Circulating Tumor Cells in the Adenocarcinoma of the Esophagus.

Circulating tumor cells (CTCs) are elements of indisputable significance as they seem to be responsible for the onset of metastasis. Despite this, research into CTCs and their clinical application have been hindered by their rarity and heterogeneity at the molecular and cellular level, and also by a lack of technical standardization. Esophageal adenocarcinoma (EAC) is a highly aggressive cancer that is often diagnosed at an advanced stage. Its incidence has increased so much in recent years that new diagnostic, prognostic and predictive biomarkers are urgently needed. Preliminary findings suggest that CTCs could represent an effective, non-invasive, real-time assessable biomarker in all stages of EAC. This review provides an overview of EAC and CTC characteristics and reports the main research results obtained on CTCs in this setting. The need to carry out further basic and translational research in this area to confirm the clinical usefulness of CTCs and to provide oncologists with a tool to improve therapeutic strategies for EAC patients was herein highlighted.

Settling the uncertainty about unconventional circulating tumor cells: Epithelial-to-mesenchymal transition, cell fusion and trogocytosis.

Circulating tumor cells (CTCs) were first described 150 years ago. The so-called "classical" CTC populations (EpCAM+/CK+/CD45-) have been fully characterized and proposed as the most representative CTC subset, with clinical relevance. Nonetheless, other "atypical" or "unconventional" CTCs have also been identified, and their critical role in metastasis formation was demonstrated. In this chapter we illustrate the studies that led to the discovery of unconventional CTCs, defined as CTCs that display both epithelial and mesenchymal markers, or both cancer and immune markers, also in the form of hybrid cancer-immune cells. We also present biological explanations for the origin of these unconventional CTCs: epithelial to mesenchymal transition, cell-cell fusion and trogocytosis. We believe that a deeper knowledge on the biology of CTCs is needed to fully elucidate their role in cancer progression and their use as cancer biomarkers.

ARID1A in cancer: Friend or foe?

ARID1A belongs to a class of chromatin regulatory proteins that function by maintaining accessibility at most promoters and enhancers, thereby regulating gene expression. The high frequency of ARID1A alterations in human cancers has highlighted its significance in tumorigenesis. The precise role of ARID1A in cancer is highly variable since ARID1A alterations can have a tumor suppressive or oncogenic role, depending on the tumor type and context. ARID1A is mutated in about 10% of all tumor types including endometrial, bladder, gastric, liver, biliopancreatic cancer, some ovarian cancer subtypes, and the extremely aggressive cancers of unknown primary. Its loss is generally associated with disease progression more often than onset. In some cancers, ARID1A loss is associated with worse prognostic features, thus supporting a major tumor suppressive role. However, some exceptions have been reported. Thus, the association of ARID1A genetic alterations with patient prognosis is controversial. However, ARID1A loss of function is considered conducive for the use of inhibitory drugs which are based on synthetic lethality mechanisms. In this review we summarize the current knowledge on the role of ARID1A as tumor suppressor or oncogene in different tumor types and discuss the strategies for treating ARID1A mutated cancers.

From phenotypical investigation to RNA-sequencing for gene expression analysis: A workflow for single and pooled rare cells.

Combining phenotypical and molecular characterization of rare cells is challenging due to their scarcity and difficult handling. In oncology, circulating tumor cells (CTCs) are considered among the most important rare cell populations. Their phenotypic and molecular characterization is necessary to define the molecular mechanisms underlying their metastatic potential. Several approaches that require cell fixation make difficult downstream molecular investigations on RNA. Conversely, the DEPArray technology allows phenotypic analysis and handling of both fixed and unfixed cells, enabling a wider range of applications. Here, we describe an experimental workflow that allows the transcriptomic investigation of single and pooled OE33 cells undergone to DEPArray analysis and recovery. In addition, cells were tested at different conditions (unfixed, CellSearch fixative (CSF)- and ethanol (EtOH)-fixed cells). In a forward-looking perspective, this workflow will pave the way for novel strategies to characterize gene expression profiles of rare cells, both single-cell and low-resolution input.

Case Report: Analysis of Plasma Extracellular Vesicles in a Triple Negative Spindle-Cell Metaplastic Breast Cancer Patient.

Metaplastic breast cancer (MpBC) is a rare tumor representing 1% of all breast malignancies. The prognosis of this histologic subtype is actually poor and there are no current clear-cut therapeutic guidelines. Hence, despite its uniqueness, its aggressive prognostic profile strongly encourages further studies to identify new markers and therapeutic targets. Herein, we report a case of 32-years-old patient affected with of triple negative spindle-shaped MpBC. The research of molecular targets on the primary tumor did not allow performing an effective therapeutic choice. Extracellular Vesicles (EVs) are under intense study as new potential pathophysiological markers and targets for therapeutic applications, in different tumors for their role in tumor onset, progression and aggressiveness. Here, we examined the involvement of EVs in this case, to look into the MpBC microenvironment willing to identify new potential molecular targets, pathways of aggressiveness, and markers of prognosis and therapeutic efficacy. Firstly, we characterized MpBC patient EV dimensions and surface proteins. Moreover, we analyzed the EV RNA cargo supposed to be delivered to nearby and distant recipient cells. Interestingly, we observed a dysregulation EV-contained miRNAs, which could determine an increased expression of oncogenes in the tumor microenvironment, probably enabling cancer progression. These data suggest that the characterization of miRNA cargo of EVs could be important for the identification of new markers and for the application of future new target therapies.

Dissecting Molecular Heterogeneity of Circulating Tumor Cells (CTCs) from Metastatic Breast Cancer Patients through Copy Number Aberration (CNA) and Single Nucleotide Variant (SNV) Single Cell Analysis.

Circulating tumor cells' (CTCs) heterogeneity contributes to counteract their introduction in clinical practice. Through single-cell sequencing we aim at exploring CTC heterogeneity in metastatic breast cancer (MBC) patients. Single CTCs were isolated using DEPArray NxT. After whole genome amplification, libraries were prepared for copy number aberration (CNA) and single nucleotide variant (SNV) analysis and sequenced using Ion GeneStudio S5 and Illumina MiSeq, respectively. CTCs demonstrate distinctive mutational signatures but retain molecular traces of their common origin. CNA profiling identifies frequent aberrations involving critical genes in pathogenesis: gains of 1q (CCND1) and 11q (WNT3A), loss of 22q (CHEK2). The longitudinal single-CTC analysis allows tracking of clonal selection and the emergence of resistance-associated aberrations, such as gain of a region in 12q (CDK4). A group composed of CTCs from different patients sharing common traits emerges. Further analyses identify losses of 15q and enrichment of terms associated with pseudopodium formation as frequent and exclusive events. CTCs from MBC patients are heterogeneous, especially concerning their mutational status. The single-cell analysis allows the identification of aberrations associated with resistance, and is a candidate tool to better address treatment strategy. The translational significance of the group populated by similar CTCs should be elucidated.

Circulating Tumor Cells as a Tool to Untangle the Breast Cancer Heterogeneity Issue.

Breast cancer (BC) is a disease characterized by high degrees of heterogeneity at morphologic, genomic, and genetic levels, even within the same tumor mass or among patients. As a consequence, different subpopulations coexist and less represented clones may have a selective advantage, significantly influencing the outcome of BC patients. Circulating tumor cells (CTCs) represent a rare population of cells with a crucial role in metastatic cascade, and in recent years have represented a fascinating alternative to overcome the heterogeneity issue as a "liquid biopsy". However, besides the raw enumeration of these cells in advanced epithelial tumors, there are no CTC-based assays applied in the clinical practice to improve personalized medicine. In this review, we report the latest findings in the field of CTCs for intra-tumoral heterogeneity unmasking in BC, supporting the need to deepen their analysis to investigate their role in metastatic process and include the molecular characterization in the clinical practice. In the future, CTCs will be helpful in monitoring patients during treatment, as well as to better address therapeutic strategies.

Case Report: Analysis of Circulating Tumor Cells in a Triple Negative Spindle-Cell Metaplastic Breast Cancer Patient.

Circulating tumor cells (CTCs) are a rare population of cells found in the bloodstream and represent key players in the metastatic cascade. Their analysis has proved to provide further core information concerning the tumor. Herein, we aim at investigating CTCs isolated from a 32-year-old patient diagnosed with triple negative spindle-shaped metaplastic breast cancer (MpBC), a rare tumor poorly responsive to therapies and with a dismal prognosis. The molecular analysis performed on the primary tumor failed to underline effective actionable targets to address the therapeutic strategy. Besides the presence of round-shaped CTCs, cells with a spindle shape were present as well, and through molecular analysis, we confirmed their malignant nature. This aspect was coherent with the primary tumor histology, proving that CTCs are released regardless of their morphology. Copy number aberration (CNA) profiling and variant analysis using next-generation sequencing (NGS) showed that these cells did not harbor the alterations exhibited by the primary tumor (PIK3CA G1049A mutation, MYC copy number gain). However, despite the great heterogeneity observed, the amplification of regions involved in metastasis emerged (8q24.22-8q24.23). Our findings support the investigation of CTCs to identify alterations that could have a role in the metastatic process. To the best of our knowledge, this is the first examination of CTCs in an MpBC patient.

Early Detection and Investigation of Extracellular Vesicles Biomarkers in Breast Cancer.

Breast cancer (BC) is the most commonly diagnosed malignant tumor in women worldwide, and the leading cause of cancer death in the female population. The percentage of patients experiencing poor prognosis along with the risk of developing metastasis remains high, also affecting the resistance to current main therapies. Cancer progression and metastatic development are no longer due entirely to their intrinsic characteristics, but also regulated by signals derived from cells of the tumor microenvironment. Extracellular vesicles (EVs) packed with DNA, RNA, and proteins, are the most attractive targets for both diagnostic and therapeutic applications, and represent a decisive challenge as liquid biopsy-based markers. Here we performed a study based on a multiplexed phenotyping flow cytometric approach to characterize BC-derived EVs from BC patients and cell lines, through the detection of multiple antigens. Our data reveal the expression of EVs-related biomarkers derived from BC patient plasma and cell line supernatants, suggesting that EVs could be exploited for characterizing and monitoring disease progression.

CNA Profiling of Single CTCs in Locally Advanced Esophageal Cancer Patients during Therapy Highlights Unexplored Molecular Pathways.

BACKGROUND: Here, we monitored the evolution of CTCs spread in 11 patients affected by locally advanced EC who were undergoing therapy. METHODS: In this perspective study, we designed multiple blood biopsies from individual patients: before and after neoadjuvant chemo-radio therapy and after surgery. We developed a multi-target array, named Grab-all assay, to estimate CTCs for their epithelial (EpCAM/E-Cadherin/Cytokeratins) and mesenchymal/stem (N-Cadherin/CD44v6/ABCG2) phenotypes. Identified CTCs were isolated as single cells by DEPArray, subjected to whole genome amplification, and copy number aberration (CNA) profiles were determined. Through bioinformatic analysis, we assessed the genomic imbalance of single CTCs, investigated specific focal copy number changes previously reported in EC and aberrant pathways using enrichment analysis. RESULTS: Longitudinal monitoring allowed the identification of CTCs in at least one time-point per patient. Through single cell CNA analysis, we revealed that CTCs showed significantly dynamic genomic imbalance during treatment. Individual CTCs from relapsed patients displayed a higher degree of genomic imbalance relative to disease-free patients' groups. Genomic aberrations previously reported in EC occurred mostly in post-neoadjuvant therapy CTCs. In-depth analysis showed that networks enrichment in all time-point CTCs were inherent to innate immune system. Transcription/gene regulation, post-transcriptional and epigenetic modifications were uniquely affected in CTCs of relapsed patients. CONCLUSIONS: Our data add clues to the comprehension of the role of CTCs in EC aggressiveness: chromosomal aberrations on genes related to innate immune system behave as relevant to the onset of CTC-status, whilst pathways of transcription/gene regulation, post-transcriptional and epigenetic modifications seem linked to patients' outcome.

Impressive long-term response with chemo-endocrine therapy in a premenopausal patient with metastatic breast cancer: A case report.

RATIONALE: Patients with, or who develop, metastatic breast cancer have a 5-year relative survival of about 25%. Endocrine therapy clearly improves outcomes in patients with estrogen receptor-positive breast cancer. In the metastatic setting, the primary goal of treatment is to maintain long-term disease control with good quality of life. Rarely, exceptional responders achieve durable disease control, and potential cures cannot be ruled out. PATIENT CONCERNS: We report the case of a 39-year-old woman with primary breast cancer and associated synchronous bone metastases, who experienced a disease response of 12 years with hormonal therapy as maintenance after first line chemotherapy, with a good toxicity profile. DIAGNOSIS: The patient was diagnosed with estrogen receptor + human epidermal growth factor receptor 2 (HER2)- metastatic breast cancer with synchronous bone metastases. INTERVENTIONS: This patient was treated with chemotherapy for 6 cycles as a first-line therapy following by endocrine treatment given as a maintenance therapy. OUTCOMES: Our patient experienced a progression-free survival >12 years with an exceptionally good quality of life. LESSONS: Our anecdotal experience highlights the existence of exceptional responders among patients with hormone receptor-positive metastatic breast cancer, who achieve clinical remission and durable disease control with endocrine therapy. Being able to identify these patients could help in the selection of the best treatment option among the many available.

Single-Cell NGS-Based Analysis of Copy Number Alterations Reveals New Insights in Circulating Tumor Cells Persistence in Early-Stage Breast Cancer.

Circulating tumor cells (CTCs) are a rare population of cells representing a key player in the metastatic cascade. They are recognized as a validated tool for the identification of patients with a higher risk of relapse, including those diagnosed with breast cancer (BC). However, CTCs are characterized by high levels of heterogeneity that also involve copy number alterations (CNAs), structural variations associated with gene dosage changes. In this study, single CTCs were isolated from the peripheral blood of 11 early-stage BC patients at different time points. A label-free enrichment of CTCs was performed using OncoQuick, and single CTCs were isolated using DEPArray. Libraries were prepared from single CTCs and DNA extracted from matched tumor tissues for a whole-genome low-coverage next-generation sequencing (NGS) analysis using the Ion Torrent S5 System. The analysis of the CNA burden highlighted that CTCs had different degrees of aberration based on the time point and subtype. CTCs were found even six months after surgery and shared CNAs with matched tumor tissue. Tumor-associated CNAs that were recurrent in CTCs were patient-specific, and some alterations involved regions associated with BC and survival (i.e., gains at 1q21-23 and 5p15.33). The enrichment analysis emphasized the involvement of aberrations of terms, associated in particular with interferon (IFN) signaling. Collectively, our findings reveal that these aberrations may contribute to understanding the molecular mechanisms involving CTC-related processes and their survival ability in occult niches, supporting the goal of exploiting their application in patients' surveillance and follow-up.

Adipocytes and microRNAs Crosstalk: A Key Tile in the Mosaic of Breast Cancer Microenvironment.

Breast cancer (BC) is a disease characterized by a high grade of heterogeneity. Consequently, despite the great achievements obtained in the last decades, most of the current therapeutic regimens still fail. The identification of new molecular mechanisms that will increase the knowledge of all steps of tumor initiation and growth is mandatory in finding new clinical strategies. The BC microenvironment, consisting of endothelial cells, fibroblasts, immune cells and adipocytes, plays an essential role in regulating BC development, and recently it has gained great attention in the scientific community. In particular, adipose tissue is emerging as an important target to investigate among mammary gland components. The mechanisms underlying BC progression driven by adipocytes are predominantly unexplored, especially that involving the switch from normal adipocytes to the so-called cancer-associated adipocytes (CAAs). MicroRNAs (miRNAs), a class of gene expression modulators, have emerged as the regulators of key oncogenes and tumor suppressor genes that affect multiple pathways of the tumor microenvironment and adipose tissue. This review concerns a presentation of the role of adipocytes in breast tissue, and describes the most recent discoveries about the interplay between adipocytes and miRNAs, which collaborate in the arrangement of a pro-inflammatory and cancerous microenvironment, laying the foundations for new concepts in the prevention and treatment of BC.

Stemness underpinning all steps of human colorectal cancer defines the core of effective therapeutic strategies.

BACKGROUND: Despite their lethality and ensuing clinical and therapeutic relevance, circulating tumor cells (CTCs) from colorectal carcinoma (CRC) remain elusive, poorly characterized biological entities. METHODS AND FINDINGS: We perfected a cell system of stable, primary lines from human CRC showing that they possess the full complement of ex- and in-vivo, in xenogeneic models, characteristics of CRC stem cells (CCSCs). Here we show how tumor-initiating, CCSCs cells can establish faithful orthotopic phenocopies of the original disease, which contain cells that spread into the circulatory system. While in the vascular bed, these cells retain stemness, thus qualifying as circulating CCSCs (cCCSCs). This is followed by the establishment of lesions in distant organs, which also contain resident metastatic CCSCs (mCCSCs). INTERPRETATION: Our results support the concept that throughout all the stages of CRC, stemness is retained as a continuous property by some of their tumor cells. Importantly, we describe a useful standardized model that can enable isolation and stable perpetuation of human CRC's CCSCs, cCCSCs and mCCSCs, providing a useful platform for studies of CRC initiation and progression that is suitable for the discovery of reliable stage-specific biomarkers and the refinement of new patient-tailored therapies. FUND: This work was financially supported by grants from "Ministero della Salute Italiano"(GR-2011-02351534, RC1703IC36 and RC1803IC35) to Elena Binda and from "Associazione Italiana Cancro" (IG-14368) Angelo L. Vescovi. None of the above funders have any role in study design, data collection, data analysis, interpretation, writing the project.

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH.

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

DNA ploidy and S-phase fraction analysis in peritoneal carcinomatosis from ovarian cancer: correlation with clinical pathological factors and response to chemotherapy.

OBJECTIVE: We investigated the correlation between ploidy or S-phase fraction (SPF) and the clinical pathological characteristics of patients with peritoneal carcinomatosis from ovarian cancer. We also assessed their relation with the in vivo and in vitro response to several chemotherapeutic agents. PATIENTS AND METHODS: Fifty-three patients with peritoneal carcinomatosis from ovarian cancer were enrolled. Frozen tumor tissue was dissociated by a detergent-trypsin method, and the resulting cell suspension was stained with RNase A and propidium iodide. Samples were then analyzed for ploidy and SPF by flow cytometry. Fresh tumor tissue was dissociated by enzymatic digestion, and cells were exposed to different concentrations of cisplatin, adriamycin, carboplatin, gemcitabine and taxol for 72 hours. In vitro drug sensitivity was then measured using the sulforhodamine B assay. RESULTS: No significant correlation was found between ploidy or SPF and patient characteristics, even though primary carcinomas were mainly hyperdiploid and more proliferative than recurrent tumors. SPF differed significantly among ploidy categories (P=0.01), and high SPF was associated with short-term survival (P=0.48). Patients with multiploid tumors were the most resistant to platinum-based chemotherapy, whereas those with hyperdiploid tumors were the most responsive. In vitro multiploid tumors were the least sensitive, while hypodiploid samples showed the highest sensitivity to the tested drugs. Sensitivity to adriamycin was significantly correlated with ploidy (P=0.03), whereas sensitivity to taxol was correlated with SPF (P=0.04). CONCLUSION: Our results indicate that ploidy and SPF could facilitate the choice of therapy for patients with peritoneal carcinomatosis.

Splicing factor ratio as an index of epithelial-mesenchymal transition and tumor aggressiveness in breast cancer.

Epithelial-to-mesenchymal transition (EMT) has been shown to be associated with tumor progression and metastasis. During this process in breast cancer, a crucial role is played by alternative splicing systems. To identify a new early prognostic marker of metastasis, we evaluated EMT-related gene expression in breast cell lines, and in primary tumor tissue from 31 patients with early breast cancer, focusing our attention on EMT-related splicing factors ESRP1, ESRP2 and RBFOX2. Results showed that the expression patterns of these genes were indicative of the onset of EMT in in-vitro models, but not in tissue samples. However, the ratio between ESRP1 or ESRP2 and RBFOX2 significantly decreased during EMT and positively correlated with the EMT-specific phenotype in cell models, representing a promising prognostic markers. Low ESRP1/RBFOX2 ratio value was associated with a higher risk of metastasis (p < 0.005) in early breast cancer patients, regardless other clinical features. A cut-off of ratio of 1.067 was determined by ROC curve analysis (AUC 0.8375; 95% CI 0.6963-0.9787). Our study show evidence that a decrease in this ratio correlates with cancer progression. The results provide a rationale for using ESRP1/RBFOX2 ratio as a new prognostic biomarker for the early prediction of metastatic potential in breast cancer.

CTCs in early breast cancer: A path worth taking.

Circulating tumor cells (CTCs) are cellular elements of undeniable significance that spread from the tumor mass into the peripheral blood and constitute one of the main vehicles for disease diffusion. Their rarity, in addition to a number of molecular and cellular features, has severely impaired research and exploitation. CTCs have been evaluated in early breast cancer (EBC), although long from being fully accepted in this field also due to a lack of technical standardization. CTCs hold promise to be a powerful non-invasive real-time measurable biomarker in all disease stages. This hypothesis is particularly appealing in the adjuvant setting of breast cancer, as it still lacks a marker that could play a central role in monitoring disease-free intervals, predicting early relapse and guiding drug selection. This review aimed to discuss CTC characteristics and show the main results of CTC-research in EBC setting, stating the urgency to continue basic and translational research in this field to definitely translate this marker from bench to bedside.

Epithelial Mesenchymal Transition: a double-edged sword.

Epithelial mesenchymal transition (EMT) is a physiological process necessary to normal embryologic development. However in genesis of pathological situations, this transition can be perverted and signaling pathways have different regulations from those of normal physiology. In cancer invasion, such a mechanism leads to generation of circulating tumor cells. Epithelial cancer cells become motile mesenchymal cells able to shed from the primary tumor and enter in the blood circulation. This is the major part of the invasive way of cancer. EMT is also implicated in chronic diseases like fibrosis and particularly renal fibrosis. In adult organisms, healing is based on EMT which is beneficial to repair wounds even if it can sometimes exceed its goal and elicit fibrosis. In this review, we delineate the clinical significance of EMT in both physiological and pathological circumstances.