Pedro C. Lara

Prediction of clinical toxicity in localized cervical carcinoma by radio-induced apoptosis study in peripheral blood lymphocytes (PBLs)

BackgroundCervical cancer is treated mainly by surgery and radiotherapy. Toxicity due to radiation is a limiting factor for treatment success. Determination of lymphocyte radiosensitivity by radio-induced apoptosis arises as a possible method for predictive test development. The aim of this study was to analyze radio-induced apoptosis of peripheral blood lymphocytes.MethodsNinety four consecutive patients suffering from cervical carcinoma, diagnosed and treated in our institution, and four healthy controls were included in the study. Toxicity was evaluated using the Lent-Soma scale. Peripheral blood lymphocytes were isolated and irradiated at 0, 1, 2 and 8 Gy during 24, 48 and 72 hours. Apoptosis was measured by flow cytometry using annexin V/propidium iodide to determine early and late apoptosis. Lymphocytes were marked with CD45 APC-conjugated monoclonal antibody.ResultsRadiation-induced apoptosis (RIA) increased with radiation dose and time of incubation. Data strongly fitted to a semi logarithmic model as follows: RIA = βln(Gy) + α. This mathematical model was defined by two constants: α, is the origin of the curve in the Y axis and determines the percentage of spontaneous cell death and β, is the slope of the curve and determines the percentage of cell death induced at a determined radiation dose (β = ΔRIA/Δln(Gy)). Higher β values (increased rate of RIA at given radiation doses) were observed in patients with low sexual toxicity (Exp(B) = 0.83, C.I. 95% (0.73-0.95), p = 0.007; Exp(B) = 0.88, C.I. 95% (0.82-0.94), p = 0.001; Exp(B) = 0.93, C.I. 95% (0.88-0.99), p = 0.026 for 24, 48 and 72 hours respectively). This relation was also found with rectal (Exp(B) = 0.89, C.I. 95% (0.81-0.98), p = 0.026; Exp(B) = 0.95, C.I. 95% (0.91-0.98), p = 0.013 for 48 and 72 hours respectively) and urinary (Exp(B) = 0.83, C.I. 95% (0.71-0.97), p = 0.021 for 24 hours) toxicity.ConclusionRadiation induced apoptosis at different time points and radiation doses fitted to a semi logarithmic model defined by a mathematical equation that gives an individual value of radiosensitivity and could predict late toxicity due to radiotherapy. Other prospective studies with higher number of patients are needed to validate these results.

IGF-1R expression predicts clinical outcome in patients with locally advanced oral squamous cell carcinoma

To assess the expression of IGF-1R in oral cavity squamous cell carcinoma patients, to explore its relation with clinical and pathologic prognostic factors and its role in predicting clinical outcome. One hundred and thirty-one consecutive patients suffering from oral cavity squamous cell carcinoma were included in this study from July 1989 to April 2005. Follow-up was closed in May 2010. The mean follow-up for survivors was 110.26±47.42 months. Patients were staged following the TNM classification. Patients in tumour stages I and II were referred to surgery. Patients in stages III-IV were referred to postoperative radiotherapy. Radiation therapy was administered up to a mean dose of 62.13±7.74 Gy in 1.8-2 Gy fractions. IGF-1R expression was studied by immunohistochemistry in paraffin-embedded tumour tissue. IGF-1R was expressed in 101 patients (77.1%). IGF-1R expression was related to tumour grade (P=0.012). Tumour stage was the most important prognostic factor for survival. Low (negative and fairly) IGF-1R tumour expression was correlated to better long-term Local Disease Free Survival (P=0.016), Disease-Free Survival (P=0.029), and Survival (P=0.009) in patients achieving tumour stages III-IV. Low IGF-1R expression was related to better long-term control in patients suffering locally advanced oral carcinoma.

Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression

Oxygen molecule modulates tumour response to radiotherapy. Higher radiation doses are required under hypoxic conditions to induce cell death. Hypoxia may inhibit the non-homologous end-joining DNA repair through down regulating Ku70/80 expression. Hypoxia induces drug resistance in clinical tumours, although the mechanism is not clearly elucidated. Vaults are ribonucleoprotein particles with a hollow barrel-like structure composed of three proteins: major vault protein (MVP), vault poly(ADP-ribose) polymerase, and telomerase associated protein-1 and small untranslated RNA. Over-expression of MVP has been associated with chemotherapy resistance. Also, it has been related to poor outcome in patients treated with radiotherapy alone. The aim of the present study was to assess the relation of Major Vault Protein expression and tumor hypoxia in clinical cervical tumors. MVP, p53 and angiogenesis, together with tumor oxygenation, were determined in forty-three consecutive patients suffering from localized cervix carcinoma. High MVP expression was related to severe hypoxia compared to low MVP expressing tumors (p = 0.022). Tumors over-expressing MVP also showed increased angiogenesis (p = 0.003). Besides it, in this study we show for the first time that severe tumor hypoxia is associated with high MVP expression in clinical cervical tumors. Up-regulation of MVP by hypoxia is of critical relevance as chemotherapy is currently a standard treatment for those patients. From our results it could be suggested that hypoxia not only induces increased genetic instability, oncogenic properties and metastatization, but through the correlation observed with MVP expression, another pathway of chemo and radiation resistance could be developed.

Constitutive gene expression profile segregates toxicity in locally advanced breast cancer patients treated with high-dose hyperfractionated radical radiotherapy.

Breast cancer patients show a wide variation in normal tissue reactions after radiotherapy. The individual sensitivity to x-rays limits the efficiency of the therapy. Prediction of individual sensitivity to radiotherapy could help to select the radiation protocol and to improve treatment results. The aim of this study was to assess the relationship between gene expression profiles of ex vivo un-irradiated and irradiated lymphocytes and the development of toxicity due to high-dose hyperfractionated radiotherapy in patients with locally advanced breast cancer. Raw data from microarray experiments were uploaded to the Gene Expression Omnibus Database http://www.ncbi.nlm.nih.gov/geo/ (GEO accession GSE15341). We obtained a small group of 81 genes significantly regulated by radiotherapy, lumped in 50 relevant pathways. Using ANOVA and t-test statistical tools we found 20 and 26 constitutive genes (0 Gy) that segregate patients with and without acute and late toxicity, respectively. Non-supervised hierarchical clustering was used for the visualization of results. Six and 9 pathways were significantly regulated respectively. Concerning to irradiated lymphocytes (2 Gy), we founded 29 genes that separate patients with acute toxicity and without it. Those genes were gathered in 4 significant pathways. We could not identify a set of genes that segregates patients with and without late toxicity. In conclusion, we have found an association between the constitutive gene expression profile of peripheral blood lymphocytes and the development of acute and late toxicity in consecutive, unselected patients. These observations suggest the possibility of predicting normal tissue response to irradiation in high-dose non-conventional radiation therapy regimens. Prospective studies with higher number of patients are needed to validate these preliminary results.

Radiation-Induced DNA Damage as a Predictor of Long-Term Toxicity in Locally Advanced Breast Cancer Patients Treated with High-Dose Hyperfractionated Radical Radiotherapy

Abstract Pinar, B., Lara, P. C., Lloret, M., Bordón, E., Núñez, M. I., Villalobos, M., Guerrero, R., Luna, J. D. and Ruiz de Almodóvar, J. M. Radiation-Induced DNA Damage as a Predictor of Long-Term Toxicity in Locally Advanced Breast Cancer Patients Treated with High-Dose Hyperfractionated Radical Radiotherapy. Radiat. Res. 168, 415–422 (2007). This 14-year-long study makes a novel contribution to the debate on the relationship between the in vitro radiosensitivity of peripheral blood lymphocytes and normal tissue reactions after radiation therapy. The aims were (1) to prospectively assess the degree and time of onset of skin side effects in 40 prospectively recruited consecutive patients with locally advanced breast cancer treated with a hyperfractionated dose-escalation radiotherapy schedule and (2) to assess whether initial radiation-induced DNA damage in peripheral blood lymphocytes of these patients could be used to determine their likelihood of suffering severe late damage to normal tissue. Initial radiation-induced DNA double-strand breaks (DSBs) were assessed in peripheral blood lymphocytes of these patients by pulsed-field electrophoresis. Acute and late cutaneous and subcutaneous toxicity was evaluated using the Radiation Therapy Oncology Group morbidity score. A wide interindividual variation was observed in toxicity grades and in radiation-induced DNA DSBs in peripheral blood lymphocytes (mean 1.61 ± 0.76 DSBs/Gy per 200 MBp, range 0.63– 4.08), which were not correlated. Multivariate analysis showed a correlation (P < 0.008) between late toxicity and higher prescribed protocol dose (81.6 Gy). Analysis of the 29 patients referred to 81.6 Gy revealed significantly (P < 0.031) more frequent late subcutaneous toxicity in those with intrinsic sensitivity to radiation-induced DNA DSBs of >1.69 DSBs/Gy per DNA unit. Our demonstration of a relationship between the sensitivity of in vitro-irradiated peripheral blood lymphocytes and the risk of developing late toxic effects opens up the possibility of predicting normal tissue response to radiation in individual patients, at least in high-dose non-conventional radiation therapy regimens.

Apoptosis in carcinoma of the bladder: relation with radiation treatment results.

PURPOSE Radiotherapy is widely used in the treatment of bladder cancer. The search for biological parameters that could select patients who will respond to radiation treatment has become essential. The aim of this study is to assess whether the pretreatment apoptotic index is useful in predicting local control and survival in a group of bladder cancer patients treated by radiotherapy. METHODS AND MATERIALS Fifty-five patients with invasive bladder carcinoma treated between 1983 and 1996 were included in this study. Radiotherapy was given to a median dose of 66 Gy, mean 63.28 Gy, in 1.8-2 Gy daily fractions. Apoptotic cells were studied in hematoxylin-eosin slides. Clinicopathological tumor characteristics were studied in relation to the apoptotic index, and as prognostic factors for local control and survival in both univariate and multivariate analysis. RESULTS Pretreatment apoptotic indexes were related to tumor stage, mitotic index, and Ki67 proliferation index. Five-year actuarial local control for the whole group was 45%. Patients with tumors showing low pretreatment apoptotic indexes had better local control (p < 0.037) and survival (p < 0.01) than highly apoptotic tumors. Tumor stage (T2 vs. T3-4) and the pretreatment apoptotic index were significant predictive factors for local control and survival in multivariate analysis. CONCLUSIONS The pretreatment apoptotic index is useful in predicting the clinical outcome of bladder cancer patients treated by radiotherapy. Assessment of biological tumor characteristics could allow the selection of patients for different treatment strategies.

Prediction of clinical toxicity in locally advanced head and neck cancer patients by radio-induced apoptosis in peripheral blood lymphocytes (PBLs).

Head and neck cancer is treated mainly by surgery and radiotherapy. Normal tissue toxicity due to x-ray exposure is a limiting factor for treatment success. Many efforts have been employed to develop predictive tests applied to clinical practice. Determination of lymphocyte radio-sensitivity by radio-induced apoptosis arises as a possible method to predict tissue toxicity due to radiotherapy. The aim of the present study was to analyze radio-induced apoptosis of peripheral blood lymphocytes in head and neck cancer patients and to explore their role in predicting radiation induced toxicity. Seventy nine consecutive patients suffering from head and neck cancer, diagnosed and treated in our institution, were included in the study. Toxicity was evaluated using the Radiation Therapy Oncology Group scale. Peripheral blood lymphocytes were isolated and irradiated at 0, 1, 2 and 8 Gy during 24 hours. Apoptosis was measured by flow cytometry using annexin V/propidium iodide. Lymphocytes were marked with CD45 APC-conjugated monoclonal antibody. Radiation-induced apoptosis increased in order to radiation dose and fitted to a semi logarithmic model defined by two constants: α and β. α, as the origin of the curve in the Y axis determining the percentage of spontaneous cell death, and β, as the slope of the curve determining the percentage of cell death induced at a determined radiation dose, were obtained. β value was statistically associated to normal tissue toxicity in terms of severe xerostomia, as higher levels of apoptosis were observed in patients with low toxicity (p = 0.035; Exp(B) 0.224, I.C.95% (0.060-0.904)). These data agree with our previous results and suggest that it is possible to estimate the radiosensitivity of peripheral blood lymphocytes from patients determining the radiation induced apoptosis with annexin V/propidium iodide staining. β values observed define an individual radiosensitivity profile that could predict late toxicity due to radiotherapy in locally advanced head and neck cancer patients. Anyhow, prospective studies with different cancer types and higher number of patients are needed to validate these results.

Immune effects of high dose radiation treatment: implications of ionizing radiation on the development of bystander and abscopal effects

Tumors grow progressively when they escape from immune surveillance. Cancer progression is mainly driven by the expansion of tumor cells, but tumor microenvironment and anti-tumor immunity may also play a role. Ionizing radiation therapy (RT), either alone or in combination with additional immune stimulators, can render cancer cells visible to the immune system. In addition to the direct effects of radiation, the ensuing immune response promotes the expression of inflammatory and immunostimulatory mediators, which act on neighboring, non-irradiated, cells. Bystander effects induced by radiation are characterized by biological responses, which are observed in non-irradiated cells that are in the vicinity of irradiated cells. Bystander effects are mediated via cell-to-cell gap junctions or through secreted, diffusible signaling molecules into the local milieu. After treatment with localized radiation, systemic effects in non-irradiated area (out-offield) may also occur. These effects are named abscopal effects and appear to be immune mediated, particularly by adaptive immunity. It has been suggested that a high single dose of RT may induce an immune response that leads to the priming of antigen-specific dendritic cells (DCs). The targeted intraoperative radiotherapy (TARGIT) method, using INTRABEAM ® , could reduce tumor recurrence, modifying the wound microenvironment, and eradicating residual tumor cells when applied immediately after surgery procedure.

MVP and vaults: a role in the radiation response

Vaults are evolutionary highly conserved ribonucleoproteins particles with a hollow barrel-like structure. The main component of vaults represents the 110 kDa major vault protein (MVP), whereas two minor vaults proteins comprise the 193 kDa vault poly(ADP-ribose) polymerase (vPARP) and the 240 kDa telomerase-associated protein-1 (TEP-1). Additionally, at least one small and untranslated RNA is found as a constitutive component. MVP seems to play an important role in the development of multidrug resistance. This particle has also been implicated in the regulation of several cellular processes including transport mechanisms, signal transmission and immune responses. Vaults are considered a prognostic marker for different cancer types. The level of MVP expression predicts the clinical outcome after chemotherapy in different tumour types. Recently, new roles have been assigned to MVP and vaults including the association with the insulin-like growth factor-1, hypoxia-inducible factor-1alpha, and the two major DNA double-strand break repair machineries: non-homologous endjoining and homologous recombination. Furthermore, MVP has been proposed as a useful prognostic factor associated with radiotherapy resistance. Here, we review these novel actions of vaults and discuss a putative role of MVP and vaults in the response to radiotherapy.

Prediction of normal tissue toxicity as part of the individualized treatment with radiotherapy in oncology patients

Normal tissue toxicity caused by radiotherapy conditions the success of the treatment and the quality of life of patients. Radiotherapy is combined with surgery in both the preoperative or postoperative setting for the treatment of most localized solid tumour types. Furthermore, radical radiotherapy is an alternative to surgery in several tumour locations. The possibility of predicting such radiation-induced toxicity would make possible a better treatment schedule for the individual patient. Radiation-induced toxicity is, at least in part, genetically determined. From decades, several predictive tests have been proposed to know the individual sensitivity of patients to the radiotherapy schedules. Among them, initial DNA damage, radiation-induced apoptosis, gene expression profiles, and gene polymorphisms have been proposed. We report here an overview of the main studies regarding to this field. Radiation-induced apoptosis in peripheral blood lymphocytes seem to be the most promising assay tested in prospective clinical trials, although they have to be validated in large clinical studies. Other promising assays, as those related with single nucleotide polymorphisms, need to be validated as well.