Nucleophosmin Phosphorylation by v-Cyclin-CDK6 Controls KSHV Latency

Nucleophosmin (NPM) is a multifunctional nuclear phosphoprotein and a histone chaperone implicated in chromatin organization and transcription control. Oncogenic Kaposi's sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). In the infected host cell KSHV displays two modes of infection, the latency and productive viral replication phases, involving extensive viral DNA replication and gene expression. A sustained balance between latency and reactivation to the productive infection state is essential for viral persistence and KSHV pathogenesis. Our study demonstrates that the KSHV v-cyclin and cellular CDK6 kinase phosphorylate NPM on threonine 199 (Thr199) in de novo and naturally KSHV-infected cells and that NPM is phosphorylated to the same site in primary KS tumors. Furthermore, v-cyclin-mediated phosphorylation of NPM engages the interaction between NPM and the latency-associated nuclear antigen LANA, a KSHV-encoded repressor of viral lytic replication. Strikingly, depletion of NPM in PEL cells leads to viral reactivation, and production of new infectious virus particles. Moreover, the phosphorylation of NPM negatively correlates with the level of spontaneous viral reactivation in PEL cells. This work demonstrates that NPM is a critical regulator of KSHV latency via functional interactions with v-cyclin and LANA

Long-term outcomes of clinical complete responders after neoadjuvant treatment for rectal cancer in the International Watch & Wait Database (IWWD): an international multicentre registry study

Background: The strategy of watch and wait (W&W) in patients with rectal cancer who achieve a complete clinical response (cCR) after neoadjuvant therapy is new and offers an opportunity for patients to avoid major resection surgery. However, evidence is based on small-to-moderate sized series from specialist centres. The International Watch & Wait Database (IWWD) aims to describe the outcome of the W&W strategy in a large-scale registry of pooled individual patient data. We report the results of a descriptive analysis after inclusion of more than 1000 patients in the registry. Methods: Participating centres entered data in the registry through an online, highly secured, and encrypted research data server. Data included baseline characteristics, neoadjuvant therapy, imaging protocols, incidence of local regrowth and distant metastasis, and survival status. All patients with rectal cancer in whom the standard of care (total mesorectal excision surgery) was omitted after neoadjuvant therapy were eligible to be included in the IWWD. For the present analysis, we only selected patients with no signs of residual tumour at reassessment (a cCR). We analysed the proportion of patients with local regrowth, proportion of patients with distant metastases, 5-year overall survival, and 5-year disease-specific survival. Findings: Between April 14, 2015, and June 30, 2017, we identified 1009 patients who received neoadjuvant treatment and were managed by W&W in the database from 47 participating institutes (15 countries). We included 880 (87%) patients with a cCR. Median follow-up time was 3·3 years (95% CI 3·1–3·6). The 2-year cumulative incidence of local regrowth was 25·2% (95% CI 22·2–28·5%), 88% of all local regrowth was diagnosed in the first 2 years, and 97% of local regrowth was located in the bowel wall. Distant metastasis were diagnosed in 71 (8%) of 880 patients. 5-year overall survival was 85% (95% CI 80·9–87·7%), and 5-year disease-specific survival was 94% (91–96%). Interpretation: This dataset has the largest series of patients with rectal cancer treated with a W&W approach, consisting of approximately 50% data from previous cohort series and 50% unpublished data. Local regrowth occurs mostly in the first 2 years and in the bowel wall, emphasising the importance of endoscopic surveillance to ensure the option of deferred curative surgery. Local unsalvageable disease after W&W was rare. Funding: European Registration of Cancer Care financed by European Society of Surgical Oncology, Champalimaud Foundation Lisbon, Bas Mulder Award granted by the Alpe d'Huzes Foundation and Dutch Cancer Society, and European Research Council Advanced Grant

Cyclin-Dependent Kinase 6 Phosphorylates NF-kappa B P65 at Serine 536 and Contributes to the Regulation of Inflammatory Gene Expression

Peer reviewe

Low-temperature heating overnight in tris-HCl buffer pH 9 is a good alternative for antigen retrieval in formalin-fixed paraffin-embedded tissue

Heat-induced retrieval of antigens masked by formalin fixation in paraffin-embedded tissue is widely used in routine surgical pathology. Microwave heating at high temperature for a relatively short period has been tested extensively. Although good results for commonly used antibodies are obtained by optimizing heating conditions and retrieval solutions, a standard protocol for all antibodies is not yet available. Other problems encountered are affected morphology and loss of tissue attachment to the slides. In this study we elaborated a previously described retrieval method applying conventional heating at a lower temperature for a prolonged time. We tested 16 routinely used antibodies on formalin-fixed, paraffin-embedded tissues, which were stained after overnight heating at 80 degrees C in retrieval solutions varying in pH value. The results were compared with standard microwave heating in a solution of Tris-HCl plus urea pH 9.5. Maximal retrieval was obtained with 14 out of 16 antibodies by overnight heating at 80 degrees C in Tris buffer pH 9. Tissue morphology was well preserved. The results were slightly superior to those obtained with microwave retrieval in pH 9.5 Tris+urea buffer. Tn conclusion, overnight heating at 80 degrees C in Tris buffer pH 9 is a good alternative for retrieval of antigens and is easy to perform

Extra nodal growth as a prognostic factor in malignant melanoma

Aim. Extra nodal growth (ENG) in lymph-node metastases may be an additional. indicator for poor prognosis and increased Loco-regional recurrence in patients with a cutaneous malignant melanoma (CMM). Most studies analyzing prognostic factors tack a proper definition or description of the histological criteria for extra nodal growth. The objective of this study was to evaluate this factor. Methods. Retrospectively 94 patients with CMM and clinically lymph-node metastases were analysed. Metastatic Lymph-nodes were evaluated for ENG and if present grouped in microscopic (2 mm) ENG. ENG was defined as metastatic tumour which clearly extends histologically through the nodal capsule into the perinodal fatty tissue or tumour involvement in the hilar region with interruption of the smooth outline of the (presumed) capsule. Results. Ninety-four patients, median age 52 (6-92) years with CMM, median Breslow thickness 2.8 (0.2-11.0) mm. In 50 patients ENG was present (macroscopic: 32, microscopic: 18). The median follow-up was 59 (range 5-325) months. The number of loco-regional recurrence was 10; 4 in the group with and 6 in the group without ENG (n.s.). Five years survival of patients with ENG was 42% and without ENG 50% (n.s.). There was no significant difference in survival or loco-regional recurrence between microscopic or macroscopic ENG. Conclusion. ENG of lymph-node metastases of CMM is of no prognostic value and has no clinical. impact. (C) 2004 Elsevier Ltd. All rights reserved