Circulating tumor cells isolation: The “post-EpCAM era”

Circulating tumor cells (CTCs) represent a submicroscopic fraction detached from a primary tumor and in transit to a secondary site. The prognostic significance of CTCs in metastatic cancer patients was demonstrated for the first time more than ten years ago. To date, it seems clear enough that CTCs are highly heterogeneous and dynamically change their shape. Thus, the inadequacy of epithelial cell adhesion molecule (EpCAM) as universal marker for CTCs detection seems unquestionable and alternative methods able to recognize a broader spectrum of phenotypes are definitely needed. In this review the pleiotropic functions of EpCAM are discussed in detail and the role of the molecule in the biology of CTCs is critically dissected

Razionale per la biopsia liquida nel carcinoma del colon-retto: focus sulle cellule tumorali circolanti

L’isolamento di cellule tumorali circolanti (CTC) e di DNA tumorale circolante (ctDNA), prende il nome di “biopsia liquida”. Rispetto alla convenzionale biopsia del tessuto, che rappresenta un’immagine istantanea e statica di un tumore, la biopsia liquida presenta il grande vantaggio di fornire informazioni in “tempo reale” sulla malattia, mettendo in luce l'evoluzione del tumore e la sua eterogeneità, in particolare, le variazioni di popolazioni di cellule tumorali durante la progressione della malattia e in risposta alle terapie. Infatti, anche se è ampiamente accettato che l’eterogeneità intratumorale possa influenzare l'efficacia della terapia a bersaglio molecolare, lo studio della farmacoresistenza è ostacolato dal fatto che la biopsia di tessuto tumorale è una procedura invasiva e non sempre ripetibile. La complessa eterogeneità tumorale si traduce in una notevole instabilità del genoma, consentendo alle cellule tumorali di adattarsi a qualsiasi ambiente ostile, comprese le terapie mirate e di guidare l'evoluzione di cloni resistenti. Si rende pertanto necessario un adeguamento delle terapie a bersaglio molecolare che rifletta le continue alterazioni che si accumulano nel genoma delle cellule tumorali. Con l’obiettivo di avvicinarsi ad una medicina di precisione e di personalizzare le terapie, ogni strumento che consenta la stratificazione dei pazienti e l’ottimizzazione delle terapie appare di fondamentale importanza nell’era dell’oncologia molecolare. Tra le malattie in cui le terapie biologiche hanno determinato un significativo miglioramento della sopravvivenza, il carcinoma metastatico del colon-retto occupa un posto di assoluto rilievo. Nonostante tali approcci abbiano di fatto modificato la storia clinica di questa neoplasia, resta ancora molto da indagare sull’identificazione e sulla validazione di biomarcatori affidabili, con significato predittivo di risposta alla terapia. La biopsia liquida potrebbe rispondere a tale esigenza. Rispetto ad altri tumori, le CTC isolate da pazienti con tumore del colon-retto sono assenti o quasi sempre al di sotto del valore soglia, pertanto non sempre correlabili con l’andamento clinico della malattia, con meccanismi non pienamente compresi. Tale discrasia tra assenza di CTC e progressione clinica di malattia nei pazienti è particolarmente evidente nei pazienti candidati a terapia con farmaci inibitori dell’angiogenesi. Sulla base dei suddetti presupposti teorici, il presente lavoro di tesi ha avuto come scopo quello di ottimizzare la biopsia liquida in pazienti affetti da carcinoma del colon-retto con i seguenti obiettivi: - analizzare sensibilità e specificità di metodiche standard di isolamento delle CTC basate su arricchimento immunomagnetico (CellSeach® ,AdnaTest) e di metodiche di filtrazione che separano le cellule tumorali circolanti dalle altre cellule ematiche in base alle dimensioni. -analizzare i meccanismi attraverso i quali i farmaci inibitori dell’angiogenesi possono impedire un efficiente isolamento delle CTC - caratterizzare il profilo molecolare delle CTC isolate con particolare riferimento a marcatori di transizione epitelio mesenchima, marcatori di staminalità e marcatori predittivi di risposta ai farmaci a bersaglio molecolare. -costruire filtri ad hoc per l’isolamento di CTC clusterizzat

Liquid Biopsy in Rare Cancers: Lessons from Hemangiopericytoma

Hemangiopericytoma (HPT) is a rare mesenchymal tumor of fibroblastic type and for its rarity is poorly studied. The most common sites of metastatic disease in patients with intracranial HPT are the bone, liver, and lung, suggestive for an hematogenous dissemination; for this reason, we investigated, for the first time, the presence of circulating tumor cells (CTCs) in hemangiopericytoma patient by CellSearch® and SceenCell® devices. Peripheral blood samples were drawn and processed by CellSearch, an EpCAM-dependent device, and ScreenCell®, a device size based. We found nontypical CTCs by CellSearch system and the immunofluorescence analysis performed on CTCs isolate by ScreenCell demonstrated the presence of single CTCs and CTC clusters. The molecular characterization of single CTCs and CTC clusters, using antibodies directed against EpCAM, CD34, cytokeratins (8, 18, and 19), and CD45, showed a great heterogeneity in CTC clusters. We believe that the present study may open a new scenario in the rare tumors: the introduction of the liquid biopsy and the molecular characterization of circulating tumor cells could lead to personalized targeted treatments and also for rare tumors

Coexistence of three EGFR mutations in an NSCLC patient: A brief report

The epidermal growth factor receptor (EGFR) represents a molecular target for tyrosine kinase inhibitors for non-small cell lung cancer (NSCLC) patients with a mutation in the EGFR gene. Mutations of the EGFR gene that occur at a single position in NSCLC tissue are found as single, whereas two or more mutations on the same allele are poorly detected and investigated

Impact of chronic exposure to bevacizumab on EpCAM-based detection of circulating tumor cells

BACKGROUND: Circulating tumor cells (CTCs) are often undetected through the immunomagnetic epithelial cell adhesion molecule (EpCAM)-based CellSearch(®) System in breast and colorectal cancer (CRC) patients treated with bevacizumab (BEV), where low CTC numbers have been reported even in patients with evidence of progression of disease. To date, the reasons for this discrepancy have not been clarified. This study was carried out to investigate the molecular and phenotypic changes in CRC cells after chronic exposure to BEV in vitro. METHODS: The human CRC cell line WiDr was exposed to a clinically relevant dose of BEV for 3 months in vitro. The expression of epithelial and mesenchymal markers and EpCAM isoforms was determined by western blotting and immunofluorescence. To evaluate the impact of EpCAM variant isoforms expression on CTC enumeration by CellSearch(®), untreated and treated colon cancer cells were spiked into 7.5 mL of blood from a healthy donor and enumerated by CellSearch(®). RESULTS: Chronic exposure of CRC cell line to BEV induced decreased expression of EpCAM 40 kDa isoform and increased expression EpCAM 42 kDa isoform, together with a decreased expression of cytokeratins (CK), while no evidence of epithelial to mesenchymal transition (EMT) in treated cells was observed. The recovery rate of cells through CellSearch(®) was gradually reduced in course of treatment with BEV, being 84%, 70% and 40% at 1, 2 and 3 months, respectively. CONCLUSIONS: We hypothesize that BEV may prevent CellSearch(®) from capturing CTCs through altering EpCAM isoforms

The rationale for liquid biopsy in colorectal cancer: focus on circulating tumor cells

Capturing circulating tumor cells (CTCs) and/or circulating tumor DNA from blood, which represents a precious source of biological material derived from both primary and metastatic tumors, has been named a 'liquid biopsy'. While the circulating tumor DNA might be more representative of the bulk of the metastatic tumor, CTCs are thought to reflect more of the metastases-initiating cells. Consequently, a liquid biopsy made of tumor cells and tumor DNA that is able to track cancer evolution, as a fingerprint of the patient's individual tumor, and is easy to perform at every stage of the disease course, sounds attractive. This article mainly focuses on the applications of CTCs to track tumor dynamics in real time using colorectal cancer as a model system. The analysis of viable CTCs at DNA, RNA and protein levels, as well as their expansion in vitro, may allow deep investigation of the features of metastases-initiating cells

Is cancer an intelligent species?

Some relevant emerging properties of intelligent systems are "adaptation to a changing environment," "reaction to unexpected situations," "capacity of problem solving," and "ability to communicate." Single cells have remarkable abilities to adapt, make adequate context-dependent decision, take constructive actions, and communicate, thus theoretically meeting all the above-mentioned requirements. From a biological point of view, cancer can be viewed as an invasive species, composed of cells that move from primary to distant sites, being continuously exposed to changes in the environmental conditions. Blood represents the first hostile habitat that a cancer cell encounters once detached from the primary site, so that cancer cells must rapidly carry out multiple adaptation strategies to survive. The aim of this review was to deepen the adaptation mechanisms of cancer cells in the blood microenvironment, particularly referring to four adaptation strategies typical of animal species (phenotypic adaptation, metabolic adaptation, niche adaptation, and collective adaptation), which together define the broad concept of biological intelligence. We provided evidence that the required adaptations (either structural, metabolic, and related to metastatic niche formation) and "social" behavior are useful principles allowing putting into a coherent frame many features of circulating cancer cells. This interpretative frame is described by the comparison with analog behavioral traits typical of various animal models

Transient disappearance of RAS mutant clones in plasma: A counterintuitive clinical use of EGFR inhibitors in RAS mutant metastatic colorectal cancer

Genomic studies performed through liquid biopsies widely elucidated the evolutionary trajectory of RAS mutant clones under the selective pressure of EGFR inhibitors in patients with wild type RAS primary colorectal tumors. Similarly, the disappearance of RAS mutant clones in plasma has been more recently reported in some patients with primary RAS mutant cancers, supporting for the first time an unexpected negative selection of RAS mutations during the clonal evolution of mCRC. To date, the extent of conversion to RAS wild type disease at the time of progression has not been clarified yet. As a proof of concept, we prospectively enrolled mCRC patients progressing under anti-VEGF based treatments. Idylla™system was used to screen RAS mutations in plasma and the wild type status of RAS was further confirmed through IT-PGM (Ion Torrent Personal Genome Machine) sequencing. RAS was found mutant in 55% of cases, retaining the same plasma mutation as in the primary tumor at diagnosis, while it was found wild-type in 45%. Four patients testing negative for RAS mutations in plasma at the time of progression of disease (PD) were considered eligible for treatment with EGFR inhibitors and treated accordingly, achieving a clinical benefit. We here propose a hypothetical algorithm that accounts for the transient disappearance of RAS mutant clones over time, which might extend the continuum of care of mutant RAS colorectal cancer patients through the delivery of a further line of therapy

Circulating Tumor Cells Identify Patients with Super-High-Risk Non-Muscle-Invasive Bladder Cancer: Updated Outcome Analysis of a Prospective Single-Center Trial

Clinical behavior of non-muscle-invasive bladder cancer (NMIBC) is largely unpredictable, and even patients treated according to European Association of Urology recommendations have a heterogeneous prognosis. High-grade T1 (HGT1) bladder cancer is the highest-risk subtype of NMIBC, with an almost 40% rate of recurrence and 20% of progression at 5 years. Nomograms predicting risk of recurrence, progression, and cancer-specific survival (CSS) are not available specifically within HGT1 bladder cancer, and the identification of robust prognostic biomarkers to better guide therapeutic strategies in this subgroup of patients is of paramount importance. Strategies to identify putative biomarkers in liquid biopsies from blood and urine collected from patients with bladder cancer have been intensively studied in the last few years