Publications by authors named "Chiara Nicolazzo"

33 Publications

True conversions from RAS mutant to RAS wild-type in circulating tumor DNA from metastatic colorectal cancer patients as assessed by methylation and mutational signature.

Cancer Lett 2021 06 18;507:89-96. Epub 2021 Mar 18.

Cancer Liquid Biopsy Unit, Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena, 324, 00161, Rome, Italy. Electronic address:

The paucity of targeted treatments available in patients with RAS mutant colorectal cancers contributes to the poor prognosis of this patient group compared to those with RAS wild-type disease. Recent liquid biopsy-driven studies have demonstrated that RAS mutant clones might disappear in plasma during the clonal evolution of the disease, opening new unforeseen perspectives for EGFR blockade in these patients. Nevertheless, the lack of detection of RAS mutations in plasma might depend on the low amount of released circulating tumor DNA (ctDNA), making it necessary a more accurate selection of patients with true RAS mutation conversions. In this liquid biopsy-based study, we assessed RAS mutational status in initially RAS-mutant patients at the time of progressive disease from any line of therapy and investigated the incidence of true conversions to plasma RAS wild-type, comparing a colon cancer specific methylation profile with a mutational signature of ctDNA. Globally, considering either mutational panel or methylation profile as reliable tests to confirm or exclude the presence of ctDNA, the percentage of "true RAS converters" was 37.5%. In our series we observed a trend toward a better PFS in patients who received anti-EGFR as second or subsequent treatment lines compared to those who did not.
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http://dx.doi.org/10.1016/j.canlet.2021.03.014DOI Listing
June 2021

Circulating Methylated DNA to Monitor the Dynamics of RAS Mutation Clearance in Plasma from Metastatic Colorectal Cancer Patients.

Cancers (Basel) 2020 Dec 4;12(12). Epub 2020 Dec 4.

Liquid Biopsy Unit, Department of Molecular Medicine, Sapienza University of Rome, 00161 Rome, Italy.

The clearance of RAS mutations in plasma circulating tumor DNA (ctDNA) from originally RAS-mutant metastatic colorectal cancer (mCRC) has been recently demonstrated. Clinical trials investigating whether RAS mutant mCRC who "convert" to wild-type in plasma might benefit from EGFR blockade are ongoing. Detection of tumor-specific DNA methylation alterations in ctDNA has been suggested as a specific tool to confirm the tumoral origin of cell-free DNA. We monitored RAS clearance in plasma from patients with RAS-mutant mCRC at baseline (pre-treatment) (T0); after 4 months of first-line therapy (T1); at the time of first (T2) and second (T3) progression. A five-gene methylation panel was used to confirm the presence of ctDNA in samples in which RAS mutation clearance was detected. At T1, ctDNA analysis revealed wild-type RAS status in 83% of samples, all not methylated, suggesting at this time point the lack of ctDNA shedding. At T2, ctDNA analysis revealed wild-type RAS status in 83% of samples, of which 62.5% were found methylated. At T3, 50% of wild-type RAS samples were found methylated. Non-methylated samples were found in patients with lung or brain metastases. This five-gene methylation test might be useful to confirm the presence of ctDNA in RAS wild-type plasma samples.
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http://dx.doi.org/10.3390/cancers12123633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761880PMC
December 2020

Baseline CD44v6-positive circulating tumor cells to predict first-line treatment failure in patients with metastatic colorectal cancer.

Oncotarget 2020 Nov 10;11(45):4115-4122. Epub 2020 Nov 10.

Department of Molecular Medicine, Liquid Biopsy Unit, Sapienza University of Rome, Rome, Italy.

CD44v6, the CD44 isoform mostly involved in cancer cell migration and invasion, has been identified as a functional biomarker and therapeutic target in colon cancer stem cells. We here provide evidence that baseline CD44v6-positive CTC predict treatment failure in patients with metastatic colorectal cancer undergoing first-line chemotherapy. We suggest that CD44v6-positive CTC can be used to early detect intrinsic drug resistance in this cancer type.
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http://dx.doi.org/10.18632/oncotarget.27794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665234PMC
November 2020

Molecular Biomarkers according to Primary Tumor Location in Colorectal Cancer: Current Standard and New Insights.

Oncology 2021 30;99(3):135-143. Epub 2020 Oct 30.

Department of Molecular Medicine, Cancer Liquid Biopsy Unit, Sapienza University of Rome, Rome, Italy,

According to the 2018 GLOBOCAN database, colorectal cancer is one of the most common malignancies and leading causes of cancer-related death worldwide. During the last decades, considerable progress has been made in understanding the complex pathogenetic mechanisms involved in this neoplastic disease. Due to the need to improve treatment responses and clinical outcomes of colorectal cancer patients, the identification of new molecular biomarkers became a crucial spot in clinical oncology. As biological indicators of a specific pathological or physiological process, molecular markers play a central role in cancer detection, diagnosis, outcome prediction, and treatment choice. Considering the existing evidence that malignancies originating from distinct colonic regions behave differently, it is clear that specific biomarkers can be associated to right- or left-sided colon carcinomas, reflecting the distinct molecular signatures of these different tumor entities. The aim of this review is to summarize the main differences among tumors arising from proximal and distal colon in terms of current and emerging biomarkers.
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http://dx.doi.org/10.1159/000510944DOI Listing
March 2021

Liquid Biopsy for Predicting Bacillus Calmette-Guérin Unresponsiveness in Non-muscle-invasive Bladder Cancer.

Eur Urol Oncol 2021 Feb 2;4(1):124-125. Epub 2020 Oct 2.

Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.euo.2020.09.003DOI Listing
February 2021

Detection of circulating tumor cells in patients with laryngeal cancer using ScreenCell: Comparative pre- and post-operative analysis and association with prognosis.

Oncol Lett 2020 Jun 10;19(6):4183-4188. Epub 2020 Apr 10.

Department of Sense Organs, Sapienza University of Rome, Rome 00186, Italy.

The presence of circulating tumor cells (CTCs) in the blood of patients with metastatic breast, colorectal and prostate cancer have been widely investigated; however, few studies have examined CTCs in patients with laryngeal cancer. The present pilot study aimed to detect pre- and postoperative CTCs in the blood of patients with laryngeal cancer and evaluate the association with prognosis. Eight patients with laryngeal squamous cell carcinoma (LSCC) at stage III were included in the present study and underwent total or subtotal laryngectomy and radical bilateral neck lymph node dissection. Blood samples were collected from all patients before and after surgery at different time-points. The following processing steps were followed; preoperative blood sampling, surgery, postoperative blood sampling at 3, 6 and 12 month follow-ups, and prognostic association analysis. CTCs were retained on ScreenCell filters for cytological characterization. The presence of CTCs was associated with a less favorable prognosis, whereas a decrease of CTCs in the postoperative sampling was observed in patients who exhibited an improved therapeutic response. The results of the present pilot study revealed a possible association between the presence of CTCs and a less favorable prognosis in patients with LSCC; therefore, these preliminary findings may encourage further research into the incorporation of a liquid biopsy in the management of LSCC, as this may help identify patients with occult metastatic disease earlier and in a non-invasive manner. In addition, this approach may represent novel independent prognostic factor for use in the clinical evaluation of patients with LSCC.
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http://dx.doi.org/10.3892/ol.2020.11528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7204636PMC
June 2020

EpCAM Circulating Tumor Cells: Gold in the Waste.

Dis Markers 2019 17;2019:1718920. Epub 2019 Sep 17.

Department of Molecular Medicine, Sapienza University of Rome, 00161 Rome, Italy.

The CellSearch® system which is still considered the gold standard for the enumeration of circulating tumor cells (CTC) utilizes antibodies against the epithelial cell adhesion molecule (EpCAM) for CTC enrichment. Recently, CTC discarded by the CellSearch® system due to their low EpCAM expression have been isolated and analyzed. We here sought to discuss technical and biological issues concerning the isolation and characterization of EpCAM CTC, highlighting the enormous potential of this subpopulation discarded by CellSearch®, which might instead reveal an unexpected clinical significance in tumor types where CTC enumeration has never been validated for prognostic and predictive purpose.
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http://dx.doi.org/10.1155/2019/1718920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766153PMC
April 2020

Circulating Tumor Cells in Right- and Left-Sided Colorectal Cancer.

Cancers (Basel) 2019 Jul 24;11(8). Epub 2019 Jul 24.

Department of Molecular Medicine, Circulating tumor cells Unit, Sapienza University of Rome, 00161 Rome, Italy.

Molecular alterations are not randomly distributed in colorectal cancer (CRC), but rather clustered on the basis of primary tumor location underlying the importance of colorectal cancer sidedness. We aimed to investigate whether circulating tumor cells (CTC) characterization might help clarify how different the patterns of dissemination might be relative to the behavior of left- (LCC) compared to right-sided (RCC) cancers. We retrospectively analyzed patients with metastatic CRC who had undergone standard baseline CTC evaluation before starting any first-line systemic treatment. Enumeration of CTC in left- and right-sided tumors were compared. The highest prognostic impact was exerted by CTC in left-sided primary cancer patients, even though the lowest median number of cells was detected in this subgroup of patients. CTC exhibit phenotypic heterogeneity, with a predominant mesenchymal phenotype found in CTC from distal compared to proximal primary tumors. Most CTC in RCC patients exhibited an apoptotic pattern. CTC in left-sided colon cancer patients exhibit a predominant mesenchymal phenotype. This might imply a substantial difference in the biology of proximal and distal cancers, associated with different patterns of tumor cells dissemination. The poor prognosis of right-sided CRC is not determined by the hematogenous dissemination of tumor cells, which appears to be predominantly a passive shedding of non-viable cells. Conversely, the subgroup of poor-prognosis left-sided CRC is reliably identified by the presence of mesenchymal CTC.
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http://dx.doi.org/10.3390/cancers11081042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721440PMC
July 2019

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

Oncologist 2019 05 3;24(5):612-616. Epub 2019 Apr 3.

Dipartimento Scienze Radiologiche, Oncologiche e Patologiche, Sapienza Università di Roma, Rome, Italy

Background: 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.

Subjects, Materials, And Methods: We here report the final analysis of a single-center prospective study aimed to investigate the impact of circulating tumor cells (CTCs) on CSS and overall survival (OS) in 102 patients with HGT1 bladder cancer, in a median follow-up of 63 months.

Results: We here demonstrate that the presence of even a single CTC is predictive of shorter CSS and OS, as compared with the standard predictive variables. Points of attention in this multivariable analysis are the long-term follow-up and the adequate number of outcome events.

Conclusion: The accurate risk stratification provided by CTCs might be essential for determining the best surveillance strategy for patients after diagnosis. A closer follow-up, an early radical surgery, or even a systemic treatment might be recommended in patients with super-high-risk non-muscle-invasive bladder cancer.

Implications For Practice: Circulating tumor cells identify patients with super-high-risk non-muscle-invasive bladder cancer who require closer monitoring for local recurrence and/or progression of disease. This super-high-risk subgroup of patients might also require more aggressive treatment interventions, which should be evaluated in large prospective cohorts.
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http://dx.doi.org/10.1634/theoncologist.2018-0784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6516110PMC
May 2019

Improving the Nuclear-Localized Androgen Receptor Splice Variant 7 Test.

JAMA Oncol 2019 03;5(3):433-434

Circulating Tumor Cells Unit, Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy.

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http://dx.doi.org/10.1001/jamaoncol.2018.6683DOI Listing
March 2019

Transient Disappearance of RAS Mutant Clones in Plasma: A Counterintuitive Clinical Use of EGFR Inhibitors in RAS Mutant Metastatic Colorectal Cancer.

Cancers (Basel) 2019 Jan 4;11(1). Epub 2019 Jan 4.

Department of Molecular Medicine, Sapienza University of Rome, V.le Regina Elena 324, 00161 Rome, Italy.

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.
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http://dx.doi.org/10.3390/cancers11010042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357143PMC
January 2019

Molecular Characterization of Circulating Tumor Cells to Study Cancer Immunoevasion.

Methods Mol Biol 2019 ;1884:247-258

Dipartimento di Medicina Molecolare, Sapienza Università di Roma, Rome, Italy.

Cancer cells leaving the primary tumor immunosuppressive microenvironment become vulnerable to active immune surveillance and require mechanisms of immunoevasion to survive in the circulation. Studies have identified several pathways by which circulating tumor cells (CTCs) might escape the immune system/immunotherapy attack. The PD-1/PD-L1 axis is an immune checkpoint regulator, playing a major role in maintaining self-tolerance. It is now well recognized that tumor cells co-opt the PD-1/PD-L1 axis of immune regulation to interfere with cytotoxic T lymphocyte function. Transcriptional changes in CTCs, leading to the upregulation of PD-L1, might enable them to survive in circulation. Very recent data revealed a previously unappreciated role of epithelial-mesenchymal transition (EMT) in reprogramming the immune response in the local tumor microenvironment and a mutual regulation between EMT and immunoevasion is becoming apparent. In this chapter, we will describe in detail both EpCAM-dependent and -independent approaches that allow the identification of PD-L1 expression and EMT-like features in circulating tumor cells.
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http://dx.doi.org/10.1007/978-1-4939-8885-3_17DOI Listing
June 2019

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

Int J Biol Markers 2018 Jun 1:1724600818782200. Epub 2018 Jun 1.

1 Dipartimento di Medicina Molecolare, Sapienza Università di Roma, Roma, Italia.

Background: 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.

Patient And Methods: We investigated the presence of the EGFR gene mutations in tumor tissue by Sanger sequencing and ion torrent sequencing in an NSCLC patient at Stage IV of disease.

Results: We found the presence of three coexisting mutations on the EGFR gene-two of which on exon 21 are present on the same allele, and the third, on exon 20, was analyzed by Sanger sequencing of the peripheral blood lymphocytes. The patient staged as cT4N0M1c (Stage IV) and started afatinib 40 mg daily 8 months ago, showing a clinical benefit.

Conclusion: In this report we describe the case of an NSCLC patient harboring three coexisting mutations on the EGFR gene, two of which are present on the same allele. This mutation pattern may represent, for patient progeny, a genetic risk of cancer development. Therefore it should be possible to obtain screening guidelines to improve the risk calculation for lung cancer susceptibility in the future.
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http://dx.doi.org/10.1177/1724600818782200DOI Listing
June 2018

Liquid Biopsy in Rare Cancers: Lessons from Hemangiopericytoma.

Anal Cell Pathol (Amst) 2018 7;2018:9718585. Epub 2018 Mar 7.

Policlinico Umerto I, II Division of Internal Medicine and Geriatrics, Sapienza University of Rome, Viale del Policlinico 155, 00161 Rome, Italy.

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.
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http://dx.doi.org/10.1155/2018/9718585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5863319PMC
October 2018

PD-L1 and epithelial-mesenchymal transition in circulating tumor cells from non-small cell lung cancer patients: A molecular shield to evade immune system

Oncoimmunology 2017;6(12):e1315488. Epub 2017 Apr 20.

Dipartimento di Medicina Molecolare, Sapienza Università di Roma Roma, Italia.

The programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) pathway has emerged as a critical inhibitory pathway regulating T-cell response in non-small-cell lung cancer (NSCLC), and the development of PD-1/PD-L1 inhibitors has changed the landscape of NSCLC therapy. Nevertheless, the high degree of non-responders demonstrates that we are still far from completely understanding the events underlying tumor immune resistance. Although the expression of PD-L1 in tumor tissue has been correlated with clinical response to anti PD-1 inhibitors, the ability of this marker to discriminate the subgroup of patients who derive benefit from immunotherapy is suboptimal. Circulating tumor cells (CTCs), as an accessible source of tumor for biologic characterization that can be serially obtained with minimally invasive procedure, hold significant promise to facilitate treatment-specific biomarkers discovery. We recently demonstrated that the presence of PD-L1 on CTCs apparently predicts resistance to the anti-PD-1 Nivolumab in metastatic NSCLC patients and that PD-L1 positive CTCs usually have an elongated morphology that can be ascribed to epithelial-mesenchymal transition (EMT). We here demonstrate for the first time that PD-L1 positive CTCs isolated from NSCLC patients are characterized by partial EMT phenotype, and hypothesize that the co-expression of PD-L1 and EMT markers might represent for these cells a possible molecular background for immune escape.
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http://dx.doi.org/10.1080/2162402X.2017.1315488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5706610PMC
April 2017

EpCAM-expressing circulating tumor cells in colorectal cancer.

Int J Biol Markers 2017 Oct 31;32(4):e415-e420. Epub 2017 Oct 31.

 Circulating Tumor Cells Unit, Department of Molecular Medicine, Sapienza University of Rome, Rome - Italy.

Background: Several studies have raised the issue of the inadequacy of CellSearch® to detect the entire pool of circulating tumor cells (CTCs) from blood of cancer patients, suggesting that cells expressing low levels of epithelial cell adhesion molecule (EpCAM) are not recognized by the capture reagent. In this exploratory study, we aimed to evaluate the status of EpCAM in CTCs isolated from a group of metastatic colorectal cancer patients, in 40% of whom, CTC had been found to be undetected by the CellSearch® system.

Methods: CTCs were analyzed using both a microfiltration method (ScreenCell) and CellSearch® in parallel. Furthermore, since EpCAM exists in 2 different variants, we investigated the presence of both its intracellular domain (EpICD) and extracellular domain (EpEX) through immunofluorescence staining of CTCs on filters.

Results: Results from immunofluorescence experiments demonstrated that, overall, EpICD and/or EpEX was expressed in 176 CTCs detected by ScreenCell, while the CellSearch® system was able to capture only 10 CTCs.

Conclusions: This is the first demonstration that the low sensitivity of CellSearch® to detect CTCs in colorectal cancer patients is not due to the lack of EpCAM.
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http://dx.doi.org/10.5301/ijbm.5000284DOI Listing
October 2017

The long-term prognostic value of survivin expressing circulating tumor cells in patients with high-risk non-muscle invasive bladder cancer (NMIBC).

J Cancer Res Clin Oncol 2017 Oct 29;143(10):1971-1976. Epub 2017 May 29.

Dipartimento Medicina Molecolare, Sapienza Università di Roma, Rome, Italy.

Objectives: Long-term follow-up study to evaluate the impact on disease-free survival and cancer-specific survival of survivin expression in tissue and CTCs from T1G3 bladder cancer patients.

Patients And Methods: The study was conducted using tumor tissue and blood samples from 54 patients with a primary diagnosis of T1G3 NMIBC. Survivin was evaluated by reverse transcription-polymerase chain reaction in tumor tissues. CTCs were isolated from blood by CELLection™ Dynabeads (Invitrogen, Carlsbad, CA, USA). Cells were lysed and cDNA was synthesized and analysed for the expression of CD45, CK8 and survivin. The endpoints of this long-termanalysis were disease-free survival, DFS and cancer-specific survival, CSS.

Results: Here, we report that, at 9 years of median follow-up, disease-free survival and cancer-specific survival are both significantly influenced by the expression of survivin in tumor tissue (p = 0.006), by the presence of CTCs (p < 0.0001) and by the expression of survivin in CTCs (p < 0.0001).

Conclusion: The statistically significant impact of survivin expressing CTCs on cancer-specific survival that we observed might be interpreted as the result of the persistence of a subpopulation of highlander cells in the blood of T1G3 bladder patients over time.
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http://dx.doi.org/10.1007/s00432-017-2449-8DOI Listing
October 2017

Monitoring PD-L1 positive circulating tumor cells in non-small cell lung cancer patients treated with the PD-1 inhibitor Nivolumab.

Sci Rep 2016 08 24;6:31726. Epub 2016 Aug 24.

Department of Molecular Medicine, Circulating tumor cells Unit, Sapienza University of Rome.

Controversial results on the predictive value of programmed death ligand 1 (PD-L1) status in lung tumor tissue for response to immune checkpoint inhibitors do not allow for any conclusive consideration. Liquid biopsy might allow real-time sampling of patients for PD-L1 through the course of the disease. Twenty-four stage IV NSCLC patients included in the Expanded Access Program with Nivolumab were enrolled. Circulating tumor cells (CTCs) were analyzed by CellSearch with anti-human B7-H1/PD-L1 PE-conjugated antibody. PD-L1 expressing CTCs were assessed at baseline, at 3 and 6 months after starting therapy, and correlated with outcome. At baseline and at 3 months of treatment, the presence of CTCs and the expression of PD-L1 on their surface were found associated to poor patients outcome. Nevertheless, the high frequency of PD-L1 expressing CTCs hampered to discriminate the role of PD-L1 in defining prognosis. Conversely although CTCs were found in all patients 6 months after treatment, at this time patients could be dichotomized into two groups based PD-L1 expression on CTCs. Patients with PD-L1 negative CTCs all obtained a clinical benefit, while patients with PD-L1 (+) CTCs all experienced progressive disease. This suggests that the persistence of PD-L1(+) CTCs might mirror a mechanism of therapy escape.
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http://dx.doi.org/10.1038/srep31726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4995431PMC
August 2016

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

Chin J Cancer Res 2015 Oct;27(5):491-6

1 Dipartimento di Medicina Molecolare, 2 Dipartimento di Medicina Sperimentale, Sapienza Università di Roma, Roma, Italy.

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.
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http://dx.doi.org/10.3978/j.issn.1000-9604.2015.04.09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4626813PMC
October 2015

Circulating tumor cells isolation: the "post-EpCAM era".

Chin J Cancer Res 2015 Oct;27(5):461-70

Dipartimento Medicina Molecolare, Sapienza Università di Roma, Roma, Italy.

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.
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http://dx.doi.org/10.3978/j.issn.1000-9604.2015.06.02DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4626820PMC
October 2015

Significance of circulating tumor cells in soft tissue sarcoma.

Anal Cell Pathol (Amst) 2015 18;2015:697395. Epub 2015 Jun 18.

Dipartimento Medicina Molecolare, Sapienza Università di Roma, Viale Regina Elena 324, 00161 Roma, Italy.

Circulating tumor cells can be detected from the peripheral blood of cancer patients. Their prognostic value has been established in the last 10 years for metastatic colorectal, breast, and prostate cancer. On the contrary their presence in patients affected by sarcomas has been poorly investigated. The discovery of EpCAM mRNA expression in different sarcoma cell lines and in a small cohort of metastatic sarcoma patients supports further investigations on these rare tumors to deepen the importance of CTC isolation. Although it is not clear whether EpCAM expression might be originally present on tumor sarcoma cells or acquired during the mesenchymal-epithelial transition, the discovery of EpCAM on circulating sarcoma cells opens a new scenario in CTC detection in patients affected by a rare mesenchymal tumor.
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http://dx.doi.org/10.1155/2015/697395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4488514PMC
March 2016

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

Expert Rev Mol Diagn 2015 9;15(7):925-32. Epub 2015 May 9.

Dipartimento di Medicina Molecolare, Sapienza Università di Roma, Roma, Italy.

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.
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http://dx.doi.org/10.1586/14737159.2015.1045491DOI Listing
March 2016

Circulating tumor cells detection has independent prognostic impact in high-risk non-muscle invasive bladder cancer.

Int J Cancer 2014 Oct 13;135(8):1978-82. Epub 2014 Mar 13.

Dipartimento Medicina Molecolare, Sapienza Università di Roma, Roma, Italy.

High-risk non-muscle invasive bladder cancer (NMIBC) progresses to metastatic disease in 10-15% of cases, suggesting that micrometastases may be present at first diagnosis. The prediction of risks of progression relies upon EORTC scoring systems, based on clinical and pathological parameters, which do not accurately identify which patients will progress. Aim of the study was to investigate whether the presence of CTC may improve prognostication in a large population of patients with Stage I bladder cancer who were all candidate to conservative surgery. A prospective single center trial was designed to correlate the presence of CTC to local recurrence and progression of disease in high-risk T1G3 bladder cancer. One hundred two patients were found eligible, all candidate to transurethral resection of the tumor followed by endovesical adjuvant immunotherapy with BCG. Median follow-up was 24.3 months (minimum-maximum: 4-36). The FDA-approved CellSearch System was used to enumerate CTC. Kaplan-Meier methods, log-rank test and multivariable Cox proportional hazard analysis was applied to establish the association of circulating tumor cells with time to first recurrence (TFR) and progression-free survival. CTC were detected in 20% of patients and predicted both decreased TFR (log-rank p < 0.001; multivariable adjusted hazard ratio [HR] 2.92 [95% confidence interval: 1.38-6.18], p = 0.005), and time to progression (log-rank p < 0.001; HR 7.17 [1.89-27.21], p = 0.004). The present findings provide evidence that CTC analyses can identify patients with Stage I bladder cancer who have already a systemic disease at diagnosis and might, therefore, potentially benefit from systemic treatment.
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http://dx.doi.org/10.1002/ijc.28830DOI Listing
October 2014

Circulating tumor cells: exploring intratumor heterogeneity of colorectal cancer.

Cancer Biol Ther 2014 May 12;15(5):496-503. Epub 2014 Feb 12.

Dipartimento Medicina Molecolare; Sapienza Università di Roma; Rome, Italy.

The hypothesis of the "liquid biopsy" using circulating tumor cells (CTCs) emerged as a minimally invasive alternative to traditional tissue biopsy to determine cancer therapy. Discordance for biomarkers expression between primary tumor tissue and circulating tumor cells (CTCs) has been widely reported, thus rendering the biological characterization of CTCs an attractive tool for biomarkers assessment and treatment selection. Studies performed in metastatic colorectal cancer (mCRC) patients using CellSearch, the only FDA-cleared test for CTCs assessment, demonstrated a much lower yield of CTCs in this tumor type compared with breast and prostate cancer, both at baseline and during the course of treatment. Thus, although attractive, the possibility to use CTCs as therapy-related biomarker for colorectal cancer patients is still limited by a number of technical issues mainly due to the low sensitivity of the CellSearch method. In the present study we found a significant discordance between CellSearch and AdnaTest in the detection of CTCs from mCRC patients. We then investigated KRAS pathway activating mutations in CTCs and determined the degree of heterogeneity for KRAS oncogenic mutations between CTCs and tumor tissues. Whether KRAS gene amplification may represent an alternative pathway responsible for KRAS activation was further explored. KRAS gene amplification emerged as a functionally equivalent and mutually exclusive mechanism of KRAS pathway activation in CTCs, possibly related to transcriptional activation. The serial assessment of CTCs may represent an early biomarker of treatment response, able to overcome the intrinsic limit of current molecular biomarkers represented by intratumor heterogeneity.
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http://dx.doi.org/10.4161/cbt.28020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026071PMC
May 2014

Effect of Serenoa repens (Permixon®) on the expression of inflammation-related genes: analysis in primary cell cultures of human prostate carcinoma.

J Inflamm (Lond) 2013 14;10:11. Epub 2013 Mar 14.

Department of Urology, Sapienza University of Rome, Rome, Italy ; Prostate Unit - Department Urology, University Sapienza, Viale Policlinico 155, 00161, Rome, Italy.

Background: To analyze the expression at basal level of inflammation-related cytokines and chemokines and the activation status of the NF-κB pathway, together with the proliferation and apoptosis indexes in two widely used in vitro tumor models, the androgen-dependent human Prostate Cancer (PC) cell line LNCaP and the androgen-independent PC3 , and in primary cultures of human PC cells. To assess in these models and primary cultures, the effects of Serenoa repens (LSESr, Permixon®) on proliferation/apoptosis ratio, inflammation-related genes expression and NF-κB pathway activation.

Methods: The expression of IL-6, CCL-5, CCL-2, COX-1, COX-2, iNOS inflammation-related genes has been evaluated at the mRNA level in two in vitro human PC models (LNCaP and PC3 cell lines) and in 40 independent human prostatic primary cultures obtained from PC patients undergoing radical prostatectomy. Tissue fragments were collected from both PC lesions and normal hyperplastic tissue counterparts for each case. All cultures were treated with two different amounts of Permixon® (44 and 88 μg/ml) for different time points (16, 24, 48 and 72 hours), depending on the cell type and the assay; the expression of inflammation-related genes, cell growth (proliferation/apoptosis ratio) and NF-κB activation has been analyzed in treated and untreated cells by means of semi-quantitative RNA-PCR, cell proliferation and immunofluorescence respectively.

Results: We detected a significant reduction (p <0.001) in PC and normal cells proliferation due to Permixon ® treatment. This result was related to an increase of the apoptotic activity showed by an increase in the number of anti-caspase-3 fluorescent cells. Almost all the inflammation-related genes (IL-6, CCL-5, CCL-2, COX-2 and iNOS) were expressed at the basal level in in vitro cultured cells and primary cultures and down-regulated by Permixon® treatment. This treatment interfered with NF-kB activation, detecting by the translocation of more than 30% of NF-κB p65 subunit to the nucleus.

Conclusions: The present study confirms the expression of inflammatory pattern in PC. We showed the effect of Permixon® on down-regulation of inflammatory-related genes in cell lines and in primary cultures. The inhibitory effect of Permixon® on cell growth could be partly associated to the down-regulation of inflammatory-related genes and to the activation of NF-κB pathway in prostate tissue.
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http://dx.doi.org/10.1186/1476-9255-10-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3653817PMC
May 2014

Circulating tumor cells and "suspicious objects" evaluated through CellSearch® in metastatic renal cell carcinoma.

Anticancer Res 2011 Dec;31(12):4219-21

Dipartimento Medicina Molecolare, Sapienza Università di Roma, Viale Regina Elena 324, 00161 Rome, Italy.

Background: Recent evidence supports the hypothesis that the CellSearch assay, used in the enumeration of circulating tumor cells (CTCs), may underestimate the number of CTCs, especially in tumors, such as renal cell carcinoma, frequently lacking cytokeratin expression. According to the CellSearch guidelines, all objects with no clear cytokeratin staining are defined as "suspicious objects", and are not counted as CTCs. The aim of this study was to investigate the presence of CTCs and "suspicious objects" in 25 patients affected by metastatic renal cell carcinoma (mRCC).

Patients And Methods: Twenty-five patients were enrolled in the study, all with a diagnosis of metastatic clear cell RCC. The CellSearch™ system was used to count the CTC in 7.5 mL of whole blood. A further 10 ml blood obtained from each patient was used to isolate CTCs through CELLection™ Dynabeads®. The expression of cytokeratin (CK) 8, 18, 19 and CD44 were evaluated by RT-PCR.

Results: Standard CTCs and suspicious objects were found in 16% and 60% of the patients, respectively. CK-8/18/19 transcripts were found in 15% and CD44 in 68% of the 19 patients with evidence of classical CTC or "suspicious objects" as assessed by Cellsearch.

Conclusion: The low number of CTCs detected through CellSearch in renal cell carcinoma may be due to the presence of a CTC population with atypical characteristics and a peculiar gene expression profile, characterized by lack of cytokeratin expression and gain of CD44.
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December 2011

Circulating tumor cells count and characterization in a male breast cancer patient.

Cancer Biol Ther 2011 Sep 1;12(5):379-82. Epub 2011 Sep 1.

Department of Molecular Medicine,apienza University of Rome, Rome, Italy.

A 64-y-old man presented to Medical Oncology Department a metastatic invasive ductal breast carcinoma, positive for estrogen (ER) and progesterone receptors (PR) and Her2/neu negative. The patient was treated with different lines of therapy, with rapid radiological progression of disease. After four courses of a third-line chemotherapy, a radiological stable disease was maintained. The patient was followed by serial blood drawings for the characterization and count of circulating tumor cells (CTC). This is the first report concerning the predictive and prognostic value of CTC in a male breast cancer patient.
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http://dx.doi.org/10.4161/cbt.12.5.16304DOI Listing
September 2011

Circulating tumour cells lacking cytokeratin in breast cancer: the importance of being mesenchymal.

J Cell Mol Med 2011 May;15(5):1066-70

Department of Molecular Medicine, Sapienza University, Viale Regina Elena, Rome, Italy.

Circulating tumour cells (CTCs) are independent predictor of prognosis in metastatic breast cancer. Nevertheless, in one third of patients, circulating tumour cells are undetected by conventional methods. Aim of the study was to assess the prognostic value of circulating tumour cells expressing mesenchymal markers in metastatic breast cancer patients. We isolated CTC from blood of 55 metastatic breast cancer patients. CTC were characterized for cytokeratins and markers of epithelial mesenchymal transition. The gain of mesenchymal markers in CTC was correlated to prognosis of patients in a follow-up of 24 months. The presence of mesenchymal markers on CTC more accurately predicted worse prognosis than the expression of cytokeratins alone. Because of the frequent loss of epithelial antigens by CTC, assays targeting epithelial antigens may miss the most invasive cell population. Thus, there is an urgent need to improve detection methods to identify CTC which undergone epithelial mesenchymal transition program.
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http://dx.doi.org/10.1111/j.1582-4934.2011.01285.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3822619PMC
May 2011
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