Building on the clinical applicability of ctDNA analysis in non-metastatic pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma represents one of the solid tumors showing the worst prognosis worldwide, with a high recurrence rate after adjuvant or neoadjuvant therapy. Circulating tumor DNA analysis raised as a promising non-invasive tool to characterize tumor genomics and to assess treatment response. In this study, surgical tumor tissue and sequential blood samples were analyzed by next-generation sequencing and were correlated with clinical and pathological characteristics. Thirty resectable/borderline pancreatic ductal adenocarcinoma patients treated at the Hospital Universitario de Navarra were included. Circulating tumoral DNA sequencing identified pathogenic variants in KRAS and TP53, and in other cancer-associated genes. Pathogenic variants at diagnosis were detected in patients with a poorer outcome, and were correlated with response to neoadjuvant therapy in borderline pancreatic ductal adneocarcinoma patients. Higher variant allele frequency at diagnosis was associated with worse prognosis, and thesum of variant allele frequency was greater in samples at progression. Our results build on the potential value of circulating tumor DNA for non-metastatic pancreatic ductal adenocarcinoma patients, by complementing tissue genetic information and as a non-invasive tool for treatment decision. Confirmatory studies are needed to corroborate these findings.

A DNA Methylation-Based Gene Signature Can Predict Triple-Negative Breast Cancer Diagnosis.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer (BC) subtype and lacks targeted treatment. It is diagnosed by the absence of immunohistochemical expression of several biomarkers, but this method still displays some interlaboratory variability. DNA methylome aberrations are common in BC, thereby methylation profiling could provide the identification of accurate TNBC diagnosis biomarkers. Here, we generated a signature of differentially methylated probes with class prediction ability between 5 non-neoplastic breast and 7 TNBC tissues (error rate = 0.083). The robustness of this signature was corroborated in larger cohorts of additional 58 non-neoplastic breast, 93 TNBC, and 150 BC samples from the Gene Expression Omnibus repository, where it yielded an error rate of 0.006. Furthermore, we validated by pyrosequencing the hypomethylation of three out of 34 selected probes (FLJ43663, PBX Homeobox 1 (PBX1), and RAS P21 protein activator 3 (RASA3) in 51 TNBC, even at early stages of the disease. Finally, we found significantly lower methylation levels of FLJ43663 in cell free-DNA from the plasma of six TNBC patients than in 15 healthy donors. In conclusion, we report a novel DNA methylation signature with potential predictive value for TNBC diagnosis.

Understanding the Molecular Mechanism of miR-877-3p Could Provide Potential Biomarkers and Therapeutic Targets in Squamous Cell Carcinoma of the Cervix.

No therapeutic targets and molecular biomarkers are available in cervical cancer (CC) management. In other cancer types, micro-RNA-877-3p (miR-877-3p) has been associated with events relevant for CC development. Thus, we aimed to determine miR-877-3p role in CC. miR-877-3p levels were examined by quantitative-PCR in 117 cervical lesions and tumors. Effects on CC cell proliferation, migration, and invasion were evaluated upon anti-miR-877-3p transfection. miR-877-3p dependent molecular mechanism was comprehensively explored by proteomics, dual-luciferase reporter assay, western blot, and immunohistochemistry. Cervical tumors expressed higher miR-877-3p levels than benign lesions. miR-877-3p promoted CC cell migration and invasion, at least partly by modulating cytoskeletal protein folding through the chaperonin-containing T-complex protein 1 complex. Notably, miR-877-3p silencing synergized with paclitaxel. Interestingly, miR-877-3p downregulated the levels of an in silico-predicted target, ZNF177, whose expression and subcellular location significantly distinguished high-grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas of the cervix (SCCCs). Cytoplasmic ZNF177 was significantly associated with worse progression-free survival in SCCC. Our results suggest that: (i) miR-877-3p is a potential therapeutic target whose inhibition improves paclitaxel effects; (ii) the expression and location of its target ZNF177 could be diagnostic biomarkers between HSIL and SCCC; and (iii) cytoplasmic ZNF177 is a poor-prognosis biomarker in SCCC.

Absence of Nuclear p16 Is a Diagnostic and Independent Prognostic Biomarker in Squamous Cell Carcinoma of the Cervix.

The tumor-suppressor protein p16 is paradoxically overexpressed in cervical cancer (CC). Despite its potential as a biomarker, its clinical value and the reasons for its failure in tumor suppression remain unclear. Our purpose was to determine p16 clinical and biological significance in CC. p16 expression pattern was examined by immunohistochemistry in 78 CC cases (high-grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas of the cervix -SCCCs). CC cell proliferation and invasion were monitored by real-time cell analysis and Transwell® invasion assay, respectively. Cytoplasmic p16 interactors were identified from immunoprecipitated extracts by liquid chromatography-tandem mass spectrometry, and colocalization was confirmed by double-immunofluorescence. We observed that SCCCs showed significantly more cytoplasmic than nuclear p16 expression than HSILs. Importantly, nuclear p16 absence significantly predicted poor outcome in SCCC patients irrespective of other clinical parameters. Moreover, we demonstrated that cytoplasmic p16 interacted with CDK4 and other unreported proteins, such as BANF1, AKAP8 and AGTRAP, which could sequester p16 to avoid nuclear translocation, and then, impair its anti-tumor function. Our results suggest that the absence of nuclear p16 could be a diagnostic biomarker between HSIL and SCCC, and an independent prognostic biomarker in SCCC; and explain why p16 overexpression fails to stop CC growth.

ADAM12 is A Potential Therapeutic Target Regulated by Hypomethylation in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and currently lacks any effective targeted therapy. Since epigenetic alterations are a common event in TNBC, DNA methylation profiling can be useful for identifying potential biomarkers and therapeutic targets. Here, genome-wide DNA methylation from eight TNBC and six non-neoplastic tissues was analysed using Illumina Human Methylation 450K BeadChip. Results were validated by pyrosequencing in an independent cohort of 50 TNBC and 24 non-neoplastic samples, where protein expression was also assessed by immunohistochemistry. The functional role of disintegrin and metalloproteinase domain-containing protein 12(ADAM12) in TNBC cell proliferation, migration and drug response was analysed by gene expression silencing with short hairpin RNA. Three genes (Von Willenbrand factor C and Epidermal Growth Factor domain-containing protein (VWCE), tetraspanin-9 (TSPAN9) and ADAM12) were found to be exclusively hypomethylated in TNBC. Furthermore, ADAM12 hypomethylation was associated with a worse outcome in TNBC tissues and was also found in adjacent-to-tumour tissue and, preliminarily, in plasma from TNBC patients. In addition, ADAM12 silencing decreased TNBC cell proliferation and migration and improved doxorubicin sensitivity in TNBC cells. Our results indicate that ADAM12 is a potential therapeutic target and its hypomethylation could be a poor outcome biomarker in TNBC.

CDH22 hypermethylation is an independent prognostic biomarker in breast cancer.

BACKGROUND: Cadherin-like protein 22 (CDH22) is a transmembrane glycoprotein involved in cell-cell adhesion and metastasis. Its role in cancer is controversial because it has been described as being upregulated in colorectal cancer, whereas it is downregulated in metastatic melanoma. However, its status in breast cancer (BC) is unknown. The purpose of our study was to determine the molecular status and clinical value of CDH22 in BC. RESULTS: We observed by immunohistochemistry that the level of CDH22 expression was lower in BC tissues than in their matched adjacent-to-tumour and non-neoplastic tissues from reduction mammoplasties. Since epigenetic alteration is one of the main causes of gene silencing, we analysed the hypermethylation of 3 CpG sites in the CDH22 promoter by pyrosequencing in a series of 142 infiltrating duct BC cases. CDH22 was found to be hypermethylated in tumoral tissues relative to non-neoplastic mammary tissues. Importantly, this epigenetic alteration was already present in adjacent-to-tumour tissues, although to a lesser extent than in tumoral samples. Furthermore, CDH22 gene regulation was dynamically modulated in vitro by epigenetic drugs. Interestingly, CDH22 hypermethylation in all 3 CpG sites simultaneously, but not expression, was significantly associated with shorter progression-free survival (p = 0.015) and overall survival (p = 0.021) in our patient series. Importantly, CDH22 hypermethylation was an independent factor that predicts poor progression-free survival regardless of age and stage (p = 0.006). CONCLUSIONS: Our results are the first evidence that CDH22 is hypermethylated in BC and that this alteration is an independent prognostic factor in BC. Thus, CDH22 hypermethylation could be a potential biomarker of poor prognosis in BC.

CHL1 hypermethylation as a potential biomarker of poor prognosis in breast cancer.

The CHL1 gene encodes a cell-adhesion molecule proposed as being a putative tumour-suppressor gene in breast cancer (BC). However, neither the underlying molecular mechanisms nor the clinical value of CHL1 downregulation in BC has been explored. The methylation status of three CpG sites in the CHL1 promoter was analysed by pyrosequencing in neoplastic biopsies from 142 patients with invasive BC and compared with that of non-neoplastic tissues. We found higher CHL1 methylation levels in breast tumours than in non-neoplastic tissues, either from mammoplasties or adjacent-to-tumour, which correlated with lower levels of protein expression in tumours measured by immunohistochemistry. A panel of five BC cell lines was treated with two epigenetic drugs, and restoration of CHL1 expression was observed, indicating in vitro dynamic epigenetic regulation. CHL1 was silenced by shRNA in immortalized but non-neoplastic mammary cells, and enhanced cell proliferation and migration, but not invasion, were found by real-time cell analysis. The prognostic value of CHL1 hypermethylation was assessed by the log-rank test and fitted in a Cox regression model. Importantly, CHL1 hypermethylation was very significantly associated with shorter progression-free survival in our BC patient series, independent of age and stage (p = 0.001). In conclusion, our results indicate that CHL1 is downregulated by hypermethylation and that this epigenetic alteration is an independent prognostic factor in BC.