Emanuela Flamini

Pathogenesis of osteoblastic bone metastases from prostate cancer.

Prostate cancer is the second leading cause of cancer‐related death in men. A typical feature of this disease is its ability to metastasize to bone. It is mainly osteosclerotic, and is caused by a relative excess of osteoblast activity, leading to an abnormal bone formation. Bone metastases are the result of a complex series of steps that are not yet fully understood and depend on dynamic crosstalk between metastatic cancer cells, cellular components of the bone marrow microenvironment, and bone matrix (osteoblasts and osteoclasts). Prostate cancer cells from primary tissue undergo an epithelial‐mesenchymal transition to disseminate and acquire a bone‐like phenotype to metastasize in bone tissue. This review discusses the biological processes and the molecules involved in the progression of bone metastases. Here we focus on the routes of osteoblast differentiation and activation, the crosstalk between bone cells and tumor cells, and the molecules involved in these processes that are expressed by both osteoblasts and tumor cells. Furthermore, this review deals with the recently elucidated role of osteoclasts in prostate cancer bone metastases. Certainly, to better understand the underlying mechanisms of bone metastasis and so improve targeted bone therapies, further studies are warranted to shed light on the probable role of the premetastatic niche and the involvement of cancer stem cells. Cancer 2010.

Serum levels of tumour necrosis factor alpha and other cytokines do not correlate with weight loss and anorexia in cancer patients

Cancer anorexia-cachexia syndrome (CACS), which is characterized by progressive weight loss (WL) and anorexia (A), is present in 50% of advanced cancer patients and in 80% of terminally ill cancer patients. One of the most controversial aspects of CACS is its oetiopathogenesis; experimental studies have identified certain cytokines [Tumour necrosis factor alpha (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), and gamma interferon (\gg-IFN)] as possible cofactors in the onset of the syndrome. The aim of our study was to investigate the correlation between serum levels of circulating cytokines and severity of CACS. The following series of parameters was indentified in 61 patients with advanced and terminal cancer: stage of disease; Karnofsky performance status (KPS) and clinical symptoms; biohumoral, anthropometric and immunological situation; level of circulating cytokines. All these parameters were evaluated for a possible link with WL/A. Our data do not show any significant correlation between circulating cytokines and WL/A. A direct correlation was identified between WL/A and nausea (P=0.03 andP<0.001, respectively) whereas inverse correlations were observed for both factors as regards arm circumference (P<0.001 for both), wrist circumference (P<0.001 for both), KPS (P<0.001 andP=0.003, respectively) and creatinine (P=0.005 andP=0.03, respectively). Other biochemical factors, such as haemoglobin, haematocrit, glycaemia, prealbumin, sodium and chlorine were also correlated with at least one of the two clinical parameters in question. Unexpected results were seen in the increases in CD20 and CD4 and in the CD4/CD8 ratio. Serum levels of these cytokines do not, therefore, appear to be critical in the onset of CACS. On the contrary, our findings confirmed the clinico-laboratory picture that is characteristic of CACS. If we consider the possibility that CACS is provoked by an aspecific response of the hosts defence mechanisms against prolonged neoplastic attack, the increase in CD4 (helper lymphocytes) could be linked to the persistent response.

Free DNA and Carcinoembryonic Antigen Serum Levels: An Important Combination for Diagnosis of Colorectal Cancer

Purpose: The identification of new molecular markers for the early detection of colorectal cancer has become an important objective. We compared the sensitivity and specificity of free circulating DNA with that of the more conventional carcinoembryonic antigen (CEA) and evaluated the two markers in combination. Experimental Design: The study was carried out on 75 healthy donors and 75 colorectal cancer patients. Free DNA was determined in serum with quantitative PCR analysis. The diagnostic accuracy of each assay was calculated using receiver operating characteristic (ROC) curves. The diagnostic relevance of the two-marker combination was analyzed by the logistic regression model. Results: Median free DNA concentration was ∼5-fold higher in patients than in healthy donors (P < 0.001). The area under the ROC curve was 0.86, and when 12.5 ng/mL was used as cutoff, 81.3% sensitivity and 73.3% specificity were observed for the overall series. As CEA and free DNA provided independent diagnostic information, they were also considered in combination. ROC curve analysis of the combined CEA and free DNA algorithms showed a higher diagnostic capacity (area under the ROC curve, 0.92) than that of markers considered singly, with 84% sensitivity and 88% specificity. Conclusions: Free circulating DNA, especially when used in combination with CEA, represents a potentially useful tool for the diagnosis of early-stage colorectal cancer.

Relationship between plasma concentrations of 5-fluorouracil and 5-fluoro-5,6-dihydrouracil and toxicity of 5-fluorouracil infusions in cancer patients.

This study investigated the relationship among the pharmacokinetics of 5-fluorouracil (5-FU) and 5-fluoro-5,6-dihydrouracil (5-FDHU); the activity of dihydropyrimidine dehydrogenase (DPD) in peripheral blood mononuclear cells; and treatment-related toxicity in 26 patients with surgically resected colorectal cancer treated with short daily infusions of 5-FU adjuvant chemotherapy, each cycle consisting of 5 consecutive days every 4 weeks. After the first chemotherapeutic cycle, severe stomatitis and diarrhea occurred in 5 patients and were related to the variations in the systemic disposition of the drug rather than to DPD activity. These patients showed a significant decrease in 5-FU clearance, and an increase in the 5-FU/5-FDHU area under the time-concentration curve (AUC) ratio, as compared with patients who experienced mild toxicities, whereas a low DPD activity was observed in only 2 patients. In conclusion, the results of this study demonstrate that the alterations in 5-FU and 5-FDHU pharmacokinetics are related to severe toxicities in patients treated with short intravenous infusion of 5-FU.

Adjuvant immunotherapy with tumor infiltrating lymphocytes and interleukin-2 in patients with resected stage III and IV melanoma.

Adoptive immunotherapy with tumor infiltrating lymphocytes (TIL) and interleukin (IL)-2 is reasonably effective in the treatment of patients with advanced melanoma. However, theoretically it should be of greater benefit as adjuvant therapy, especially in high-risk stages (resected stages III and IV). In a preliminary study, 25 patients (aged 23–72 years) with stage III–IV melanoma who underwent resection of metachronous metastases were reinfused with TIL cultivated and expanded in vitro with IL-2 from surgically removed metastases. IL-2 (starting dose 12 × 106 IU/m2) was co-administered as a continuous infusion according to Wests scheme. A total of 8/22 (36.3%) evaluable patients were disease-free (DF) at a median follow-up of 5 years. DF survival (DFS) and overall survival (OS) rates were 44% and 37%, respectively, at 2 years, and 52% and 45% at 3 years. The CNS was the only site of disease recurrence in 57% of patients who relapsed. DF patients received a higher median dose of IL-2 than those who progressed (total dose 110 × 106 versus 86 × 106 IU/m2, respectively). The progressive reduction in IL-2 dosage allowed all patients to complete treatment without permanent grade 4 toxicity. Analysis of tumor immunosuppression factors in lymphocytes inside the tumor (TCR ζ and ε chains, p56lck, FAS, and FAS-ligand) confirmed that the immunologic potential of TIL, depressed at the time of metastasectomy, was significantly restored after in vitro culture with IL-2. Adoptive immunotherapy with TIL and IL-2 could improve DFS and OS, although further work is required to determine its role in the treatment of patients with high-risk melanoma.

Adjuvant adoptive immunotherapy with tumour-infiltrating lymphocytes and modulated doses of interleukin-2 in 22 patients with melanoma, colorectal and renal cancer, after radical metastasectomy, and in 12 advanced patients

Abstract Adoptive tumour infiltrating lymphocytes (TIL) in combination with a modulated dosage of interleukin-2 (IL-2) can be used with acceptable toxicity in the treatment of immunogenic tumours. Following an experience of reinfusion in advanced melanoma, colorectal and renal cancer patients, treatment was given to disease-free patients after metastasectomy. The high risk of relapse and favourable ratio between reinfused TIL and possible microscopic residual disease determined this choice of adjuvant treatment. A group of 12 patients with advanced disease (7 melanoma, 4 colorectal carcinoma, 1 kidney carcinoma) were treated with TIL (median 5.8×1010 cells) and IL-2 (West’s schedule) modulated towards a lower dosage (from 12 to 6 MIU/day) in order to maintain an acceptable level of toxicity. As treatment was well tolerated, it was offered to another 22 patients in an adjuvant setting after metastasectomy (11 melanoma, 10 colorectal carcinoma, 1 renal cancer), the median dose of TIL reinfused being 4.95×1010 cells. No objective response was observed in advanced patients: all patients progressed after a median of 1.5 months (0–8 months) and median survival was 8 months (3–22+ months). Thirteen patients from the second group are still disease-free after a median of 23+ months (9+–47+ months). The remaining 9 patients relapsed after a median of 5 months (3–18 months). Toxicity was moderate as clinical and hepatic/renal function parameters were used to assess the need for dose reductions. Consequently, there was great diversity in IL-2 dosages administered. In particular, there seemed to be a difference in IL-2 doses administered between disease-free cases and those who progressed (17.5 MIU/day versus 7 MIU/day in melanoma patients; 11.2 MIU/day versus 7.1 MIU/day in colorectal cancer patients). By contrast, no differences were observed between number of TIL reinfused and clinical response. Phenotypical characteristics of reinfused TIL were similar to those reported in the literature: 97% were CD3 and 92% were CD8. Aspecific cytolytic activity was evaluated on 12 cases whereas, in 2 melanoma cases, autologous tumour tissue was available for the specific cytotoxicity test. Perforin levels in TIL measured at the end of culture were generally high or very high. Cytokine levels were measured on the supernatant at the end of culture, with an estreme variability in results. Finally, ζ chain and p56lck were histologically assessed on the resected tissue from which TIL were cultivated. There were virtually none of the former and a complete absence of the latter, which concurs with data reported in the literature. The same immunocytochemical analysis was carried out on TIL at the end of culture. This time an almost complete restoration of both functions was seen, especially in melanoma patients, who are still free from disease. The study is on-going and it has been decided to focus on disease-free patients after metastasectomy in order to increase the number and possibility of clinical and histological correlations.

Tumor infiltrating lymphocytes and continuous infusion interleukin-2 after metastasectomy in 61 patients with melanoma, colorectal and renal carcinoma.

Aims and background Adoptive immunotherapy with tumor infiltrating lymphocyte (TIL) reinfusion plus continuous interleukin-2 (IL-2) infusion could represent an innovative way of treating immunogenic tumors. This study therefore recruited melanoma, colorectal and renal carcinoma patients whose metastases had been surgically removed. Study design The treatment was initially given to 22 patients with advanced disease and more recently to 39 disease-free (DF) patients after radical metastasectomy. The latter group was selected in view of a theoretically better lymphocyte/tumor cell ratio and with the aim to improve disease-free and overall survival (DFS-OS) in very high risk patients. The starting IL-2 dose was 12 MlU/day (Wests schedule); doses were modulated on the bases of toxicity parameters. Even though patients received different total amounts of IL-2, all of them completed the treatment. Results The treatment was offered to 22 advanced-stage cancer patients (12 melanomas, 9 colorectal carcinomas, 1 kidney carcinoma). Few and short stabilizations were observed with a median survival of 12 months (range, 3-29). Subsequently, another 39 patients were treated in an adjuvant setting after radical metastasectomy (18 melanomas, 19 colorectal carcinomas, 2 kidney cancers). Eleven out of 17 DF melanoma patients (64.7%) are still free of disease after a median of 37+ months (range, 5+ - 69+). In the group of DF colorectal cancer patients eight (44.4%) are still DF after a median of 21+ months (range, 7+ - 67+ months). One of the two patients with kidney cancer is still DF after 28+ months. Two patients (1 melanoma and 1 colorectal cancer) had just been treated and were therefore not evaluable. Severe toxicity occurred in three cases but was rapidly resolved. There was a great diversity in IL-2 doses administered; comparison of the total IL-2 dose administered between the patients who are still DF and those who progressed revealed no difference between the two groups of colorectal cancer patients, whereas melanoma patients who progressed rebeived an average IL-2 dose of 6.5 MlU/day versus 15.8 MlU/day in DF patients. No differences were observed in any of the groups between the number of TILs reinfused and clinical response. Conclusions The study is still ongoing; it has been decided to focus on DF melanoma patients after radical metastasectomy, for whom the data seem to be encouraging. Further endpoints of the study are the role of IL-2 dosage in the adjuvant setting, and the possibility to make correlations between biological parameters and clinical results.

Bone metastases detection by circulating biomarkers: OPG and RANK-L

Osteoprotegerin (OPG) is a decoy receptor of the receptor activator of nuclear factor-κB ligand (RANK-L) and plays an important role in the formation of metastatic bone lesions. We evaluated the usefulness of circulating OPG and RANK-L for the detection of bone metastases. We enrolled 143 individuals in the study: 30 healthy donors (HD) and 113 breast cancer patients. Among patients, 49 had no evidence of disease (NEDP), 54 had bone metastases (BMP) at first diagnosis, and 10 had visceral metastases (VMP). Both transcripts were determined in peripheral blood samples using quantitative PCR. Receiver operating characteristic (ROC) curve analysis was used to calculate the diagnostic accuracy of OPG, RANK-L, CEA and CA15-3. OPG and RANK-L median values were significantly lower in BMP (median 0.5, range 0.1-5.7, p<0.001 and median 0.5, range 0.1-4.5, p=0.024, respectively) compared to NEDP (median 1.7, range 0.4-8.9 and median 0.8, range 0.2-3.8, respectively), regardless of the number and type of bone lesions or the presence of visceral metastases. The area under the ROC curve (NEDP vs. BMP) was higher for OPG (82.5, 95% CI 74.5-90.6) than for RANK-L (69.2, 95% CI 59.0-79.40). Specificity for OPG was 87.7% (95% CI 75.7-94.2) and sensitivity was 74.1% (95% CI 60.4-85.0), both values increasing when considered together with CEA and CA15-3. For VMP, OPG and RANK-L were expressed in only one patient. Our results highlight the potentially important role of circulating OPG in the diagnosis of bone metastases. A confirmatory study on a larger case series is ongoing.

Comparison between single saturating dose ligand binding assay and enzyme immunoassay for low-salt extractable oestrogen and progesterone receptors in breast cancer: A multicentre study

An excellent correlation between ligand binding assay (LBA) and enzyme immunoassay (EIA) for both oestrogen (ER) and progesterone (PR) receptors has been reported. Nevertheless, considering that the clinical value of any discrepancy between LBA and EIA probably varies with the receptor level, we undertook a collaborative study in which a single saturating dose (SSD) LBA and EIA were compared in different ER and PR dose ranges. The values of ER measured by EIA were higher in tumours with low or intermediate receptor content, causing a misclassification of ER status in 9% of cases (ER+: 77.5%, EIA, 68.8% SSD). In the case of ER, EIA values tended to be higher than SSD in all centres. For PR, EIA and SSD were generally more comparable (PR+: 66.0% EIA, 72.0% SSD, discordance rate 6%), with EIA showing, however, different trends in different centres. PR concentration was not significantly different in ER SSD-/EIA+ and in ER SSD+/EIA+ cases, suggesting that EIA detects at least in part integer ER. We conclude that although EIA may be a reliable methodological alternative to SSD, the two methods are not interchangeable until effective cut-off levels for clinical decisions are assessed for EIA.

Time-dependent pharmacokinetics of 5-fluorouracil and association with treatment tolerability in the adjuvant setting of colorectal cancer.

The authors evaluated the influence of 5‐fluorouracil (5‐FU) on treatment tolerability in 81 colorectal cancer patients given adjuvant 5‐FU intravenously plus folinic acid for 6 cycles. The pharmacokinetics of 5‐FU and its inactive metabolite 5‐fluoro‐5,6‐dihydrouracil (5‐FDHU) were measured on days 1 and 5 of the first chemotherapy cycle, 5 and 45 minutes after bolus administration. 5‐FU clearance was significantly lower on day 5 (62.64 ± 20.16 L/h/m2) than on day 1 (74.83 ± 31.61 L/h/m2). The lower 5‐FU clearance values also predicted the side effects during the entire course of chemotherapy. In particular, patients with low clearance values on day 1 had a further reduction in this parameter on day 5, associated with severe toxicities. In conclusion, 5‐FU alters its clearance, which could be partly due to a reduction in 5‐FDHU biotransformation. These findings have safety implications for poor metabolizers whose drug clearance may fall below the threshold during repeated treatment cycles.