Association of Patient Portal Messaging with Survival Among Radiation Oncology Patients.

PURPOSE: The shift to electronic health records has led to both patient portal messaging and large amounts of digital, real-world data for research. The objective of this study was to examine the association between portal messaging and survival among radiation oncology patients, using real-world data. METHODS AND MATERIALS: This retrospective cohort study included patients at least 21 years old and seen by radiation oncology providers between January 14, 2014, and April 23, 2023, at the University of California, San Francisco. We developed Cox proportional hazards models for the outcome of death and examined factors associated with portal messaging using logistic regression models. RESULTS: Among 25,367 patients, the median age was 64 (interquartile range [IR], 54-72), 13,175 (52%) were White, and 14,389 (57%) were male. Overall, as the first message in a thread, 8986 (35%) patients sent messages to radiation oncology providers, and 4218 (17%) patients were sent messages from radiation oncology providers. Patients with head and neck or genitourinary malignancies were more likely than those with other diagnoses to send portal messages to and be sent portal messages from radiation oncology providers. Both sending portal messages to radiation oncology providers (hazard ratio [HR], 0.90; 95% confidence interval [CI], 0.84-0.96; P = .001) and being sent messages from radiation oncology providers (HR, 0.77; CI, 0.70-0.84; P < .001) as the first message in a thread were associated with patient survival after adjusting for socioeconomic, disease, and treatment characteristics. There were disparities among patients sending portal messages to radiation oncology providers, including for Black versus White patients (odds ratio [OR], 0.60; CI, 0.51-0.69; P < .001) and for Medicaid versus Medicare patients (OR, 0.70; CI, 0.62-0.79; P < .001). There were also disparities among patients being sent portal messages by radiation oncology providers, including for Black versus White patients (OR, 0.77; CI, 0.64-0.91; P = .003), for Medicaid versus Medicare patients (OR, 0.76; CI, 0.65-0.89; P < .001), and for patients with female versus male providers (OR, 1.47; CI 1.34-1.62; P < .001). CONCLUSIONS: Sending portal messages to and being sent portal messages from radiation oncology providers were associated with better survival. Future studies should elucidate how best to support patient and provider engagement.

Nonspecific deidentification of date-like text in deidentified clinical notes enables reidentification of dates.

To facilitate the secondary usage of electronic health record data for research, the University of California, San Francisco (UCSF) recently implemented a clinical data warehouse including, among other data, deidentified clinical notes and reports, which are available to UCSF researchers without Institutional Review Board approval. For deidentification of these notes, most of the Health Insurance Portability and Accountability Act identifiers are redacted, but dates are transformed by shifting all dates for a patient back by the same random number of days. We describe an issue in which nonspecific (ie, excess) transformation of nondate, date-like text by this deidentification process enables reidentification of all dates, including birthdates, for certain patients. This issue undercuts the common assumption that excess deidentification is a safe tradeoff to protect patient privacy. We present this issue as a caution to other institutions that may also be considering releasing deidentified notes for research.

Relative Incidence of Emergency Department Visits After Treatment for Prostate Cancer With Radiation Therapy or Radical Prostatectomy.

PURPOSE: Side effect profiles play an important role in treatment decisions for localized prostate cancer. Emergency department (ED) visits, which may be due to side effects from treatment, can be measured in real-world, structured, electronic health record (EHR) data. The goal of this study was to determine whether treatments for localized prostate cancer are associated with ED visits, as a measure of side effects, using EHR data. METHODS AND MATERIALS: We used a self-controlled case series study design, including patients treated at an urban academic medical center with radiation therapy (RT) or radical prostatectomy (RP) for prostate cancer between 2011 and 2020 who had visits documented for ≥6 months before and after treatment and ≥1 ED visit. We estimated relative incidences (RI) of ED visits, comparing incidence in the exposed and unexposed periods, with the exposed period being between start of treatment and 1 month after completion, and the unexposed period consisting of all other documented time. RESULTS: Among men who had at least 1 ED visit and after adjusting for age, there were higher rates of ED visits after RP (RI, 20.4; 95% confidence interval [CI], 15.4-27.0; P < .001), RT overall (RI, 2.4; CI, 1.7-3.4; P < .001), intensity modulated radiation therapy with high dose-rate brachytherapy (RI, 3.4; CI, 1.7-6.8; P < .001) or stereotactic body radiation therapy boost (RI, 7.1; CI, 3.4-14.8; P < .001), and high dose rate brachytherapy alone (RI, 16.3; CI, 7.2-36.9; P < .001) compared with unexposed time. The number needed to harm to result in an ED visit was less for RP (17; CI, 13-23) than RT overall (43; CI, 25-126), but varied by RT modality. CONCLUSIONS: In summary, relative rates of ED visits vary by treatment type, suggesting differing severities of side effects. These data may aid in selecting treatments and demonstrate the feasibility of using the self-controlled case series study design on ED visits in real-world, structured EHR data to better understand side effects of treatment.

Multimodal single-cell analysis reveals distinct radioresistant stem-like and progenitor cell populations in murine glioma.

Radiation therapy is part of the standard of care for gliomas and kills a subset of tumor cells, while also altering the tumor microenvironment. Tumor cells with stem-like properties preferentially survive radiation and give rise to glioma recurrence. Various techniques for enriching and quantifying cells with stem-like properties have been used, including the fluorescence activated cell sorting (FACS)-based side population (SP) assay, which is a functional assay that enriches for stem-like tumor cells. In these analyses, mouse models of glioma have been used to understand the biology of this disease and therapeutic responses, including the radiation response. We present combined SP analysis and single-cell RNA sequencing of genetically-engineered mouse models of glioma to show a time course of cellular response to radiation. We identify and characterize two distinct tumor cell populations that are inherently radioresistant and also distinct effects of radiation on immune cell populations within the tumor microenvironment.

Concurrent genome and epigenome editing by CRISPR-mediated sequence replacement.

BACKGROUND: Recent advances in genome editing have facilitated the direct manipulation of not only the genome, but also the epigenome. Genome editing is typically performed by introducing a single CRISPR/Cas9-mediated double-strand break (DSB), followed by non-homologous end joining (NHEJ)- or homology-directed repair-mediated repair. Epigenome editing, and in particular methylation of CpG dinucleotides, can be performed using catalytically inactive Cas9 (dCas9) fused to a methyltransferase domain. However, for investigations of the role of methylation in gene silencing, studies based on dCas9-methyltransferase have limited resolution and are potentially confounded by the effects of binding of the fusion protein. As an alternative strategy for epigenome editing, we tested CRISPR/Cas9 dual cutting of the genome in the presence of in vitro methylated exogenous DNA, with the aim of driving replacement of the DNA sequence intervening the dual cuts via NHEJ. RESULTS: In a proof of concept at the HPRT1 promoter, successful replacement events with heavily methylated alleles of a CpG island resulted in functional silencing of the HPRT1 gene. Although still limited in efficiency, our study demonstrates concurrent epigenome and genome editing in a single event. CONCLUSIONS: This study opens the door to investigations of the functional consequences of methylation patterns at single CpG dinucleotide resolution. Our results furthermore support the conclusion that promoter methylation is sufficient to functionally silence gene expression.

Increased HOXA5 expression provides a selective advantage for gain of whole chromosome 7 in IDH wild-type glioblastoma.

Glioblastoma is the most frequently occurring and invariably fatal primary brain tumor in adults. The vast majority of glioblastomas is characterized by chromosomal copy number alterations, including gain of whole chromosome 7 and loss of whole chromosome 10. Gain of whole chromosome 7 is an early event in gliomagenesis that occurs in proneural-like precursor cells, which give rise to all isocitrate dehydrogenase (IDH) wild-type glioblastoma transcriptional subtypes. Platelet-derived growth factor A (PDGFA) is one gene on chromosome 7 known to drive gliomagenesis, but, given its location near the end of 7p, there are likely several other genes located along chromosome 7 that select for its increased whole-chromosome copy number within glioblastoma cells. To identify other potential genes that could select for gain of whole chromosome 7, we developed an unbiased bioinformatics approach that identified homeobox A5 (HOXA5) as a gene whose expression correlated with gain of chromosome 7 and a more aggressive phenotype of the resulting glioma. High expression of HOXA5 in glioblastoma was associated with a proneural gene expression pattern and decreased overall survival in both human proneural and PDGF-driven mouse glioblastoma. Furthermore, HOXA5 overexpression promoted cellular proliferation and potentiated radioresistance. We also found enrichment of HOXA5 expression in recurrent human and mouse glioblastoma at first recurrence after radiotherapy. Overall, this study implicates HOXA5 as a chromosome 7-associated gene-level locus that promotes selection for gain of whole chromosome 7 and an aggressive phenotype in glioblastoma.

Pre-plan parameters predict post-implant D90 ≥ 140 Gy for (125)I permanent prostate implants.

PURPOSE: To find permanent prostate implant (PPI) pre-plan dosimetric parameters that predict post-implant D90 ≥ 140 Gy. MATERIAL AND METHODS: Pre-plans were evaluated for 504 patients undergoing PPI with (125)I seeds for low or intermediate risk prostate cancer. Baseline patient and disease factors, numbers of seeds, ratios of number of seeds to available positions (occupancy proportion), and distances between the 100% isodose line and edge of the prostate (margin) planned for the whole prostate (WP), superior (S), inferior (I), anterior (A), and posterior (P) halves, SA, SP, IA, and IP quarters, and superior (ST), inferior (IT), and middle (MT) thirds, and anterior (AT) and posterior (PT) middle one-sixth segments were analyzed by post-implant D90 subset (≥ 140 Gy vs. < 140 Gy). RESULTS: 20% had post-implant D90 < 140 Gy (mean: 128.0 Gy, range: 97.5-139.2) vs. ≥ 140 Gy (mean: 154.4 Gy, range: 140.0-193.5). The D90 ≥ 140 Gy subset had larger AT and IA segment mean numbers of seeds (p = 0.01, 0.046), larger WP, S, A, SA, ST, AT, and MT segment mean margins (p = 0.01, 0.01, 0.001, 0.0001, 0.03, 0.005, 0.02), and lower PT segment occupancy proportion (p = 0.004). On multivariate analysis, independent predictors of post-implant D90 ≥ 140 Gy were increased SA mean margin, no pre-implant 5-α-reductase inhibitor, higher pre-plan D90, decreased P occupancy proportion, no pre-implant hormone therapy, and decreased SP mean margin. CONCLUSIONS: Higher occupancy proportion and larger margins anteriorly and reduced occupancy proportion, and smaller margins posteriorly on PPI pre-plans predict post-implant D90 ≥ 140 Gy.

Spatial exclusivity combined with positive and negative selection of phosphorylation motifs is the basis for context-dependent mitotic signaling.

The timing and localization of events during mitosis are controlled by the regulated phosphorylation of proteins by the mitotic kinases, which include Aurora A, Aurora B, Nek2 (never in mitosis kinase 2), Plk1 (Polo-like kinase 1), and the cyclin-dependent kinase complex Cdk1/cyclin B. Although mitotic kinases can have overlapping subcellular localizations, each kinase appears to phosphorylate its substrates on distinct sites. To gain insight into the relative importance of local sequence context in kinase selectivity, identify previously unknown substrates of these five mitotic kinases, and explore potential mechanisms for substrate discrimination, we determined the optimal substrate motifs of these major mitotic kinases by positional scanning oriented peptide library screening (PS-OPLS). We verified individual motifs with in vitro peptide kinetic studies and used structural modeling to rationalize the kinase-specific selection of key motif-determining residues at the molecular level. Cross comparisons among the phosphorylation site selectivity motifs of these kinases revealed an evolutionarily conserved mutual exclusion mechanism in which the positively and negatively selected portions of the phosphorylation motifs of mitotic kinases, together with their subcellular localizations, result in proper substrate targeting in a coordinated manner during mitosis.

Linear motif atlas for phosphorylation-dependent signaling.

Systematic and quantitative analysis of protein phosphorylation is revealing dynamic regulatory networks underlying cellular responses to environmental cues. However, matching these sites to the kinases that phosphorylate them and the phosphorylation-dependent binding domains that may subsequently bind to them remains a challenge. NetPhorest is an atlas of consensus sequence motifs that covers 179 kinases and 104 phosphorylation-dependent binding domains [Src homology 2 (SH2), phosphotyrosine binding (PTB), BRCA1 C-terminal (BRCT), WW, and 14-3-3]. The atlas reveals new aspects of signaling systems, including the observation that tyrosine kinases mutated in cancer have lower specificity than their non-oncogenic relatives. The resource is maintained by an automated pipeline, which uses phylogenetic trees to structure the currently available in vivo and in vitro data to derive probabilistic sequence models of linear motifs. The atlas is available as a community resource (http://netphorest.info).

Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate.

Polo-like kinase-1 (Plk1) phosphorylates a number of mitotic substrates, but the diversity of Plk1-dependent processes suggests the existence of additional targets. Plk1 contains a specialized phosphoserine-threonine binding domain, the Polo-box domain (PBD), postulated to target the kinase to its substrates. Using the specialized PBD of Plk1 as an affinity capture agent, we performed a screen to define the mitotic Plk1-PBD interactome by mass spectrometry. We identified 622 proteins that showed phosphorylation-dependent mitosis-specific interactions, including proteins involved in well-established Plk1-regulated processes, and in processes not previously linked to Plk1 such as translational control, RNA processing, and vesicle transport. Many proteins identified in our screen play important roles in cytokinesis, where, in mammalian cells, the detailed mechanistic role of Plk1 remains poorly defined. We go on to characterize the mitosis-specific interaction of the Plk1-PBD with the cytokinesis effector kinase Rho-associated coiled-coil domain-containing protein kinase 2 (Rock2), demonstrate that Rock2 is a Plk1 substrate, and show that Rock2 colocalizes with Plk1 during cytokinesis. Finally, we show that Plk1 and RhoA function together to maximally enhance Rock2 kinase activity in vitro and within cells, and implicate Plk1 as a central regulator of multiple pathways that synergistically converge to regulate actomyosin ring contraction during cleavage furrow ingression.

Diagnostic markers that distinguish colon and ovarian adenocarcinomas: identification by genomic, proteomic, and tissue array profiling.

Colon and ovarian cancers can be difficult to distinguish in the abdomen, and the distinction is important because it determines which drugs will be used for therapy. To identify molecular markers for that differential diagnosis, we developed a multistep protocol starting with the 60 human cancer cell lines used by the National Cancer Institute to screen for new anticancer agents. The steps included: (a) identification of candidate markers using cDNA microarrays; (b) verification of clone identities by resequencing; (c) corroboration of transcript levels using Affymetrix oligonucleotide chips; (d) quantitation of protein expression by "reverse-phase" protein microarray; and (e) prospective validation of candidate markers on clinical tumor sections in tissue microarrays. The two best candidates identified were villin for colon cancer cells and moesin for ovarian cancer cells. Because moesin stained stromal elements in both types of cancer, it would probably not have been identified as a marker if we had started with mRNA or protein profiling of bulk tumors. Villin appears at least as useful as the currently used colon cancer marker cytokeratin 20, and moesin also appears to have utility. The multistep process introduced here has the potential to produce additional markers for cancer diagnosis, prognosis, and therapy.

Apoptotic susceptibility of cancer cells selected for camptothecin resistance: gene expression profiling, functional analysis, and molecular interaction mapping.

To study the molecular mechanisms by which drug resistance develops, we compared DU145 humanprostate cancer cells with a subline selected for resistance to camptothecin. Differences in gene expression level were assessed by hybridizing the two cell types against each other using quadruplicate "Oncochip" cDNA microarrays that included 1648 cancer-related genes. Expression levels differing by a factor of >1.5 were detected for 181 of the genes. These differences were judged statistically reliable on the basis of a stratum-adjusted Kruskal-Wallis test, after taking into account a dye-dependent variable. The 181 expression-altered genes included a larger than expected number of the "apoptosis-related" genes (P = 0.04). To assess whether this observation reflected a generalized resistance of RCO.1 to apoptosis, we exposed the cells to a range of stresses (cisplatin, staurosporine, UV, ionizing radiation, and serum starvation) and found greatly reduced apoptotic responses for RC0.1 (relative to DU145) using flow cytometric Annexin V and terminal deoxynucleotidyl transferase-mediated nick end labeling assays. We next examined the apoptosis-related genes in the context of a molecular interaction map and found expression differences in the direction "expected" on the basis of the apoptosis-resistance of RC0.1 for BAD, caspase-6, and genes that signal via the Akt pathway. Exposure of the cells to wortmannin, an inhibitor of the Akt effector phosphatidylinositol 3-kinase, provided functional support for involvement of the Akt pathway. However, closer examination of the molecular interaction map revealed a paradox: many of the expression differences observed for apoptosis-related genes were in the direction "contrary" to that expected given the resistance of RC0.1. The map indicated that most of these unexpected expression differences were associated with genes involved in the nuclear factor kappa B and transforming growth factor beta pathways. Overall, the patterns that emerged suggested a two-step model for the selection process that led to resistance in RC0.1 cells. The first hypothesized step would involve a decrease in apoptotic susceptibility through changes in the apoptosis-control machinery associated with the Bcl-2 and caspase gene families, and also in antiapoptotic pathways operating through Akt/PKB. The second step would involve changes in multifunctional upstream genes (including some genes in the nuclear factor kappa B and transforming growth factor beta pathways) that can facilitate apoptosis but that would also tend to contribute to cell proliferation in the presence of drug. Thus, we propose that a downstream blockade of apoptosis was "permissive" for the selection of upstream pathway changes that would otherwise have induced apoptosis. This model is analogous to one suggested previously for the relationship between oncogene function and apoptosis in carcinogenesis.