Notch-1 regulates transcription of the epidermal growth factor receptor through p53.

The Notch pathway plays a key role in the development and is increasingly recognized for its importance in cancer. We demonstrated previously the overexpression of Notch-1 and its ligands in gliomas and showed that their knockdown inhibits glioma cell proliferation and survival. To elucidate the mechanisms downstream of Notch-1 in glioma cells, we performed microarray profiling of glioma cells transfected with Notch-1 small interfering RNA. Notable among downregulated transcripts was the epidermal growth factor receptor (EGFR), known to be overexpressed or amplified in gliomas and prominent in other cancers as well. Further studies confirmed that Notch-1 inhibition decreased EGFR messenger RNA (mRNA) and EGFR protein in glioma and other cell lines. Transfection with Notch-1 increased EGFR expression. Additionally, we found a significant correlation in levels of EGFR and Notch-1 mRNA in primary high-grade human gliomas. Subsequent experiments showed that p53, an activator of the EGFR promoter, is regulated by Notch-1. Experiments with p53-positive and -null cell lines confirmed that p53 partially mediates the effects of Notch-1 on EGFR expression. These results show for the first time that Notch-1 upregulates EGFR expression and also demonstrate Notch-1 regulation of p53 in gliomas. These observations have significant implications for understanding the mechanisms of Notch in cancer and development.

Expression of Notch-1 and its ligands, Delta-like-1 and Jagged-1, is critical for glioma cell survival and proliferation.

The Notch family of proteins plays an integral role in determining cell fates, such as proliferation, differentiation, and apoptosis. We show that Notch-1 and its ligands, Delta-like-1 and Jagged-1, are overexpressed in many glioma cell lines and primary human gliomas. Immunohistochemistry of a primary human glioma tissue array shows the presence in the nucleus of the Notch-1 intracellular domain, indicating Notch-1 activation in situ. Down-regulation of Notch-1, Delta-like-1, or Jagged-1 by RNA interference induces apoptosis and inhibits proliferation in multiple glioma cell lines. In addition, pretreatment of glioma cells with Notch-1 or Delta-like-1 small interfering RNA significantly prolongs survival in a murine orthotopic brain tumor model. These results show, for the first time, the dependence of cancer cells on a single Notch ligand; they also suggest a potential Notch juxtacrine/autocrine loop in gliomas. Notch-1 and its ligands may present novel therapeutic targets in the treatment of glioma.

Marginal recurrence requiring salvage radiotherapy after stereotactic body radiotherapy for spinal metastases.

INTRODUCTION: We sought to quantify and identify risk factors associated with margin recurrence (MR) requiring salvage radiotherapy after stereotactic body radiation therapy (SBRT) for spinal metastases. METHODS: We retrospectively reviewed patients with spinal metastases who were treated with single-fraction SBRT between 2006 and 2009. Gross tumor was contoured, along with either the entire associated vertebral body(ies) or the posterior elements, and included in the planning target volume. No additional margins were used. MR was defined as recurrent tumor within one vertebral level above or below the treated lesion that required salvage radiotherapy. Only patients who presented for 3-month post-SBRT follow-up were included in the analysis. Fine and Gray competing risk regression models were generated to identify variables associated with higher risks of MR. MR was plotted using cumulative incidence analysis. RESULTS: SBRT was delivered to 208 lesions in 149 patients. Median follow-up was 8.6 months, and median survival was 12.8 months. The median prescribed dose was 14 Gy (10-16 Gy). MR occurred in 26 (12.5%) treated lesions, at a median time of 7.7 months after SBRT. Patients with paraspinal disease at the time of SBRT (20.8% vs. 7.6% of patients; p = 0.02), and those treated with <16 Gy (16.3% vs. 6.3% of patients, p = 0.14) had higher rates of MR. Both variables were associated with significantly higher risk of MR on multivariate analysis. CONCLUSION: SBRT for spinal metastases results in a low overall rate of MR. The presence of paraspinal disease at the time of SBRT and a dose of <16 Gy were associated with higher risks of MR.

External beam radiotherapy followed by 90Y ibritumomab tiuxetan in relapsed or refractory bulky follicular lymphoma.

PURPOSE: We combined external beam radiotherapy (EBRT) with yttrium-90 ibritumomab tiuxetan ((90)Y-IT) in an attempt to improve therapeutic response in patients with relapsed or refractory bulky follicular lymphoma (RRBFL). METHODS AND MATERIALS: Between February 2006 and September 2007, 11 patients with RRBFL were treated with EBRT followed by (90)Y-IT. Bulky disease (BD) was defined as >5 cm. EBRT was delivered to BD as 2,400 cGy in eight fractions using computed tomography (CT)-based planning. BD was contoured as the gross tumor volume. A planning margin of 1 to 2 cm was added depending on anatomical location. After recovery of complete blood counts (CBC), (90)Y-IT was administered at a dose of 0.3 or 0.4 mCi/kg depending on platelet counts. Hematologic toxicity was monitored through weekly CBC. Response was measured by positron emission tomography/CT or CT 3-4 months after (90)Y-IT. RESULTS: Only 2 patients required prolonged breaks between EBRT and (90)Y-IT. The median time after (90)Y-IT for platelets to recover to >100,000/ml was 55 days (range, 41-128 days). Platelet counts for 1 patient, who had received 4 previous chemotherapy regimens, never reached 100,000/ml. The complete and overall responses to combined therapy as measured 3-4 months after (90)Y-IT were 64%. No patients relapsed within the EBRT field. With a median follow-up of 36.1 months, 6 patients have relapsed, 2 of whom have died. Median progression-free survival was 17.5 months. CONCLUSIONS: In contrast to prior failure analysis data for RRBFL patients treated with (90)Y-IT alone, a brief course of EBRT prevented relapse in sites of BD. EBRT used to pretreat bulky sites may improve clinical outcomes and potentially extend survival when combined with (90)Y-IT.

Maximum standardized uptake value from staging FDG-PET/CT does not predict treatment outcome for early-stage non-small-cell lung cancer treated with stereotactic body radiotherapy.

PURPOSE: To perform a retrospective review to determine whether maximum standardized uptake values (SUV(max)) from staging 2-deoxy-2- [(18)F] fluoro-D-glucose (FDG) positron emission tomography/computed tomography (PET/CT) studies are associated with outcomes for early-stage non-small-cell lung cancer (NSCLC) treated with stereotactic body radiotherapy (SBRT). METHODS AND MATERIALS: Seventy-two medically inoperable patients were treated between October 17, 2003 and August 17, 2007 with SBRT for T1-2N0M0 NSCLC. SBRT was administered as 60 Gy in 3 fractions, 50 Gy in 5 fractions, or 50 Gy in 10 fractions using abdominal compression and image-guided SBRT. Cox proportional hazards regression was performed to determine whether PET SUV(max) and other variables influenced outcomes: mediastinal failure (MF), distant metastases (DM), and overall survival (OS). RESULTS: Biopsy was feasible in 49 patients (68.1%). Forty-nine patients had T1N0 disease, and 23 had T2N0 disease. Median SUV(max) was 6.55 (range, 1.5-21). Median follow-up was 16.9 months (range, 0.1-37.9 months). There were 3 local failures, 8 MF, 19 DM, and 30 deaths. Two-year local control, MF, DM, and OS rates were 94.0%, 10.4%, 30.1%, and 61.3%, respectively. In univariate analysis, PET/CT SUV(max), defined either as a continuous or dichotomous variable, did not predict for MF, DM, or OS. On multivariable analysis, the only predictors for overall survival were T1 stage (hazard ratio = 0.331 [95% confidence interval, 0.156-0.701], p = 0.0039) and smoking pack-year history (hazard ratio = 1.015 [95% confidence interval, 1.004-1.026], p = 0.0084). CONCLUSIONS: Pretreatment PET SUV(max) did not predict for MF, DM, or OS in patients treated with SBRT for early-stage NSCLC.

Update on the rational use of tositumomab and iodine-131 tositumomab radioimmunotherapy for the treatment of non-Hodgkin's lymphoma.

Targeted radioimmunotherapy in non-Hodgkin's B-cell lymphoma (NHL) offers an efficacious therapy and minimal toxicity compared to conventional chemotherapy. Iodine 131 tositumomab ((131)I-TST) is a murine monoclonal antibody against the CD20 cell surface protein and is directly covalently conjugated to (131)I, a radioactive beta and gamma emitter. While initially approved for use in relapsed, refractory, or transformed low grade B-cell NHL, investigational uses with promising results include autologous stem cell transplant, intermediate grade NHL, and the frontline management of indolent NHL. This review summarizes the (131)I-TST literature on mechanism of action, treatment indications, treatment delivery, efficacy, investigational uses, and future prospects.

Comparison of biochemical relapse-free survival between primary Gleason score 3 and primary Gleason score 4 for biopsy Gleason score 7 prostate cancer.

PURPOSE: To determine whether the primary grade (PG) of biopsy Gleason score (GS) 7 prostate cancer (CaP) was predictive for biochemical relapse-free survival (bRFS). Most of the present data regarding the PG of GS7 CaP refer to surgical specimens. Our goal was to determine whether the biopsy GS used at the time of medical decision making predicted for the biochemical outcome. METHODS AND MATERIALS: We reviewed the data from 705 patients with biopsy GS7 CaP, from a prospectively maintained database, who had been treated at our institution between September 1996 and March 2005 with radical prostatectomy (n = 310), external beam radiotherapy (n = 268), or prostate radioactive seed implantation (n = 127). The bRFS rates were estimated using the Kaplan-Meier method. Cox proportional hazards regression analysis was used for univariate and multivariate analyses examining these factors in relation to bRFS: PG of biopsy GS, initial prostate-specific antigen level, clinical T stage, use of androgen deprivation, risk group (high or intermediate), and treatment modality. RESULTS: The 5-year bRFS rate was 78% and 71% (p = 0.0108) for biopsy GS7 PG3 CaP and biopsy GS7 PG4 CaP, respectively. Comparing PG3 and PG4 within treatment modalities, only prostate implantation patients had a significant difference in the 5-year bRFS rate, 88% vs. 76%, respectively (p = 0.0231). On multivariate analysis, the PG of biopsy GS remained an independent predictor of bRFS, with PG3 having better bRFS than PG4 (relative risk, 0.655; 95% confidence interval, 0.472-0.909; p = 0.0113). CONCLUSION: Biopsy GS7 PG4 CaP carries a worse bRFS than biopsy GS7 PG3 CaP.

A single glycosylation site within the receptor-binding domain of the avian sarcoma/leukosis virus glycoprotein is critical for receptor binding.

Retroviral envelope proteins are heavily glycosylated. In some cases, glycosylation has been shown to be important for folding, protein stability, immune evasion, or receptor usage. The receptor-binding subunit (SU or gp85) of the envelope protein (EnvA) of the avian sarcoma/leukosis virus, subtype A (ASLV-A), contains 11 potential N-linked glycosylation sites (NXS/T). To address the importance of N-linked glycosylation for the function of EnvA, we prepared a series of EnvA proteins lacking one or more of these carbohydrate addition sites. Using site-directed mutagenesis, we mutated the S or T in each NXS/T glycosylation sequon to A. We also prepared EnvAs bearing selected double and triple mutations. We examined each mutant EnvA for its ability to be expressed at the cell surface, proteolytically processed into gp85 and gp37, incorporated into MLV pseudotyped virions, and to support infection of cells expressing the ASLV-A receptor, Tva. Eight single mutations were well tolerated, and, in general, EnvA was able to tolerate double mutations of these glycosylation sites. Triple mutations were more variable in their effects. Of the three glycosylation sites important for EnvA function, two are important for folding (EnvA production and processing were severely impaired). For the third, although EnvA processing was impaired, significant amounts of processed EnvA were expressed at the cell surface and incorporated into virions. Nonetheless, this mutant EnvA, EnvADeltaNg10, was unable to support infection. Further examination of EnvADeltaNg10 revealed that it was unable to bind Tva and was severely impaired for binding to a monoclonal antibody which inhibits receptor binding. This work has therefore identified a single N-linked glycosylation site in the SU domain of EnvA that is critical for binding between EnvA and its receptor, Tva.