Cristina Raimondi

Epithelial-mesenchymal transition and stemness features in circulating tumor cells from breast cancer patients.

Currently used methods to detect and enumerate circulating tumor cells (CTCs) rely on the expression of the epithelial cell adhesion molecule (EpCAM) and cytokeratins. This selection may exclude cells that have undergone intrinsic modifications of their phenotype, as epithelial-mesenchymal transition (EMT). Aim of the study was to investigate the expression of EMT and stemness markers in CTCs from breast cancer patients in all stages of disease. 92 female breast cancer patients were enrolled. CTCs were isolated by CELLection™ Dynabeads® coated with the monoclonal antibody toward EpCam. Samples found positive for CTCs presence (CD45−/CK+) were evaluated for the expression of ER alpha, HER2, ALDH1, vimentin, and fibronectin. Samples negative for CTCs presence (CD45−/CK−) were also evaluated for the expression of vimentin and fibronectin, used as markers of EMT. CTCs were found in 66% of patients. The distribution of CTCs presence according to stage and grade of disease was found statistically significant. The expression of ALDH1 on CTCs was found to correlate to stage of disease and to the expression of vimentin and fibronectin. In 34% of patients, we detected cells with negative CK/CD45 expression but positive expression of vimentin and fibronectin. There is an urgent need for optimizing CTCs detection methods through the inclusion of EMT markers. The detection of cells in mesenchymal transition, retaining EMT and stemness features, may contribute to discover additional therapeutic targets useful to eradicate micrometastatic disease in breast cancer.

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

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.

Prognostic value of circulating tumor cells in nonmuscle invasive bladder cancer: a CellSearch analysis

BACKGROUND Circulating tumor cells (CTCs) provide prognostic information in patients with metastatic tumors. Recent studies have shown that CTCs are released in circulation in an early phase of cancer disease so that their presence is under investigation in the adjuvant setting. Few studies investigated the prognostic significance of CTCs enumeration in patients with metastatic and advanced bladder cancer. The current study has analyzed the presence of CTC in patients with nonmuscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS Forty-four NMIBC patients were enrolled and included in a 24-month follow-up program. Blood drawings were carried out in all patients at the first diagnosis. CellSearch system (Veridex; LLC, Raritan, NJ) was used for CTCs enumeration. RESULTS CTC were detectable in 8/44 patients (18%). Presence of CTC was found significantly associated to shorter time to first recurrence (6.5 versus 21.7 months, P < 0.001). Median time to progression was not reached, due to the short follow-up period. CTC presence was found associated to concomitant carcinoma in situ and higher T category. CONCLUSION The detection of CTC in this setting of disease may allow to distinguish patients with high risk of recurrence from those with high risk of progression, as well as to early identify patients candidate for adjuvant treatment.BACKGROUND Circulating tumor cells (CTCs) provide prognostic information in patients with metastatic tumors. Recent studies have shown that CTCs are released in circulation in an early phase of cancer disease so that their presence is under investigation in the adjuvant setting. Few studies investigated the prognostic significance of CTCs enumeration in patients with metastatic and advanced bladder cancer. The current study has analyzed the presence of CTC in patients with nonmuscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS Forty-four NMIBC patients were enrolled and included in a 24-month follow-up program. Blood drawings were carried out in all patients at the first diagnosis. CellSearch system (Veridex; LLC, Raritan, NJ) was used for CTCs enumeration. RESULTS CTC were detectable in 8/44 patients (18%). Presence of CTC was found significantly associated to shorter time to first recurrence (6.5 versus 21.7 months, P<0.001). Median time to progression was not reached, due to the short follow-up period. CTC presence was found associated to concomitant carcinoma in situ and higher T category. CONCLUSION The detection of CTC in this setting of disease may allow to distinguish patients with high risk of recurrence from those with high risk of progression, as well as to early identify patients candidate for adjuvant treatment.

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

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.

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

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.

Cancer Stem Cells and Epithelial-Mesenchymal Transition: Revisiting Minimal Residual Disease

The cancer stem cell (CSC) hypothesis provides an attractive model of tumour development and progression, holding that solid tumours are hierarchically organized and sustained by a minority of the tumour cell population with stem cell properties, such as self-renewal, tumorigenicity and multilineage differentiation capacity. Therapeutic resistance, underlying tumour recurrence and the lack of curative treatments in metastatic disease, raise the question if conventional anticancer therapies target the right cells. Indeed, these treatments might miss CSCs, which represent a more chemoresistant and radioresistant subpopulation within cancer. Recently, a direct link between the epithelial-mesenchymal transition process and the gain of stem cell competence were demonstrated in cultured breast cells. In particular, it was shown that the induction of EMT program not only allow cancer cells to disseminate from the primary tumor, but also promotes their self-renewal capability. Furthermore, the expression of stemness and EMT markers in CTCs were associated with resistance to conventional anti-cancer therapies and treatment failure, highlighting the urgency of improving tools for detecting and eliminating minimal residual disease.

Circulating tumor cells: Exploring intratumor heterogeneity of colorectal cancer

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.

Circulating tumor cells, colon cancer and bevacizumab: the meaning of zero

It has been recently observed that in metastatic breast cancer patients treated with bevacizumab, circulating tumor cells (CTC) are often undetected [1]. Due to the large use of bevacizumab as first-line treatment for metastatic colorectal cancer (mCRC), we investigated the predictive value of CTC count in patients with mCRC treated with a first-line chemotherapy plus bevacizumab compared with those treated with chemotherapy plus cetuximab. The predictive value of CTC was defined as the effect of change in CTC number after 2–3 months of treatment on response to therapies evaluated by RECIST criteria. CTC count was carried out through CellSearch system (Veriplex Corporation, Warren, NJ). In a group of 27 patients treated with bevacizumab, the median number of baseline CTC was 2.7 (range 0–37). After 6–12 weeks of treatment, CTC count dropped to 0 in 24 patients (89%) and 1 in 3 patients (11%). Surprisingly, 56% of patients with 0 CTC at follow-up evaluation had a progressive disease (PD) (Table 1, A).

Clinical utility of circulating tumor cell counting through CellSearch ® : the dilemma of a concept suspended in Limbo

To date, 10 years after the first demonstration of circulating tumor cells (CTCs), prognostic significance in metastatic breast cancer using the US Food and Drug Administration–cleared system CellSearch®, the potential utility of CTCs in early clinical development of drugs, their role as a surrogate marker of response to therapy, and their molecular analysis for patient stratification for targeted therapies are still major unsolved questions. Great expectations are pinned on the ongoing interventional trials aimed to demonstrate that CTCs might be of value for guiding treatment of patients and predicting cancer progression. To fill the gap between theory and practice with regard to the clinical utility of CTCs, a bridge is needed, taking into account innovative design for clinical trials, a revised definition of traditional CTCs, next-generation CTC technology, the potential clinical application of CTC analysis in non-validated settings of disease, and finally, expanding the number of patients enrolled in the studies. In this regard, the results of the first European pooled analysis definitely validated the independent prognostic value of CTC counting in metastatic breast cancer patients.

How circulating tumor cells escape from multidrug resistance: translating molecular mechanisms in metastatic breast cancer treatment.

Resistance to anthracyclines is responsible for treatment failure in most patients with metastatic breast cancer. According to recent studies, the expression of specific drug transporters (MRPs) on circulating tumor cells is predictive of prognosis in different cancer types. We observed that patients whose circulating tumor cells expressed MRP1 and MRP2, two drug-export pumps responsible for anthracyclines efflux, who received conventional anthracyclines had a significantly shorter time to progression compared with patients sharing same characteristics who received non pegylated liposomal doxorubicin (P < 0.005). These results may highlight a new appealing role of the liposomal doxorubicin formulation, not only because of its reduced cardiac toxicity but especially referring to its theoretical efficacy in anthracycline-resistant breast cancer patients.