NCCN Guidelines® Insights: Melanoma: Cutaneous, Version 2.2021.

Over the past few years, the NCCN Guidelines for Melanoma: Cutaneous have been expanded to include pathways for treatment of microscopic satellitosis (added in v2.2020), and the following Principles sections: Molecular Testing (added in v2.2019), Systemic Therapy Considerations (added in v2.2020), and Brain Metastases Management (added in v3.2020). The v1.2021 update included additional modifications of these sections and notable revisions to Principles of: Pathology, Surgical Margins for Wide Excision of Primary Melanoma, Sentinel Lymph Node Biopsy, Completion/Therapeutic Lymph Node Dissection, and Radiation Therapy. These NCCN Guidelines Insights discuss the important changes to pathology and surgery recommendations, as well as additions to systemic therapy options for patients with advanced disease.

Ovarian Cancer, Version 2.2020, NCCN Clinical Practice Guidelines in Oncology.

Epithelial ovarian cancer is the leading cause of death from gynecologic cancer in the United States and is the country's fifth most common cause of cancer mortality in women. A major challenge in treating ovarian cancer is that most patients have advanced disease at initial diagnosis. These NCCN Guidelines discuss cancers originating in the ovary, fallopian tube, or peritoneum, as these are all managed in a similar manner. Most of the recommendations are based on data from patients with the most common subtypes─high-grade serous and grade 2/3 endometrioid. The NCCN Guidelines also include recommendations specifically for patients with less common ovarian cancers, which in the guidelines include the following: carcinosarcoma, clear cell carcinoma, mucinous carcinoma, low-grade serous, grade 1 endometrioid, borderline epithelial, malignant sex cord-stromal, and malignant germ cell tumors. This manuscript focuses on certain aspects of primary treatment, including primary surgery, adjuvant therapy, and maintenance therapy options (including PARP inhibitors) after completion of first-line chemotherapy.

Cutaneous Melanoma, Version 2.2019, NCCN Clinical Practice Guidelines in Oncology.

The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Cutaneous melanoma have been significantly revised over the past few years in response to emerging data on immune checkpoint inhibitor therapies and BRAF-targeted therapy. This article summarizes the data and rationale supporting extensive changes to the recommendations for systemic therapy as adjuvant treatment of resected disease and as treatment of unresectable or distant metastatic disease.

NCCN Guidelines Insights: Ovarian Cancer, Version 1.2019.

Epithelial ovarian cancer is the leading cause of death from gynecologic cancer in the United States, with less than half of patients living >5 years from diagnosis. A major challenge in treating ovarian cancer is that most patients have advanced disease at initial diagnosis. The best outcomes are observed in patients whose primary treatment includes complete resection of all visible disease plus combination platinum-based chemotherapy. Research efforts are focused on primary neoadjuvant treatments that may improve resectability, as well as systemic therapies providing improved long-term survival. These NCCN Guidelines Insights focus on recent updates to neoadjuvant chemotherapy recommendations, including the addition of hyperthermic intraperitoneal chemotherapy, and the role of PARP inhibitors and bevacizumab as maintenance therapy options in select patients who have completed primary chemotherapy.

NCCN Guidelines Insights: Cancer-Associated Venous Thromboembolic Disease, Version 2.2018.

Venous thromboembolism (VTE) is common in patients with cancer and increases morbidity and mortality. VTE prevention and treatment are more complex in patients with cancer. The NCCN Guidelines for Cancer-Associated Venous Thromboembolic Disease outline strategies for treatment and prevention of VTE in adult patients diagnosed with cancer or in whom cancer is clinically suspected. These NCCN Guidelines Insights explain recent changes in anticoagulants recommended for the treatment of cancer-associated VTE.

Merkel Cell Carcinoma, Version 1.2018, NCCN Clinical Practice Guidelines in Oncology.

This selection from the NCCN Guidelines for Merkel Cell Carcinoma (MCC) focuses on areas impacted by recently emerging data, including sections describing MCC risk factors, diagnosis, workup, follow-up, and management of advanced disease with radiation and systemic therapy. Included in these sections are discussion of the new recommendations for use of Merkel cell polyomavirus as a biomarker and new recommendations for use of checkpoint immunotherapies to treat metastatic or unresectable disease. The next update of the complete version of the NCCN Guidelines for MCC will include more detailed information about elements of pathology and addresses additional aspects of management of MCC, including surgical management of the primary tumor and draining nodal basin, radiation therapy as primary treatment, and management of recurrence.

Cancer-Associated Venous Thromboembolic Disease, Version 1.2015.

The NCCN Guidelines for Cancer-Associated Venous Thromboembolic Disease outline strategies for treatment and prevention of venous thromboembolism (VTE) in adult patients with a diagnosis of cancer or for whom cancer is clinically suspected. VTE is a common complication in patients with cancer, which places them at greater risk for morbidity and mortality. Therefore, risk-appropriate prophylaxis is an essential component for the optimal care of inpatients and outpatients with cancer. Critical to meeting this goal is ensuring that patients get the most effective medication in the correct dose. Body weight has a significant impact on blood volume and drug clearance. Because obesity is a common health problem in industrialized societies, cancer care providers are increasingly likely to treat obese patients in their practice. Obesity is a risk factor common to VTE and many cancers, and may also impact the anticoagulant dose needed for safe and effective prophylaxis. These NCCN Guidelines Insights summarize the data supporting new dosing recommendations for VTE prophylaxis in obese patients with cancer.

Central Nervous System Cancers, Version 1.2015.

The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Central Nervous System (CNS) Cancers provide interdisciplinary recommendations for managing adult CNS cancers. Primary and metastatic brain tumors are a heterogeneous group of neoplasms with varied outcomes and management strategies. These NCCN Guidelines Insights summarize the NCCN CNS Cancers Panel's discussion and highlight notable changes in the 2015 update. This article outlines the data and provides insight into panel decisions regarding adjuvant radiation and chemotherapy treatment options for high-risk newly diagnosed low-grade gliomas and glioblastomas. Additionally, it describes the panel's assessment of new data and the ongoing debate regarding the use of alternating electric field therapy for high-grade gliomas.

The mechanism of fast-gate opening in ClC-0.

ClC-0 is a chloride channel whose gating is sensitive to both voltage and chloride. Based on analysis of gating kinetics using single-channel recordings, a five-state model was proposed to describe the dependence of ClC-0 fast-gate opening on voltage and external chloride (Chen, T.-Y., and C. Miller. 1996. J. Gen. Physiol. 108:237-250). We aimed to use this five-state model as a starting point for understanding the structural changes that occur during gating. Using macroscopic patch recordings, we were able to reproduce the effects of voltage and chloride that were reported by Chen and Miller and to fit our opening rate constant data to the five-state model. Upon further analysis of both our data and those of Chen and Miller, we learned that in contrast to their conclusions, (a) the features in the data are not adequate to rule out a simpler four-state model, and (b) the chloride-binding step is voltage dependent. In order to be able to evaluate the effects of mutants on gating (described in the companion paper, see Engh et al. on p. 351 of this issue), we developed a method for determining the error on gating model parameters, and evaluated the sources of this error. To begin to mesh the kinetic model(s) with the known CLC structures, a model of ClC-0 was generated computationally based on the X-ray crystal structure of the prokaryotic homolog ClC-ec1. Analysis of pore electrostatics in this homology model suggests that at least two of the conclusions derived from the gating kinetics analysis are consistent with the known CLC structures: (1) chloride binding is necessary for channel opening, and (2) chloride binding to any of the three known chloride-binding sites must be voltage dependent.

The role of a conserved lysine in chloride- and voltage-dependent ClC-0 fast gating.

ClC-0 is a chloride channel whose gating is sensitive to voltage, chloride, and pH. In a previous publication, we showed that the K149C mutation causes a +70-mV shift in the voltage dependence of ClC-0 fast gating. In this paper we analyze the effects of a series of mutations at K149 on the voltage and chloride dependence of gating. By fitting our data to the previously proposed four-state model for ClC-0 fast gating, we show which steps in fast-gate opening are likely to be affected by these mutations. Computational analysis of mutant ClC-0 homology models show electrostatic contributions to chloride binding that may partially account for the effects of K149 on gating. The analysis of gating kinetics in combination with the available structural information suggests some of the structural changes likely to underpin fast-gate opening.

Cysteine accessibility in ClC-0 supports conservation of the ClC intracellular vestibule.

ClC chloride channels, which are ubiquitously expressed in mammals, have a unique double-barreled structure, in which each monomer forms its own pore. Identification of pore-lining elements is important for understanding the conduction properties and unusual gating mechanisms of these channels. Structures of prokaryotic ClC transporters do not show an open pore, and so may not accurately represent the open state of the eukaryotic ClC channels. In this study we used cysteine-scanning mutagenesis and modification (SCAM) to screen >50 residues in the intracellular vestibule of ClC-0. We identified 14 positions sensitive to the negatively charged thiol-modifying reagents sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) or sodium 4-acetamido-4'-maleimidylstilbene-2'2-disulfonic acid (AMS) and show that 11 of these alter pore properties when modified. In addition, two MTSES-sensitive residues, on different helices and in close proximity in the prokaryotic structures, can form a disulfide bond in ClC-0. When mapped onto prokaryotic structures, MTSES/AMS-sensitive residues cluster around bound chloride ions, and the correlation is even stronger in the ClC-0 homology model developed by Corry et al. (2004). These results support the hypothesis that both secondary and tertiary structures in the intracellular vestibule are conserved among ClC family members, even in regions of very low sequence similarity.

Diversity in the rates of transcript elongation by single RNA polymerase molecules.

Single-molecule measurements of the activities of a variety of enzymes show that rates of catalysis may vary markedly between different molecules in putatively homogeneous enzyme preparations. We measured the rate at which purified Escherichia coli RNA polymerase moves along a approximately 2650-bp DNA during transcript elongation in vitro at 0.5 mm nucleoside triphosphates. Individual molecules of a specifically biotinated RNA polymerase derivative were tagged with 199-nm diameter avidin-coated polystyrene beads; enzyme movement along a surface-linked DNA molecule was monitored by observing changes in bead Brownian motion by light microscopy. The DNA was derived from a naturally occurring transcription unit and was selected for the absence of regulatory sequences that induce lengthy pausing or termination of transcription. With rare exceptions, individual enzyme molecules moved at a constant velocity throughout the transcription reaction; the distribution of velocities across a population of 140 molecules was unimodal and was well fit by a Gaussian. However, the width of the Gaussian, sigma = 6.7 bp/s, was considerably larger than the precision of the velocity measurement (1 bp/s). The observations show that different transcription complexes have differences in catalytic rate (and thus differences in structure) that persist for thousands of catalytic turnovers. These differences may provide a parsimonious explanation for the complex transcription kinetics observed in bulk solution.

Simultaneous, coincident optical trapping and single-molecule fluorescence.

We constructed a microscope-based instrument capable of simultaneous, spatially coincident optical trapping and single-molecule fluorescence. The capabilities of this apparatus were demonstrated by studying the force-induced strand separation of a dye-labeled, 15-base-pair region of double-stranded DNA (dsDNA), with force applied either parallel ('unzipping' mode) or perpendicular ('shearing' mode) to the long axis of the region. Mechanical transitions corresponding to DNA hybrid rupture occurred simultaneously with discontinuous changes in the fluorescence emission. The rupture force was strongly dependent on the direction of applied force, indicating the existence of distinct unbinding pathways for the two force-loading modes. From the rupture force histograms, we determined the distance to the thermodynamic transition state and the thermal off rates in the absence of load for both processes.