Characterization of CDKN2A(p16) methylation and impact in colorectal cancer: systematic analysis using pyrosequencing.

BACKGROUND: The aim of this study is to analyse CDKN2A methylation using pyrosequencing on a large cohort of colorectal cancers and corresponding non-neoplastic tissues. In a second step, the effect of methylation on clinical outcome is addressed. METHODS: Primary colorectal cancers and matched non-neoplastic tissues from 432 patients underwent CDKN2A methylation analysis by pyrosequencing (PyroMarkQ96). Methylation was then related to clinical outcome, microsatellite instability (MSI), and BRAF and KRAS mutation. Different amplification conditions (35 to 50 PCR cycles) using a range of 0-100% methylated DNA were tested. RESULTS: Background methylation was at most 10% with ≥35 PCR cycles. Correlation of observed and expected values was high, even at low methylation levels (0.02%, 0.6%, 2%). Accuracy of detection was optimal with 45 PCR cycles. Methylation in normal mucosa ranged from 0 to >90% in some cases. Based on the maximum value of 10% background, positivity was defined as a ≥20% difference in methylation between tumor and normal tissue, which occurred in 87 cases. CDKN2A methylation positivity was associated with MSI (p = 0.025), BRAF mutation (p < 0.0001), higher tumor grade (p < 0.0001), mucinous histology (p = 0.0209) but not with KRAS mutation. CDKN2A methylation had an independent adverse effect (p = 0.0058) on prognosis. CONCLUSION: The non-negligible CDKN2A methylation of normal colorectal mucosa may confound the assessment of tumor-specific hypermethylation, suggesting that corresponding non-neoplastic tissue should be used as a control. CDKN2A methylation is robustly detected by pyrosequencing, even at low levels, suggesting that this unfavorable prognostic biomarker warrants investigation in prospective studies.

The impact of CpG island methylator phenotype and microsatellite instability on tumour budding in colorectal cancer.

AIMS: In colorectal cancer, tumour budding, a process likened to epithelial mesenchymal transition, is an adverse prognostic factor which is rarely found in tumours with high-level microsatellite instability (MSI-H). Cases with MSI-H or high-level CpG island methylator phenotype (CIMP-H) have similar histomorphological features, yet seemingly opposite prognosis. We hypothesized that tumour budding is related to CIMP, thus partially explaining this prognostic difference. METHODS AND RESULTS: MSI, KRAS, BRAF, CIMP and 0(6)-methylguanine-DNA methyltransferase (MGMT) were investigated in tissues from 127 colorectal cancer patients. Tumour budding was scored using pan-cytokeratin-stained whole tissue sections within the densest area of buds (×40). Tumour budding was not associated with KRAS, BRAF, MGMT or CIMP, but was correlated inversely with MSI-H (P = 0.0049). Multivariate survival time analysis revealed that tumour budding was independent of all five molecular features and was predicted by MSI status [odds ratio (OR): 4.29, 95% confidence interval (CI) 1.5-12.1; P = 0.006)], but not CIMP (OR: 0.81, 95% CI 0.3-2.5; P = 0.714). CONCLUSIONS: These findings underline that MSI, rather than CIMP, plays a role in conferring a tumour budding phenotype. Budding retains its unfavourable prognostic effect independently of these five molecular features. Continued efforts to standardize the assessment of tumour budding are necessary to integrate this feature into daily diagnostic routine.

Comprehensive analysis of CpG island methylator phenotype (CIMP)-high, -low, and -negative colorectal cancers based on protein marker expression and molecular features.

CpG island methylator phenotype (CIMP) is being investigated for its role in the molecular and prognostic classification of colorectal cancer patients but is also emerging as a factor with the potential to influence clinical decision-making. We report a comprehensive analysis of clinico-pathological and molecular features (KRAS, BRAF and microsatellite instability, MSI) as well as of selected tumour- and host-related protein markers characterizing CIMP-high (CIMP-H), -low, and -negative colorectal cancers. Immunohistochemical analysis for 48 protein markers and molecular analysis of CIMP (CIMP-H: ≥ 4/5 methylated genes), MSI (MSI-H: ≥ 2 instable genes), KRAS, and BRAF were performed on 337 colorectal cancers. Simple and multiple regression analysis and receiver operating characteristic (ROC) curve analysis were performed. CIMP-H was found in 24 cases (7.1%) and linked (p < 0.0001) to more proximal tumour location, BRAF mutation, MSI-H, MGMT methylation (p = 0.022), advanced pT classification (p = 0.03), mucinous histology (p = 0.069), and less frequent KRAS mutation (p = 0.067) compared to CIMP-low or -negative cases. Of the 48 protein markers, decreased levels of RKIP (p = 0.0056), EphB2 (p = 0.0045), CK20 (p = 0.002), and Cdx2 (p < 0.0001) and increased numbers of CD8+ intra-epithelial lymphocytes (p < 0.0001) were related to CIMP-H, independently of MSI status. In addition to the expected clinico-pathological and molecular associations, CIMP-H colorectal cancers are characterized by a loss of protein markers associated with differentiation, and metastasis suppression, and have increased CD8+ T-lymphocytes regardless of MSI status. In particular, Cdx2 loss seems to strongly predict CIMP-H in both microsatellite-stable (MSS) and MSI-H colorectal cancers. Cdx2 is proposed as a surrogate marker for CIMP-H.

Combined analysis of specific KRAS mutation, BRAF and microsatellite instability identifies prognostic subgroups of sporadic and hereditary colorectal cancer.

Confounding effects of specific KRAS gene alterations on colorectal cancer (CRC) prognosis stratified by microsatellite instability (MSI) and BRAF(V600E) have not yet been investigated. The aim of our study was to evaluate the combined effects of MSI, BRAF(V600E) and specific KRAS mutation (Gly → Asp; G12D, Gly → Asp, G13D; Gly → Val; G12V) on prognosis in 404 sporadic and 94 hereditary CRC patients. MSI status was determined according to the Bethesda guidelines. Mutational status of KRAS and BRAF(V600E) was assessed by direct DNA sequencing. In sporadic CRC, KRAS G12D mutations had a negative prognostic effect compared to G13D and wild-type cancers (p = 0.038). With MSI, specific KRAS and BRAF(V600E) mutations, 3 distinct prognostic subgroups were observed in univariate (p = 0.006) and multivariable (p = 0.051) analysis: patients with (i) KRAS mutation G12D, G12V or BRAF(V600E) mutation, (ii) KRAS/BRAF(V600E) wild-type or KRAS G13D mutations in MSS/MSI-L and (iii) MSI-H and KRAS G13D mutations. Moreover, none of the sporadic MSI-H or hereditary patients with KRAS G13 mutations had a fatal outcome. Specific KRAS mutation is an informative prognostic factor in both sporadic and hereditary CRC and applied in an algorithm with BRAF(V600E) and MSI may identify sporadic CRC patients with poor clinical outcome.

KIT, PDGFRalpha and EGFR analysis in nephroblastoma.

Nephroblastoma prognosis has dramatically improved, but an unfavourable prognostic subgroup warrants development of novel therapeutic strategies. Selective KIT, PDGFRalpha and epidermal growth factor receptor (EGFR) tyrosine kinase inhibition evolved as powerful targeted therapy for gastrointestinal stromal tumours and non-small-cell lung cancer. To investigate a potential role for tyrosine kinase inhibition, we analyzed 209 nephroblastomas for immunohistochemical KIT and EGFR expression, 63 nephroblastomas for mutations in KIT exons 9, 11, 13, EGFR exons 18, 19, 20 and 21, and all 209 nephroblastomas for PDGFRalpha exons 12, 14 and 18. Twenty-two tumours (10.5%) expressed KIT, 31 (14.8%) EGFR, and 10 (4.8%) both KIT and EGFR, respectively. KIT expression was relatively more common among high-risk tumours (6/27; 22.3%) compared to low-/intermediate-risk tumours (26/181; 14.4%). Nine patients deceased, four of which had high-risk tumours with KIT expression in two of four and EGFR expression in one of four. There were no KIT, PDGFRalpha or EGFR mutations. Our results suggest no significant contribution of KIT, EGFR or PDGFRalpha mutations to nephroblastoma pathogenesis. Despite a trend towards association of immunohistochemical KIT and EGFR expression with poor outcome in high-risk nephroblastomas, statistical analysis did not yield significant correlations in this subgroup. Therefore, it remains open if KIT, PDGFRalpha or EGFR tyrosine kinase inhibition constitute a therapeutic target in nephroblastoma in the absence of KIT, PDGFRalpha or EGFR mutations.

Human Papillomavirus (HPV) Detection in Cytologic Specimens: Similarities and Differences of Available Methodology.

Accumulating evidence regarding the causative role of human papillomavirus (HPV) in a wide range of malignant and nonmalignant diseases highlights the importance of HPV testing. This study describes and discusses the efficacy and characteristics of 4 well-established and commercially available tests. Here, 181 cytologic specimens from cervical smears were analyzed using the HPV SIGN PQ (Diatech) and the Linear Array (Roche) method. Discrepant results were further studied with the Real Time High-Risk HPV (Abbott) method and the INNO-LiPA (Fujirebio) method. Of 181 cytologic specimens, 61 (34%) showed discrepant results. High-risk HPV was not detected in 9 cases by HPV SIGN PQ, in 16 cases by Linear Array, in 10 cases by Real Time High-Risk HPV, and in 6 cases by INNO-LiPA, respectively. Lack of DNA detection or problems in interpreting the result were seen in 9 cases with HPV SIGN PQ, 8 cases with Linear Array, 3 cases with Real Time High-Risk HPV, and 3 cases with INNO-LiPA, respectively. This study indicates that the choice of HPV detection method has a substantial influence on the HPV risk classification of tested PAP smears and clinical follow-up decisions.

Stratification and Prognostic Relevance of Jass's Molecular Classification of Colorectal Cancer.

BACKGROUND: The current proposed model of colorectal tumorigenesis is based primarily on CpG island methylator phenotype (CIMP), microsatellite instability (MSI), KRAS, BRAF, and methylation status of 0-6-Methylguanine DNA Methyltransferase (MGMT) and classifies tumors into five subgroups. The aim of this study is to validate this molecular classification and test its prognostic relevance. METHODS: Three hundred two patients were included in this study. Molecular analysis was performed for five CIMP-related promoters (CRABP1, MLH1, p16INK4a, CACNA1G, NEUROG1), MGMT, MSI, KRAS, and BRAF. Methylation in at least 4 promoters or in one to three promoters was considered CIMP-high and CIMP-low (CIMP-H/L), respectively. RESULTS: CIMP-H, CIMP-L, and CIMP-negative were found in 7.1, 43, and 49.9% cases, respectively. One hundred twenty-three tumors (41%) could not be classified into any one of the proposed molecular subgroups, including 107 CIMP-L, 14 CIMP-H, and two CIMP-negative cases. The 10 year survival rate for CIMP-high patients [22.6% (95%CI: 7-43)] was significantly lower than for CIMP-L or CIMP-negative (p = 0.0295). Only the combined analysis of BRAF and CIMP (negative versus L/H) led to distinct prognostic subgroups. CONCLUSION: Although CIMP status has an effect on outcome, our results underline the need for standardized definitions of low- and high-level CIMP, which clearly hinders an effective prognostic and molecular classification of colorectal cancer.

KRAS mutation testing in colorectal cancer: comparison of the results obtained using 3 different methods for the analysis of codons G12 and G13.

Targeting the epidermal growth factor receptor (EGFR) is a new therapeutic option for patients with metastatic colorectal or lung carcinoma. However, the therapy efficiency highly depends on the KRAS mutation status in the given tumour. Therefore a reliable and secure KRAS mutation testing is crucial. Here we investigated 100 colorectal carcinoma samples with known KRAS mutation status (62 mutated cases and 38 wild type cases) in a comparative manner with three different KRAS mutation testing techniques (Pyrosequencing, Dideoxysequencing and INFINITI) in order to test their reliability and sensitivity. For the large majority of samples (96/100, 96%), the KRAS mutation status obtained by all three methods was the same. Only two cases with clear discrepancies were observed. One case was reported as wild type by the INFINITI method while the two other methods detected a G13C mutation. In the second case the mutation could be detected by the Pyrosequencing and INFINITI method (15% and 15%), while no signal for mutation could be observed with the Dideoxysequencing method. Additional two unclear results were due to a detection of a G12V with the INFINITI method, which was below cut-off when repeated and which was not detectable by the other two methods and very weak signals in a G12V mutated case with the Dideoxy- and Pyroseqencing method compared to the INFINITI method, respectively. In summary all three methods are reliable and robust methods in detecting KRAS mutations. INFINITI, however seems to be slightly more sensitive compared to Dideoxy- and Pyrosequencing.

Molecular and immunohistochemical characterization of B-cell lymphoma-2-negative follicular lymphomas.

Aberrant expression of the antiapoptotic protein BCL (B-cell lymphoma)-2 in neoplastic germinal centers is one of the diagnostic hallmarks of follicular lymphoma. If BCL-2 cannot be detected by immunohistochemistry, the distinction between florid follicular hyperplasia and follicular lymphoma might become a diagnostic challenge. Most of those cases also lack the typical t(14;18), and the underlying pathophysiologic conditions of follicular lymphoma that lack BCL-2 protein expression are largely unknown. Here, we collected 18 BCL-2-negative follicular lymphoma cases from 5 different institutions. After restaining, 9 cases proved to be truly BCL-2 negative (6 follicular lymphoma grade 2, 2 follicular lymphoma grade 3a, and 1 follicular lymphoma grade 3b). In 4 additional cases, BCL-2 was very faint (all grade 2). Of the 9 BCL-2-negative follicular lymphoma cases, 2 were negative for CD10 (22%); all showed expression of BCL-6. Apoptotic level as determined by caspase 3 was the lowest in the BCL-2-positive follicular lymphoma group (15 ± 8 mm(2)), the highest in the normal/reactive group (n = 7, 60 ± 12 mm(2)) and very similar in the BCL-2 low follicular lymphoma and BCL-2-negative follicular lymphoma groups (25 ± 13 and 33 ± 19 mm(2), respectively), assuming an intermediate position between reactive follicles and BCL-2-positive neoplastic follicles (P < .001 [Kruskal-Wallis]). Also noted was a difference in proliferation fractions between the BCL-2-positive follicular lymphoma (27% ± 15%), the BCL-2 low follicular lymphoma (30% ± 20%) and the BCL-2-negative follicular lymphoma groups (30% ± 22%). Regarding the network of follicular dendritic cells, 8 (89%) of 9 cases from the BCL-2-negative subgroup showed disrupted, weakly developed networks, whereas all of the follicular lymphoma BCL-2 low-expression cases showed a well-defined and strongly developed follicular dendritic cell network. Among BCL-2-negative follicular lymphoma, BCL-2 and BCL-6 breaks were found in 1 case each, whereas in the follicular lymphoma BCL-2 low group, only 1 case with a BCL-6 break was recorded. No statistically significant result was achieved upon assessment of BCL-2α or BCL-2ß RNA or the ratio of α/ß isolated by real-time-polymerase chain reaction. Taken together, BCL-2-negative follicular lymphoma did not show a BCL-2 break on the genetic level and showed both increased apoptotic and proliferation rates compared with BCL-2-positive follicular lymphoma. In our series, BCL-6 breaks were infrequent in BCL-2-negative follicular lymphoma.

A comprehensive analysis of p16 expression, gene status, and promoter hypermethylation in surgically resected non-small cell lung carcinomas.

INTRODUCTION: The role of p16 is gaining importance in non-small cell lung cancer (NSCLC) because of epigenetic therapy options. Further insight into the significance of protein expression, gene status and promoter methylation is needed and has the potential to optimize existing treatment strategies. METHODS: This population-based study analyzes p16 in 383 surgically resected non-small cell lung carcinomas brought into a standardized tissue microarray platform. Immunohistochemistry and fluorescence in situ hybridization were performed. For selected cases, p16 promoter hypermethylation was assessed by a pyrosequencing assay. Extensive clinical data and a postoperative follow-up period of 15 years enabled detailed correlations. RESULTS: Loss of p16 expression is a common event in NSCLC (232/365, 64%), especially in squamous cell carcinomas (97/115, 84%) in contrast to adenocarcinomas (93/186, 50%). Loss of p16 expression was associated with poorer survival time for the entire cohort and for certain subgroups including men, age younger than 65 years, smokers, early tumor stage, adenocarcinoma, and large-cell carcinoma. Promoter hypermethylation was absent for cases expressing p16 but was commonly observed when (heterozygous) p16 gene deletions were present and in cases negative for p16. CONCLUSION: Our comprehensive data would be compatible with a two-step process leading to loss of p16 expression in NSCLC. Hypermethylation of the promoter region may represent an early event, followed by heterozygous deletion of the p16 locus. Because of the possibility of detection of hypermethylated gene regions, these data may lead to the identification of specific patient subgroups more likely to benefit from upcoming demethylating treatment strategies.