Julie L. Ryan

Epstein-Barr Virus Quantitation by Real-Time PCR Targeting Multiple Gene Segments: A Novel Approach to Screen for the Virus in Paraffin-Embedded Tissue and Plasma

Epstein-Barr Virus (EBV) infects nearly all humans and then persists for the life of the host. In some people who later develop cancer, EBV DNA is present within malignant cells and circulates at elevated levels in the plasma. In the current study, we validated five novel quantitative polymerase chain reaction (Q-PCR) assays targeting disparate but highly conserved segments of the EBV genome (BamH1W, EBNA1, LMP1, LMP2, and BZLF1). Each assay was sensitive to as few as 50 copies of EBV DNA per reaction and was linear across at least four orders of magnitude. When applied to paraffin-embedded tissues in concert with EBV-encoded RNA (EBER) in situ hybridization, the BamH1W and EBNA1 assays were the most informative, while use of the entire battery of EBV PCR assays may help identify genomic polymorphisms or deletions. Higher viral loads were found in the 17 EBER-positive compared with the 13 EBER-negative tumors (means 84,978 versus 22 copies of EBV per 100,000 cells, respectively). The five Q-PCR assays were also informative in plasma samples where EBV was measurable in all nine patients with lymphoma or infectious mononucleosis, whereas EBV was undetectable in all nine healthy controls. The findings suggest that Q-PCR is an effective method of distinguishing disease-associated virus from incidental virus in paraffin-embedded tissue and in plasma samples.

Curcumin for Radiation Dermatitis: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial of Thirty Breast Cancer Patients

Radiation dermatitis occurs in approximately 95% of patients receiving radiotherapy (RT) for breast cancer. We conducted a randomized, double-blind, placebo-controlled clinical trial to assess the ability of curcumin to reduce radiation dermatitis severity in 30 breast cancer patients. Eligible patients were adult females with noninflammatory breast cancer or carcinoma in situ prescribed RT without concurrent chemotherapy. Randomized patients took 2.0 grams of curcumin or placebo orally three times per day (i.e., 6.0 grams daily) throughout their course of RT. Weekly assessments included Radiation Dermatitis Severity (RDS) score, presence of moist desquamation, redness measurement, McGill Pain Questionnaire-Short Form and Symptom Inventory questionnaire. The 30 evaluable patients were primarily white (90%) and had a mean age of 58.1 years. Standard pooled variances t test showed that curcumin reduced RDS at end of treatment compared to placebo (mean RDS = 2.6 vs. 3.4; P = 0.008). Fishers exact test revealed that fewer curcumin-treated patients had moist desquamation (28.6% vs. 87.5%; P = 0.002). No significant differences were observed between arms for demographics, compliance, radiation skin dose, redness, pain or symptoms. In conclusion, oral curcumin, 6.0 g daily during radiotherapy, reduced the severity of radiation dermatitis in breast cancer patients.

Epstein-Barr Virus (EBV) DNA in plasma is not encapsidated in patients with EBV-related malignancies.

Epstein-Barr Virus (EBV), a ubiquitous gamma herpes virus, infects more than 95% of the human population before adulthood. Life-long persistence, usually without adverse health consequences, relies on a balance between viral latency, viral replication, and host immune response. Patients with EBV-related disease often have high levels of EBV DNA in their plasma. This study addresses whether this circulating, cell-free EBV DNA is encapsidated in virions or exists as naked genomes. First, an assay was developed, combining DNase I and quantitative real-time PCR, to discriminate encapsidated from naked EBV DNA. EBV DNA was almost always naked in the plasma of AIDS-related lymphoma patients (n = 11) and immunosuppressed/posttransplantation patients (n = 8). In contrast, infectious mononucleosis patients (n = 30) often had a mixture of encapsidated and naked EBV DNA. These findings may be important in understanding how viral load relates to disease status and in predicting response to nucleoside analogs and other antiviral therapies.

High Levels of Epstein-Barr Virus DNA in Latently Infected Gastric Adenocarcinoma

Gastric adenocarcinoma is the second leading cause of cancer death worldwide. Epstein–Barr virus (EBV) is present in the malignant cells of approximately 10% of cases. It is unclear whether EBV is being missed in some gastric adenocarcinomas due to insensitive test methods or partial EBV genome loss. In this study, we screened 113 gastric adenocarcinomas from low- and high-incidence regions (United States and Central America) for the presence of EBV using a battery quantitative real-time PCR (Q-PCR) assays targeting disparate segments of the EBV genome (BamH1W, EBNA1, LMP1, LMP2, BZLF1, EBER1) and histochemical stains targeting EBV-encoded RNA (EBER), the latent proteins LMP1 and LMP2, and the lytic proteins BMRF1 and BZLF1. EBV DNA was detected by Q-PCR in 48/75 United States cancers (64%) and in 38/38 Central American cancers (100%), which was a significant differrence. EBER was localized to malignant epithelial cells in 8/48 (17%) United States and 3/38 (8%) Central American cancers. Viral loads were considerably higher for EBER-positive vs EBER-negative cancers (mean 162 986 vs 62 EBV DNA copies per 100 000 cells). A viral load of 2000 copies per 100 000 cells is recommended as the threshold distinguishing EBER-positive from EBER-negative tumors. One infected cancer selectively failed to amplify the LMP2 gene because of a point mutation, whereas another cancer had an atypical pattern of Q-PCR positivity suggesting deletion of large segments of the EBV genome. Three different viral latency profiles were observed in the cancers based on constant expression of EBER and focal or variable expression of LMP1 or LMP2, without lytic protein expression. We conclude that EBV DNA levels generally reflect EBER status, and a panel of at least two Q-PCR assays is recommended for sensitive identification of infected cancers.

Clonal evolution of lymphoblastoid cell lines

Lymphoblastoid cell lines represent Epstein–Barr virus (EBV)-immortalized B lymphocytes, which are typically prepared by in vitro culture of normal blood cells. In this study, we evaluated the kinetics of clonal evolution in lymphoblastoid cell lines (LCLs) established from five different donors. Immunoglobulin heavy chain (IGH) and T-cell receptor gamma (TRG) gene rearrangements were tracked over time using PCR, while EBV clonality was tracked using Southern blot analysis of the viral terminal repeat fragment. All five cultures evolved towards monoclonal B cells within 8 weeks, while T lymphocytes disappeared over the same period. No significant association was found between the rapidity of clonal emergence and either the proliferation rate or the size of the EBV terminal repeat fragment, suggesting the random nature of clonal selection. Our results suggest that EBV-driven B lymphocytes rapidly progress from polyclonal to virtually monoclonal, which has implications for the pace at which lymphoma might evolve in vivo. In addition, our findings indicate that established lymphobastoid cell lines are not an ideal ‘normal control’ for human B lymphocytes because they do not represent the spectrum of polyclonal B cells found in healthy humans; instead, they primarily represent the progeny of a single B lymphocyte.

Informational Needs of Patients and Perceived Adequacy of Information Available Before and After Treatment of Cancer

Abstract To examine the various concerns of patients after being diagnosed with cancer as well as the availability of information to address concerns from the time of diagnosis to the completion of treatment, we analyzed data from a longitudinal study of 731 adults recently diagnosed with cancer. Concerns about the effectiveness and side effects of treatment and family stress were most common after diagnosis. Information about the diagnosis and treatment plan was readily available to patients, but information addressing social, lifestyle, and financial concerns was less available. Significantly more information was desired regarding the long-term implications of treatment and disease.

Cancer-Related Stress and Complementary and Alternative Medicine: A Review

A cancer diagnosis elicits strong psychophysiological reactions that characterize stress. Stress is experienced by all patients but is usually not discussed during patient-healthcare professional interaction; thus underdiagnosed, very few are referred to support services. The prevalence of CAM use in patients with history of cancer is growing. The purpose of the paper is to review the aspects of cancer-related stress and interventions of commonly used complementary and alternative techniques/products for amelioration of cancer-related stress. Feasibility of intervention of several CAM techniques and products commonly used by cancer patients and survivors has been established in some cancer populations. Efficacy of some CAM techniques and products in reducing stress has been documented as well as stress-related symptoms in patients with cancer such as mindfulness-based stress reduction, yoga, Tai Chi Chuan, acupuncture, energy-based techniques, and physical activity. Much of the research limitations include small study samples and variety of intervention length and content. Efficacy and safety of many CAM techniques and some herbs and vitamin B and D supplements need to be confirmed in further studies using scientific methodology. Several complementary and alternative medicine therapies could be integrated into standard cancer care to ameliorate cancer-related stress.

A Phase II/III Randomized, Placebo-Controlled, Double-Blind Clinical Trial of Ginger (Zingiber officinale) for Nausea Caused by Chemotherapy for Cancer: A Currently Accruing URCC CCOP Cancer Control Study

Despite the widespread use of 5-HT3 receptor antagonist antiemetics such as ondansetron and granistron, up to 70% of patients with cancer receiving highly emetogenic chemotherapy agents experience postchemotherapy nausea and vomiting. Delayed postchemotherapy nausea (nausea that occurs >/= 24 hours after chemotherapy administration) and anticipatory nausea (nausea that develops before chemotherapy administration, in anticipation of it) are poorly controlled by currently available antiemetic agents. Scientific studies suggest that ginger (Zingiber officinale) might have beneficial effects on nausea and vomiting associated with motion sickness, surgery, and pregnancy. In 2 small studies of patients with cancer receiving chemotherapy, addition of ginger to standard antiemetic medication further reduced the severity of postchemotherapy nausea. This article describes a phase II/III randomized, dose-finding, placebo-controlled, double-blind clinical trial to assess the efficacy of ginger for nausea associated with chemotherapy for cancer. The study is currently being conducted by private practice oncology groups that are funded by the National Cancer Institutes Community Clinical Oncology Program and affiliated with the University of Rochester Cancer Center Community Clinical Oncology Program Research Base.

Post-treatment skin reactions reported by cancer patients differ by race, not by treatment or expectations

Cancer patients may experience skin problems while undergoing chemotherapy and radiation therapy. Frequency of skin reactions may be influenced by skin pigmentation and psychological factors. A Symptom Inventory completed by 656 cancer patients nationwide before and after chemotherapy, radiation therapy, or chemotherapy plus radiation therapy was analysed to determine if treatment type, race (Black vs White), and pretreatment expectations influenced post-treatment skin reactions. Subsequent analysis of a local Symptom Inventory completed weekly for 5 weeks by 308 patients receiving radiation therapy examined severity of reported skin reactions. Significantly more patients receiving radiation therapy had stronger expectations of skin problems (62%) than patients receiving chemotherapy (40%, P=0.001) or chemotherapy plus radiation therapy (45%, P=0.003). Overall, expectations did not correlate with patient reported post-treatment skin problems in white (r=0.014, P=0.781) or black (r=0.021, P=0.936) patients. Although no significant difference was found between black and white patients in their pretreatment expectations of skin problems (P=0.32), black patients (10 out of 18, 56%) reported more skin problems than white patients (90 out of 393, 23%, P=0.001). Similarly, the local study showed that significantly more black patients (1 out of 5, 20%) reported severe skin reactions at the treatment site than white patients (12 out of 161, 8%). A direct correlation was observed between severity of skin problems and pain at the treatment site (r=0.541, P<0.001). Total radiation exposure did not significantly correlate with the report of skin problems at the treatment site for white or black patients. Overall, black patients reported more severe post-treatment skin problems than white patients. Our results suggest that symptom management for post-treatment skin reactions in cancer patients receiving radiation treatment could differ depending on their racial background.

Exposure to Ionizing Radiation Induces the Migration of Cutaneous Dendritic Cells by a CCR7-Dependent Mechanism

In the event of a deliberate or accidental radiological emergency, the skin would likely receive substantial ionizing radiation (IR) poisoning, which could negatively impact cellular proliferation, communication, and immune regulation within the cutaneous microenvironment. Indeed, as we have previously shown, local IR exposure to the murine ear causes a reduction of two types of cutaneous dendritic cells (cDC), including interstitial dendritic cells of the dermis and Langerhans cells of the epidermis, in a dose- and time-dependent manner. These APCs are critical regulators of skin homeostasis, immunosurveillance, and the induction of T and B cell-mediated immunity, as previously demonstrated using conditional cDC knockout mice. To mimic a radiological emergency, we developed a murine model of sublethal total body irradiation (TBI). Our data would suggest that TBI results in the reduction of cDC from the murine ear that was not due to a systemic response to IR, as a loss was not observed in shielded ears. We further determined that this reduction was due, in part, to the upregulation of the chemoattractant CCL21 on lymphatic vessels as well as CCR7 expressed on cDC. Migration as a potential mechanism was confirmed using CCR7−/− mice in which cDC were not depleted following TBI. Finally, we demonstrated that the loss of cDC following TBI results in an impaired contact hypersensitivity response to hapten by using a modified contact hypersensitivity protocol. Taken together, these data suggest that IR exposure may result in diminished immunosurveillance in the skin, which could render the host more susceptible to pathogens.