Peroxidasin Enhances Basal Phenotype and Inhibits Branching Morphogenesis in Breast Epithelial Progenitor Cell Line D492

The human breast is composed of terminal duct lobular units (TDLUs) that are surrounded by stroma. In the TDLUs, basement membrane separates the stroma from the epithelial compartment, which is divided into an inner layer of luminal epithelial cells and an outer layer of myoepithelial cells. Stem cells and progenitor cells also reside within the epithelium and drive a continuous cycle of gland remodelling that occurs throughout the reproductive period. D492 is an epithelial cell line originally isolated from the stem cell population of the breast and generates both luminal and myoepithelial cells in culture. When D492 cells are embedded into 3D reconstituted basement membrane matrix (3D-rBM) they form branching colonies mimicking the TDLUs of the breast, thereby providing a well-suited in vitro model for studies on branching morphogenesis and breast development. Peroxidasin (PXDN) is a heme-containing peroxidase that crosslinks collagen IV with the formation of sulfilimine bonds. Previous studies indicate that PXDN plays an integral role in basement membrane stabilisation by crosslinking collagen IV and as such contributes to epithelial integrity. Although PXDN has been linked to fibrosis and cancer in some organs there is limited information on its role in development, including in the breast. In this study, we demonstrate expression of PXDN in breast epithelium and stroma and apply the D492 cell line to investigate the role of PXDN in cell differentiation and branching morphogenesis in the human breast. Overexpression of PXDN induced basal phenotype in D492 cells, loss of plasticity and inhibition of epithelial-to-mesenchymal transition as is displayed by complete inhibition of branching morphogenesis in 3D culture. This is supported by results from RNA-sequencing which show significant enrichment in genes involved in epithelial differentiation along with significant negative enrichment of EMT factors. Taken together, we provide evidence for a novel role of PXDN in breast epithelial differentiation and mammary gland development.

A GATA6-centred gene regulatory network involving HNFs and ΔNp63 controls plasticity and immune escape in pancreatic cancer

ObjectiveMolecular taxonomy of tumours is the foundation of personalised medicine and is becoming of paramount importance for therapeutic purposes. Four transcriptomics-based classification systems of pancreatic ductal adenocarcinoma (PDAC) exist, which consistently identified a subtype of highly aggressive PDACs with basal-like features, including ΔNp63 expression and loss of the epithelial master regulator GATA6. We investigated the precise molecular events driving PDAC progression and the emergence of the basal programme.DesignWe combined the analysis of patient-derived transcriptomics datasets and tissue samples with mechanistic experiments using a novel dual-recombinase mouse model for Gata6 deletion at late stages of KRasG12D-driven pancreatic tumorigenesis (Gata6LateKO).ResultsThis comprehensive human-to-mouse approach showed that GATA6 loss is necessary, but not sufficient, for the expression of ΔNp63 and the basal programme in patients and in mice. The concomitant loss of HNF1A and HNF4A, likely through epigenetic silencing, is required for the full phenotype switch. Moreover, Gata6 deletion in mice dramatically increased the metastatic rate, with a propensity for lung metastases. Through RNA-Seq analysis of primary cells isolated from mouse tumours, we show that Gata6 inhibits tumour cell plasticity and immune evasion, consistent with patient-derived data, suggesting that GATA6 works as a barrier for acquiring the fully developed basal and metastatic phenotype.ConclusionsOur work provides both a mechanistic molecular link between the basal phenotype and metastasis and a valuable preclinical tool to investigate the most aggressive subtype of PDAC. These data, therefore, are important for understanding the pathobiological features underlying the heterogeneity of pancreatic cancer in both mice and human.

Retrospective analysis of an alternative immuno-score in clinical management of patients with pT2 urothelial carcinoma.

e17038 Background: Within the mapping of the genome through the TCGA collaborative project, the urothelial carcinoma (UC) has recently revealed major intrinsic molecular subtypes, Basal, Luminal and Neural muscle-invasive UC. Here we propose a fast and standardized immuno-phenotypical score classification (Piescore), as a surrogate, which may discriminate Luminal, Basal or Neural UC and may correlate with histological and clinical variables in a mono-institutional cohort of patients treated with trans-urethral resection (TURB) and radical cystectomy (RC). Methods: This is a retrospective study of TURB specimens that harbored foci of HG pT2 (MIBC) UC from 116 pts who underwent RC. All the samples were assessed for immunohistochemical pattern, using relevant gene-expression-based markers for Basal type (CD44, CK5/6) and Luminal type (CK20 and pPARg). Piescore, investigated in both superficial and muscle-invasive component of the tumors, divided Basal and Luminal UC-types when at least 3 of the 4 markers were consistent with a specific phenotype, Mixed if two luminal and two basal markers were present simultaneously, and Neural when all four markers were negative. Results: Overall in muscle-invasive component, Piescore identified Basal phenotypes in 49 pts (42,2%), Luminal in 38 pts (32,7%), and Mixed in 9 pts (7,8%). No expression was identified in 20 pts (17,2%): 7 cases with morphological neuroendocrine differentiation and 13 cases with classical urothelial histology, all consistent with Neural phenotype. In 26,7% of cases (31/116 pts) we observed an immuno-phenotypical switch from superficial to deep component: 16 of 31 (51,6%) switched to Basal, 10 (32,2%) switched to Neural, and 5 cases (16,2%) to Mixed phenotype. No cases switched to Luminal. No cases have lost Basal phenotype from superficial to deep component, with the exception of one (switched to Neural). No statistically significant differences in terms of staging, DFS, and OS were observed in the different phenotypes. The presence of phenotypical switch did not affect angioinvasion, staging, DFS, and OS compared to non-switched cases. Conclusions: Piescore immunophenotyping (CD44, CK5/6, CK20 and pPARg) could be a simple surrogate able to stratify UC-TURB patients between Luminal vs Basal type. To our knowledge, preliminary results using the Piescore identify for the first time a phenotypical switch (to basal, Mixed or Neural) between superficial and deeper side of the same tumor, although this phenomenon did not show any prognostic implication.

A GATA6-centered gene regulatory network involving HNFs and ΔNp63 controls plasticity and immune escape in pancreatic cancer

ObjectiveMolecular taxonomy of tumors is the foundation of personalized medicine and is becoming of paramount importance for therapeutic purposes. Four transcriptomics-based classification systems of pancreatic ductal adenocarcinoma (PDAC) exist, which consistently identified a subtype of highly aggressive PDACs with basal-like features, including ΔNp63 expression and loss of the epithelial master regulator GATA6. We investigated the precise molecular events driving PDAC progression and the emergence of the basal program.DesignWe combined the analysis of patient-derived transcriptomics datasets and tissue samples with mechanistic experiments using a novel dual-recombinase mouse model for Gata6 deletion at late stages of KRasG12D-driven pancreatic tumorigenesis (Gata6LateKO).ResultsThis comprehensive human-to-mouse approach allowed us to show that GATA6 loss is necessary, but not sufficient, for the expression of a basal program in patients and in mice. The concomitant loss of HNF1A and HNF4A, likely through epigenetic silencing, is required for the full phenotype switch. Moreover, Gata6 deletion in mice dramatically increased the metastatic rate, with a propensity for lung metastases. Through RNA-Seq analysis of primary cells isolated from mouse tumors, we show that Gata6 inhibits tumor cell plasticity and immune evasion, suggesting that it works as a barrier for acquiring the fully developed basal and metastatic phenotype.ConclusionsOur work provides both a mechanistic molecular link between the basal phenotype and metastasis and a valuable preclinical tool to investigate the most aggressive subtype of PDAC. These data, therefore, are important for understanding the pathobiological features underlying the heterogeneity of pancreatic cancer in both mice and human.What is already known about this subject?Multiple transcriptomics-based studies have identified a basal-like subtype of pancreatic ductal adenocarcinoma (PDAC) with especially poor prognosis.Loss of GATA6 in PDAC cells is associated with altered differentiation, including ectopic expression of basal markers such as KRT14.Aberrant expression of the ΔNp63 transcription factor can drive the expression of the basal transcriptional program.What are the new findings?Loss of GATA6 expression is necessary but not sufficient for the expression of ΔNp63 and the basal phenotype.Concomitant silencing of HNF4A and HNF1A, possibly through epigenetic mechanisms, is required for the full-blown phenotype.Gata6 deletion in established murine tumors favors the basal and metastatic phenotype, with a lung tropism, in a next-generation model of KRasG12D-driven PDAC.Loss of GATA6 expression is associated with features of immune escape in mouse and human PDAC cells.How might it impact on clinical practice in the foreseeable future?The combined analysis of GATA6, HNFs, and TP63 expression in patient-derived samples will provide a more precise classification of PDAC.Restoration of the classical PDAC phenotype may not only reduce metastatic potential but also increase immune recognition of tumor cells.

Phase III Trial of Adjuvant Capecitabine After Standard Neo-/Adjuvant Chemotherapy in Patients With Early Triple-Negative Breast Cancer (GEICAM/2003-11_CIBOMA/2004-01)

PURPOSE Operable triple-negative breast cancers (TNBCs) have a higher risk of relapse than non-TNBCs with standard therapy. The GEICAM/2003-11_CIBOMA/2004-01 trial explored extended adjuvant capecitabine after completion of standard chemotherapy in patients with early TNBC. PATIENTS AND METHODS Eligible patients were those with operable, node-positive—or node negative with tumor 1 cm or greater—TNBC, with prior anthracycline- and/or taxane-containing chemotherapy. After central confirmation of TNBC status by immunohistochemistry, patients were randomly assigned to either capecitabine or observation. Stratification factors included institution, prior taxane-based therapy, involved axillary lymph nodes, and centrally determined phenotype (basal v nonbasal, according to cytokeratins 5/6 and/or epidermal growth factor receptor positivity by immunohistochemistry). The primary objective was to compare disease-free survival (DFS) between both arms. RESULTS Eight hundred seventy-six patients were randomly assigned to capecitabine (n = 448) or observation (n = 428). Median age was 49 years, 55.9% were lymph node negative, 73.9% had a basal phenotype, and 67.5% received previous anthracyclines plus taxanes. Median length of follow-up was 7.3 years. DFS was not significantly prolonged with capecitabine versus observation [hazard ratio (HR), 0.82; 95% CI, 0.63 to 1.06; P = .136]. In a preplanned subgroup analysis, nonbasal patients seemed to derive benefit from the addition of capecitabine with a DFS HR of 0.53 versus 0.94 in those with basal phenotype (interaction test P = .0694) and an HR for overall survival of 0.42 versus 1.23 in basal phenotype (interaction test P = .0052). Tolerance of capecitabine was as expected, with 75.2% of patients completing the planned 8 cycles. CONCLUSION This study failed to show a statistically significant increase in DFS by adding extended capecitabine to standard chemotherapy in patients with early TNBC. In a preplanned subset analysis, patients with nonbasal phenotype seemed to obtain benefit with capecitabine, although this will require additional validation.

Imaging Biomarkers as Predictors for Breast Cancer Death

Background. To differentiate the risk of breast cancer death in a longitudinal cohort using imaging biomarkers of tumor extent and biology, specifically, the mammographic appearance, basal phenotype, histologic tumor distribution, and conventional tumor attributes.Methods. Using a prospective cohort study design, 498 invasive breast cancer patients diagnosed between 1996 and 1998 were used as the test cohort to assess the independent effects of the imaging biomarkers and other predictors on the risk of breast cancer death. External validation was performed with a cohort of 848 patients diagnosed between 2006 and 2010.Results. Mammographic tumor appearance was an independent predictor of risk of breast cancer death (P=0.0003) when conventional tumor attributes and treatment modalities were controlled. The casting type calcifications and architectural distortion were associated with 3.13-fold and 3.19-fold risks of breast cancer death, respectively. The basal phenotype independently conferred a 2.68-fold risk compared with nonbasal phenotype. The observed deaths did not differ significantly from expected deaths in the validation cohort. The application of imaging biomarkers together with other predictors classified twelve categories of risk for breast cancer death.Conclusion. Combining imaging biomarkers such as the mammographic appearance of the tumor with the histopathologic distribution and basal phenotype, accurately predicted long-term risk of breast cancer death. The information may be relevant for determining the need for molecular testing, planning treatment, and determining the most appropriate clinical surveillance schedule for breast cancer patients.

The Genetic Basis of Transcriptional and Spatial Heterogeneity of Squamous Features in Pancreatic Ductal Adenocarcinoma

SummaryRecent studies indicate that pancreatic cancer expression profiles are variable and largely reflect a classical or basal-type phenotype. We performed genetic sequencing, RNA-seq, and histologic review of multiregion sampled pancreatic cancers and found that squamous and squamoid features, indicators of poor prognosis, correlate with a “basal-like” expressional type. Cancers with squamous features were more likely to have truncal mutations in chromatin modifier genes and intercellular heterogeneity for MYC amplification that was associated with entosis. In most patients the basal phenotype coexisted with a glandular component, and phylogenetic studies indicated that it arose from a subclonal population in the tumor. These data provide a unifying paradigm for understanding the interrelationship of basal-type features, squamous histology, and somatic mutations in chromatin modifier genes in the context of the clonal evolution of pancreatic cancer.