Alberto Riccardi

Long-term survival of stage I multiple myeloma given chemotherapy just after diagnosis or at progression of the disease: a multicentre randomized study

We conducted a randomized trial to evaluate whether melphalan-prednisone (MPH-P) treatment administered just after diagnosis improves survival of stage I multiple myeloma (MM). Between January 1987 and March 1993, 145 consecutive previously untreated patients with stage I MM were randomized between treatment with MPH-P (administered for 4 days every 6 weeks) just after diagnosis and treatment only at disease progression. Survival was not influenced by MPH-P treatment either administered just after diagnosis or at disease progression (64 vs 71 months respectively). Comparing the first with the second group the odds ratio of death is 1.17 (95% confidence interval 0.57–2.42;P = 0.64). Disease progression occurred within a year in about 50% of patients who were initially untreated. Response rate was similar in both groups, but duration of response was shorter in patients who were treated at disease progression (48 vs 79 months, P = 0.044). Patients actually treated at disease progression (34/70) survived shorter than those who had neither disease progression nor treatment (56 vs > 92 months;P = 0.005). Starting MPH-P just after diagnosis does not improve survival and response rate in stage I MM, with respect to deferring therapy until disease progression. However, patients with stage I MM randomized to have treatment delayed and who actually progressed and were treated had shorter survival than those with stable disease and no treatment. Biologic or other disease features could identify these subgroups of patients.

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.

Changing clinical presentation of multiple myeloma

We compared the presentation features of three series of patients with multiple myeloma diagnosed between 1960 and 1971 (Kyle R, Mayo Clin Proc, 1975, 50, 29, n = 869), 1972 and 1986 (Clinica Medica, University of Pavia, n = 345) and 1987 and 1990 (Cooperative Group for Study and Treatment of Multiple Myeloma, n = 341). In the most recently diagnosed patients, the percentage of those who had symptoms related to multiple myeloma (i.e. any of bone pain, systemic symptoms, disturbances related to hypercalcemia, neurological involvement and hyperviscosity) was reduced (90 vs. 86 vs. 66%) (P less than 0.001), while the percentage of asymptomatic patients diagnosed by chance was increased (not reported, and 14 vs. 34%). In the most recent series, a lower percentage of spontaneous bone pain (68 vs. 60 vs. 37%, P less than 0.001) paralleled a lower incidence of advanced bone disease (osteolyses and pathological fractures, 60 vs. 64 vs. 34%), and renal failure (serum creatinine greater than 1.2 mg/dl) was also less common (56 vs. 44 vs. 33%, P less than 0.01), at least partially due to a decreased incidence of both hypercalcemia (30 vs. 20 vs. 18%, P less than 0.001) and of hyperuricemia (serum uric acid greater than 7 mg/dl, 47 vs. 32 vs. 26%, P less than 0.01). Systemic symptoms (weakness, infections, fever or weight loss) were reported more seldom by recently diagnosed patients, due to a decreased frequency of anaemia (haemoglobin less than 12 g/dl), leukopenia and thrombocytopenia, as well as of the systemic effects of bone pain and of renal insufficiency. These data indicate that multiple myeloma is diagnosed earlier now than in the past, and this must be taken into account when comparing survival data in treated series.

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.

A prognostic index for multiple myeloma

The current prognostic systems have failed to identify multiple myeloma (MM) patients who require aggressive therapy. These staging systems do not reliably distinguish patients with different prognoses. This paper explores the possibility of improving the prognostic forecast in MM by considering some clinical characteristics at diagnosis together with response to first-line chemotherapy. A total of 231 patients were prospectively randomised in a multicentre trial to no therapy vs melphalan + prednisone (MP) for stage I, MP in stage II, and MP vs peptichemio, vincristine and prednisone for stage III. The clinical features of these groups were evaluated for prognostic variables predictive of overall survival by means of univariate and multivariate analysis. The independently significant variables were incorporated into a model that identified three groups of patients with different risks of death and different overall survival. Three variables retained statistical significance: the staging system proposed by the British Medical Research Council, a composite parameter integrating the percentage of bone marrow plasma cells with cytological features of the infiltrating elements (plasma cell vs plasmablast), and response to 6 months of first-line chemotherapy. These three variables led the proposal of a scoring system able to identify three different risk classes (with median overall survival of 52, 28 and 13 months respectively) and to estimate individual patient prognosis more flexibly. The proposed risk classes, drawn from both diagnostic and therapeutic parameters, are thought to be a clinical and investigational instrument for separating MM patients into comparable groups, for selecting the best available therapy and for evaluating response with respect to the disease of each new patient.

Adding Gemcitabine to Paclitaxel/Carboplatin Combination Increases Survival in Advanced Non–Small-Cell Lung Cancer: Results of a Phase II-III Study

PURPOSE Paclitaxel/carboplatin (PC) is one of the reference combinations in the treatment of non-small-cell lung cancer (NSCLC). No triplet novel agent combination has until now shown superiority over a two-drug combination for advanced NSCLC. We therefore conducted a clinical trial to test if paclitaxel/carboplatin/gemcitabine (PCG) increases overall survival (OS) and response rate (RR) over PC. METHODS Stage IIIB patients not suitable for radical radiation treatment and stage IV chemotherapy-naive patients with measurable disease and performance status of 0 to 2 were randomly assigned to PC arm (paclitaxel 200 mg/m2 and carboplatin area under the concentration-time curve 6 day 1/q21 days) or the PCG arm (paclitaxel 200 mg/m(2) and carboplatin area under the concentration-time curve 6 day 1, and gemcitabine 1,000 mg/m2 days 1 and 8 every 21 days). RESULTS A total of 324 patients were randomly assigned to the two arms. The RR for PC arm and PCG arm were 20.2% and 43.6% [corrected] (P < .0001). The median time to the progression was 5.1 months in the PC group and 7.6 months in the PCG group (P = .012; hazard ratio [HR] 1.34; 95% CI: 1.06 to 1.72). Median OS was 8.3 months and 10.8 months (P = .032; HR 1.309; 95% CI: 1.03 to 1.67) in favor of the PCG arm. One-year survival was 34% (PC arm) and 45% (PCG arm; P = .032). Only hematologic toxicity (neutropenia, thrombocytopenia, and anemia) was significantly increased in the PCG arm and the experimental arm required more platelet and red blood cell transfusions, and more granulocyte colony-stimulating factor usage. No toxic/early deaths were observed. CONCLUSION The PCG regimen offers a significant survival advantage over PC in advanced NSCLC, making PCG a treatment option for advanced NSCLC patients.

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.

Proliferative activity of bone marrow cells in primary dysmyelopoietic (preleukemic) syndromes

The proliferative activity of bone marrow cells was studied in 24 patients with primary dysmyelopoiesis by means of flow cytometry and 3H‐IdR autoradiography. Abnormal DNA content was found in two cases with aneuploid karyotypes. DNA content typical of a diploid population was observed in all patients with normal karyotype and in three patients with chromosomal aberrations. The fraction of bone marrow cells in S‐and G2‐phase was higher in primary acquired sideroblastic anemia and refractory anemia (without excess of blasts) than in refractory anemia with excess of blasts and chronic myelomonocytic leukemia. Regardless to the diagnosis, the patients with low fraction of cells in S‐ and G2‐phase had short survival time and showed high rate of evolution into acute nonlymphoblastic leukemia. The labeling (LI) and mitotic (MI) indexes of both erythroblasts and granulocytic cells were decreased in nearly all patients. The lowest values of LI and MI of the granulocytic compartment were found in the patients who subsequently developed acute leukemia. These data suggest that cytokinetic analysis allows investigators to achieve useful information on the stage of disease in the dysmyelopoietic syndromes.

Predicting and preventing cardiotoxicity in the era of breast cancer targeted therapies. Novel molecular tools for clinical issues

Treatment of breast cancer (BC) has changed over the last decade with the advent of targeted therapies. Whereas traditional chemotherapy was directed toward all rapidly dividing cells (cancerous or not), several new anti-cancer drugs are mainly tailored to specific genetic pathways of cancer cells. Ideally, the goal of these new therapies is to improve the management of cancer with a specific targeting of the malignant cell and fewer side effects than traditional chemotherapy. Due to the initial success of this approach, an increasing number of targeted drugs entered into clinical development. However, unanticipated side effects of the new drugs, such as cardiotoxicity and heart failure, emerged from several clinical trials. The mechanisms of cardiotoxicity due to traditional chemotherapy and the one due to new drugs seem to be inherently different. In the case of BC, available targeted therapies are probably associated with the abrogation of normal molecular pathways involved in cardiomyocytes and endothelial cells survival/proliferation. The cardiac safety profile of these new drugs asks for a careful patient monitoring and follow up. Herein we will review the cardiotoxicity of BC patients receiving antiERBB2 treatment (Trastuzumab, Lapatinib), VEGF inhibitors (Bevacizumab) and tirosin-kinase inhibitors (Sorafenib, Sunitinib). We will discuss the molecular mechanisms that underlie the risk of cardiotoxicity, and we will examine the molecular tools useful for prediction of heart failure and for identification of subgroups of BC patients more susceptible to cardiac side effects induced by targeted therapies. Attention will be paid in particular to ERBB2 gene and its polymorphisms, as well as to the possible genetic risk stratification of BC patients. Finally, we will discuss the possible clinical strategies to prevent and minimizing the cardiotoxicity of targeted therapies in BC patients, focusing in particular on new drugs combination and on the emerging role of a tight partnership between cardiologists and oncologists.