Next-generation sequencing of bile cell-free DNA for the early detection of patients with malignant biliary strictures.

OBJECTIVE: Despite significant progresses in imaging and pathological evaluation, early differentiation between benign and malignant biliary strictures remains challenging. Endoscopic retrograde cholangiopancreatography (ERCP) is used to investigate biliary strictures, enabling the collection of bile. We tested the diagnostic potential of next-generation sequencing (NGS) mutational analysis of bile cell-free DNA (cfDNA). DESIGN: A prospective cohort of patients with suspicious biliary strictures (n=68) was studied. The performance of initial pathological diagnosis was compared with that of the mutational analysis of bile cfDNA collected at the time of first ERCP using an NGS panel open to clinical laboratory implementation, the Oncomine Pan-Cancer Cell-Free assay. RESULTS: An initial pathological diagnosis classified these strictures as of benign (n=26), indeterminate (n=9) or malignant (n=33) origin. Sensitivity and specificity of this diagnosis were 60% and 100%, respectively, as on follow-up 14 of the 26 and eight of the nine initially benign or indeterminate strictures resulted malignant. Sensitivity and specificity for malignancy of our NGS assay, herein named Bilemut, were 96.4% and 69.2%, respectively. Importantly, one of the four Bilemut false positives developed pancreatic cancer after extended follow-up. Remarkably, the sensitivity for malignancy of Bilemut was 100% in patients with an initial diagnosis of benign or indeterminate strictures. Analysis of 30 paired bile and tissue samples also demonstrated the superior performance of Bilemut. CONCLUSION: Implementation of Bilemut at the initial diagnostic stage for biliary strictures can significantly improve detection of malignancy, reduce delays in the clinical management of patients and assist in selecting patients for targeted therapies.

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.

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.

Radiopotentiation of enzalutamide over human prostate cancer cells as assessed by real-time cell monitoring.

AIM: To evaluate the radiopotentiation of enzalutamide in human prostate cancer cells. BACKGROUND: While radiotherapy is the first line of treatment for prostate cancer, androgen blockade therapies are demonstrating significant survival benefit as monotherapies. As androgen blockade can cause cell death by apoptosis, it is likely that androgen blockade will potentiate the cytotoxic activities of radiotherapy. MATERIALS AND METHODS: Here, we tested the potential synergistic effects of these two treatments over two human metastatic prostate cancer cells by real-time cell analysis (RTCA), androgen-sensitive LNCaP cells (Lymph Node Carcinoma of the Prostate) and androgen-independent PC-3. Both cell lines were highly resistant to high doses of radiotherapy. RESULTS: A pre-treatment of LNCaP cells with IC50 concentrations of enzalutamide significantly sensitized them to radiotherapy through enhanced apoptosis. In contrast, enzalutamide resistant PC-3 cells were not sensitized to radiotherapy by androgen blockade. CONCLUSIONS: These results provide evidence that the enzalutamide/radiotherapy combination could maximize therapeutic responses in patients with enzalutamide-sensitive prostate cancer.

Differential role of gene hypermethylation in adenocarcinomas, squamous cell carcinomas and cervical intraepithelial lesions of the uterine cervix.

Cervical cancer is the third most common cancer in women worldwide. The hypermethylation of P16, TSLC-1 and TSP-1 genes was analyzed in squamous cell carcinomas (SCC), cervical intraepithelial lesions (CIN) and adenocarcinomas (ADC) of the uterine cervix (total 181 lesions). Additionally human papillomavirus (HPV) type, EPB41L3, RASSF1 and RASSF2 hypermethylation were tested in ADC and the results were compared with those obtained previously by our group in SCC. P16, TSLC-1 and TSP-1 hypermethylation was more frequent in SCCs than in CINs. These percentages and the corresponding ones for EPB41L3, RASSF1 and RASSF2 genes were also higher in SCCs than in ADCs, except for P16. The presence of HPV in ADCs was lower than reported previously in SCC and CIN. Patients with RASSF1A hypermethylation showed significantly longer disease-free survival (P = 0.015) and overall survival periods (P = 0.009) in ADC patients. To our knowledge, this is the first description of the EPB41L3 and RASSF2 hypermethylation in ADCs. These results suggest that the involvement of DNA hypermethylation in cervical cancer varies depending on the histological type, which might contribute to explaining the different prognosis of patients with these types of tumors.

Primary Cutaneous Acral CD8-Positive Lymphoproliferative Disorder: A Case Report With Nonacral Presentation.

In this study, we describe a patient of primary cutaneous acral CD8-positive lymphoproliferative disorder located in a nonacral region. A 65-year-old male presented with an ill-defined lesion of rubbery consistency and a maximum diameter of 2.5 cm localized in the right thigh. Histologically, it was composed of a diffuse dermal infiltration of medium-sized atypical lymphocytes that expressed CD3, CD8, and TIA-1. In addition, a characteristic paranuclear positivity with CD68 was observed. During the follow-up, the patient had a recurrence of the disease in the abdomen with a lesion showing similar morphology and phenotype. To our knowledge, < 20 patients of primary cutaneous acral CD8-positive lymphoproliferative disorder with a nonacral presentation have been described in English literature. Although rare, its identification is essential to differentiate it from other T-cell lymphoma that express CD8 and cytotoxic markers, and whose clinical courses are very aggressive.

Biphasic papillary (biphasic squamoid alveolar) renal cell carcinoma: a clinicopathologic and molecular study of 17 renal cell carcinomas including 10 papillary adenomas.

Biphasic papillary renal cell carcinoma (synonymous with biphasic squamoid alveolar renal cell carcinoma) is considered within the spectrum of papillary renal cell carcinoma (PRCC). With < 70 reported cases of biphasic PRCC, there is limited data on the pathologic spectrum and clinical course. Seventeen biphasic PRCC cases and 10 papillary adenomas with similar biphasic morphology were assessed. The mean age of the biphasic PRCC patients was 62 years (male to female ratio of 1.8:1), from 10 partial nephrectomies, 6 radical nephrectomies, and 1 biopsy. The mean tumor size was 3.6 cm (range 1.6-8 cm), with 24% showing multifocality. Fifteen out of 17 cases were limited to the kidney (one of which was staged as pT2a but had lung metastases at diagnosis) and 2/17 cases were staged as T3a. All tumors showed typical biphasic morphology with an extent of squamoid foci widely variable from 10 to 95%. Emperipolesis was identified in 88% of cases. All biphasic PRCC tested exhibited positivity for PAX8 (16/16), keratin 7 (17/17), EMA (15/15), AMACR (17/17), and vimentin (12/12) in both large and small cells; cyclin D1 was only expressed in the large cells (16/16). The 10 papillary adenomas showed a similar immunoprofile to biphasic PRCC. NGS testing performed on 13 biphasic PRCC revealed 4 (31%) harboring MET SNVs. In 1/5 (20%) papillary adenomas, a pathogenic MET SNV was identified. Biphasic PRCC is rare with a generally similar immunoprofile to "type 1" PRCC but with notable strong positivity for cyclin D1 in the large cell component. Although most of the biphasic PRCC cases were of small size, low stage, and with an indolent behavior, one patient had metastatic disease and one patient died of the disease.

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.

RASSF2 hypermethylation is present and related to shorter survival in squamous cervical cancer.

Ras association (RalGDS/AF-6) domain family member 2 (RASSF2) is a gene involved in the progression of several human cancers, including breast, colorectal and lung cancer. The aims of this study were to determine the hypermethylation of the gene in squamous cervical cancer and precursor lesions, along with that of RASSF1 and the recently described EPB41L3, and to analyze the potential prognostic role of these genes. Methylation-specific PCR and bisulfite sequencing were used to analyze the methylation status of RASSF2 and EPB41L3 gene in 60 squamous cervical cancer, 76 cervical intraepithelial neoplasias grade III, 16 grade II, 14 grade I and 13 cases of normal tissue adjacent to cervical intraepithelial neoplasia. RASSF2 expression was evaluated by immunohistochemistry and the re-expression of RASSF2 and EPB41L3 was analyzed by quantitative reverse-transcription PCR in HeLa, SiHa, C33A and A431 cell lines treated with 5-aza-2'-deoxycytidine and/or trichostatin. RASSF1 hypermethylation and human papillomavirus type were also analyzed in all the cases by methylation-specific PCR and reverse line blot, respectively. RASSF2 hypermethylation was predominant in squamous cervical cancer (60.9%) compared with cervical intraepithelial neoplasias (4.2%) and was associated with a lower level of RASSF2 expression and vascular invasion in squamous cervical cancer. EPB41L3 and RASSF1 hypermethylations were also more frequent in cancer than in precursor lesions. Patients with RASSF2 hypermethylation had shorter survival time, independent of tumor stage (hazard ratio: 6.0; 95% confidence interval: 1.5-24.5). Finally, the expressions of RASSF2 and EPB41L3 were restored in several cell lines treated with 5-aza-2'-deoxycytidine. Taken together, our results suggest that RASSF2 potentially functions as a new tumor-suppressor gene that is inactivated through hypermethylation in cervical cancer and is related to the bad prognosis of these patients.

Differential gene hypermethylation in genital lichen sclerosus and cancer: a comparative study.

AIMS: Lichen sclerosus (LS) is a chronic inflammatory disease of the genital skin of unknown aetiology. The role of LS in penile squamous cell carcinogenesis is not well characterized. HPV has been implicated in both, as have epigenetic changes. The presence of HPV and hypermethylation of the MGMT, p16, RASSF1, RASSF2, TSLC1 and TSP1 genes were studied in penile LS; MGMT, RASSF2 and TSLC1 hypermethylation in penile cancer and TSLC1 hypermethylation in vulvar LS and cancer extends previous results reported by our group. METHODS AND RESULTS: Thirty-seven HPV genotypes and hypermethylation were evaluated by PCR/reverse-line-blot and methylation-specific PCR respectively, in 27 preputial LS, 24 penile SCC, 30 vulvar SCC, 21 vulvar LS and 22 normal skin cases. HPV66 was present in 3.7% of penile LS cases, and p16 and RASSF2 hypermethylation were more frequent in penile cancer than in penile LS. p16, RASSF1, RASSF2 and TSP1 hypermethylation were similar in penile and vulvar LS. CONCLUSIONS: Gene hypermethylation is a common event in penile LS, and occurs approximately as frequently as in vulvar LS. Certain genes can be hypermethylated as an early or late event in LS or cancer, respectively. This suggests a possible sequential role for these alterations in the transition from benign to malignant lesions.

ZEB1 hypermethylation is associated with better prognosis in patients with colon cancer.

BACKGROUND: Colon cancer (CC) is a heterogeneous disease that is categorized into four Consensus Molecular Subtypes (CMS) according to gene expression. Patients with loco-regional CC (stages II/III) lack prognostic factors, making it essential to analyze new molecular markers that can delineate more aggressive tumors. Aberrant methylation of genes that are essential in crucial mechanisms such as epithelial mesenchymal transition (EMT) contributes to tumor progression in CC. We evaluate the presence of hyper- and hypomethylation in subrogate IHC markers used for CMS classification (CDX2, FRMD6, HTR2B, ZEB1) of 144 stage II/III patients and CC cell lines by pyrosequencing. ZEB1 expression was also studied in control and shRNA-silenced CC cell lines and in paired normal tissue/tumors by quantitative PCR. The pattern of ZEB1 staining was also analyzed in methylated/unmethylated tumors by immunohistochemistry. RESULTS: We describe for the first time the hypermethylation of ZEB1 gene and the hypomethylation of the FRMD6 gene in 32.6% and 50.9% of tumors, respectively. Additionally, we confirm the ZEB1 re-expression by epigenetic drugs in methylated cell lines. ZEB1 hypermethylation was more frequent in CMS1 patients and, more importantly, was a good prognostic factor related to disease-free survival (p = 0.015) and overall survival (p = 0.006) in our patient series, independently of other significant clinical parameters such as patient age, stage, lymph node involvement, and blood vessel and perineural invasion. CONCLUSIONS: Aberrant methylation is present in the subrogate genes used for CMS classification. Our results are the first evidence that ZEB1 is hypermethylated in CC and that this alteration is an independent factor of good prognosis.

Pleomorphic carcinoma of the breast with expression of macrophage markers: report of two cases.

Pleomorphic ductal carcinoma of the breast is a rare variant included in the morphological group of infiltrating ductal carcinoma. The pleomorphic carcinoma is composed predominantly of epithelial and multinucleated tumor giant cells. We report here two cases presenting a lesion composed microscopically of a proliferation of large pleomorphic cells with a predominance of multinucleated giant cells. These lesions were negative for estrogen receptor, progesterone receptor and Her2-neu (triple-negative phenotype). Basal markers (cytokeratin 5/6, cytokeratin 17 and epidermal growth factor receptor [EGFR]) were present, accompanied by the presence of histiocyte marker CD163 in most neoplastic giant cells. High-grade pleomorphic breast carcinomas with the triple-negative phenotype and expression of basal markers might be included in the basal subtype. This is the first report about the co-expression of macrophage marker CD163, with tumor (P53) or epithelial markers (CAM5.2), as indicated by double immunohistochemistry in pleomorphic ductal carcinoma of the breast.

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.

PD-L1 as a Prognostic Factor in Early-Stage Colon Carcinoma within the Immunohistochemical Molecular Subtype Classification.

BACKGROUND: There is a patent need to better characterize early-stage colorectal cancer (CRC) patients. PD-1 ligand (PD-L1) expression has been proposed as a prognostic factor but yields mixed results in different settings. The Consensus Molecular Subtype (CMS) classification has yet to be integrated into clinical practice. We sought to evaluate the prognostic value of PD-L1 expression overall and within CMS in early-stage colon cancer patients, in the hope of assisting treatment choice in this setting. METHODS: Tissue-microarrays were constructed from tumor samples of 162 stage II/III CRC patients. They underwent automatic immunohistochemical staining for PD-L1 and the proposed CMS panel. Primary endpoints were overall survival (OS) and disease-free survival (DFS). RESULTS: PD-L1 expression was significantly and independently associated with better prognosis (HR = 0.46 (0.26-0.82), p = 0.009) and was mostly seen in immune cells of the tumor-related stroma. CMS4 five-folds the risk of mortalitycompared with CMS1 (HR = 5.58 (1.36, 22.0), p = 0.034). In the subgroup CMS2/CMS3 analysis, PD-L1 expression significantly differentiated individuals with better OS (p = 0.004) and DFS (p < 0.001). CONCLUSIONS: Our study suggests that PD-L1 expression is an independent prognostic factor in patients with stage II/III colon cancer. Additionally, it successfully differentiates patients with better prognosis in the CMS2/CMS3 group and may prove significant for the clinical relevance of the CMS classification.

A Novel Prognostic Biomarker Panel for Early-Stage Colon Carcinoma.

Molecular characterization of colorectal cancer has helped us understand better the biology of the disease. However, previous efforts have yet to provide significant clinical value in order to be integrated into clinical practice for patients with early-stage colon cancer (CC). The purpose of this study was to assess PD-L1, GLUT-1, e-cadherin, MUC2, CDX2, and microsatellite instability (dMMR) and to propose a risk-panel with prognostic capabilities. Biomarkers were immunohistochemically assessed through tissue microarrays in a cohort of 144 patients with stage II/III colon cancer. A biomarker panel consisting of PD-L1, GLUT-1, dMMR, and potentially CDX2 was constructed that divided patients into low, medium, and high risk of overall survival or disease-free survival (DFS) in equally sized groups. Compared with low-risk patients, medium-risk patients have almost twice the risk of death (HR = 2.10 (0.99-4.46), p = 0.054), while high-risk patients have almost four times the risk (HR = 3.79 (1.77-8.11), p = 0.001). The multivariate goodness of fit was 0.756 and was correlated with Kaplan-Meier curves (p = 0.002). Consistent results were found for DFS. This study provides a critical basis for the future development of an immunohistochemical assessment capable of discerning early-stage CC patients as a function of their prognosis. This tool may aid with treatment personalization in daily clinical practice and improve survival outcomes.

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.

A core of kinase-regulated interactomes defines the neoplastic MDSC lineage.

Myeloid-derived suppressor cells (MDSCs) differentiate from bone marrow precursors, expand in cancer-bearing hosts and accelerate tumor progression. MDSCs have become attractive therapeutic targets, as their elimination strongly enhances anti-neoplastic treatments. Here, immature myeloid dendritic cells (DCs), MDSCs modeling tumor-infiltrating subsets or modeling non-cancerous (NC)-MDSCs were compared by in-depth quantitative proteomics. We found that neoplastic MDSCs differentially expressed a core of kinases which controlled lineage-specific (PI3K-AKT and SRC kinases) and cancer-induced (ERK and PKC kinases) protein interaction networks (interactomes). These kinases contributed to some extent to myeloid differentiation. However, only AKT and ERK specifically drove MDSC differentiation from myeloid precursors. Interfering with AKT and ERK with selective small molecule inhibitors or shRNAs selectively hampered MDSC differentiation and viability. Thus, we provide compelling evidence that MDSCs constitute a distinct myeloid lineage distinguished by a "kinase signature" and well-defined interactomes. Our results define new opportunities for the development of anti-cancer treatments targeting these tumor-promoting immune cells.