Evaluation of Culture Conducive to Academic Success by Gender at a Comprehensive Cancer Center.

INTRODUCTION: The primary objective of this study was to determine whether workplace culture in academic oncology differed by gender, during the COVID-19 pandemic. MATERIALS AND METHODS: We used the Culture Conducive to Women's Academic Success (CCWAS), a validated survey tool, to investigate the academic climate at an NCI-designated Cancer Center. We adapted the CCWAS to be applicable to people of all genders. The full membership of the Cancer Center was surveyed (total faculty = 429). The questions in each of 4 CCWAS domains (equal access to opportunities, work-life balance, freedom from gender bias, and leadership support) were scored using a 5-point Likert scale. Median score and interquartile ranges for each domain were calculated. RESULTS: A total of 168 respondents (men = 58, women = 106, n = 4 not disclosed) submitted survey responses. The response rate was 39% overall and 70% among women faculty. We found significant differences in perceptions of workplace culture by gender, both in responses to individual questions and in the overall score in the following domains: equal access to opportunities, work-life balance, and leader support, and in the total score for the CCWAS. CONCLUSIONS: Our survey is the first of its kind completed during the COVID-19 pandemic at an NCI-designated Cancer Center, in which myriad factors contributed to burnout and workplace challenges. These results point to specific issues that detract from the success of women pursuing careers in academic oncology. Identifying these issues can be used to design and implement solutions to improve workforce culture, mitigate gender bias, and retain faculty.

Design-Based Bone Marrow Biopsy Training.

INTRODUCTION: Hematology/oncology fellows must achieve bone marrow biopsy proficiency. However, opportunities for fellows to perform bone marrow biopsies on patients are highly dependent on clinical volume. An easily accessible and feasible system to practice these procedures repetitively has not been described. Other specialties use 3-dimensional (3D)-printed models to practice procedures, but hematology/oncology has not yet incorporated this novel medical education tool, which has the potential to provide such an accessible and feasible system for procedural practice. METHODS: We used design thinking to develop and pilot a bone marrow biopsy simulation using 3D-printed pelvis models. We printed and optimized 2 models through iterative prototyping. In July 2019, we conducted a 1-hour session with 9 fellows. After an anatomy review, fellows practiced biopsies using the models with faculty feedback. To evaluate feasibility, we reviewed session evaluations, measured fellow comfort, surveyed supervising attendings, and gathered fellow and attending feedback. RESULTS: Fellows rated the 3D session highly. Fellow comfort improved after orientation. Supervisors noted no difference between the 2019 fellows and prior years. Fellows praised the opportunity to rehearse mechanics, receive feedback, and internalize anatomy. Fellows suggested incorporating a female pelvis and more soft tissue. Attending feedback on the model aligned with fellow feedback. We implemented the session again in 2020 with adjustments based on feedback. CONCLUSIONS: Three-dimensional printing represents an accessible and feasible educational tool. Three-dimensional-printed models provide opportunities for iterative practice, feedback, and anatomy visualization. Future iterations should continue to incorporate user feedback to optimize model utility.

An Elusive Seizure.

We present a case of a healthy 62-year-old woman who developed recurrent seizures preceded by subacute cognitive slowing, ataxia, night sweats, and weight loss. She was found to have cytopenias, multifocal T2/FLAIR hyperintensities on magnetic resonance imaging (MRI), and magnetic susceptibility artifact lesions on susceptibility weighted imaging (SWI). Her symptoms, imaging and laboratory abnormalities all improved with high-doses of steroids and intravenous immunoglobulin (IVIG). But recurred several weeks after completing treatment. Despite extensive work-up, she required multiple hospitalizations and repeat diagnostic studies to arrive at a diagnosis. With an expert discussant in hematology and oncology, we review the differential diagnosis and stepwise approach of unexplained neuro-inflammatory syndromes with cytopenias and systemic symptoms. Our case highlights how time, empiric treatment response, and repeated diagnostic studies refine differential diagnoses and subsequent evaluation. After revealing the diagnosis, we discuss the heterogenous clinical manifestations of this disease process.

A single center evaluation of applicant experiences in virtual interviews across eight internal medicine subspecialty fellowship programs.

Due to the COVID-19 pandemic, most graduate medical education (GME) training programs conducted virtual interviews for prospective trainees during the 2020-2021 application cycle. Many internal medicine (IM) subspecialty fellowship programs hosted virtual interviews for the first time with little published data to guide best practices.To evaluate how IM subspecialty fellowship applicants perceived the virtual interview day experience.We designed a 38-item questionnaire that was sent via email to applicants in eight IM subspecialty programs at a single tertiary academic medical center (University of California, San Francisco) from September-November, 2020.Seventy-five applicants completed the survey (75/244, 30.7%), including applicants from all eight fellowship programs. Most survey respondents agreed that the length of the virtual interview day (mean = 6.4 hours) was long enough to gather the information they needed (n = 65, 86.7%) and short enough to prevent fatigue (n = 55, 73.3%). Almost all survey respondents agreed that they could adequately assess the clinical experience (n = 71, 97.3%), research opportunities (n = 72, 98.6%), and program culture (n = 68, 93.2%). Of the respondents who attended a virtual educational conference, most agreed it helped to provide a sense of the program's educational culture (n = 20, 66.7%). Areas for improvement were identified, with some survey respondents reporting that the virtual interview day was too long (n = 11) or that they would have preferred to meet more fellows (n = 10).Survey respondents indicated that the virtual interview was an adequate format to learn about fellowship programs. These findings can inform future virtual interviews for GME training programs.

Integrating Concept Maps into a Medical Student Oncology Curriculum.

Expanding frontiers of knowledge have prompted medical schools to reconsider how best to promote learning in the face of information overload. Concept mapping (CM) promotes knowledge retention and integration. Students have perceived CM positively in prior studies, but the feasibility and utility of integrating CM into a medical student oncology curriculum as a learning and assessment tool have not been described. At the University of California, San Francisco, 152 medical students in a second-year hematology/oncology course produced concept maps about a single cancer type over 4 weeks. We collected student evaluations about CM. Two of three graders independently scored each map using a standard rubric. We compared CM scores with USMLE Step 1 scores and clerkship grades using regression. All students produced a concept map. Student perception was mixed, and students provided feedback to improve CM utility as a learning tool. Grading was feasible, and inter-rater reliability was high. CM scores did not predict USMLE Step 1 scores or clerkship grades. CM was feasible as a learning tool, and we present strategies based on student feedback and literature review to improve utility. CM was feasible and reliable as an assessment tool; additional validity evidence may improve utility. Future studies should explore whether CM integrated into medical student oncology curricula early, serially, and collaboratively, with iterative practice and feedback, may predict meaningful learning and performance outcomes.

Virtual Interviews at Graduate Medical Education Training Programs: Determining Evidence-Based Best Practices.

The COVID-19 pandemic has had a profound impact on the nation's health care system, including on graduate medical education (GME) training programs. Traditionally, residency and fellowship training program applications involve in-person interviews conducted on-site, with only a minority of programs offering interviews remotely via a virtual platform. However, in light of the COVID-19 pandemic, it is anticipated that most interviews will be conducted virtually for the 2021 application cycle and possibly beyond. Therefore, GME training programs need to prepare for the transition to virtual interviews using evidence-based practices. At the University of California, San Francisco, a multidisciplinary task force was convened to review existing literature about virtual interviews and determine best practices. This article summarizes these findings, first discussing the advantages and disadvantages of the virtual interview format and then providing evidence-based best practices for GME training programs. Specifically, the authors make the following recommendations: develop a detailed plan for the interview process, consider using standardized interview questions, recognize and respond to potential biases that may be amplified with the virtual interview format, prepare your own trainees for virtual interviews, develop electronic materials and virtual social events to approximate the interview day, and collect data about virtual interviews at your own institution. With adequate preparation, the virtual interview experience can be high yield, positive, and equitable for both applicants and GME training programs.

Circulating proteolytic signatures of chemotherapy-induced cell death in humans discovered by N-terminal labeling.

It is known that many chemotherapeutics induce cellular apoptosis over hours to days. During apoptosis, numerous cellular proteases are activated, most canonically the caspases. We speculated that detection of proteolytic fragments released from apoptotic cells into the peripheral blood may serve as a unique indicator of chemotherapy-induced cell death. Here we used an enzymatic labeling process to positively enrich free peptide α-amines in the plasma of hematologic malignancy patients soon after beginning treatment. This N-terminomic approach largely avoids interference by high-abundance proteins that complicate traditional plasma proteomic analyses. Significantly, by mass spectrometry methods, we found strong biological signatures of apoptosis directly in the postchemotherapy plasma, including numerous caspase-cleaved peptides as well as relevant peptides from apoptotic and cell-stress proteins second mitochondria-derived activator of caspases, HtrA serine peptidase 2, and activating transcription factor 6. We also treated hematologic cancer cell lines with clinically relevant chemotherapeutics and monitored proteolytic fragments released into the media. Remarkably, many of these peptides coincided with those found in patient samples. Overall, we identified 153 proteolytic peptides in postchemotherapy patient plasma as potential indicators of cellular apoptosis. Through targeted quantitative proteomics, we verified that many of these peptides were indeed increased post- vs. prechemotherapy in additional patients. Our findings reveal that numerous proteolytic fragments are released from dying tumor cells. Monitoring posttreatment proteolysis may lead to a novel class of inexpensive, rapid biomarkers of cell death.

The DegraBase: a database of proteolysis in healthy and apoptotic human cells.

Proteolysis is a critical post-translational modification for regulation of cellular processes. Our lab has previously developed a technique for specifically labeling unmodified protein N termini, the α-aminome, using the engineered enzyme, subtiligase. Here we present a database, called the DegraBase (http://wellslab.ucsf.edu/degrabase/), which compiles 8090 unique N termini from 3206 proteins directly identified in subtiligase-based positive enrichment mass spectrometry experiments in healthy and apoptotic human cell lines. We include both previously published and unpublished data in our analysis, resulting in a total of 2144 unique α-amines identified in healthy cells, and 6990 in cells undergoing apoptosis. The N termini derive from three general categories of proteolysis with respect to cleavage location and functional role: translational N-terminal methionine processing (∼10% of total proteolysis), sites close to the translational N terminus that likely represent removal of transit or signal peptides (∼25% of total), and finally, other endoproteolytic cuts (∼65% of total). Induction of apoptosis causes relatively little change in the first two proteolytic categories, but dramatic changes are seen in endoproteolysis. For example, we observed 1706 putative apoptotic caspase cuts, more than double the total annotated sites in the CASBAH and MEROPS databases. In the endoproteolysis category, there are a total of nearly 3000 noncaspase nontryptic cleavages that are not currently reported in the MEROPS database. These studies significantly increase the annotation for all categories of proteolysis in human cells and allow public access for investigators to explore interesting proteolytic events in healthy and apoptotic human cells.

Quantitative profiling of caspase-cleaved substrates reveals different drug-induced and cell-type patterns in apoptosis.

Proapoptotic drugs are a mainstay of cancer drug treatment. These drugs stress cells and ultimately trigger the activation of caspases, cysteine-class proteases that cleave after aspartic acid and deconstruct the cell. It is well known that cells respond differently to proapoptotic cancer drug treatments. Here, using a global and unbiased quantitative N-terminomics technology, we show that ~500 products of caspase cleavage and their kinetics vary dramatically between cell type and cytotoxic drug treatment. It is likely that variations arise from differences in baseline proteome composition of the cell type and the alterations induced by drug treatments to yield a unique cohort of proteins that caspases finally target. Many targets are specific to both drug treatment and cell type, providing candidate-specific biomarkers for apoptosis. For example, in multiple myeloma cells treated with the proteasome inhibitor bortezomib, levels of activating transcription factor-4 increase dramatically early in drug treatment and then decrease upon cleavage by activated caspases. Thus, caspase-derived cleavage products are a sensitive reflection of cell-type and drug-induced stress, and provide useful fingerprints for mechanisms of drug action and response.

Suppression of Rev3, the catalytic subunit of Pol{zeta}, sensitizes drug-resistant lung tumors to chemotherapy.

Platinum-based chemotherapeutic drugs are front-line therapies for the treatment of non-small cell lung cancer. However, intrinsic drug resistance limits the clinical efficacy of these agents. Recent evidence suggests that loss of the translesion polymerase, Polζ, can sensitize tumor cell lines to cisplatin, although the relevance of these findings to the treatment of chemoresistant tumors in vivo has remained unclear. Here, we describe a tumor transplantation approach that enables the rapid introduction of defined genetic lesions into a preclinical model of lung adenocarcinoma. Using this approach, we examined the effect of impaired translesion DNA synthesis on cisplatin response in aggressive late-stage lung cancers. In the presence of reduced levels of Rev3, an essential component of Polζ, tumors exhibited pronounced sensitivity to cisplatin, leading to a significant extension in overall survival of treated recipient mice. Additionally, treated Rev3-deficient cells exhibited reduced cisplatin-induced mutation, a process that has been implicated in the induction of secondary malignancies following chemotherapy. Taken together, our data illustrate the potential of Rev3 inhibition as an adjuvant therapy for the treatment of chemoresistant malignancies, and highlight the utility of rapid transplantation methodologies for evaluating mechanisms of chemotherapeutic resistance in preclinical settings.

Dissecting the differences between the alpha and beta anomers of the oxidative DNA lesion FaPydG.

The oxidative DNA lesion, FaPydG rapidly anomerizes to form a mixture of the alpha and beta anomer. To investigate the mutagenic potential of both forms, we prepared stabilized bioisosteric analogues of both configurational isomers and incorporated them into oligonucleotides. These were subsequently used for thermodynamic melting-point studies and for primer-extension experiments. While the beta compound, in agreement with earlier data, prefers cytidine as the pairing partner, the alpha compound is not able form a stable base pair with any natural base. In primer-extension studies with the high-fidelity polymerase Bst Pol I, the polymerase was able to read through the lesion. The beta compound showed no strong mutagenic potential. The alpha compound, in contrast, strongly destabilized DNA duplexes and also blocked all of the tested DNA polymerases, including two low-fidelity polymerases of the Y-family.

Structure of a high fidelity DNA polymerase bound to a benzo[a]pyrene adduct that blocks replication.

Of the carcinogens to which humans are most frequently exposed, the polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) is one of the most ubiquitous. BP is a byproduct of grilled foods and tobacco and fuel combustion and has long been linked to various human cancers, particularly lung and skin. BP is metabolized to diol epoxides that covalently modify DNA bases to form bulky adducts that block DNA synthesis by replicative or high fidelity DNA polymerases. Here we present the structure of a high fidelity polymerase from a thermostable strain of Bacillus stearothermophilus (Bacillus fragment) bound to the most common BP-derived N2-guanine adduct base-paired with cytosine. The BP adduct adopts a conformation that places the polycyclic BP moiety in the nascent DNA minor groove and is the first structure of a minor groove adduct bound to a polymerase. Orientation of the BP moiety into the nascent DNA minor groove results in extensive disruption to the interactions between the adducted DNA duplex and the polymerase. The disruptions revealed by the structure of Bacillus fragment bound to a BP adduct provide a molecular basis for rationalizing the potent blocking effect on replication exerted by BP adducts.

Observing translesion synthesis of an aromatic amine DNA adduct by a high-fidelity DNA polymerase.

Aromatic amines have been studied for more than a half-century as model carcinogens representing a class of chemicals that form bulky adducts to the C8 position of guanine in DNA. Among these guanine adducts, the N-(2'-deoxyguanosin-8-yl)-aminofluorene (G-AF) and N-2-(2'-deoxyguanosin-8-yl)-acetylaminofluorene (G-AAF) derivatives are the best studied. Although G-AF and G-AAF differ by only an acetyl group, they exert different effects on DNA replication by replicative and high-fidelity DNA polymerases. Translesion synthesis of G-AF is achieved with high-fidelity polymerases, whereas replication of G-AAF requires specialized bypass polymerases. Here we have presented structures of G-AF as it undergoes one round of accurate replication by a high-fidelity DNA polymerase. Nucleotide incorporation opposite G-AF is achieved in solution and in the crystal, revealing how the polymerase accommodates and replicates past G-AF, but not G-AAF. Like an unmodified guanine, G-AF adopts a conformation that allows it to form Watson-Crick hydrogen bonds with an opposing cytosine that results in protrusion of the bulky fluorene moiety into the major groove. Although incorporation opposite G-AF is observed, the C:G-AF base pair induces distortions to the polymerase active site that slow translesion synthesis.

Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase.

Aerobic respiration generates reactive oxygen species that can damage guanine residues and lead to the production of 8-oxoguanine (8oxoG), the major mutagenic oxidative lesion in the genome. Oxidative damage is implicated in ageing and cancer, and its prevalence presents a constant challenge to DNA polymerases that ensure accurate transmission of genomic information. When these polymerases encounter 8oxoG, they frequently catalyse misincorporation of adenine in preference to accurate incorporation of cytosine. This results in the propagation of G to T transversions, which are commonly observed somatic mutations associated with human cancers. Here, we present sequential snapshots of a high-fidelity DNA polymerase during both accurate and mutagenic replication of 8oxoG. Comparison of these crystal structures reveals that 8oxoG induces an inversion of the mismatch recognition mechanisms that normally proofread DNA, such that the 8oxoG.adenine mismatch mimics a cognate base pair whereas the 8oxoG.cytosine base pair behaves as a mismatch. These studies reveal a fundamental mechanism of error-prone replication and show how 8oxoG, and DNA lesions in general, can form mismatches that evade polymerase error-detection mechanisms, potentially leading to the stable incorporation of lethal mutations.