Characterization of chelator-mediated recovery of pancreatic islets from barium-stabilized alginate microcapsules.

INTRODUCTION: Islet recovery from within alginate-based microcapsules is necessary for certain analytical assays like flow cytometry; however, this technology has not been widely characterized. In this study, we explore the ability of EDTA, EGTA, and sodium citrate to induce reverse alginate polymerization via chelation and assess the toxicity of each chelator on pancreatic islets. METHODS: EDTA, EGTA, and sodium citrate were used to dissolve single-layered Ba2+ alginate encapsulated islets and the rate of capsule breakdown calculated from analysis of imaging data. The effect of chelator exposure on islet viability and recovery was assessed using flow cytometry, while glucose-stimulated insulin release (GSIR) assay was used to measure effects on islet function. RESULTS: EGTA demonstrated the most rapid microcapsule dissolving rate followed by EDTA and sodium citrate. Islet recovery was significantly better when encapsulated islets were treated with EDTA than EGTA and Na+ citrate. A decrease in viability and increase in apoptotic cells were observed when encapsulated islets were treated with Na+ citrate compared to islets treated with EDTA and EGTA. Islets treated with EDTA and EGTA demonstrated comparable stimulation index values to non-treated control. Conversely, islets treated with Na+ citrate exhibited significantly decreased SI values compared to control. All chelator groups showed significantly lower insulin secretion than non-treated islets. CONCLUSION: Islet recovery from alginate microcapsule is possible using common chelators like Na+ citrate, EDTA, and EGTA. Chelation of encapsulated islets using EDTA demonstrated the most efficient dissolving capabilities with the least toxicity toward islet recovery and health.

Assigning clinical meaning to somatic and germ-line whole-exome sequencing data in a prospective cancer precision medicine study.

PURPOSE: Implementing cancer precision medicine in the clinic requires assessing the therapeutic relevance of genomic alterations. A main challenge is the systematic interpretation of whole-exome sequencing (WES) data for clinical care. METHODS: One hundred sixty-five adults with metastatic colorectal and lung adenocarcinomas were prospectively enrolled in the CanSeq study. WES was performed on DNA extracted from formalin-fixed paraffin-embedded tumor biopsy samples and matched blood samples. Somatic and germ-line alterations were ranked according to therapeutic or clinical relevance. Results were interpreted using an integrated somatic and germ-line framework and returned in accordance with patient preferences. RESULTS: At the time of this analysis, WES had been performed and results returned to the clinical team for 165 participants. Of 768 curated somatic alterations, only 31% were associated with clinical evidence and 69% with preclinical or inferential evidence. Of 806 curated germ-line variants, 5% were clinically relevant and 56% were classified as variants of unknown significance. The variant review and decision-making processes were effective when the process was changed from that of a Molecular Tumor Board to a protocol-based approach. CONCLUSION: The development of novel interpretive and decision-support tools that draw from scientific and clinical evidence will be crucial for the success of cancer precision medicine in WES studies.Genet Med advance online publication 26 January 2017.

Gut resident Escherichia coli profile predicts the eighteen-month probability and antimicrobial susceptibility of urinary tract infections.

Background: Community-acquired UTI is the most common bacterial infection managed in general medical practice that can lead to life-threatening outcomes. While UTIs are primarily caused by Escherichia coli colonizing the patient's gut, it is unclear whether the gut resident E. coli profiles can predict the person's risks for UTI and optimal antimicrobial treatments. Thus, we conducted an eighteen-month long community-based observational study of fecal E. coli colonization and UTI in women aged 50 years and above. Methods and Findings: We enrolled a total of 1,804 women distributed among age groups 50-59 yo (437 participants), 60-69 yo (632), 70-79 yo (532), and above 80 yo (203), lacking antibiotic prescriptions for at least one year. The provided fecal samples were plated for the presence of E. coli and other enterobacteria resistant to trimethoprim/sulfamethoxazole (TMP/STX), ciprofloxacin (CIP) and 3rd generation cephalosporins (3GC). E. coli was also characterized as belonging to the pandemic multi-drug resistant clonal groups ST131 (subclone H30) and ST1193. Following sample collection, the women were monitored for 18 months for occurrence of UTI.E. coli was cultured from 90.8% fecal samples, with 24.1% containing bacteria resistant to TMP/STX, 19.4% to CIP, and 7.9% to 3GC. In 62.5% samples, only all-susceptible E. coli were present. Overall, there were no age-related differences in resistance prevalence. However, while the total E. coli H30 and ST1193 carriage rates were similar (4.3% and 4.2%, respectively), there was a notable increase of H30 carriage with age (P = .001), while carriage decreased with age for ST1193 (P = .057).Within 18 months, 184 women (10.2%) experienced at least one episode of UTI - 10.9% among the gut E. coli carriers and 3.0% among the non-carriers (P=.0013). The UTI risk among carriers of E. coli H30 but not ST1193 was significantly above average (24.3%, P = .0004). The UTI probability increased with age, occurring in 6.4% of 50-59 yo and 19.7% of 80+ yo (P<.001), with the latter group being especially at high risk for UTI, if they were colonized by E. coli H30 (40.0%, P<.001).E. coli was identified in 88.1% of urine samples, with 16.1% resistant to TMP/STX, 16.1% to CIP, 4.2% to 3GC and 73.1% to none of the antibiotics. Among tested urinary E. coli resistant to antibiotics, 86.1% matched the resistance profile of E. coli in the fecal samples, with the clonotyping and whole genome sequencing confirming the matching strains' identity. Positive predictive value (PPV) of using gut resistance profiles to predict UTI pathogens' susceptibility to TMP/STX, CIP, 3GC and all three antibiotics were 98.4%, 98.3%, 96.6% and 95.3%, respectively. Corresponding negative predictive values (NPV) were 63.0%, 54.8%, 44.4% and 75.8%, respectively. The AUC ROC curve values for the accuracy of fecal diagnostic testing for the prediction of UTI resistance ranged .86-.89. The fecal test-guided drug-bug mismatch rate for empirical (pre-culture) prescription of TMP-SXT or CIP is reduced to ≤2% in 89.6% of patients and 94.8% of patients with an optional 3GC prescription. Conclusion: The resistance profile and clonal identity of gut colonizing E. coli, along with the carrier's age, can inform personalized prediction of a patients' UTI risk and the UTI pathogen's antibiotic susceptibility within an 18-month period.

What are the barriers and facilitators to community handwashing with water and soap? A systematic review.

Handwashing with water and soap (HWWS) is an effective method of cleaning and disinfecting the surface of the hands. HWWS is effective in infection control and prevention transmission, such as in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). However, rates of handwashing compliance vary globally. This systematic review aimed to identify the barriers and facilitators to community HWWS globally. We conducted a comprehensive search strategy in OVID Medline, OVID Embase, Web of Science Core Collection, and Scopus using keywords and subject headings related to handwashing. Studies were excluded if they reported hand hygiene among healthcare or food service workers, considered the use of alcohol rubs, or involved an intervention in a healthcare or food preparation setting. The quality of eligible studies was assessed using the Mixed Methods Appraisal Tool, and data were extracted from the articles and analyzed using the Theoretical Domains Framework and inductive thematic analysis. The search strategy yielded a total of 11,696 studies, and 46 studies met the eligibility criteria. Study dates ranged from 2003 to 2020 and included 26 countries; the most frequently represented were Bangladesh, India, and Kenya. A total of 21 barriers and 23 facilitators to HWWS were identified and organized into the Theoretical Domains Framework. The most frequently cited domains were environmental context and resources, goals, and knowledge. Nine themes emerged from these barriers and facilitators: resource availability, cost and affordability, handwash station design and infrastructure, accessibility, gender roles, champions, health promotion, time management, and knowledge, beliefs, and behaviors. This review uncovered multiple barriers and facilitators around a determinant framework to observe and create an in-depth, multidimensional image of a community-based hand hygiene situation. New comprehensive interventions and implementation strategies can be developed using the findings to target the contextual barriers and facilitators to improve and increase HWWS rates. Stakeholders (i.e., practitioners, researchers, policymakers) can use the findings to revise, design, or evaluate new or existing projects, interventions, and policies to improve HWWS. Registration: A protocol for this systematic review was developed and uploaded onto the PROSPERO-International prospective register of systematic reviews database (Registration number: CRD42020221210).

Increase in the Rate of Gut Carriage of Fluoroquinolone-Resistant Escherichia coli despite a Reduction in Antibiotic Prescriptions.

Background : Fluoroquinolone use for urinary tract infections has been steadily declining. Gut microbiota is the main reservoir for uropathogenic Escherichia coli but whether the carriage of fluoroquinolone-resistant E. coli has been changing is unknown. Methods . We determined the frequency of isolation and other characteristics of E. coli nonsuceptible to fluoroquinolones (at ³0.5 mg/L of ciprofloxacin) in 515 and 1605 E. coli -positive fecal samples collected in 2015 and 2021, respectively, from non-antibiotic- taking women of age 50+ receiving care in the Seattle area Kaiser Permanente Washington healthcare system. Results . Between 2015 and 2021 the prescription of fluoroquinolones dropped nearly three-fold in the study population. During the same period, the rates of gut carriage of fluoroquinolone-resistant E. coli increased from 14.4 % to 19.9% (P=.005), driven by a significant increase of isolates from the recently emerged, pandemic multi-drug resistant clonal group ST1193 (1.7% to 4.3%; P=.007) and those with an incomplete set of or no fluoroquinolone-resistance determining mutations (2.3% to 7.5%; P<.001). While prevalence of the resistance-associated mobile genes among the isolates dropped from 64.1% to 32.6% (P<.001), co-resistance to third generation cephalosporins has increased 21.5% to 33.1%, P=.044). Conclusion . Despite reduction in fluoroquinolone prescriptions, gut carriage of fluoroquinolone-resistant uropathogenic E. coli increased with a rise of previously sporadic lineages and co-resistance to third generation cephalosporins. Thus, to reduce the rates of antibiotic resistant urinary tract infections, greater focus should be on controlling the gut carriage of resistant bacteria.

Increase in the community circulation of ciprofloxacin-resistant Escherichia coli despite reduction in antibiotic prescriptions.

BACKGROUND: Community circulating gut microbiota is the main reservoir for uropathogenic Escherichia coli, including those resistant to antibiotics. Ciprofloxacin had been the primary antibiotic prescribed for urinary tract infections, but its broad use has been discouraged and steadily declined since 2015. How this change in prescriptions affected the community circulation of ciprofloxacin-resistant E. coli is unknown. METHODS: We determined the frequency of isolation and other characteristics of E. coli resistant to ciprofloxacin in 515 and 1604 E. coli-positive fecal samples collected in 2015 and 2021, respectively. The samples were obtained from non-antibiotic-taking women of age 50+ receiving care in the Kaiser Permanente Washington healthcare system. RESULTS: Here we show that despite a nearly three-fold drop in the prescription of ciprofloxacin between 2015 and 2021, the rates of gut carriage of ciprofloxacin-resistant E. coli increased from 14.2 % to 19.8% (P = .004). This is driven by a significant increase of isolates from the pandemic multi-drug resistant clonal group ST1193 (1.7% to 4.2%; P = .009) and isolates with relatively few ciprofloxacin-resistance determining chromosomal mutations (2.3% to 7.4%; P = .00003). Though prevalence of isolates with the plasmid-associated ciprofloxacin resistance dropped (59.0% to 30.9%; P = 2.7E-06), the isolates co-resistance to third generation cephalosporins has increased from 14.1% to 31.5% (P = .002). CONCLUSIONS: Despite reduction in ciprofloxacin prescriptions, community circulation of the resistant uropathogenic E. coli increased with a rise of co-resistance to third generation cephalosporins. Thus, to reduce the rates of urinary tract infections refractory to antibiotic treatment, greater focus should be on controlling the resistant bacteria in gut microbiota.

The alarming rise of bacteria causing infections that are difficult to treat with antibiotics, known as multidrug-resistant bacteria, is a major problem in medicine. The reduction in the use of antibiotics has been encouraged to control the spread of antibiotic-resistant bacteria. Some multidrug-resistant bacteria reside in the gut of healthy individuals and can cause various forms of urinary tract infections (UTIs). Ciprofloxacin is an antibiotic that was widely used to treat UTIs, but strong recommendations to reduce its prescription have been recently introduced. We compared the presence of bacteria in the gut that could not be killed by ciprofloxacin in women aged 50 and above who do not use antibiotics and reside in the Seattle area. Despite a nearly three-fold drop in the prescription of ciprofloxacin between 2015 and 2021, antibiotic-resistant bacteria in the gut were found more frequently, affecting one in five women. Our study demonstrates that antibiotic-resistant bacteria continue to be present even when antibiotic prescriptions are reduced, demonstrating the need to undertake further similar studies.

Oncogenic switch and single-agent MET inhibitor sensitivity in a subset of EGFR-mutant lung cancer.

The clinical efficacy of epidermal growth factor receptor (EGFR)­targeted therapy in EGFR-mutant non­small cell lung cancer is limited by the development of drug resistance. One mechanism of EGFR inhibitor resistance occurs through amplification of the human growth factor receptor (MET) proto-oncogene, which bypasses EGFR to reactivate downstream signaling. Tumors exhibiting concurrent EGFR mutation and MET amplification are historically thought to be codependent on the activation of both oncogenes. Hence, patients whose tumors harbor both alterations are commonly treated with a combination of EGFR and MET tyrosine kinase inhibitors (TKIs). Here, we identify and characterize six patient-derived models of EGFR-mutant, MET-amplified lung cancer that have switched oncogene dependence to rely exclusively on MET activation for survival. We demonstrate in this MET-driven subset of EGFR TKI-refractory cancers that canonical EGFR downstream signaling was governed by MET, even in the presence of sustained mutant EGFR expression and activation. In these models, combined EGFR and MET inhibition did not result in greater efficacy in vitro or in vivo compared to single-agent MET inhibition. We further identified a reduced EGFR:MET mRNA expression stoichiometry as associated with MET oncogene dependence and single-agent MET TKI sensitivity. Tumors from 10 of 11 EGFR inhibitor­resistant EGFR-mutant, MET-amplified patients also exhibited a reduced EGFR:MET mRNA ratio. Our findings reveal that a subset of EGFR-mutant, MET-amplified lung cancers develop dependence on MET activation alone, suggesting that such patients could be treated with a single-agent MET TKI rather than the current standard-of-care EGFR and MET inhibitor combination regimens.

Optimal Time to Ship Human Islets Post Tissue Culture to Maximize Islet.

Access to functional high-quality pancreatic human islets is critical to advance diabetes research. The Integrated Islet Distribution Program (IIDP), a major source for human islet distribution for over 15 years, conducted a study to evaluate the most advantageous times to ship islets postisolation to maximize islet recovery. For the evaluation, three experienced IIDP Islet Isolation Centers each provided samples from five human islet isolations, shipping 10,000 islet equivalents (IEQ) at four different time periods postislet isolation (no 37°C culture and shipped within 0 to 18 hours; or held in 37°C culture for 18 to 42, 48 to 96, or 144 to 192 hours). A central evaluation center compared samples for islet quantity, quality, and viability for each experimental condition preshipment and postshipment, as well as post 37°C culture 18 to 24 hours after shipment receipt. Additional evaluations included measures of functional potency by static glucose-stimulated insulin release (GSIR), represented as a stimulation index. Comparing the results of the four preshipment holding periods, the greatest IEQ loss postshipment occurred with the shortest preshipment times. Similar patterns emerged when comparing preshipment to postculture losses. In vitro islet function (GSIR) was not adversely impacted by increased tissue culture time. These data indicate that allowing time for islet recovery postisolation, prior to shipping, yields less islet loss during shipment without decreasing islet function.

Discrimination of Germline EGFR T790M Mutations in Plasma Cell-Free DNA Allows Study of Prevalence Across 31,414 Cancer Patients.

Purpose: Plasma cell-free DNA (cfDNA) analysis is increasingly used clinically for cancer genotyping, but may lead to incidental identification of germline-risk alleles. We studied EGFR T790M mutations in non-small cell lung cancer (NSCLC) toward the aim of discriminating germline and cancer-derived variants within cfDNA.Experimental Design: Patients with EGFR-mutant NSCLC, some with known germline EGFR T790M, underwent plasma genotyping. Separately, deidentified genomic data and buffy coat specimens from a clinical plasma next-generation sequencing (NGS) laboratory were reviewed and tested.Results: In patients with germline T790M mutations, the T790M allelic fraction (AF) in cfDNA approximates 50%, higher than that of EGFR driver mutations. Review of plasma NGS results reveals three groups of variants: a low-AF tumor group, a heterozygous group (∼50% AF), and a homozygous group (∼100% AF). As the EGFR driver mutation AF increases, the distribution of the heterozygous group changes, suggesting increased copy number variation from increased tumor content. Excluding cases with high copy number variation, mutations can be differentiated into somatic variants and incidentally identified germline variants. We then developed a bioinformatic algorithm to distinguish germline and somatic mutations; blinded validation in 21 cases confirmed a 100% positive predictive value for predicting germline T790M. Querying a database of 31,414 patients with plasma NGS, we identified 48 with germline T790M, 43 with nonsquamous NSCLC (P < 0.0001).Conclusions: With appropriate bioinformatics, plasma genotyping can accurately predict the presence of incidentally detected germline risk alleles. This finding in patients indicates a need for genetic counseling and confirmatory germline testing. Clin Cancer Res; 23(23); 7351-9. ©2017 AACR.

Association Between Younger Age and Targetable Genomic Alterations and Prognosis in Non-Small-Cell Lung Cancer.

IMPORTANCE: Non-small-cell lung cancer (NSCLC) diagnosed in young patients is rare, and the genomics and clinical characteristics of this disease are poorly understood. In contrast, the diagnosis of other cancers at a young age has been demonstrated to define unique disease biology. Herein, we report on the association of young age with targetable genomic alterations and prognosis in a cohort of 2237 patients with NSCLC. OBJECTIVE: To determine the relationship between young age at diagnosis and the presence of a potentially targetable genomic alteration, disease prognosis, and natural history. DESIGN, SETTING, AND PARTICIPANTS: A cohort of all 2237 patients with NSCLC who were genotyped at the Dana-Farber Cancer Institute between January 2002 and December 2014 were identified. Tumor genotype, patient characteristics, and clinical outcomes were collected and studied at a National Cancer Institute-designated comprehensive cancer center. Multivariate logistic regression was used to analyze the relationship between age and mutation status, and multivariate Cox proportional hazard models were fitted for survival analysis. MAIN OUTCOMES AND MEASURES: The frequency of targetable genomic alterations by defined age categories as well as the association of these age groups with survival. Age categories used in this analysis were younger than 40, 40 to 49, 50 to 59, 60 to 69, and 70 years or older. RESULTS: A cohort of 2237 patients with NSCLC was studied. Of the 2237 participants, 1939 (87%) had histologically confirmed adenocarcinoma, 269 (12%) had NSCLC not otherwise specified, and 29 (1%) had squamous histologic findings; 1396 (63%) had either stage IIIB or IV cancers; and median (range) age was 62 (20-95) years. We found that gene mutations for EGFR (P = .02) and ALK (P < .001) were associated with cancer diagnosis at a younger age, and a similar trend existed for ERBB2 (P = .15) and ROS1 (P = .10) but not BRAF V600E (P = .43). Among patients tested for all 5 targetable genomic alterations (n = 1325), younger age was associated with an increased frequency of a targetable genotype (P < .001). Those diagnosed at 50 years or younger have a 59% increased likelihood of harboring a targetable genotype. While presence of a potentially targetable genomic alteration treated with a targeted agent was associated with improved survival, the youngest and oldest age groups had similarly poor outcomes even when a targetable genotype was present. CONCLUSIONS AND RELEVANCE: Younger age is associated with an increased likelihood of harboring a targetable genotype and is an underappreciated clinical biomarker in NSCLC. The survival of young patients with NCSLC is unexpectedly poor compared with other age groups, suggesting more aggressive disease biology. These findings underscore the importance of comprehensive genotyping, including next-generation sequencing, in younger patients with lung cancer.

MET Exon 14 Mutations in Non-Small-Cell Lung Cancer Are Associated With Advanced Age and Stage-Dependent MET Genomic Amplification and c-Met Overexpression.

PURPOSE: Non-small-cell lung cancers (NSCLCs) harboring mutations in MET exon 14 and its flanking introns may respond to c-Met inhibitors. We sought to describe the clinical, pathologic, and genomic characteristics of patients with cancer with MET exon 14 mutations. PATIENTS AND METHODS: We interrogated next-generation sequencing results from 6,376 cancers to identify those harboring MET exon 14 mutations. Clinical characteristics of MET exon 14 mutated NSCLCs were compared with those of NSCLCs with activating mutations in KRAS and EGFR. Co-occurring genomic mutations and copy number alterations were identified. c-Met immunohistochemistry and real-time polymerase chain reaction to detect exon 14 skipping were performed where sufficient tissue was available. RESULTS: MET exon 14 mutations were identified in 28 of 933 nonsquamous NSCLCs (3.0%) and were not seen in other cancer types in this study. Patients with MET exon 14-mutated NSCLC were significantly older (median age, 72.5 years) than patients with EGFR-mutant (median age, 61 years; P < .001) or KRAS-mutant NSCLC (median age, 65 years; P < .001). Among patients with MET exon 14 mutations, 68% were women, and 36% were never-smokers. Stage IV MET exon 14-mutated NSCLCs were significantly more likely to have concurrent MET genomic amplification (mean ratio of MET to chromosome 7, 4.3) and strong c-Met immunohistochemical expression (mean H score, 253) than stage IA to IIIB MET exon 14-mutated NSCLCs (mean ratio of MET to chromosome 7, 1.4; P = .007; mean H score, 155; P = .002) and stage IV MET exon 14-wild-type NSCLCs (mean ratio of MET to chromosome 7, 1.2; P < .001; mean H score, 142; P < .001). A patient whose lung cancer harbored a MET exon 14 mutation with concurrent genomic amplification of the mutated MET allele experienced a major partial response to the c-Met inhibitor crizotinib. CONCLUSION: MET exon 14 mutations represent a clinically unique molecular subtype of NSCLC. Prospective clinical trials with c-Met inhibitors will be necessary to validate MET exon 14 mutations as an important therapeutic target in NSCLC.

Clinical Implications of Variant ALK FISH Rearrangement Patterns.

INTRODUCTION: Break-apart fluorescence in situ hybridization (FISH) is the FDA-approved assay for detecting anaplastic lymphoma kinase (ALK) rearrangements in non-small-cell lung cancer (NSCLC), identifying patients who can gain dramatic benefit from ALK kinase inhibitors. Assay interpretation can be technically challenging, and either splitting of the 5' and 3' probes or loss of the 5' probe constitute rearrangement. We hypothesized that there may be clinical differences depending on rearrangement pattern on FISH. METHODS: An IRB-approved database of NSCLC patients at Dana-Farber Cancer Institute was queried for ALK rearrangement. Clinical characteristics and response to crizotinib were reviewed. Immunohistochemistry (IHC) and targeted next-generation sequencing (NGS) were obtained when available. RESULTS: Of 1614 NSCLC patients with ALK testing, 82 patients (5.1%) had ALK rearrangement by FISH: 30 patients with split signals, 25 patients with 5' deletion, and 27 patients with details unavailable. Patients with 5' deletion were older (p = 0.01) and tended to have more extensive smoking histories (p = 0.08). IHC was positive for ALK rearrangement in all 27 patients with FISH split signals, whereas three of 21 patients with FISH 5' deletion had negative IHC (p = 0.05). Targeted NGS on two of three cases with discordant FISH and IHC results did not identify ALK rearrangement, instead finding driver mutations in EGFR and KRAS. Patients with 5' deletion treated with crizotinib had a smaller magnitude of tumor response (p = 0.03). CONCLUSIONS: Patients with 5' deletion on ALK FISH harbor features less typical of ALK-rearranged tumors, potentially indicating that some cases with this variant are false positives. Corroborative testing with IHC or NGS may be beneficial.