Functional recombinant human complement C1q with different affinity tags.

Complement C1q is a multifunctional protein able to sense pathogens and immune molecules such as immunoglobulins and pentraxins, and to trigger the classical complement pathway through activation of its two associated proteases, C1r and C1s. C1q is a multimeric protein composed of three homologous yet distinct polypeptide chains A, B, and C, each composed of an N-terminal collagen-like sequence and a C-terminal globular gC1q module, that assemble into six heterotrimeric (A-B-C) subunits. This hexameric structure exhibits the characteristic shape of a bouquet of flowers, comprising six collagen-like triple helices, each terminating in a trimeric C-terminal globular head. We have produced previously functional recombinant full-length C1q in stably transfected HEK 293-F cells, with a FLAG tag inserted at the C-terminal end of C1qC chain. We report here the generation of additional recombinant C1q proteins, with a FLAG tag fused to the C-terminus of C1qA or C1qB chains, or to the N-terminus of the C1qC chain. Two other variants harboring a Myc or a 6-His tag at the C-terminal end of C1qC were also produced. We show that all C1q variants, except for the His-tagged protein, can be produced at comparable yields and are able to bind with similar affinities to either IgM, a ligand of the globular regions, or to the C1r2-C1s2 tetramer, and to trigger IgM-mediated serum complement activation. These new recombinant C1q variants provide additional tools to investigate the multiple functions of C1q.

Characterization of transgenic mouse lines for selectively targeting satellite glial cells and macrophages in dorsal root ganglia.

The importance of glial cells in the modulation of neuronal processes is now generally accepted. In particular, enormous progress in our understanding of astrocytes and microglia physiology in the central nervous system (CNS) has been made in recent years, due to the development of genetic and molecular toolkits. However, the roles of satellite glial cells (SGCs) and macrophages-the peripheral counterparts of astrocytes and microglia-remain poorly studied despite their involvement in debilitating conditions, such as pain. Here, we characterized in dorsal root ganglia (DRGs), different genetically-modified mouse lines previously used for studying astrocytes and microglia, with the goal to implement them for investigating DRG SGC and macrophage functions. Although SGCs and astrocytes share some molecular properties, most tested transgenic lines were found to not be suitable for studying selectively a large number of SGCs within DRGs. Nevertheless, we identified and validated two mouse lines: (i) a CreERT2 recombinase-based mouse line allowing transgene expression almost exclusively in SGCs and in the vast majority of SGCs, and (ii) a GFP-expressing line allowing the selective visualization of macrophages. In conclusion, among the tools available for exploring astrocyte functions, a few can be used for studying selectively a great proportion of SGCs. Thus, efforts remain to be made to characterize other available mouse lines as well as to develop, rigorously characterize and validate new molecular tools to investigate the roles of DRG SGCs, but also macrophages, in health and disease.

Rapid Fabrication of Protein Microarrays via Autogeneration and on-Chip Purification of Biotinylated Probes.

A streamlined approach toward the rapid fabrication of streptavidin-biotin-based protein microarrays was investigated. First, using our engineered versatile plasmid (pBADcM-tBirA) and an optimal coexpression strategy for biotin ligase and biotin acceptor peptide (BAP) chimeric recombinant protein, an autogeneration system for biotinylated probes was developed. This system permitted an advantageous biotinylation of BAP chimeric recombinant proteins, providing a strategy for the high-throughput synthesis of biotinylated probes. Then, to bypass the conventional rate-limiting steps, we employed an on-chip purification process to immobilize the biotinylated probes with high-throughput recombinant lysates. The integration of the autogeneration of probes and on-chip purification not only contributed to the effective and reliable fabrication of the protein microarray, but also enabled simplification of the process and an automated throughput format. This labor- and cost-effective approach may facilitate the use of protein microarrays for diagnosis, pharmacology, proteomics, and other laboratory initiatives.

Monitoring protease activity in biological tissues using antibody prodrugs as sensing probes.

Proteases have been implicated in the development of many pathological conditions, including cancer. Detection of protease activity in diseased tissues could therefore be useful for diagnosis, prognosis, and the development of novel therapeutic approaches. Due to tight post-translational regulation, determination of the expression level of proteases alone may not be indicative of protease activities, and new methods for measuring protease activity in biological samples such as tumor biopsies are needed. Here we report a novel zymography-based technique, called the IHZTM assay, for the detection of specific protease activities in situ. The IHZ assay involves imaging the binding of a protease-activated monoclonal antibody prodrug, called a Probody® therapeutic, to tissue. Probody therapeutics are fully recombinant, masked antibodies that can only bind target antigen after removal of the mask by a selected protease. A fluorescently labeled Probody molecule is incubated with a biological tissue, thereby enabling its activation by tissue endogenous proteases. Protease activity is measured by imaging the activated Probody molecule binding to antigen present in the sample. The method was evaluated in xenograft tumor samples using protease specific substrates and inhibitors, and the measurements correlated with efficacy of the respective Probody therapeutics. Using this technique, a diverse profile of MMP and serine protease activities was characterized in breast cancer patient tumor samples. The IHZ assay represents a new type of in situ zymography technique that can be used for the screening of disease-associated proteases in patient samples from multiple pathological conditions.

rBC2LCN lectin as a potential probe of early-stage HER2-positive breast carcinoma.

The recombinant N-terminal domain of BC2L-C lectin (rBC2LCN) is useful for detecting not only human pluripotent stem cells but also some cancers. However, the cancer types and stages that can be detected by rBC2LCN remain unclear. In this study, we identified the human breast carcinoma subtypes and stages that can be detected by rBC2LCN. Compared with rBC2LCN-negative breast carcinoma cell lines, the rBC2LCN-positive cells expressed higher levels of human epidermal growth factor receptor 2 (HER2) and epithelial marker genes. Importantly, rBC2LCN histochemical staining of human breast carcinoma tissues demonstrated the utility of rBC2LCN in detecting breast carcinoma types that express HER2 and have not spread much in the early phase of growth. We conclude that rBC2LCN may have potential as a detection probe and a drug delivery vehicle to identify and treat early-stage HER2-positive breast carcinoma.

Methods for measuring HMGB1 release during immunogenic cell death.

The exodus of the alarmin high mobility group box 1 (HMGB1) from the nucleus constitutes a crucial cellular danger signal and manifests as a sequential process in which HMGB1 first exits the nucleus into the cytoplasm and then is secreted or passively released through the permeabilized plasma membrane. Extracellular HMGB1 can interact with pattern recognition receptors to stimulate innate immune responses. Here, we describe a discovery pipeline for the identification of pharmacological agents endowed with HMGB1 releasing properties. The "retention using selective hooks" (RUSH) system in which a streptavidin-NLS3 fusion protein serves as a nuclear hook to sequester streptavidin-binding peptide (SBP) fused with HMGB1 and green fluorescent protein (GFP) allowed for synchronizing HMGB1 increase. Thus, exclusively in the presence of biotin, which liberates HMGB1-SBP-GFP from its nuclear hook, immunogenic cell death (ICD) inducers such as anthracyclines are able to cause the nucleo-cytoplasmic translocation of HMGB1-SBP-GFP. This system facilitates the identification of HMGB1 releasing agents in medium- to high-throughput screening assays.

Profiling surface proteins on individual exosomes using a proximity barcoding assay.

Exosomes have been implicated in numerous biological processes, and they may serve as important disease markers. Surface proteins on exosomes carry information about their tissues of origin. Because of the heterogeneity of exosomes it is desirable to investigate them individually, but this has so far remained impractical. Here, we demonstrate a proximity-dependent barcoding assay to profile surface proteins of individual exosomes using antibody-DNA conjugates and next-generation sequencing. We first validate the method using artificial streptavidin-oligonucleotide complexes, followed by analysis of the variable composition of surface proteins on individual exosomes, derived from human body fluids or cell culture media. Exosomes from different sources are characterized by the presence of specific combinations of surface proteins and their abundance, allowing exosomes to be separately quantified in mixed samples to serve as markers for tissue-specific engagement in disease.

Gene Mosaicism Screening Using Single-Molecule Molecular Inversion Probes in Routine Diagnostics for Systemic Autoinflammatory Diseases.

Diagnosis of systemic autoinflammatory diseases (SAIDs) is often difficult to achieve and can delay the start of proper treatments and result in irreversible organ damage. In several patients with dominantly inherited SAID, postzygotic mutations have been detected as the disease-causing gene defects. Mutations with allele frequencies <5% have been detected, even in patients with severe phenotypes. Next-generation sequencing techniques are currently used to detect mutations in SAID-associated genes. However, even if the genomic region is highly covered, this approach is usually not able to distinguish low-grade postzygotic variants from background noise. We, therefore, developed a sensitive deep sequencing assay for mosaicism detection in SAID-associated genes using single-molecule molecular inversion probes. Our results show the accurate detection of postzygotic variants with allele frequencies as low as 1%. The probability of calling mutations with allele frequencies ≥3% exceeds 99.9%. To date, we have detected three patients with mosaicism, two carrying likely pathogenic NLRP3 variants and one carrying a likely pathogenic TNFRSF1A variant with an allele frequency of 1.3%, confirming the relevance of the technology. The assay shown herein is a flexible, robust, fast, cost-effective, and highly reliable method for mosaicism detection; therefore, it is well suited for routine diagnostics.

Imaging-based pooled CRISPR screening reveals regulators of lncRNA localization.

Pooled-library CRISPR screening provides a powerful means to discover genetic factors involved in cellular processes in a high-throughput manner. However, the phenotypes accessible to pooled-library screening are limited. Complex phenotypes, such as cellular morphology and subcellular molecular organization, as well as their dynamics, require imaging-based readout and are currently beyond the reach of pooled-library CRISPR screening. Here we report an all imaging-based pooled-library CRISPR screening approach that combines high-content phenotype imaging with high-throughput single guide RNA (sgRNA) identification in individual cells. In this approach, sgRNAs are codelivered to cells with corresponding barcodes placed at the 3' untranslated region of a reporter gene using a lentiviral delivery system with reduced recombination-induced sgRNA-barcode mispairing. Multiplexed error-robust fluorescence in situ hybridization (MERFISH) is used to read out the barcodes and hence identify the sgRNAs with high accuracy. We used this approach to screen 162 sgRNAs targeting 54 RNA-binding proteins for their effects on RNA localization to nuclear compartments and uncovered previously unknown regulatory factors for nuclear RNA localization. Notably, our screen revealed both positive and negative regulators for the nuclear speckle localization of a long noncoding RNA, MALAT1, suggesting a dynamic regulation of lncRNA localization in subcellular compartments.

The rBC2LCN-positive subpopulation of PC-3 cells exhibits cancer stem-like properties.

The recombinant lectin rBC2LCN is a useful marker for discriminating the undifferentiated status of human induced or embryonic stem cells. Recently, rBC2LCN has also been used for detecting some cancers and niche cells. However, the generality of which types of cells are detected by rBC2LCN is unclear. In this study, we demonstrated the potential of rBC2LCN as a probe for detecting and isolating cancer stem-like cells. Interestingly, flow cytometric analysis of various human cell lines indicated that the human prostate cancer cell line PC-3 consisted of rBC2LCN-positive and -negative subpopulations. Compared with the rBC2LCN-negative subpopulation, the rBC2LCN-positive subpopulation possessed representative features of cancer stem cells and malignancy, such as slow proliferation, increased cell motility, anchorage-independent growth, and drug resistance. The comprehensive expression profiles revealed that the rBC2LCN-positive subpopulation expressed higher levels of cancer stem cell markers. These findings indicate that rBC2LCN is useful for detecting not only pluripotent stem cells but also the cancer stem-like subpopulation of PC-3 cells. Pluripotent and cancer cells with rBC2LCN positivity would be important for future stem cell research.

Supramolecular Microgels with Molecular Beacons at the Interface for Ultrasensitive, Amplification-Free, and SNP-Selective miRNA Fluorescence Detection.

In this study, a supramolecular structure with femtomolar biorecognition properties is proposed for use in analytical devices. It is obtained by an innovative interface between synthetic hydrogel polymers and molecular beacon (mb) probes. Supramolecularly structured microgels are synthetized with a core-shell architecture with specific dyes polymerized in a desired compartment. Mb probes are opportunely conjugated at the microgel interface so that their recognition mechanism is preserved and their spatial distribution is optimized to avoid crowding effects. The miR-21, a microRNA involved in various biological processes and usually used as a biomarker in early cancer diagnosis, has been selected as the target. The results demonstrate that by tuning the spatial distribution of molecular probes immobilized on the microgel and/or the amount of microgels, the assay shows scalable sensitivity reaching a limit of detection down to about 10 fM, without amplification steps and with detection time as short as 1 h. The assay results specific toward single mutated targets, and it is stable in the presence of high-interfering oligonucleotides concentrations. The miRNA target is also detected in human serum with performances similar to those observed in PBS buffer because of microgel antifouling properties without the need of any surface treatment. All tests were performed in a low sample volume (20 µL). As a result, mb-microgel represents an innovative biosensor to precisely quantify microRNAs in a direct (mix&read), scalable, and selective way. Such an approach paves the way for creating innovative biosensing interfaces with other probes, such as hairpins, aptamers, and PNA.

Color-coded molecular beacons for multiplex PCR screening assays.

The number of different fluorescent colors that can be distinguished in a PCR screening assay restricts the number of different targets that can be detected. If only six colors can be distinguished, and the probe for each target is labeled with a unique color, then only six different targets can be identified. Yet, it is often desirable to identify more targets. For instance, the rapid identification of which bacterial species (if any) is present in a patient's normally sterile blood sample, out of a long list of species, would enable appropriate actions to be taken to prevent sepsis. We realized that the number of different targets that can be identified in a screening assay can be increased significantly by utilizing a unique combination of two colors for the identification of each target species. We prepared a demonstration assay in which 15 different molecular beacon probe pairs were present, each pair specific for the same identifying sequence in the 16S ribosomal RNA gene of a different bacterial species, and each pair labeled with a unique combination of two fluorophores out of the six differently colored fluorophores that our PCR instrument could distinguish. In a set of PCR assays, each containing all 30 color-coded molecular beacons, and each containing DNA from a different bacterial species, the only two colors that arose in each real-time assay identified the species-specific target sequence that was present. Due to the intrinsic low background of molecular beacon probes, these reactions were specific and extremely sensitive, and the threshold cycle reflected the abundance of the target sequence present in each sample.

In Vitro Selection of a DNA Aptamer by Cell-SELEX as a Molecular Probe for Cervical Cancer Recognition and Imaging.

Aptamers have become the most promising recognition reagents in terms of early diagnosis and effective treatment of cancers. In this study, using cervical cancer as a model, we have identified a DNA aptamer specifically binding to cervical cancer cells with high affinity using the cell-SELEX (systematic evolution of ligands by exponential enrichment) method, in which a negative selection was carried out using normal epithelial cells as control. The binding abilities of 6 selected truncated aptamers were determined by laser confocal fluorescence microscopy and flow cytometry, while most of them only recognize the target cells and do not bind the control cells, and the aptamer C-9S with 51-mer shows the best binding affinity to Ca Ski cells (target cells) with a dissociation constant value of 19.3 ± 2.9 nM. Moreover, at physiological temperature, C-9S remains its specific recognition capability to Ca Ski cells as well. Meanwhile, C-9S shows a similar binding ability to another cervical cancer cells (HeLa). Therefore, on the basis of its excellent targeting properties and inherent functional versatility of aptamer, C-9S holds great potential to be a molecular probe for early detection, in vivo imaging, and targeted delivery for further researches in cancer.

Comparative evaluation of dimeric and monomeric forms of ADAPT scaffold protein for targeting of HER2-expressing tumours.

ADAPTs are small engineered non-immunoglobulin scaffold proteins, which have demonstrated very promising features as vectors for radionuclide tumour targeting. Radionuclide imaging of human epidermal growth factor 2 (HER2) expression in vivo might be used for stratification of patients for HER2-targeting therapies. ADAPT6, which specifically binds to HER2, has earlier been shown to have very promising features for in vivo targeting of HER2 expressing tumours. In this study we tested the hypothesis that dimerization of ADAPT6 would increase the apparent affinity to HER2 and accordingly improve tumour targeting. To find an optimal molecular design of dimers, a series of ADAPT dimers with different linkers, -SSSG- (DiADAPT6L1), -(SSSG)2- (DiADAPT6L2), and -(SSSG)3- (DiADAPT6L3) was evaluated. Dimers in combination with optimal linker lengths demonstrated increased apparent affinity to HER2. The best variants, DiADAPT6L2 and DiADAPT6L3 were site-specifically labelled with 111In and 125I, and compared with a monomeric ADAPT6 in mice bearing HER2-expressing tumours. Despite higher affinity, both dimers had lower tumour uptake and lower tumour-to-organ ratios compared to the monomer. We conclude that improved affinity of a dimeric form of ADAPT does not compensate the disadvantage of increased size. Therefore, increase of affinity should be obtained by affinity maturation and not by dimerization.

A Novel Engineered Small Protein for Positron Emission Tomography Imaging of Human Programmed Death Ligand-1: Validation in Mouse Models and Human Cancer Tissues.

PURPOSE: To design and evaluate a small engineered protein binder targeting human programmed death-1 ligand (hPD-L1) in vivo for PET imaging in four mouse tumor models, and in situ in human cancer specimens.Experimental Design: The hPD-L1 protein binder, FN3hPD-L1, was engineered using a 12-kDa human fibronectin type-3 domain (FN3) scaffold. The binder's affinity was assayed in CT26 mouse colon carcinoma cells stably expressing hPD-L1 (CT26/hPD-L1). 64Cu-FN3hPD-L1 was assayed for purity, specific activity, and immunoreactivity. Four groups of NSG mice (n = 3-5/group) were imaged with 64Cu-FN3hPD-L1 PET imaging (1-24 hours postinjection of 3.7 MBq/7 µg of Do-FN3 in 200 µL PBS): Nod SCID Gamma (NSG) mice bearing (i) syngeneic CT26/hPD-L1tumors, (ii) CT26/hPD-L1 tumors blocked (blk) by preinjected nonradioactive FN3hPD-L1 binder, (iii) hPD-L1-negative Raji xenografts, and (iv) MDA-MB-231 xenografts. The FN3hPD-L1 binder staining was evaluated against validated hPD-L1 antibodies by immunostaining in human cancer specimens. RESULTS: FN3hPD-L1 bound hPD-L1 with 1.4 ± 0.3 nmol/L affinity in CT26/hPD-L1 cells. 64Cu-FN3hPD-L1 radiotracer showed >70% yield and >95% purity. 64Cu-FN3hPD-L1 PET imaging of mice bearing CT26/hPD-L1 tumors showed tumor-to-muscle ratios of 5.6 ± 0.9 and 13.1 ± 2.3 at 1 and 4 hours postinjection, respectively. The FN3hPD-L1 binder detected hPD-L1 expression in human tissues with known hPD-L1 expression status based on two validated antibodies. CONCLUSIONS: The 64Cu-FN3hPD-L1 radiotracer represents a novel, small, and high-affinity binder for imaging hPD-L1 in tumors. Our data support further exploration and clinical translation of this binder for noninvasive identification of cancer patients who may respond to immune checkpoint blockade therapies.

Metabolism-Driven High-Throughput Cancer Identification with GLUT5-Specific Molecular Probes.

Point-of-care applications rely on biomedical sensors to enable rapid detection with high sensitivity and selectivity. Despite advances in sensor development, there are challenges in cancer diagnostics. Detection of biomarkers, cell receptors, circulating tumor cells, gene identification, and fluorescent tagging are time-consuming due to the sample preparation and response time involved. Here, we present a novel approach to target the enhanced metabolism in breast cancers for rapid detection using fluorescent imaging. Fluorescent analogs of fructose target the fructose-specific transporter GLUT5 in breast cancers and have limited to no response from normal cells. These analogs demonstrate a marked difference in adenocarcinoma and premalignant cells leading to a novel detection approach. The vastly different uptake kinetics of the analogs yields two unique signatures for each cell type. We used normal breast cells MCF10A, adenocarcinoma cells MCF7, and premalignant cells MCF10AneoT, with hepatocellular carcinoma cells HepG2 as the negative control. Our data indicated that MCF10AneoT and MCF7 cells had an observable difference in response to only one of the analogs. The response, observed as fluorescence intensity, leads to a two-point assessment of the cells in any sample. Since the treatment time is 10 min, there is potential for use in rapid on-site high-throughput diagnostics.

A genetically encoded 19F NMR probe for lysine acetylation.

Advances in acetylated protein-protein/DNA interactions depend on the development of a novel NMR (nuclear magnetic resonance) probe to study the conformational changes of acetylated proteins. However, the method for detecting the acetylated protein conformation is underdeveloped. Herein, an acetyllysine mimic has been exploited for detecting the conformational changes of acetylated p53-protein/DNA interactions by genetic code expansion and 19F NMR. This 19F NMR probe shows high structural similarity to acetyllysine and could not be deacetylated by sirtuin deacetylase in vitro/vivo. Moreover, acetylation of p53 K164 is reported to be deacetylated by SIRT2 for the first time.

A molecular beacon based on DNA-templated silver nanoclusters for the highly sensitive and selective multiplexed detection of virulence genes.

In this work, we develop a fluorescent molecular beacon based on the DNA-templated silver nanoclusters (DNA-Ag NCs). The skillfully designed molecular beacon can be conveniently used for detection of diverse virulence genes as long as the corresponding recognition sequences are embedded. Importantly, the constructed detection system allows simultaneous detection of multiple nucleic acids, which is attributed to non-overlapping emission spectra of the as-synthesized silver nanoclusters. Based on the target-induced fluorescence enhancement, three infectious disease-related genes HIV, H1N1, and H5N1 are detected, and the corresponding detection limits are 3.53, 0.12 and 3.95nM, respectively. This design allows specific, versatile and simultaneous detection of diverse targets with easy operation and low cost.

Evaluation of HER2-specific peptide ligand for its employment as radiolabeled imaging probe.

HER2 transmembrane receptor is an important target in immunotherapy treatment of breast and gastroesophageal cancer. Molecular imaging of HER2 expression may provide essential prognostic and predictive information concerning disseminated cancer and aid in selection of an optimal therapy. Radiolabeled low molecular weight peptide ligands are particularly attractive as probes for molecular imaging, since they reach and bind to the target and clear from non-target organs and blood stream faster than bulky antibodies. In this study, we evaluated a potential HER2-imaging probe, an A9 nonapeptide, derived from the trastuzumab-Fab portion. Its cellular uptake was investigated by mass spectrometry analysis of the cytoplasmic cellular extracts. Moreover, based on in-silico modeling, DTPA chelator was conjugated to N-terminus of A9. 111In-labeled A9 demonstrated nanomolar affinity to HER2-expressing BT474 cells and favorable biodistribution profile in NMRI mice. This study suggests that the peptide A9 represents a good lead candidate for development of molecular probe, to be used for imaging purposes and for the delivery of cytotoxic agents.

Impact of an alternative chromosome 17 probe and the 2013 American Society of Clinical Oncology and College of American Pathologists guidelines on fluorescence in situ hybridization for the determination of HER2 gene amplification in breast cancer.

BACKGROUND: The dual-probe fluorescence in situ hybridization (FISH) assay for human epidermal growth factor receptor 2 (HER2) gene amplification in breast cancer provides an HER2:CEP17 (centromere enumeration probe for chromosome 17) ratio. Copy number alteration (CNA) in CEP17 may skew this ratio. The authors analyzed the impact of the 2013 American Society of Oncology/College of American Pathologists (ASCO/CAP) guidelines and an alternative chromosome 17 probe on HER2 status in tumor specimens with CEP17 CNA. METHODS: Specimens with CEP17 CNA (n = 310) were selected from 3048 tumor samples that were received from January 2013 to June 2015 for testing with the alternative chromosome 17 probe D17S122. Reclassification of HER2 status was assessed using the 2007 and 2013 ASCO/CAP guidelines. RESULTS: The alternative chromosome 17 probe reclassified 82 of 310 (26.5%) and 87 of 310 (28.1%) tumors using the 2007 and 2013 guidelines, respectively. Of the 41 of 310 tumors (13.2%) that were reclassified from nonamplified to amplified according to 2007 guidelines, 28 of 41 (68.3%) had an average HER2 copy number ≥4.0 and <6.0. The 39 of 310 tumors (12.6%) that were reclassified from equivocal to amplified according to 2013 guidelines had a mean HER2 copy number between ≥4.0 and <6.0. Most of these patients had stage I, hormone receptor-positive, lymph node-negative tumors, which is an unusual clinicopathologic profile for HER2-amplified tumors, and most received HER2-targeted therapy in addition to endocrine therapy. CONCLUSIONS: Reflex testing with an alternative chromosome 17 probe using the 2013 ASCO/CAP guidelines reclassified 28.1% of tumor samples that had CEP17 CNA, converting nearly one-half from equivocal to amplified. The benefit of HER2-targeted therapy in this patient population requires further study. Cancer 2017;123:2230-2239. © 2017 American Cancer Society.