, C11orf53 and POU2AF1) mapping to this region makes the association likely to be a consequence of non-coding effects on gene expression.

A recent study described a recessive ATPase activating germ-line mutation in smooth-muscle myosin ( smmhc / myh11 ) underlying the zebrafish meltdown ( mlt ) phenotype. The mlt zebrafish develops intestinal abnormalities reminiscent of human Peutz–Jeghers syndrome (PJS) and juvenile polyposis (JP). To examine the role of MYH11 in human intestinal neoplasia, we searched for MYH11 mutations in patients with colorectal cancer (CRC), PJS and JP. We found somatic protein-elongating frameshift mutations in 55% of CRCs displaying microsatellite instability and in the germ-line of one individual with PJS. Additionally, two somatic missense mutations were found in one microsatellite stable CRC. These two missense mutations, R501L and K1044N, and the frameshift mutations were functionally evaluated. All mutations resulted in unregulated molecules displaying constitutive motor activity, similar to the mutant myosin underlying mlt . Thus, MYH11 mutations appear to contribute also to human intestinal neoplasia. Unregulated MYH11 may affect the cellular energy balance or disturb cell lineage decisions in tumor progenitor cells. These data challenge our view on MYH11 as a passive differentiation marker functioning in muscle contraction and add to our understanding of intestinal neoplasia.

To identify colorectal cancer (CRC) susceptibility alleles, we conducted a genome-wide association study. In phase 1, we genotyped 550,163 tagSNPs in 940 familial colorectal tumor cases (627 CRC, 313 high-risk adenoma) and 965 controls. In phase 2, we genotyped 42,708 selected SNPs in 2,873 CRC cases and 2,871 controls. In phase 3, we evaluated 11 SNPs showing association at P < 10(-4) in a joint analysis of phases 1 and 2 in 4,287 CRC cases and 3,743 controls. Two SNPs were taken forward to phase 4 genotyping (10,731 CRC cases and 10,961 controls from eight centers). In addition to the previously reported 8q24, 15q13 and 18q21 CRC risk loci, we identified two previously unreported associations: rs10795668, located at 10p14 (P = 2.5 x 10(-13) overall; P = 6.9 x 10(-12) replication), and rs16892766, at 8q23.3 (P = 3.3 x 10(-18) overall; P = 9.6 x 10(-17) replication), which tags a plausible causative gene, EIF3H. These data provide further evidence for the 'common-disease common-variant' model of CRC predisposition.

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.

Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were recently shown to cause susceptibility to pituitary adenoma predisposition. The purpose of this review is to briefly recapitulate the current knowledge on hereditary susceptibility to pituitary adenomas and what led to the identification of AIP as a novel predisposition gene. We will then concentrate on the data on AIP mutations and pituitary adenoma predisposition phenotype that have accumulated since the gene was identified. Major future challenges, as well as the possibilities for clinical practice based on this recent finding, will also be discussed.

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.

Somatic interstitial deletions of chromosome segment 7q22-q31 in uterine leiomyomas are a frequent event, thought to be indicative of a tumor suppressor gene in the region. Previous LOH and CGH studies have refined this region to 7q22.3-q31, although the target gene has not been identified. Here, we have used tiling-path resolution microarray CGH to further refine the region and to identify homozygous deletions in fibroids. Furthermore, we have screened all manually annotated genes in the region for mutations. We have refined the minimum deleted region at 7q22.3-q31 to 2.79 Mbp and identified a second region of deletion at 7q34. However, we identified no pathogenic coding variation.

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 p27Kip1, was reported in one suspected MEN1 family with two acromegalic patients. Objective: Our objective was to evaluate the role of CDKN1B/p27Kip1 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/p27Kip1 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/p27Kip1 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/p27Kip1 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.

Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were recently identified in individuals with pituitary adenoma predisposition (PAP). These patients have prolactin (PRL) or growth hormone (GH) oversecreting pituitary adenomas, the latter exhibiting acromegaly or gigantism. Loss-of-heterozygosity (LOH) analysis revealed that AIP is lost in PAP tumours, suggesting that it acts as a tumour-suppressor gene. Aryl hydrocarbon receptor interacting protein is involved in several pathways, but it is best characterised as a cytoplasmic partner of the aryl hydrocarbon receptor (AHR). To examine the possible role of AIP in the genesis of common cancers, we performed somatic mutation screening in a series of 373 colorectal cancers (CRCs), 82 breast cancers, and 44 prostate tumour samples. A missense R16H (47G>A) change was identified in two CRC samples, as well as in the respective normal tissues, but was absent in 209 healthy controls. The remaining findings were silent, previously unreported, changes of the coding, non-coding, or untranslated regions of AIP. These results suggest that somatic AIP mutations are not common in CRC, breast, and prostate cancers.