Pixelwise H-score: A novel digital image analysis-based metric to quantify membrane biomarker expression from immunohistochemistry images.

Immunohistochemistry (IHC) assays play a central role in evaluating biomarker expression in tissue sections for diagnostic and research applications. Manual scoring of IHC images, which is the current standard of practice, is known to have several shortcomings in terms of reproducibility and scalability to large scale studies. Here, by using a digital image analysis-based approach, we introduce a new metric called the pixelwise H-score (pix H-score) that quantifies biomarker expression from whole-slide scanned IHC images. The pix H-score is an unsupervised algorithm that only requires the specification of intensity thresholds for the biomarker and the nuclear-counterstain channels. We present the detailed implementation of the pix H-score in two different whole-slide image analysis software packages Visiopharm and HALO. We consider three biomarkers P-cadherin, PD-L1, and 5T4, and show how the pix H-score exhibits tight concordance to multiple orthogonal measurements of biomarker abundance such as the biomarker mRNA transcript and the pathologist H-score. We also compare the pix H-score to existing automated image analysis algorithms and demonstrate that the pix H-score provides either comparable or significantly better performance over these methodologies. We also present results of an empirical resampling approach to assess the performance of the pix H-score in estimating biomarker abundance from select regions within the tumor tissue relative to the whole tumor resection. We anticipate that the new metric will be broadly applicable to quantify biomarker expression from a wide variety of IHC images. Moreover, these results underscore the benefit of digital image analysis-based approaches which offer an objective, reproducible, and highly scalable strategy to quantitatively analyze IHC images.

Association of clinical factors and recent anticancer therapy with COVID-19 severity among patients with cancer: a report from the COVID-19 and Cancer Consortium.

BACKGROUND: Patients with cancer may be at high risk of adverse outcomes from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We analyzed a cohort of patients with cancer and coronavirus 2019 (COVID-19) reported to the COVID-19 and Cancer Consortium (CCC19) to identify prognostic clinical factors, including laboratory measurements and anticancer therapies. PATIENTS AND METHODS: Patients with active or historical cancer and a laboratory-confirmed SARS-CoV-2 diagnosis recorded between 17 March and 18 November 2020 were included. The primary outcome was COVID-19 severity measured on an ordinal scale (uncomplicated, hospitalized, admitted to intensive care unit, mechanically ventilated, died within 30 days). Multivariable regression models included demographics, cancer status, anticancer therapy and timing, COVID-19-directed therapies, and laboratory measurements (among hospitalized patients). RESULTS: A total of 4966 patients were included (median age 66 years, 51% female, 50% non-Hispanic white); 2872 (58%) were hospitalized and 695 (14%) died; 61% had cancer that was present, diagnosed, or treated within the year prior to COVID-19 diagnosis. Older age, male sex, obesity, cardiovascular and pulmonary comorbidities, renal disease, diabetes mellitus, non-Hispanic black race, Hispanic ethnicity, worse Eastern Cooperative Oncology Group performance status, recent cytotoxic chemotherapy, and hematologic malignancy were associated with higher COVID-19 severity. Among hospitalized patients, low or high absolute lymphocyte count; high absolute neutrophil count; low platelet count; abnormal creatinine; troponin; lactate dehydrogenase; and C-reactive protein were associated with higher COVID-19 severity. Patients diagnosed early in the COVID-19 pandemic (January-April 2020) had worse outcomes than those diagnosed later. Specific anticancer therapies (e.g. R-CHOP, platinum combined with etoposide, and DNA methyltransferase inhibitors) were associated with high 30-day all-cause mortality. CONCLUSIONS: Clinical factors (e.g. older age, hematological malignancy, recent chemotherapy) and laboratory measurements were associated with poor outcomes among patients with cancer and COVID-19. Although further studies are needed, caution may be required in utilizing particular anticancer therapies. CLINICAL TRIAL IDENTIFIER: NCT04354701.

Indications of proton-dominated cosmic-ray composition above 1.6 EeV.

We report studies of ultrahigh-energy cosmic-ray composition via analysis of depth of air shower maximum (X(max)), for air shower events collected by the High-Resolution Fly's Eye (HiRes) observatory. The HiRes data are consistent with a constant elongation rate d/d[log(E)] of 47.9+/-6.0(stat)+/-3.2(syst) g/cm2/decade for energies between 1.6 and 63 EeV, and are consistent with a predominantly protonic composition of cosmic rays when interpreted via the QGSJET01 and QGSJET-II high-energy hadronic interaction models. These measurements constrain models in which the galactic-to-extragalactic transition is the cause of the energy spectrum ankle at 4x10(18) eV.

First observation of the Greisen-Zatsepin-Kuzmin suppression.

The High Resolution Fly's Eye (HiRes) experiment has observed the Greisen-Zatsepin-Kuzmin suppression (called the GZK cutoff) with a statistical significance of five standard deviations. HiRes' measurement of the flux of ultrahigh energy cosmic rays shows a sharp suppression at an energy of 6 x 10(19) eV, consistent with the expected cutoff energy. We observe the ankle of the cosmic-ray energy spectrum as well, at an energy of 4 x 10(18) eV. We describe the experiment, data collection, and analysis and estimate the systematic uncertainties. The results are presented and the calculation of the statistical significance of our observation is described.

Measurement of the flux of ultrahigh energy cosmic rays from monocular observations by the High Resolution Fly's Eye experiment.

We have measured the cosmic ray spectrum above 10(17.2) eV using the two air-fluorescence detectors of the High Resolution Fly's Eye observatory operating in monocular mode. We describe the detector, phototube, and atmospheric calibrations, as well as the analysis techniques for the two detectors. We fit the spectrum to a model consisting of galactic and extragalactic sources.

Tissue- and agonist-specific regulation of human and murine plasminogen activator inhibitor-1 promoters in transgenic mice.

Numerous studies have described regulatory factors and sequences that control transcriptional responses in vitro. However, there is a paucity of information on the qualitative and quantitative regulation of heterologous promoters using transgenic strategies. In order to investigate the physiological regulation of human plasminogen activator inhibitor type-1 (hPAI-1) expression in vivo compared to murine PAI-1 (mPAI-1) and to test the physiological relevance of regulatory mechanisms described in vitro, we generated transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the proximal -2.9 kb of the hPAI-1 promoter. Transgenic animals were treated with Ang II, TGF-beta1 and lipopolysaccharide (LPS) to compare the relative activation of the human and murine PAI-1 promoters. Ang II increased EGFP expression most effectively in brain, kidney and spleen, while mPAI-1 expression was quantitatively enhanced most prominently in heart and spleen. TGF-beta1 failed to induce activation of the hPAI-1 promoter but potently stimulated mPAI-1 in kidney and spleen. LPS administration triggered robust expression of mPAI-1 in liver, kidney, pancreas, spleen and lung, while EGFP was induced only modestly in heart and kidney. These results indicate that the transcriptional response of the endogenous mPAI-1 promoter varies widely in terms of location and magnitude of response to specific stimuli. Moreover, the physiological regulation of PAI-1 expression likely involves a complex interaction of transcription factors and DNA sequences that are not adequately replicated by in vitro functional studies focused on the proximal -2.9 kb promoter.

Student Research Award 1999: comparative contrast of voice measurements.

Currently, a variety of objective and subjective measures are available to describe voice and voice function. Despite these various tools, there is no standard measure of voice function that incorporates both objective and subjective measures. The goal of this research was to study the relationship between objective, subjective, and patient-based measures of voice function. Objective voice function was measured with 4 laboratory-based parameters (subglottic pressure, airflow at the lips, maximum phonation time, and vocal efficiency), subjective function with the GRBAS (grade, rough, breathy, asthenic, strained) scale, and patient-based function according to an overall global rating of quality. The objective and subjective measures were significantly related to each other (P < 0.05); the objective and patient-based measures were also related (P = 0.019), but the subjective and patient-based measures were not related. We demonstrate a significant relationship between some but not all measures of voice function. We believe that subjective measures provide additional valuable information not obtained from objective measures alone.

EXP3174, the AII antagonist human metabolite of losartan, but not losartan nor the angiotensin-converting enzyme inhibitor captopril, prevents the development of lethal ischemic ventricular arrhythmias in a canine model of recent myocardial infarction.

OBJECTIVES: The antiarrhythmic efficacies of the competitive angiotensin II (AII) antagonist losartan, losartan's more potent noncompetitive AII antagonist human metabolite EXP3174 and the angiotensin-converting enzyme inhibitor captopril were assessed in a canine model of recent myocardial infarction. BACKGROUND: Multiple hemodynamic and electrophysiologic effects of AII may contribute to cardiac electrical instability. In the recent Losartan Heart Failure Study, Evaluation of Losartan in the Elderly (ELITE), a 722-patient trial primarily designed to assess effects on renal function, an unexpected survival benefit was observed with losartan compared with captopril, with the lower mortality using losartan primarily confined to a reduction in sudden cardiac death. METHODS: Intravenous losartan (1 mg/kg + 0.03 mg/kg/min), EXP3174 (0.1 mg/kg + 0.01 mg/kg/min), captopril (1 mg/kg + 0.5 mg/kg/h) or vehicle were infused in anesthetized dogs with recent (8.1 +/- 0.4 days) anterior myocardial infarction. Electrolytic injury of the left circumflex coronary artery to induce thrombotic occlusion and posterolateral ischemia was initiated 1 h after the start of treatment. RESULTS: Losartan, EXP3174 and captopril elevated plasma renin activities and comparably and significantly reduced mean arterial pressure. No significant electrocardiographic or cardiac electrophysiologic effects were noted with any treatment. Incidences of acute posterolateral ischemia-induced lethal arrhythmias were: vehicle, 7/9 (77%); losartan, 6/8 (75%); EXP3174, 2/8 (25%; p < 0.05 vs. vehicle control); captopril, 7/10 (70%). There were no among-group differences in time to onset of acute posterolateral ischemia or underlying anterior infarct size. CONCLUSIONS: EXP3174, but not losartan nor captopril, reduced the incidence of lethal ischemic ventricular arrhythmia in this preparation. The antiarrhythmic efficacy of EXP3174 may be due to an attenuation of deleterious effects of local cardiac AII formed during acute myocardial ischemia or, alternatively, a non-AII-related activity specific to EXP3174. These findings suggest that in humans, metabolic conversion of losartan to EXP3174 may afford antiarrhythmic protection.

Immunocytochemical and in situ hybridization studies of gastrin after cisplatin treatment.

Cisplatin treatment (9 mg/kg) causes bloating of the stomach, an increase in gastric acid, and ulceration in rats. Gastrin, a gut peptide, plays an important role in regulating gastric acid production. To study the role of gastrin in this increased gastric acid production after cisplatin treatment, male Wistar rats (100-150 g) were treated with cisplatin (9 mg/kg) in five divided doses over 5 consecutive days. The rats were sacrificed 1, 6, 10, or 15 days after the last treatment. As measured by immunocytochemistry, in situ hybridization, Northern blot, and dot-blot techniques, gastrin was found to be below detectable limits just 1 day after cisplatin treatment. However, 10-15 days after the last injection, the levels for both gastrin and its mRNA gradually recovered to normal. Northern blot studies showed that decreased somatostatin mRNA parallels the changes of gastrin and its mRNA. These results suggest that after cisplatin treatment the increased gastric acid production in rat stomach is independent of gastrin. This decrease of gastrin production is not under the influence of somatostatin, which also decreased after cisplatin treatment.

Assessment of two objective voice function indices.

In the care of patients with voice disorders, physicians, speech pathologists, and other health care professionals routinely make diagnoses, recommend treatment, and evaluate outcomes. Although objective and subjective measures exist, unfortunately, there is no widely accepted, valid method for classifying voice disorders and assessing outcome after voice treatment. In the present research, the relationship between two previously created multivariate objective voice function indices, the weighted odds ratio index and the multivariate logistic regression index, and subjective assessment of voice function was evaluated. Twenty-three adult patients presenting to a speech science laboratory for evaluation of voice disorders were studied in this prospective observational study together with 12 normal volunteers as controls. Vocal function was measured on 14 different parameters with a protocol that included a multichannel input for simultaneous assessment of acoustic and physiological parameters. Each patient was recorded reading the standard passage "The North Wind and the Sun," and recordings were then evaluated by the GRBAS scale. Overall, there was a statistically significant relationship between the weighted odds ratio index and multivariate logistic regression index and mean GRBAS scores. This research demonstrates that the voice function values calculated from two different multivariate objective voice function indices are significantly associated with subjective voice assessments. These multivariate objective voice indices may be appropriate for use in clinical trials and outcomes research on treatment effectiveness for voice disorders.

Multivariate analysis of objective vocal function.

No standard and valid multidimensional index of objective voice function has been developed that integrates the information generated from the multiple objective parameters of voice function. The goals of this research were 1) to identify important objective voice parameters and 2) to create a multidimensional voice function index by combining relevant parameters. We evaluated 97 dysphonic patients and 35 normal volunteers on 14 objective voice parameters. Three multidimensional voice indices were created and evaluated: 1) nonweighted univariate index, 2) weighted odds ratio index, and 3) weighted multivariate regression index. The univariate index required all 14 parameters, while the odds ratio and logistic regression models required only 4 parameters (frequency range, airflow at lips, maximum phonation time, and subglottic pressure). The chi2 values for the 3 models were 37.8, 37.6, and 46.0, respectively. All 3 indices were able to satisfactorily classify voice function as normal or abnormal. However, the regression index performed best.

Skill acquisition and competency testing for physical agent modality use.

OBJECTIVE: The use of physical agent modalities is not considered an entry-level skill and requires postprofessional education, according to the American Occupational Therapy Association. The purpose of this survey was to determine how occupational therapy practitioners who use physical agents modalities are trained. METHOD: Two hundred fifty occupational therapists were randomly selected from the American Occupational Therapy Association's Physical Disabilities Special Interest Section. The practitioners were surveyed about their use of, education in, competency testing for, and opinions on eight physical agent modalities. RESULTS: Results were based on 100 responses (40% response rate). Of the eight modalities, the most commonly used were hot and cold packs, and the least were transcutaneous electrical nerve stimulators. The most common means of education was on-the-job training, and the least common was higher level accredited education. A majority (71) of respondents reported that no competency testing was being performed at their facilities. In the remaining facilities (29), the majority performed competency tests with no routine frequency, using no particular guidelines for testing and no formal methods for maintaining standards for physical agent modality use. Eighty-five respondents indicated they would be interested in attending continuing education programs on the use of physical agent modalities, and 88 believed that functional activities should follow the use of physical agent modalities within the same treatment session. CONCLUSION: The occupational therapy profession may need specific educational and competency guidelines to assure the qualifications of therapists using physical agent modalities.

Residual Alkaline Phosphatase Activity in Milks Subjected to Various Time-Temperature Treatments.

Milk is routinely tested for proper pasteurization. The Scharer and Fluorophos methods, among others, test for residual alkaline phosphatase (ALP) activity to assure proper pasteurization. Until recently there were no tests available to accurately detect residual ALP activity levels below the U.S. legal limit of 1 µg of phenol or 350 mU of ALP per liter of milk. The new Fluorophos method can detect accurately residual ALP activity levels as low as 10 mU/liter. The Fluorophos method was used to investigate residual ALP activity levels in several fluid milk products. The milk products were thermally processed under various time and temperature protocols below, at, and above current U.S. Food and Drug Administration-mandated heat treatments for fluid milk and milk products. The data established values for residual ALP activity in milks pasteurized under high-temperature short-time (HTST) and low-temperature long-time (LTLT) treatments. The mean ALP activities for whole, 2% lowfat, 1% lowfat, skim, half and half, and chocolate-flavored milks thermally processed at the legal minimum HTST pasteurization treatment are 169.7 ± 12.3, 145.2 ± 9.3, 98.6 ± 8.9, 72.5 ± 4.2, 38.4 ± 4.6 and 157.3 ± 6.5 mU/liter, respectively. The mean ALP activities generated at the legal minimum LTLT pasteurization treatment are 81.8 ± 4.8, 66.4 ± 5.9, 56.4 ± 2.1, 39.1 ± 3.9, 35.0 ± 1.2 and 91.3 ± 7.7 mU/liter, respectively. The values for all milks pasteurized at the legal minimum heat treatment were significantly below the current legal cutoff for residual ALP activity of 350 mU/liter of milk or milk product.

Characterization and chromosomal localization of PTPRO, a novel receptor protein tyrosine phosphatase, expressed in hematopoietic stem cells.

Hematopoietic stem cells (HSCs) support blood cells throughout life by utilizing their self-renewing and multilineage differentiating capabilities. Hematopoietic growth factors mediate their effects on stem cells by the tyrosine phosphorylation of proteins. Regulation of tyrosine phosphorylation is partially mediated by protein tyrosine phosphatases (PTPases). A possible mechanism by which hematopoietic stem cells maintain their self-renewing capacity and undifferentiated state is by controlling the balanced and opposing actions of protein tyrosine kinases (PTKs), receptors for growth factors, and PTPases. We have characterized the expression of PTPases in 5-fluorouracil (5-FU)-treated murine bone marrow cells, which represent a very primitive population of progenitors enriched for reconstituting stem cells, by using a consensus polymerase chain reaction (PCR) method. Several PTPases were expressed abundantly in the 5-FU-treated bone marrow stem cells. A novel PTP, termed protein tyrosine phosphatase receptor omicron (PTPRO), which is related to the homotypically adhering kappa, mu and PCP-2 receptor-type tyrosine phosphatases, was identified and characterized. We have cloned the murine and full-length human PTPRO cDNAs which share 89% homology, indicating that PTPRO is highly conserved between these species. The human PTPRO cDNA clone encodes a polypeptide of 1439 amino acids (aa) and has a calculated molecular mass of approximately 162 kDa. PTPRO consists of an extracellular segment containing a MAM domain, an immunoglobulin (Ig) domain, four fibronectin-type III (FN-III) repeats, a transmembrane segment, and two tandem intracellular PTP domains. The human PTPRO gene was assigned to human chromosome 1p35-pter using Southern blot analyses of genomic DNAs from rodent/human somatic hybrid cell lines containing human chromosome 1 or the p35-pter region of the chromosome. The mouse Ptpro gene was mapped to chromosome 4, closely linked to D4Mit16 and Elp1 (elliptocytosis-1), by using genomic DNAs from a (C57BL/6J x Mus spretus)F1 x Mus spretus backcross. In fetal tissues, PTPRO expression was observed in the brain and lung, whereas lower levels were observed in the kidney. In adult tissues, PTPRO was less restricted and was observed in the lung, heart, skeletal muscle, prostate, testis, and in various areas of the brain, indicating that PTPRO expression is developmentally regulated. Expression of PTPRO was also observed in human CD34+ bone marrow cells and 5-FU-treated murine primitive stem cells. These results suggest a potential role for PTPRO in stem cell adhesion and in mediating homophilic cell-cell interactions in other cell types.

Effect of hematocrit on systemic O2 transport in hypoxic and normoxic exercise in rats.

The effect of hematocrit (Hct) on O2 transport in hypoxic [inspired PO2 (PIO2) approximately 70 Torr] and normoxic (PIO2 approximately 145 Torr) exercise was studied in rats acclimatized to 3 wk of PIO2 at approximately 70 Torr (A rats) and in nonacclimatized littermates (NA rats). Isovolumic exchange transfusion of plasma or red blood cells was used to lower Hct in A rats from approximately 60 to 45% and to raise Hct of NA rats from 45 to 60%: Controls were A and NA rats exchange transfused with whole blood at constant Hct. Lowering Hct of A rats lowered the arterial O2 concentration (CaO2) and the arterial-mixed venous O2 difference and increased the maximal cardiac output (Qmax) without changes in maximal O2 uptake (VO2 max) or in the product of Qmax x CaO2, circulatory O2 convection at maximal exercise (TO2 max). Raising Hct in NA rats produced the opposite changes in CaO2, arterial-mixed venous O2 difference, and Qmax, but VO2 max and TO2 max increased significantly, both in hypoxia and normoxia, because of relatively small changes in Qmax. In NA rats, a steeper slope of the line relating VO2 max to calculated mean capillary PO2 at high Hct suggested a higher tissue O2 diffusing capacity with high Hct. For a given Hct and Qmax, systemic arterial pressure was higher in A rats. The data suggest that 1) the effect of Hct on systemic hemodynamics is different in A and NA rats, resulting in different effects on VO2 max; 2) factors in addition to Hct contribute to the high systemic vascular resistance of A rats; and 3) increased diffusive conductance for O2, as well as increased TO2 max, could be responsible for the effect of Hct on VO2 max of NA rats.

Regulation of phagocytosis and [Ca2+]i flux by distinct regions of an Fc receptor.

The binding of multivalent immunoglobulin G complexes to Fc receptors (Fc gamma Rs) on macrophages activates multiple immune functions. A murine macrophage cell line, but not a fibroblast cell line, that was transfected with human Fc gamma RIIA mediated phagocytosis and an intracellular Ca2+ concentration ([Ca2+]i) flux upon cross-linking of human Fc gamma RIIA. Transfected macrophages that expressed a truncated receptor lacking 17 carboxy-terminal amino acids phagocytosed small antibody complexes. However, only wild-type transfectants phagocytosed labeled erythrocytes and fluxed [Ca2+]i. Thus, the cytoplasmic domain of human Fc gamma RIIA contains distinct functional regions.

Semi-automated voice evaluation.

This report describes a voice evaluation procedure that in some way parallels the audiologic tests used for hearing and has multiple uses both clinically and in research. It uses a simultaneous eight-channel input, is not difficult to use, requiring between 12 and 25 minutes to administer, and provides the physician with a printout in standardized form before the patient leaves the room. This three-page report includes 15 abstracted or calculated values, normal ranges by sex for each value, notes that draw attention to deviations from the normal, a summary profile, a graphic representation of the evaluation, and raw data waveforms.

The laryngeal vestibule, voice quality and paralinguistic markers.

The laryngeal component of voice quality markers has been quantified in the present study, suggesting that the laryngeal vestibule and lower pharynx play an important role in voice quality. Findings also show that voice quality can be partly described in terms of laryngeal configurations and that a knowledge of these configurations may be useful to the laryngologist, speech pathologist and singer. Twenty-five voice qualities were videorecorded, using a nasal fiberscope. Still photographs were taken for each voice quality and distance measurements made on each one for 15 laryngeal parameters. The raw data were normalized, sorted from high to low, turned into scalar values and processed to establish which parameters exhibited similar functions, which photographs were essentially identical, and in what respect any two photographs were different. Each voice quality was seen to be associated with a different, describable and quantifiable laryngeal configuration.

Laryngeal configuration of 99 dysphonic patients.

The aim of this investigation was to study a wide range of dysphonic patients and determine the best matches among the laryngeal configurations on phonation for each patient and those previously established for control data, which were obtained from a professional voice user producing the whole range of voice qualities. Ninety-nine patients were selected and laryngeal photographs were produced for each patient. Fifteen laryngeal parameters were quantified and normalized. The data were sorted, scalar values assigned and a measure of similarity between configurations applied. The best, second-best, third-best and worst matches between each patient separately and the control data were then examined. Although 41% of the patients did not have particularly unusual configurations, 59% exhibited a narrowing of the laryngeal vestibule caused by epiglottic retraction, cuneiform fronting and/or false fold adduction. This suggests that clinical reports for patients with dysphonia should contain information not only on any lesions present but also on laryngeal configurations and, in particular, vestibular narrowing.

Electroglottogram waveform types of untrained speakers.

Electroglottography is a useful, non-invasive technique that can assist in the assessment of vocal fold dysfunction. However, if it is to become a useful clinical tool, there is a need for normative studies of the electroglottogram waveform types that characterize different groups of speakers. This report compares the electroglottogram waveform types characterizing one trained professional voice user phonating in 15 experimental sessions under various fundamental frequencies, intensities and voice qualities with those obtained from 52 untrained non-professional speakers.