Costimulation Blockade Disrupts CD4+ T Cell Memory Pathways and Uncouples Their Link to Decline in ß-Cell Function in Type 1 Diabetes.

We previously reported that costimulation blockade by abatacept limits the decline of ß-cell function and the frequency of circulating CD4+ central memory T cells (TCM) (CD45RO+CD62L+) in new-onset type 1 diabetes. In human subjects receiving placebo, we found a significant association between an increase in CD4+ TCM cells and the decline of ß-cell function. To extend and refine these findings, we examined changes in human CD4+ and CD8+ naive and memory T cell subsets at greater resolution using polychromatic flow and mass cytometry. In the placebo group, we successfully reproduced the original finding of a significant association between TCM and ß-cell function and extended this to other T cell subsets. Furthermore, we show that abatacept treatment significantly alters the frequencies of a majority of CD4+ conventional and regulatory T cell subsets; in general, Ag-naive subsets increase and Ag-experienced subsets decrease, whereas CD8+ T cell subsets are relatively resistant to drug effects, indicating a lesser reliance on CD28-mediated costimulation. Importantly, abatacept uncouples the relationship between changes in T cell subsets and ß-cell function that is a component of the natural history of the disease. Although these data suggest immunological markers for predicting change in ß-cell function in type 1 diabetes, the finding that abatacept blunts this relationship renders the biomarkers nonpredictive for this type of therapy. In sum, our findings point to a novel mechanism of action for this successful immunotherapy that may guide other disease-modifying approaches for type 1 diabetes.

Peripheral immune circadian variation, synchronisation and possible dysrhythmia in established type 1 diabetes.

AIMS/HYPOTHESIS: The circadian clock influences both diabetes and immunity. Our goal in this study was to characterise more thoroughly the circadian patterns of immune cell populations and cytokines that are particularly relevant to the immune pathology of type 1 diabetes and thus fill in a current gap in our understanding of this disease. METHODS: Ten individuals with established type 1 diabetes (mean disease duration 11 years, age 18-40 years, six female) participated in a circadian sampling protocol, each providing six blood samples over a 24 h period. RESULTS: Daily ranges of population frequencies were sometimes large and possibly clinically significant. Several immune populations, such as dendritic cells, CD4 and CD8 T cells and their effector memory subpopulations, CD4 regulatory T cells, B cells and cytokine IL-6, exhibited statistically significant circadian rhythmicity. In a comparison with historical healthy control individuals, but using shipped samples, we observed that participants with type 1 diabetes had statistically significant phase shifts occurring in the time of peak occurrence of B cells (+4.8 h), CD4 and CD8 T cells (~ +5 h) and their naive and effector memory subsets (~ +3.3 to +4.5 h), and regulatory T cells (+4.1 h). An independent streptozotocin murine experiment confirmed the phase shifting of CD8 T cells and suggests that circadian dysrhythmia in type 1 diabetes might be an effect and not a cause of the disease. CONCLUSIONS/INTERPRETATION: Future efforts investigating this newly described aspect of type 1 diabetes in human participants are warranted. Peripheral immune populations should be measured near the same time of day in order to reduce circadian-related variation.

Efficacy of GAD-alum immunotherapy associated with HLA-DR3-DQ2 in recently diagnosed type 1 diabetes.

AIMS/HYPOTHESIS: The aim of this study was to determine if retention of C-peptide following immunotherapy using recombinant GAD65 conjugated to aluminium hydroxide (GAD-alum) is influenced by HLA risk haplotypes DR3-DQ2 and DR4-DQ8. METHODS: HLA-dependent treatment effect of GAD-alum therapy on C-peptide retention in individuals with recent-onset type 1 diabetes was evaluated using individual-level patient data from three placebo-controlled, randomised clinical trials using a mixed repeated measures model. RESULTS: A significant and dose-dependent effect was observed in individuals positive for the genotypes that include HLA-DR3-DQ2 but not HLA-DR4-DQ8 and in the broader subgroup of individuals positive for all genotypes that include HLA-DR3-DQ2 (i.e. including those also positive for HLA-DR4-DQ8). Higher doses (three or four injections) showed a treatment effect ratio of 1.596 (95% CI 1.132, 2.249; adjusted p = 0.0035) and 1.441 (95% CI 1.188, 1.749; adjusted p = 0.0007) vs placebo for the two respective HLA subgroups. CONCLUSIONS/INTERPRETATION: GAD65-specific immunotherapy has a significant effect on C-peptide retention in individuals with recent-onset type 1 diabetes who have the DR3-DQ2 haplotype. Graphical abstract.

GAD vaccine reduces insulin loss in recently diagnosed type 1 diabetes: findings from a Bayesian meta-analysis.

AIMS/HYPOTHESIS: GAD is a major target of the autoimmune response that occurs in type 1 diabetes mellitus. Randomised controlled clinical trials of a GAD + alum vaccine in human participants have so far given conflicting results. METHODS: In this study, we sought to see whether a clearer answer to the question of whether GAD65 has an effect on C-peptide could be reached by combining individual-level data from the randomised controlled trials using Bayesian meta-analysis to estimate the probability of a positive biological effect (a reduction in C-peptide loss compared with placebo approximately 1 year after the GAD vaccine). RESULTS: We estimate that there is a 98% probability that 20 µg GAD with alum administered twice yields a positive biological effect. The effect is probably a 15-20% reduction in the loss of C-peptide at approximately 1 year after treatment. This translates to an annual expected loss of between -0.250 and -0.235 pmol/ml in treated patients compared with an expected 2 h AUC loss of -0.294 pmol/ml at 1 year for untreated newly diagnosed patients. CONCLUSIONS/INTERPRETATION: The biological effect of this vaccination should be developed further in order to reach clinically desirable reductions in insulin loss in patients recently diagnosed with type 1 diabetes.

Statistical considerations when analyzing biomarker data.

Biomarkers have become, and will continue to become, increasingly important to clinical immunology research. Yet, biomarkers often present new problems and raise new statistical and study design issues to scientists working in clinical immunology. In this paper I discuss statistical considerations related to the important biomarker problems of: 1) The design and analysis of clinical studies which seek to determine whether changes from baseline in a biomarker are associated with changes in a metabolic outcome; 2) The conditions that are required for a biomarker to be considered a "surrogate"; 3) Considerations that arise when analyzing whether or not a predictive biomarker could act as a surrogate endpoint; 4) Biomarker timing relative to the clinical endpoint; 5) The problem of analyzing studies that measure many biomarkers from few subjects; and, 6) The use of statistical models when analyzing biomarker data arising from count data.

Heterogeneity in recent-onset type 1 diabetes - a clinical trial perspective.

BACKGROUND: Type 1 diabetes (T1D) TrialNet is a National Institutes of Health-sponsored clinical trial network aimed at altering the disease course of T1D. The purpose of this study is to evaluate age-dependent heterogeneity in clinical, metabolic and immunologic characteristics of individuals with recent-onset T1D, to identify cohorts of interest and to aid in planning of future studies. METHODS: Eight hundred eighty-three individuals with recent-onset T1D involved in five TrialNet studies were categorized by age as follows: ≥18 years, 12-17 years, 8-12 years and <8 years. Data were compared with healthy age-matched subjects in the National Health and Nutrition Examination Survey. RESULTS: Only 2.0% of the individuals overall were excluded from trial participation because of insufficient C-peptide values (<0.2 pmol/mL). A disproportionate number of these subjects were <8 years old. Leukopenia was present in 21.2% of individuals and lymphopenia in 11.6%; these frequencies were markedly higher than age-matched healthy National Health and Nutrition Examination Survey population. Of the cohort, 24.5% were overweight or obese. Neither high-risk human leukocyte antigen type DR3 nor DR4 was present in 31% of adults and 21% of children. CONCLUSIONS: The ability of recent-onset T1D patients to meet key entry criteria for TrialNet studies, including C-peptide >0.2 pmol/mL, varies by age. Lower C-peptide level requirements for younger participants and other aspects of heterogeneity of recent-onset T1D patients, such as white blood cell count abnormalities and body mass index should be considered in the design of future clinical studies.

Pioglitazone versus glimepiride on coronary artery calcium progression in patients with type 2 diabetes mellitus: a secondary end point of the CHICAGO study.

OBJECTIVE: To compare coronary artery calcium (CAC) progression between 2 treatment groups, pioglitazone versus glimepiride. METHODS AND RESULTS: The CHICAGO (Carotid Intima-Media Thickness in Atherosclerosis Using Pioglitazone) study demonstrated that pioglitazone significantly decreased carotid intima-media thickness progression compared with glimepiride in patients with type 2 diabetes mellitus. The CAC level was determined at baseline and at the end of 72 weeks of treatment in the pioglitazone (n=146) and glimepiride (n=153) treatment groups using electron beam computed tomography. There was no difference in CAC progression between the treatment groups. By using backward and forward selection models, age, race/ethnicity, and baseline apolipoprotein B level predicted CAC progression. There was no relationship between carotid intima-media thickness and CAC progression during the study. CONCLUSIONS: There was no difference in CAC progression in patients with type 2 diabetes mellitus treated with pioglitazone or glimepiride. Age, race/ethnicity, and baseline apolipoprotein B level predicted CAC progression in patients with type 2 diabetes mellitus.

Making the diagnosis of acute appendicitis: do more preoperative CT scans mean fewer negative appendectomies? A 10-year study.

PURPOSE: To determine the frequency of preoperative computed tomography (CT) in the evaluation of patients suspected of having appendicitis at one institution during the past 10 years and to determine whether changes in CT utilization were associated with changes in the negative appendectomy rate. MATERIALS AND METHODS: Institutional review board approval was obtained, and a waiver of informed consent was granted for this HIPAA-compliant study. A surgical database search yielded medical record numbers of 925 patients (526 [ 56.9%] men and 399 [43.1%] women; mean age, 38 years (range, 18-95 years]) who underwent urgent appendectomy between January 1998 and September 2007. Patients who were younger than 18 years of age at the time of surgery were excluded. CT, pathology, and surgery reports were reviewed. By using logistic regression, changes in the proportion of patients undergoing CT and in the proportion of patients undergoing each year appendectomy in which the appendix was healthy were evaluated. Subgroup analyses based on patient age ( 45 years) and sex also were performed. RESULTS: Prior to urgent appendectomy, 18.5% of patients underwent preoperative CT in 1998 compared with 93.2% of patients in 2007. The negative appendectomy rate for women 45 years of age and younger decreased from 42.9% in 1998% to 7.1% in 2007. However, the timing of the decline in negative appendectomy rates for women 45 years and younger could not be proved to be associated with the increase in CT use. There was no significant trend toward a lower negative appendectomy rate for men regardless of age or for women older than 45 years of age with increased use of preoperative CT. The shift from single-detector CT to multidetector CT and the use of decreasing section thickness also correlated with a reduction in false-positive diagnoses. CONCLUSION: Rising utilization of preoperative CT and advances in technology coincided with a decrease in the negative appendectomy rate for women 45 years and younger but not in men of any age or women older than 45 years.

Combined antioxidant carotenoids and the risk of persistent human papillomavirus infection.

Persistent infection with human papillomavirus (HPV) is the primary etiologic factor for cervical cancer. The synergistic effect of carotenoids on HPV persistence has not been examined. To explore these potential synergies, we developed 2 measures of carotenoid status using circulating and dietary intake nutrients in which each nutrient was given equal weighting. We then compared persistent HPV infection with its counterpart, intermittent infection. In the analysis using the Crude Index, no association was observed between circulating nutrients and persistent infection with oncogenic HPV [odds ratio (OR)(adjusted) = 0.8, 95% confidence interval (CI) = 0.3-2.2)] or any type HPV (OR(adjusted) = 0.8, 95% CI = 0.3-2.1). Similar results were obtained using the Cumulative Index. However, associations between dietary intake and persistent infection were observed using both indexes. When the analysis was restricted to oncogenic HPV, a 50% higher risk was observed for women with low dietary carotenoid status using the Crude Index (OR(adjusted) = 1.5, 95% CI = 0.6-3.7). In the analysis using any type HPV, the adjusted OR for women with low dietary intake of combined carotenoids using the Cumulative Index was 2.4 (95% CI = 1.1-5.2). These results may be consistent with the hypothesis that low levels of carotenoids may increase the risk of persistent HPV infection.

Synchronization of the Normal Human Peripheral Immune System: A Comprehensive Circadian Systems Immunology Analysis.

In this study, we sought to fill an important gap in fundamental immunology research by conducting a comprehensive systems immunology analysis of daily variation in the normal human peripheral immune system. Although variation due to circadian rhythmicity was not a significant source of variation in daily B-cell levels or any CD4+ functional subset, it accounted for more than 25% of CD4+ regulatory T-cell variation and over 50% of CD8+ central memory variation. Circadian rhythmicity demonstrated phase alignment within functional phenotypes. In addition, we observed that previously-described mechanistic relationships can also appear in the peripheral system as phase shifting in rhythmic patterns. We identified a set of immune factors which are ubiquitously correlated with other factors and further analysis also identified a tightly-correlated "core" set whose relational structure persisted after analytically removing circadian-related variation. This core set consisted of CD8+ and its subpopulations and the NK population. In sum, the peripheral immune system can be conceptualized as a dynamic, interconnected wave-field repeating its pattern on a daily basis. Our data provide a comprehensive inventory of synchronization and correlation within this wave-field and we encourage use of our data to discover unknown mechanistic relationships which can then be tested in the laboratory.

Cell adhesion to fibronectin (CAM-DR) influences acquired mitoxantrone resistance in U937 cells.

Cell adhesion to fibronectin is known to confer a temporally related cell adhesion-mediated drug resistance (CAM-DR). However, it is unknown whether cell adhesion during drug selection influences the more permanent form of acquired drug resistance. To examine this question, we compared the acquisition of mitoxantrone resistance in U937 cells adhered to fibronectin versus cells selected in a traditional suspension culture. Our data show that acquired drug resistance levels of resistance to mitoxantrone are 2- to 3-fold greater for cells adhered to fibronectin compared with cells in suspension culture. We also compared mechanism(s) of resistance associated with drug selection in suspension versus fibronectin-adherent cultures. Drug resistance in both suspension and fibronectin-adhered cultures correlated with reduced drug-induced DNA damage and diminished topoisomerase II levels and activity; however, mechanisms regulating topoisomerase II levels differed depending on culture conditions. In suspension cultures, a reduction in topoisomerase IIbeta levels was detected at both RNA and protein levels. Furthermore, the decreased expression of topoisomerase IIbeta mRNA levels correlated with decreased expression of NF-YA. In contrast, in spite of no changes in NF-YA or topoisomerase IIbeta RNA expression, topoisomerase IIbeta protein levels were decreased in fibronectin-adherent, drug-resistant cells. In addition, topoisomerase IIalpha protein levels (but not RNA levels) were reduced in drug resistance cells selected on fibronectin; however, no change in topoisomerase IIalpha was observed in cells selected with mitoxantrone in suspension culture. Taken together, our results suggest that the development of drug resistance models must consider interactions with the microenvironment to identify clinically relevant targets and mechanisms associated with acquired drug resistance.

Autoreactive T effector memory differentiation mirrors ß cell function in type 1 diabetes.

In type 1 diabetes, cytotoxic CD8+ T cells with specificity for ß cell autoantigens are found in the pancreatic islets, where they are implicated in the destruction of insulin-secreting ß cells. In contrast, the disease relevance of ß cell-reactive CD8+ T cells that are detectable in the circulation, and their relationship to ß cell function, are not known. Here, we tracked multiple, circulating ß cell-reactive CD8+ T cell subsets and measured ß cell function longitudinally for 2 years, starting immediately after diagnosis of type 1 diabetes. We found that change in ß cell-specific effector memory CD8+ T cells expressing CD57 was positively correlated with C-peptide change in subjects below 12 years of age. Autoreactive CD57+ effector memory CD8+ T cells bore the signature of enhanced effector function (higher expression of granzyme B, killer-specific protein of 37 kDa, and CD16, and reduced expression of CD28) compared with their CD57- counterparts, and network association modeling indicated that the dynamics of ß cell-reactive CD57+ effector memory CD8+ T cell subsets were strongly linked. Thus, coordinated changes in circulating ß cell-specific CD8+ T cells within the CD57+ effector memory subset calibrate to functional insulin reserve in type 1 diabetes, providing a tool for immune monitoring and a mechanism-based target for immunotherapy.

Activation of stat3 in primary tumors from high-risk breast cancer patients is associated with elevated levels of activated SRC and survivin expression.

PURPOSE: Constitutive activation of signal transducer and activator of transcription 3 (Stat3) protein has been observed in a wide variety of tumors, including breast cancer, and contributes to oncogenesis at least in part by prevention of apoptosis. In a study of 45 patients with high-risk breast cancer enrolled in a phase II neoadjuvant chemotherapy trial with docetaxel and doxorubicin, we evaluated the levels of Stat3 activation and potentially associated molecular biomarkers in invasive breast carcinoma compared with matched nonneoplastic tissues. EXPERIMENTAL DESIGN: Using immunohistochemistry and image analysis, we quantified the levels of phospho-Stat3 (pY-Stat3), phospho-Src (pY-Src), epidermal growth factor receptor, HER2/neu, Ki-67, estrogen receptor, Bcl-2, Bcl-xL, Survivin, and apoptosis in formalin-fixed, paraffin-embedded sections from invasive carcinomas and their paired nonneoplastic parenchyma. The levels of molecular biomarkers in nonneoplastic and tumor tissues were analyzed as continuous variables for statistically significant correlations. RESULTS: Levels of activated pY-Stat3 and pY-Src measured by immunohistochemistry were significantly higher in invasive carcinoma than in nonneoplastic tissue (P < 0.001). In tumors, elevated levels of pY-Stat3 correlated with those of pY-Src and Survivin. Levels of pY-Stat3 were higher in partial pathologic responders than in complete pathologic responders. In partial pathologic responders, pY-Stat3 levels correlated with Survivin expression. CONCLUSIONS: Our findings suggest important roles for elevated activities of Stat3 and Src, as well as Survivin expression, in malignant progression of breast cancer. Furthermore, elevated Stat3 activity correlates inversely with complete pathologic response. These findings suggest that specific Stat3 or Src inhibitors could offer clinical benefits to patients with breast cancer.

Persistent activation of stat3 signaling induces survivin gene expression and confers resistance to apoptosis in human breast cancer cells.

PURPOSE: Signal transducer and activator of transcription 3 (Stat3) protein is persistently activated in breast cancer and promotes tumor cell survival. To gain a better understanding of the role of constitutive Stat3 signaling in breast cancer progression, we evaluated the expression profile of potential Stat3-regulated genes that may confer resistance to apoptosis. EXPERIMENTAL DESIGN: Stat3 signaling was blocked with antisense oligonucleotides in human MDA-MB-435s breast cancer cells and Affymetrix GeneChip microarray analysis was done. The candidate Stat3 target gene Survivin was further evaluated in molecular assays using cultured breast cancer cells and immunohistochemistry of breast tumor specimens. RESULTS: Survivin, a member of the inhibitor of apoptosis protein family, was identified as a potential Stat3-regulated gene by microarray analysis. This was confirmed in Survivin gene promoter studies and chromatin immunoprecipitation assays showing that Stat3 directly binds to and regulates the Survivin promoter. Furthermore, direct inhibition of Stat3 signaling blocked the expression of Survivin protein and induced apoptosis in breast cancer cells. Direct inhibition of Survivin expression also induced apoptosis. Increased Survivin protein expression correlates significantly (P = 0.001) with elevated Stat3 activity in primary breast tumor specimens from high-risk patients who were resistant to chemotherapy treatment. CONCLUSIONS: We identify Survivin as a direct downstream target gene of Stat3 in human breast cancer cells that is critical for their survival in culture. Our findings suggest that activated Stat3 signaling contributes to breast cancer progression and resistance to chemotherapy by, at least in part, inducing expression of the antiapoptotic protein, Survivin.

Expansion of human umbilical cord blood SCID-repopulating cells using chromatin-modifying agents.

OBJECTIVE: We investigated whether the addition of two chromatin-modifying agents, 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA), to cord blood (CB) CD34(+) cells in culture results in expansion of the numbers of severe combined immunodeficient (SCID) repopulating cells (SRC). MATERIALS AND METHODS: Human CB CD34(+) cells were cultured with cytokines in the presence or absence of 5azaD/TSA. After 9 days of culture, the fold expansion of CD34(+) and CD34(+)CD90(+) cell numbers, colony-forming unit (CFU)-mix, cobblestone area-forming cell (CAFC), and SRC numbers were determined. RESULTS: A 12.5-fold expansion of CD34(+)CD90(+) cells was observed in the 5azaD/TSA-treated cultures in comparison to the input cell numbers. Expansion of CD34(+)CD90(+) cells was associated with a 9.8-fold increase in the numbers of CFU-mix and 11.5-fold increase in CAFC. 5azaD/TSA treatment of the CB CD34(+) cells resulted in a 9.6-fold expansion of the absolute number of SRC following 9 days of culture as determined by limiting dilution analysis. Expansion of cells maintaining CD34(+)CD90(+) phenotype was not due to the retention of a quiescent population of cells because all of the CD34(+)CD90(+) cells in the culture had undergone cellular division. 5azaD/TSA-treated CD34(+)CD90(+) cells, but not CD34(+)CD90(-) cells were responsible for in vivo hematopoietic repopulation potential of nonobese diabetic/SCID mice. CONCLUSION: Ex vivo expansion strategy using chromatin-modifying agents provides a potential avenue by which to expand the number of hematopoietic stem cells (HSC) with a single CB unit for use as an alternative source of HSC grafts for adult recipients.

Association of volume and volume-independent factors with accuracy in screening mammogram interpretation.

BACKGROUND: Early detection of breast cancer is associated with the accurate reading of screening mammograms, but factors that influence reading accuracy are not well understood. We thus investigated whether reading volume and other factors were independently associated with accuracy in reading screening mammograms in a population of U.S. radiologists. METHODS: A random selection of 110 of 292 radiologists who agreed to participate, if selected, interpreted screening mammograms from 148 randomly selected women. Original index mammograms (i.e., mediolateral oblique and craniocaudal views of each breast) were used; comparison original mammograms were provided when available. Radiologist-level and facility-level factors were surveyed. Two standard metrics of screening accuracy, both based on receiver operating characteristic curves, were analyzed. The influence of volume on accuracy after controlling for other factors was assessed with multiple regression analysis. RESULTS: Current reading volume was not statistically significantly associated with interpretive accuracy. More recently trained radiologists interpreted mammograms more accurately than those trained earlier (-0.76% [95% confidence interval (CI) = -1.75% to -0.02%] reduction in sensitivity per year since residency). Facility-level factors that were statistically significantly and independently associated with better accuracy were the number of diagnostic breast imaging examinations and image-guided breast interventional procedures performed (0.55% [95% CI = 0.11% to 2.40%] increase in accuracy per examination or procedure offered), being classified as a comprehensive breast diagnostic and/or screening center or freestanding mammography center (1.39% [95% CI = 0.15% to 3.82%] higher than a hospital radiology department or multispecialty medical clinic), and being a facility that practiced double reading (1.61% [95% CI = 1.99% to 11.65%]) higher than in a facility without such practice). CONCLUSIONS: Individual radiologists' current reading volume was not statistically significantly associated with accuracy in reading screening mammograms, but several other factors were. Expertise reflects a complex multifactorial process that needs further clarification.

Genotypic and phenotypic comparisons of de novo and acquired melphalan resistance in an isogenic multiple myeloma cell line model.

Cancer cell adhesion confers a transient, de novo drug-resistant phenotype referred to as cell adhesion-mediated drug resistance (CAM-DR). In this report, we extend the CAM-DR phenotype to primary specimens from patients with myeloma, providing further evidence that CAM-DR is a viable clinical form of drug resistance. To examine mechanisms of cellular resistance to melphalan, we compared genotypic and phenotypic profiles of acquired and de novo melphalan resistance in an isogenic human myeloma cell line. Acquired melphalan resistance (8226/LR5) was associated with decreased drug-induced DNA damage and a complex gene expression profile showing that genes involved in the Fanconi anemia DNA repair pathway are increased in the LR5 cells compared with drug-sensitive or adherent cells. In contrast, cells adhered to fibronectin accumulate similar amounts of DNA damage compared with drug-sensitive cells but are protected from melphalan-induced mitochondrial perturbations and caspase activation. Levels of the proapoptotic protein Bim were significantly reduced in adherent cells. Gene expression changes associated with de novo resistance were significantly less complex compared with acquired resistance, but a significant overlap in gene expression was noted involving cholesterol synthesis. We propose that myeloma cell adhesion promotes a form of de novo drug resistance by protecting cells from melphalan-induced cytotoxic damage and that this transient protection allows cells to acquire a more permanent and complex drug resistance phenotype associated with a reduction in drug induced DNA damage.

Perceptually based FROC analysis.

RATIONALE AND OBJECTIVES: Analysis of reading data when cases have multiple targets and/or the reader is required to localize targets is difficult. One approach to this free-response operating characteristic (FROC) problem is for images to be segmented (eg, with quadrants) by the investigator and a segment-level analysis be conducted with the case as a nesting factor. In this report, we introduce an alternative method that uses the visual scan path of the reader to segment the image. We evaluate the new method by applying it to data from a mammography reading experiment. MATERIALS AND METHODS: The gaze scan path of one radiologist was recorded as she scanned 40 mammograms for masses and microcalcifications. The observer is an experienced mammographer and was not one of the authors. In addition, the reader provided a rating indicating the degree of suspicion for any suspected targets she identified and localized. We then established "perceptual regions" by using a clustering algorithm on the visual fixations. We combined ratings given to specific locations indicated by the reader with the segmentation from the visual scan to generate a series of ratings classified for whether the perceptually based region associated with the rating contained or did not contain a known target. We analyzed data generated by our method from all 40 cases by using the conventional maximum-likelihood method based on the binormal model. Finally, we tested goodness-of-fit of the binormal model to the data by using chi-square. RESULTS: Maximum-likelihood estimation led to a model that did not fit the data (P < .001). However, examination of the observed and expected counts suggests that the binormal assumption does not hold for segments that contain targets and a bimodal distribution model might be preferred. CONCLUSION: Our new method provides an alternative approach to analysis of the FROC experiment. It needs to be developed further. Specifically, we propose that a mixture model extension of the binormal model be developed for ratings data arising from perceptually based FROC experiments. A disadvantage to our method is the requirement to record the scan path of the reader. However, we believe that adding such information to receiver operating characteristic (ROC) curve analysis will pay off when appropriate statistical models have been identified because we believe our data support our hypothesis that the perceptual scanning of images by humans deconvolves interpretation correlation. If true, this hypothesis implies that conventional statistical methods for ROC analysis based on independent data can be applied to the analysis of FROC data after conditioning on the scan path of the observer.

Use of Dried Capillary Blood Sampling for Islet Autoantibody Screening in Relatives: A Feasibility Study.

BACKGROUND: Islet autoantibody testing provides the basis for assessment of risk of progression to type 1 diabetes. We set out to determine the feasibility and acceptability of dried capillary blood spot-based screening to identify islet autoantibody-positive relatives potentially eligible for inclusion in prevention trials. MATERIALS AND METHODS: Dried blood spot (DBS) and venous samples were collected from 229 relatives participating in the TrialNet Pathway to Prevention Study. Both samples were tested for glutamic acid decarboxylase, islet antigen 2, and zinc transporter 8 autoantibodies, and venous samples were additionally tested for insulin autoantibodies and islet cell antibodies. We defined multiple autoantibody positive as two or more autoantibodies in venous serum and DBS screen positive if one or more autoantibodies were detected. Participant questionnaires compared the sample collection methods. RESULTS: Of 44 relatives who were multiple autoantibody positive in venous samples, 42 (95.5%) were DBS screen positive, and DBS accurately detected 145 of 147 autoantibody-negative relatives (98.6%). Capillary blood sampling was perceived as more painful than venous blood draw, but 60% of participants would prefer initial screening using home fingerstick with clinic visits only required if autoantibodies were found. CONCLUSIONS: Capillary blood sampling could facilitate screening for type 1 diabetes prevention studies.

A new approach for diagnosing type 1 diabetes in autoantibody-positive individuals based on prediction and natural history.

OBJECTIVE: We assessed whether type 1 diabetes (T1D) can be diagnosed earlier using a new approach based on prediction and natural history in autoantibody-positive individuals. RESEARCH DESIGN AND METHODS: Diabetes Prevention Trial-Type 1 (DPT-1) and TrialNet Natural History Study (TNNHS) participants were studied. A metabolic index, the T1D Diagnostic Index60 (Index60), was developed from 2-h oral glucose tolerance tests (OGTTs) using the log fasting C-peptide, 60-min C-peptide, and 60-min glucose. OGTTs with Index60 ≥2.00 and 2-h glucose <200 mg/dL (Ind60+Only) were compared with Index60 <2.00 and 2-h glucose ≥200 mg/dL (2hglu+Only) OGTTs as criteria for T1D. Individuals were assessed for C-peptide loss from the first Ind60+Only OGTT to diagnosis. RESULTS: Areas under receiver operating characteristic curves were significantly higher for Index60 than for the 2-h glucose (P < 0.001 for both DPT-1 and the TNNHS). As a diagnostic criterion, sensitivity was higher for Ind60+Only than for 2hglu+Only (0.44 vs. 0.15 in DPT-1; 0.26 vs. 0.17 in the TNNHS) OGTTs. Specificity was somewhat higher for 2hglu+Only OGTTs in DPT-1 (0.97 vs. 0.91) but equivalent in the TNNHS (0.98 for both). Positive and negative predictive values were higher for Ind60+Only OGTTs in both studies. Postchallenge C-peptide levels declined significantly at each OGTT time point from the first Ind60+Only OGTT to the time of standard diagnosis (range -22 to -34% in DPT-1 and -14 to -27% in the TNNHS). C-peptide and glucose patterns differed markedly between Ind60+Only and 2hglu+Only OGTTs. CONCLUSIONS: An approach based on prediction and natural history appears to have utility for diagnosing T1D.