Best of the Radiosurgery Society® Scientific Meeting 2014: stereotactic radiosurgery/stereotactic body radiotherapy treatment of extracranial and intracranial lesions.

The SRS/SBRT Scientific Meeting 2014, Minneapolis, MN, USA, 7-10 May 2014. The Radiosurgery Society(®), a professional medical society dedicated to advancing the field of stereotactic radiosurgery (SRS) and stereotactic body radiotherapy (SBRT), held the international Radiosurgery Society Scientific Meeting, from 7-10 May 2014 in Minneapolis (MN, USA). This year's conference attracted over 400 attendants from around the world and featured over 100 presentations (46 oral) describing the role of SRS/SBRT for the treatment of intracranial and extracranial malignant and nonmalignant lesions. This article summarizes the meeting highlights for SRS/SBRT treatments, both intracranial and extracranial, in a concise review.

Elevated E2F1 inhibits transcription of the androgen receptor in metastatic hormone-resistant prostate cancer.

Activation of E2F transcription factors, through disruption of the retinoblastoma (Rb) tumor-suppressor gene, is a key event in the development of many human cancers. Previously, we showed that homozygous deletion of Rb in a prostate tissue recombination model exhibits increased E2F activity, activation of E2F-target genes, and increased susceptibility to hormonal carcinogenesis. In this study, we examined the expression of E2F1 in 667 prostate tissue cores and compared it with the expression of the androgen receptor (AR), a marker of prostate epithelial differentiation, using tissue microarray analysis. We show that E2F1 expression is low in benign and localized prostate cancer, modestly elevated in metastatic lymph nodes from hormone-naïve patients, and significantly elevated in metastatic tissues from hormone-resistant prostate cancer patients (P = 0.0006). In contrast, strong AR expression was detected in benign prostate (83%), localized prostate cancer (100%), and lymph node metastasis (80%), but decreased to 40% in metastatic hormone-resistant prostate cancer (P = 0.004). Semiquantitative reverse transcription-PCR analysis showed elevated E2F1 mRNA levels and increased levels of the E2F-target genes dihyrofolate reductase and proliferating cell nuclear antigen in metastatic hormone-independent prostate cancer cases compared with benign tissues. To identify a role of E2F1 in hormone-independent prostate cancer, we examined whether E2F1 can regulate AR expression. We show that exogenous expression of E2F1 significantly inhibited AR mRNA and AR protein levels in prostate epithelial cells. E2F1 also inhibited an AR promoter-luciferase construct that was dependent on the transactivation domain of E2F1. Furthermore, using chromatin immunoprecipitation assays, we show that E2F1 and the pocket protein family members p107 and p130 bind to the AR promoter in vivo. Taken together, these results show that elevated E2F1, through its ability to repress AR transcription, may contribute to the progression of hormone-independent prostate cancer.

Regulation of DNA methyltransferase 1 by the pRb/E2F1 pathway.

Tumor suppressor gene silencing by DNA hypermethylation contributes to tumorigenesis in many tumor types. This aberrant methylation may be due to increased expression and activity of DNA methyltransferases, which catalyze the transfer of methyl groups from S-adenosylmethionine to cytosines in CpG dinucleotides. Elevated expression of the maintenance DNA methyltransferase, DNA methyltransferase 1 (DNMT-1), has been shown in carcinomas of the colon, lung, liver, and prostate. Based on the nearly ubiquitous alterations of both DNA methylation and the retinoblastoma protein (pRb) pathway found in human cancer, we investigated a potential regulatory pathway linking the two alterations in murine and human prostate epithelial cells. Analysis of DNA methyltransferase levels in Rb-/- murine prostate epithelial cell lines revealed elevated Dnmt-1 levels. Genomic DNA sequence analysis identified conserved E2F consensus binding sites in proximity to the transcription initiation points of murine and human Dnmt-1. Furthermore, the Dnmt-1 promoter was shown to be regulated by the pRb/E2F pathway in murine and human cell lines of epithelial and fibroblast origin. In the absence of pRb, Dnmt-1 transcripts exhibited aberrant cell cycle regulation and Rb-/- cells showed aberrant methylation of the paternally expressed gene 3 (Peg3) tumor suppressor gene. These findings show a link between inactivation of the pRb pathway and induction of DNA hypermethylation of CpG island-containing genes in tumorigenesis.

Disruption of Rb/E2F pathway results in increased cyclooxygenase-2 expression and activity in prostate epithelial cells.

The loss of the retinoblastoma tumor suppressor gene (RB) is common in many human cancers, including prostate. We previously reported that engineered deletion of RB in prostate epithelial cells results in sustained cell growth in serum-free media, a predisposition to develop hyperplasia and dysplasia in prostate tissue recombinant grafts, and sensitization to hormonal carcinogenesis. Examining the molecular consequence of RB loss in this system, we show that cyclooxygenase-2 (COX-2) is significantly up-regulated following RB deletion in prostate tissue recombinants. To study the effect of RB deletion on COX-2 regulation, we generated wild-type (PrE) and Rb-/- (Rb-/-PrE) prostate epithelial cell lines rescued by tissue recombination. We show elevated COX-2 mRNA and protein expression in Rb-/-PrE cell lines with increased prostaglandin synthesis. We also find that loss of Rb leads to deregulated E2F activity, with increased expression of E2F target genes, and that exogenous expression of E2F1 results in elevated COX-2 mRNA and protein levels. COX-2 promoter studies reveal that E2F1 transcriptionally activates COX-2, which is dependent on the transactivation and DNA-binding domains of E2F1. Further analysis revealed that the E2F1 target gene, c-myb, is elevated in Rb-/-PrE cells and E2F1-overexpressing cells, whereas ectopic overexpression of c-myb activates the COX-2 promoter in prostate epithelial cells. Additionally, cotransfection with E2F1 and a dominant-negative c-myb inhibited E2F1 activation of the COX-2 promoter. Taken together, these results suggest activation of a transcriptional cascade by which E2F1 regulates COX-2 expression through the c-myb oncogene. This study reports a novel finding describing that deregulation of the Rb/E2F complex results in increased COX-2 expression and activity.

Stereotactic body radiotherapy for centrally located early-stage non-small cell lung cancer or lung metastases from the RSSearch(®) patient registry.

BACKGROUND: The purpose of this study was to evaluate treatment patterns and outcomes of stereotactic body radiotherapy (SBRT) for centrally located primary non-small cell lung cancer (NSCLC) or lung metastases from the RSSearch(®) Patient Registry, an international, multi-center patient registry dedicated to radiosurgery and SBRT. METHODS: Eligible patients included those with centrally located lung tumors clinically staged T1-T2 N0, M0, biopsy-confirmed NSCLC or lung metastases treated with SBRT between November 2004 and January 2014. Descriptive analysis was used to report patient demographics and treatment patterns. Overall survival (OS) and local control (LC) were determined using Kaplan-Meier method. Toxicity was reported using the Common Terminology Criteria for Adverse Events version 3.0. RESULTS: In total, 111 patients with 114 centrally located lung tumors (48 T1-T2,N0,M0 NSCLC and 66 lung metastases) were treated with SBRT at 19 academic and community-based radiotherapy centers in the US and Germany. Median follow-up was 17 months (range, 1-72). Median age was 74 years for primary NSCLC patients and 65 years for lung metastases patients (p < 0.001). SBRT dose varied from 16 - 60 Gy (median 48 Gy) delivered in 1-5 fractions (median 4 fractions). Median dose to centrally located primary NSCLC was 48 Gy compared to 37.5 Gy for lung metastases (p = 0.0001) and median BED10 was 105.6 Gy for primary NSCLC and 93.6 Gy for lung metastases (p = 0.0005). Two-year OS for T1N0M0 and T2N0M0 NSCLC was 79 and 32.1 %, respectively (p = 0.009) and 2-year OS for lung metastases was 49.6 %. Two-year LC was 76.4 and 69.8 % for primary NSCLC and lung metastases, respectively. Toxicity was low with no Grade 3 or higher acute or late toxicities. CONCLUSION: Overall, patients with centrally located primary NSCLC were older and received higher doses of SBRT than those with lung metastases. Despite these differences, LC and OS was favorable for patients with central lung tumors treated with SBRT. Reported toxicity was low, although low grade toxicities were observed in patients where dose tolerances approached or exceeded published guidelines. Prospective studies are needed to further define the optimal SBRT dose for this cohort of patients. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT01885299.

Stereotactic body radiotherapy for early-stage non-small cell lung cancer: clinical outcomes from a National Patient Registry.

OBJECTIVES: Stereotactic body radiotherapy (SBRT) is a definitive local treatment option for patients with stage I non-small cell lung cancer (NSCLC) who are not surgical candidates and patients who refuse surgery. The purpose of this study was to assess the impact of SBRT on T1-T2 NSCLC from a national registry, reflecting practices and outcomes in a real-world setting. METHODS: The RSSearch® Patient Registry was screened for T1-T2N0M0 NSCLC patients treated from May 2004 to May 2013 with SBRT. Descriptive analyses were used for patient, tumor, and treatment characteristics. Overall survival (OS) and local control (LC) were calculated using the Kaplan-Meier method. RESULTS: In total, 723 patients with 517 T1 and 224 T2 lesions were treated with SBRT. Median follow-up was 12 months (1-87 months) with a median age of 76 years. Median SBRT dose was 54 Gy (range 10-80 Gy) delivered in a median of 3 fractions (range 1-5), and median biological equivalent dose (BED10) was 151.2 Gy (range 20-240 Gy). Median OS was 30 and 26 months for T1 and T2 tumors, respectively (p = 0.019). LC was associated with higher BED10 for T2 tumors, but not in T1 tumors at a median follow-up of 17 months. Seventeen-month LC for T2 tumors treated with BED10 < 105 Gy, BED10 105-149, and BED10 ≥ 150 Gy was 43, 74, and 95 %, respectively (p = 0.011). Local failure rates for T2 tumors treated with BED10 < 105 Gy, 105-149 Gy, and ≥150 Gy were 32, 21, and 8 % (p = 0.029), respectively. Median OS for patients with T2 tumors treated with BED10 < 105 Gy was 17 vs. 32 months for T2 tumors treated with BED10 105-149 Gy (p = 0.062). CONCLUSION: SBRT for T1-T2 NSCLC is feasible and effective in the community setting. OS was greater for patients with T1 lesions compared to T2 lesions. An improvement in LC was observed in patients with T2 lesions treated with BED10 > 105 Gy.

The convergence of hormone regulation and cell cycle in prostate physiology and prostate tumorigenesis.

Despite the intense research focused on prostate cancer, it remains the most frequently diagnosed malignancy in men over 40-yr-of-age, and the second most frequent cause of cancer-related deaths in men in the United States (1). In 1990, the National Cancer Institute convened 50 experts and leaders from various disciplines in the prostate cancer field to discuss research directions that would help elucidate the molecular basis of this disease and reduce the incidence and mortality of prostate cancer (2). Critical issues identified at this meeting included the role of androgens and the regulation of cell cycle in prostate tumorigenesis and its progression to androgen-independence. Hormones and cell cycle clearly play important roles in normal and cancerous prostate physiology; however, little information has emerged that clearly delineates their function in the etiology of prostate cancer. Some of the mutational events that occur during prostate tumorigenesis and its progression to androgen-independence involve alterations to normal growth, developmental and apoptotic programs regulated by androgen, and the cell cycle. As such, the delineation of events by which prostate cancer cells circumvent these regulatory mechanisms will be central to our understanding of prostate tumorigenesis and to the development of new modalities to treat this disease. This article is then intended to summarize the functional convergence of androgen regulation and cell cycle in normal prostate physiology and prostate tumorigenesis.

The RSSearch™ Registry: patterns of care and outcomes research on patients treated with stereotactic radiosurgery and stereotactic body radiotherapy.

BACKGROUND: The RSSearch™ Registry is a multi-institutional, observational, ongoing registry established to standardize data collection from patients treated with stereotactic radiosurgery (SRS) and/or stereotactic body radiotherapy (SBRT). This report describes the design, patient demographics, lesion characteristics, and SRS/SBRT treatment patterns in RSSearch™. Illustrative patient-related outcomes are also presented for two common treatment sites--brain metastases and liver metastases. MATERIALS AND METHODS: Thirty-nine US centers participated in RSSearch™. Patients screened for SRS/SBRT were eligible to be enrolled. Descriptive analyses were performed to assess patient characteristics, physician treatment practices, and clinical outcomes. Kaplan-Meier analysis was used to determine overall survival (OS), local progression-free (LPFS), and distant disease-free survival (DDFS). RESULTS: From January, 2008-January, 2013, 11,457 patients were enrolled. The median age was 67 years (range 7-100 years); 51% male and 49% female. Forty-six percent had no prior treatment, 22% had received chemotherapy, 19% radiation therapy and 17% surgery. There were 11,820 lesions from 65 treatment locations; 54% extracranial and 46% intracranial. The most common treatment locations were brain/cranial nerve/spinal cord, lung, prostate and liver. Metastatic lesions accounted for the majority of cases (41.6%), followed by primary malignant (32.9%), benign (10.9%), recurrent (9.4%), and functional diseases (4.3%). SRS/SBRT was used with a curative intent in 39.8% and palliative care in 44.8% of cases. The median dose for all lesions was 30 Gy (range < 1-96.7 Gy) delivered in a median number of 3 fractions. The median dose for lesions in the brain/cranial nerve/spinal cord, lung, liver, pancreas and prostate was 24, 54, 45, 29 and 36.25 Gy, respectively. In a subset analysis of 799 patients with 952 brain metastases, median OS was 8 months. For patients with a Karnofsky performance score (KPS) > 70, OS was 11 months vs. 4 months for KPS ≤ 70. Six-month and 12-month local control was 79% and 61%, respectively for patients with KPS ≤ 70, and 85% and 74%, respectively for patients with KPS > 70. In a second subset analysis including 174 patients with 204 liver metastases, median OS was 22 months. At 1-year, LPFS and DDFS rates were 74% and 53%, respectively. LPFS CONCLUSION: This study demonstrates that collective patterns of care and outcomes research for SRS/SBRT can be performed and reported from data entered by users in a common database. The RSSearch™ dataset represents SRS/SBRT practices in a real world setting, providing a useful resource for expanding knowledge of SRS/SBRT treatment patterns and outcomes and generating robust hypotheses for randomized clinical studies.

Repression of androgen receptor transcription through the E2F1/DNMT1 axis.

Although androgen receptor (AR) function has been extensively studied, regulation of the AR gene itself has been much less characterized. In this study, we observed a dramatic reduction in the expression of androgen receptor mRNA and protein in hyperproliferative prostate epithelium of keratin 5 promoter driven E2F1 transgenic mice. To confirm an inhibitory function for E2F1 on AR transcription, we showed that E2F1 inhibited the transcription of endogenous AR mRNA, subsequent AR protein, and AR promoter activity in both human and mouse epithelial cells. E2F1 also inhibited androgen-stimulated activation of two AR target gene promoters. To elucidate the molecular mechanism of E2F-mediated inhibition of AR, we evaluated the effects of two functional E2F1 mutants on AR promoter activity and found that the transactivation domain appears to mediate E2F1 repression of the AR promoter. Because DNMT1 is a functional intermediate of E2F1 we examined DNMT1 function in AR repression. Repression of endogenous AR in normal human prostate epithelial cells was relieved by DNMT1 shRNA knock down. DNMT1 was shown to be physically associated within the AR minimal promoter located 22 bps from the transcription start site; however, methylation remained unchanged at the promoter regardless of DNMT1 expression. Taken together, our results suggest that DNMT1 operates either as a functional intermediary or in cooperation with E2F1 inhibiting AR gene expression in a methylation independent manner.

Expression of prostate-specific antigen is transcriptionally regulated by genistein in prostate cancer cells.

Prostate cancer is the second-leading cause of cancer-related deaths in men in the United States. Unfortunately, there is no effective therapy when prostate cancer becomes metastatic and refractory to conventional treatments. For this reason, the identification and exploration of new agents that reduce prostate cancer cell growth are of paramount importance. High consumption of plant-derived phytoestrogens is inversely associated with the incidence and mortality rate of prostate cancer. Previous studies, including our own, have shown that the phytoestrogen genistein inhibits prostate cancer cell growth in vitro and in vivo and decreases secreted and intracellular levels of the androgen-regulated protein prostate-specific antigen (PSA), but the role of genistein as an agonist/antagonist for hormone receptors remains unclear. To elucidate the mechanism by which genistein modulates PSA protein expression in prostate cancer cells, we investigated the effects of genistein on androgen-mediated and estrogen-mediated transcriptional regulation of PSA, androgen receptor (AR) mRNA and protein expression, and the ability of nuclear proteins to bind to androgen-response elements (AREs) in LNCaP cells. We showed that genistein decreased the transcriptional activation of PSA by both androgen-dependent and androgen-independent methods in LNCaP cells. The reduction of androgen-mediated transcriptional activation of PSA was correlated with decreased AR protein and mRNA levels and decreased binding to AREs. In contrast, genistein had differential effects on 17beta-estradiol-mediated PSA expressions. Low concentrations of genistein enhanced 17beta-estradiol-mediated PSA expressions, whereas high concentrations of genistein inhibited estrogen-mediated PSA expression in LNCaP cells. Genistein did not inhibit AR protein expression in the presence of 17beta-estradiol. These results suggest that ligand-dependent differences in the ability to activate PSA expression may contribute to the agonistic/antagonistic responses observed with genistein in prostate cancer cells.

Rescue of embryonic epithelium reveals that the homozygous deletion of the retinoblastoma gene confers growth factor independence and immortality but does not influence epithelial differentiation or tissue morphogenesis.

The ability to rescue viable prostate precursor tissue from retinoblastoma-deficient (Rb-/-) fetal mice has allowed for the isolation and characterization of the first Rb-/- prostate epithelial cell line. This cell line, designated Rb-/-PrE, was utilized for experiments examining the consequences of Rb loss on an epithelial population. These findings demonstrated that Rb deletion has no discernible effect on prostatic histodifferentiation in Rb-/-PrE cultures. When Rb-/-PrE cells were recombined with embryonic rat urogenital mesenchyme and implanted into athymic male, nude mouse hosts, the recombinants developed into fully differentiated and morphologically normal prostate tissue. The Rb-/-PrE phenotype was characterized by serum independence in culture and immortality in vivo, when compared with wild type controls. Cell cycle analysis revealed elevated S phase DNA content accompanied by increased expression of cyclin E1 and proliferating cell nuclear antigen. Rb-/-PrE cultures also exhibited a diminished ability to growth arrest under high density culture conditions. We believe that the development of Rb-/- prostate tissue and cell lines has provided a unique experimental platform with which to investigate the consequences of Rb deletion in epithelial cells under various physiological conditions. Additionally, the development of this technology will allow similar studies in other tissues and cell populations rescued from Rb-/- fetuses.