Immune surveillance and therapy of lymphomas driven by Epstein-Barr virus protein LMP1 in a mouse model.

B cells infected by Epstein-Barr virus (EBV), a transforming virus endemic in humans, are rapidly cleared by the immune system, but some cells harboring the virus persist for life. Under conditions of immunosuppression, EBV can spread from these cells and cause life-threatening pathologies. We have generated mice expressing the transforming EBV latent membrane protein 1 (LMP1), mimicking a constitutively active CD40 coreceptor, specifically in B cells. Like human EBV-infected cells, LMP1+ B cells were efficiently eliminated by T cells, and breaking immune surveillance resulted in rapid, fatal lymphoproliferation and lymphomagenesis. The lymphoma cells expressed ligands for a natural killer (NK) cell receptor, NKG2D, and could be targeted by an NKG2D-Fc fusion protein. These experiments indicate a central role for LMP1 in the surveillance and transformation of EBV-infected B cells in vivo, establish a preclinical model for B cell lymphomagenesis in immunosuppressed patients, and validate a new therapeutic approach.

Role of the RB1 family in stabilizing histone methylation at constitutive heterochromatin.

Here, we show a role for the RB1 family proteins in directing full heterochromatin formation. Mouse embryonic fibroblasts that are triply deficient for RB1 (retinoblastoma 1), RBL1 (retinoblastoma-like 1) and RBL2 (retinoblastoma-like 2) - known as TKO cells - show a marked genomic instability, which is coincidental with decreased DNA methylation, increased acetylation of histone H3 and decreased tri-methylation of histone H4 at lysine 20 (H4K20). Chromatin immunoprecipitation showed that H4K20 tri-methylation was specifically decreased at pericentric and telomeric chromatin. These defects are independent of E2F family function. Indeed, we show a direct interaction between the RB1 proteins and the H4K20 tri-methylating enzymes Suv4-20h1 and Suv4-20h2, indicating that the RB1 family has a role in controlling H4K20 tri-methylation by these histone methyltransferases. These observations indicate that the RB1 family is involved in maintaining overall chromatin structure and, in particular, that of constitutive heterochromatin, linking tumour suppression and the epigenetic definition of chromatin.

Salvage radiation in men after prostate-specific antigen failure and the risk of death.

BACKGROUND: A survival benefit has been observed with salvage radiation therapy (RT) for prostate-specific antigen (PSA) failure after radical prostatectomy (RP) in men with rapid rises in PSA doubling time (DT, < 6 months). Whether such a benefit exits in men with a protracted PSA rise in DT (≥ 6 months) is unclear and was examined in the current study. METHODS: Of 4036 men who underwent RP at Duke University between 1988 and 2008, 519 experienced a PSA failure, had complete data, and were the subjects of this study. Univariate and multivariate Cox regression analyses were performed to evaluate whether salvage RT in men with either a rapid (< 6 months) or a protracted (≥ 6 months) PSA DT was associated with the risk of all-cause mortality adjusting for age at the time of PSA failure, known prostate cancer prognostic factors, and cardiac comorbidity. RESULTS: After a median follow-up of 11.3 years after PSA failure, 195 men died. Salvage RT was associated with a significant reduction in all-cause mortality for men with either a PSA DT of < 6 months (adjusted hazard ratio [AHR], 0.53; P = .02) or a PSA DT of ≥ 6 months (AHR, 0.52; P = .003). In a subset of patients with comorbidity data at the time of PSA failure, salvage RT remained associated with a significant reduction in all-cause mortality for both men with a PSA DT of < 6 months (AHR, 0.35; P = .042) or a PSA DT of ≥ 6 months (AHR, 0.60; P = .04). CONCLUSIONS: Salvage RT for PSA DTs less than or in excess of 6 months is associated with a decreased risk in all-cause mortality.

Structural basis for the differential binding affinities of the HsfBD1 and HsfBD2 domains in the Haemophilus influenzae Hsf adhesin.

Haemophilus influenzae is a human-specific gram-negative coccobacillus that causes a variety of human infections ranging from localized respiratory infections to invasive diseases. Hsf is the major nonpilus adhesin in encapsulated strains of H. influenzae and belongs to the trimeric autotransporter family of proteins. The Hsf protein contains two highly homologous binding domains, designated HsfBD1 and HsfBD2. In this study we characterized the differential binding properties of HsfBD1 and HsfBD2. In assays using HeLa cells, we found that bacteria expressing either full-length Hsf or HsfBD1 by itself adhered at high levels, while bacteria expressing HsfBD2 by itself adhered at low levels. Immunofluorescence microscopy and a cellular enzyme-linked immunosorbent assay using purified proteins revealed that the binding affinity was significantly higher for HsfBD1 than for HsfBD2. Purified HsfBD1 was able to completely block adherence by bacteria expressing either HsfBD1 or HsfBD2, while purified HsfBD2 was able to block adherence by bacteria expressing HsfBD2 but had minimal activity against bacteria expressing HsfBD1. Conversion of the residue at position 1935 in the HsfBD1 binding pocket from Asp to Glu resulted in HsfBD2-like binding properties, and conversion of the residue at position 569 in the HsfBD2 binding pocket from Glu to Asp resulted in HsfBD1-like binding properties, as assessed by adherence assays with recombinant bacteria and by immunofluorescence microscopy with purified proteins. This work demonstrates the critical role of a single amino acid in the core of the binding pocket in determining the relative affinities of the HsfBD1 and HsfBD2 binding domains.

Trimeric autotransporters: a distinct subfamily of autotransporter proteins.

Autotransporter proteins are a large family of gram-negative bacterial extracellular proteins. These proteins have a characteristic arrangement of functional domains, including an N-terminal signal peptide, an internal passenger domain, and a C-terminal translocator domain. Recent studies have identified a novel subfamily of autotransporters, defined by a short trimeric C-terminal translocator domain and known as trimeric autotransporters. In this article, we review our current knowledge of the structural and functional characteristics of trimeric autotransporters, highlighting the distinctions between this subfamily and conventional autotransporters. We speculate that trimeric autotransporters evolved to enable high-affinity multivalent adhesive interactions with host surfaces and circulating host molecules to take place.

FDG-PET/CT in the evaluation of anal carcinoma.

PURPOSE: Surgical staging and treatment of anal carcinoma has been replaced by noninvasive staging studies and combined modality therapy. In this study, we compare computed tomography (CT) and physical examination to [(18)F]-fluoro-2-deoxy-D-glucose-positron emission tomography/computed tomography (FDG-PET/CT) in the staging of carcinoma of the anal canal, with special emphasis on determination of spread to inguinal lymph nodes. METHODS AND MATERIALS: Between July 2003 and July 2005, 41 consecutive patients with biopsy-proved anal carcinoma underwent a complete staging evaluation including physical examination, CT, and 2-FDG-PET/CT. Patients ranged in age from 30 to 89 years. Nine men were HIV-positive. Treatment was with standard Nigro regimen. RESULTS: [(18)F]-fluoro-2-deoxy-D-glucose-positron emission tomography/computed tomography (FDG-PET/CT) detected 91% of nonexcised primary tumors, whereas CT visualized 59%. FDG-PET/CT detected abnormal uptake in pelvic nodes of 5 patients with normal pelvic CT scans. FDG-PET/CT detected abnormal nodes in 20% of groins that were normal by CT, and in 23% without abnormality on physical examination. Furthermore, 17% of groins negative by both CT and physical examination showed abnormal uptake on FDG-PET/CT. HIV-positive patients had an increased frequency of PET-positive lymph nodes. CONCLUSION: [(18)F]-fluoro-2-deoxy-D-glucose-positron emission tomography/computed tomography detects the primary tumor more often than CT. FDG-PET/CT detects substantially more abnormal inguinal lymph nodes than are identified by standard clinical staging with CT and physical examination.

Palliative treatment for in-transit cutaneous metastases of Merkel cell carcinoma using surface-mold computer-optimized high-dose-rate brachytherapy.

PURPOSE: The objective of this study was to evaluate the palliative treatment benefit of surface-mold computer-optimized high-dose-rate brachytherapy (SMBT) for in-transit cutaneous metastases of Merkel cell carcinoma (MCC). METHODS: Ten patients with in-transit cutaneous MCC metastases were treated with SMBT at the Dana-Farber/Brigham & Women's Cancer Center between 2006 and 2012. RESULTS: The median age at diagnosis was 76 years (range, 63-87 years). Seven patients had in-transit metastases on the lower extremities (70%), 2 patients on the head and neck (20%), and 1 patient on an upper extremity (10%). A total of 152 metastatic MCC lesions were treated with SMBT. All SMBT-treated lesions resolved clinically within a few weeks of therapy. The median follow-up was 34 months (range, 22-85 months). Two of 152 treated lesions recurred during the study period for a local control rate of 99%. Eight patients (80%) developed additional in-transit metastases outside the original SMBT fields. Five of these 8 patients underwent additional SMBT. At study conclusion, 3 patients (30%) are alive without disease, 3 patients (30%) are alive with disease, and 4 patients (40%) died of MCC. DISCUSSION: Surface-mold computer-optimized high-dose-rate brachytherapy offers effective and durable palliation for cutaneous metastases of MCC, although it does not appear to alter disease course.

Proton radiotherapy for solid tumors of childhood.

The increasing efficacy of pediatric cancer therapy over the past four decades has produced many long-term survivors that now struggle with serious treatment related morbidities affecting their quality of life. Radiation therapy is responsible for a significant proportion of these late effects, but a relatively new and emerging modality, proton radiotherapy hold great promise to drastically reduce these treatment related late effects in long term survivors by sparing dose to normal tissues. Dosimetric studies of proton radiotherapy compared with best available photon based treatment show significant dose sparing to developing normal tissues. Furthermore, clinical data are now emerging that begin to quantify the benefit in decreased late treatment effects while maintaining excellent cancer control rates.

Proton radiotherapy for pediatric bladder/prostate rhabdomyosarcoma: clinical outcomes and dosimetry compared to intensity-modulated radiation therapy.

PURPOSE: In this study, we report the clinical outcomes of 7 children with bladder/prostate rhabdomyosarcoma (RMS) treated with proton radiation and compare proton treatment plans with matched intensity-modulated radiation therapy (IMRT) plans, with an emphasis on dose savings to reproductive and skeletal structures. METHODS AND MATERIALS: Follow-up consisted of scheduled clinic appointments at our institution or direct communication with the treating physicians for referred patients. Each proton radiotherapy plan used for treatment was directly compared to an IMRT plan generated for the study. Clinical target volumes and normal tissue volumes were held constant to facilitate dosimetric comparisons. Each plan was optimized for target coverage and normal tissue sparing. RESULTS: Seven male patients were treated with proton radiotherapy for bladder/prostate RMS at the Massachusetts General Hospital between 2002 and 2008. Median age at treatment was 30 months (11-70 months). Median follow-up was 27 months (10-90 months). Four patients underwent a gross total resection prior to radiation, and all patients received concurrent chemotherapy. Radiation doses ranged from 36 cobalt Gray equivalent (CGE) to 50.4 CGE. Five of 7 patients were without evidence of disease and with intact bladders at study completion. Target volume dosimetry was equivalent between the two modalities for all 7 patients. Proton radiotherapy led to a significant decrease in mean organ dose to the bladder (25.1 CGE vs. 33.2 Gy; p=0.03), testes (0.0 CGE vs. 0.6 Gy; p=0.016), femoral heads (1.6 CGE vs. 10.6 Gy; p=0.016), growth plates (21.7 CGE vs. 32.4 Gy; p=0.016), and pelvic bones (8.8 CGE vs. 13.5 Gy; p=0.016) compared to IMRT. CONCLUSIONS: This study provides evidence of significant dose savings to normal structures with proton radiotherapy compared to IMRT and is well tolerated in this patient population. The long-term impact of these reduced doses can be tested in future studies incorporating extended follow-up, objective outcome measures, and quality-of-life analyses.

Management of complications of prostate cancer treatment.

Prostate cancer is the most commonly diagnosed noncutaneous cancer in men in the United States. Treatment of men with prostate cancer commonly involves surgical, radiation, or hormone therapy. Most men with prostate cancer live for many years after diagnosis and may never suffer morbidity or mortality attributable to prostate cancer. The short-term and long-term adverse consequences of therapy are, therefore, of great importance. Adverse effects of radical prostatectomy include immediate postoperative complications and long-term urinary and sexual complications. External beam or interstitial radiation therapy in men with localized prostate cancer may lead to urinary, gastrointestinal, and sexual complications. Improvements in surgical and radiation techniques have reduced the incidence of many of these complications. Hormone treatment typically consists of androgen deprivation therapy, and consequences of such therapy may include vasomotor flushing, anemia, and bone density loss. Numerous clinical trials have studied the role of bone antiresorptive therapy for prevention of bone density loss and fractures. Other long-term consequences of androgen deprivation therapy may include adverse body composition changes and increased risk of insulin resistance, diabetes, and cardiovascular disease. Ongoing and planned clinical trials will continue to address strategies to prevent treatment-related side effects and improve quality of life for men with prostate cancer.

Trimeric autotransporters require trimerization of the passenger domain for stability and adhesive activity.

In recent years, structural studies have identified a number of bacterial, viral, and eukaryotic adhesive proteins that have a trimeric architecture. The prototype examples in bacteria are the Haemophilus influenzae Hia adhesin and the Yersinia enterocolitica YadA adhesin. Both Hia and YadA are members of the trimeric-autotransporter subfamily and are characterized by an internal passenger domain that harbors adhesive activity and a short C-terminal translocator domain that inserts into the outer membrane and facilitates delivery of the passenger domain to the bacterial surface. In this study, we examined the relationship between trimerization of the Hia and YadA passenger domains and the capacity for adhesive activity. We found that subunit-subunit interactions and stable trimerization are essential for native folding and stability and ultimately for full-level adhesive activity. These results raise the possibility that disruption of the trimeric architecture of trimeric autotransporters, and possibly other trimeric adhesins, may be an effective strategy to eliminate adhesive activity.

Architecture and adhesive activity of the Haemophilus influenzae Hsf adhesin.

Haemophilus influenzae type b is an important cause of meningitis and other serious invasive diseases and initiates infection by colonizing the upper respiratory tract. Among the major adhesins in H. influenzae type b is a nonpilus protein called Hsf, a large protein that forms fiber-like structures on the bacterial surface and shares significant sequence similarity with the nontypeable H. influenzae Hia autotransporter. In the present study, we characterized the structure and adhesive activity of Hsf. Analysis of the predicted amino acid sequence of Hsf revealed three regions with high-level homology to the HiaBD1 and HiaBD2 binding domains in Hia. Based on examination of glutathione S-transferase fusion proteins corresponding to these regions, two of the three had adhesive activity and one was nonadhesive in assays with cultured epithelial cells. Structural modeling demonstrated that only the two regions with adhesive activity harbored an acidic binding pocket like the binding pocket identified in the crystal structure of HiaBD1. Consistent with these results, disruption of the acidic binding pockets in the adhesive regions eliminated adhesive activity. These studies advance our understanding of the architecture of Hsf and the family of trimeric autotransporters and provide insight into the structural determinants of H. influenzae type b adherence.

Structural basis for host recognition by the Haemophilus influenzae Hia autotransporter.

Haemophilus influenzae is an important human pathogen that initiates infection by colonizing the upper respiratory tract. The H. influenzae Hia autotransporter is an adhesive protein that promotes adherence to respiratory epithelial cells. Hia adhesive activity resides in two homologous binding domains, called HiaBD1 and HiaBD2. These domains interact with the same host cell receptor, but bind with different affinities. In this report, we describe the crystal structure of the high-affinity HiaBD1 binding domain, which has a novel trimeric architecture with three-fold symmetry and a mushroom shape. The subunit constituents of the trimer are extensively intertwined. The receptor-binding pocket is formed by an acidic patch that is present on all three faces of the trimer, providing potential for a multivalent interaction with the host cell surface, analogous to observations with the trimeric tumor necrosis factor superfamily of proteins. Hia is a novel example of a bacterial trimeric adhesin and may be the prototype member of a large family of bacterial virulence proteins with a similar architecture.