The histone acetyltransferase inhibitor Nir regulates epidermis development.

In addition to its function as an inhibitor of histone acetyltransferases, Nir (Noc2l) binds to p53 and TAp63 to regulate their activity. Here, we show that epidermis-specific ablation of Nir impairs epidermal stratification and barrier function, resulting in perinatal lethality. Nir-deficient epidermis lacks appendages and remains single layered during embryogenesis. Cell proliferation is inhibited, whereas apoptosis and p53 acetylation are increased, indicating that Nir is controlling cell proliferation by limiting p53 acetylation. Transcriptome analysis revealed that Nir regulates the expression of essential factors in epidermis development, such as keratins, integrins and laminins. Furthermore, Nir binds to and controls the expression of p63 and limits H3K18ac at the p63 promoter. Corroborating the stratification defects, asymmetric cell divisions were virtually absent in Nir-deficient mice, suggesting that Nir is required for correct mitotic spindle orientation. In summary, our data define Nir as a key regulator of skin development.

Exome Sequencing Identifies Biallelic MSH3 Germline Mutations as a Recessive Subtype of Colorectal Adenomatous Polyposis.

In ∼30% of families affected by colorectal adenomatous polyposis, no germline mutations have been identified in the previously implicated genes APC, MUTYH, POLE, POLD1, and NTHL1, although a hereditary etiology is likely. To uncover further genes with high-penetrance causative mutations, we performed exome sequencing of leukocyte DNA from 102 unrelated individuals with unexplained adenomatous polyposis. We identified two unrelated individuals with differing compound-heterozygous loss-of-function (LoF) germline mutations in the mismatch-repair gene MSH3. The impact of the MSH3 mutations (c.1148delA, c.2319-1G>A, c.2760delC, and c.3001-2A>C) was indicated at the RNA and protein levels. Analysis of the diseased individuals' tumor tissue demonstrated high microsatellite instability of di- and tetranucleotides (EMAST), and immunohistochemical staining illustrated a complete loss of nuclear MSH3 in normal and tumor tissue, confirming the LoF effect and causal relevance of the mutations. The pedigrees, genotypes, and frequency of MSH3 mutations in the general population are consistent with an autosomal-recessive mode of inheritance. Both index persons have an affected sibling carrying the same mutations. The tumor spectrum in these four persons comprised colorectal and duodenal adenomas, colorectal cancer, gastric cancer, and an early-onset astrocytoma. Additionally, we detected one unrelated individual with biallelic PMS2 germline mutations, representing constitutional mismatch-repair deficiency. Potentially causative variants in 14 more candidate genes identified in 26 other individuals require further workup. In the present study, we identified biallelic germline MSH3 mutations in individuals with a suspected hereditary tumor syndrome. Our data suggest that MSH3 mutations represent an additional recessive subtype of colorectal adenomatous polyposis.

Genomic and transcriptomic heterogeneity of colorectal tumours arising in Lynch syndrome.

Colorectal cancer (CRC) arising in Lynch syndrome (LS) comprises tumours with constitutional mutations in DNA mismatch repair genes. There is still a lack of whole-genome and transcriptome studies of LS-CRC to address questions about similarities and differences in mutation and gene expression characteristics between LS-CRC and sporadic CRC, about the molecular heterogeneity of LS-CRC, and about specific mechanisms of LS-CRC genesis linked to dysfunctional mismatch repair in LS colonic mucosa and the possible role of immune editing. Here, we provide a first molecular characterization of LS tumours and of matched tumour-distant reference colonic mucosa based on whole-genome DNA-sequencing and RNA-sequencing analyses. Our data support two subgroups of LS-CRCs, G1 and G2, whereby G1 tumours show a higher number of somatic mutations, a higher amount of microsatellite slippage, and a different mutation spectrum. The gene expression phenotypes support this difference. Reference mucosa of G1 shows a strong immune response associated with the expression of HLA and immune checkpoint genes and the invasion of CD4+ T cells. Such an immune response is not observed in LS tumours, G2 reference and normal (non-Lynch) mucosa, and sporadic CRC. We hypothesize that G1 tumours are edited for escape from a highly immunogenic microenvironment via loss of HLA presentation and T-cell exhaustion. In contrast, G2 tumours seem to develop in a less immunogenic microenvironment where tumour-promoting inflammation parallels tumourigenesis. Larger studies on non-neoplastic mucosa tissue of mutation carriers are required to better understand the early phases of emerging tumours. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Activating ERBB2/HER2 mutations indicate susceptibility to pan-HER inhibitors in Lynch and Lynch-like colorectal cancer.

OBJECTIVE: Microsatellite instability (MSI) is detected in approximately 15% of all colorectal cancers (CRC) and virtually in all cases with Lynch syndrome. The MSI phenotype is caused by dysfunctional mismatch repair (MMR) and leads to accumulation of DNA replication errors. Sporadic MSI CRC often harbours BRAF(V600E); however, no consistent data exist regarding targeted treatment approaches in BRAF(wt) MSI CRC. DESIGN: Mutations and quantitative MSI were analysed by deep sequencing in 196 formalin fixed paraffin embedded (FFPE) specimens comprising Lynch and Lynch-like CRCs from the German Hereditary Nonpolyposis Colorectal Cancer registry. Functional relevance of recurrent ERBB2/HER2 mutations was investigated in CRC cell lines using reversible and irreversible HER-targeting inhibitors, EGFR-directed antibody cetuximab, HER2-directed antibody trastuzumab and siRNA-mediated ERBB2/HER2 knockdown. RESULTS: Quantification of nucleotide loss in non-coding mononucleotide repeats distinguished microsatellite status with very high accuracy (area under curve=0.9998) and demonstrated progressive losses with deeper invasion of MMR-deficient colorectal neoplasms (p=0.008). Characterisation of BRAF(wt) MSI CRC revealed hot-spot mutations in well-known oncogenic drivers, including KRAS (38.7%), PIK3CA (36.5%), and ERBB2 (15.0%). L755S and V842I substitutions in ERBB2 were highly recurrent. Functional analyses in ERBB2-mutated MSI CRC cell lines revealed a differential response to HER-targeting compounds and superiority of irreversible pan-HER inhibitors. CONCLUSIONS: We developed a high-throughput deep sequencing approach for concomitant MSI and mutational analyses in FFPE specimens. We provided novel insights into clinically relevant alterations in MSI CRC and a rationale for targeting ERBB2/HER2 mutations in Lynch and Lynch-like CRC.

Phosphorylation of histone H3T6 by PKCbeta(I) controls demethylation at histone H3K4.

Demethylation at distinct lysine residues in histone H3 by lysine-specific demethylase 1 (LSD1) causes either gene repression or activation. As a component of co-repressor complexes, LSD1 contributes to target gene repression by removing mono- and dimethyl marks from lysine 4 of histone H3 (H3K4). In contrast, during androgen receptor (AR)-activated gene expression, LSD1 removes mono- and dimethyl marks from lysine 9 of histone H3 (H3K9). Yet, the mechanisms that control this dual specificity of demethylation are unknown. Here we show that phosphorylation of histone H3 at threonine 6 (H3T6) by protein kinase C beta I (PKCbeta(I), also known as PRKCbeta) is the key event that prevents LSD1 from demethylating H3K4 during AR-dependent gene activation. In vitro, histone H3 peptides methylated at lysine 4 and phosphorylated at threonine 6 are no longer LSD1 substrates. In vivo, PKCbeta(I) co-localizes with AR and LSD1 on target gene promoters and phosphorylates H3T6 after androgen-induced gene expression. RNA interference (RNAi)-mediated knockdown of PKCbeta(I) abrogates H3T6 phosphorylation, enhances demethylation at H3K4, and inhibits AR-dependent transcription. Activation of PKCbeta(I) requires androgen-dependent recruitment of the gatekeeper kinase protein kinase C (PKC)-related kinase 1 (PRK1). Notably, increased levels of PKCbeta(I) and phosphorylated H3T6 (H3T6ph) positively correlate with high Gleason scores of prostate carcinomas, and inhibition of PKCbeta(I) blocks AR-induced tumour cell proliferation in vitro and cancer progression of tumour xenografts in vivo. Together, our data establish that androgen-dependent kinase signalling leads to the writing of the new chromatin mark H3T6ph, which in consequence prevents removal of active methyl marks from H3K4 during AR-stimulated gene expression.

Reduced migration of MLH1 deficient colon cancer cells depends on SPTAN1.

INTRODUCTION: Defects in the DNA mismatch repair (MMR) protein MLH1 are frequently observed in sporadic and hereditary colorectal cancers (CRC). Affected tumors generate much less metastatic potential than the MLH1 proficient forms. Although MLH1 has been shown to be not only involved in postreplicative MMR but also in several MMR independent processes like cytoskeletal organization, the connection between MLH1 and metastasis remains unclear. We recently identified non-erythroid spectrin αII (SPTAN1), a scaffolding protein involved in cell adhesion and motility, to interact with MLH1. In the current study, the interaction of MLH1 and SPTAN1 and its potential consequences for CRC metastasis was evaluated. METHODS: Nine cancer cell lines as well as fresh and paraffin embedded colon cancer tissue from 12 patients were used in gene expression studies of SPTAN1 and MLH1. Co-expression of SPTAN1 and MLH1 was analyzed by siRNA knock down of MLH1 in HeLa, HEK293, MLH1 positive HCT116, SW480 and LoVo cells. Effects on cellular motility were determined in MLH1 deficient HCT116 and MLH1 deficient HEK293T compared to their MLH1 proficient sister cells, respectively. RESULTS: MLH1 deficiency is clearly associated with SPTAN1 reduction. Moreover, siRNA knock down of MLH1 decreased the mRNA level of SPTAN1 in HeLa, HEK293 as well as in MLH1 positive HCT116 cells, which indicates a co-expression of SPTAN1 by MLH1. In addition, cellular motility of MLH1 deficient HCT116 and MLH1 deficient HEK293T cells was impaired compared to the MLH1 proficient sister clones. Consequently, overexpression of SPTAN1 increased migration of MLH1 deficient cells while knock down of SPTAN1 decreased cellular mobility of MLH1 proficient cells, indicating SPTAN1-dependent migration ability. CONCLUSIONS: These data suggest that SPTAN1 levels decreased in concordance with MLH1 reduction and impaired cellular mobility in MLH1 deficient colon cancer cells. Therefore, aggressiveness of MLH1-positive CRC might be related to SPTAN1.

Alveolar echinococcosis as a cause of vertebral osteomyelitis and soft tissue infection with recurrent cutaneous fistula formation.

Echinococcus multilocularis is endemic in Germany. However, alveolar echinococcosis is a rare disease. Most commonly the parasite affects the liver, behaving like a malignant tumour. Bones are affected in less than 2% of cases. We report a case of vertebral osteomyelitis accompanied by recurrent cutaneous fistula formation.

Teenage-Onset Colorectal Cancers in a Digenic Cancer Predisposition Syndrome Provide Clues for the Interaction between Mismatch Repair and Polymerase δ Proofreading Deficiency in Tumorigenesis.

Colorectal cancer (CRC) in adolescents and young adults (AYA) is very rare. Known predisposition syndromes include Lynch syndrome (LS) due to highly penetrant MLH1 and MSH2 alleles, familial adenomatous polyposis (FAP), constitutional mismatch-repair deficiency (CMMRD), and polymerase proofreading-associated polyposis (PPAP). Yet, 60% of AYA-CRC cases remain unexplained. In two teenage siblings with multiple adenomas and CRC, we identified a maternally inherited heterozygous PMS2 exon 12 deletion, NM_000535.7:c.2007-786_2174+493del1447, and a paternally inherited POLD1 variant, NP_002682.2:p.Asp316Asn. Comprehensive molecular tumor analysis revealed ultra-mutation (>100 Mut/Mb) and a large contribution of COSMIC signature SBS20 in both siblings' CRCs, confirming their predisposition to AYA-CRC results from a high propensity for somatic MMR deficiency (MMRd) compounded by a constitutional Pol δ proofreading defect. COSMIC signature SBS20 as well as SBS26 in the index patient's CRC were associated with an early mutation burst, suggesting MMRd was an early event in tumorigenesis. The somatic second hits in PMS2 were through loss of heterozygosity (LOH) in both tumors, suggesting PPd-independent acquisition of MMRd. Taken together, these patients represent the first cases of cancer predisposition due to heterozygous variants in PMS2 and POLD1. Analysis of their CRCs supports that POLD1-mutated tumors acquire hypermutation only with concurrent MMRd.

MET-FISH Evaluation Algorithm: Proposal of a Simplified Method.

MET amplifications (METamp) occur in 5% of NSCLC and represent in most case mechanisms of resistance to ALK and/or EGFR-targeted therapies. METamp detection can be performed using different techniques, although Fluorescence In-Situ Hybridization (FISH) remains the gold-standard, especially in the context of subclonality. To date current evaluation algorithms of MET amplifications are time consuming. Aim of the study was to identify a faster, equally reliable diagnostic algorithm for the detection of METamp, which is currently classified in negativity and low/intermediate/high-level amplification. N=497 NSCLC cases with available MET-FISH data had been selected. The results based on the first evaluated 20 cells had been re-calculated and compared with the definitive results based on 60 cells. For n=464 (93.4%) identical results had been obtained when counting 20 cells instead of 60 cells. Thirty-three cases (5.6%) showed a discrepancy, leading to an incorrect upgrade to a higher diagnostic category (n=25) and to an incorrect downgrade (n=8). We propose a simplified, yet equally reliable MET FISH-algorithm: after accurate screening of the whole tumor slide, twenty tumor cells have to be evaluated and results calculated: If the result is negative, or if all criteria of high-level METamp are fulfilled, the case can be signed out as such. All other cases should be considered as equivocal and additional 40 cells have to be counted. Given that, reliable results can be obtained by counting 20 cells only and an "equivocal" category for cases that need further investigation have been clearly defined.

FHL2 expression in peritumoural fibroblasts correlates with lymphatic metastasis in sporadic but not in HNPCC-associated colon cancer.

Four and a half LIM domain protein-2 (FHL2) is a component of the focal adhesion structures and has been suggested to have an important role in cancer progression. This study analyses the role of FHL2 in peritumoural fibroblasts of sporadic and hereditary non-polyposis colorectal cancer (HNPCC). Tissue specimens of 48 sporadic and 49 hereditary colon cancers, respectively, were stained immunohistochemically for FHL2, transforming growth factor (TGF)-ß1 ligand and α-SMA. Myofibroblasts at the tumour invasion front co-expressed α-SMA and FHL2. Sporadic colon cancer but not HNPCC cases showed a correlation between TGF-ß1 expression of the invading tumour cells and FHL2 staining of peritumoural myofibroblasts. Overexpression of FHL2 in peritumoural myofibroblasts correlated to lymphatic metastasis in sporadic colon cancer but not in HNPCC. In cultured mouse fibroblasts, TGF-ß1 treatment induced myofibroblast differentiation, stimulated FHL2 protein expression and elevated number of migratory cells in transwell motility assays, suggesting that FHL2 is regulated downstream of TGF-ß. Physical contact of colon cancer cells and myofibroblasts via FHL2-positive focal adhesions was detected in human colon carcinoma tissue and in co-culture assays using sporadic as well as HNPCC-derived tumour cell lines. Our data provide strong evidence for an important role of FHL2 in the progression of colon cancers. Tumour-secreted TGF-ß1 stimulates FHL2 protein expression in peritumoural fibroblasts, probably facilitating the invasion of tumour glands into the surrounding tissue by enhanced myofibroblast migration and tight connection of fibroblasts to tumour cells via focal adhesions. These findings are absent in HNPCC-associated colon cancers in vivo and may contribute to a less invasive and more protruding tumour margin of microsatellite instable carcinomas.

Phosphatidylinositol-3'-kinase/AKT signaling is essential in synovial sarcoma.

Synovial sarcomas account for 5-10% of all malignant soft tissue tumors. They have been shown to express different membranous growth factor receptors, many of them signaling via intracellular kinase cascades. In our study, the functional role of PI3K/AKT signals in synovial sarcoma is analyzed with regard to tumor biology and therapeutic applicability. Immunohistochemical stainings of (Ser473)-phosphorylated (p)-AKT, its targets p-(Ser9)-GSK-3ß and p-(Ser2448)-mTOR and the cell cycle regulators Cyclin D1 and p27(KIP1) were performed in 36 synovial sarcomas. The PIK3CA gene was screened for mutations. In vitro, four synovial sarcoma cell lines were treated with the PI3K inhibitor LY294002. Phosphorylation of AKT, GSK-3ß and mTOR was assessed, and cellular proliferation and apoptosis were analyzed to functionally characterize the effects of PI3K inhibition. Finally, coincubations of LY294002 with cytotoxic drugs were performed. Most tumors showed significant expression levels of p-AKT, p-GSK-3ß and p-mTOR, indicating activation of the PI3K/AKT signaling cascade in synovial sarcomas; Cyclin D1 and p27(KIP1) were differentially expressed. Mutations in the PIK3CA gene could be excluded. In vitro, PI3K inhibition diminished synovial sarcoma cell growth accompanied by reduced phosphorylation of AKT, GSK-3ß and mTOR. Mechanistically, PI3K pathway inhibition lead to enhanced apoptosis and decreased cellular proliferation linked to reduced Cyclin D1 and increased p27(KIP1) levels. Simultaneous treatment of synovial sarcoma cell lines with LY294002 and cytotoxic drugs resulted in additive effects. In summary, PI3K signaling plays an essential role in growth control of synovial sarcomas and might be successfully targeted in multimodal therapeutic strategies.

Efficacy of annual colonoscopic surveillance in individuals with hereditary nonpolyposis colorectal cancer.

BACKGROUND & AIMS: Individuals with hereditary nonpolyposis colorectal cancer (HNPCC; Lynch syndrome) have a high risk for developing colorectal cancer (CRC). We evaluated the efficacy of annual surveillance colonoscopies to detect adenomas and CRCs. METHODS: In a prospective, multicenter cohort study, 1126 individuals underwent 3474 colonoscopies. We considered individuals from 3 groups of HNPCC families: those with a pathogenic germline mutation in a mismatch repair gene (MUT group), those without a mutation but with microsatellite instability (MSI group), and those who fulfilled the Amsterdam criteria without microsatellite instability (MSS group). RESULTS: Compliance to annual intervals was good, with 81% of colonoscopies completed within 15 months. Ninety-nine CRC events were observed in 90 patients. Seventeen CRCs (17%) were detected through symptoms (8 before baseline colonoscopy, 8 at intervals >15 months to the preceding colonoscopy, and 1 interval cancer). Only 2 of 43 CRCs detected by follow-up colonoscopy were regionally advanced. Tumor stages were significantly lower among CRCs detected by follow-up colonoscopies compared with CRCs detected by symptoms (P = .01). Cumulative CRC risk at the age of 60 years was similar in the MUT and MSI groups (23.0% combined; 95% confidence interval [CI], 14.8%-31.2%) but considerably lower in the MSS group (1.8%; 95% CI, 0.0%-5.1%). Adenomas at baseline colonoscopy predicted an earlier occurrence of subsequent adenoma (hazard ratio, 2.6; 95% CI, 1.7-4.0) and CRC (hazard ratio, 3.9; 95% CI, 1.7-8.5), providing information about interindividual heterogeneity of adenomas and kinetics of CRC formation. CONCLUSIONS: Annual colonoscopic surveillance is recommended for individuals with HNPCC. Less intense surveillance might be appropriate for MSS families.

Foxp3(+) cell infiltration and granzyme B(+)/Foxp3(+) cell ratio are associated with outcome in neoadjuvant chemotherapy-treated ovarian carcinoma.

Preoperative neoadjuvant chemotherapy (NAC) can significantly reduce tumour burden in patients with primarily unresectable chemosensitive tumours, allowing a more complete cytoreduction during debulking surgery and facilitating evaluation of tumour chemosensitivity, identification of appropriate treatment options and improvement of intervention protocols. In this study, we investigate, using immunohistochemistry, the impact of platinum/taxane-based NAC (NAC) on tumour-infiltrating lymphocytes (TILs) in advanced epithelial ovarian cancer (EOC) and their relationship with clinical outcome. All patients had clinical response, as shown by ascites volume and CA125 levels compared to pre-treatment findings. NAC intervention significantly increased CD4(+), CD8(+) and granzyme B(+) infiltration while Foxp3(+) accumulation remained unaffected. TILs were prognostically neutral for both progression-free survival (PFS) and overall survival (OS) before NAC. In contrast, after NAC, elevated granzyme B(+) infiltration displayed a tendency for improved PFS (log-rank 0.064). Further, low Foxp3(+) cell density was associated with longer PFS, as compared with strong Foxp3(+) infiltration (median 20.94 vs. 11.24 months; log-rank 0.0001) and with improved OS (median 30.75 vs. 16.04 months, respectively; log-rank 0.056), demonstrating clear prognostic significance for PFS. In addition, high granzyme B(+)/Foxp3(+) ratio post-NAC strongly correlated with improved PFS compared to low granzyme B(+)/Foxp3(+) cell ratio (median 17.88 vs. 11.24 months, respectively), and showed to be a favourable prognostic factor for PFS (log-rank 0.014). Our findings indicate that NAC elicited an immunologic profile in which low immunosuppressive Foxp3(+) infiltration and elevated numbers of activated granzyme B(+) cells were significantly associated with EOC-specific PFS, suggesting a contribution of immunologic effects to improved clinical outcome.

In vivo intratumor angiogenic treatment effects during taxane-based neoadjuvant chemotherapy of ovarian cancer.

BACKGROUND: The aim of our study was to analyze the effect of taxane-based chemotherapy on tumor angiogenesis in patients with advanced epithelial ovarian cancer. METHODS: Within a prospective phase II trial, 32 patients with stage IIIC and IV ovarian cancer were treated with either two or three cycles of neoadjuvant chemotherapy prior to cytoreductive surgery. Carboplatin (AUC5) and docetaxel (75 mg/m2) were administered intravenously in a 3-weekly schedule. Changes in intratumor microvessel density (MVD) were assessed with immunohistochemistry by staining pre- and posttreatment surgical tumor specimens with panendothelial, neovascular and lymphatic vessel markers. RESULTS: Mean values of MVD defined by CD31, CD34, CD105 and D2-40 antibodies showed 12.3, 21.0, 2.7 and 3.1 vessels per high power field (HPF) before chemotherapy and increased after treatment to 15.3, 21.8, 4.8 and 3.6 per HPF, respectively. These changes were significant for CD31 (p = 0.04) and for CD105 (p = 0.02). CONCLUSION: Taxane-based chemotherapy appears to promote tumor vascularization when administered every 3 weeks. A possible explanation is the secondary recovery of MVD in response to immediate cytotoxic and antiangiogenic effects of the chemotherapy. If confirmed prospectively, these findings favor shorter treatment intervals of taxane-based chemotherapy to counteract proangiogenic recovery.

Changes in Ki-67 labeling indices during neoadjuvant chemotherapy for advanced ovarian cancer are associated with survival.

OBJECTIVE: To evaluate changes in Ki-67 expression during neoadjuvant chemotherapy (NACT) in advanced ovarian cancer. MATERIALS AND METHODS: Patients with International Federation of Gynecology and Obstetrics stage IIIC or IV and large-volume ascites were treated with NACT within a phase 2 trial. The expression of Ki-67 was evaluated by immunohistochemistry on paraffin-embedded tissue samples and classified by percentage of stained cells. Survival curves were plotted using the Kaplan-Meier method. RESULTS: Comparison of 40 individual paired results from pretreatment and posttreatment samples revealed a median difference of -0.11 in the Ki-67 index (95% confidence interval, -0.20 to -0.01; P = 0.005, signed rank test). Univariate analysis identified a high Ki-67 index as well as an increasing Ki-67 index after NACT as significant prognostic markers for progression-free survival (P = 0.004 and P = 0.001; log-rank test). Six of 12 patients with an increased Ki-67 index after NACT developed recurrence within 6 months after therapy. CONCLUSIONS: Changes of the Ki-67 index during NACT are associated with progression-free survival. If confirmed in prospective trials, an increasing Ki-67 index during preoperative treatment may serve as an indicator for resistance to chemotherapy.

DNA methylation in patients with colorectal cancer--correlation with some clinical and morphological features and with local tumour invasion.

AIM: To study quantitatively the promoter methylation of hMLH1, p16INK, TIMP3 and TPEF genes in patients with colorectal cancer and synchronous polyps, and correlate it with some clinicomorphological features. METHODS: DNA was extracted from all studied tumours and the corresponding normal mucosa. Microsatellite instability was analysed using two mononucleotide (BAT 25 and BAT 26) and three dinucleotide markers (D2S123, D5S356, D17S250) and automated DNA sequencing. Quantitative analysis of methylation was performed using DNA bisulfite modification, PCR with biotinylated primers, visualisation by 2% agarose gel electrophoresis and pyrosequencing. RESULTS: High methylation levels of hMLH1 and p16INK were found in elderly patients (mean age 73.8 +/- 9.5 years and 65.7 +/- 16.6 years, p < 0.03, t-test). Proximal tumours were more often associated with microsatellite instability (p < 0.05, Fisher's test) and higher level of methylation of hMLH1, p16INK and TIMP3 (p < 0.02, Kruskal-Wallis test), while tumours with poor differentiation tended to have higher methylation of the p16INK gene (p < 0.02, Kruskal-Wallis test). Local tumour invasion was correlated with the level of methylation of hMLH1, TIMP3 and the CpG island methylator phenotype (CIMP) status. Tumours with liver metastases showed a lower level of TIMP3 methylation than tumours with no systemic invasion (p < 0.05, Kruskal-Wallis test). We found concordance of methylation in 56% of the cases with colonic cancer and synchronous adenomas; the remaining 44% were discordant. CONCLUSIONS: Tumours with microsatellite instability, high level methylation and CIMP have distinctive clinical and morphological features. The level of hMLH1 and TIMP3 methylation and CIMP status can be correlated with the local tumour invasion. Different mechanisms, even for one and the same patient, can be responsible for the development of more than one third of the synchronous polyps and carcinomas.

Enhanced Tumoral MLH1-Expression in MLH1-/PMS2-Deficient Colon Cancer Is Indicative of Sporadic Colon Cancer and Not HNPCC.

Hereditary Non-Polyposis Colorectal Cancer (HNPCC) is caused by germline mutations of mismatch-repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. MLH1-/PMS2-deficient colorectal carcinomas might be HNPCC-associated but also caused by MLH1-promoter methylation in sporadic colon carcinoma. This study analyzed semiquantitatively whether the MLH1 staining pattern might be indicative of sporadic or HNPCC-associated colorectal cancer. Using a semiquantitative score ranging from 0 (negative) to 12 (maximum immunopositivity) we analyzed MLH1 expression patterns in 130 MLH1-/PMS2-deficient colorectal cancers. The collective consisted of 70 HNPCC-associated colorectal cancers and 60 sporadic colon cancers. In tumor cells of 70 HNPCC-associated colorectal cancers, 64 cases (91.43%) showed no MLH1 staining, 5 cases weak (7.14%) and 1 case (1.43%) stronger staining intensity. In contrast, in tumor cells of 60 sporadic colorectal cancers 45 cases (75.0%) showed no MLH1 staining, 10 cases weak (16.67%) and 5 cases (8.33%) stronger staining intensity to a varying extent. In immunopositive cases, MLH1 showed a characteristic dot-like nuclear staining pattern in the tumor cells. We compared cases with absent to weak MLH1-staining (immunoscores 0 to 2) to cases with elevated immunoscores (3 to 12) detecting a statistically significant difference between HNPCC-associated and sporadic colon cancers (p value = 0.0031, Fisher's exact test). Taken together, enhanced tumoral MLH1 expression in MLH1-/PMS2-deficient colorectal carcinomas seems to be indicative of sporadic origin. In contrast, HNPCC-associated colorectal cancer showed absent or very weak MLH1 immunopositivity. Therefore, this semiquantitative and easy to exert MLH1 immunoscore might help to identify sporadic MLH1-/PMS2-deficient colorectal cancer cases prior to time-consuming methylation analysis.

The low-affinity neurotrophin receptor, p75, is upregulated in ganglioneuroblastoma/ganglioneuroma and reduces tumorigenicity of neuroblastoma cells in vivo.

Neuroblastoma, the most common extracranial tumor of childhood, is derived from neural crest progenitor cells that fail to differentiate along their predefined route to sympathetic neurons or sympatho-adrenergic adrenal cells. Although expression of the high-affinity neurotrophin receptors, TrkA and TrkB, is of major importance in neuroblastoma, the significance of the expression of the low-affinity neurotrophin receptor, p75, is unclear. Here, we analyzed immunohistochemically expression of p75 on a tissue microarray of 93 primary neuroblastic tumors and assessed the functional consequences of p75 expression in neuroblastoma cell lines. We found the p75 receptor protein to be expressed in neuroblastic cells of ganglioneuromas/ganglioneuroblastomas as well as differentiating neuroblastomas, but not in poorly differentiated neuroblastomas. In an unrelated cohort of 110 neuroblastic tumors, p75 mRNA expression levels correlated with differentiation, and patients with tumors that expressed p75 at high levels had an increased event-free and overall survival. In addition, we did not detect p75 expression in 8 established neuroblastoma cell lines examined with FACS analysis. These cell lines exhibited an undifferentiated morphology, and were all derived from aggressive, high-stage neuroblastomas. Ectopic p75 expression in the SH-SY5Y neuroblastoma cell line significantly reduced proliferation, increased the fraction of apoptotic cells in vitro and resulted in a loss of tumorigenicity in nude mice. Taken together, our data suggest that expression of the p75 low-affinity neurotrophin receptor is correlated with a reduced level of tumorigenicity, and that induction of p75 expression may be an option to revert features of an aggressive tumor phenotype.

Deficiency in the LIM-only protein FHL2 impairs assembly of extracellular matrix proteins.

We have described the scaffolding protein FHL2 as a component of focal adhesion structures, to which it is recruited via binding to both alpha- or beta-integrin subunits. Using mesenchymal stem cells from wild-type and FHL2-knockout mice, we show here that inactivation of FHL2 leads to impaired assembly of extracellular matrix proteins on the cell surface and to impaired bundling of focal adhesions. Both altered properties can be restored by reexpression of recombinant FHL2 protein in FHL2-null cells. Molecular analysis of integrin-mediated signaling revealed a higher phosphorylation of FAK at tyrosine 925 in FHL2-knockout cells compared to their wild-type counterpart. Consequently, the activation of the mitogenic kinase ERK was more pronounced in knockout cells on cell adhesion. The growth factor-induced activation of ERK, however, was not altered. The perturbed organization of extracellular matrix on FHL2-null cells was improved when the increased activation of MAPK was inhibited. Our findings point to a role of FHL2 in bundling of focal adhesion structures, in integrin-mediated ERK activation, and subsequently in proper allocation of matrix proteins on the cell surface.