Evaluation of an interprofessional care coordination model: Benefits to health professions students and the community served.

BACKGROUND/PURPOSE: An innovative care coordination program was developed to enhance wellness among low-income older adults living in subsidized apartment buildings and to provide rich interprofessional education experiences for health professions students. METHODS: Program effectiveness for the residents was measured through an evaluation of participation, services used, and healthcare utilization. Educational effectiveness was measured through a change in health concepts and perceptions of interprofessional practice. FINDINGS: Health care utilization among participating residents showed an 8.6% reduction in emergency department visits and 9.8% reduction in hospital admissions. Students demonstrated improved knowledge in motivational interviewing (p = .02); diabetes (p = .02); hypertension (p≤.01); and frailty (p≤.01). Changes in students perception of interprofessional practice were significant in two areas; Teamwork and Collaboration (p≥.00); and Person Centeredness (p = .00). DISCUSSION: This care coordination model may be an effective approach to reduce care resource utilization among medically complex lower income older adults and provides a rich interprofessional learning experience for students.

Mitotically Active Nevus and Nevoid Melanoma: A Clinicopathological and Molecular Study.

ABSTRACT: The distinction between nevoid melanoma and a mitotically active nevus can be challenging at the microscopic level. In this study, we performed cytogenetic testing on a cohort of 25 mitotically active melanocytic proliferations resembling common melanocytic nevus from 25 patients. Based on cytogenetic findings, the lesions were classified as "nevoid melanoma" (n = 13) or "mitotically active nevus" (n = 12). Subsequently, we compared the clinicopathological features between these 2 groups. Nevoid melanomas occurred in older patients (P = 0.007); however, there were no significant differences in gender, size, or anatomical distribution between the 2 groups. Histologically, deep/marginal mitoses (P = 0.006), lack of maturation with depth (P = 0.036), and pseudo-maturation (P = 0.006) were significantly more common in nevoid melanomas. Immunohistochemically, complete loss of p16 was an important divisive feature (P = 0.0004), seen in 70% of nevoid melanomas, and highly correlated with loss of CDKN2A gene (chromosome 9p21). Our findings suggest that such reproducible immunomorphological differences can be of value in distinguishing nevoid melanoma from mitotically active nevus. Nevoid melanomas demonstrated a spectrum of chromosomal aberrations similar to those seen in common subtypes of melanoma, which can serve as a powerful adjunct diagnostic tool in morphologically challenging lesions.

Detection of Low-level EGFR c.2369 C > T (p.Thr790Met) Resistance Mutation in Pre-treatment Non-small Cell Lung Carcinomas Harboring Activating EGFR Mutations and Correlation with Clinical Outcomes.

Increasing evidence points to the presence of low-level de novo T790M mutations in patients with non-small cell lung carcinoma (NSCLC) harboring activating EGFR mutations. We utilized digital PCR (dPCR), a highly sensitive gene mutation detection method, to detect pre-treatment T790M mutations in NSCLC tumor samples and correlated the T790M status with clinical features and patient outcomes. DNA extracted from pre-treatment NSCLC tumor tissue with known activating EGFR mutations, diagnosed between October 2010 and May 2017 at PathWest laboratory, was used to perform targeted dPCR for quantitative detection of T790M mutations. T790M was detected in 42 of 109 pre-treatment samples (38.5%). Median variant allele frequency was 0.14% (range 0.02-28.5%). Overall response rate to first generation EGFR tyrosine kinase inhibitors (TKI) was 67% regardless of T790M status. The median progression free survival was 10.7 (IQR 5.6-19.9) versus 6.7 (IQR 3.5-20.8) months in T790M negative and positive patients respectively. T790M positivity correlated with increased rate of early disease progression. It also correlated with increased mortality (HR 3.1 95%CI 1.2-8.1, p = 0.022) in patients who did not respond to TKI treatment. We detected a significant rate of low-level pre-treatment T790M mutations in NSCLC using highly sensitive dPCR. Low-level pre-treatment T790M did not impact treatment response rate or overall survival, but was associated with increased rate of early progression on TKI therapy.

Invasive lobular carcinoma of the breast: assessment of proliferative activity using automated Ki-67 immunostaining.

Invasive lobular carcinoma (ILC) is almost always classified as Nottingham histological grade 2. Despite this, prognosis is markedly varied, with some ILCs behaving more akin to grade 3 invasive ductal carcinoma (IDC). Methods to separate these aggressive ILCs are needed. Digital image analysis (DIA) of the Ki-67 biomarker has potential in this regard; thus, we sought to determine the feasibility of its use for automated evaluation of ILC. An initial pilot study demonstrated no ILC specific changes were required to our Ki-67 DIA algorithm for reproducible results. Subsequently, 42 consecutive cases of ILC were evaluated by visual mitosis counting in H&E stained sections and by DIA on Ki-67 stained sections. Ki-67 proliferative index (PI) DIA showed significant correlation with visual mitosis counting on H&E stained sections (rs=0.63; p<0.05), significant strong correlation (rs=0.78; p<0.05) and substantial agreement (κ=0.62) with manual/visual Ki-67 assessment and significant positive associations with grade, nodal status and 'pleomorphic' ILC subtype, and a wide stratification of values in classical/grade 2 ILC. In conclusion, DIA of Ki-67 PI in ILC is feasible, correlates with mitotic index, manual/visual Ki-67 PI and clinico-pathological variables. The broad stratification of Ki-67 PI in classical/grade 2 ILC supports its practicability as a biomarker with prognostic and predictive potential, although large studies with outcome data are required for validation.

Laboratory validation studies in Ki-67 digital image analysis of breast carcinoma: a pathway to routine quality assurance.

Ki-67 proliferative index (PI) has prognostic and predictive value in invasive breast carcinoma (IBC), but clinical uptake has been hampered by suboptimal accuracy, reproducibility and standardisation. Published guidelines have addressed pre-analytical and analytical factors to improve Ki-67 PI utility; however, practicalities of ongoing monitoring of Ki-67 PI quality in IBC reporting have not been established. We aimed to evaluate the internal and external quality of our established digital Ki-67 PI IBC reporting practice at a tertiary institution. In the 5 years since initial validation work, we've completed a series of internal and external quality assurance (QA) projects: (1) an interobserver agreement study, (2) a two site interlaboratory agreement study, (3) determination of the error of our Ki-67 results, (4) an audit of the year-to-year Ki-67 values, (5) an audit of Ki-67 in neoadjuvant chemotherapy (NAC) treated cases, and (6) comparison of our Ki-67 datasets with similar published datasets. There was excellent concordance (intra-class correlation = 0.98) and good agreement [kappa (κ) = 0.76-0.96] between pathologists, excellent concordance [Pearson correlation (R) = 0.94] and very good agreement (κ = 0.80) between laboratories and excellent concordance (R = 0.92-0.95) and good agreement (κ = 0.67-1.0) over time for our Ki-67 results. No significant difference was observed in Ki-67 data from year-to-year. Expected associations with clinico-pathological prognosticators, pathological complete response following NAC and mitotic index were evident. The median Ki-67 values from the overall and NAC treated datasets were within the range reported in other studies, and our data could be separated into similarly proportioned 'high' and 'low' Ki-67 PI groups when dichotomised as per protocols in other studies. Collectively, our work provides evidence of adequate internal and external quality control for our digital Ki-67 PI IBC reporting protocols. Given the paucity of formal Ki-67 QA programs, our approach could be emulated, and results compared between laboratories as a framework for internal and external Ki-67 QA.

Central Nervous System Processing of Emotions in Children with Fecal Incontinence and Constipation.

OBJECTIVE: Fecal incontinence and constipation are common disorders in childhood. The enteric nervous system and the central nervous system are highly interactive along the brain-gut axis. The interaction is mainly afferent. These afferent pathways include centers that are involved in the central nervous processing of emotions as the mid/posterior insula and the anterior cingulate cortex. A previous study revealed altered processing of emotions in children with fecal incontinence. The present study replicates these results. METHODS: In order to analyze the processing of emotions, we compared the event-related potentials of 25 children with fecal incontinence and constipation to those of 15 control children during the presentation of positive, negative, and neutral pictures. RESULTS: Children with fecal incontinence and constipation showed altered processing of emotions, especially in the parietal and central cortical regions. CONCLUSIONS: The main study results of the previous study were replicated, increasing the certainty and validity of the findings.

Detection of copy number variations in melanocytic lesions utilising array based comparative genomic hybridisation.

Distinction between melanocytic naevi and melanoma occasionally poses a diagnostic challenge in ambiguous cases showing overlapping histological features. Melanomas are characterised by the presence of multiple genomic copy number variants (CNVs), while this is not a feature of naevi. We assessed the feasibility and utility of array-based comparative genomic hybridisation (aCGH) to assess CNVs in melanocytic lesions. DNA was extracted from formalin fixed, paraffin embedded (FFPE) sections of unambiguous naevi (n=19) and melanomas (n=19). The test DNA and gender mismatched human reference DNA were differentially labelled with fluorophores. Equal quantities of the two DNA samples were mixed and co-hybridised to a SurePrint G3 Human CGH 8x60K array, and digitally scanned to capture and quantify the relative fluorescence intensities. The ratio of the fluorescence intensities was analysed by Cytogenomics software (Agilent). Frequent large CNVs were identified in 94.7% of melanoma samples, including losses of 9p (73.6%), 9q (52.6%), 10q (36.8%), 11q (36.8%), 3p (21%), and 10p (21%), and gains of 6p (42.1%), 7p (42.1%), 1q (36.8%), 8q (31.5%) and 20q (21%). Only one naevus showed two large copy number changes. Overall aCGH showed a specificity and sensitivity of 94.7% in separating naevi from melanomas. Based on our results, aCGH can be successfully used to analyse CNVs of melanocytic lesions utilising FFPE derived biopsy samples, providing a potentially useful adjunctive test for the classification of diagnostically challenging melanocytic proliferations.

Expression of proliferation genes in formalin-fixed paraffin-embedded (FFPE) tissue from breast carcinomas. Feasibility and relevance for a routine histopathology laboratory.

AIM: Breast carcinoma proliferative activity, histological grade and commercial molecular tests are all important in prognostication and treatment. There is a particular need for improved, standardised techniques for subclassification of grade 2 breast cancers into low-risk and high-risk prognostic groups. In this study we investigated whether gene expression profiling of five proliferation genes was feasible using breast cancer tissue in a clinical setting and whether these profiles could enhance pathological assessment. METHODS: Expression of five proliferation gene mRNAs; Ki-67, STK 15, CCNB1, CCND1 and MYBL2, was quantified in 27 breast carcinomas and compared with Ki-67 proliferation index (PI) and Nottingham mitotic score. RESULTS: Expression of Ki-67, STK15 and MYBL2 mRNA showed moderate Spearman's correlation with Ki-67 PI (p<0.01), but CCND1 and CCNB1 showed weak, non-significant correlation. Individual gene expression did not associate with mitotic score but combined mRNA expression correlated with both Ki-67 PI (p=0.018) and mitotic score (p=0.03; 0.007). CONCLUSIONS: This study confirms mRNA analysis in breast carcinoma formalin-fixed, paraffin-embedded samples is feasible and suggests gene expression profiling, using a small set of five proliferation genes, has potential in aiding histological grading or assessment of proliferative activity of breast cancers. To fully evaluate the clinical applicability of this approach, a larger cohort study with long-term follow-up data is required.

Coordination of Diagnostic Efforts in the Great Plains: Wheat Virus Survey and Modeling of Disease Onset.

Following the discovery of two new wheat virus diseases in the United States, the Great Plains region (Colorado, Kansas, Montana, Nebraska, North Dakota, Oklahoma, South Dakota, Texas, and Wyoming) of the National Plant Diagnostic Network (NPDN) initiated a project to measure the prevalence of five wheat diseases using indirect ELISA. Wheat streak mosaic virus (WSMV), Wheat mosaic virus (WMoV), and Triticum mosaic virus (TriMV) were found in all nine states. WSMV was the most prevalent, averaging 23 to 47% of samples each year. TriMV and WMoV were detected with WSMV (in up to 76% of the samples). All three mite-transmitted viruses were present in 26% or fewer of the samples. Aphid-transmitted viruses in the barley yellow dwarf complex Barley yellow dwarf virus, and Cereal yellow dwarf virus-RPV were less frequent (fewer than 65% of the samples). This paper presents the first case-control methodology paper using plant diagnostic laboratory data and the first signed diagnostic data-sharing agreement between the NPDN and its regulatory stakeholders. Samples collected when <700 cumulative degree-days base 0°C, were twice as likely to be virus negative. This proof-of-concept effort highlights the potential of the NPDN and its National Data Repository to develop knowledge about emerging diseases.

BRAF mutation detection in hairy cell leukaemia from archival haematolymphoid specimens.

Hairy cell leukaemia (HCL) is a rare, indolent chronic B-cell leukaemia accounting for approximately 2% of all adult leukaemias. The recent association of the BRAF p.Val600Glu (V600E) mutation in HCL makes it a valuable molecular diagnostic marker. We compared the ability of Sanger sequencing, fluorescent single-strand conformational polymorphism (F-SSCP) and high resolution melting (HRM) analysis to detect BRAF mutations in 20 cases of HCL consisting of four archival Romanowsky stained air-dried peripheral blood and bone marrow aspirate smears, 12 mercury fixed decalcified bone marrow trephine biopsies, three formalin fixed, paraffin embedded (FFPE) splenectomy samples and one fresh peripheral blood sample. DNA was amplified and BRAF mutation status determined by the three methods above. V600E mutation was identified in 94%, 89% and 72% of HCL cases by F-SSCP, HRM and Sanger sequencing, respectively. In one case, in addition to the p.Val600Glu mutation, a p.Lys601Thr (K601T) mutation was identified. DNA from archival slide scrapings, mercury-fixed and FFPE tissue can be used to identify BRAF mutations with high sensitivity, especially using HRM/F-SSCP. The V600E mutation can be used as a supplementary molecular marker to aid in the diagnosis of HCL and the presence of the mutation may provide a target for therapy.

Reconsidering Leaf Wetness Duration Determination for Plant Disease Management.

Relationships between leaf wetness and plant diseases have been studied for centuries. The progress and risk of many bacterial, fungal, and oomycete diseases on a variety of crops have been linked to the presence of free water on foliage and fruit under temperatures favorable to infection. Whereas the rate parameters for infection or epidemic models have frequently been linked with temperature during the wet periods, leaf wetness periods of specific time duration are necessary for the propagule germination of most phytopathogenic fungi and for their penetration of plant tissues. Using these types of relationships, disease-warning systems were developed and are now being used by grower communities for a variety of crops. As a component of Integrated Pest Management, disease-warning systems provide growers with information regarding the optimum timing for chemical or biological management practices based on weather variables most suitable for pathogen dispersal or host infection. Although these systems are robust enough to permit some errors in the estimates or measurements of leaf wetness duration, the need for highly accurate leaf wetness duration data remains a priority to achieve the most efficient disease management.

Practical issues concerning the implementation of Ki-67 proliferative index measurement in breast cancer reporting.

Commercial molecular tests which rely heavily on proliferation markers to stratify breast cancer are in increasing demand, but are expensive and not widely available. There is heightened interest in the use of Ki-67 immunohistochemistry as a marker of proliferation. This study sought to examine practical issues in the incorporation of Ki-67 measurement into breast cancer reporting.We conducted a prospective study of Ki-67 proliferative activity in 85 breast carcinomas in 70 patients. We considered whether dual staining with cytokeratin and Ki-67 was necessary to exclude background cells in automated digital image analysis (DIA) and how well a semi-quantitative assessment (SQA) method of Ki-67 proliferation and formal manual counting by two pathologists correlated with DIA.Our study showed good correlation between single and dual stained specimens by DIA (Spearman correlation coefficient 0.8), with a kappa statistic of 0.51 (moderate agreement) but with significantly fewer positive cells identified in dual stained sections. There was fair correlation between SQA and DIA by two pathologists (Spearman correlation coefficient 0.7 and 0.7). Using a ≥10% cut-off to define cases with a 'low' and 'high' proliferative index gave a kappa statistic of 0.25 and 0.32 (fair agreement). There was fair correlation between formal manual counts between two pathologists (Spearman correlation coefficient 0.7; kappa 0.32). Repeat DIA on all cases showed excellent correlation (Spearman coefficient 0.98; kappa 1.0).Automated digital analysis of Ki-67 PI is likely to be more accurate and consistent than semi-quantitative assessment and more practicable than formal manual counting. There remain challenges in standardisation of technique within and across laboratories, interpretation of results and in evaluating clinical relevance.

Coil fraction-dependent phase behaviour of a model globular protein-polymer diblock copolymer.

The self-assembly of the model globular protein-polymer block copolymer mCherry-b-poly(N-isopropyl acrylamide) is explored across a range of polymer coil fractions from 0.21 to 0.82 to produce a phase diagram for these materials as a function of molecular composition. Overall, four types of morphologies were observed: hexagonally packed cylinders, perforated lamellae, lamellae, and disordered nanostructures. Across all coil fractions and morphologies, a lyotropic re-entrant order-disorder transition in water was observed, with disordered structures below 30 wt% and above 70 wt% and well-ordered morphologies at intermediate concentrations. Solid state samples prepared by solvent evaporation show moderately ordered structures similar to those observed in 60 wt% solutions, suggesting that bulk structures result from kinetic trapping of morphologies which appear at lower concentrations. While highly ordered cylindrical nanostructures are observed around a bioconjugate polymer volume fraction of 0.3 and well-ordered lamellae are seen near a volume fraction of 0.6, materials at lower or higher coil fractions become increasingly disordered. Notable differences between the phase behaviour of globular protein-polymer block copolymers and coil-coil diblock copolymers include the lack of spherical nanostructures at either high or low polymer coil fractions as well as shifted phase boundaries between morphologies which result in an asymmetric phase diagram.

The nature of protein interactions governing globular protein-polymer block copolymer self-assembly.

The effects of protein surface potential on the self-assembly of protein-polymer block copolymers are investigated in globular proteins with controlled shape through two approaches: comparison of self-assembly of mCherry-poly(N-isopropylacrylamide) (PNIPAM) bioconjugates with structurally homologous enhanced green fluorescent protein (EGFP)-PNIPAM bioconjugates, and mutants of mCherry with altered electrostatic patchiness. Despite large changes in amino acid sequence, the temperature-concentration phase diagrams of EGFP-PNIPAM and mCherry-PNIPAM conjugates have similar phase transition concentrations. Both materials form identical phases at two different coil fractions below the PNIPAM thermal transition temperature and in the bulk. However, at temperatures above the thermoresponsive transition, mCherry conjugates form hexagonal phases at high concentrations while EGFP conjugates form a disordered micellar phase. At lower concentration, mCherry shows a two-phase region while EGFP forms homogeneous disordered micellar structures, reflecting the effect of changes in micellar stability. Conjugates of four mCherry variants with changes to their electrostatic surface patchiness also showed minimal change in phase behavior, suggesting that surface patchiness has only a small effect on the self-assembly process. Measurements of protein/polymer miscibility, second virial coefficients, and zeta potential show that these coarse-grained interactions are similar between mCherry and EGFP, indicating that coarse-grained interactions largely capture the relevant physics for soluble, monomeric globular protein-polymer conjugate self-assembly.

Effect of small molecule osmolytes on the self-assembly and functionality of globular protein-polymer diblock copolymers.

Blending the small molecule osmolytes glycerol and trehalose with the model globular protein-polymer block copolymer mCherry-b-poly(N-isopropyl acrylamide) (mCherry-b-PNIPAM) is demonstrated to improve protein functionality in self-assembled nanostructures. The incorporation of either additive into block copolymers results in functionality retention in the solid state of 80 and 100% for PNIPAM volume fractions of 40 and 55%, respectively. This represents a large improvement over the 50-60% functionality observed in the absence of any additive. Furthermore, glycerol decreases the thermal stability of block copolymer films by 15-20 °C, while trehalose results in an improvement in the thermal stability by 15-20 °C. These results suggest that hydrogen bond replacement is responsible for the retention of protein function but suppression or enhancement of thermal motion based on the glass transition of the osmolyte primarily determines thermal stability. While both osmolytes are observed to have a disordering effect on the nanostructure morphology with increasing concentration, this effect is less pronounced in materials with a larger polymer volume fraction. Glycerol preferentially localizes in the protein domains and swells the nanostructures, inducing disordering or a change in morphology depending on the PNIPAM coil fraction. In contrast, trehalose is observed to macrophase separate from the block copolymer, which results in nanodomains becoming more disordered without changing significantly in size.

Kinetically controlled nanostructure formation in self-assembled globular protein-polymer diblock copolymers.

Aqueous processing of globular protein-polymer diblock copolymers into solid-state materials and subsequent solvent annealing enables kinetic and thermodynamic control of nanostructure formation to produce block copolymer morphologies that maintain a high degree of protein fold and function. When model diblock copolymers composed of mCherry-b-poly(N-isopropylacrylamide) are used, orthogonal control over solubility of the protein block through changes in pH and the polymer block through changes in temperature is demonstrated during casting and solvent annealing. Hexagonal cylinders, perforated lamellae, lamellae, or hexagonal and disordered micellar phases are observed, depending on the coil fraction of the block copolymer and the kinetic pathway used for self-assembly. Good solvents for the polymer block produce ordered structures reminiscent of coil-coil diblock copolymers, while an unfavorable solvent results in kinetically trapped micellar structures. Decreasing solvent quality for the protein improves long-range ordering, suggesting that the strength of protein interactions influences nanostructure formation. Subsequent solvent annealing results in evolution of the nanostructures, with the best ordering and the highest protein function observed when annealing in a good solvent for both blocks. While protein secondary structure was found to be almost entirely preserved for all processing pathways, UV-vis spectroscopy of solid-state films indicates that using a good solvent for the protein block enables up to 70% of the protein to be retained in its functional form.