CHEK2 Founder Variants and Thyroid Cancer Risk.

Background: Germline pathogenic variants in CHEK2 are associated with a moderate increase in the lifetime risk for breast cancer. Increased risk for other cancers, including non-medullary thyroid cancer (NMTC), has also been suggested. To date, data implicating CHEK2 variants in NMTC predisposition primarily derive from studies within Poland, driven by a splice site variant (c.444 + 1G>A) that is uncommon in other populations. In contrast, the predominant CHEK2 variants in non-Polish populations are c.1100del and c.470T>C/p.I157T, representing 61.1% and 63.8%, respectively, of all CHEK2 pathogenic variants in two large U.S.-based commercial laboratory datasets. To further delineate the impact of common CHEK2 variants on thyroid cancer, we aimed to investigate the association of three CHEK2 founder variants (c.444 + 1G>A, c.1100del, and c.470T>C/p.Ile157Thr) on NMTC susceptibility in three groups of unselected NMTC patients. Methods: The presence of three CHEK2 founder variants was assessed within three groups: (1) 1544 NMTC patients (and 1593 controls) from previously published genome-wide association study (GWAS) analyses, (2) 789 NMTC patients with germline exome sequencing (Oncology Research Information Exchange Network [ORIEN] Avatar), and (3) 499 NMTC patients with germline sequence data available in The Cancer Genome Atlas (TCGA). A case-control study design was utilized with odds ratios (ORs) calculated by comparison of all three groups with the Ohio State University GWAS control group. Results: The predominant Polish variant (c.444 + 1G>A) was present in only one case. The proportion of patients with c.1100del was 0.92% in the GWAS group, 1.65% in the ORIEN Avatar group, and 0.80% in the TCGA group. The ORs (with 95% confidence intervals [CIs]) for NMTC associated with c.1100del were 1.71 (0.73-4.29), 2.64 (0.95-7.63), and 2.5 (0.63-8.46), respectively. The proportion of patients with c.470T>C/p.I157T was 0.91% in the GWAS group, 0.76% in the ORIEN Avatar group, and 0.80% in the TCGA group, respectively. The ORs (with CIs) for NMTC associated with c.470T>C/p.I157T were 1.75 (0.74-4.39), 1.52 (0.42-4.96), and 2.31 (0.58-7.90), respectively. Conclusions: Our analyses of unselected patients with NMTC suggest that CHEK2 variants c.1100del and c.470T>C/p.I157T have only a modest impact on thyroid cancer risk. These results provide important information for providers regarding the relatively low magnitude of thyroid cancer risk associated with these CHEK2 variants.

PDPR Gene Variants Predisposing to Papillary Thyroid Cancer.

Background: Papillary thyroid cancer (PTC) is the predominant subtype of thyroid cancer (THCA), and it can cluster in families with an autosomal dominant (AD) inheritance pattern. The aim of this study was to identify novel genes and mechanisms underlying PTC susceptibility. Methods: Our previous investigation of 17 AD PTC families led us to conduct a deeper analysis on one family (Family Q) with whole-genome sequencing data from 3 PTC-affected individuals. In addition, 323 sporadic THCA cases from Avatar data and 12 familial adenomatous polyposis (FAP) individuals with secondary THCA were screened for pyruvate dehydrogenase phosphatase regulatory (PDPR) variants. CRISPR-Cas9 was used to create PDPR-deficient THCA (TPC1) and transformed normal thyroid cell lines (N-Thyori3-1) to study the metabolic consequences of PDPR loss. Results: We found truncating PDPR splice donor variants (NM_017990.4:c.361 + 1G>C) in all affected PTC Family Q members, and another PDPR splice donor variant (NM_017990.4:c.443 + 1G>C) in a sporadic PTC case. In addition, an ultra-rare missense variant was found in an FAP-PTC patient. The PDPR-deficient cells presented with elevated phosphorylation of pyruvate dehydrogenase and altered glucose metabolism, implying that PDPR plays an essential part in regulating glucose metabolism in thyroid cells. Conclusions: Our finding of novel truncating germline variants in PDPR in Family Q and additional cohorts suggests a role for PDPR loss in PTC predisposition. Also, somatic and RNA sequencing from the thyroid carcinoma (Firehouse Legacy) data showed that PDPR gene expression is much lower in THCA tumor tissue compared with matching normal tissue. Thus, PDPR appears to have a loss of function effect on THCA tumorigenesis.

Mono- and biallelic germline variants of DNA glycosylase genes in colon adenomatous polyposis families from two continents.

Recently, biallelic germline variants of the DNA glycosylase genes MUTYH and NTHL1 were linked to polyposis susceptibility. Significant fractions remain without a molecular explanation, warranting searches for underlying causes. We used exome sequencing to investigate clinically well-defined adenomatous polyposis cases and families from Finland (N=34), Chile (N=21), and Argentina (N=12), all with known susceptibility genes excluded. Nine index cases (13%) revealed germline variants with proven or possible pathogenicity in the DNA glycosylase genes, involving NEIL1 (mono- or biallelic) in 3 cases, MUTYH (monoallelic) in 3 cases, NTHL1 (biallelic) in 1 case, and OGG1 (monoallelic) in 2 cases. NTHL1 was affected with the well-established, pathogenic c.268C>T, p.(Gln90Ter) variant. A recurrent heterozygous NEIL1 c.506G>A, p.(Gly169Asp) variant was observed in two families. In a Finnish family, the variant occurred in trans with a truncating NEIL1 variant (c.821delT). In an Argentine family, the variant co-occurred with a genomic deletion of exons 2 - 11 of PMS2. Mutational signatures in tumor tissues complied with biological functions reported for NEIL1. Our results suggest that germline variants in DNA glycosylase genes may occur in a non-negligible proportion of unexplained colon polyposis cases and may predispose to tumor development.

A novel essential splice site variant in SPTB in a large hereditary spherocytosis family.

BACKGROUND: We studied a large family with 22 individuals affected with autosomal dominant hereditary spherocytosis (HS). METHODS: Genome-wide linkage, whole-genome sequencing (WGS), Sanger sequencing, RT-PCR, and ToPO TA cloning analyses were performed. RESULTS: We revealed a heterozygous G>A transition in the 14q23 locus, at position +1 of the intron 8 donor splice site of the spectrin beta, erythrocytic (SPTB) gene. This splice variant (SPTB c.1064+1G>A) was confirmed by Sanger sequencing and showed complete co-segregation with HS in the family. Further RT-PCR reactions and sequencing analysis indicated that the variant leads to the exclusion of exon 8 and subsequent frameshift in exon 9 and a premature stop codon in SPTB. Translation of the altered allele would lead to a truncation with a loss of all spectrin repeat domains in SPTB protein. CONCLUSION: This variant is novel and has not been found in any databases. We propose that this splice variant explains the spherocytosis phenotype observed in this large family.

Updates in the field of hereditary nonpolyposis colorectal cancer.

INTRODUCTION: Up to one third of colorectal cancers show familial clustering and 5% are hereditary single-gene disorders. Hereditary non-polyposis colorectal cancer comprises DNA mismatch repair-deficient and -proficient subsets, represented by Lynch syndrome (LS) and familial colorectal cancer type X (FCCTX), respectively. Accurate knowledge of molecular etiology and genotype-phenotype correlations are critical for tailored cancer prevention and treatment. AREAS COVERED: The authors highlight advances in the molecular dissection of hereditary non-polyposis colorectal cancer, based on recent literature retrieved from PubMed. Future possibilities for novel gene discoveries are discussed. EXPERT COMMENTARY: LS is molecularly well established, but new information is accumulating of the associated clinical and tumor phenotypes. FCCTX remains poorly defined, but several promising candidate genes have been discovered and share some preferential biological pathways. Multi-level characterization of specimens from large patient cohorts representing multiple populations, combined with proper bioinformatic and functional analyses, will be necessary to resolve the outstanding questions.

Molecular Basis of Mismatch Repair Protein Deficiency in Tumors from Lynch Suspected Cases with Negative Germline Test Results.

Some 10-50% of Lynch-suspected cases with abnormal immunohistochemical (IHC) staining remain without any identifiable germline mutation of DNA mismatch repair (MMR) genes. MMR proteins form heterodimeric complexes, giving rise to distinct IHC patterns when mutant. Potential reasons for not finding a germline mutation include involvement of an MMR gene not predicted by the IHC pattern, epigenetic mechanism of predisposition, primary mutation in another DNA repair or replication-associated gene, and double somatic MMR gene mutations. We addressed these possibilities by germline and tumor studies in 60 Lynch-suspected cases ascertained through diagnostics (n = 55) or research (n = 5). All cases had abnormal MMR protein staining in tumors but no point mutation or large rearrangement of the suspected MMR genes in the germline. In diagnostic practice, MSH2/MSH6 (MutS Homolog 2/MutS Homolog 6) deficiency prompts MSH2 mutation screening; in our study, 3/11 index individuals (27%) with this IHC pattern revealed pathogenic germline mutations in MSH6. Individuals with isolated absence of MSH6 are routinely screened for MSH6 mutations alone; we found a predisposing mutation in MSH2 in 1/7 such cases (14%). Somatic deletion of the MSH2-MSH6 region, joint loss of MSH6 and MSH3 (MutS Homolog 3) proteins, and hindered MSH2/MSH6 dimerization offered explanations to misleading IHC patterns. Constitutional epimutation hypothesis was pursued in the MSH2 and/or MSH6-deficient cases plus 38 cases with MLH1 (MutL Homolog 1)-deficient tumors; a primary MLH1 epimutation was identified in one case with an MLH1-deficient tumor. We conclude that both MSH2 and MSH6 should be screened in MSH2/6- and MSH6-deficient cases. In MLH1-deficient cases, constitutional epimutations of MLH1 warrant consideration.

Thyroid Carcinomas That Occur in Familial Adenomatous Polyposis Patients Recurrently Harbor Somatic Variants in APC, BRAF, and KTM2D.

Background: Familial adenomatous polyposis (FAP) is a condition typically caused by pathogenic germline mutations in the APC gene. In addition to colon polyps, individuals with FAP have a substantially increased risk of developing papillary thyroid cancer (PTC). Little is known about the events underlying this association, and the prevalence of somatic "second-hit" mutations in APC is controversial. Methods: Whole-genome sequencing was performed on paired thyroid tumor and normal DNA from 12 FAP patients who developed PTC. Somatic mutation profiles were compared with clinical characteristics and previously sequenced sporadic PTC cases. Germline variant profiling was performed to assess the prevalence of variants in genes previously shown to have a role in PTC predisposition. Results: All 12 patients harbored germline mutations in APC, consistent with FAP. Seven patients also had somatic mutations in APC, and seven patients harbored somatic mutations in KMT2D, which encodes a lysine methyl transferase. Mutation of these genes is extremely rare in sporadic PTCs. Notably, only two of the tumors harbored the somatic BRAF p.V600E mutation, which is the most common driver mutation found in sporadic PTCs. Six tumors displayed a cribriform-morular variant of PTC (PTC-CMV) histology, and all six had somatic mutations in APC. Additionally, nine FAP-PTC patients had rare germline variants in genes that were previously associated with thyroid carcinoma. Conclusions: Our data indicate that FAP-associated PTCs typically have distinct mutations compared with sporadic PTCs. Roughly half of the thyroid cancers that arise in FAP patients have somatic "second-hits" in APC, which is associated with PTC-CMV histology. Somatic BRAF p.V600E variants also occur in some FAP patients, a novel finding. We speculate that in carriers of heterozygous pathogenic mutations of tumor suppressor genes such as APC, a cooperating second-hit somatic variant may occur in a different gene such as KTM2D or BRAF, leading to differences in phenotypes. The role of germline variance in genes other than APC (9 of the 12 patients in this series) needs further research.

A Truncating Germline Mutation of TINF2 in Individuals with Thyroid Cancer or Melanoma Results in Longer Telomeres.

Background: Our genome sequencing analysis revealed a frameshift mutation in the shelterin gene TINF2 in a large family with individuals affected with papillary thyroid carcinoma (PTC) and melanoma. Here, we further characterized the mutation and screened for coding variants in the 6 shelterin genes in 24 families. Methods: Sanger sequencing was performed to screen for the TINF2 mutation in the key family. Quantitative reverse transcription-polymerase chain reaction (PCR) was used for TINF2 gene expression analysis. Exogenous expression and co-immunoprecipitation techniques were used for assessing TINF2 binding to TERF1. Relative telomere length (RTL) was quantified in DNAs from lymphocytes by using quantitative real-time PCR. Whole exome sequencing (WES) was performed in seven families with individuals affected with PTC and other cancer types. Screening for DNA variants in shelterin genes was performed by using whole genome sequencing data from 17 families and WES data from 7 further families. Results: The TINF2 mutation (TINF2 p.Trp198fs) showed complete co-segregation with PTC and melanoma in the key family. The mutation is not reported in databases and not identified in 23 other families we screened. The expression of TINF2 was borderline reduced in individuals with the mutation. The truncated TINF2 protein showed abolished binding to TERF1. The RTL in the individuals with the mutation was significantly longer when compared with those without the mutation from the same family as well as compared with 62 healthy controls. Among the 24 families, we identified 3 missense and 1 synonymous variant(s) in 2 shelterin genes (TINF2 and ACD). Conclusions: The rare frameshift mutation in the TINF2 gene and the associated longer telomere length suggest that dysregulated telomeres could be a mechanism predisposing to PTC and melanoma. DNA coding variants in shelterin genes are rare. Further studies are required to evaluate the roles of variants in shelterin genes in thyroid cancer and melanoma.

Identification of Rare Variants Predisposing to Thyroid Cancer.

Background: Familial non-medullary thyroid cancer (NMTC) accounts for a relatively small proportion of thyroid cancer cases, but it displays strong genetic predisposition. So far, only a few NMTC susceptible genes and low-penetrance variants contributing to NMTC have been described. This study aimed to identify rare germline variants that may predispose individuals to NMTC by sequencing a cohort of 17 NMTC families. Methods: Whole-genome sequencing and genome-wide linkage analysis were performed in 17 NMTC families. MendelScan and BasePlayer were applied to screen germline variants followed by customized filtering. The remaining candidate variants were subsequently validated by Sanger sequencing. A panel of 277 known cancer predisposition genes was also screened in these families. Results: A total of 41 rare coding candidate variants in 40 genes identified by whole-genome sequencing are reported, including 24 missense, five frameshift, five splice change, and seven nonsense variants. Sanger sequencing confirmed all 41 rare variants and proved their co-segregation with NMTC in the extended pedigrees. In silico functional analysis of the candidate genes using Ingenuity Pathway Analysis showed that cancer was the top category of "Diseases and Disorders." Additionally, a targeted search displayed six variants in known cancer predisposition genes, including one frameshift variant and five missense variants. Conclusions: The data identify rare germline variants that may play important roles in NMTC predisposition. It is proposed that in future research including functional characterization, these variants and genes be considered primary candidates for thyroid cancer predisposition.

Biallelic germline nonsense variant of MLH3 underlies polyposis predisposition.

PURPOSE: Some 10% of familial adenomatous polyposis (FAP) and 80% of attenuated polyposis (AFAP) cases remain molecularly unexplained. We scrutinized such cases by exome-wide and targeted methods to search for novel susceptibility genes. METHODS: Exome sequencing was conducted on 40 unexplained (mainly sporadic) cases with FAP or AFAP from Finland. The DNA mismatch repair (MMR) gene MLH3 (MutL Homolog 3) was pinpointed and prompted a subsequent screen of ~1000 Swedish patients referred to clinical panel sequencing for colon tumor susceptibility. RESULTS: Three homozygous carriers of a truncating variant in MLH3, c.3563C>G, p.Ser1188Ter, were identified among the index cases from the Finnish series. An additional biallelic carrier of the same variant was present in the Swedish series. All four patients shared a 0.8-Mb core haplotype around MLH3, suggesting a founder variant. Colorectal polyps from variant carriers showed no instability at mono-, di-, tri-, or tetranucleotide repeats, in agreement with previous findings of a minor role of MLH3 in MMR. Multiple loci were affected by loss of heterozygosity, suggesting chromosomal instability. CONCLUSION: Our results show that a biallelic nonsense variant of MLH3 underlies a novel syndrome with susceptibility to classical or attenuated adenomatous polyposis and possibly extracolonic tumors, including breast cancer.

Sequencing of Lynch syndrome tumors reveals the importance of epigenetic alterations.

Genomic instability and epigenetic aberrations are important classifiers of human tumors, yet, their interrelations are poorly understood. We used Lynch syndrome (LS) to address such relationships. Forty-five tumors (11 colorectal adenomas, 18 colorectal carcinomas, and 16 ovarian carcinomas) were profiled for CpG Island Methylator Phenotype (CIMP) and somatic mutations. All tumors showed high-degree microsatellite instability. Panel sequencing of 578 cancer-relevant genes revealed the average number of 1433, 1124, and 657 non-synonymous somatic mutations per colorectal adenoma, colorectal carcinoma, and ovarian carcinoma, respectively. Genes harboring mutations with allele frequency 25 % or higher in at least 31 % of tumors were regarded to be possible drivers. Among 72 and 10 such genes identified in colorectal and ovarian tumors, respectively, the most frequently mutated genes BRD4 and MLL2 (62 % of colorectal tumors) and ARID1A (50 % of ovarian carcinomas) are involved in epigenetic regulation. The total number of somatic mutations or mutant genes per tumor were significantly associated with CIMP. Our results suggest that even in an inherited disease, tumor type-specific epigenetic changes are significant and may result from regulatory changes (CIMP) or structural events (mutations of epigenetic regulatory genes). The findings are clinically relevant since many of the affected pathways can be therapeutically targeted.

Pseudoexons provide a mechanism for allele-specific expression of APC in familial adenomatous polyposis.

Allele-specific expression (ASE) of the Adenomatous Polyposis Coli (APC) gene occurs in up to one-third of families with adenomatous polyposis (FAP) that have screened mutation-negative by conventional techniques. To advance our understanding of the genomic basis of this phenomenon, 54 APC mutation-negative families (21 with classical FAP and 33 with attenuated FAP, AFAP) were investigated. We focused on four families with validated ASE and scrutinized these families by sequencing of the blood transcriptomes (RNA-seq) and genomes (WGS). Three families, two with classical FAP and one with AFAP, revealed deep intronic mutations associated with pseudoexons. In all three families, intronic mutations (c.646-1806T>G in intron 6, c.1408+729A>G in intron 11, and c.1408+731C>T in intron 11) created new splice donor sites resulting in the insertion of intronic sequences (of 127 bp, 83 bp, and 83 bp, respectively) in the APC transcript. The respective intronic mutations were absent in the remaining polyposis families and the general population. Premature stop of translation as the predicted consequence as well as co-segregation with polyposis supported the pathogenicity of the pseudoexons. We conclude that next generation sequencing on RNA and genomic DNA is an effective strategy to reveal and validate pseudoexons that are regularly missed by traditional screening methods and is worth considering in apparent mutation-negative polyposis families.

Desmoid tumor patients carry an elevated risk of familial adenomatous polyposis.

BACKGROUND: The prevalence of desmoid tumors among patients with familial adenomatous polyposis (FAP) is at least 10%, and the prevalence of FAP among desmoid patients varies between 7.5-16%. METHODS: Data included 106 desmoid patients identified from the database of the Department of Pathology, Helsinki University Hospital, years 2000-2012. We evaluated the risk of FAP among patients by using endoscopy and we identified individuals with attenuated FAP by APC gene mutation test. We compared sporadic desmoid patients' and FAP patients' clinical characteristics. RESULTS: Ten of 106 patients already had FAP diagnosis before the desmoid. Eleven patients had had FAP screening already earlier due to desmoid and three of them were found to have FAP. Total of 52 patients participated into prospective screening of FAP. No new cases of FAP were found. The risk of FAP among desmoid tumor patients was 4.8%. In the FAP desmoid group, there were more males and patients were younger than in the sporadic group. Intra-abdominal desmoids were more common in the FAP group. CONCLUSIONS: Patients with desmoid carry an elevated risk of FAP and therefore screening is indicated. Asymptomatic patients with desmoid situated in extra truncal region may not need to be screened.

Methyltransferase expression and tumor suppressor gene methylation in sporadic and familial colorectal cancer.

Molecular mechanisms underlying coordinated hypermethylation of multiple CpG islands in cancer remain unclear and studies of methyltransferase enzymes have arrived at conflicting results. We focused on DNMT1 and DNMT3B, DNA methyltransferases responsible for (de novo) methylation, and EZH2, histone (H3K27) methyltransferase, and examined their roles in tumor suppressor gene (TSG) methylation patterns we have previously established in sporadic and familial cancers. Our investigation comprised 165 tumors, stratified by tissue of origin (117 colorectal and 48 endometrial carcinomas) and sporadic vs. familial disease (57 sporadic vs. 60 familial, mainly Lynch syndrome, colorectal carcinomas). By immunohistochemical evaluation, EZH2 protein expression was associated with a TSG methylator phenotype. DNMT1, DNMT3B, and EZH2 were expressed at significantly higher levels in tumor vs. normal tissues. DNMT1 and EZH2 expression were positively correlated and higher in microsatellite-unstable vs. microsatellite-stable tumors, whether sporadic or hereditary. Ki-67 expression mirrored the same pattern. Promoter methylation of the methyltransferase genes themselves was addressed as a possible cause behind their altered expression. While DNMT1 or EZH2 did not show differential methylation between normal and tumor tissues, DNMT3B analysis corroborated the regulatory role of a distal promoter region. Our study shows that methyltransferase expression in cancer depends on the tissue of origin, microsatellite-instability status, cellular proliferation, and--in the case of DNMT3B--promoter methylation of the respective gene. Translation of methyltransferase expression into DNA methylation appears complex as suggested by the fact that except for EZH2, no clear association between methyltransferase protein expression and TSG methylation was observed.

Germline mutation of RPS20, encoding a ribosomal protein, causes predisposition to hereditary nonpolyposis colorectal carcinoma without DNA mismatch repair deficiency.

Little is known about the genetic factors that contribute to familial colorectal cancer type X (FCCX), characterized by hereditary nonpolyposis colorectal carcinoma with no mismatch repair defects. Genetic linkage analysis, exome sequencing, tumor studies, and functional investigations of 4 generations of a FCCX family led to the identification of a truncating germline mutation in RPS20, which encodes a component (S20) of the small ribosomal subunit and is a new colon cancer predisposition gene. The mutation was associated with a defect in pre-ribosomal RNA maturation. Our findings show that mutations in a gene encoding a ribosomal protein can predispose individuals to microsatellite-stable colon cancer. Evaluation of additional FCCX families for mutations in RPS20 and other ribosome-associated genes is warranted.

Promoter-specific alterations of APC are a rare cause for mutation-negative familial adenomatous polyposis.

n familial adenomatous polyposis (FAP), 20% of classical and 70% of attenuated/atypical (AFAP) cases remain mutation-negative after routine testing; yet, allelic expression imbalance may suggest an APC alteration. Our aim was to determine the proportion of families attributable to genetic or epigenetic changes in the APC promoter region. We studied 51 unrelated families/cases (26 with classical FAP and 25 with AFAP) with no point mutations in the exons and exon/intron borders and no rearrangements by multiplex ligation-dependent probe amplification (MLPA, P043-B1). Promoter-specific events of APC were addressed by targeted resequencing, MLPA (P043-C1), methylation-specific MLPA, and Sanger sequencing of promoter regions. A novel 132-kb deletion encompassing the APC promoter 1B and upstream sequence occurred in a classical FAP family with allele-specific APC expression. No promoter-specific point mutations or hypermethylation were present in any family. In conclusion, promoter-specific alterations are a rare cause for mutation-negative FAP (1/51, 2%). The frequency and clinical correlations of promoter 1B deletions are poorly defined. This investigation provides frequencies of 1/26 (4%) for classical FAP, 0/25 (0%) for AFAP, and 1/7 (14%) for families with allele-specific expression of APC. Clinically, promoter 1B deletions may associate with classical FAP without extracolonic manifestations.

Loss of p15INK4b expression in colorectal cancer is linked to ethnic origin.

Colorectal cancers remain to be a common cause of cancer-related death. Early-onset cases as well as those of various ethnic origins have aggressive clinical features, the basis of which requires further exploration. The aim of this work was to examine the expression patterns of p15INK4b and SMAD4 in colorectal carcinoma of different ethnic origins. Fifty-five sporadic colorectal carcinoma of Egyptian origin, 25 of which were early onset, and 54 cancers of Finnish origin were immunohistochemically stained with antibodies against p15INK4b and SMAD4 proteins. Data were compared to the methylation status of the p15INK4b gene promotor. p15INK4b was totally lost or deficient (lost in ≥ 50% of tumor cell) in 47/55 (85%) tumors of Egyptian origin as compared to 6/50 (12%) tumors of Finnish origin (p=7e-15). In the Egyptian cases with p15INK4b loss and available p15INK4b promotor methylation status, 89% of cases which lost p15INK4b expression were associated with p15INK4b gene promotor hypermethylation. SMAD4 was lost or deficient in 25/54 (46%) tumors of Egyptian origin and 28/48 (58%) tumors of Finnish origin. 22/54 (41%) Egyptian tumors showed combined loss/deficiency of both p15INK4b and SMAD4, while p15INK4b was selectively lost/deficient with positive SMAD4 expression in 24/54 (44%) tumors. Loss of p15INK4b was associated with older age at presentation (>50 years) in the Egyptian tumors (p=0.04). These data show for the first time that p15INK4b loss of expression marks a subset of colorectal cancers and ethnic origin may play a role in this selection. In a substantial number of cases, the loss was independent of SMAD4 but rather associated with p15INK4b gene promotor hypermethylation and old age which could be related to different environmental exposures.