Targeted Quantitative Mass Spectrometry Analysis of Protein Biomarkers From Previously Stained Single Formalin-Fixed Paraffin-Embedded Tissue Sections.

Formalin-fixed, paraffin-embedded tissues represent a majority of all biopsy specimens commonly analyzed by histologic or immunohistochemical staining with adhesive coverslips attached. Mass spectrometry (MS) has recently been used to precisely quantify proteins in samples consisting of multiple unstained formalin-fixed, paraffin-embedded sections. Here, we report an MS method to analyze proteins from a single coverslipped 4-µm section previously stained with hematoxylin and eosin, Masson trichrome, or 3,3'-diaminobenzidine-based immunohistochemical staining. We analyzed serial unstained and stained sections from non-small cell lung cancer specimens for proteins of varying abundance (PD-L1, RB1, CD73, and HLA-DRA). Coverslips were removed by soaking in xylene, and after tryptic digestion, peptides were analyzed by targeted high-resolution liquid chromatography with tandem MS with stable isotope-labeled peptide standards. The low-abundance proteins RB1 and PD-L1 were quantified in 31 and 35 of 50 total sections analyzed, respectively, whereas higher abundance CD73 and HLA-DRA were quantified in 49 and 50 sections, respectively. The inclusion of targeted ß-actin measurement enabled normalization in samples where residual stain interfered with bulk protein quantitation by colorimetric assay. Measurement coefficient of variations for 5 replicate slides (hematoxylin and eosin stained vs unstained) from each block ranged from 3% to 18% for PD-L1, from 1% to 36% for RB1, 3% to 21% for CD73, and 4% to 29% for HLA-DRA. Collectively, these results demonstrate that targeted MS protein quantification can add a valuable data layer to clinical tissue specimens after assessment for standard pathology end points.

Capacity of Primary Care to Deliver Telehealth in the United States.

BACKGROUND: Because of the Coronavirus disease 2019 (COVID 19) pandemic, many primary care practices have transitioned to telehealth visits to keep patients at home and decrease the transmission of the disease. Yet, little is known about the nationwide capacity for delivering primary care services via telehealth. METHODS: Using the 2016 National Ambulatory Medical Survey we estimated the number and proportion of reported visits and services that could be provided via telehealth. We also performed cross-tabulations to calculate the number and proportion of physicians providing telephone visits and e-mail/internet encounters. RESULTS: Of the total visits (nearly 400 million) to primary care physicians, 42% were amenable to telehealth and 73% of the total services rendered could be delivered through telehealth modalities. Of the primary care physicians, 44% provided telephone consults and 19% provided e-consults. DISCUSSION: This study underscores how and where primary care services could be delivered. It provides the first estimates of the capacity of primary care to provide telehealth services for COVID-19 related illness, and for several other acute and chronic medical conditions. It also highlights the fact that, as of 2016, most outpatient telehealth visits were done via telephone. CONCLUSIONS: This study provides an estimate of the primary care capacity to deliver telehealth and can guide practices and payers as care delivery models change in a post-COVID 19 environment.

Not Telehealth: Which Primary Care Visits Need In-Person Care?

The Coronavirus disease 2019 (COVID 19) pandemic has resulted in a rapid shift to telehealth and many services that need in-person care have been avoided. Yet, as practices and payment policies return to a new normal, there will be many questions about what proportion of visits should be done in-person vs telehealth. Using the 2016 National Ambulatory Medical Survey (NAMCS), we estimated what proportion of visits were amenable to telehealth before COVID-19 as a guide. We divided services into those that needed in-person care and those that could be done via telehealth. Any visit that included at least 1 service where in-person care was needed was counted as an in-person only visit. We then calculated what proportion of reported visits and services in 2016 could have been provided via telehealth, as well as what proportion of in-person only services were done by primary care. We found that 66% of all primary care visits reported in NAMCS in 2016 required an in-person service. 90% of all wellness visits and immunizations were done in primary care offices, as were a quarter of all Papanicolaou smears. As practices reopen, patients will need to catch up on many of the in-person only visits that were postponed such as Papanicolaou smears and wellness visits. At the same time, patients and clinicians now accustomed to telehealth may have reservations about returning to in-person only visits. Our estimates may provide a guide to practices as they navigate how to deliver care in a post-COVID-19 environment.

Practice, clinician, and patient factors associated with the adoption of lung cancer screening.

OBJECTIVES: Lung cancer remains the leading cause of cancer-related deaths in the United States. In 2013, the US Preventive Services Task Force recommended annual screening for lung cancer with low-dose computed tomography in adults meeting certain criteria. This study seeks to assess lung cancer screening uptake in three health systems. SETTING: This study was part of a randomized controlled trial to engage underserved populations in preventive care and includes 45 primary care practices in eight states. METHODS: Practice and clinician characteristics were manually collected. Lung cancer was measured from electronic health record data. A generalized linear mixed model was used to assess characteristics associated with screening. RESULTS: Patient records between 2012 and 2016 were examined. Lung cancer screening uptake overall increased only slightly after the guideline change (2.8-5.6%, p < 0.01). One health system did not show an increase in uptake (0.2-0.1%, p = 0.32), another had a clinically insignificant increase (1.5-2.9%, p < 0.01), and the third nearly doubled its higher baseline screening rate (10.4-19.1%, p < 0.01). Within the third health system, patients more likely to be screened were older, male, had more comorbid conditions, visited the office more frequently, were seen in practices closer to the screening clinic, or were uninsured or covered by Medicare or Medicaid. CONCLUSIONS: Certain patients appeared more likely to be screened. The only health system with increased lung cancer screening explicitly promoted screening rather than relying on clinicians to implement the new guideline. Systems approaches may help increase the low uptake of lung cancer screening.

"You Will Miss Your Grandmother's Funeral": Surviving Medical School, Just in Time to Burnout.

The abounding negativity of my first year of medical school was best exemplified by a preparatory class entitled "How to Survive the Spring Semester." Physician burnout is an epidemic that may begin in the classrooms and cadaver labs of medical schools with scare tactics, fatalistic messaging, and a pervasive "culture of burnout." Medical education's axiom-"if I suffered through it, so should you"-is a persistent disservice to students who must survive medical school, just in time to burnout as physicians. I wonder if combating physician burnout could start with medical schools, which must stop sowing the seeds of burnout in students. Medical educators can start by reevaluating the words and tone they strike with the physicians of the future.

Care Quality and Spending Among Commercially Insured Children With Disabilities.

OBJECTIVE: To identify opportunities to improve care value for children with disabilities (CWD), we examined CWD prevalence within a commercially insured population and compared outpatient care quality and annual health plan spending levels for CWD relative to children with complex medical conditions without disabilities; children with chronic conditions that are not complex; and children without disabling, complex, or chronic conditions. METHODS: This cross-sectional study comprised 1,118,081 person-years of Blue Cross Blue Shield Massachusetts data for beneficiaries aged 1 to 19years old during 2008 to 2012. We combined the newly developed and validated Children with Disabilities Algorithm with the Pediatric Medical Complexity Algorithm to identify CWD and non-CWD subgroups. We used 14 validated or National Quality Forum-endorsed measures to assess outpatient care quality and paid claims to examine annual plan spending levels and components. RESULTS: CWD constituted 4.5% of all enrollees. Care quality for CWD was between 11% and 59% for 8 of 14 quality measures and >80% for the 6 remaining measures and was generally comparable to that for non-CWD subgroups. Annual plan spending among CWD was a median and mean 23% and 53% higher than that for children with complex medical conditions without disabilities, respectively; CWD mean and median values were higher than for all other groups as well. CONCLUSIONS: CWD were prevalent in our commercially insured population. CWD experienced suboptimal levels of care, but those levels were comparable to non-CWD groups. Improving the care value for CWD involves a deeper understanding of what higher spending delivers and additional aspects of care quality.

Racial, Ethnic, and Socioeconomic Disparities in Patient Safety Events for Hospitalized Children.

OBJECTIVES: Previous studies have revealed racial/ethnic and socioeconomic disparities in quality of care and patient safety. However, these disparities have not been examined in a pediatric inpatient environment by using a measure of clinically confirmed adverse events (AEs). In this study, we do so using the Global Assessment of Pediatric Patient Safety (GAPPS) Trigger Tool. METHODS: GAPPS was applied to medical records of randomly selected pediatric patients discharged from 16 hospitals in the Pediatric Research in Inpatient Settings Network across 4 US regions from January 2007 to December 2012. Disparities in AEs for hospitalized children were identified on the basis of patient race/ethnicity (black, Latino, white, or other; N = 17 336 patient days) and insurance status (public, private, or self-pay/no insurance; N = 19 030 patient days). RESULTS: Compared with hospitalized non-Latino white children, hospitalized Latino children experienced higher rates of all AEs (Latino: 30.1 AEs per 1000 patient days versus white: 16.9 AEs per 1000 patient days; P ≤ .001), preventable AEs (Latino: 15.9 AEs per 1000 patient days versus white: 8.9 AEs per 1000 patient days; P = .002), and high-severity AEs (Latino: 12.6 AEs per 1000 patient days versus white: 7.7 AEs per 1000 patient days; P = .02). Compared with privately insured children, publicly insured children experienced higher rates of preventable AEs (public: 12.1 AEs per 1000 patient days versus private: 8.5 AEs per 1000 patient days; P = .02). No significant differences were observed among other groups. CONCLUSIONS: The GAPPS analysis revealed racial and/or ethnic and socioeconomic disparities in rates of AEs experienced by hospitalized children across a broad range of geographic and hospital settings. Further investigation may reveal underlying mechanisms of these disparities and could help hospitals reduce harm.

Cancer Survivorship for Primary Care Annotated Bibliography.

Long-term cancer survivorship care is a relatively new and rapidly advancing field of research. Increasing cancer survivorship rates have created a huge population of long-term cancer survivors whose cancer-specific needs challenge healthcare infrastructure and highlight a significant deficit of knowledge and guidelines in transitional care from treatment to normalcy/prolonged survivorship. As the paradigm of cancer care has changed from a fixation on the curative to the maintenance on long-term overall quality of life, so to, has the delineation of responsibility between oncologists and primary care physicians (PCPs). As more patients enjoy long-term survival, PCPs play a more comprehensive role in cancer care following acute treatment. To this end, this annotated bibliography was written to provide PCPs and other readers with an up-to-date and robust base of knowledge on long-term cancer survivorship, including definitions and epidemiological information as well as specific considerations and recommendations on physical, psychosocial, sexual, and comorbidity needs of survivors. Additionally, significant information is included on survivorship care, specifically Survivorship Care Plans (SPCs) and their evolution, utilization by oncologists and PCPs, and current gaps, as well as an introduction to patient navigation programs. Given rapid advancements in cancer research, this bibliography is meant to serve as current baseline reference outlining the state of the science.

Reduced p53 in peripheral blood mononuclear cells from patients with rheumatoid arthritis is associated with loss of radiation-induced apoptosis.

OBJECTIVE: Patients with autoimmune disorders exhibit highly reproducible gene expression profiles in their peripheral blood mononuclear cells. This profile includes, at least in part, a collection of underexpressed genes that encode proteins that inhibit cell cycle progression and stimulate apoptosis. We aimed to determine whether this gene expression profile confers functional liability on lymphocytes from patients with rheumatoid arthritis (RA). METHODS: Viability studies in response to a panel of proapoptotic stimuli revealed that T lymphocytes from patients with RA were resistant to gamma radiation-induced apoptosis, a process known to be dependent on p53. To assess p53 function in RA peripheral blood mononuclear cells, baseline levels of p53 protein and TP53 transcript were measured in patients with RA and controls. The cellular p53 response to gamma radiation was also assessed by immunoblotting. RESULTS: Lymphocytes from patients with RA had lower baseline levels of TP53 messenger RNA (mRNA) and p53 protein than did those from control subjects and were deficient in their ability to increase p53 after exposure to gamma radiation. A subgroup of patients with RA had a second biochemical defect characterized by expression of very low baseline levels of checkpoint kinase 2 mRNA and protein. CONCLUSION: We conclude that defects in the expression of TP53 mRNA and, in a subgroup, defects in expression of CHK2 mRNA, lead to severe defects in apoptosis in patients with RA. We hypothesize that this liability may contribute to autoimmunity.

Ultraviolet radiation induces phosphorylation and ubiquitin-mediated degradation of DeltaNp63alpha.

DeltaNp63alpha, a homologue of the tumor suppressor p53, acts as a transcriptional repressor with dominant negative effects towards p53. Additionally, DeltaNp63alpha is overexpressed in a number of squamous cell carcinomas, suggesting a potential role in oncogenesis. However, the mechanisms regulating p63 have yet to be elucidated. The goal of the current study was to determine the effect of various genotoxic stresses on DeltaNp63alpha posttranslational modification and stability in normal and transformed squamous epithelial cells. We found that DeltaNp63alpha protein levels decreased after ultraviolet radiation and paclitaxel treatment of both normal and transformed cells. After UV and paclitaxel treatment, DeltaNp63alpha phosphorylation was significantly modulated. Additionally, DeltaNp63alpha protein levels were regulated in a proteasome-dependent manner in control and UV treated cells with increased DeltaNp63alpha ubiquitination after UV treatment or proteasome inhibition. Our studies provide insight to a mechanism for DeltaNp63alpha regulation during normal cell proliferation and, in particular, after stress. Further, the inverse regulation of p53 and DeltaNp63alpha protein levels after cell stress through opposing regulation of proteasome-mediated degradation may allow for rapid transcriptional changes of specific target genes that are consistent with the roles of these family members in tumor suppression and cell growth.

NDRG1 is necessary for p53-dependent apoptosis.

Although a number of target genes for the tumor suppressor p53 have been described, the mechanism of p53-dependent apoptosis is incompletely understood. Thus, it is essential to identify and characterize additional target genes that could mediate apoptosis. In the study reported here, we isolated a p53-regulated gene named NDRG1 (N-Myc down-regulated gene 1). Its expression is induced by DNA damage in a p53-dependent fashion. The promoter region of the NDRG1 gene contains a p53 binding site that confers p53-dependent transcriptional activation via a heterologous reporter. RNA interference and inducible gene expression approaches suggest that NDRG1 is necessary but not sufficient for p53-mediated caspase activation and apoptosis. This report further supports the notion that p53 controls a network of genes that are required for its apoptotic function.

p63: Molecular complexity in development and cancer.

Discovery of the p53 homologs p63 and p73 has brought new excitement to the p53 field. Identification of homologous genes coding for several proteins with similar and antagonistic properties towards p53 has been both intriguing and perplexing. A multitude of properties have been attributed to these new homologs and this review will focus on the biochemical and biological aspects of one family member, p63. Although the most ancient member of the p53 family, p63 is the most recently discovered and the least is known about this family member. Unlike p53, whose protein expression is not readily detectable in epithelial cells unless they are exposed to various stress conditions, p63 is expressed in select epithelial cells at high levels under normal conditions. p63 is highly expressed in embryonic ectoderm and in the nuclei of basal regenerative cells of many epithelial tissues in the adult including skin, breast myoepithelium, oral epithelium, prostate and urothelia. In contrast to the tumor suppressive function of p53, over-expression of select p63 splice variants is observed in many squamous carcinomas suggesting that p63 may act as an oncogene. Undoubtedly, the biochemical and biological activities attributed to p63 over the next several years will be diverse and regulation of the p63 gene and its several protein products complex. The use of various model systems and the study of human disease should continue to lead to rapid advances in our understanding of the role of p63 in development, epithelial cell maintenance and tumorigenesis.

Significance of p63 amplification and overexpression in lung cancer development and prognosis.

The fight against lung cancer is greatly compromised by the lack of effective early detection strategies. Genomic abnormalities and specifically the amplification of chromosomal region 3q26-3qter in lung cancer represent a major signature of neoplastic transformation. Here, we address the significance of p53 homologue p63 mapping to 3q27 in lung tumorigenesis. We analyzed p63 gene copy number (CN) by fluorescence in situ hybridization and expression by immunohistochemistry in tissue microarrays of 217 non-small cell lung cancers (NSCLCs) and correlated them with survival. We additionally characterized our findings in a subset of 24 NSCLCs by reverse transcription-PCR and Western blotting. We analyzed p63 CN and protein expression in 41 preinvasive squamous lesions. The p63 genomic sequence was amplified in 88% of squamous carcinomas, in 42% of large cell carcinomas, and in 11% of adenocarcinomas of the lung. The predominant splice variant of p63 expressed was DeltaNp63alpha. Western analyses revealed DeltaNp63alpha expression in normal bronchus and squamous carcinomas but not in normal lung or in adenocarcinomas. Furthermore, p63genomic amplification and protein staining intensity associated with better survival. We found a significant increase in CN in preinvasive lesions graded severe dysplasia or higher. Our data demonstrate that there is early and frequent genomic amplification of p63 in the development of squamous carcinoma of the lung and that patients with NSCLC showing amplification and overexpression of p63 have prolonged survival. These observations suggest that p63 genomic amplification has an early role in lung tumorigenesis and deserves additional evaluation as a biomarker for lung cancer progression.

The Delta Np63 alpha phosphoprotein binds the p21 and 14-3-3 sigma promoters in vivo and has transcriptional repressor activity that is reduced by Hay-Wells syndrome-derived mutations.

p63 is a recently identified homolog of p53 that is found in the basal layer of several stratified epithelial tissues such as the epidermis, oral mucosa, prostate, and urogenital tract. Studies with p63(-/-) mice and analysis of several human autosomal-dominant disorders with germ line p63 mutations suggest p63 involvement in maintaining epidermal stem cell populations. The p63 gene encodes six splice variants with reported transactivating or dominant-negative activities. The goals of the current study were to determine the splice variants that are expressed in primary human epidermal keratinocytes (HEKs) and the biochemical activity p63 has in these epithelial cell populations. We found that the predominant splice variant expressed in HEKs was Delta Np63 alpha, and it was present as a phosphorylated protein. During HEK differentiation, Delta Np63 alpha and p53 levels decreased, while expression of p53 target genes p21 and 14-3-3 sigma increased. Delta Np63 alpha had transcriptional repressor activity in vitro, and this activity was reduced in Delta Np63 alpha proteins containing point mutations, corresponding to those found in patients with Hay-Wells syndrome. Further, we show that Delta Np63 alpha and p53 can bind the p21 and 14-3-3 sigma promoters in vitro and in vivo, with decreased binding of p63 to these promoters during HEK differentiation. These data suggest that Delta Np63 alpha acts as a transcriptional repressor at select growth regulatory gene promoters in HEKs, and this repression likely plays an important role in the proliferative capacity of basal keratinocytes.

Cell-cycle dysregulation and anticancer therapy.

Cell-cycle dysregulation is a hallmark of tumor cells. The ability of normal cells to undergo cell-cycle arrest after damage to DNA is crucial for the maintenance of genomic integrity. The biochemical pathways that stop the cell cycle in response to cellular stressors are called checkpoints. Defective checkpoint function results in genetic modifications that contribute to tumorigenesis. The regulation of checkpoint signaling also has important clinical implications because the abrogation of checkpoint function can alter the sensitivity of tumor cells to chemotherapeutics. Here, we provide an overview of the mechanisms that regulate the cell cycle, current anticancer therapies that target checkpoint signaling pathways, and strategies for the development of novel chemotherapeutic agents.

Stabilization and functional impairment of the tumor suppressor p53 by the human papillomavirus type 16 E7 oncoprotein.

The p53 tumor suppressor is stabilized in cells expressing the human papillomavirus type 16 (HPV-16) E7 oncoprotein. In contrast, expression of the HPV-16 E6 protein inactivates p53 by targeting it for proteasomal degradation. Since p53 activation is associated with protein accumulation we investigated the biochemical mechanisms and biological consequences of p53 stabilization in HPV-16 E7-expressing cells. Transcriptional reporter assays, expression profiling studies using cDNA arrays, and immunoblot analyses of known p53 target genes suggest that p53 remains transcriptionally inert in E7-expressing cells. The stabilized p53 in E7-expressing cells is in a wild-type conformation and the same number of phospho-forms is present. Furthermore, E7 expression does not alter p53 localization or generally block nuclear export or proteasomal degradation of p53. Moreover, the stabilized p53 remains susceptible to mdm2-induced proteasome-mediated degradation, and exogenous transfected p53 is transcriptionally active in E7-expressing cells. Taken together, these results suggest that E7 can interfere with the normal turnover of p53 but that the resulting increase of p53 has no detectable transcriptional consequences on the p53 targets that we investigated.