Marco Danova

Objective Response to Chemotherapy As a Potential Surrogate End Point of Survival in Metastatic Breast Cancer Patients

PURPOSE To assess the validity of objective response to chemotherapy as a surrogate end point for survival in metastatic breast cancer. PATIENTS AND METHODS We carried out a meta-analysis on individual data from 2,126 metastatic breast cancer patients who were enrolled onto 10 randomized trials comparing standard versus intensified epirubicin-containing chemotherapy. RESULTS The intensified chemotherapy was associated with a significantly higher tumor response rate compared with standard chemotherapy (pooled odds ratio for nonresponse, 0.60; 95% CI, 0.51 to 0.72). The intensified regimens also led to better (although not significant) survival (pooled odds ratio, 0.94; 95% CI, 0.86 to 1.04; P = .22). Tumor response was a highly significant predictor of survival (P < .0001). When tumor response was introduced in the Cox model, the hazard ratio in favor of experimental treatment changed from 0.94 to 1.005 (95% CI, 0.91 to 1.11; P = .92), indicating that no residual effect of the experimental treatment on survival was present once tumor response was adjusted for. This suggests that the overall survival benefit of intensified epirubicin was a result of the increase in response rate. The median survival time of patients with complete response and partial response was 28.8 months (95% CI, 25.4 to 45.3 months) and 21.3 months (95% CI, 19.2 to 22.4 months), respectively; whereas, the median survival time of patients with no response was 14.6 months (95% CI, 13.9 to 15.4 months). CONCLUSION These results support the hypothesis that the achievement of an objective response to chemotherapy in metastatic breast cancer is associated with a true survival benefit. The potential role of objective response as a surrogate end point for survival in chemotherapy trials of metastatic breast cancer warrants further investigation.

All-trans retinoic acid (ATRA)-induced apoptosis is preceded by G1 arrest in human MCF-7 breast cancer cells.

In this study the effects of all-trans retinoic acid (ATRA) on cell cycle and apoptosis of MCF-7 human breast cancer cells were investigated to elucidate the mechanisms underlying the antineoplastic potential of this retinoid in breast cancer. The antiproliferative effect of ATRA was evaluated by DNA content measurements and dual-parameter flow cytometry of bromodeoxyuridine (BrdU) incorporation and of the expression of cell cycle-related proteins (Ki-67 as proliferation marker and statin as quiescence marker) vs DNA content. Apoptosis was also studied by flow cytometry of either DNA content or Annexin V labelling. After 10(-6) M ATRA treatment, the fraction of S-phase cells decreased significantly, and cells accumulated in the G0/G1 range of DNA contents. Dual-parameter flow cytograms showed a decrease in the percentage of Ki-67-labelled cells (after 10 days, only 20% of the cells were still positive for Ki-67 compared with 95% in controls), while the fraction of statin-positive cells increased slightly. From 3 days of treatment onwards, apoptosis was found to occur. These results show that ATRA-induced inhibition of MCF-7 cell growth is related to two mechanisms, i.e. the block of cell proliferation, mostly in a pre-S phase, and the induction of apoptosis. These results should be taken into account when attempting to design treatment programmes that associate ATRA with antineoplastic compounds of different cell cycle specificity.

Expression of p53 protein during the cell cycle measured by flow cytometry in human leukemia

The nuclear protein p53 has been reported to be associated with cell transformation and/or proliferation so that the study of p53 expression in human malignancy has potentially important clinical implications. We have analyzed the p53 expression in mitogen-stimulated and nonstimulated human lymphocytes, in several human leukemia cell lines (Molt-4, Raji, Daudi, HL-60, KG-1, K562 and U937) and in fresh bone marrow (BM) cells. Simultaneous differential staining of p53 (identified by a FITC-labeled monoclonal antibody) versus DNA (stained with propidium iodide, PI), followed by bivariate analysis with flow cytometry (FCM) made it possible to evaluate p53 expression with respect to cell position during the cell cycle. The data show that in stimulated lymphocytes p53 is progressively accumulated during the G1, S and G2-phases, while in non-stimulated conditions most cells are remaining in G0/G1 and express p53 to a lesser degree. This suggests that expression of p53 is more correlated with cell growth than with entrance into (or progression through particular phases of) the cell cycle. Cells from acute lymphoblastic leukemia (ALL) and Burkitts lymphoma cell lines express elevated levels of p53, while all examined human acute myeloid leukemia cell lines synthesize negligible p53 protein. Understanding the variations in p53 expression in different types of human leukemia may provide some insight into the biologic roles of p53 in normal and malignant cells.

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.

Flow cytometric DNA index in the prognosis of colorectal cancer

The authors investigated the relationship between flow cytometric DNA index (DI, defined as the ratio of the DNA content of malignant cells to that of normal cells) and other prognostic factors (grade and stage, anatomical site, age and sex) with the survival of 115 patients with colorectal cancer. Multiple biopsy specimens from 62 patients were taken during colonoscopy before surgery. Additional samples from 53 patients were obtained from paraffin‐embedded material. All patients were treated with surgery only. Fresh–frozen material gave higher incidence of DNA aneuploidy than paraffin‐embedded material (79% versus 41%). The patients with DNA diploid tumors (DI = 1) had a better overall survival than those with DNA aneuploid tumors (DI > 1). Among DNA aneuploid tumors, those with DI > 1.2 (excluding DI = 2) were worse than those with DI > 1.2 (excluding DI = 1) and DI = 2. Coxs regression analysis showed that pathologic stage was more important for prognosis than DNA index, whereas age, sex, histologic grade, and anatomic site were removed from the analysis as not relevant for prognosis. Relative risks of death (RR), in reference to patients with DI = 1 and Stages A + B (RR = 1), were RR = 1.8 for patients with carcinomas with Stage C, RR = 2.7 for patients with carcinomas with DNA near‐diploid and DNA tetraploid tumors, RR = 3.5 for those with DI > 1.2 (excluding DI = 2), and RR = 8.0 for those with Stage D. These data indicate that flow cytometrically evaluated DI values have a relevant independent power for predicting the clinical outcome of colorectal cancer patients.

Saporin, a ribosome-inactivating protein used to prepare immunotoxins, induces cell death via apoptosis.

The plant toxin saporin is a ribosome‐inactivating protein which inhibits protein synthesis and growth of both normal and tumour cells. Its cytotoxic activity can be increased by coupling with antibodies recognizing cell surface antigens. In this work we performed experiments to test the hypothesis that saporin induces cell death via apoptosis. Exposure to saporin induced apoptosis in different cellular models, such as human peripheral blood B lymphocytes and neutrophils, in the Daudi B‐cell line, and in the haemopoietic cell lines HL‐60 and TF‐1. This was indicated by: (a) the appearance of typical morphological features such as chromatin condensation, nuclear fragmentation and blebbing of plasma membranes; (b) DNA degradation into oligonucleosomal fragments; (c) the appearance of apoptotic cells on DNA flow cytometry as a cell population with reduced DNA content (A0 region). The fraction of cells showing features of apoptosis ranged from 19 ± 5% for TF‐1 cells to 35 ± 8% for neutrophils. In experiments with normal peripheral blood B lymphocytes or with Daudi cells, we compared the activity of native saporin with that of an immunotoxin hybrid molecule obtained by binding the toxin to two bispecific antibodies recognizing both saporin and the B lymphocyte‐specific antigen CD22 (Sap/BsAb complexes). Saporin bound to the antibodies was 2–3 logs more effective than native saporin in inducing apoptosis, with maximal inhibitions being observed at concentrations of 10−6 M for native saporin and 10−9–10−8 M for the hybrid molecules. These findings indicate that treatment with saporin results in apoptosis of target cells and suggest that this may be relevant to the therapeutic use of saporin‐containing immunotoxins. In fact, if used in vivo as an immunotoxin, its cytotoxic activity could be devoid of more extensive and non‐specific tissue damaging effects as would be the case if saporin induced necrosis of target cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) allows acceleration and dose intensity increase of CEF chemotherapy: a randomised study in patients with advanced breast cancer.

A randomised study was conducted in 62 patients with advanced breast cancer to assess whether granulocyte-macrophage colony-stimulating factor (GM-CSF) would yield an increase in the dose intensity of a standard-dose CEF regimen through an acceleration of chemotherapy administration. Patients received CEF (cyclophosphamide 600 mg m-2, epidoxorubicin 60 mg m-2 and fluorouracil 600 mg m-2) i.v. on day 1 or the same chemotherapy, plus GM-CSF 10 micrograms kg-1 s.c. starting from day 4, repeated as soon as haematopoietic recovery from nadir occurred. Patients in the CEF + GM-CSF group received chemotherapy at a median interval of 16 days compared with 20 days in the control group. This led to a significant increase (P = 0.02) in the dose intensity actually administered in the third, fourth and sixth cycles: +28%, +25%, +20% respectively. Non-haematological toxicity was mild. GM-CSF had to be reduced or suspended in 50% of patients because of toxicity. Haematological toxicity, mainly cumulative anaemia and thrombocytopenia, was manageable. An increase in response rate for patients with measurable disease, of borderline statistical significance (P = 0.088, P for trend = 0.018), from 42% in the CEF group to 69% in the CEF + GM-CSF group, was observed. This randomised trial indicates that GM-CSF is useful for chemotherapy acceleration. Accelerated CEF + GM-CSF is a moderately dose-intensive regimen that can be administered in an outpatient clinic and is associated with a high objective response.

Retrospective exploratory analysis of VEGF polymorphisms in the prediction of benefit from first-line FOLFIRI plus bevacizumab in metastatic colorectal cancer

BackgroundMolecular predictors of bevacizumab efficacy in colorectal cancer have not been identified yet. Specific VEGF polymorphisms may affect gene transcription and therefore indirectly influence the efficacy of bevacizumab.MethodsGenomic DNA of 111 consecutive metastatic colorectal cancer patients treated with first-line FOLFIRI plus bevacizumab was obtained from blood samples. VEGF -2578 C/A, -1498 C/T, + 405 C/G, + 936 C/T polymorphisms were analyzed by means of PCR-RFLP. DNA samples from 107 patients treated with FOLFIRI alone served as historical control group. The relation of VEGF polymorphisms with PFS, evaluated through Kaplan-Meier method and log-rank test, was the primary end-point. An interaction test with a Cox model has been performed in order to demonstrate the heterogeneity of the effect of VEGF -1498 C/T polymorphism between bevacizumab-and control group.ResultsIn the bevacizumab-group median PFS and OS of patients carrying VEGF -1498 C/C, C/T and T/T allelic variants were, respectively, 12.8, 10.5, 7.5 months (p = 0.0046, log-rank test) and 27.3, 20.5, 18.6 months (p = 0.038, log-rank test). VEGF -1498 T/T genotype was associated with shorter PFS (HR = 2.13, [1.41-5.10], p = 0.0027). In the control group no significant association of VEGF -1498 C/T allelic variants and PFS or OS was found. Interaction between VEGF -1498 C/T variants and treatment effect suggested that the relation of VEGF -1498 T/T genotype with shorter PFS was caused by the effect of bevacizumab (p = 0.011). Other investigated polymorphisms did not affect the outcome.ConclusionsThese data suggest a possible role for VEGF -1498 C/T variants in predicting the efficacy of bevacizumab in the up-front treatment of metastatic colorectal cancer patients. A molecular tool for selecting subjects candidate to benefit from the anti-VEGF could be important for clinical practice. The retrospective and exploratory design of the present study, coupled with the non-randomized nature of the comparison between treated and untreated patients, imply that these results should be considered as hypothesis generators. A prospective validating trial is currently ongoing.

Cisplatin and gemcitabine with either vinorelbine or paclitaxel in the treatment of carcinomas of unknown primary site : Results of an Italian multicenter, randomized, phase II study

To date, the standard treatment for patients who have carcinoma of unknown primary site has not been established.

Primary solitary intracranial melanoma: Case report and review of the literature

Among CNS tumors, intracranial melanomas represent a subject of interest for neurooncologists and neurosurgeons because clinical and radiological patterns of these tumors can mimic the presence of meningiomas, and in spite of their malignant behavior they can be satisfactorily treated. In the present report we describe a new case of primary intracranial melanoma that displayed some radiological features of meningioma; we review the clinical features of 80 previously well-documented cases. The importance of neuroradiological and histochemical (S-100 protein, antimelanin antibodies, proliferating cell nuclear antigen staining) methods and of flow cytometry in helping with histopathological examination is stressed. Review of the clinical histories demonstrates that surgical excision is recommended in most cases, depending on tumor location, and that if total removal is performed, long-term disease-free periods can be attained.