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

Henríquez Hernández LA, Lara PC, Pinar B, Bordón E, Rodríguez Gallego C, Bilbao C, Fernández Pérez L, Flores Morales A - Radiat Oncol (2009)

Bottom Line: Those genes were gathered in 4 significant pathways.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.Prospective studies with higher number of patients are needed to validate these preliminary results.

View Article: PubMed Central - HTML - PubMed

Affiliation: Canary Foundation of Investigation and Health, Spain. lhenriquez@dcc.ulpgc.es

ABSTRACT
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.

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Experimental design. RNA from lymphocytes treated with 0 and 2 Gy dose of radiation were compared against a human universal RNA. SAM analyses were performed to disclose significant regulated genes in these two ways. In order to explore genes modulated by radiation, a two-class paired test was performed using SAM. To discriminate genes that could be significantly associated with RT toxicity, non-supervised hierarchical clustering, in MeV, was used to visualize the whole set of significant genes modulated before and after X-ray exposure in patients with and without acute/late toxicity.
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Figure 1: Experimental design. RNA from lymphocytes treated with 0 and 2 Gy dose of radiation were compared against a human universal RNA. SAM analyses were performed to disclose significant regulated genes in these two ways. In order to explore genes modulated by radiation, a two-class paired test was performed using SAM. To discriminate genes that could be significantly associated with RT toxicity, non-supervised hierarchical clustering, in MeV, was used to visualize the whole set of significant genes modulated before and after X-ray exposure in patients with and without acute/late toxicity.

Mentions: Twelve consecutive patients treated between 1991 and 1997 by a hyperfractionated dose-escalation radiation therapy schedule at the Hospital Dr. Negrín suffering from LABC were prospectively recruited and inform consent was given. The study was approved by the Research and Ethics Committee of our institution. Blood samples were extracted and tested during 2005 and follow up was closed on December 2008. Characteristics of the patients are shown in Table 1. Early toxicity was evaluated during and at the end of RT, and late toxicity was evaluated at 6-month follow-up examination. The RTOG morbidity score system was used to classify the toxicity of patients into three levels: grades 1, 2 and 3–4 (Table 2). All patients were referred to recieve 60 Gy to the whole breast over a period of 5 weeks in two daily fractions of 1.2 Gy separated by at least 6 h on 5 days each week, and followed by a boost of 21.6 Gy to a total dose of 81.6 Gy. Culture of lymphocytes and radiation protocol details were previously reported [11]. Twenty four independent hybridizations were performed to compare lymphocytes from twelve patients, before and after 2 Gy irradiation, against a human RNA universal control. A microarray containing 35.327 human 70-mer oligo probe sets, produced at the SweGene DNA Microarray Resource Center (Lund University, Sweden) was used. Array scanning, image analysis and data normalization were performed as previously described [12,13]. Identification of differentially-expressed genes was performed using the SAM (Significance Analysis for Microarrays) statistical technique [14]. A q value was assigned for each of the detectable genes in the array measuring the lowest false discovery rate (FDR). Genes with a FDR of less than 10% were considered to present significant differential expression. Thus, we studied gene expression profile of lymphocytes treated with 0 and 2 Gy separately. To explore genes modulated by radiation, we also compared gene expression profiles of lymphocytes treated with 0 versus 2 Gy. T-test and ANOVA test [15,16] were used to compare the set of genes significantly regulated, in un-irradiated and in 2 Gy-irradiated lymphocytes, with toxicity. Non-supervised hierarchical clustering [17] was made using MultiExperiment Viewer (The Institute for Genomic Research, ). A genetic signature that could separate toxicity and non toxicity in a constitutive and in a modulated-by-radiation way was performed (Figure 1). Functional classification and pathway analysis of expressed genes were performed by using the web-based tools Onto-Express (OE) and Pathway-Express (PE) (Intelligence Systems and Bioinformatics Laboratory, Wayne University, Detroit, MI. ) [18,19]. OE classify genes in order to biological process (BP), cellular component and molecular function, and it is able to estimate statistical differences between different gene ontology terms [20]. PE is based on a novel method that uses a system biology approach that includes important biological factors that describes how these genes interacts and the type of signaling interactions between them [21,22].


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

Henríquez Hernández LA, Lara PC, Pinar B, Bordón E, Rodríguez Gallego C, Bilbao C, Fernández Pérez L, Flores Morales A - Radiat Oncol (2009)

Experimental design. RNA from lymphocytes treated with 0 and 2 Gy dose of radiation were compared against a human universal RNA. SAM analyses were performed to disclose significant regulated genes in these two ways. In order to explore genes modulated by radiation, a two-class paired test was performed using SAM. To discriminate genes that could be significantly associated with RT toxicity, non-supervised hierarchical clustering, in MeV, was used to visualize the whole set of significant genes modulated before and after X-ray exposure in patients with and without acute/late toxicity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698866&req=5

Figure 1: Experimental design. RNA from lymphocytes treated with 0 and 2 Gy dose of radiation were compared against a human universal RNA. SAM analyses were performed to disclose significant regulated genes in these two ways. In order to explore genes modulated by radiation, a two-class paired test was performed using SAM. To discriminate genes that could be significantly associated with RT toxicity, non-supervised hierarchical clustering, in MeV, was used to visualize the whole set of significant genes modulated before and after X-ray exposure in patients with and without acute/late toxicity.
Mentions: Twelve consecutive patients treated between 1991 and 1997 by a hyperfractionated dose-escalation radiation therapy schedule at the Hospital Dr. Negrín suffering from LABC were prospectively recruited and inform consent was given. The study was approved by the Research and Ethics Committee of our institution. Blood samples were extracted and tested during 2005 and follow up was closed on December 2008. Characteristics of the patients are shown in Table 1. Early toxicity was evaluated during and at the end of RT, and late toxicity was evaluated at 6-month follow-up examination. The RTOG morbidity score system was used to classify the toxicity of patients into three levels: grades 1, 2 and 3–4 (Table 2). All patients were referred to recieve 60 Gy to the whole breast over a period of 5 weeks in two daily fractions of 1.2 Gy separated by at least 6 h on 5 days each week, and followed by a boost of 21.6 Gy to a total dose of 81.6 Gy. Culture of lymphocytes and radiation protocol details were previously reported [11]. Twenty four independent hybridizations were performed to compare lymphocytes from twelve patients, before and after 2 Gy irradiation, against a human RNA universal control. A microarray containing 35.327 human 70-mer oligo probe sets, produced at the SweGene DNA Microarray Resource Center (Lund University, Sweden) was used. Array scanning, image analysis and data normalization were performed as previously described [12,13]. Identification of differentially-expressed genes was performed using the SAM (Significance Analysis for Microarrays) statistical technique [14]. A q value was assigned for each of the detectable genes in the array measuring the lowest false discovery rate (FDR). Genes with a FDR of less than 10% were considered to present significant differential expression. Thus, we studied gene expression profile of lymphocytes treated with 0 and 2 Gy separately. To explore genes modulated by radiation, we also compared gene expression profiles of lymphocytes treated with 0 versus 2 Gy. T-test and ANOVA test [15,16] were used to compare the set of genes significantly regulated, in un-irradiated and in 2 Gy-irradiated lymphocytes, with toxicity. Non-supervised hierarchical clustering [17] was made using MultiExperiment Viewer (The Institute for Genomic Research, ). A genetic signature that could separate toxicity and non toxicity in a constitutive and in a modulated-by-radiation way was performed (Figure 1). Functional classification and pathway analysis of expressed genes were performed by using the web-based tools Onto-Express (OE) and Pathway-Express (PE) (Intelligence Systems and Bioinformatics Laboratory, Wayne University, Detroit, MI. ) [18,19]. OE classify genes in order to biological process (BP), cellular component and molecular function, and it is able to estimate statistical differences between different gene ontology terms [20]. PE is based on a novel method that uses a system biology approach that includes important biological factors that describes how these genes interacts and the type of signaling interactions between them [21,22].

Bottom Line: Those genes were gathered in 4 significant pathways.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.Prospective studies with higher number of patients are needed to validate these preliminary results.

View Article: PubMed Central - HTML - PubMed

Affiliation: Canary Foundation of Investigation and Health, Spain. lhenriquez@dcc.ulpgc.es

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus