The Association of Mitochondrial Copy Number With Sarcopenia in Adult Survivors of Childhood Cancer.

BACKGROUND: Adult childhood cancer survivors are at risk for frailty, including low muscle mass and weakness (sarcopenia). Using peripheral blood mitochondrial DNA copy number (mtDNAcn) as a proxy for functional mitochondria, this study describes cross-sectional associations between mtDNAcn and sarcopenia among survivors. METHODS: Among 1762 adult childhood cancer survivors (51.6% male; median age = 29.4 years, interquartile range [IQR] = 23.3-36.8), with a median of 20.6 years from diagnosis (IQR = 15.2-28.2), mtDNAcn estimates were derived from whole-genome sequencing. A subset was validated by quantitative polymerase chain reaction and evaluated cross-sectionally using multivariable logistic regression for their association with sarcopenia, defined by race-, age-, and sex-specific low lean muscle mass or weak grip strength. All statistical tests were 2-sided. RESULTS: The prevalence of sarcopenia was 27.0%, higher among female than male survivors (31.5% vs 22.9%; P < .001) and associated with age at diagnosis; 51.7% of survivors with sarcopenia were diagnosed ages 4-13 years (P = .01). Sarcopenia was most prevalent (39.0%) among central nervous system tumor survivors. Cranial radiation (odds ratio [OR] = 1.84, 95% confidence interval [CI] = 1.32 to 2.59) and alkylating agents (OR = 1.34, 95% CI = 1.04 to 1.72) increased, whereas glucocorticoids decreased odds (OR = 0.72, 95% CI = 0.56 to 0.93) of sarcopenia. mtDNAcn decreased with age (ß = -0.81, P = .002) and was higher among female survivors (ß = 9.23, P = .01) and among survivors with a C allele at mt.204 (ß = -17.9, P = .02). In adjusted models, every standard deviation decrease in mtDNAcn increased the odds of sarcopenia 20% (OR = 1.20, 95% CI = 1.07 to 1.34). CONCLUSIONS: A growing body of evidence supports peripheral blood mtDNAcn as a biomarker for adverse health outcomes; however, this study is the first to report an association between mtDNAcn and sarcopenia among childhood cancer survivors.

The Mouse Universal Genotyping Array: From Substrains to Subspecies.

Genotyping microarrays are an important resource for genetic mapping, population genetics, and monitoring of the genetic integrity of laboratory stocks. We have developed the third generation of the Mouse Universal Genotyping Array (MUGA) series, GigaMUGA, a 143,259-probe Illumina Infinium II array for the house mouse (Mus musculus). The bulk of the content of GigaMUGA is optimized for genetic mapping in the Collaborative Cross and Diversity Outbred populations, and for substrain-level identification of laboratory mice. In addition to 141,090 single nucleotide polymorphism probes, GigaMUGA contains 2006 probes for copy number concentrated in structurally polymorphic regions of the mouse genome. The performance of the array is characterized in a set of 500 high-quality reference samples spanning laboratory inbred strains, recombinant inbred lines, outbred stocks, and wild-caught mice. GigaMUGA is highly informative across a wide range of genetically diverse samples, from laboratory substrains to other Mus species. In addition to describing the content and performance of the array, we provide detailed probe-level annotation and recommendations for quality control.

Analyses of allele-specific gene expression in highly divergent mouse crosses identifies pervasive allelic imbalance.

Complex human traits are influenced by variation in regulatory DNA through mechanisms that are not fully understood. Because regulatory elements are conserved between humans and mice, a thorough annotation of cis regulatory variants in mice could aid in further characterizing these mechanisms. Here we provide a detailed portrait of mouse gene expression across multiple tissues in a three-way diallel. Greater than 80% of mouse genes have cis regulatory variation. Effects from these variants influence complex traits and usually extend to the human ortholog. Further, we estimate that at least one in every thousand SNPs creates a cis regulatory effect. We also observe two types of parent-of-origin effects, including classical imprinting and a new global allelic imbalance in expression favoring the paternal allele. We conclude that, as with humans, pervasive regulatory variation influences complex genetic traits in mice and provide a new resource toward understanding the genetic control of transcription in mammals.

Status and access to the Collaborative Cross population.

The Collaborative Cross (CC) is a panel of recombinant inbred lines derived from eight genetically diverse laboratory inbred strains. Recently, the genetic architecture of the CC population was reported based on the genotype of a single male per line, and other publications reported incompletely inbred CC mice that have been used to map a variety of traits. The three breeding sites, in the US, Israel, and Australia, are actively collaborating to accelerate the inbreeding process through marker-assisted inbreeding and to expedite community access of CC lines deemed to have reached defined thresholds of inbreeding. Plans are now being developed to provide access to this novel genetic reference population through distribution centers. Here we provide a description of the distribution efforts by the University of North Carolina Systems Genetics Core, Tel Aviv University, Israel and the University of Western Australia.

Accelerating the inbreeding of multi-parental recombinant inbred lines generated by sibling matings.

Inbred model organisms are powerful tools for genetic studies because they provide reproducible genomes for use in mapping and genetic manipulation. Generating inbred lines via sibling matings, however, is a costly undertaking that requires many successive generations of breeding, during which time many lines fail. We evaluated several approaches for accelerating inbreeding, including the systematic use of back-crosses and marker-assisted breeder selection, which we contrasted with randomized sib-matings. Using simulations, we explored several alternative breeder-selection methods and monitored the gain and loss of genetic diversity, measured by the number of recombination-induced founder intervals, as a function of generation. For each approach we simulated 100,000 independent lines to estimate distributions of generations to achieve full-fixation as well as to achieve a mean heterozygosity level equal to 20 generations of randomized sib-mating. Our analyses suggest that the number of generations to fully inbred status can be substantially reduced with minimal impact on genetic diversity through combinations of parental backcrossing and marker-assisted inbreeding. Although simulations do not consider all confounding factors underlying the inbreeding process, such as a loss of fecundity, our models suggest many viable alternatives for accelerating the inbreeding process.

High-resolution genetic mapping using the Mouse Diversity outbred population.

The JAX Diversity Outbred population is a new mouse resource derived from partially inbred Collaborative Cross strains and maintained by randomized outcrossing. As such, it segregates the same allelic variants as the Collaborative Cross but embeds these in a distinct population architecture in which each animal has a high degree of heterozygosity and carries a unique combination of alleles. Phenotypic diversity is striking and often divergent from phenotypes seen in the founder strains of the Collaborative Cross. Allele frequencies and recombination density in early generations of Diversity Outbred mice are consistent with expectations based on simulations of the mating design. We describe analytical methods for genetic mapping using this resource and demonstrate the power and high mapping resolution achieved with this population by mapping a serum cholesterol trait to a 2-Mb region on chromosome 3 containing only 11 genes. Analysis of the estimated allele effects in conjunction with complete genome sequence data of the founder strains reduced the pool of candidate polymorphisms to seven SNPs, five of which are located in an intergenic region upstream of the Foxo1 gene.

Reducing health care-associated infections (HAIs): lessons learned from a national collaborative of regional HAI programs.

BACKGROUND: Health care-associated infections (HAIs) are a leading cause of death in United States health care settings, with an overall estimated annual incidence of 1.7 million. As antimicrobial resistance has increased, so too have efforts to reduce HAI rates. The objective of this study was to identify commonly cited lessons learned across a wide variety of HAI projects and hospital settings. METHODS: Thirty-three hospitals participated in 5 different regional collaboratives supported by the Agency for Healthcare Research and Quality (AHRQ). Data on hospitals' successes, challenges, and lessons learned were collected via key informant interviews and structured, standardized case report forms. RESULTS: Seven commonly cited themes were identified: foster change by first understanding resistance; commit to regular strategic communication and join a collaborative; start small and tailor implementation to local needs and cultures; engage frontline staff by involving them in the project and enlisting champions; educate and reeducate; convince administration to provide leadership, funds, and dedicated staff and assign accountability; and provide timely, relevant feedback and celebrate successes. CONCLUSION: Despite the diversity of hospital settings, cultures, personnel, and HAI reduction projects, we found that hospitals encounter similar challenges and facilitators across projects. We offer a model of 7 process elements shown to be important to successful implementation.

Implementing the MRSA bundle in ICUs: one citywide collaborative's key lessons learned.

Because bundle implementation in intensive care units to reduce methicillin-resistant Staphylococcus aureus is challenging, we conducted in-depth interviews with implementation teams at 5 participating hospitals. Key lessons learned across hospitals included the following: maintain management support, engage frontline staff, build the right multidisciplinary team, conduct process mapping, and commit to data collection and feedback.

A national collaborative for reducing health care‒associated infections: current initiatives, challenges, and opportunities.

There has been increasing interest and an upsurge in efforts to prevent hospital-associated infections (HAIs), a leading cause of death in the United States. This study was conducted to assess current strategies and efforts of HAI reduction initiatives in hospitals. HAI reduction initiatives and factors influencing institutional participation in these initiatives were categorized. Data were collected via open-ended questions on surveys performed in 5 different HAI collaboratives. Thematic analysis of the coded qualitative data was conducted. A total of 1,212 health care professionals from 33 different hospitals participated. Improving hand hygiene was the most frequently mentioned HAI reduction initiative implemented in the previous year. Initiatives for reducing central line or central venous catheter infections and ventilator-associated pneumonia also were commonly cited. The most frequently mentioned challenges to implementing HAI reduction initiatives included poor adherence, insufficient resources, staffing problems, lack of culture change, no impetus to change, and issues related to staff and patient education. Many respondents identified engaging physicians as particularly challenging.These findings suggest that consistently improving hand hygiene remains a widespread problem for reducing HAIs and sustaining this type of behavioral change is difficult. Furthermore, ensuring staff and physician engagement and compliance in HAI reduction efforts remains challenging for most hospitals.

Subspecific origin and haplotype diversity in the laboratory mouse.

Here we provide a genome-wide, high-resolution map of the phylogenetic origin of the genome of most extant laboratory mouse inbred strains. Our analysis is based on the genotypes of wild-caught mice from three subspecies of Mus musculus. We show that classical laboratory strains are derived from a few fancy mice with limited haplotype diversity. Their genomes are overwhelmingly Mus musculus domesticus in origin, and the remainder is mostly of Japanese origin. We generated genome-wide haplotype maps based on identity by descent from fancy mice and show that classical inbred strains have limited and non-randomly distributed genetic diversity. In contrast, wild-derived laboratory strains represent a broad sampling of diversity within M. musculus. Intersubspecific introgression is pervasive in these strains, and contamination by laboratory stocks has played a role in this process. The subspecific origin, haplotype diversity and identity by descent maps can be visualized using the Mouse Phylogeny Viewer (see URLs).

Final results of a phase II study of nab-paclitaxel, bevacizumab, and gemcitabine as first-line therapy for patients with HER2-negative metastatic breast cancer.

In order to examine the efficacy and safety of nanoparticle albumin-bound paclitaxel (nab-P) in combination with bevacizumab (B) and gemcitabine (G) for the first-line treatment of patients with HER2-negative metastatic breast cancer (MBC). In this single-center, open-label phase II trial, patients with HER2-negative MBC received gemcitabine 1500 mg/m(2), nab-paclitaxel 150 mg/m(2), and bevacizumab 10 mg/kg (each administered intravenously) on days 1 and 15 of a 28-day cycle. The primary end point was progression free survival (PFS); secondary end points were overall response rate (ORR), complete (CR) and partial (PR) response rates, clinical benefit (ORR + stable disease), overall survival (OS), and safety. Thirty patients were enrolled. One patient was ineligible and was not included in analysis. Median PFS was 10.4 months (95% CI: 5.6-15.2 months). ORR was 75.9%, comprising eight (27.6%) CRs and 14 (48.3%) PRs; five patients had stable disease (SD) and two patients (6.9%) had progressive disease (PD) as their best response. The clinical benefit rate was 93.1% (27/29) in the overall group and 84.6% in the triple-negative cohort (11/13). The 18-month survival rate was 77.2% (95% CI: 51.1-90.5%). Eight (27.6%) patients experienced grade 3 or 4 toxicity: grade 4 neutropenic fever (n = 1) and grade 3 infection (n = 6), leukopenia, thrombocytopenia, peripheral neuropathy, seizure, shortness of breath, hematuria, and cardiac tamponade (one each). First-line therapy with nab-P, B, and G demonstrated a median PFS of 10.4 months and a 75.9% ORR with acceptable toxicity; this novel combination warrants investigation in a randomized study.

Barriers and facilitators to nursing handoffs: Recommendations for redesign.

During a handoff, communication errors can lead to adverse events and suboptimal patient care. As a result, many institutions want to redesign their handoff processes, but have little specific guidance from the literature. We examined two approaches to nursing end-of-shift reports both taped and written, to identify specific factors limiting and facilitating such handoffs. Twenty nurses were interviewed using a semistructured format. They were asked about the current reporting process, the limitations, the elements that helped, and ideas for improvement. Analyses revealed that inadequate information, inconsistent quality, limited opportunity to ask questions, equipment malfunction, insufficient time to generate reports, and interruptions, limited handoffs. Facilitators were "pertinent" content, notes and space for notes, face-to-face interaction, and structured form/checklist. Recommendations for redesign are defining content pertinent to the unit, structuring handoffs so that information is received in a standard way, embedding an opportunity for questions into the process, planning for all 3 handoff subprocesses, and conducting peer evaluations and education.

A Phase II trial of split, low-dose docetaxel and low-dose capecitabine: a tolerable and efficacious regimen in the first-line treatment of patients with HER2/neu-negative metastatic breast cancer.

BACKGROUND: Successful therapeutic regimens in metastatic breast cancer (MBC) must balance efficacy and tolerability. Docetaxel/capecitabine is an active and commonly used doublet in this setting. Docetaxel upregulates thymidine phosphorylase and thus potentiates the antitumor effects of capecitabine. A schedule with split, low-dose docetaxel in combination with low-dose capecitabine could improve the therapeutic index of this regimen without compromising its clinical activity. PATIENTS AND METHODS: Patients with previously untreated HER2/neu-negative MBC were eligible. Treatment consisted of docetaxel 25 mg/m2 on days 1 and 8 in combination with capecitabine 750 mg/m2 twice daily on days 1-14 of a 3-week cycle. Thirty-nine women were enrolled. Median age was 55 years (range, 36-75 years). Fourteen patients had triple-negative disease. Sites of metastasis were as follows: bone (n = 27); liver (n = 15); lung (n = 17); nonregional chest (n = 4); lymph nodes (n = 2); and skin (n = 1). Six patients had bone-only disease. All subjects had a performance status of 0/1. A total of 329 cycles were administered (median, 6; range, 1-50). RESULTS: Of 37 patients who received study treatment, 32 had evaluable disease, 1 had a complete response, and 15 had a partial response (overall response rate was 50% in evaluable patients and 43% in the intent-to-treat analysis). Six patients had stable disease. The overall clinical benefit rate was 69% for patients with evaluable disease and 60% overall. Fifteen patients had disease that progressed or had been withdrawn from study at the time of first evaluation. With a median follow-up of 25 months, median time to treatment failure was 4.25 months (95% CI, 1.5-7 months), and median overall survival has not yet been reached. Toxicity was moderate: 15 patients (41%) had grade 3/4 adverse events. CONCLUSION: Split, low-dose docetaxel and low-dose capecitabine is an active combination in the first-line treatment of patients with MBC. Toxicity with this schedule was manageable, making it an attractive regimen for further study in combination with targeted agents.

Methodology for preservation of high molecular-weight RNA in paraffin-embedded tissue: application for laser-capture microdissection.

Laser-capture microdissection techniques have enhanced the ability to perform molecular studies of pure-cell populations. Although many technical factors affect the outcome of the procedure, none is more critical than the appropriate handling of the tissue. Because extraction of intact RNA from paraffin-embedded tissue is a difficult and inconsistent process, frozen sections with their attendant problems are used for this purpose. The major limitation of frozen section is its inferior morphologic quality compared with paraffin-embedded sections that may complicate accurate identification of cells during microdissection. We have developed a procedure that provides both high-quality histomorphology and RNA preservation in paraffin-embedded tissue. It is based on the use of a methanol-based fixative coupled with microwave-assisted rapid tissue processing. This technology in conjunction with a modified hematoxylin-eosin stain and a RNA extraction method allows isolation of high molecular-weight RNA from laser-capture microdissected, hematoxylin and eosin-stained paraffin sections. The high quality of the extracted RNA was confirmed by capillary electrophoresis and RT-PCR. The combination of a methanol-based fixative, rapid microwave tissue processing, and a modified hematoxylin and eosin stain produces paraffin sections that yield high molecular-weight RNA upon microdissection. This methodology opens the door for a wide range of gene expression analyses using paraffin-embedded tissue.

Weekly docetaxel/carboplatin as primary systemic therapy for HER2-negative locally advanced breast cancer.

To evaluate the effectiveness and safety of weekly docetaxel/carboplatin as primary systemic therapy (PST) for locally advanced breast cancer, we conducted a phase II study. Forty-four patients with HER2-negative locally advanced or inflammatory breast cancer (IBC) received docetaxel 35 mg/m(2) and carboplatin to an area under the curve of 2 mg/mL/min for 3 of 4 weeks over 16 weeks. After completion of PST, patients had breast surgery and then received 4 cycles of adjuvant cyclophosphamide/doxorubicin, standard radiation therapy, and, for hormone receptorpositive tumors, tamoxifen. The mean tumor size was 9.3 cm (range, 5-24 cm). Thirty-seven patients (85%) had palpable lymph nodes; 13 patients (30%) had matted or fixed nodes (N2). Eight patients had IBC. There were 11 clinical complete responses (25%) and 29 clinical partial responses (66%), resulting in 40 objective responses (91% [95% CI, 78%-96%]). Invasive disease disappeared (pathologic complete response) from the breast and axilla in 6 patients (14% [95% CI, 5%-27%]) and from the axilla in 17 patients (39% [95% CI, 24%-55%]). The only significant adverse hematologic event was grade 3 neutropenia in 4 patients (9%). The most common adverse nonhematologic events were fatigue (84% of patients) and alopecia (84%), which were usually grade 1/2. Weekly docetaxel/carboplatin appears to be active and feasible as PST in patients with large breast tumors.

Deregulation of the Rho GTPase, Rac1, suppresses cyclin-dependent kinase inhibitor p21(CIP1) levels in androgen-independent human prostate cancer cells.

Abnormally suppressed levels of cyclin-dependent kinase inhibitors (CKIs) are associated with aggressive androgen-independent prostate cancer and contribute to uncontrolled proliferation. The androgen-independent human prostate cancer cell lines, LNCaP-104R1, ALVA31 and PC-3, express low levels of the CKI, p21(CIP1), compared to the less-malignant, androgen-dependent LNCaP cells. We investigated the mechanism underlying this suppression by examining the role of Rho GTPases, signaling proteins that play important roles in cell cycle progression, at least in part through regulation of CKIs. Inhibition of Rac1 induced p21 expression in androgen-independent lines but had no effect on the higher p21 levels characteristic of LNCaP cells. This induction of p21 was functionally significant as evidenced by inhibition of cyclin-dependent kinase 2 activity and decreased cell proliferation. Conversely, overexpression of constitutively active Rac1 suppressed the higher p21 levels seen in LNCaP cells. Thus, Rac1 activity is both necessary and sufficient for suppression of p21 in prostate cancer cells. Furthermore, Rac1 activity was significantly higher in all three androgen-independent cell lines compared to LNCaP cells. Thus in three models of aggressive human prostate cancer, hyperactivity of Rac1 corresponds to suppressed levels of p21. These results are unique in describing a role for Rac1 in p21 regulation and may implicate the Rac1 signaling pathway as a potential therapeutic target for controlling prostate cancer cell growth following progression to androgen independence.

Rho GTPases as key transducers of proliferative signals in g1 cell cycle regulation.

Mitogenic growth factor- and integrin-dependent signaling pathways cooperate to control the proliferation of nontransformed cells. As integral mediators of these networks, the Rho family of GTPases play a pivotal role in G1 cell cycle progression, primarily through regulation of cyclin D1 expression, as well as the levels of the cyclin-dependent kinase inhibitors p21cip1 and p27kip1. Such dual control of both the critical positive and negative regulators of G1 progression make the Rho GTPases prime candidates to target the autonomous proliferation which typifies cancer cells. Cyclin D1 has been identified as an important oncogene and the cdk inhibitors as tumor suppressors in human breast carcinogenesis. Evidence pointing to the potential role of Rho-dependent pathways and their interaction with oncogenic Ras in contributing to such cell cycle abnormalities that characterize human breast cancer is also presented.