Relative contribution of clinicopathological variables, genomic markers, transcriptomic subtyping and microenvironment features for outcome prediction in stage II/III colorectal cancer.

BACKGROUND: It remains unknown to what extent consensus molecular subtype (CMS) groups and immune-stromal infiltration patterns improve our ability to predict outcomes over tumor-node-metastasis (TNM) staging and microsatellite instability (MSI) status in early-stage colorectal cancer (CRC). PATIENTS AND METHODS: We carried out a comprehensive retrospective biomarker analysis of prognostic markers in adjuvant chemotherapy-untreated (N = 1656) and treated (N = 980), stage II (N = 1799) and III (N = 837) CRCs. We defined CMS scores and estimated CD8+ cytotoxic lymphocytes (CytoLym) and cancer-associated fibroblasts (CAF) infiltration scores from bulk tumor tissue transcriptomes (CMSclassifier and MCPcounter R packages); constructed a stratified multivariable Cox model for disease-free survival (DFS); and calculated the relative proportion of explained variation by each marker (clinicopathological [ClinPath], genomics [Gen: MSI, BRAF and KRAS mutations], CMS scores [CMS] and microenvironment cells [MicroCells: CytoLym+CAF]). RESULTS: In multivariable models, only ClinPath and MicroCells remained significant prognostic factors, with both CytoLym and CAF infiltration scores improving survival prediction beyond other markers. The explained variation for DFS models of ClinPath, MicroCells, Gen markers and CMS4 scores was 77%, 14%, 5.3% and 3.7%, respectively, in stage II; and 55.9%, 35.1%, 4.1% and 0.9%, respectively, in stage III. Patients whose tumors were CytoLym high/CAF low had better DFS than other strata [HR=0.71 (0.6-0.9); P = 0.004]. Microsatellite stable tumors had the strongest signal for improved outcomes with CytoLym high scores (interaction P = 0.04) and the poor prognosis linked to high CAF scores was limited to stage III disease (interaction P = 0.04). CONCLUSIONS: Our results confirm that tumor microenvironment infiltration patterns represent potent determinants of the risk for distant dissemination in early-stage CRC. Multivariable models suggest that the prognostic value of MSI and CMS groups is largely explained by CytoLym and CAF infiltration patterns.

Characterization of early recurrences following liver resection by ALPPS and two stage hepatectomy in patients with colorectal liver-metastases and small future liver remnants; a translational substudy of the LIGRO-RCT.

BACKGROUND: Associated liver partition and portal vein ligation in staged hepatectomy (ALPPS) is an alternative resection method to portal vein embolization (PVE) in patients with small future liver remnants (FLR) but has been associated with early tumor recurrences. METHODS: Twenty-four patients with colorectal liver metastases (CRLM) patients from the randomized multicenter LIGRO trial comparing outcome of ALPPS (n = 13) vs PVE (n = 11) were included in the study. Mutational analyses of the KRAS, NRAS, BRAF, PIC3CA and TP53 genes of the metastases were performed in 21 patients and correlated to early tumor recurrence. RESULTS: Within 12 months, 13 patients experienced recurrences (6 in TSH group and 7 in ALPPS group). Nine of 13 patients with recurrences had mutations in the TP53 gene, while 3 of 8 patients without recurrence carried the same mutation. Only sporadic cases of the other mutations studied were identified. CONCLUSIONS: ALPPS did not appear to be associated with higher rate of rapid recurrences than PVE following radical resection of colorectal liver metastases. Mutations in genes associated with negative oncologic outcome after surgical resection most likely play a role for tumor recurrences in these patients.

CMS-dependent prognostic impact of KRAS and BRAFV600E mutations in primary colorectal cancer.

Background: The prognostic impact of KRAS and BRAFV600E mutations in primary colorectal cancer (CRC) varies with microsatellite instability (MSI) status. The gene expression-based consensus molecular subtypes (CMSs) of CRC define molecularly and clinically distinct subgroups, and represent a novel stratification framework in biomarker analysis. We investigated the prognostic value of these mutations within the CMS groups. Patients and methods: Totally 1197 primary tumors from a Norwegian series of CRC stage I-IV were analyzed for MSI and mutation status in hotspots in KRAS (codons 12, 13 and 61) and BRAF (codon 600). A subset was analyzed for gene expression and confident CMS classification was obtained for 317 samples. This cohort was expanded with clinical and molecular data, including CMS classification, from 514 patients in the publically available dataset GSE39582. Gene expression signatures associated with KRAS and BRAFV600E mutations were used to evaluate differential impact of mutations on gene expression among the CMS groups. Results: BRAFV600E and KRAS mutations were both associated with inferior 5-year overall survival (OS) exclusively in MSS tumors (BRAFV600E mutation versus KRAS/BRAF wild-type: Hazard ratio (HR) 2.85, P < 0.001; KRAS mutation versus KRAS/BRAF wild-type: HR 1.30, P = 0.013). BRAFV600E-mutated MSS tumors were strongly enriched and associated with metastatic disease in CMS1, leading to negative prognostic impact in this subtype (OS: BRAFV600E mutation versus wild-type: HR 7.73, P = 0.001). In contrast, the poor prognosis of KRAS mutations was limited to MSS tumors with CMS2/CMS3 epithelial-like gene expression profiles (OS: KRAS mutation versus wild-type: HR 1.51, P = 0.011). The subtype-specific prognostic associations were substantiated by differential effects of BRAFV600E and KRAS mutations on gene expression signatures according to the MSI status and CMS group. Conclusions: BRAFV600E mutations are enriched and associated with metastatic disease in CMS1 MSS tumors, leading to poor prognosis in this subtype. KRAS mutations are associated with adverse outcome in epithelial (CMS2/CMS3) MSS tumors.

Prognostic markers for colorectal cancer: estimating ploidy and stroma.

Background: We report here the prognostic value of ploidy and digital tumour-stromal morphometric analyses using material from 2624 patients with early stage colorectal cancer (CRC). Patients and methods: DNA content (ploidy) and stroma-tumour fraction were estimated using automated digital imaging systems and DNA was extracted from sections of formalin-fixed paraffin-embedded (FFPE) tissue for analysis of microsatellite instability. Samples were available from 1092 patients recruited to the QUASAR 2 trial and two large observational series (Gloucester, n = 954; Oslo University Hospital, n = 578). Resultant biomarkers were analysed for prognostic impact using 5-year cancer-specific survival (CSS) as the clinical end point. Results: Ploidy and stroma-tumour fraction were significantly prognostic in a multivariate model adjusted for age, adjuvant treatment, and pathological T-stage in stage II patients, and the combination of ploidy and stroma-tumour fraction was found to stratify these patients into three clinically useful groups; 5-year CSS 90% versus 83% versus 73% [hazard ratio (HR) = 1.77 (95% confidence interval (95% CI): 1.13-2.77) and HR = 2.95 (95% CI: 1.73-5.03), P < 0.001]. Conclusion: A novel biomarker, combining estimates of ploidy and stroma-tumour fraction, sampled from FFPE tissue, identifies stage II CRC patients with low, intermediate or high risk of CRC disease specific death, and can reliably stratify clinically relevant patient sub-populations with differential risks of tumour recurrence and may support choice of adjuvant therapy for these individuals.

Prediction of overall survival in stage II and III colon cancer beyond TNM system: a retrospective, pooled biomarker study.

Background: TNM staging alone does not accurately predict outcome in colon cancer (CC) patients who may be eligible for adjuvant chemotherapy. It is unknown to what extent the molecular markers microsatellite instability (MSI) and mutations in BRAF or KRAS improve prognostic estimation in multivariable models that include detailed clinicopathological annotation. Patients and methods: After imputation of missing at random data, a subset of patients accrued in phase 3 trials with adjuvant chemotherapy (n = 3016)-N0147 (NCT00079274) and PETACC3 (NCT00026273)-was aggregated to construct multivariable Cox models for 5-year overall survival that were subsequently validated internally in the remaining clinical trial samples (n = 1499), and also externally in different population cohorts of chemotherapy-treated (n = 949) or -untreated (n = 1080) CC patients, and an additional series without treatment annotation (n = 782). Results: TNM staging, MSI and BRAFV600E mutation status remained independent prognostic factors in multivariable models across clinical trials cohorts and observational studies. Concordance indices increased from 0.61-0.68 in the TNM alone model to 0.63-0.71 in models with added molecular markers, 0.65-0.73 with clinicopathological features and 0.66-0.74 with all covariates. In validation cohorts with complete annotation, the integrated time-dependent AUC rose from 0.64 for the TNM alone model to 0.67 for models that included clinicopathological features, with or without molecular markers. In patient cohorts that received adjuvant chemotherapy, the relative proportion of variance explained (R2) by TNM, clinicopathological features and molecular markers was on an average 65%, 25% and 10%, respectively. Conclusions: Incorporation of MSI, BRAFV600E and KRAS mutation status to overall survival models with TNM staging improves the ability to precisely prognosticate in stage II and III CC patients, but only modestly increases prediction accuracy in multivariable models that include clinicopathological features, particularly in chemotherapy-treated patients.

Prognostic impact of genomic instability in colorectal cancer.

BACKGROUND: The prognostic impact of an indication of chromosomal instability (CIN) is evaluated in a consecutive series of 952 colorectal cancer patients treated at Aker University Hospital, Norway, during 1993-2003. Microsatellite instability (MSI) in this case series has recently been reported and made it possible to find the co-occurrence and compare the prognostic significance of CIN and MSI. METHODS: Data sets for overall survival (OS; n=855) and time to recurrence (TTR; n=579) were studied. To reveal CIN we used automated image cytometry (ICM). Non-diploid histograms were taken as indicative of the presence of CIN. PCR-based measures of MSI in this material have already been described. RESULTS: As with MSI, CIN was found to be an independent predictor of early relapse and death among stage II patients (TTR: n=278: HR 2.19 (95% CI: 1.35-3.55), P=0.002). Of the MSI tumours (16%), 71% were found to be DNA diploid, 21% were DNA tetraploid and 8% were DNA aneuploid. Among microsatellite stable tumours, 24% were DNA diploid, 15% were DNA tetraploid and 61% were DNA aneuploid. CONCLUSION: For patients presenting with stage II disease, genomic instability as detected by DNA image cytometry has the potential to provide a useful biomarker for relapse and cancer-related death following surgery with curative intent.

Microsatellite instability has a positive prognostic impact on stage II colorectal cancer after complete resection: results from a large, consecutive Norwegian series.

BACKGROUND: Microsatellite instability (MSI) was suggested as a marker for good prognosis in colorectal cancer in 1993 and a systematic review from 2005 and a meta-analysis from 2010 support the initial observation. We here assess the prognostic impact and prevalence of MSI in different stages in a consecutive, population-based series from a single hospital in Oslo, Norway. PATIENTS AND METHODS: Of 1274 patients, 952 underwent major resection of which 805 were included in analyses of MSI prevalence and 613 with complete resection in analyses of outcome. Formalin-fixed tumor tissue was used for PCR-based MSI analyses. RESULTS: The overall prevalence of MSI was 14%, highest in females (19%) and in proximal colon cancer (29%). Five-year relapse-free survival (5-year RFS) was 67% and 55% (P = 0.030) in patients with MSI and MSS tumors, respectively, with the hazard ratio (HR) equal to 1.60 (P = 0.045) in multivariate analysis. The improved outcome was confined to stage II patients who had 5-year RFS of 74% and 56% respectively (P = 0.010), HR = 2.02 (P = 0.040). Examination of 12 or more lymph nodes was significantly associated with proximal tumor location (P < 0.001). CONCLUSIONS: MSI has an independent positive prognostic impact on stage II colorectal cancer patients after complete resection.

CLC and IFNAR1 are differentially expressed and a global immunity score is distinct between early- and late-onset colorectal cancer.

Colorectal cancer (CRC) incidence increases with age, and early onset of the disease is an indication of genetic predisposition, estimated to cause up to 30% of all cases. To identify genes associated with early-onset CRC, we investigated gene expression levels within a series of young patients with CRCs who are not known to carry any hereditary syndromes (n=24; mean 43 years at diagnosis), and compared this with a series of CRCs from patients diagnosed at an older age (n=17; mean 79 years). Two individual genes were found to be differentially expressed between the two groups, with statistical significance; CLC was higher and IFNAR1 was less expressed in early-onset CRCs. Furthermore, genes located at chromosome band 19q13 were found to be enriched significantly among the genes with higher expression in the early-onset samples, including CLC. An elevated immune content within the early-onset group was observed from the differentially expressed genes. By application of outlier statistics, H3F3A was identified as a top candidate gene for a subset of the early-onset CRCs. In conclusion, CLC and IFNAR1 were identified to be overall differentially expressed between early- and late-onset CRC, and are important in the development of early-onset CRC.

The testicular germ cell tumour transcriptome.

Testicular germ cell tumours (TGCTs) are characterized by young age of onset and a complex pattern of histological subtypes. Transcriptomic studies have tried to uncover the gene expression patterns underlying this. Here, we present a systematic review of transcriptome studies of TGCTs of adolescents and young adults and identify genes common across the various studies, both for TGCTs in general as well as the histological subtypes, hence elucidating both transcriptional changes associated with malignant transformation and differentiation patterns. A meta-analysis of this type adds power and significance to the genes thus found, where most studies have included only a limited number of samples. Both known (KRAS, MYCN and TPD52) and novel (CCT6A, IGFBP3 and SALL2) cancer genes are implicated in TGC tumorigenesis. Gene expression patterns characteristic to embryonic stem cells are also found deregulated in TGC tumorigenesis. This is reflected in how pluripotent embryonal carcinoma cells commonly differentiate into a variety of embryonic and extra-embryonic histological types, each with unique transcriptomes. The embryonal carcinomas in particular are found to overexpress pluripotency genes, while gene signatures for seminomas, teratomas and yolk sac tumours were also identified. This underlines the distinctive transcriptomic programme across histological subtypes, especially striking given that the TGCT genome is largely similar across the same subtypes.

Molecular genetic studies of tumor suppressor gene regions on chromosomes 13 and 17 in colorectal tumors.

BACKGROUND: In the majority of colorectal carcinomas, both copies of the tumor suppressor gene TP53 (tumor protein 53) are known to be inactivated. In contrast to a loss of tumor suppressor function, it has been suggested that an increased copy number of the RB1 gene is involved in the progression of these tumors. PURPOSE: To determine genetic alterations at chromosomes 13 and 17 in colorectal tumors, we have studied several loci on these chromosomes, with special focus on the RB1 and TP53 genes at both the level of DNA sequence and the level of gene expression. METHODS: Restriction fragment length polymorphism analysis was performed after alkaline Southern blotting of the DNA fragments and hybridization (in 7% sodium dodecyl sulfate and 0.5 M NaPO4) of the nylon membranes with multiprimed, radioactively labeled probes. Total RNA was extracted from tissue biopsy specimens by homogenization of the samples in guanidinium thiocyanate followed by separation in a CsCl gradient. By use of an image-processing system, x-ray film signals were measured densitometrically. Point mutations within the TP53 gene were detected by use of polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis. Direct sequencing of PCR products revealed the exact nature of the mutations. Protein expression of TP53 was seen by immunostaining of sections from paraffin-embedded material using a mouse monoclonal antibody. The two-sided Fisher's Exact Test was used for statistical analysis. RESULTS: An increase in allelic copy number at 13q loci was seen in 10 (32%) of 31 tumors. In the majority of the cases, this increase probably reflected a change in the diploid status of chromosome 13; in some cases, however, only part of the 13q seemed to be involved. The RB1 gene showed an elevated level of RNA compared with the beta-actin signal. Fourteen (48%) of 29 tumors showed loss of heterozygosity at loci on 17p, and base mutations within the TP53 gene were seen in 14 (42%) of 33 tumors. RNA and protein analyses of TP53 revealed an increased level of expression in the tumors compared with normal mucosa. Allelic variations seen at 13q and 17p were not associated (P = .7). CONCLUSIONS: Our results suggest that, in addition to aneuploidy, gain of specific chromosome 13 sequences is involved in the tumorigenesis of the colon and rectum. In addition, they confirm the importance of TP53 mutations for the progression of such tumors and support the view that accumulation of events is more important than the order of events. The genetic changes observed at chromosome arms 13q and 17p seem to be independent of each other.

Genomic instability in colorectal cancer: relationship to clinicopathological variables and family history.

Recent reports have suggested that one or more genes may cause replication errors (RER) during colorectal tumorigenesis. Additional alleles are seen in the tumors when analyzing random microsatellite loci. We have studied seven dinucleotide repeat loci, located on seven different chromosomes, by use of polymerase chain reaction amplification and denaturing polyacrylamide gel electrophoresis. We found that 16.5% (40 of 243) colorectal cancers showed RER at one or several loci (RER+). This includes 31% (4 of 13) among cases with a strong positive family history according to previously published criteria and 17% (35 of 207) among cases with no history of familial cancer. Interestingly, no significant association was found between RER+ tumors and a general familial clustering of cancer. Microsatellite instability was significantly associated with DNA diploid status of the tumor (P < 0.001), with the location of the tumor in the proximal colon (P < 0.001), and with poorly differentiated tumor phenotype (P < 0.001). Patients with RER+ at > or = 2 loci tumors had an increased survival (P = 0.05). We further analyzed 84 breast cancers and 86 male germ cell cancers using the same seven markers. None of the tumors were RER+, indicating that this phenomenon may be specific to certain types of tumors.

Microsatellite instability is associated with tumors that characterize the hereditary non-polyposis colorectal carcinoma syndrome.

Microsatellite instability implying multiple replication errors (RER+ phenotype) characterizes a proportion of colorectal carcinomas, particularly those from patients with the hereditary non-polyposis colorectal carcinoma syndrome. We studied the incidence of microsatellite instability in more than 500 sporadic tumors representing 6 different types of cancer. Apart from colorectal carcinoma [see the paper by Lothe et al. (Cancer Res., 53:5849-5852, 1993)] the RER+ phenotype was found in 18% (6 of 33) of gastric carcinomas and 22% (4 of 18) of endometrial carcinomas. In contrast, no evidence of this abnormality was detected in cancers of the lung (N = 85), breast (N = 84), and testis (N = 86). Importantly, the first three cancers, as opposed to the latter three, are characteristic of the hereditary non-polyposis colorectal carcinoma syndrome. These findings suggest that the cancers belonging to the hereditary non-polyposis colorectal carcinoma tumor spectrum may have essential pathogenetic steps in common, including a tendency to multiple replication errors.

Gain of 17q24-qter detected by comparative genomic hybridization in malignant tumors from patients with von Recklinghausen's neurofibromatosis.

The genetic changes leading to the development of malignant peripheral nerve sheath tumors (MPNSTs) are largely unknown. The few tumors that have been investigated cytogenetically had highly complex karyotypes and no consistent rearrangements, and the attempts to pinpoint consistent DNA-level changes have met with only limited success. We used comparative genomic hybridization to analyze seven MPNSTs and one dermatofibrosarcoma protuberans from eight patients with von Recklinghausen's disease (neurofibromatosis type 1), as well as three sporadic MPNSTs. Gains and losses of DNA sequences were found in all tumors, with an average of four losses (range, 0-14) and two gains (range, 0-5) per tumor. Two striking observations were made: (a) an increase in copy number of the distal part of the long arm of chromosome 17, with the smallest region of overlap 17q24-qter, was seen in five of seven MPNSTs and in the only dermatofibrosarcoma protuberans, all of which were from patients with neurofibromatosis, whereas none of the three sporadic MPNSTs had this alteration; and (b) loss of 13q, with the smallest region of overlap 13q14-q21, was found in 6 of 10 MPNSTs. The consistent involvement of these two chromosomal regions probably reflects two different pathogenetic mechanisms for MPNSTs.

DNA copy number changes in malignant ovarian germ cell tumors.

Malignant ovarian germ cell tumors (OGCTs) include immature teratomas (ITs), dysgerminomas (DGs), endodermal sinus tumors (ESTs), choriocarcinomas, and embryonal carcinomas. Knowledge about the genetic changes associated with malignant OGCT development is sparse. We therefore analyzed 25 OGCTs (12 DGs, 4 ESTs, and 9 ITs) for gains and losses by comparative genomic hybridization. In total, more gains than losses were observed, and the number of alterations ranged from 0-20 per tumor. The average number of changes among DGs, ESTs, and ITs was 10, 6, and 1.4, respectively. The most common changes in DGs were gains from chromosome arms 1p (33%), 6p (33%), 12p (67%), 12q (75%), 15q (42%), 20q (50%), 21q (67%), and 22q (58%); gains of the whole of chromosomes 7 (42%), 8 (42%), 17 (42%), and 19 (50%); and losses from 13q (58%). Two of three DGs with a gonadoblastoma component showed gains of 3p21 and loss of 5p, whereas none of the nine pure DGs had these changes, suggesting that they might be characteristic either of gonadoblastoma or of DG developing from a gonadoblastoma. Gain of 12p and gain from 1q were seen in three of four ESTs, whereas gains from 3p, 11q, and Xp and loss from 18q were each found in two tumors. Five of the ITs revealed changes (range, 1-4 changes/tumor), with gains from 1p, 16p, 19, and 22q each being found in two tumors. We conclude that ovarian DGs and ESTs seem to develop via the same genetic pathways that are already known for testicular germ cell tumors. On the other hand, ITs do not exhibit gain of 12p and also typically show fewer changes than other malignant OGCTs, indicating that they arise via different pathogenetic mechanisms.

The APC gene I1307K variant is rare in Norwegian patients with familial and sporadic colorectal or breast cancer.

Recently, a T-to-A transversion creating an 8-base mononucleotide tract in the APC gene, resulting in substitution of lysine for isoleucine at codon 1307 (I1307K), was found in a subset of Ashkenazi Jews. This sequence variant was most frequent in colorectal cancer patients with a positive family history of colorectal cancer. To determine whether the I1307K variant plays a role in colorectal or breast cancer predisposition in the Norwegian population, we have analyzed blood samples from 210 colorectal cancer patients and 183 breast cancer patients by PCR and direct sequencing. Thirty-seven of the colorectal cancer patients had a positive family history of cancer. Among the breast cancer patients, 24 had a family history of colorectal cancer and 75 a family history of breast and/or ovarian cancer. Only one colorectal cancer patient who belonged to a Jewish family was found to carry the A variant. Our data show that the I1307K variant is rare in the Norwegian population and should not be viewed as a candidate for susceptibility testing for colorectal cancer.

Somatic mutations in LKB1 are rare in sporadic colorectal and testicular tumors.

Germ-line mutations in a serine/threonine kinase gene, LKB1, were recently shown to underlie Peutz-Jeghers syndrome (PJS), a hereditary disorder that predisposes to benign and malignant tumors of multiple organ systems. Most mutations that have been described thus far dramatically change the predicted protein and are likely to be of an inactivating nature. This observation and a previous observation that the LKB1 locus is often deleted in PJS polyps suggest that the gene may function as a tumor suppressor. We examined whether somatic mutations in this gene are present in sporadic carcinomas of the colon and testis, tumors that are characteristic of PJS. First, 20 randomly selected colorectal and 28 testicular tumors were analyzed by single-strand conformation polymorphism analysis. No mutations in LKB1 were found in colorectal tumors. One testicular tumor displayed a heterozygous missense type variant, in which glycine 163 was changed to aspartic acid. This change was absent in the DNA of normal tissue. To better focus our efforts, we tested 75 additional colon carcinomas for loss of heterozygosity at 19p, where LKB1 is localized. Of 75 samples analyzed, 50 were informative with a closely linked marker, D19S886, and 13 (26%) of these displayed loss of heterozygosity. The 13 tumors were scrutinized for LKB1 mutations by genomic sequencing. This analysis revealed no changes. Together, these findings suggest that somatic mutations of LKB1 are not frequent in colorectal and testicular cancer.

Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes.

Alternative splicing is a widespread process contributing to structural transcript variation and proteome diversity. In cancer, the splicing process is commonly disrupted, resulting in both functional and non-functional end-products. Cancer-specific splicing events are known to contribute to disease progression; however, the dysregulated splicing patterns found on a genome-wide scale have until recently been less well-studied. In this review, we provide an overview of aberrant RNA splicing and its regulation in cancer. We then focus on the executors of the splicing process. Based on a comprehensive catalog of splicing factor encoding genes and analyses of available gene expression and somatic mutation data, we identify cancer-associated patterns of dysregulation. Splicing factor genes are shown to be significantly differentially expressed between cancer and corresponding normal samples, and to have reduced inter-individual expression variation in cancer. Furthermore, we identify enrichment of predicted cancer-critical genes among the splicing factors. In addition to previously described oncogenic splicing factor genes, we propose 24 novel cancer-critical splicing factors predicted from somatic mutations.

Variable mutation frequencies in coding repeats of TCF-4 and other target genes in colon, gastric and endometrial carcinoma showing microsatellite instability.

Frameshift mutations in genes containing mononucleotide repeats are often observed in cancers exhibiting a high frequency of microsatellite instability (MSI-H). Several tumor types, including colorectal, gastric, and endometrial carcinomas, display this phenotype in a significant proportion of cases. We recently showed in a large series of MSI-H colorectal tumors that approximately 40% of them exhibited frameshift mutations in an (A)9 tract within the coding region of the TCF-4 gene, a crucial member of the APC/beta-catenin/TCF pathway. In the present study, we have examined MSI-H cancers from other primary tumor sites for mutations in this new target gene. Two of 22 (9%) MSI-H primary gastric cancers and none of 23 MSI-H endometrial primary tumors and cell lines were found to have a 1 bp deletion in the TCF-4 repeat. In the same series of tumors we also looked for frameshift mutations in other coding repeats localized within the TGF beta-RII, BAX, IGFIIR, hMSH3 and hMSH6 genes. Our results suggest that the TCF-4 gene, in a similar manner to some of these latter genes, is differentially altered in MSI-H tumors from different primary sites.

Epigenetic inactivation of LKB1 in primary tumors associated with the Peutz-Jeghers syndrome.

Germ-line mutations of the LKB1 gene cause Peutz-Jeghers syndrome (PJS) characterized by mucocutaneous pigmentation, predisposition to benign hamartomas of the gastrointestinal tract and also to several types of tumors. However, somatic mutations of this gene are very rare. To examine inactivation of LKB1 by epigenetic mechanisms, we investigated a series of primary tumors and cancer cell lines, for hypermethylation affecting the CpG island located in the 5' region of the LKB1 gene using Methylation-specific PCR (MSP). First, we screened 51 cancer cell lines. Only three colorectal and one cervical carcinoma cell lines were methylated at LKB1, and loss of the LKB1 transcript was demonstrated. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored LKB1 expression. To address the incidence of LKB1 epigenetic inactivation in primary tumors, we analysed colorectal, breast, gastric, pancreatic, thyroid, bladder and testicular carcinomas (n=195). Normal tissues from the mentioned organs were unmethylated in this region. Among the described tumors, only one colorectal carcinoma and three testicular tumors displayed LKB1 promoter hypermethylation. Further study of those histological types more commonly associated with PJS, demonstrated that LKB1 promoter hypermethylation was present in five of 11 (45%) papillary breast carcinomas. Finally, in three patients with a strong family story suggestive of PJS disease, abnormal LKB1 methylation was found in four of 22 (18%) hamartomatous polyps lesions. Our findings provide an alternative pathway for inactivation of the LKB1 tumor suppressor gene involving promoter hypermethylation.

Frequent promoter hypermethylation of the O6-Methylguanine-DNA Methyltransferase (MGMT) gene in testicular cancer.

Testicular germ cell tumours are classified into two major histological subgroups, seminomas and nonseminomas. All tumours display several recurrent chromosomal aberrations, but few target genes have been identified. Previous studies have shown that genome-wide hypermethylation of CpG islands is significantly more prevalent in nonseminomas than in seminomas. We have studied two potential target genes in testicular cancer. A series of 70 tumours were analysed for methylation of CpG sites in the O(6)-methylguanine-DNA methyltransferase (MGMT) gene promoter, and in exon 1alpha of the cyclin-dependent kinase inhibitor 2A gene (CDKN2A). In addition, eight microsatellite markers within and flanking these genes at chromosome arms 10q and 9p, respectively, were analysed for allelic imbalances. Allele alterations were frequently seen at 9p loci (47 out of 70, 67%), but none of the tumours (none out of 55) showed methylation of CDKN2A. On the other hand, a high frequency of MGMT promoter methylation (32 out of 69, 46%) was found, as well as allelic imbalances at 10q markers (50 out of 70, 71%). A significantly higher methylation frequency was found in nonseminomas (24 out of 35, 69%) compared to seminomas (eight out of 33, 24%) (P=0.0003, Fisher's exact test). Immunohistochemical analysis of the MGMT protein in a subgroup (n=20) of the testicular tumours supported the hypothesis of gene silencing being the functional consequence of the promoter methylation. In summary, our data suggest that inactivation of MGMT contributes to development of nonseminomatous testicular cancer.