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.

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.

OBJECTIVE: 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. PATIENTS AND METHODS: The study included 54 patients with T1G3 non-muscle-invasive bladder cancer. Additional inclusion criteria were: tumour size <3 cm, absence of carcinoma in situ and multifocality. The planned follow-up was 24 months. Survivin was evaluated by reverse transcription-polymerase chain reaction in tumour tissues. CTCs were isolated from blood by CELLection Dynabeads (Invitrogen, Carlsbad, CA, USA) coated with the monoclonal antibody towards the human epithelial cell adhesion molecule. Cells were lysed and Dynabeads Oligo(dT) was used to capture poly A + mRNA. cDNA was synthesized and analysed for the expression of CD45, CK8 and survivin. The primary endpoint was disease-free survival (DFS); the favourable group at 24 months was defined as that with no clinical evidence of disease; the unfavourable group was that with evidence of recurrent disease or progressive disease. Tumour survivin expression and presence of CTC were correlated with DFS. Multivariate analysis was used to investigate whether the presence of CTC was an independent indicator of DFS. RESULTS: Survivin was found in half of the tumours; patients with survivin-negative tumours had a longer DFS than those with survivin-positive tumours (chi-square, P = 0.029). CTCs were found in 24/54 patients (44%); 92% of CTC expressed survivin. The difference in DFS between CTC-ve and CTC+ve patients was statistically significant (chi-square, P < 0.001). The presence of CTC was an independent prognostic factor for DFS (P < 0.001). CONCLUSION: The presence of CTC is an independent prognostic factor in patients with T1G3 bladder cancer.

A chemosensitivity test to individualize intravesical treatment for non-muscle-invasive bladder cancer.

OBJECTIVE: To describe the design of a new chemosensitivity assay based on the expression of genes involved in the resistance to standard intravesical regimens, to allow individualization of therapy for high-risk non-muscle-invasive bladder cancer. PATIENTS AND METHODS: To date, 35 patients with high-risk non-muscle-invasive bladder cancer have been enrolled, all candidates for transurethral resection of the bladder (TURB) followed by intravesical treatment. The intravesical regimen was chosen according to the risk profile of each patient. All patients were evaluated by cystoscopy 3 and 6 months after TURB. According to the molecular characterization of each tumour, our team of molecular oncologists determined for each patient a molecular profile of chemosensitivity to BCG, mitomycin c, anthracyclines and gemcitabine. This profile was then correlated to the response to intravesical therapy 6 months after TURB. RESULTS: This chemosensitivity test was able to predict response to treatment in 96% of patients. The assay is easy to perform, inexpensive and quick. CONCLUSION: Our results, although preliminary, are encouraging for the future of an individualized therapeutic approach, with the aim to provide a higher treatment success rate while sparing patients unnecessary toxicity from drugs that are not suited for their tumours.

Failure of apoptosis and activation on NFkappaB by celecoxib and aspirin in lung cancer cell lines.

Recent studies have demonstrated that antineoplastic activity of Cox-2 inhibitors may depend on targets other than Cox: among those, nuclear factor kappaB (NFkappaB) seems the most promising. Although preclinical studies have suggested that aspirin and Cox-2 inhibitors may influence the progression of lung cancer, the molecular mechanisms of these protective effects in this tumor type has not been fully elucidated. We investigated the effects of celecoxib and aspirin in the induction of apoptosis and in the ability to activate NFkappaB in three non-small cell lung cancer cell lines. Apoptosis was evaluated by FACS, caspase activation assay and expression of apoptosis-related genes by RT-PCR, while NFkappaB activation was assessed by immunofluorescence. No apoptotic response was observed after treatment with both high and low dose of celecoxib. Nevertheless, celecoxib at both concentrations induced a strong NFkappaB activation, with increased expression of NFkappaB-dependent genes, such as bcl-2, bcl-XL and survivin. Similarly, aspirin at both concentrations did not induce any apoptotic response, but activated NFkappaB in a dose-dependent manner. This study supports the hypothesis that NFkappaB activation is an important effect of NSAIDs in lung cancer, leading to apoptosis resistance. This effect of both aspirin and celecoxib may be considered undesirable in lung cancer chemoprevention.

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.

Comparison of extracellular matrix and apoptotic markers between benign lesions and carcinomas in human breast.

The expression of the extracellular matrix-related genes, such as fibronectin, laminin and tenascin C, and apoptosis-related genes, such as bax, bcl2 and survivin, was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and by immunohistochemistry in normal breast tissue and benign and malignant breast tumors and then correlated to several clinical parameters: estrogen and progesterone receptors, Ki67, ErbB2, tumor size, lymph node status and grading. Seventy-three breast tissue samples were examined. After RNA extraction, an RT-PCR was performed to detect fibronectin, laminin, tenascin C, bax, bcl2 and survivin gene expression. Thirty-two samples were evaluated also by immunohistochemistry at the protein level to detect fibronectin, laminin, tenascin C, bax and survivin. We found a significant correlation (P=0.025) between fibronectin gene expression and lymph node status, and a significant negative correlation (P=0.049) between laminin gene expression and Ki67. In addition, we found a statistically significant increase in survivin transcription in malign tumors compared to fibroadenomas (P=0.024). The negative correlation between laminin transcription and Ki67 could suggest that laminin impacts negatively on tumor proliferation, and the positive correlation between fibronectin and lymph node status may lead to consider fibronectin as predictive of long distance metastasis.

Tenascin C and epidermal growth factor receptor as markers of circulating tumoral cells in bladder and colon cancer.

Tenascin C has been recently suggested as a tumor marker, however, its levels in serum has been evaluated only in patients with head and neck cancer and melanoma. In this study, we investigated Tenascin C expression in blood samples from colorectal and bladder cancer patients, compared to that of epidermal growth factor receptor (EGFR), a known circulating tumor marker in these cancer types. RT-PCR specific for Tenascin C and EGFR was performed on RNAs extracted from blood samples of 60 patients affected by colon or bladder cancer. We then investigated the statistical association between Tenascin C, EGFR expression and disease-free survival using the Kaplan-Meier method. Furthermore, in order to select which variable between EGFR and Tenascin C was the most predictive for recurrence, a Cox model for proportional risk was applied. Among all patients analysed, a significantly higher disease-free time was found in the group negative for both EGFR and Tenascin C expression; EGFR expression was significantly correlated to disease progression in stages III and IV, whereas in all patients with stage I and II disease Tenascin C correlated better with prognosis. Negative expression of both EGFR and Tenascin C identifies a group of patients with poor tendency to disease recurrence and longer relapse-free time. While Tenascin C expression seems to influence prognosis in patients with low-stage disease, EGFR appears a marker of worse prognosis in patients with high-staged tumors.

UDP-glucuronosyltransferases 1A expression in human urinary bladder and colon cancer by immunohistochemistry.

UDP-glucuronosyltrasferases (UGTs) are detoxifying enzymes, which convert endogenous substrates, dietary constituents and potential carcinogens to inactive hydrophilic glucuronides. Although the liver is considered the most important organ for glucuronidation, UGTs are also expressed in extrahepatic tissues. Since UGTs may be important to protect cells from cancer in organs naturally exposed to potential carcinogens such as smoking and diet derivatives, we investigated the UGT expression in normal and malignant tissues from urinary bladder and large intestine. The study was carried out by immunohistochemistry, using an antiserum recognizing all the UGT1A isoforms. Our results showed that UGTs were highly expressed on the surfaces of the normal bladder as well as in normal large bowel mucosa. The neoplastic counterpart showed a general protein down-regulation associated with a different cellular localization. We found that 3/11 papillary and 3/6 invasive urothelial carcinomas were virtually negative, whereas 2/2 papillomas and 8/11 papillary bladder carcinomas still expressed the protein. In colon cancer the enzyme down-regulation was even more dramatic. In fact, a faint diffused cytoplasmic expression or scattered cell positivity was observed only in adenomas with low grade dysplasia (5/5) and in 2/11 carcinomas. Interestingly, 5/5 adenomas with high grade dysplasia, 9 carcinomas, the lymph nodes and liver metastases were UGT-negative, suggesting that the loss of UGT is associated with the early phase of neoplastic transformation. Based on our results we suggest that UGTs constitutive expression in the normal mucosa could protect these organs from carcinogens released in the bladder or introduced directly with the diet in the colon.

Persistence of epidermal growth factor receptor and interleukin 10 in blood of colorectal cancer patients after surgery identifies patients with high risk to relapse.

PURPOSE: Despite the great number of studies performed to detect circulating markers of disease progression in colorectal cancer, few have shown a clinical use; among those, epidermal growth factor receptor (EGFR) and, more recently, interleukin (IL)-10. In this article, we sought to investigate how primary surgery could affect expression levels of EGFR, IL-6, and IL-10 in blood from colorectal cancer patients. EXPERIMENTAL DESIGN: We investigated by reverse transcriptase-PCR assay the expression at mRNA level of EGFR, IL-6, and IL-10 in blood samples taken from 56 colorectal cancer patients. Each gene expression was evaluated 1 day before and 20 days after primary surgery. Persistence of each gene in blood after surgery was then correlated to the relapse free time in a follow-up of 3 years. RESULTS: In blood samples taken before surgery, EGFR, IL-6, and IL-10 were found expressed in 62, 100, and 100% of patients, respectively. EGFR expression, but not IL-6 and IL-10, correlates with stage of disease. In the group of 41 patients who underwent follow-up studies, EGFR was found persistently high in 67%; 94% of them had relapse. Persistence of IL-10 after surgery also identifies relapses in 89% of cases. IL-6 persistence was not found to significantly correlate to progression of disease. CONCLUSIONS: Persistence of both EGFR and IL-10 in blood of colorectal cancer patients after surgery identifies patients with high propensity to relapse. These findings may suggest a clinical use of preoperative EGFR/IL-10 reverse transcriptase-PCR assay in the prediction of tumor recurrence.

Beyond the Immune Suppression: The Immunotherapy in Prostate Cancer.

Prostate cancer (PCa) is the second most common cancer in men. As well in many other human cancers, inflammation and immune suppression have an important role in their development. We briefly describe the host components that interact with the tumor to generate an immune suppressive environment involved in PCa promotion and progression. Different tools provide to overcome the mechanisms of immunosuppression including vaccines and immune checkpoint blockades. With regard to this, we report results of most recent clinical trials investigating immunotherapy in metastatic PCa (Sipuleucel-T, ipilimumab, tasquinimod, Prostvac-VF, and GVAX) and provide possible future perspectives combining the immunotherapy to the traditional therapies.

Synthesis and pharmacological screening of a large library of 1,3,4-thiadiazolines as innovative therapeutic tools for the treatment of prostate cancer and melanoma.

Antimitotic agents are widely used in cancer chemotherapy but the numerous side effects and the onset of resistance limit their clinical efficacy. Therefore, with the purpose of discovering more selective and efficient anticancer agents to be administered alone or in combination with traditional drugs, we synthesized a large library of 1,3,4-thiadiazoline analogues, maintaining the pharmacophoric structure of an antiproliferative compound known as K858: this is a new inhibitor of kinesin Eg5, able to induce the mitotic arrest in colorectal cancer cells and in xenograft ovarian cancer cells. We screened 103 compounds to assess their antiproliferative activity on PC3 prostate cancer cell line. Two derivatives, compounds 32 (corresponding to K858) and 33, have shown to be the most effective against prostate tumor cells and also towards two melanoma cell lines (SK-MEL-5 and SK-MEL-28) at low micromolar concentrations, confirming the pharmacological activity of this scaffold and revealing the potential role of 1,3,4-thiadiazolines in the management of cancer.

Raloxifene modulates interleukin-6 and tumor necrosis factor-alpha synthesis in vivo: results from a pilot clinical study.

Raloxifene (RAL), a selective estrogen receptor modulator, is indicated for the prevention and treatment of postmenopausal osteoporosis. RAL, by decreasing bone turnover, prevents bone loss and microarchitecture damage, reducing the incidence of osteoporotic fractures. Our previous in vitro data demonstrated that RAL modulates osteoclast activity by, at least in part, an IL-6- and TNF-alpha-dependent mechanism. In this study we evaluated the effects of RAL treatment (60 mg/d) on circulating levels of these cytokines in 14 postmenopausal women with osteoporosis. Lumbar bone density (determined by dual energy x-ray absorptiometry) and IL-6 and TNF-alpha levels were measured before and after 6 and 24 months of therapy. After 24 months, RAL increased bone density. IL-6 and TNF-alpha expression, elevated before treatment, significantly decreased (50% and 30%, respectively) after 6 months. This effect was sustained up to the end of the treatment (75% and 35%, respectively). Thus, our data show that RAL can modulate circulating levels of cytokines involved in osteoclastogenesis and bone resorption, suggesting that modulation of soluble factors could play a pivotal role in the mechanisms of the osteoprotective effect of RAL.

Detection of CK19, CK20 and EGFR mRNAs in peripheral blood of carcinoma patients: correlation with clinical stage of disease.

Expression of genes such as cytokeratin 19 (CK19), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) has been investigated at mRNA level in peripheral blood of carcinoma patients to detect the presence of circulating tumor cells (CTC). We performed this study because recent literature emphasizes that the importance of CK19, 20 and EGFR mRNAs in CTC as prognostic factors remains unclear especially for breast, head and neck and colon cancer patients. Reverse transcriptase polymerase chain reaction (RT-PCR) followed by Southern blot hybridization was performed in blood samples from 47 subjects (12 colorectal, 15 head and neck and 20 breast carcinoma patients), as well as in 35 healthy donors. The CK19 expression was found in 36/47 patients (9 colorectal, 9 head and neck and 18 breast cancer), two patients (one affected by colorectal and one by head and neck cancer) were positive for CK20 whereas EGFR was found expressed in 9 patients (3 colorectal, 5 head and neck and one breast cancer). Seven of 35 and 4/35 healthy donors displayed positivity for the expression of CK19 and CK20 genes respectively, whereas no EGFR mRNA was found in this group. The correlation of the detection of CTC in peripheral blood with progression of the disease in a follow-up period of 40 months did not show any prognostic value to the presence of mRNAs of these biomarkers in blood. We believe that research should be addressed, at least for breast cancer, to the identification of occult metastases in sentinel lymph nodes, such as recently performed in melanoma patients.

Gemcitabine-induced apoptosis in 5637 cell line: an in-vitro model for high-risk superficial bladder cancer.

Recent data suggest that new treatment options for superficial bladder cancer are necessary, owing to the high recurrence rate after conventional treatment, especially in T1G3 and Bacillus Calmette-Guerin-refractory patients. Phase I and II studies have demonstrated that gemcitabine may represent a candidate for intravesical therapy in superficial bladder cancer. Despite clinical trials, the in-vitro cytotoxic and proapoptotic effects of gemcitabine have been poorly investigated. In the present study, we investigated how gemcitabine affects apoptosis in bladder cancer cell line 5637, which has the same molecular features of high-risk superficial bladder cancer. Apoptosis was evaluated by DNA fragmentation, flow cytometry and caspase activation. bcl-2, bcl-X, bax, survivin and fas gene expression were also evaluated by reverse-transcriptase polymerase chain reaction. Nuclear factor-kappa B activation was assessed by immunofluorescence. Gemcitabine induced apoptosis in 5637 cells in a time-dependent manner, with activation of caspase-3, -8 and -9. Expression of bcl-2, bax, survivin and bcl-X was not affected by treatment, whereas fas strongly increased after 24 h of treatment. After treatment, we failed to find any nuclear localization of nuclear factor-kappa B. As gemcitabine-induced apoptosis involves fas upregulation, these results may encourage the investigation of intravesical gemcitabine in fas-negative bladder tumors. Furthermore, as nuclear factor-kappa B activation by cisplatin, doxorubicin and adriamycin may result in enhanced proliferation, migration, immortality and inhibition of apoptosis, the observation that gemcitabine does not activate nuclear factor-kappa B may have implications in intravesical therapy of high-risk superficial bladder cancer.