Angela Gradilone

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.

bcl-2/bax mRNA expression ratio as prognostic factor in low-grade urinary bladder cancer

Apoptotic cell death represents an important mechanism for the precise regulation of cell numbers, and a defence mechanism against tumoral cells. bcl‐2 and bax genes are known to be involved in the control of apoptotic pathways; in particular, the ratio between bcl‐2 and bax represents a cell rheostat that is able to predict a cells response toward life or death to an apoptotic stimulus. In the present study we investigated the role of bcl‐2 and bax gene expression in a panel of 37 low‐grade tumours of the urinary bladder, and correlated the expression of these genes to the prognosis of patients in a follow‐up of more than one year. We found that levels of bax expression higher than bcl‐2 in bladder tumours well correlates to a better outcome for patients. Early relapses are much more frequently observed in those patients whose tumours express more bcl‐2 than bax mRNA. We conclude that the bcl‐2/bax expression ratio may be considered as a marker for disease progression in low grade bladder tumours, independently of clinical staging and histological grading.

Survivin, bcl-2, bax, and bcl-X Gene Expression in Sentinel Lymph Nodes From Melanoma Patients

PURPOSE The expression of apoptosis-related genes, such as survivin, bcl-2, bcl-X, and bax, has been evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry in sentinel lymph nodes (SLNs) from melanoma patients and then correlated to the outcome of patients. PATIENTS AND METHODS Thirty-six SLNs were examined. After RNA extraction, an RT-PCR followed by Southern blot hybridization was performed to detect survivin, bcl-2, bcl-X, and bax mRNA. bcl-2, survivin, and bax gene expression was evaluated, whenever possible, also by immunohistochemistry at the protein level. RESULTS We found a significant correlation (P <.005) between survivin expression and outcome of patients; in fact, 61.5% of patients expressing survivin gene progressed or died because of the disease, whereas 38.5% are currently disease-free. Among patients negative for survivin expression, 100% are disease-free after a median follow-up time of 52.9 months. We did not find a significant correlation between bcl-2, bax, and bcl-X gene expression and outcome of patients. In fact, these genes were found equally expressed in patients with disease progression and in disease-free patients. CONCLUSION Our findings show a variable expression of apoptosis-related genes in SLNs of melanoma patients; more interestingly, we found that survivin expression correlates to outcome of patients in a statistically significant way, whereas the expression of other genes, such as bcl-2, bax, and bcl-X, did not seem to correlate to progression of disease. We suggest that the detection of survivin gene expression by RT-PCR in SLNs may be a useful prognostic indicator.

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.

Chemosensitivity profile assay of circulating cancer cells: prognostic and predictive value in epithelial tumors

The prognostic value associated with the detection of circulating tumor cells (CTCs) in metastatic breast cancer by the CellSearch™ technology raise additional issues regarding the biological value of this information. We postulated that a drug‐resistance profile of CTCs may predict response to chemotherapy in cancer patients and therefore could be used for patient selection. One hundred 5 patients with diagnosis of carcinoma were enrolled in a prospective trial. CTCs were isolated from peripheral blood, and positive samples were evaluated for the expression of a panel of genes involved in anticancer drugs resistance. The drug‐resistance profile was correlated with disease‐free survival (DFS; patients in adjuvant setting) and time to progression (TTP; metastatic patients) in a 24‐months follow‐up. Objective response correlation was a secondary end point. Fifty‐one percent of patients were found positive for CTCs while all blood samples from healthy donors were negative. The drug‐resistance profile correlates with DFS and TTP (p < 0.001 in both). Sensitivity of the test: able to predict treatment response in 98% of patients. Specificity of the test: 100%; no sample from healthy subject was positive for the presence of CTCs. Positive and negative predictive values were found to be 96.5 and 100%, respectively. We identified a drug‐resistance profile of CTCs, which is predictive of response to chemotherapy, independent of tumor type and stage of disease. This approach may represent a first step toward the individualization of chemotherapy in cancer patients.

Prognostic significance of survivin‐expressing circulating tumour cells in T1G3 bladder cancer

To evaluate the prognostic significance of survivin in tumour tissues and that of survivin‐expressing circulating tumour cells (CTCs) in T1G3 bladder tumours, as the prognosis of T1G3 bladder cancer is highly variable and unpredictable from clinical and pathological prognostic factors.

Prevalence of human papillomavirus, cytomegalovirus, and Epstein-Barr virus in the cervix of healthy women.

The prevalence of some sexually transmitted viruses, possibly involved in cervical carcinogenesis, was studied in the cervix of women with normal cytology. The presence of human papillomaviruses (HPV) type 16 and 18, cytomegalovirus (CMV) and Epstein‐Barr virus (EBV) genomes in cervical cells taken from 143 healthy Italian women was investigated using the polymerase chain reaction (PCR). The study population was divided into four groups with respect to age as follows: group I, 17 to 25 years, n = 48 women; group II, 26 to 35 years, n = 30; group III, 36 to 50 years, n = 32; and group IV, 51 to 70 years, n = 33. In the first age group prevalence rates of HPV 16, CMV and EBV infection of 23%, 21% and 19% were found respectively. The infection rates of HPV 16 and CMV were shown to decrease with age, with prevalences of HPV 16 at 10% in the second group, 6% in the third and 3% in the fourth and of CMV at 13% in the second and third and 6% in the fourth groups. The prevalence of EBV infection did not decrease with increasing age (19% in the first and third groups, 20% in the second and 18% in the fourth). The occurrence of HPV 18 genome was very low (0‐3%) and independent of age. In the first age group a higher percentage of double infections (16.6%) was found than in the three other age groups (6% in the second and third and 3% in the fourth). The finding of multiple infections in younger women requires further study in order to clarify the implications of such viral infections in healthy women and their contribution to the development of genital tract malignancies.