Irradiation, cisplatin, and 5-azacytidine upregulate cytomegalovirus promoter in tumors and muscles: Implementation of non-invasive fluorescence imaging

Purpose: The cytomegalovirus (CMV) promoter is one of the most commonly used promoters for expression of transgenes in mammalian cells. The aim of our study was to evaluate the role of methylation and upregulation of the CMV promoter by irradiation and the chemotherapeutic agent cisplatin in vivo using non-invasive fluorescence in vivo imaging. Procedures: Murine fibrosarcoma LPB and mammary carcinoma TS/A cells were stably transfected with plasmids encoding CMV and p21 promoter-driven green fluorescent protein (GFP) gene. Solid TS/A tumors were induced by subcutaneous injection of fluorescent tumor cells, while leg muscles were transiently transfected with plasmid encoding GFP under the control of the CMV promoter. Cells, tumors, and legs were treated either by DNA methylation inhibitor 5-azacytidine, irradiation, or cisplatin. GFP expression was determined using a fluorescence microplate reader in vitro and by non-invasive fluorescence imaging in vivo. Results: Treatment of cells, tumors, and legs with 5-azacytidine (re)activated the CMV promoter. Furthermore, treatment with irradiation or cisplatin resulted in significant upregulation of GFP expression both in vitro and in vivo. Conclusions: Observed alterations in the activity of the CMV promoter limit the usefulness of this widely used promoter as a constitutive promoter. On the other hand, inducibility of CMV promoters can be beneficially used in gene therapy when combined with standard cancer treatment, such as radiotherapy and chemotherapy. © 2010 The Author(s).

Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10-5), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10-4) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10-9). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.

The impact of inter-fraction set-up errors on the probability of pulmonary and cardiac complication in left-sided breast cancer patients

Purpose This study evaluated the impact of patient set-up errors on the probability of pulmonary and cardiac complications in the irradiation of left-sided breast cancer. Methods and Materials Using the CMS XiO Version 4.6 (CMS Inc., St Louis, MO) radiotherapy planning system's NTCP algorithm and the Lyman -Kutcher-Burman (LKB) model, we calculated the DVH indices for the ipsilateral lung and heart and the resultant normal tissue complication probabilities (NTCP) for radiation-induced pneumonitis and excess cardiac mortality in 12 left-sided breast cancer patients. Results Isocenter shifts in the posterior direction had the greatest effect on the lung V20, heart V25, mean and maximum doses to the lung and the heart. Dose volume histograms (DVH) results show that the ipsilateral lung V20 tolerance was exceeded in 58% of the patients after 1cm posterior shifts. Similarly, the heart V25 tolerance was exceeded after 1cm antero-posterior and left-right isocentric shifts in 70% of the patients. The baseline NTCPs for radiation-induced pneumonitis ranged from 0.73% - 3.4% with a mean value of 1.7%. The maximum reported NTCP for radiation-induced pneumonitis was 5.8% (mean 2.6%) after 1cm posterior isocentric shift. The NTCP for excess cardiac mortality were 0 % in 100% of the patients (n=12) before and after setup error simulations. Conclusions Set-up errors in left sided breast cancer patients have a statistically significant impact on the Lung NTCPs and DVH indices. However, with a central lung distance of 3cm or less (CLD <3cm), and a maximum heart distance of 1.5cm or less (MHD<1.5cm), the treatment plans could tolerate set-up errors of up to 1cm without any change in the NTCP to the heart.

MicroRNAs in breast cancer progression and DNA damage response

Os tumores de mama são caracterizados pela sua alta heterogeneidade. O câncer de mama é uma doença complexa, que possui o seu desenvolvimento fortemente influenciado por fatores ambientais, combinada a uma progressiva acumulação de mutações genéticas e desregulação epigenética de vias críticas. Alterações nos padrões de expressão gênica podem ser resultado de uma desregulação no controle de eventos epigenéticos, assim como, na regulação pós-transcricional pelo mecanismo de RNA de interferência endógeno via microRNA (miRNA). Estes eventos são capazes de levar à iniciação, à promoção e à manutenção da carcinogênese, como também ter implicações no desenvolvimento da resistência à terapia Os miRNAs formam uma classe de RNAs não codificantes, que durante os últimos anos surgiram como um dos principais reguladores da expressão gênica, através da sua capacidade de regular negativamente a atividade de RNAs mensageiros (RNAms) portadores de uma seqüencia parcialmente complementar. A importância da regulação mediada por miRNAs foi observada pela capacidade destas moléculas em regular uma vasta gama de processos biológicos incluindo a proliferação celular, diferenciação e a apoptose. Para avaliar a expressão de miRNAs durante a progressão tumoral, utilizamos como modelo experimental a série 21T que compreende 5 linhagens celulares originárias da mesma paciente diagnosticada com um tumor primário de mama do tipo ErbB2 e uma posterior metástase pulmonar. Essa série é composta pela linhagem obtida a partir do tecido normal 16N, pelas linhagens correspondentes ao carcinoma primário 21PT e 21NT e pelas linhagens obtidas um ano após o diagnóstico inicial, a partir da efusão pleural no sítio metastatico 21MT1 e 21MT2. O miRNAoma da série 21T revelou uma redução significativa nos níveis de miR-205 e nos níveis da proteina e-caderina e um enriquecimento do fator pró-metastático ZEB-1 nas células 21MT. Considerando a importância dos miRNAs na regulação da apoptose, e que a irradiação em diferentes espectros é comumente usada em procedimentos de diagnóstico como mamografia e na radioterapia, avaliamos a expressão de miRNAs após irradiação de alta e baixa energia e do tratamento doxorrubicina. Para os ensaios foram utilizados as linhagens não tumorais MCF-10A e HB-2 e as linhagens de carcinoma da mama MCF-7 e T-47D. Observou-se que raios-X de baixa energia são capazes de promover quebras na molécula do DNA e apoptose assim como, alterar sensivelmente miRNAs envolvidos nessas vias como o let-7a, miR-34a e miR-29b. No que diz respeito à resposta a danos genotóxicos, uma regulação positiva sobre a expressão de miR-29b, o qual em condições normais é regulado negativamente foi observada uma regulação positiva sobre miR-29b expressão após todos os tratamentos em células tumorais. Nossos resultados indicam que miR-29b é um possível biomarcador de estresse genotóxico e que miR-205 pode participar no potencial metastático das células 21T.

Potentiality of phosphorylation of BRCA1 at Ser 1524 to activate p21 in response to X-ray irradiation

The breast and ovarian cancer susceptibility gene BRCA1 encodes a nuclear phosphoprotein, which functions as a tumor suppressor gene. Many studies suggested that multiple functions of BRCA1 may contribute to its tumor suppressor activity, including roles in cell cycle checkpoints, apoptosis and transcription. It is postulated that phosphorylation of BRCA1 is an important means by which its cellular functions are regulated. In this study, we employed phospho-Ser-specific antibody recognizing Ser-1524 to study BRCA1 phosphorylation under conditions of DNA damage and the effects of phosphorylation on BRCA1 functions. The results showed that 10 Gy X-ray treatment significantly induced phosphorylation of Ser-1524 but not total BRCA1 protein levels. The expression both of p53 and p21 increased after irradiation, but ionizing radiation (IR) -induced activation of p21 was prior to that of p53. The percentages of G0/G1 phase remarkably increased after IR. In addition, no detectable levels of 89 kDa fragment of PARP, a marker of apoptotic cells, were observed. Data implied that IR-induced phosphorylation of BRCA1 at Ser-1524 might activatep21 protein, by which BRCA1 regulated cell cycle, but play no role in apoptosis.

Evaluation of organ-specific peripheral doses after 2-dimensional, 3-dimensional and hybrid intensity modulated radiation therapy for breast cancer based on Monte Carlo and convolution/superposition algorithms: implications for secondary cancer risk assessment.

To make a comprehensive evaluation of organ-specific out-of-field doses using Monte Carlo (MC) simulations for different breast cancer irradiation techniques and to compare results with a commercial treatment planning system (TPS). Three breast radiotherapy techniques using 6MV tangential photon beams were compared: (a) 2DRT (open rectangular fields), (b) 3DCRT (conformal wedged fields), and (c) hybrid IMRT (open conformal+modulated fields). Over 35 organs were contoured in a whole-body CT scan and organ-specific dose distributions were determined with MC and the TPS. Large differences in out-of-field doses were observed between MC and TPS calculations, even for organs close to the target volume such as the heart, the lungs and the contralateral breast (up to 70% difference). MC simulations showed that a large fraction of the out-of-field dose comes from the out-of-field head scatter fluence (>40%) which is not adequately modeled by the TPS. Based on MC simulations, the 3DCRT technique using external wedges yielded significantly higher doses (up to a factor 4-5 in the pelvis) than the 2DRT and the hybrid IMRT techniques which yielded similar out-of-field doses. In sharp contrast to popular belief, the IMRT technique investigated here does not increase the out-of-field dose compared to conventional techniques and may offer the most optimal plan. The 3DCRT technique with external wedges yields the largest out-of-field doses. For accurate out-of-field dose assessment, a commercial TPS should not be used, even for organs near the target volume (contralateral breast, lungs, heart).

Evaluation of frozen-section analysis of surgical margins in the treatment of breast cancer

Objective: To evaluate surgical margins in cases of ductal carcinoma through a histopathological exam using frozen sections. Materials and Methods: Retrospective study encompassing 242 conservative surgeries, 179 of which included intraoperative frozensection histopathology and 63 intraoperative nonfreezing techniques (macroscopy/gross examination and cytology). The results of such analyses were compared with those of the histology processing following paraffin embedment and hematoxylin and eosin (H & E) staining. A margin was deemed free when the distance between the tumor and the surgical border was equal to or greater than two millimeters. The factors given consideration for possibly affecting the results were: age, surgical aspects (skin removal and widening of surgical margins), histopathological findings (size, affected lymph nodes, and angiolymphatic invasion), and extensive intraductal and immunohistochemical components (estrogen, progesterone, Ki-67, and HER-2 receptors). In the statistical analyses, the chi-square test was used and negative predictive values were calculated. Results: The negative predictive values were 87.1% and 79.3% for frozen and nonfrozen sections, respectively. There was no significant difference between the two groups (p = 0.14). The factors under consideration had no influence on the results of the intraoperative exam of the margins. Conclusion: The present study allowed to conclude that the intraoperative exam of the surgical margins by frozen section is not superior to a macroscopy and / or cytology exam.

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

[EN] 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.

Treatment of premenopausal women with early breast cancer: old challenges and new opportunities

Breast cancer occurring in women before the age of menopause continues to be a major medical and psychological challenge. Endocrine therapy has emerged as the mainstay of adjuvant treatment for women with estrogen receptor-positive tumours. Although the suppression of ovarian function (by oophorectomy, irradiation of the ovaries or gonadotropin releasing factor analogues) is effective as adjuvant therapy if used alone, its value has not been proven after chemotherapy. This is presumably because of the frequent occurrence of chemotherapy-induced amenorrhoea. Tamoxifen reduces the risk of recurrence by approximately 40%, irrespective of age and the ovarian production of estrogens. The worth of ovarian function suppression in combination with tamoxifen is unproven and is being investigated in an intergroup randomised clinical trial (SOFT [Suppression of Ovarian Function Trial]). Aromatase inhibitors are more effective than tamoxifen in postmenopausal women but are only being investigated in younger patients. The use of chemotherapies is identical in younger and older patients; however, at present the efficacy of chemotherapy in addition to ovarian function suppression plus tamoxifen is unknown in premenopausal patients with endocrine responsive disease. 'Targeted' therapies such as monoclonal antibodies to human epidermal growth factor receptor (HER)-2, HER1 and vascular endothelial growth factor, 'small molecule' inhibitors of tyrosine kinases and breast cancer vaccines are rapidly emerging. Their use depends on the function of the targeted pathways and is presently limited to clinical trials. Premenopausal patients are best treated in the framework of a clinical trial.

Adjuvant exemestane with ovarian suppression in premenopausal breast cancer.

BACKGROUND Adjuvant therapy with an aromatase inhibitor improves outcomes, as compared with tamoxifen, in postmenopausal women with hormone-receptor-positive breast cancer. METHODS In two phase 3 trials, we randomly assigned premenopausal women with hormone-receptor-positive early breast cancer to the aromatase inhibitor exemestane plus ovarian suppression or tamoxifen plus ovarian suppression for a period of 5 years. Suppression of ovarian estrogen production was achieved with the use of the gonadotropin-releasing-hormone agonist triptorelin, oophorectomy, or ovarian irradiation. The primary analysis combined data from 4690 patients in the two trials. RESULTS After a median follow-up of 68 months, disease-free survival at 5 years was 91.1% in the exemestane-ovarian suppression group and 87.3% in the tamoxifen-ovarian suppression group (hazard ratio for disease recurrence, second invasive cancer, or death, 0.72; 95% confidence interval [CI], 0.60 to 0.85; P<0.001). The rate of freedom from breast cancer at 5 years was 92.8% in the exemestane-ovarian suppression group, as compared with 88.8% in the tamoxifen-ovarian suppression group (hazard ratio for recurrence, 0.66; 95% CI, 0.55 to 0.80; P<0.001). With 194 deaths (4.1% of the patients), overall survival did not differ significantly between the two groups (hazard ratio for death in the exemestane-ovarian suppression group, 1.14; 95% CI, 0.86 to 1.51; P=0.37). Selected adverse events of grade 3 or 4 were reported for 30.6% of the patients in the exemestane-ovarian suppression group and 29.4% of those in the tamoxifen-ovarian suppression group, with profiles similar to those for postmenopausal women. CONCLUSIONS In premenopausal women with hormone-receptor-positive early breast cancer, adjuvant treatment with exemestane plus ovarian suppression, as compared with tamoxifen plus ovarian suppression, significantly reduced recurrence. (Funded by Pfizer and others; TEXT and SOFT ClinicalTrials.gov numbers, NCT00066703 and NCT00066690, respectively.).

Abrogation of the G2 cell cycle checkpoint associated with overexpression of HSIX1: A possible mechanism of breast carcinogenesis

While conducting a search for cell cycle-regulated genes in human mammary carcinoma cells, we identified HSIX1, a recently discovered member of a new homeobox gene subfamily. HSIX1 expression was absent at the onset of and increased toward the end of S phase. Since its expression pattern is suggestive of a role after S phase, we investigated the effect of HSIX1 in the G2 cell cycle checkpoint. Overexpression of HSIX1 in MCF7 cells abrogated the G2 cell cycle checkpoint in response to x-ray irradiation. HSIX1 expression was absent or very low in normal mammary tissue, but was high in 44% of primary breast cancers and 90% of metastatic lesions. In addition, HSIX1 was expressed in a variety of cancer cell lines, suggesting an important function in multiple tumor types. These data support the role for homeobox genes in tumorigenesis/tumor progression, possibly through a cell cycle function.

The impact of gamma irradiation on the cytotoxic properties and phenolic composition of Thymus vulgaris L. and Menta x piperita L.

Irradiation has been increasingly recognized as an effective decontamination technique, also ensuring the chemical and organoleptic quality of medicinal and aromatic plants 1 . The use of medicinal plants in the prevention and or treatment of several diseases has revealed satisfactory results as anti-inflammatory, antimutagenic, anti-cancer and antioxidant agents 2 . The aim of the present study was to evaluate the effects of gamma irradiation on the cytotoxic properties and phenolic composition of Thymus vulgaris L. and Menta x piperita L. (methanolic extracts). Phenolic compounds were analyzed by HPLC-DAD-ESI MS, while the cytotoxicity of the samples was assessed in MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), HeLa (cervical carcinoma), HepG2 (hepatocellular carcinoma) cell lines, as also in non-tumor cells (PLP2). Thirteen and fourteen phenolic compounds were detected in T. vulgaris and M. piperita, respectively, but none of them was affected by the irradiation up to a dose of 10 kGy. However, despite there were no changes in the cytotoxic properties of irradiated peppermint samples in tumor cell lines, the thyme samples irradiated with 10 kGy showed higher cytotoxicity in comparison with the samples submitted to other doses (2 and 5 kGy). This highlights that 10 kGy can be a suitable dose to ensure the sanitary treatment, without modifying the bioactive composition and properties of these aromatic plants.