Multiomic analysis of HER2-enriched and AR-positive breast carcinoma with apocrine differentiation and an oligometastatic course: a case report.

Breast carcinoma is the most prevalent cancer among women globally. It has variable clinical courses depending on the stage and clinical-biological features. This case report describes a 56-year-old female with invasive breast cancer without estrogen or progesterone receptor expression, with apocrine differentiation, and with no germline variants in the BRCA1 and BRCA2 genes. Throughout the clinical course, the patient exhibited discordant results for HER2 in immunohistochemistry and in situ hybridization. During the second relapse, the disease displayed apocrine microscopic features. The tumor underwent analysis for the androgen receptor, GCDFP-15, RNA-seq, and whole-genome sequencing (WGS) to identify the breast cancer subtype and to characterize the cancer genome. Our bioinformatic analysis revealed 20,323 somatic SNV/Indels, including five mutations in cancer-related genes that are believed to be responsible for the tumor's development. Two of these mutations were found in the PIK3CA and TP53 genes. Furthermore, the tumor tissue exhibited large copy number alterations to the chromosomes, which could impact gene expression through complex mechanisms and contribute to the tumor phenotype. Clustering algorithms applied on RNA-sequencing data categorized this cancer as a HER2+ subtype. The second-line capecitabine chemotherapy treatment is ongoing, and the patient is responding well. Bioinformatic results support the current treatment decision and open the way to further treatments.

Decellularized extracellular matrix as scaffold for cancer organoid cultures of colorectal peritoneal metastases.

Peritoneal metastases (PM) from colorectal cancer (CRC) are associated with poor survival. The extracellular matrix (ECM) plays a fundamental role in modulating the homing of CRC metastases to the peritoneum. The mechanisms underlying the interactions between metastatic cells and the ECM, however, remain poorly understood, and the number of in vitro models available for the study of the peritoneal metastatic process is limited. Here, we show that decellularized ECM of the peritoneal cavity allows the growth of organoids obtained from PM, favoring the development of three-dimensional (3D) nodules that maintain the characteristics of in vivo PM. Organoids preferentially grow on scaffolds obtained from neoplastic peritoneum, which are characterized by greater stiffness than normal scaffolds. A gene expression analysis of organoids grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. An impact of the ECM on the response to standard chemotherapy treatment for PM was also observed. The ex vivo 3D model, obtained by combining patient-derived decellularized ECM with organoids to mimic the metastatic niche, could be an innovative tool to develop new therapeutic strategies in a biologically relevant context to personalize treatments.

miRNome profiling of lung cancer metastases revealed a key role for miRNA-PD-L1 axis in the modulation of chemotherapy response.

Locally advanced non-small cell lung cancer (NSCLC) is frequent at diagnosis and requires multimodal treatment approaches. Neoadjuvant chemotherapy (NACT) followed by surgery is the treatment of choice for operable locally advanced NSCLC (Stage IIIA). However, the majority of patients are NACT-resistant and show persistent lymph nodal metastases (LNmets) and an adverse outcome. Therefore, the identification of mechanisms and biomarkers of NACT resistance is paramount for ameliorating the prognosis of patients with Stage IIIA NSCLC. Here, we investigated the miRNome and transcriptome of chemo-naïve LNmets collected from patients with Stage IIIA NSCLC (N = 64). We found that a microRNA signature accurately predicts NACT response. Mechanistically, we discovered a miR-455-5p/PD-L1 regulatory axis which drives chemotherapy resistance, hallmarks metastases with active IFN-γ response pathway (an inducer of PD-L1 expression), and impacts T cells viability and relative abundances in tumor microenvironment (TME). Our data provide new biomarkers to predict NACT response and add molecular insights relevant for improving the management of patients with locally advanced NSCLC.

Towards population-based genetic screenings for breast and ovarian cancer: A comprehensive review from economic evaluations to patient perspectives.

Genetic testing for hereditary breast and ovarian cancer following genetic counseling is based on guidelines that take into account particular features of the personal and family history, and clinical criteria conferring a probability of having a BRCA mutation greater than 10% as a threshold for accessing the test. However, besides reducing mortality and social impact, the extension of screening programs also for healthy family members would allow a huge saving of the rising costs associated with these pathologies, supporting the choice of the "Test" strategy versus a "No Test" one. Analyses of different health care systems show that by applying the "Test" strategy on patients and their families, a decrease in breast and ovarian cancer cases is achieved, as well as a substantial decrease in costs of economic resources, including the costs of the clinical management of early detected tumors. In this review, we analyzed the most recent papers published on this topic and we summarized the findings on the economic evaluations related to breast and ovarian cancer population screenings. These results proved and validated that the population-wide testing approach is a more accurate screening and preventive intervention than traditional guidelines based on personal/family history and clinical criteria to reduce breast and ovarian cancer risk.

A self-sustaining endocytic-based loop promotes breast cancer plasticity leading to aggressiveness and pro-metastatic behavior.

The subversion of endocytic routes leads to malignant transformation and has been implicated in human cancers. However, there is scarce evidence for genetic alterations of endocytic proteins as causative in high incidence human cancers. Here, we report that Epsin 3 (EPN3) is an oncogene with prognostic and therapeutic relevance in breast cancer. Mechanistically, EPN3 drives breast tumorigenesis by increasing E-cadherin endocytosis, followed by the activation of a ß-catenin/TCF4-dependent partial epithelial-to-mesenchymal transition (EMT), followed by the establishment of a TGFß-dependent autocrine loop that sustains EMT. EPN3-induced partial EMT is instrumental for the transition from in situ to invasive breast carcinoma, and, accordingly, high EPN3 levels are detected at the invasive front of human breast cancers and independently predict metastatic rather than loco-regional recurrence. Thus, we uncover an endocytic-based mechanism able to generate TGFß-dependent regulatory loops conferring cellular plasticity and invasive behavior.

Low-Dose Aspirin in High-Risk Individuals With Screen-Detected Subsolid Lung Nodules: A Randomized Phase II Trial.

Lung cancer screening by helical low-dose computed tomography detects nonsolid nodules that may be lung adenocarcinoma precursors. Aspirin's anti-inflammatory properties make it an attractive target for prevention of multiple cancers, including lung cancer. Therefore, we conducted a phase IIb trial (NCT02169271) to study the efficacy of low-dose aspirin to reduce the size of subsolid lung nodules (SSNs). A total of 98 current or former smokers (67.3% current) undergoing annual low-dose computed tomography screening with persistent SSNs were randomly assigned to receive aspirin 100 mg/day or placebo for 1 year. There was no difference in change in the sum of the longest diameters of target nodules in the placebo and aspirin arm after 12 months of treatment (-0.12 mm [SD = 1.55 mm] and +0.30 mm [SD= 2.54 mm], respectively; 2-sided P = .33 primary endpoint). There were no changes observed in subgroup analyses by individual characteristics or nodule type. One year of low-dose aspirin did not show any effect on lung SSNs. SSNs regression may not be the proper target for aspirin, and/or longer duration may be needed to see SSNs modifications.

Identification and clinical validation of a multigene assay that interrogates the biology of cancer stem cells and predicts metastasis in breast cancer: A retrospective consecutive study.

BACKGROUND: Breast cancers show variations in the number and biological aggressiveness of cancer stem cells that correlate with their clinico-prognostic and molecular heterogeneity. Thus, prognostic stratification of breast cancers based on cancer stem cells might help guide patient management. METHODS: We derived a 20-gene stem cell signature from the transcriptional profile of normal mammary stem cells, capable of identifying breast cancers with a homogeneous profile and poor prognosis in in silico analyses. The clinical value of this signature was assessed in a prospective-retrospective cohort of 2, 453 breast cancer patients. Models for predicting individual risk of metastasis were developed from expression data of the 20 genes in patients randomly assigned to a training set, using the ridge-penalized Cox regression, and tested in an independent validation set. FINDINGS: Analyses revealed that the 20-gene stem cell signature provided prognostic information in Triple-Negative and Luminal breast cancer patients, independently of standard clinicopathological parameters. Through functional studies in individual tumours, we correlated the risk score assigned by the signature with the proliferative and self-renewal potential of the cancer stem cell population. By retraining the 20-gene signature in Luminal patients, we derived the risk model, StemPrintER, which predicted early and late recurrence independently of standard prognostic factors. INTERPRETATION: Our findings indicate that the 20-gene stem cell signature, by its unique ability to interrogate the biology of cancer stem cells of the primary tumour, provides a reliable estimate of metastatic risk in Triple-Negative and Luminal breast cancer patients independently of standard clinicopathological parameters.

HOXB7 overexpression in lung cancer is a hallmark of acquired stem-like phenotype.

HOXB7 is a homeodomain (HOX) transcription factor involved in regional body patterning of invertebrates and vertebrates. We previously identified HOXB7 within a ten-gene prognostic signature for lung adenocarcinoma, where increased expression of HOXB7 was associated with poor prognosis. This raises the question of how HOXB7 overexpression can influence the metastatic behavior of lung adenocarcinoma. Here, we analyzed publicly available microarray and RNA-seq lung cancer expression datasets and found that HOXB7-overexpressing tumors are enriched in gene signatures characterizing adult and embryonic stem cells (SC), and induced pluripotent stem cells (iPSC). Experimentally, we found that HOXB7 upregulates several canonical SC/iPSC markers and sustains the expansion of a subpopulation of cells with SC characteristics, through modulation of LIN28B, an emerging cancer gene and pluripotency factor, which we discovered to be a direct target of HOXB7. We validated this new circuit by showing that HOXB7 enhances reprogramming to iPSC with comparable efficiency to LIN28B or its target c-MYC, which is a canonical reprogramming factor.

MicroRNA expression profile in primary lung cancer cells lines obtained by endobronchial ultrasound transbronchial needle aspiration.

BACKGROUND: Novel cancer biomarkers like microRNA (miRNA) are promising tools to gain a better understanding of lung cancer pathology and yield important information to guide therapy. In recent years, new less invasive methods for the diagnosis and staging of NSCLC have become key tools in thoracic oncology and the worldwide spread of endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA). However, appropriate specimen handling is mandatory to achieve adequate results and reproducibility. The aim of this single centre prospective study was to evaluate the feasibility of a complete miRNA expression profile in fresh NSCLC cell lines obtained by EBUS-TBNA. METHODS: Patients with proven NSCLC underwent EBUS-TBNA for the diagnosis of suspect lymph node metastasis, and cytological specimens were collected for epithelial cell culture and miRNA expression analysis. To validate the miRNA expression profile, we compared the results from EBUS-TBNA NSCLC specimens with those obtained from formalin-fixed paraffin-embedded (FFPE) mediastinoscopy specimens. RESULTS: Analysis of the miRNA expression profiles of three independent EBUS-TBNA-derived primary cell lines allowed the screening of 377 different human miRNAs. One hundred and fifty miRNAs were detected in all cell lines. Analysis of the miRNA expression profile in mediastinoscopy specimens showed a strong similarity in the clusters analysed. CONCLUSIONS: The miRNA expression profile is feasible and reliable in EBUS-TBNA specimens. Validation of this protocol in fresh cytological specimens represents an effective and reproducible method to correlate translational and clinical research.

A Numb-Mdm2 fuzzy complex reveals an isoform-specific involvement of Numb in breast cancer.

Numb functions as an oncosuppressor by inhibiting Notch signaling and stabilizing p53. This latter effect depends on the interaction of Numb with Mdm2, the E3 ligase that ubiquitinates p53 and commits it to degradation. In breast cancer (BC), loss of Numb results in a reduction of p53-mediated responses including sensitivity to genotoxic drugs and maintenance of homeostasis in the stem cell compartment. In this study, we show that the Numb-Mdm2 interaction represents a fuzzy complex mediated by a short Numb sequence encompassing its alternatively spliced exon 3 (Ex3), which is necessary and sufficient to inhibit Mdm2 and prevent p53 degradation. Alterations in the Numb splicing pattern are critical in BC as shown by increased chemoresistance of tumors displaying reduced levels of Ex3-containing isoforms, an effect that could be mechanistically linked to diminished p53 levels. A reduced level of Ex3-less Numb isoforms independently predicts poor outcome in BCs harboring wild-type p53. Thus, we have uncovered an important mechanism of chemoresistance and progression in p53-competent BCs.

Indications for a High-Rigidity Break in the Cosmic-Ray Diffusion Coefficient.

Using cosmic-ray boron to carbon ratio (B/C) data recently released by the Ams-02 experiment, we find indications (decisive evidence, in Bayesian terms) in favor of a diffusive propagation origin for the broken power-law spectra found in protons (p) and helium nuclei (He). The result is robust with respect to currently estimated uncertainties in the cross sections, and in the presence of a small component of primary boron, expected because of spallation at the acceleration site. Reduced errors at high energy as well as further cosmic ray nuclei data (as absolute spectra of C, N, O, Li, Be) may definitively confirm this scenario.

An Aggressive Subtype of Stage I Lung Adenocarcinoma with Molecular and Prognostic Characteristics Typical of Advanced Lung Cancers.

PURPOSE: The National Lung Cancer Screening Trial has confirmed that lung cancer mortality can be reduced if tumors are diagnosed early, that is, at stage I. However, a substantial fraction of stage I lung cancer patients still develop metastatic disease within 5 years from surgery. Prognostic biomarkers are therefore needed to identify patients at risk of an adverse outcome, who might benefit from multimodality treatment. EXPERIMENTAL DESIGN: We extensively validated a 10-gene prognostic signature in a cohort of 507 lung adenocarcinoma patients using formalin-fixed paraffin-embedded samples. Furthermore, we performed an integrated analysis of gene expression, methylation, somatic mutations, copy number variations, and proteomic profiles on an independent cohort of 468 patients from The Cancer Genome Atlas (TCGA). RESULTS: Stage I lung cancer patients (N = 351) identified as high-risk by the 10-gene signature displayed a 4-fold increased risk of death [HR = 3.98; 95% confidence interval (CI), 1.73-9.14], with a 3-year overall survival of 84.2% (95% CI, 78.7-89.7) compared with 95.6% (92.4-98.8) in low-risk patients. The analysis of TCGA cohort revealed that the 10-gene signature identifies a subgroup of stage I lung adenocarcinomas displaying distinct molecular characteristics and associated with aggressive behavior and poor outcome. CONCLUSIONS: We validated a 10-gene prognostic signature capable of identifying a molecular subtype of stage I lung adenocarcinoma with characteristics remarkably similar to those of advanced lung cancer. We propose that our signature might aid the identification of stage I patients who would benefit from multimodality treatment. Clin Cancer Res; 23(1); 62-72. ©2016 AACR.

The pseudophosphatase STYX targets the F-box of FBXW7 and inhibits SCFFBXW7 function.

The F-box protein FBXW7 is the substrate-recruiting subunit of an SCF ubiquitin ligase and a major tumor-suppressor protein that is altered in several human malignancies. Loss of function of FBXW7 results in the stabilization of numerous proteins that orchestrate cell proliferation and survival. Little is known about proteins that directly regulate the function of this protein. In the current work, we have mapped the interactome of the enigmatic pseudophosphatase STYX We reasoned that a catalytically inactive phosphatase might have adopted novel mechanisms of action. The STYX interactome contained several F-box proteins, including FBXW7. We show that STYX binds to the F-box domain of FBXW7 and disables its recruitment into the SCF complex. Therefore, STYX acts as a direct inhibitor of FBXW7, affecting the cellular levels of its substrates. Furthermore, we find that levels of STYX and FBXW7 are anti-correlated in breast cancer patients, which affects disease prognosis. We propose the STYX-FBXW7 interaction as a promising drug target for future investigations.

RAB2A controls MT1-MMP endocytic and E-cadherin polarized Golgi trafficking to promote invasive breast cancer programs.

The mechanisms of tumor cell dissemination and the contribution of membrane trafficking in this process are poorly understood. Through a functional siRNA screening of human RAB GTPases, we found that RAB2A, a protein essential for ER-to-Golgi transport, is critical in promoting proteolytic activity and 3D invasiveness of breast cancer (BC) cell lines. Remarkably, RAB2A is amplified and elevated in human BC and is a powerful and independent predictor of disease recurrence in BC patients. Mechanistically, RAB2A acts at two independent trafficking steps. Firstly, by interacting with VPS39, a key component of the late endosomal HOPS complex, it controls post-endocytic trafficking of membrane-bound MT1-MMP, an essential metalloprotease for matrix remodeling and invasion. Secondly, it further regulates Golgi transport of E-cadherin, ultimately controlling junctional stability, cell compaction, and tumor invasiveness. Thus, RAB2A is a novel trafficking determinant essential for regulation of a mesenchymal invasive program of BC dissemination.

Sensitive and affordable diagnostic assay for the quantitative detection of anaplastic lymphoma kinase (ALK) alterations in patients with non-small cell lung cancer.

Accurate detection of altered anaplastic lymphoma kinase (ALK) expression is critical for the selection of lung cancer patients eligible for ALK-targeted therapies. To overcome intrinsic limitations and discrepancies of currently available companion diagnostics for ALK, we developed a simple, affordable and objective PCR-based predictive model for the quantitative measurement of any ALK fusion as well as wild-type ALK upregulation. This method, optimized for low-quantity/-quality RNA from FFPE samples, combines cDNA pre-amplification with ad hoc generated calibration curves. All the models we derived yielded concordant predictions when applied to a cohort of 51 lung tumors, and correctly identified all 17 ALK FISH-positive and 33 of the 34 ALK FISH-negative samples. The one discrepant case was confirmed as positive by IHC, thus raising the accuracy of our test to 100%. Importantly, our method was accurate when using low amounts of input RNA (10 ng), also in FFPE samples with limited tumor cellularity (5-10%) and in FFPE cytology specimens. Thus, our test is an easily implementable diagnostic tool for the rapid, efficacious and cost-effective screening of ALK status in patients with lung cancer.

Mining cancer gene expression databases for latent information on intronic microRNAs.

Around 50% of all human microRNAs reside within introns of coding genes and are usually co-transcribed. Gene expression datasets, therefore, should contain a wealth of miRNA-relevant latent information, exploitable for many basic and translational research aims. The present study was undertaken to investigate this possibility. We developed an in silico approach to identify intronic-miRNAs relevant to breast cancer, using public gene expression datasets. This led to the identification of a miRNA signature for aggressive breast cancer, and to the characterization of novel roles of selected miRNAs in cancer-related biological phenotypes. Unexpectedly, in a number of cases, expression regulation of the intronic-miRNA was more relevant than the expression of their host gene. These results provide a proof of principle for the validity of our intronic miRNA mining strategy, which we envision can be applied not only to cancer research, but also to other biological and biomedical fields.

Memo is a copper-dependent redox protein with an essential role in migration and metastasis.

Memo is an evolutionarily conserved protein with a critical role in cell motility. We found that Memo was required for migration and invasion of breast cancer cells in vitro and spontaneous lung metastasis from breast cancer cell xenografts in vivo. Biochemical assays revealed that Memo is a copper-dependent redox enzyme that promoted a more oxidized intracellular milieu and stimulated the production of reactive oxygen species (ROS) in cellular structures involved in migration. Memo was also required for the sustained production of the ROS O2- by NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase 1 (NOX1) in breast cancer cells. Memo abundance was increased in >40% of the primary breast tumors tested, was correlated with clinical parameters of aggressive disease, and was an independent prognostic factor of early distant metastasis.

The fragile X protein binds mRNAs involved in cancer progression and modulates metastasis formation.

The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression.

Selective high-level expression of epsin 3 in gastric parietal cells, where it is localized at endocytic sites of apical canaliculi.

Epsin is a ubiquitin-binding endocytic adaptor, which is highly concentrated at clathrin-coated pits and coordinates acquisition of bilayer curvature with coat recruitment and cargo selection. Epsin is encoded by three distinct genes in mammals. Epsin 1 and 2 have broad tissue distribution with high-level expression in the brain. In contrast, epsin 3 was reported to be expressed primarily in immature keratinocytes. Here, we show that epsin 3 is selectively expressed at high levels in the stomach (including the majority of gastric cancers), where it is concentrated in parietal cells. In these cells, epsin 3 is enriched and colocalized with clathrin around apical canaliculi, the sites that control acidification of the stomach lumen via the exo-endocytosis of vesicles containing the H/K ATPase. Deletion of the epsin 3 gene in mice did not result in obvious pathological phenotypes in either the stomach or other organs, possibly because of overlapping functions of the other two epsins. However, levels of EHD1 and EHD2, two membrane tubulating proteins with a role in endocytic recycling, were elevated in epsin 3 knock-out stomachs, pointing to a functional interplay of epsin 3 with EHD proteins in the endocytic pathway of parietal cells. We suggest that epsin 3 cooperates with other bilayer binding proteins with curvature sensing/generating properties in the specialized traffic and membrane remodeling processes typical of gastric parietal cells.