Camillo Almici

Association between single nucleotide polymorphisms in the XRCC1 and RAD51 genes and clinical radiosensitivity in head and neck cancer

PURPOSE Individual variability in radiosensitivity is large in cancer patients. Single nucleotide polymorphisms (SNPs) in genes involved in DNA repair and in protection against reactive oxygen species (ROS) could be responsible for such cases of radiosensitivity. We investigated the association between the occurrence of acute reactions in 101 patients with squamous cell carcinoma of the head and neck (SCCHN) after radiotherapy (RT) and five genetic polymorphisms: XRCC1 c.1196A>G, XRCC3 c.722C>T, RAD51 (c.-3429G>C, c.-3392G>T), and GSTP1 c.313A>G. MATERIALS AND METHODS Genetic polymorphisms were detected by high resolution melting analysis (HRMA). The development of acute reactions (oral mucositis, skin erythema and dysphagia) associated with genetic polymorphisms was modeled using Cox proportional hazards, accounting for biologically effective dose (BED). RESULTS Development of grade ≥2 mucositis was increased in all patients (chemo-radiotherapy and radiotherapy alone) with XRCC1-399Gln allele (HR=1.72). The likelihood of developing grade ≥2 dysphagia was higher in carriers of RAD51 c.-3429 CC/GC genotypes (HR=4.00). The presence of at least one SNP or the co-presence of both SNPs in XRCC1 p.Gln399Arg /RAD51 c.-3429 G>C status were associated to higher likelihood of occurrence of acute toxicities (HR=2.03). CONCLUSIONS Our findings showed an association between genetic polymorphisms, XRCC1 c.1196A>G and RAD51 c.-3429 G>C, and the development of radiation-induced toxicities in SCCHN patients.

Circulating Tumour Cells in locally advanced head and neck cancer: Preliminary report about their possible role in predicting response to non-surgical treatment and survival

BACKGROUND AND PURPOSE The mechanism of dissemination of locally advanced head and neck cancer (LAHNC) is far to be resolved. Circulating tumour cells (CTC) have been identified as a prognostic factor in metastatic breast and prostate cancer. This prospective multi-centric analysis studied the possible role of CTC identification in LAHNC. MATERIALS AND METHODS CTC were searched in 73 patients with LAHNC (oropharynx, n=39; nasopharynx, n=10; larynx, n=10; paranasal sinuses, n=6, of whom 3 with sinonasal undifferentiated carcinoma, SNUC; hypopharynx, n=5; oral cavity, n=3). All of them (apart from SNUC) had squamous cell cancers. The relationship between CTC positivity and other clinical prognostic factors has been investigated. Response to treatment and survival has been related with changes in CTC number during the treatment. RESULTS CTC were frequently identified in oro- and hypopharyngeal cancer and in SNUC. They were more frequent in stage IV than in stages I-III disease (18% versus 6%, p=NS (not significant)). Partial or complete response (CR) was related with the absence or disappearance of CTC during treatment (p=0.017). A decrease in the CTC number or their absence throughout the treatment seems also related with non-progressive disease, after both complete or incomplete remission and with the proportion of patients alive and NED (no evidence of disease) (p=0.009). CONCLUSIONS These preliminary data suggest a possible role of CTC determination in head and neck cancer. Additional and longer follow up data need to be collected to confirm these findings.

A case of acute promyelocytic leukaemia following mitoxantrone treatment of multiple sclerosis

A case of acute promyelocytic leukaemia following mitoxantrone treatment of multiple sclerosis

Clonogenic capacity and ex vivo expansion potential of umbilical cord blood progenitor cells are not impaired by cryopreservation

Umbilical cord blood (UCB) progenitor cells have been demonstrated to possess significant advantages over bone marrow (BM), in terms of proliferative capacity and immunologic reactivity. Therefore, UCB has been recently considered an attractive potential alternative to BM as a source of hematopoietic progenitor cells for clinical applications. Since several programs throughout the world are currently evaluating the feasibility of large-scale UCB banking for unrelated transplants, it was the aim of this study to evaluate whether cryopreservation procedures might heavily impair the clonogenic capacity, the feasibility of CD34+ selection and the ex vivo expansion potential of UCB progenitor cells. UCB samples were collected and cryopreserved as unseparated (n = 21) or mononuclear (MNC) cells (n = 15) within 12 h from delivery, and evaluated for viability, immunophenotype, cell and progenitor numbers after a minimum stay in liquid nitrogen of 6 months (range 6–14 months). Viability was always >97% and no statistically significant difference was detected by flow cytometric analysis. Clonogenic recovery from unseparated cells was 80–87% for HPP-CFC, CFU-GEMM, BFU-E and CFU-GM, and from MNC cells ranged from 82 to 91% for LTC-IC, CFU-GEMM, BFU-E and CFU-GM. CD34+ selection (n = 8) was performed on fresh and cryopreserved MNC cells using the MiniMACS immunomagnetic separation device, showing no difference in yield (68 ± 7% vs 57 ± 4%, P ⩽ 0.4) or in purity (89 ± 2% vs 81 ± 6%, ⩽ 0.4), for fresh in comparison to cryopreserved MNC cells. After 14 days of liquid culture in the presence of different combinations of SCF, IL-3, IL-6 and G-CSF no statistically significant difference was detected in CFC fold-expansion for fresh or cryopreserved MNC cells and for CD34+ cells, either selected and cultured from fresh or cryopreserved MNC cells. In conclusion we can state that UCB is a potential source of primitive progenitor cells that can be cryopreserved unmanipulated or after physical separation without major losses in clonogenic capacity and immunophenotypic composition. Moreover, CD34+ selection from cryopreserved MNC cells is feasible and ex vivo expansion is not impaired. These results have important implications in the large scale UCB banking, in view of the potential applications of ex vivo expanded hematopoietic progenitor cells for the engraftment of adult patients.

Biologic and phenotypic analysis of early hematopoietic progenitor cells in umbilical cord blood

Umbilical cord blood (UCB) is an attractive potential alternative to bone marrow (BM) as a source of hematopoietic progenitor cells since the number of progenitors in UCB is similar or even greater than that in normal BM. It was the aim of the present study to analyze the degree of immaturity of UCB progenitor cells. UCB mononuclear (MNC) and/or CD34+ cells were tested for surface antigen phenotype, expression of cytokines receptor, effect of stem cell factor (SCF) on colony growth, resistance to mafosfamide and replating potential. We have found that 34.9 ± 3.4% and 77.9 ± 2.6% of UCB CD34+ cells did not express CD38 and CD45RA antigens, respectively, suggesting that UCB contains a high proportion of immature progenitor cells. By means of three-color analysis, the receptor for SCF was detected on the majority of the CD34+HLA-DR+ subpopulation; in fact, 81.8% ± 4.3% of CD34+HLA-DR+ cells were defined as SCFlow and 8.1 ± 1.5% as SCFhigh. Colony growth of MNC and CD34+ cells was enhanced by the addition of SCF to methylcellulose mixture, resulting in a statistically significant increase in CFU-GM and CFU-GEMM but not in BFU-E numbers. UCB progenitor cells showed a higher resistance to mafosfamide treatment, in comparison to BM; the addition of SCF to the culture medium resulted in a statistically significant increase in mafosfamide concentration required to inhibit 95% of colony growth (P ⩽ 0.05). Moreover, as shown by single colony transfer assays, the presence of SCF in primary cultures promoted a significantly higher replating potential for both untreated (42 ± 3.3% vs 21 ± 4.6%, P ⩽ 0.018) and mafosfamide-treated samples (62 ± 5.6% vs 44 ± 6.1%, P ⩽ 0.018). In conclusion, UCB is a source of progenitor cells with immature characteristics in terms of surface antigen expression, distribution of SCF receptor, resistance to mafosfamide and replating potential. Therefore, UCB progenitor cells represent an ideal candidate population for experimental programs involving gene transfer and ex vivo stem cell expansion.

Endothelial progenitor cells, defined by the simultaneous surface expression of VEGFR2 and CD133, are not detectable in healthy peripheral and cord blood

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual‐platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re‐evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.

Circulating Tumor Cells in Patients with Recurrent or Metastatic Head and Neck Carcinoma: Prognostic and Predictive Significance

Introduction We investigated the frequency of detection and the prognostic and predictive significance of circulating tumor cells (CTCs) in patients with recurrent/metastatic (R/M) head and neck carcinoma (HNC) before starting systemic therapy. Patients and methods Using the CellSearch technology, CTCs were assessed prospectively in peripheral blood of 53 R/M-HNC patients. We performed spiking experiments to test the diagnostic performance of the CellSearch platform in identifying squamous carcinoma cells. Results CTCs were identified in 14 (26%) and 22 (41%) patients at baseline and at any time point, respectively. In univariate analysis ≥2 CTCs had a poorer prognostic role than 0–1 CTC. In multivariate analysis, the presence of one CTC or more was associated with a poor prognosis both in terms of progression-free survival (PFS) [Hazard Ratio (HR): 3.068, 95% confidence interval (CI): 1.53–6.13, p 0.002] and overall survival (OS) [HR: 3.0, 95% CI: 1.48–6.0, p 0.002]. A disease control after systemic therapy was obtained in 8% of CTC-positive patients as opposed to 45% in CTC-negative ones (p 0.03). The epidermal growth factor receptor (EGFR) expression was identified in 45% of CTC-positive patients. Discussion In conclusion, CTCs are detected in one out of three patients with RM-HNC. CTC detection is a strong prognostic parameter and may be predictive of treatment efficacy. The frequency of EGFR expression in CTCs seems to be lower than that expected in the primary tumor.

Effect of the protein tyrosine kinase inhibitor genistein on normal and leukaemic haemopoietic progenitor cells

Receptor and nonreceptor protein tyrosine kinases (PTKs) play a key role in the control of normal and neoplastic cell growth. The availability of PTK inhibitors prompted us to evaluate the effects of genistein, a natural inhibitor of PTKs, on in vitro colony formation by normal multilineage colony‐forming units (CFU‐Mix), erythroid bursts (BFU‐E), granulocyte‐macrophage colony‐forming units (CFU‐GM), long‐term culture‐initiating cells (LTC‐IC) and acute myelogenous leukaemia colony‐forming units (CFU‐AML). Continuous exposure of normal marrow and blood mononuclear non‐adherent cells, blood CD34+CD45RA− cells, and leukaemic blasts to increasing doses of genistein (1–100 μM) resulted in a statistically significant (P ≤ 0.05) dose‐dependent suppression of CFU‐Mix, BFU‐E, CFU‐GM and CFU‐AML growth. Regression analysis showed that growth inhibition was linearly related to genistein concentration. Genistein dose causing 50% inhibition (ID50) of CFU‐AML was significantly lower compared to CFU‐GM ID50 for marrow (19 v 32 μMP  ≤0.017), unseparated blood (19 v 44 μMP ≤ 0.028) or CD34+CD45RA− blood (19 v 36, P ≤ 0.04). Preincubation of leukaemic blasts with genistein (200 μM) for 1–2 h confirmed that CFU‐AML were significantly more sensitive than normal marrow and blood CFU‐GM to genistein. Preincubation conditions which maximally suppressed leukaemic and normal colony growth spared a substantial percentage of marrow (29 ± 4%) and blood (40 ± 3%) LTC‐IC. In conclusion, our data demonstrate that: (a) genistein strongly inhibits the growth of normal and leukaemic haemopoietic progenitors; (b) growth inhibition is dose‐ and time‐dependent; (c) leukaemic progenitors are more sensitive than normal progenitors to genistein‐induced growth inhibition; (d) genistein exerts a direct toxic effect on haemopoietic cells while sparing a substantial proportion of LTC‐IC. The potent CFU‐AML growth inhibition associated with the relative resistance of normal LTC‐IC strongly supports the use of genistein for marrow purging.

Density Separation and Cryopreservation of Umbilical Cord Blood Cells: Evaluation of Recovery in Short- and Long-Term Cultures

The clonogenic capacity of human umbilical cord blood (UCB) has been evaluated in several studies, which have shown high numbers of primitive hematopoietic progenitor cells. Recently, UCB progenitor cells were demonstrated to possess significant advantages over bone marrow (BM) in terms of proliferative capacity and immunologic reactivity. Therefore, UCB has been considered an attractive source of hematopoietic stem cells for both research and clinical applications. Previous reports have documented a significant loss of progenitor cells by any manipulation other than cryopreservation. We have evaluated the feasibility of fractionating and cryopreserving UCB samples with minimal loss of progenitor cells. We compared separation over three different densities of Percoll (1.069, 1.077 and 1.084 g/ml), sedimentation over poligeline (Emagel), and sedimentation over poligeline followed by separation over Ficoll/Hypaque (F/H). Separated samples (n = 25) were analyzed for recovery of CD34+ cells and progenitor cells (CFU-GEMM, BFU-E, CFU-GM). Separation by sedimentation over poligeline followed by F/H allowed the highest depletion of RBCs (hematocrit of the final cellular suspension 0.4 +/- 0.1%), while maintaining a high recovery of CD34+ cells (85.3%) and total recovery of CFU-GEMM, BFU-E and CFU-GM. After cryopreservation, recovery of clonogenic progenitors was 82% for CFU-GEMM, 94% for BFU-E, 82% for CFU-GM and 90% for colony-forming units after 5 weeks of longterm culture. Moreover, the presence of Stem cell factor significantly increased CFU-GEMM (14 +/- 4 vs. 49 +/- 5, p < or = 0.0005) and CFU-GM (112 +/- 18 vs. 178 +/- 19, p < or = 0.025), but not B FU-E (42 +/- 7 vs. 53 +/- 7, p < or = 0.375) growth. In conclusion, RBC depletion of UCB can be accomplished with minimal loss of committed and primitive hematopoietic progenitors. This procedure may have important implications in the large-scale banking of UCB and in ex vivo expansion/gene therapy protocols.

Circulating tumor cells as predictors of prognosis in metastatic breast cancer: clinical application outside a clinical trial

AIMS AND BACKGROUND Circulating tumor cells have a prognostic role in metastatic breast cancer. The aim of the study was to confirm the ability of circulating tumor cells, detected by the US Food and Drug Administration approved Cell Search assay, to predict the outcome of patients treated in a community general hospital. PATIENTS AND METHODS A prospective mono-institutional study was conducted at the Department of Medical Oncology at Spedali Civili, Brescia, Italy, from January 2009 to September 2010. A total of 93 consecutive patients with metastatic breast cancer were enrolled. Patients underwent a blood sample collection to detect circulating tumor cells at baseline and, subsequently, at the first follow-up examination (after 3-4 weeks from the beginning of a systemic therapy). A third sample was drawn at disease progression (at the beginning of a subsequent new course of therapy). The prognostic cutoff value of circulating tumor cells was fixed at 5 cells/7.5 ml of blood. RESULTS At baseline, median overall survival and progression-free survival in the subgroup ≥5 circulating tumor cells/7.5 ml of blood were significantly shorter (5 months and 3 months, respectively) than in the subgroup with <5 circulating tumor cells (8 months and 7 months, respectively) (P = 0.003 and P <0.001). At the first follow-up, the subgroup with more than 5 circulating tumor cells/7.5 ml of blood had a median overall survival of 4 months versus 8 months in the subgroup with <5 circulating tumor cells (P <0.001) and a median progression-free survival of 3 months versus 7 months respectively (P <0.001). At multivariate analysis, the level of circulating tumor cells at the first follow-up and at baseline remained significant as a predictor of progression-free and overall survival. The number of metastatic sites was significantly associated with overall and progression-free survival and correlated with the number of circulating tumor cells. CONCLUSIONS Our study confirms the role of circulating tumor cells as predictors of prognosis in metastatic breast cancer patients treated in general clinical practice.