Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein.

Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells.

Treatment strategies in elderly breast cancer patients: Is there a need for surgery?

BACKGROUND: The aim of this study was to determine the role of surgery in elderly patients with breast cancer. METHODS: Between 1999 and 2009, 153 consecutive women, ≥80 years old with breast cancer were treated at our hospital. Surgically and non-surgically treated patients were compared with respect to characteristics and survival. RESULTS: Treatment was surgical in 102 patients (67%). The non-surgically treated patients were older than surgically treated patients, had more co-morbidity and were more often diagnosed with a clinically T3/T4 tumour and distant metastasis. Patients not receiving surgery, had an 11% overall survival rate at 5-year versus 48% in surgically treated patients (P < 0.001). Independent factors for survival were clinical N0 status, M0 status at presentation and surgery. CONCLUSION: One in three patients of 80 years and older did not have surgical treatment for breast cancer. Patient not treated surgically are older, have more severe co-morbidity and are diagnosed with more advanced disease than patients who underwent surgery.The selection of patients, who have a poor prognosis, is made on clinical grounds not measurable with a common co-morbidity survey. Better and evidence-based selection criteria for surgical and non-surgical treatment in these patients are needed.

Metastasis from breast cancer to an endometrial polyp; treatment options and follow-up. Report of a case and review of the literature.

INTRODUCTION: The female genital tract is rarely involved by metastatic tumors. The most common anatomic locations are the ovaries and the vagina. A case is presented of metastatic breast carcinoma to the vulva and endometrial polyp, both exceptional. CASE REPORT: We report the case of an 83-year-old female who presented with vaginal bleeding. Lobular breast carcinoma was diagnosed earlier and during follow-up vulvar metastasis was detected. Hysteroscopic examination because of postmenopausal bleeding revealed an endometrial polyp which was resected. The morphology and immunohistochemistry of the polyp were consistent with lobular breast cancer: metastatic breast cancer to an endometrial polyp. After reviewing the literature 15 cases of metastatic breast carcinoma to endometrial polyps have been reported. The clinical presentation and course, risk factors, treatment and follow-up are discussed. CONCLUSION: Metastasis of a breast carcinoma to the vulva and an endometrial polyp are extremely rare, but clinicians should be aware of both phenomena.

Increased prevalence of risk factors for cardiovascular disease in long-term survivors of acute lymphoblastic leukemia and Wilms tumor treated with radiotherapy.

BACKGROUND: Only a few studies have assessed cardiovascular risk factors (CRFs) in childhood cancer survivors. We determined the prevalence of CRFs in long-term survivors of acute lymphoblastic leukemia (ALL) and Wilms tumor. PROCEDURE: Adult survivors of ALL and Wilms tumor treated with radiotherapy and chemotherapy (RT + CT) or treated with chemotherapy alone (CT) were compared with sibling controls. CRFs (hypertension, diabetes mellitus, hypercholesterolemia, obesity, renal insufficiency) and hormonal deficiencies were assessed in each participant. Multivariate logistic regression analysis was used to evaluate the association between CRFs and treatment. RESULTS: Seventy-nine ALL, 62 Wilms tumor survivors, and 69 control subjects (mean ages 24.5, 25.9, and 26 years, respectively) were enrolled. Mean follow-up time since cancer treatment was 20.8 years. In the Wilms RT + CT group significantly more survivors had hypertension (21.6% vs. 1.4%, P < 0.001) and renal insufficiency (8.1% vs. 0%, P = 0.016) compared to controls. There were also more patients with multiple CRFs in the Wilms RT + CT group (16.2% vs. 2.9% in controls, P = 0.019). Almost 15% of ALL RT + CT survivors had growth hormone deficiency. Hypogonadism was seen in 18.9% of survivors in the Wilms RT + CT group. We observed no significant differences between CT-treated survivors of both malignancies and controls. The adjusted odds ratio for the occurrence of at least one CRF was 2.6 increased for survivors following abdominal radiotherapy. Treatment with CT alone was not associated with the occurrence of multiple CRFs. CONCLUSIONS: Long-term survivors of ALL and Wilms tumor have unfavorable CRFs due to previous RT not CT.

Hypertension in long-term survivors of childhood cancer: a nested case-control study.

AIM OF THE STUDY: To examine risk factors for developing hypertension in childhood cancer survivors (CCS). METHODS: We conducted a nested case-control study of risk for hypertension within a cohort of 1362 childhood cancer survivors treated between 1966 and 1996 in the Emma's Children's Hospital/Academic Medical Center in the Netherlands. Detailed information on treatment and several lifestyle factors was collected for 44 cases with hypertension and 123 matched controls. Odds ratios (ORs) for hypertension were calculated by conditional logistic regression analysis. RESULTS: Body Mass Index (BMI) was the only significant risk factor associated with the occurrence of hypertension (OR 3.95; 95% confidence interval (CI) 1.7-9.1 for BMI25kg/m(2) compared to BMI<25kg/m(2)). However, cisplatin, cyclophosphamide and radiotherapy (RT) to the abdominal region were all associated with non-significant risk increases (ORs of 4.3, 2.1, and 1.8, respectively). CONCLUSION: Our results show that BMI is the most important risk factor for hypertension following treatment of childhood cancer, emphasising the need for CCS to maintain a normal weight.

Development of a national protocol to screen Dutch cancer survivors on late cancer treatment effects.

PURPOSES: The development of a national protocol to formalize the screening of Dutch cancer survivors on potential late cancer treatment effects and the medical terminology used in describing the patient follow up procedures. METHODS: A combined evidence-based and qualitative approach, the Glaser's State of the Art Strategy, was used to reach consensus on how to screen Dutch cancer survivors on late cancer treatment effects. A core working group set up a first proposal of a screening protocol and a handbook of medical term definitions by incorporating available research evidence (1980-2003), clinical expertise and definitions from Dutch medical dictionaries and textbooks. External experts reviewed this proposal in a cycle of two postal and two discussion rounds. The follow-up procedures and medical term definitions described in the draft screening protocol were to be accepted if consensus among external experts was > or =50%. RESULTS: A protocol for screening cancer survivors on late cancer treatment effects was developed describing the follow-up procedures for cancer survivors according to previous therapeutic exposures. Four hundred and twenty one medical terms were used in describing these follow-up procedures. One hundred and fifteen of these terms were classified as multi-interpretable and 101 of these terms were defined. No definitions could be found for the remaining 14 medical terms. CONCLUSIONS: We succeeded in reaching consensus throughout The Netherlands on a protocol to screen cancer survivors on late cancer treatment effects. This protocol is now in use by all Dutch outpatient clinics and warrants that the screening of cancer survivors is consistent across The Netherlands. The screening protocol specifies in detail how screening of cancer survivors should take place and can therefore be used by clinicians who were not involved in the consensus study.

Frequent adverse events after treatment for childhood-onset differentiated thyroid carcinoma: a single institute experience.

Since the mortality rate for childhood differentiated thyroid carcinoma is nearly zero, the focus must be to minimise morbidity following treatment. Our aim was to analyse early and late adverse events. Twenty-five of 26 children treated between 1962 and 2002 were evaluated. Median follow-up was 14.2 years (range 0.9-39.4 years). All underwent total thyroidectomy, 15 (60%) with lymph node dissection and 15 (60%) with adjuvant radio-iodide therapy. Mortality was zero. Seven developed recurrent disease, two developed a third recurrence. Twenty-one (84%) had > or =1 adverse event. Eight had permanent hypoparathyroidism (PH), six permanent recurrent nerve paralysis (PRNP) and two Horner's syndrome. Risk factors for PH and PRNP were total thyroidectomy with lymph node dissection (RR: 6.45, P = 0.015) and recurrent nerve tumour encasement (RR: 8.00, P = 0.001), respectively. Other adverse events were fatigue (n = 5), scar problems (n = 4) and chronic myeloid leukaemia (n = 1). These results emphasise the need to improve treatment strategies.

Screening for late effects in survivors of childhood cancer: growth hormone deficiency from a pediatric oncologist's point of view.

At the Emma Kinderziekenhuis/Academic Medical Center in Amsterdam, survivors of childhood cancer are screened annually or biennially for the occurrence of late treatment effects. The screening procedures are based on previously used treatment modalities. The data gathered at the outpatient clinic are registered in the database PLEKsys. Evaluation of the data concerning over 1000 cancer survivors screened since the start of the clinic once more illustrated the relation between cranial irradiation and the development of central endocrine abnormalities. Surprisingly, at least a proportion of the growth hormone (GH)-deficient cancer survivors were registered as not being on a replacement therapy regimen. The reasons for survivors not to be on replacement therapy are currently being evaluated. The late-effects outpatient clinic and the PLEKsys database provide a platform for additional research in fields including endocrinology, which should be aimed at improving the care for and the health status of the survivors of childhood cancer.

The use of cognitive methods in analyzing clinicians' task behavior.

To ensure the acceptance and routine use of information systems in healthcare tight coupling is required between the work practices of potential endusers and the systems functionalities and presentation of these functionalities via the user interface. The application of methods from cognitive engineering during requirement analysis may contribute to the support of healthcare work practice by computer systems. We applied the think aloud method in combination with video analysis during the requirement analysis phase in designing a user interface for a patient information retrieval system. These methods provided a detailed insight in the information needs of physicians and the way in which they search through this information in preparing a patient visit. Using these insights in endusers work practices in the early phase of user interface development may lead to a better fit between physicians' work practices and the supporting computer system.