Silvia Brugnatelli

Circulating endothelial cells and endothelial progenitors as predictive markers of clinical response to bevacizumab-based first-line treatment in advanced colorectal cancer patients

BACKGROUND Despite the consistent clinical results demonstrated by studies on anti-angiogenic drugs targeted against the vascular endothelial growth factor in metastatic colorectal cancer (mCRC) patients, no specific direct/indirect biomarker of their efficacy has been validated. In this field, circulating endothelial cells (CECs) and endothelial progenitor cells (CEPs) have recently been proposed as noninvasive biomarkers. PATIENTS AND METHODS The absolute numbers of CEPs, total CECs (tCECs) and their resting (rCECs) and activated subsets were evaluated by multiparameter flow cytometry in 40 mCRC patients at baseline and before the administration of the third and sixth course of a bevacizumab-based first-line treatment. Fifty healthy subjects were utilized as control. RESULTS The overall response rate was 80%, overall clinical benefit was 90% and median progression-free survival (PFS) was 13.8 months. In our patients, tCECs and rCECs were significantly increased compared with healthy subjects. The patients who achieved a radiological response showed, at baseline, a significant decrease of rCECs and a trend in decrease of tCECs in comparison with patients not achieving response. Finally, a baseline absolute number of tCEC and rCEC <40 cells/ml was evidenced in patients with a longer PFS. No correlation was found regarding CEP. CONCLUSIONS Our study suggests significant correlations between both tCEC and rCEC baseline levels and the antitumor efficacy of a bevacizumab-based combination therapy in mCRC patients, thus confirming that these biomarkers could be used in the clinical setting as an early predictor of tumor response.

Dendritic cells and vascular endothelial growth factor in colorectal cancer: correlations with clinicobiological findings.

Objective: Dendritic cells (DC) are central to the development of immune system responses. In a cohort of 54 patients affected by colorectal cancer, we prospectively investigated the number of peripheral blood (PB) DC type 1 (DC1) and type 2 (DC2) and correlated their counts and functionality to the stage of the disease and to vascular endothelial growth factor (VEGF) levels. Results: At diagnosis, compared with healthy controls, patients presented reduced PBDC1 and PBDC2 numbers (p < 0.001). Moreover, in cancer patients, PBDC showed low levels of DC-associated antigens (HLA DR, p = 0.004; CD11c, p < 0.001; CD83, p = 0.01; CD86, p = 0.007 and Mannose receptor, p = 0.029), an upregulation of CXCR4 (p = 0.017) and a reduced T cell stimulation capability (p < 0.001). DC1 and DC2 loss was higher in stage D versus stage ABC patients (p = 0.003 and p = 0.002, respectively); surgery and chemotherapy appeared to attenuate a DC defect, although the restoration of normal PBDC levels is completed only at 6 and 12 months after diagnosis, respectively. In this series of patients, PBDC1 and PBDC2 numbers inversely correlated with VEGF serum levels (p < 0.001), suggesting a possible effect of this cytokine on DC compartment. In culture, the exposure of monocyte-derived DC to VEGF produced a dramatic alteration of DC differentiation by (1) induction of apoptosis, (2) alteration of DC immunophenotypic profile and (3) increased CXCR4 expression. Exposure to anti-VEGF blocking antibodies reversed VEGF inhibitory effects in all cases. Conclusions: These findings suggest that in colorectal cancer patients there is a numerical and functional impairment of PBDC compartment possibly related to the stage of the disease and to VEGF levels.

Combined use of intraoperative ultrasound and indocyanine green fluorescence imaging to detect liver metastases from colorectal cancer

OBJECTIVES Surgical excision is the standard strategy for managing liver metastases from colorectal carcinoma. The achievement of negative (R0) margins is a major determinant of disease-free survival in these patients. Current imaging techniques are of limited value in achieving this goal. A new approach to the intraoperative detection of colorectal liver metastatic tissue based on the emission of indocyanine green (ICG) fluorescence was evaluated. METHODS A total of 25 consecutive patients with liver metastases from primary colorectal cancers who were eligible for liver resection received a bolus of ICG (0.5 mg/kg body weight) 24 h before surgery. During surgery, ICG fluorescence, which accumulates around lesions as a result of defective biliary clearance, was detected with a near-infrared camera system, the Photodynamic Eye (PDE). Numbers of lesions detected by, respectively, PDE + ICG, intraoperative ultrasound (IOUS) and preoperative computed tomography (CT) were recorded. RESULTS The near-infrared camera plus ICG revealed a total of 77 metastatic liver nodules. Preoperative CT demonstrated 45 (58.4%) and IOUS showed 55 (71.4%). Preoperative CT and IOUS alone were inferior to the combined use of PDE + ICG and IOUS in the detection of lesions of ≤ 3 mm in size. CONCLUSIONS This experience suggests that PDE + ICG, combined with IOUS, may represent a safe and effective tool for ensuring the complete surgical eradication of liver metastases from colorectal cancer.

Cell kinetics of human brain tumors: in vivo study with bromodeoxyuridine and flow cytometry

Bromodeoxyuridine (BUDR) is a thymidine analog which is incorporated into the DNA of proliferating cells. Since the dose of BUDR needed to label cells is not toxic, cell labelling can be accomplished in vivo, by infusing the substance in patients. A monoclonal antibody against BUDR is then used to identify BUDR-labelled cells. The same cell population can also be stained for DNA content with propidium iodide (PI). Using bivariate flow cytometry (FCM) for measurements, both the percentage of BUDR-labelled cells and their total DNA content can be evaluated. This technique allows one to obtain the labelling index (LI) and the DNA synthesis time (TS). The potential doubling time (Tpot) and the fractional turnover rate (FTR) can be mathematically derived, so that a complete picture of tumor growth can be obtained. Our aim was to ascertain whether this method is clinically applicable and whether the kinetic values obtained are reliable. We studied 22 patients with benign and malignant brain tumors, and observed no immediate toxicity from BUDR administration. The BUDRLI obtained ranged from 0.9% to 3.9% (median: 2.0%) in meningiomas and from 3.8% to 7.6% (median: 6.3%) in malignant gliomas (P less than 0.01). The fraction of S-phase cells determined with the BUDR FCM technique was statistically similar to that found by single DNA flow cytometric analysis performed on duplicate samples of both benign and malignant brain tumors. The TS obtained in malignant gliomas ranged from 10.5 to 227 h (median: 12.8). The calculated Tpot ranged from 7.6 to 26.8 days (median: 11.6), and the calculated FTR ranged from 3.7 to 13.1 cells/100 cells/day (median: 8.8). These data suggest that in vivo BUDR infusion coupled with FCM can be performed in clinical settings, and it is reliable and can easily be used for kinetic studies in clinical trials aimed at evaluating the prognostic relevance of proliferative parameters and in planning tumor treatment.

Cell kinetics with in vivo bromodeoxyuridine and flow cytometry: Clinical significance in acute non-lymphoblastic leukaemia

From 1986 to 1988, 54 consecutive previously untreated patients with acute non-lymphoblastic leukaemia (ANLL), median age 54 years, were treated for remission (CR) induction with vincristine and intravenous medium-dose cytarabine sequentially followed by daunomycin and infusion cytarabine. CR patients received intensive consolidation. Bone marrow blast kinetics was studied before therapy with in vivo bromodeoxyuridine and bivariate flow cytometry. CR rate was 70.2%, median CR was 13.2 months, responsive patient survival was 16.9 months and overall survival was 9.2 months. Besides lower median age, the 33 responsive patients also had shorter potential doubling time (Tpot) and greater cell production rate (PR) than the 14 unresponsive patients (mean values = 10.9 vs. 25.4 days, P less than 0.05, and 14.7 vs. 8.9 cells/100 cells/day, P less than 0.02, respectively), due to a higher mean labelling index (7.0 vs. 5.1%, P less than 0.05) and/or to a shorter mean DNA synthesis time (13.6 vs. 18.6 hours, P less than 0.05). Besides lower white blood cell count and bone marrow blast percentage, patients who experienced CR longer than 13.2 months had shorter Tpot (P less than 0.05) and a greater PR (P less than 0.02) than those who relapsed before this time. These data indicate that kinetic parameters have prognostic relevance in ANLL patients treated with sequential vincristine, cytarabine and daunomycin for inducing CR and with intensive consolidation after CR, a high proliferative activity being a favourable factor for both CR achievement and its duration.

Weekly Administration of Gemcitabine Plus Docetaxel in Patients with Advanced Breast Cancer: A Phase 1 Study

Objective: This study was designed to determine the maximum tolerable dose (MTD) of gemcitabine plus docetaxel, both given on a weekly schedule, in patients with pretreated metastatic breast cancer (MBC). Methods: Heavily pretreated patients with MBC, aged 18–75 years with World Health Organization performance status of 0–2 were enrolled. Three escalating weekly doses of docetaxel (30, 35 and 40 mg/m2) followed by a weekly fixed dose of gemcitabine, 800 mg/m2, were administered on days 1, 8 and 15 of a 28-day cycle. Dose-limiting toxicity (DLT) included grade >3 hematologic toxicity and grade >2 stomatitis, asthenia, diarrhea or organ-specific toxicity (except alopecia). Dose escalation was stopped if ≧3 of 5 patients at any dose level experienced DLT. Results: Eighteen patients (median age 56 years) received a mean of 4.1 (range 1–6) cycles. Asthenia, stomatitis and leukopenia were the main DLTs. One of 5 patients had DLT at dose level 1 and 2 of 5 patients at dose level 2. At dose level 3, 3 of 5 patients had DLTs. Three additional patients treated at dose level 3 confirmed that the MTD had been reached. Therefore, the recommended docetaxel dose in combination with gemcitabine 800 mg/m2 for phase II studies was established at the next lower dose, 35 mg/m2. Of 12 evaluable patients, 7 (58%) achieved an objective response. Conclusions: Gemcitabine 800 mg/m2 plus docetaxel 35 mg/m2 on days 1, 8 and 15 of a 28-day cycle is a safe regimen which shows activity in heavily pretreated patients with MBC. Further phase II investigations with this combination are now warranted.

Single-dose palonosetron and dexamethasone in preventing nausea and vomiting induced by moderately emetogenic chemotherapy in breast and colorectal cancer patients.

AIMS AND BACKGROUND Palonosetron, a unique second-generation 5-HT3 receptor antagonist, has been demonstrated to control emesis related to chemotherapy-induced nausea and vomiting (CINV). The aim of this study was to evaluate the efficacy and tolerability of palonosetron followed by a single dose of dexamethasone in patients with breast cancer (BC) or colorectal cancer (CRC) receiving moderate emetogenic chemotherapy (MEC). METHODS AND STUDY DESIGN Chemotherapy-naive BC and CRC patients were given MEC as adjuvant or first-line treatment. Palonosetron (0.25 mg IV) and dexamethasone (8 mg IV) were administered before chemotherapy on day 1. The primary endpoint was complete response (CR; no vomiting and no use of rescue medication) during the overall study period (days 1-5). The antiemetic response was evaluated during the acute (day 1) and delayed (days 2-5) phases. RESULTS Sixty-eight patients were enrolled (median age 61 years, 56 females; BC = 40, CRC = 28). CR was observed in 46 of 68 patients (67.6%), while CR during the acute and delayed phases was 75.0% in each cancer group. The antiemetic regimen was well tolerated. CONCLUSIONS A single administration of palonosetron and dexamethasone on day 1 in BC and CRC patients adequately controls CINV during the entire period of emetic risk.

UFT as Maintenance Therapy in Patients with Advanced Colorectal Cancer Responsive to the FOLFOX4 Regimen

Background: In advanced colorectal cancer (ACC), FOLFOX4 has been accepted as a standard chemotherapeutic regimen. Due to the neurotoxicity induced by oxaliplatin, which occurs in about 50% of patients during the 6-month FOLFOX4 regimen, and the frequent need for hospitalization, alternative regimens may be required. We aimed to determine whether a ‘maintenance’ therapy with oral UFT (uracil-tegafur) in patients responding to FOLFOX4 is able to maintain the response and improve the quality of life (QoL) as a result of the outpatient regimen and lower psychological distress. Methods: Untreated patients with ACC who did not progress after 6 months of FOLFOX4 received oral UFT until disease progression or unacceptable toxicity. The aim of the study was to maintain the response obtained with the FOLFOX4 regimen for at least 6 months. The secondary objective was to evaluate QoL during the two different treatment regimens utilizing the 36-item Short Form Health Survey (SF-36). Results: From January 2003 to August 2004, out of the enrolled 30 patients [22 males and 8 females; 2 patients with a complete response (CR), 14 patients with a partial response (PR) and 6 patients in stable disease (SD) after 6 months of FOLFOX4] 22 continued therapy with UFT until progression without significant toxicity; the remaining 8 patients (27%) had progressive disease (PD) during or at the end of FOLFOX4 and were treated with other regimen. After 6 months of UFT, 4 patients (13%) had CR, 6 patients (20%) PR and 4 patients (13%) SD; 16 patients (53%) progressed. Median follow-up was 31 months [interquartile range (IQR): 20–31 months]; 14 patients died of PD. The median time to progression was 13.9 (IQR: 7.7–20.1) months and the median survival time was 31 months (IQR: 20–31 months). Evaluation of QoL demonstrated a trend towards better QoL during UFT treatment. Conclusions: These results support the feasibility of maintaining good response and improving QoL (measured by SF-36) with an oral fluoropyrimidine after combination chemotherapy in ACC patients; moreover, since UFT can be used orally, patient compliance is increased and the duration of hospitalization can be decreased.

Liver involvement as the hallmark of aggressive disease in light chain amyloidosis: distinctive clinical features and role of light chain type in 225 patients

Previous studies have linked liver involvement to a poor prognosis in immunoglobulin light chain (AL) amyloidosis. However, the reason for this dismal outcome remains unclear. We compared the clinical presentation and outcome of 225 patients with and 643 subjects without liver involvement from a series of 868 consecutive patients with AL amyloidosis diagnosed between 1986 and 2007. Patients with liver involvement and l clones had a more severe cardiac dysfunction as assessed by biomarkers and shorter median survival (1.4 years) compared to subjects with hepatic amyloidosis and k clones (4.8 years) and to patients without liver involvement (4.4 years). The poor outcome of patients with hepatic amyloidosis compared with subjects without liver involvement among those with l clones was confirmed in the subgroups of patients with heart involvement, with heart failure and without heart involvement. Introduction: The liver is involved in approximately 25% of patients with light chain (AL) amyloidosis, although one autopsy series found histological evidence of hepatic amyloidosis in 70% of patients [1,2]. The clinical clues that help physicians to recognize liver involvement include hepatomegaly (particularly in the absence of congestive heart failure) and the elevated concentration of serum alkaline phosphatase [3], whereas liver biopsy is not recommended, in order to avoid the risk of bleeding [4]. Previous studies linked liver involvement with a very short survival (median 8.5 months) in AL amyloidosis [4,5]. Severe intrahepatic cholestasis, concomitant hyposplenism, and congestive heart failure at diagnosis have been reported as predictive of short survival in patients with liver involvement [4,6,7]. However, the reason for the dismal outcome of patients with hepatic AL amyloidosis remains unclear, and there is no large series comparing patients with liver involvement to other subjects with AL amyloidosis. In the present study, we compare the clinical presentation and outcome of subjects with and without liver involvement from a series of 868 consecutive patients evaluated at the Pavia Amyloidosis Research and Treatment Center. Methods: We identified 225 patients with liver involvement and 643 subjects without liver involvement from a series of 868 consecutive patients with AL amyloidosis between 1986 and 2007. All the patients had biopsy-proven AL amyloidosis. Hereditary amyloidoses were excluded by DNA analysis. The assessment of organ involvement and of hematologic response was based on the 2005 International Society of Amyloidosis consensus criteria [3]. In particular, patients were deemed to have hepatic amyloidosis if total liver span was 415 cm in the absence of heart failure, or alkaline phosphatase was 41.5 times the upper limit of normal. Differences in quantitative variables between subgroups were tested for significance by the nonparametric Mann–Whitney U test. The Fisher exact test or the w test were used to compare categorical variables, as appropriate. Survival curves were plotted according to Kaplan-Meier, and differences in survival were tested for significance by the logrank test. To identify independent predictors of survival, a multivariable cox model was fitted. Results and discussion: Six hundred forty-three patients without liver involvement were compared with 225 subjects with hepatic AL amyloidosis. Kappa clones were more frequently observed in hepatic amyloidosis (33% vs. 22%, p1⁄4 0.002). The heart was involved in 152 (67%) of the 225 subjects with liver involvement and in 391 (61%) of the 643 patients without amyloidosis of the liver. Patients with hepatic involvement had higher NT-proBNP (median 3018 vs. 1806 ng/l, p1⁄4 0.01) and cTnI (median 120 vs. 40 ng/l, p1⁄4 0.008) compared with patients without liver involvement. However, if subjects with k and l clones were considered separately, the difference remained significant only for l patients (NT-proBNP 3134 vs. 1661 ng/l, p1⁄4 0.03; cTnI 130 vs. 45 ng/l, p1⁄4 0.02). There was no significant difference between patients with and without liver involvement in the time between the onset of symptoms and diagnosis, in the frequency of heart involvement and heart failure, as well as in mean left ventricular wall thickness and ejection fraction at echocardiography, irrespective of the type of the amyloidogenic light chain. The severity of liver involvement, as assessed by liver size and alkaline phosphatase concentration, was not different 92

Systematic analysis of human oncogenic viruses in colon cancer revealed EBV latency in lymphoid infiltrates

BackgroundEnvironmental factors may play a role in colon cancer. In this view, several studies investigated tumor samples for the presence of various viral DNA with conflicting results.FindingsWe undertook a systematic DNA analysis of 44 consecutive, prospectively collected primary tumor samples by real time and qualitative PCR for viruses of known or potential oncogenic role in humans, including polyomavirus (JCV, BKV, Merkel cell polyomavirus), HPV, HTLV, HHV-8 and EBV. Negative controls consisted of surgical resection margins. No evidence of genomic DNA fragments from tested virus were detected, except for EBV, which was found in a significant portion of tumors (23/44, 52%). Real-time PCR showed that EBV DNA was present at a highly variable content (median 258 copies in 105 cells, range 15–4837). Presence of EBV DNA had a trend to be associated with high lymphocyte infiltration (p = 0.06, χ 2 test), and in situ hybridization with EBER1-2 probes revealed latency in a fraction of these lymphoid cells, with just a few scattered plasma cells positive for BZLF-1, an immediate early protein expressed during lytic replication. LMP-1 expression was undetectable by immunohistochemistry.ConclusionsThese results argue against a significant involvement of the tested oncogenic viruses in established colon cancer.