Genetic and Epigenetic Characterization of Colorectal and Endometrial Cancer

Colorectal cancer is one of the three most common cancers today, for both men and women. Approximately 90% of the cases are sporadic while the remaining 10% is hereditary. Among this 10% is hereditary nonpolyposis colorectal cancer (HNPCC), an autosomal dominant disease, accounting for up to 13% of these cases. HNPCC is associated with germline mutations in four mismatch repair (MMR) genes, MLH1, MSH2, MSH6, and PMS2, and is characterized by a familial accumulation of endometrial, gastric, urological, and ovarian tumors, in addition to colorectal cancer. An important etiological characteristic of HNPCC is the presence of microsatellite instability (MSI), caused by mutations of the MMR genes. Approximately 15% of sporadic cases share the MSI+ trait. Colon cancer is believed to be a consequence of an accumulation of mutations in tumor suppressor genes and oncogenes, eventually resulting in tumor development. This phenomena is accelerated in HNPCC due the presence of an inherited mutation in the MMR genes, accounting for one of the two hits proposed to be needed by Knudson (1971) in order for the manifestation of the MSI phenotype. MMR alterations alone, however, do not occur in the majority of sporadic colon cancers, prompting searches for other mechanisms. One such mechanism found to play a role in colon cancer development was DNA methylation, which is known to play a role in MLH1 inactivation. Our objective was clarification of mechanisms associated with tumor development in both HNPCC and sporadic colorectal cancer in relation to tumorigenic mechanisms. Of particular interest were underlying mechanisms of MSI in sporadic colorectal cancers, with attention to DNA methylation changes and their correlation to MSI. Of additional interest were the genetic and epigenetic events leading to the HNPCC tumor spectrum, chiefly colon and endometrial cancers, in regards to what extent the somatic changes in target tissue explained this phenomenon. We made a number of important findings pertaining to these questions. First, MSI tumor development differs epigenetically from stable tumor development, possibly underlying developmental pathway differences. Additionally, while epigenetic modification, principally DNA methylation, is a major mechanism in sporadic MSI colorectal cancer MLH1 inactivation it does not play a significant role in HNPCC tumors with germline MLH1 mutations. This is possibly an explanation for tumorigenic pathways and clinicopathological characteristic differences between sporadic and hereditary MSI colorectal cancers. Finally, despite indistinguishable genetic predisposition for endometrial and colorectal cancers, instability profiles highlighting organ-specific differences, may be important HNPCC tumor spectrum determinants.

Genetic Basis of Pituitary Adenoma Predisposition

Much of the global cancer research is focused on the most prevalent tumors; yet, less common tumor types warrant investigation, since A rare disorder is not necessarily an unimportant one . The present work discusses a rare tumor type, the benign adenomas of the pituitary gland, and presents the advances which, during the course of this thesis work, contributed to the elucidation of a fraction of their genetic background. Pituitary adenomas are benign neoplasms of the anterior pituitary lobe, accounting for approximately 15% of all intracranial tumors. Pituitary adenoma cells hypersecrete the hormones normally produced by the anterior pituitary tissue, such as growth hormone (GH) and prolactin (PRL). Despite their non-metastasizing nature, these adenomas can cause significant morbidity and have to be adequately treated; otherwise, they can compromise the patient s quality of life, due to conditions provoked by hormonal hypersecretion, such as acromegaly in the case of GH-secreting adenomas, or due to compressive effects to surrounding tissues. The vast majority of pituitary adenomas arise sporadically, whereas a small subset occur as component of familial endocrine-related tumor syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 is caused by germline mutations in the MEN1 tumor suppressor gene (11q13), whereas the majority of CNC cases carry germline mutations in the PRKAR1A gene (17q24). Pituitary adenomas are also encountered in familial settings outside the context of MEN1 and CNC, but unlike in the latter syndromes, their genetic background until recently remained elusive. Evidence in previous literature supported the notion that a tumor suppressor gene on 11q13, residing very close to but still distinct from MEN1, causes genetic susceptibility to pituitary tumors. The aim of the study was to identify the genetic cause of a low penetrance form of Pituitary Adenoma Predisposition (PAP) in families from Northern Finland. The present work describes the methodological approach that led to the identification of aryl hydrocarbon receptor interacting protein (AIP) as the gene causing PAP. Combining chip-based technologies (SNP and gene expression arrays) with traditional gene mapping methods and genealogy data, we showed that germline AIP mutations cause PAP in familial and sporadic settings. PAP patients were diagnosed with mostly adenomas of the GH/PRL-secreting cell lineage. In Finland, two AIP mutations accounted for 16% of all patients diagnosed with GH-secreting adenomas, and for 40% of patients being younger than 35 years of age at diagnosis. AIP is suggested to act as a tumor suppressor gene, a notion supported by the nature of the identified mutations (most are truncating) and the biallelic inactivation of AIP in the tumors studied. AIP has been best characterized as a cytoplasmic interaction partner of aryl hydrocarbon receptor (AHR), also known as dioxin receptor, but it has other partners as well. The mechanisms that underlie AIP-mediated pituitary tumorigenesis are to date largely unknown and warrant further investigation. Because AIP was identified in the genetically homogeneous Finnish population, it was relevant to examine its contribution to PAP in other, more heterogeneous, populations. Analysis of pituitary adenoma patient series of various ethnic origins and differing clinical settings revealed germline AIP mutations in all cohorts studied, albeit with low frequencies (range 0.8-7.4%). Overall, PAP patients were typically diagnosed at a young age (range 8-41 years), mainly with GH-secreting adenomas, without strong family history of endocrine disease. Because many PAP patients did not display family history of pituitary adenomas, detection of the condition appeared challenging. AIP immunohistochemistry was tested as a molecular pre-screening tool on mutation-positive versus mutation-negative tumors, and proved to be a potentially useful predictor of PAP. Mutation screening of a large cohort of colorectal, breast, and prostate tumors did not reveal somatic AIP mutations. These tumors, apart from being the most prevalent among men and women worldwide, have been associated with acromegaly, particularly colorectal neoplasia. In this material, AIP did not appear to contribute to the pathogenesis of these common tumor types and other genes seem likely to play a role in such tumorigenesis. Finally, the contribution of AIP in pediatric onset pituitary adenomas was examined in a unique population-based cohort of sporadic pituitary adenoma patients from Italy. Germline AIP mutations may account for a subset of pediatric onset GH-secreting adenomas (in this study one of seven GH-secreting adenoma cases or 14.3%), and appear to be enriched among young (≤25 years old) patients. In summary, this work reveals a novel tumor susceptibility gene, namely AIP, which causes genetic predisposition to pituitary adenomas, in particular GH-secreting adenomas. Moreover, it provides molecular tools for identification of individuals predisposed for PAP. Further elaborate studies addressing the functional role of AIP in normal and tumor cells will hopefully expand our knowledge on endocrine neoplasia and reveal novel cellular mechanisms of pituitary tumorigenesis, including potential drug targets.

Molecular basis of colorectal cancer predisposition

Colorectal cancer (CRC) is one of the most frequent malignancies in Western countries. Inherited factors have been suggested to be involved in 35% of CRCs. The hereditary CRC syndromes explain only ~6% of all CRCs, indicating that a large proportion of the inherited susceptibility is still unexplained. Much of the remaining genetic predisposition for CRC is probably due to undiscovered low-penetrance variations. This study was conducted to identify germline and somatic changes that contribute to CRC predisposition and tumorigenesis. MLH1 and MSH2, that underlie Hereditary non-polyposis colorectal cancer (HNPCC) are considered to be tumor suppressor genes; the first hit is inherited in the germline and somatic inactivation of the wild type allele is required for tumor initiation. In a recent study, frequent loss of the mutant allele in HNPCC tumors was detected and a new model, arguing against the two-hit hypothesis, was proposed for somatic HNPCC tumorigenesis. We tested this hypothesis by conducting LOH analysis on 25 colorectal HNPCC tumors with a known germline mutation in the MLH1 or MSH2 genes. LOH was detected in 56% of the tumors. All the losses targeted the wild type allele supporting the classical two-hit model for HNPCC tumorigenesis. The variants 3020insC, R702W and G908R in NOD2 predispose to Crohn s disease. Contribution of NOD2 to CRC predisposition has been examined in several case-control series, with conflicting results. We have previously shown that 3020insC does not predispose to CRC in Finnish CRC patients. To expand our previous study the variants R702W and G908R were genotyped in a population-based series of 1042 Finnish CRC patients and 508 healthy controls. Association analyses did not show significant evidence for association of the variants with CRC. Single nucleotide polymorphism (SNP) rs6983267 at chromosome 8q24 was the first CRC susceptibility variant identified through genome-wide association studies. To characterize the role of rs6983267 in CRC predisposition in the Finnish population, we genotyped the SNP in the case-control material of 1042 cases and 1012 controls and showed that G allele of rs6983267 is associated with the increased risk of CRC (OR 1.22; P=0.0018). Examination of allelic imbalance in the tumors heterozygous for rs6983267 revealed that copy number increase affected 22% of the tumors and interestingly, it favored the G allele. By utilizing a computer algorithm, Enhancer Element Locator (EEL), an evolutionary conserved regulatory motif containing rs6983267 was identified. The SNP affected the binding site of TCF4, a transcription factor that mediates Wnt signaling in cells, and has proven to be crucial in colorectal neoplasia. The preferential binding of TCF4 to the risk allele G was showed in vitro and in vivo. The element drove lacZ marker gene expression in mouse embryos in a pattern that is consistent with genes regulated by the Wnt signaling pathway. These results suggest that rs6983267 at 8q24 exerts its effect in CRC predisposition by regulating gene expression. The most obvious target gene for the enhancer element is MYC, residing ~335 kb downstream, however further studies are required to establish the transcriptional target(s) of the predicted enhancer element.

Genetic basis of hereditary colorectal cancers: Hereditary nonpolyposis colorectal cancer and Familial adenomatous polyposis

Hereditary nonpolyposis colorectal cancer (HNPCC) and familial adenomatous polyposis (FAP) are characterized by a high risk and early onset of colorectal cancer (CRC). HNPCC is due to a germline mutation in one of the following MMR genes: MLH1, MSH2, MSH6 and PMS2. A majority of FAP and attenuated FAP (AFAP) cases are due to germline mutations of APC, causing the development of multiple colorectal polyps. To date, over 450 MMR gene mutations and over 800 APC mutations have been identified. Most of these mutations lead to a truncated protein, easily detected by conventional mutation detection methods. However, in about 30% of HNPCC and FAP, and about 90% of AFAP families, mutations remain unknown. We aimed to clarify the genetic basis and genotype-phenotype correlation of mutation negative HNPCC and FAP/AFAP families by advanced mutation detection methods designed to detect large genomic rearrangements, mRNA and protein expression alterations, promoter mutations, phenotype linked haplotypes, and tumoral loss of heterozygosity. We also aimed to estimate the frequency of HNPCC in Uruguayan CRC patients. Our expression based analysis of mutation negative HNPCC divided these families into two categories: 1) 42% of families linked to the MMR genes with a phenotype resembling that of mutation positive, and 2) 58% of families likely to be associated with other susceptibility genes. Unbalanced mRNA expression of MLH1 was observed in two families. Further studies revealed that a MLH1 nonsense mutation, R100X was associated with aberrant splicing of exons not related to the mutation and an MLH1 deletion (AGAA) at nucleotide 210 was associated with multiple exon skipping, without an overall increase in the frequency of splice events. APC mutation negative FAP/AFAP families were divided into four groups according to the genetic basis of their predisposition. Four (14%) families displayed a constitutional deletion of APC with profuse polyposis, early age of onset and frequent extracolonic manifestations. Aberrant mRNA expression of one allele was observed in seven (24%) families with later onset and less frequent extracolonic manifestations. In 15 (52%) families the involvement of APC could neither be confirmed nor excluded. In three (10%) of the families a germline mutation was detected in genes other than APC: AXIN2 in one family, and MYH in two families. The families with undefined genetic basis and especially those with AXIN2 or MYH mutations frequently displayed AFAP or atypical polyposis. Of the Uruguayan CRC patients, 2.6% (12/461) fulfilled the diagnostic criteria for HNPCC and 5.6% (26/461) were associated with increased risk of cancer. Unexpectedly low frequency of molecularly defined HNPCC cases may suggest a different genetic profile in the Uruguayan population and the involvement of novel susceptibility genes. Accurate genetic and clinical characterization of families with hereditary colorectal cancers, and the definition of the genetic basis of "mutation negative" families in particular, facilitate proper clinical management of such families.

Molecular classification of familial colorectal and endometrial carcinoma

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common known clearly hereditary cause of colorectal and endometrial cancer (CRC and EC). Dominantly inherited mutations in one of the known mismatch repair (MMR) genes predispose to HNPCC. Defective MMR leads to an accumulation of mutations especially in repeat tracts, presenting microsatellite instability. HNPCC is clinically a very heterogeneous disease. The age at onset varies and the target tissue may vary. In addition, families that fulfill the diagnostic criteria for HNPCC but fail to show any predisposing mutation in MMR genes exist. Our aim was to evaluate the genetic background of familial CRC and EC. We performed comprehensive molecular and DNA copy number analyses of CRCs fulfilling the diagnostic criteria for HNPCC. We studied the role of five pathways (MMR, Wnt, p53, CIN, PI3K/AKT) and divided the tumors into two groups, one with MMR gene germline mutations and the other without. We observed that MMR proficient familial CRC consist of two molecularly distinct groups that differ from MMR deficient tumors. Group A shows paucity of common molecular and chromosomal alterations characteristic of colorectal carcinogenesis. Group B shows molecular features similar to classical microsatellite stable tumors with gross chromosomal alterations. Our finding of a unique tumor profile in group A suggests the involvement of novel predisposing genes and pathways in colorectal cancer cohorts not linked to MMR gene defects. We investigated the genetic background of familial ECs. Among 22 families with clustering of EC, two (9%) were due to MMR gene germline mutations. The remaining familial site-specific ECs are largely comparable with HNPCC associated ECs, the main difference between these groups being MMR proficiency vs. deficiency. We studied the role of PI3K/AKT pathway in familial ECs as well and observed that PIK3CA amplifications are characteristic of familial site-specific EC without MMR gene germline mutations. Most of the high-level amplifications occurred in tumors with stable microsatellites, suggesting that these tumors are more likely associated with chromosomal rather than microsatellite instability and MMR defect. The existence of site-specific endometrial carcinoma as a separate entity remains equivocal until predisposing genes are identified. It is possible that no single highly penetrant gene for this proposed syndrome exists, it may, for example be due to a combination of multiple low penetrance genes. Despite advances in deciphering the molecular genetic background of HNPCC, it is poorly understood why certain organs are more susceptible than others to cancer development. We found that important determinants of the HNPCC tumor spectrum are, in addition to different predisposing germline mutations, organ specific target genes and different instability profiles, loss of heterozygosity at MLH1 locus, and MLH1 promoter methylation. This study provided more precise molecular classification of families with CRC and EC. Our observations on familial CRC and EC are likely to have broader significance that extends to sporadic CRC and EC as well.

Genetic alterations in microsatellite-unstable colorectal cancer

Colorectal cancer (CRC) is the third most common cancer in Finland. Of all CRC tumors, 15% display microsatellite-instability (MSI) caused by defective cellular mismatch repair. Cells displaying MSI accumulate a high number of mutations genome-wide, especially in short repeat areas, microsatellites. When targeting genes essential for cell growth or death, MSI can promote tumorigenesis. In non-coding areas, microsatellite mutations are generally considered as passenger events. Since the discovery of MSI and its linkage to cancer, more that 200 genes have been investigated for a role in MSI tumorigenesis. Although various criteria have been suggested for MSI target gene identification, the challenge has been to distinguish driver mutations from passenger mutations. This study aimed to clarify these key issues in the research field of MSI cancer. Prior to this, background mutation rate in MSI cancer has not been studied in a large-scale. We investigated the background mutation rate in MSI CRC by analyzing the spectrum of microsatellite mutations in non-coding areas. First, semenogelin I was studied for a possible role in MSI carcinogenesis. The intronic T9 repeat of semenogelin I was frequently mutated but no evidence for selection during tumorigenesis was obtained. Second, a sequencing approach was utilized to evaluate the general background mutation rate in MSI CRC. Both intronic and intergenic repeats harbored extremely high mutation rates of ≤ 87% and intergenic repeats were more unstable than the intronic repeats. As mutation rates of presumably neutral microsatellites can be high in MSI CRC in the absence of apparent selection pressure, high mutation frequency alone is not sufficient evidence for identification of driver MSI target genes. Next, an unbiased approach was designed to identify the mutatome of MSI CRC. By combining expression array data and a database search we identified novel genes possibly related to MSI CRC carcinogenesis. One of the genes was studied further. In the functional analysis this gene was observed to cause an abnormal cancer-prone cellular phenotype, possibly through altered responses to DNA damage. In our recent study, smooth muscle myosin heavy chain 11 (MYH11) was identified as a novel MSI CRC gene. Additionally, MYH11 has a well established role in acute myeloid leukemia (AML) through an oncogenic fusion protein CBFB-MYH11. We investigated further the role of MYH11 in AML by sequencing. Three novel missense variants of MYH11 were identified. None of the variants were present in the population-based control material. One of the identified variants, V71A, lies in the N-terminal SH3-like domain of MYH11 of unknown function. The other two variants, K1059E and R1792Q are located in the coil-coiled myosin rod essential for the regulation and filament formation of MYH11. The variant K1059E lies in the close proximity of the K1044N that has been functionally assessed in our earlier work of CRC and has been reported to cause total loss of MYH11 protein regulation. As the functional significance of the three novel variants examined in this work remains unknown, future studies should clarify the further role of MYH11 in AML leukaemogenesis and in other malignancies.

Novel Insights into the Molecular Genetic Background of Colorectal Tumors

Many of the genes predisposing to highly penetrant colorectal cancer (CRC) syndromes, including hereditary non-polyposis colorectal cancer (MLH1, MSH2, MSH6, PMS2), familial adenomatous polyposis (APC), Peutz-Jeghers syndrome (LKB1), juvenile polyposis (SMAD4, BMPR1A), MYH-associated polyposis (MYH), and Cowden syndrome (PTEN) have already been discovered. Identification of these genes has allowed a more precise classification of the hereditary CRC syndromes and provided a means for predictive genetic testing and surveillance. Some of the genes are also involved in sporadic cancer forms, and therefore the investigation of the rare CRC syndromes has been a breakthrough for general cancer research. Despite the accumulating knowledge on hereditary cancer syndromes, a significant number of familial CRCs remain molecularly unexplained after genetic testing, reflecting the possibility of other predisposing genes or existence of novel syndromes. Moreover, genetic variants conferring low-penetrance risk are still largely unknown. In this study, we examined the role of some new high- and low-penetrance alleles on CRC predisposition. We identified disease causing MYH mutations in a subset (9%) of patients with APC and AXIN2 mutation negative adenomatous polyposis. Due to differences in the pattern of inheritance and clinical manifestation, screening for mutations in MYH is beneficial in view of genetic counselling and surveillance. A novel functionally deficient MYH founder mutation A459D was identified in the Finnish population, and this finding had immediate clinical implications for genetic counselling of at risk families. Many patients with hamartomatous polyposis remain without molecular diagnosis due to atypical phenotypes. We therefore sought to classify 49 patients with unexplained hamartomatous or hyperplastic/mixed polyposis by extensive molecular analyses of PTEN, LKB1, BMPR1A, SMAD4, ENG, BRAF, MYH, and BHD along with revision of polyp histology. Mutations were identified in 11/49 (22%) of the patients. In 6 cases the molecular diagnosis was re-classified guiding surveillance and decisions for prophylactic surgery. Re-evaluation of polyp histology with subsequent more accurate selection of candidate gene analyses is beneficial and can be recommended for patients with unexplained polyposis. Furthermore, germline mutations in ENG underlying juvenile polyposis were described for the first time, characterizing a possible novel genetically defined form of hereditary CRC. Association analyses on two putative low-penetrance alleles, NOD2 3020insC and MDM2 SNP309 were performed in a population-based series of 1042 Finnish CRC patients and in cancer-free controls. In contrast to previous results, NOD2 3020insC did not associate with CRC or age at disease onset in the Finnish population. These data suggest that NOD2 3020insC alone might not be sufficient for CRC predisposition. MDM2 SNP309 was as common in the CRC cohort as in the healthy controls. Interesting trends, however, were observed, which after correction for multiple testing did not reach statistical significance. SNP309 was more common in female CRC patients and a trend towards an earlier age at disease onset was observed in women with SNP309. Subsequent studies have supported this observation and SNP309 could affect gender- or hormone-related tumorigenesis. Finally, a large-scale unbiased effort was designed to characterize the complete mutatome of CRC with microsatellite instability (MSI). Using an approach combining expression microarray and genome database searches, we were able to identify putative MSI target genes. Further characterization of one of the genes suggested that it might play a role also in microsatellite stable CRC and Peutz-Jeghers syndrome pathogenesis.

Surveillance in hereditary nonpolyposis colorectal cancer syndrome

Hereditary nonpolyposis colorectal cancer (HNPCC) is an inherited cancer predisposition syn-drome characterized by early onset colorectal cancer (CRC) and several other extra-colonic cancers, most commonly endometrial cancer (EC) and gastric cancer. Our aim was to evaluate the efficiency and results of the ongoing CRC and EC surveillance programs and to investigate the grounds for future gastric cancer screening by comparing the gastric biopsies of mutation positive and negative siblings in search for premalignant lesions. We also compared a new surveillance method, computerized tomographic colonoscopy (CTC) with optic colonoscopy. The patient material consisted of 579 family members from 111 Finnish HNPCC families al-most all harboring a known mismatch repair gene mutation. The efficacy of CRC and EC surveillance programs on HNPCC patients was evaluated by comparing the stage and survival of cancer cases detected with surveillance versus without. The performance of a new technique, CTC, was explored using a same-day colonoscopy as a reference standard. The use of intrauterine aspiration biopsies for EC surveillance was intro-duced for the first time in a HNPCC setting. Upper GI endoscopies were performed and biop-sies taken from mutation carriers and their mutation-negative siblings. The present surveillance program for CRC proved to be efficient. The CRC cases detected by surveillance were at a significantly more favorable stage than those in the non-surveilled group. This advantage was reflected in a significantly higher CRC-specific survival in the sur-veilled group. The performance of a new technique, CTC was found insufficient for polyp detection in this population in which every polyp, no matter the size, should be detected and removed. Colono-scopy was confirmed as a better surveillance modality than CTC. We could not observe any of the assumed differences in the gastric mucosa from mutation carriers and their mutation-negative siblings and no cases of gastric cancer were detected. The results gave no support for gastric surveillance. The EC surveillance program (transvaginal ultrasound and intra-uterine biopsy every 2-3 years) seemed to be efficient. It yielded several asymptomatic cancer cases and premalignant lesions. The stage distribution of the endometrial cancers in the group under surveillance tended to be more favorable than that of the mutation-positive, symptomatic EC patients who had no surveillance. None of the surveilled EC patients died of EC compared to six in the non-surveilled patients during the follow up. The improvement was, however, not statistically sig-nificant, thus far. Another observation was the good performance of endometrial aspiration biopsies used in this setting for the first time.