Methylation profiles of exocrine and neuroendocrine colorectal carcinomas using methylation-specific multiple ligation-dependent probe amplification.

In this study, we employed Methylation-Specific Multiple Ligation-dependent Probe Amplification (MS-MLPA) to evaluate the methylation status of 34 genes in a series 104 formalin-fixed and paraffin-embedded specimens, including 83 exocrine adenocarcinomas (CRCs) and 21 neuroendocrine cancers (NECs), previously examined for clinico-pathological and molecular features, as well as in 25 colorectal mucosae from 13 CRC patients and from12 non-neoplastic patients. We found higher levels of promoter methylation in normal colonic tissue of CRC-patients with respect to the control group. The CpG Island Methylation Phenotype (CIMP) was observed with similar frequency in exocrine CRCs and in NECs, but different methylation profiles were present in the two tumour types. Microsatellite instability was the marker most strongly associated with CIMP. In both CRCs and NECs, CIMP+/MSI+ tumours represented a homogeneous clinicopathological entity, associated with a very good prognosis compared with the other three classes of cancers. Our results demonstrated that MS-MLPA is a rapid and sensitive method to analyse the methylation status of multiple genes simultaneously and presents innovative aspects that may have important scientific and clinical implications. The use of DNA methylation alterations as a molecular marker system could potentially be a powerful approach to population-based screening for the early detection and for risk assessment of colorectal cancer

Methylation profiles of exocrine and neuroendocrine colorectal carcinomas using methylation-specific multiple ligation-dependent probe amplification.

In this study, we employed Methylation-Specific Multiple Ligation-dependent Probe Amplification (MS-MLPA) to evaluate the methylation status of 34 genes in a series 104 formalin-fixed and paraffin-embedded specimens, including 83 exocrine adenocarcinomas (CRCs) and 21 neuroendocrine cancers (NECs), previously examined for clinico-pathological and molecular features, as well as in 25 colorectal mucosae from 13 CRC patients and from12 non-neoplastic patients. We found higher levels of promoter methylation in normal colonic tissue of CRC-patients with respect to the control group. The CpG Island Methylation Phenotype (CIMP) was observed with similar frequency in exocrine CRCs and in NECs, but different methylation profiles were present in the two tumour types. Microsatellite instability was the marker most strongly associated with CIMP. In both CRCs and NECs, CIMP+/MSI+ tumours represented a homogeneous clinicopathological entity, associated with a very good prognosis compared with the other three classes of cancers. Our results demonstrated that MS-MLPA is a rapid and sensitive method to analyse the methylation status of multiple genes simultaneously and presents innovative aspects that may have important scientific and clinical implications. The use of DNA methylation alterations as a molecular marker system could potentially be a powerful approach to population-based screening for the early detection and for risk assessment of colorectal cancer

Aberrant DNA methylation profiles of inherited and sporadic colorectal cancer

Background: Aberrant DNA methylation has been widely investigated in sporadic colorectal carcinomas (CRCs), and extensive work has been performed to characterize different methylation profiles of CRC. Less information is available about the role of epigenetics in hereditary CRC and about the possible clinical use of epigenetic biomarkers in CRC, regardless of the etiopathogenesis. Long interspersed nucleotide element 1 (LINE-1) hypomethylation and gene-specific hypermethylation of 38 promoters were analyzed in multicenter series of 220 CRCs including 71 Lynch (Lynch colorectal cancer with microsatellite instability (LS-MSI)), 23 CRCs of patients under 40 years in which the main inherited CRC syndromes had been excluded (early-onset colorectal cancer with microsatellite stability (EO-MSS)), and 126 sporadic CRCs, comprising 28 cases with microsatellite instability (S-MSI) and 98 that were microsatellite stable (S-MSS). All tumor methylation patterns were integrated with clinicopathological and genetic characteristics, namely chromosomal instability (CIN), TP53 loss, BRAF, and KRAS mutations. Results: LS-MSI mainly showed absence of extensive DNA hypo-and hypermethylation. LINE-1 hypomethylation was observed in a subset of LS-MSI that were associated with the worse prognosis. Genetically, they commonly displayed G:A transition in the KRAS gene and absence of a CIN phenotype and of TP53 loss. S-MSI exhibited a specific epigenetic profile showing low rates of LINE-1 hypomethylation and extensive gene hypermethylation. S-MSI were mainly characterized by MLH1 methylation, BRAF mutation, and absence of a CIN phenotype and of TP53 loss. By contrast, S-MSS showed a high frequency of LINE-1 hypomethylation and of CIN, and they were associated with a worse prognosis. EO-MSS were a genetically and epigenetically heterogeneous group of CRCs. Like LS-MSI, some EO-MSS displayed low rates of DNA hypo-or hypermethylation and frequent G:A transitions in the KRAS gene, suggesting that a genetic syndrome might still be unrevealed in these patients. By contrast, some EO-MSS showed similar features to those observed in S-MSS, such as LINE-1 hypomethylation, CIN, and TP53 deletion. In all four classes, hypermethylation of ESR1, GATA5, and WT1 was very common. Conclusions: Aberrant DNA methylation analysis allows the identification of different subsets of CRCs. This study confirms the potential utility of methylation tests for early detection of CRC and suggests that LINE-1 hypomethylation may be a useful prognostic marker in both sporadic and inherited CRCs

Methylation profiles of exocrine and neuroendocrine colorectal carcinomas using methylation-specific multiple ligation-dependent probe amplification.

In this study, we employed Methylation-Specific Multiple Ligation-dependent Probe Amplification (MS-MLPA) to evaluate the methylation status of 34 genes in a series 104 formalin-fixed and paraffin-embedded specimens, including 83 exocrine adenocarcinomas (CRCs) and 21 neuroendocrine cancers (NECs), previously examined for clinico-pathological and molecular features, as well as in 25 colorectal mucosae from 13 CRC patients and from12 non-neoplastic patients. We found higher levels of promoter methylation in normal colonic tissue of CRC-patients with respect to the control group. The CpG Island Methylation Phenotype (CIMP) was observed with similar frequency in exocrine CRCs and in NECs, but different methylation profiles were present in the two tumour types. Microsatellite instability was the marker most strongly associated with CIMP. In both CRCs and NECs, CIMP+/MSI+ tumours represented a homogeneous clinicopathological entity, associated with a very good prognosis compared with the other three classes of cancers. Our results demonstrated that MS-MLPA is a rapid and sensitive method to analyse the methylation status of multiple genes simultaneously and presents innovative aspects that may have important scientific and clinical implications. The use of DNA methylation alterations as a molecular marker system could potentially be a powerful approach to population-based screening for the early detection and for risk assessment of colorectal cancer

AKAP2-anchored protein phosphatase 1 controls prostatic neuroendocrine carcinoma cell migration and invasion

Prostate cancer (PC) is the second leading cause of cancer-related death in men. The growth of primary prostate cancer cells relies on circulating androgens and thus the standard therapy for the treatment of localized and advanced PC is the androgen deprivation therapy. Prostatic neuroendocrine carcinoma (PNEC) is an aggressive and highly metastatic subtype of prostate cancer, which displays poor prognosis and high lethality. Most of PNECs develop from prostate adenocarcinoma in response to androgen deprivation therapy, however the mechanisms involved in this transition and in the elevated biological aggressiveness of PNECs are poorly defined. Our current findings indicate that AKAP2 expression is dramatically upregulated in PNECs as compared to non-cancerous prostate tissues. Using a PNEC cell model, we could show that AKAP2 is localized both intracellularly and at the cell periphery where it colocalizes with F-actin. AKAP2 and F-actin interact directly through a newly identified actin-binding domain located on AKAP2. RNAi-mediated silencing of AKAP2 promotes the phosphorylation and deactivation of cofilin, a protein involved in actin turnover. This effect correlates with a significant reduction in cell migration and invasion. Co-immunoprecipitation experiments and proximity ligation assays revealed that AKAP2 forms a complex with the catalytic subunit of protein phosphatase 1 (PP1) in PNECs. Importantly, AKAP2-mediated anchoring of PP1 to the actin cytoskeleton regulates cofilin dephosphorylation and activation, which, in turn, enhances F-actin dynamics and favors migration and invasion. In conclusion, this study identified AKAP2 as an anchoring protein overexpressed in PNECs that controls cancer cell invasive properties by regulating cofilin phosphorylation

Molecular Features and Methylation Status in Early Onset ( 6440 Years) Colorectal Cancer: A Population Based, Case-Control Study

Colorectal cancer is usually considered a disease of the elderly. However, a small fraction of patients develops colorectal cancer earlier. The aim of our study was to define the frequency of known hereditary colorectal syndromes and to characterise genetic and epigenetic features of early nonhereditary tumors. Thirty-three patients 6440 years with diagnosis of colorectal cancer and 41 patients with disease at >60 years of age were investigated for MSI, Mismatch Repair proteins expression, KRAS and BRAF mutations, hypermethylation, and LINE-1 hypomethylation. Detection of germline mutations was performed in Mismatch Repair, APC and MUTYH genes. Early onset colorectal cancer showed a high incidence of hereditary forms (18%). KRAS mutations were detected in 36% of early nonhereditary tumors. Early onset colorectal cancer disclosed an average number of methylated genes significantly lower when compared to the controls (p = 0.02). Finally both of the two groups were highly methylated in ESR1, GATA5, and WT1 genes and were similar for LINE-1 hypomethylation. The genetic make-up of carcinomas differs from young to elderly patients. Early onset tumors showed more frequently a constitutional defective of Mismatch Repair System and a minor number of methylated genes. Hypermethylation of ESR1, GATA5, and WT1 genes suggests possible markers in the earlier diagnosis of colorectal tumorigenesis

Androgen receptor is frequently expressed in HER2-positive, ER/PR-negative breast cancers

The estrogen receptor (ER)/progesterone receptor (PR)-negative breast carcinomas (BCs) encompass three molecular subtypes: one with human epidermal growth factor receptor 2 (HER) overexpression, one normal like, and the triple negative. The androgen receptor (AR) is expressed in 7090% of invasive BCs. The aim of our study is to detect the expression of AR in a series of ER/PR-negative BCs to ascertain if there is clinical significance in relation to BC molecular subtypes. A immunohistochemical study for all receptors and cytokeratin expression was performed in 232 cases of ER/PR-negative BCs. According to cytokeratin expression, BCs were classified into two groups: luminal-type BCs (44.2%) and basal-like-type BCs (55.8%). According to the expression of HER2, 59.3% were triple-negative BCs (when ER, PR, and HER2 were negative) and 40.7% were HER2-positive BCs. AR expression was observed in 128 tumors (56.6%). One hundred and ten cases (48.8%) had >10% and 18 (7.8%) had <10% of positively stained cells. AR immunoreactivity was found in 31.2% basal-like BCs, while in the luminal group 71.1% of cases were positive, showing highly significant correlation (p<108). Regarding HER2 status, 76.7% of HER2-positive BC cases were AR positive compared with only 30.4% of triple-negative BC types, showing a strong statistically significant correlation. In conclusion, we show that AR is frequently expressed in ER/PR-negative BCs and that expression of HER2 and AR is highly correlated (p<0.005). Our results point out the role of AR and HER2 in the pathogenesis of BCs and suggest the potential role of AR in clinical management of ER/PR-negative BC

Disruption of the APC gene by t(5;7) translocation in a Turcot family

Turcot syndrome (TS) refers to the combination of colorectal polyps and primary tumours of the central nervous system. TS is a heterogeneous genetic condition due to APC and/or mismatch repair germline mutations. When APC is involved the vast majority of mutations are truncating, but in approximately 20%-30% of patients with familial polyposis no germline mutation can be found. A 30-year-old Caucasian woman with a positive pedigree for TS was referred to our Genetic Counselling Service. She was negative for APC and MUTYH but showed a reciprocal balanced translocation t(5;7)(q22;p15) at chromosome analysis. FISH analysis using specific BAC probes demonstrated that 5q22 breakpoint disrupted the APC gene. Transcript analysis by MLPA and digital PCR revealed that the cytogenetic rearrangement involving the 3' end of the APC gene caused a defective expression of a truncated transcript. This result allowed cytogenetic analysis to be offered to all the other family members and segregation analysis clearly demonstrated that all the carriers were affected, whereas non-carriers did not have the polyposis. A cytogenetic approach permitted the identification of the mutation-causing disease in this family, and the segregation analysis together with the transcript study supported the pathogenetic role of this mutation. Karyotype analysis was used as a predictive test in all members of this family. This family suggests that clinically positive TS and FAP cases, which test negative with standard molecular analysis, could be easily and cost-effectively resolved by a classical and molecular cytogenetic approach

EBV+ and MSI Gastric Cancers Harbor High PD-L1/PD-1 Expression and High CD8+ Intratumoral Lymphocytes

Both EBV+ and MSI gastric cancers (GCs) have high lymphoid infiltration which is rare in MSS/EBV− cancers. PD-L1/PD-1 interaction leads to a down-regulated immune response and it is one of the most promising targets for gastric cancer immunotherapy. PD-L1/PD-1 and CD8 expression were immunohistochemically investigated in a series of 169 FFPE GCs, including 33 EBV+, 59 MSI and 77 MSS/EBV− cases. PD-L1 membrane immunoreactivity in more than 5% of tumor cells was present in 31/169 GCs and was associated with high levels of CD8 intraepithelial lymphocytes (TILs; p &lt; 0.001). PD-L1+ cases were mainly poorly differentiated (71%), intestinal type (85%) and high lymphoid response (HLR; 90%) tumors. PD-L1 expression was only present in EBV⁺ (46%), MSI (24%) and rare MSS/EBV− (3%) GCs with high CD8+ TILs (p &lt; 0.001). Despite being associated with a better prognosis both in the whole series (p &lt; 0.05) and in the MSI subset, PD-L1 is not an independent prognostic factor. PD-L1 gene amplification was detected in 3/17 cases, including 2/7 EBV+ and 1/8 MSI GC. PD-1⁺ TILs were significantly higher in EBV⁺ than MSI and MSS/EBV− cases. PD-L1/PD-1 pathway is selectively activated in HLR GCs and could be considered an emerging therapeutic target, particularly for EBV and MSI GCs