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Objective: Recent evidence suggests that the fimbriated end of the fallopian tube harbors the precursor cells for many high-grade ovarian cancers, opening the door for development of better screening methods that directly assess the fallopian tube in women at risk for malignancy. Previously we have shown that the karyometric signature is abnormal in the fallopian tube epithelium in women at hereditary risk of ovarian cancer. In this study, we sought to determine whether the karyometric signature in serous tubal intraepithelial carcinoma (STIC) is significantly different from normal, and whether an abnormal karyometric signature can be detected in histologically normal tubal epithelial cells adjacent to STIC lesions., Methods: The karyometric signature was measured in epithelial cells from the proximal and fimbriated portion of the fallopian tube in fallopian tube specimens removed from women at: 1) average risk for ovarian cancer undergoing surgery for benign gynecologic indications (n = 37), 2) hereditary risk of ovarian cancer (germline BRCA alterations) undergoing risk-reducing surgery (n = 44), and 3) diagnosed with fimbrial STICs (n = 17)., Results: The karyometric signature in tubes with fimbrial STICs differed from that of tubes with benign histology. The degree of karyometric alteration increased with increasing proximity to fimbrial STICs, ranging from moderate in the proximal portion of the tube, to greatest in both normal appearing fimbrial cells near STICs as well as in fimbrial STIC lesions., Conclusion: These data demonstrate an abnormal karyometric signature in STICs that may extend beyond the STIC, potentially providing an opportunity for early detection of fallopian tube neoplasia., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Difference-in-difference (DID) estimators are a valuable method for identifying causal effects in the public health researcher's toolkit. A growing methods literature points out potential problems with DID estimators when treatment is staggered in adoption and varies with time. Despite this, no practical guide exists for addressing these new critiques in public health research. We illustrate these new DID concepts with step-by-step examples, code, and a checklist. We draw insights by comparing the simple 2 × 2 DID design (single treatment group, single control group, two time periods) with more complex cases: additional treated groups, additional time periods of treatment, and treatment effects possibly varying over time. We outline newly uncovered threats to causal interpretation of DID estimates and the solutions the literature has proposed, relying on a decomposition that shows how the more complex DIDs are an average of simpler 2 × 2 DID subexperiments.

Significance: Most cases of high-grade serous ovarian carcinoma originate as serous tubal intraepithelial carcinoma (STIC) lesions in the fallopian tube epithelium (FTE), enabling early endoscopic detection., Aim: The cell-acquiring fallopian endoscope (CAFE) was built to meet requirements for locating potentially pathological tissue indicated by an alteration in autofluorescence or presence of a targeted fluorophore. A channel was included for directed scrape biopsy of cells from regions of interest., Approach: Imaging resolution and fluorescence sensitivity were measured using a standard resolution target and fluorescence standards, respectively. A prototype was tested in ex vivo tissue, and collected cells were counted and processed., Results: Measured imaging resolution was 88  μm at a 5-mm distance, and full field of view was ∼45  deg in air. Reflectance and fluorescence images in ex vivo porcine reproductive tracts were captured, and fit through human tracts was verified. Hemocytometry counts showed that on the order of 105  cells per scrape biopsy could be collected from ex vivo porcine tissue., Conclusions: All requirements for viewing STIC in the FTE were met, and collected cell counts exceeded input requirements for relevant analyses. Our benchtop findings suggest the potential utility of the CAFE device for in vivo imaging and cell collection in future clinical trials.

A chemopreventive effect of aspirin (ASA) on lung cancer risk is supported by epidemiologic and preclinical studies. We conducted a randomized, double-blinded study in current heavy smokers to compare modulating effects of intermittent versus continuous low-dose ASA on nasal epithelium gene expression and arachidonic acid (ARA) metabolism. Fifty-four participants were randomized to intermittent (ASA 81 mg daily for one week/placebo for one week) or continuous (ASA 81 mg daily) for 12 weeks. Low-dose ASA suppressed urinary prostaglandin E2 metabolite (PGEM; change of -4.55 ± 11.52 from baseline 15.44 ± 13.79 ng/mg creatinine for arms combined, P = 0.02), a surrogate of COX-mediated ARA metabolism, but had minimal effects on nasal gene expression of nasal or bronchial gene-expression signatures associated with smoking, lung cancer, and chronic obstructive pulmonary disease. Suppression of urinary PGEM correlated with favorable changes in a smoking-associated gene signature ( P < 0.01). Gene set enrichment analysis (GSEA) showed that ASA intervention led to 1,079 enriched gene sets from the Canonical Pathways within the Molecular Signatures Database. In conclusion, low-dose ASA had minimal effects on known carcinogenesis gene signatures in nasal epithelium of current smokers but results in wide-ranging genomic changes in the nasal epithelium, demonstrating utility of nasal brushings as a surrogate to measure gene-expression responses to chemoprevention. PGEM may serve as a marker for smoking-associated gene-expression changes and systemic inflammation. Future studies should focus on NSAIDs or agent combinations with broader inhibition of pro-inflammatory ARA metabolism to shift gene signatures in an anti-carcinogenic direction., (©2019 American Association for Cancer Research.)

A large body of epidemiologic evidence has shown that use of progestin-containing preparations lowers ovarian cancer risk. The purpose of the current study was to gather further preclinical evidence supporting progestins as cancer chemopreventives by demonstrating progestin-activation of surrogate endpoint biomarkers pertinent to cancer prevention in the genital tract of women at increased risk of ovarian cancer. There were 64 women enrolled in a multi-institutional randomized trial who chose to undergo risk-reducing bilateral salpingo-oophorectomy (BSO) and to receive the progestin levonorgestrel or placebo for 4 to 6 weeks prior to undergoing BSO. The ovarian and fallopian tube epithelia (FTE) were compared immunohistochemically for effects of levonorgestrel on apoptosis (primary endpoint). Secondary endpoints included TGFβ isoform expression, proliferation, and karyometric features of nuclear abnormality. In both the ovary and fallopian tube, levonorgestrel did not confer significant changes in apoptosis or expression of the TGFβ1, 2, or 3 isoforms. In the ovarian epithelium, treatment with levonorgestrel significantly decreased the proliferation index. The mean ovarian Ki-67 value in the placebo arm was 2.027 per 100 cells versus 0.775 per 100 cells in the levonorgestrel arm (two-sided P value via Mann-Whitney U test = 0.0114). The karyometric signature of nuclei in both the ovarian and FTE deviated significantly from normal controls (women at average risk of ovarian cancer), but was significantly less abnormal in women treated with levonorgestrel. These karyometric data further support the idea that progestins may clear genetically abnormal cells and act as chemopreventive agents against ovarian and fallopian tube cancer., (©2019 American Association for Cancer Research.)

Prevention of nonmelanoma skin cancers remains a health priority due to high costs associated with this disease. Diclofenac and difluoromethylornithine (DFMO) have demonstrated chemopreventive efficacy for cutaneous squamous cell carcinomas. We designed a randomized study of the combination of DFMO and diclofenac in the treatment of sun-damaged skin. Individuals with visible cutaneous sun damage were eligible. Subjects were randomized to one of the three groups: topical DFMO applied twice daily, topical diclofenac applied daily, or DFMO plus diclofenac. The treatment was limited to an area on the left forearm, and the duration of use was 90 days. We hypothesized that combination therapy would have increased efficacy compared with single-agent therapy. The primary outcome was change in karyometric average nuclear abnormality (ANA) in the treated skin. Individuals assessing the biomarkers were blinded regarding the treatment for each subject. A total of 156 subjects were randomized; 144 had baseline and end-of-study biopsies, and 136 subjects completed the study. The ANA unexpectedly increased for all groups, with higher values correlating with clinical cutaneous inflammation. Nearly all of the adverse events were local cutaneous effects. One subject had cutaneous toxicity that required treatment discontinuation. Significantly more adverse events were seen in the groups taking diclofenac. Overall, the study indicated that the addition of topical DFMO to topical diclofenac did not enhance its activity. Both agents caused inflammation on a cellular and clinical level, which may have confounded the measurement of chemopreventive effects. More significant effects may be observed in subjects with greater baseline cutaneous damage., (©2015 American Association for Cancer Research.)

Objectives: Treatment for atypical endometrial hyperplasia (AEH) is based on pathologic diagnosis. About 40% of AEH is found to be carcinoma at surgery. This study's objective is to derive an objective characterization of nuclei from cases diagnosed as AEH or superficially invasive endometrial cancer (SIEC)., Methods: Cases from GOG study 167A were classified by a central pathology committee as AEH (n=39) or SIEC (n=39). High resolution digitized images of cell nuclei were recorded. Features of the nuclear chromatin pattern were computed. Classification rules were derived by discriminant analysis., Results: Nuclei from cases of AEH and SIEC occupy the same range on a progression curve for endometrial lesions. Cases of AEH and SIEC both comprise nuclei of two phenotypes: hyperplastic characteristics and premalignant/neoplastic characteristics. The principal difference between AEH and SIEC is the percentage of premalignant/neoplastic nuclei. When this percentage approaches 50-60% superficial invasion is likely. SIEC may develop already from lesions at the low end of the progression curve., Conclusions: AEH comprises cases which may constitute a low risk group involving <40% of AEH cases. These cases hold a percentage of <20% of nuclei of a preneoplastic phenotype. AEH cases from the central and high end of progression have >40% of nuclei of preneoplastic phenotype. Nuclei of the preneoplastic phenotype in AEH lesions are almost indistinguishable from nuclei in SIEC, where this percentage exceeds 60%. The percentage of nuclei of the preneoplastic phenotype in AEH esions might serve as criterion for assessment of risk for the development of invasive disease., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: To establish the karyometric characteristics of the two main nuclear phenotypes in cutaneous squamous cell cancer (cSCC) lesions., Study Design: The clinical materials comprised 75 cases of cSCC, 38 with aggressive lesions and 37 with nonaggressive lesions. High-resolution images of 100 nuclei per case were recorded. Data were partitioned into four subgroups covering the range of lesion progression. Four discriminant functions were derived to distinguish aggressive from nonaggressive lesions. The most typical nuclei from the phenotype predominant in aggressive lesions and nonaggressive lesions were separated out by thresholding on the discriminant function score axes. For these homogeneous sets of nuclei the karyometric features were computed., Results: The nuclear populations in cSCC lesions are a very heterogeneous set. There are two axes of dispersion, along the line of lesion progression and between aggressive and nonaggressive lesions. The analysis faces the difficulty that lesions from both diagnostic categories contain nuclei of the same two phenotypes with the difference between categories consisting only of differences in proportion of the two phenotypes., Conclusion: The nuclei of the aggressive phenotype I and nonaggressive phenotype II have substantially different chromatin patterns and can be distinguished with > 90% correct recognition rate.

Objective: To determine whether low-dose topical applications of difluoromethylornithine (DFMO) with or without Triamcinolone (Fougena, Melville, New York, U.S.A.) to moderately sun-damaged skin with actinic skin keratoses are efficacious., Study Design: There were 4 topically administered, 6-month treatments, DFMO + Eucerin (Beiersdorf Inc., Hamburg, Germany), DFMO + Triamcinolone, Triamcinolone + Eucerin and Eucerin + Eucerin (to serve as double placebo). Participant eligibility included evidence of at least 2 actinic keratoses on each posterolateral forearm as well as moderate to severe evidence of sun-damaged skin, as evaluated by a board certified dermatologist. High resolution digitized imagery of nuclei from histologic sections of 4-mm punch biopsies from sun-damaged skin on the posterolateral forearms was recorded, at baseline and at the end of 6 months of study., Results: With 102 participants and 185 skin biopsies, a total of 16,395 skin cell nuclei were recorded. The nuclei were analyzed to assess the changes in the pattern of the nuclear chromatin. Two specific measures of end point evaluation were computed, including the percentage of nuclei with high values of nuclear abnormality and the reduction of the percentage of nuclei assigned by a discriminant function to the baseline data set. All 3 active interventions, including low-dose topical DFMO, topical Triamcinolone and topical DFMO + Triamcinolone, led to statistically significant reductions of both the number of nuclei with high nuclear abnormality as well as the number of nuclei assigned to the baseline data set. These reductions were found for all 3 treatments involving DFMO or Triamcinolone. For the placebo data sets only small, statistically insignificant increases or decreases of these percentages were observed., Conclusion: The low-dose, topical drug interventions were all effective in reducing skin biopsy nuclear abnormality by a statistically significant 15-20%, whereas there was no evidence of a double placebo effect by karyometric assessment. These effects were greater than the case-to-case sampling error.

Aromatase inhibitors are currently being evaluated as preventive agents in post-menopausal women at high risk for breast cancer. A phase II trial of 42 women on hormone replacement therapy (HRT) treated with letrozole for 6 months showed Ki-67 was reduced by 66% but showed no change in cytomorphology or Masood score. Subsequent image analytical procedures (karyometry) conducted on a subset of the samples captured subvisual information that showed reduced cellular abnormality after 6 months of letrozole. In the present study we expanded on the preliminary karyometry study to determine if the change in karyometric measurements corresponded to changes in risk biomarkers quantified in the Phase II trial; and secondly, whether these biomarkers might be used together to serve as markers of response in individual cases. Pap stained slides from the Phase II trial were used. Epithelial cell images were digitized on a CCD video-microphotometer and the nuclei were segmented from the field using a semiautomatic algorithm. Nine out of 37 cases analyzed showed a numerical decrease in all three markers, although only three of these exhibited changes substantial enough to be considered as an improvement. However, 12 cases showed improvement by cytology (a decrease in Masood score of at least 2), an additional 13 cases demonstrated a reduction in Ki-67 expression by 50% of the median baseline value, and an additional five cases exhibited a decrease of at least 10% in abnormal cells by nuclear morphometry. Thus, a total of 30 of 37 cases (81%) showed improvement in at least one marker. There was no correlation between changes in Ki-67%, karyometric abnormality, and Masood score change other than specimens that exhibited an improvement in cytology also displayed greater decreases in nuclear morphometry abnormalities. Given the heterogeneity of mechanisms leading to malignancy, the quantitative analysis of nuclear chromatin patterns may be valuable as a global, or integrating, biomarker of change in chemoprevention studies in conjunction with additional markers. Correlation with long term clinical outcome is needed to validate meaningful combinations of informative biomarkers.

Objective: To assess the changes in the nuclear chromatin pattern concomitant with progressive sun damage in skin biopsies ranging from sun-exposed, normal-appearing skin to squamous cell carcinoma (SCC)., Study Design: Biopsies were taken from 140 cases with sun-exposed but histopathologically normal skin, from 20 cases visually assessed as pre-actinic keratosis (pre-AK) or early AK, from 30 cases of AK, and from 21 cases of SCC. A total of 21,094 nuclei were recorded from these biopsies. High-resolution digital imagery was recorded, and features descriptive of the nuclear chromatin pattern were computed. Both supervised learning and unsupervised learning algorithms were employed to derive progression plots., Results: With increased sun exposure, the proportion of nuclei exhibiting changes in the nuclear chromatin pattern rises notably. Using karyometry, no significant differences could be substantiated between nuclei collected from early AK sites and AK lesions. Cases of SCC fell into 2 distinct groups. A larger group (approximately 66.7% of cases) had characteristics similar to AK. A smaller group (approximately 33.3% of cases) represented much more progressed lesions., Conclusion: Karyometric assessment can provide a numeric measure of progression for sun damage and of the deviation from normal in both AK and SCC lesions.

Objective: To establish measures of sun damage in histopathologically normal skin., Study Design: Biopsies were taken from the upper inner arm, representing skin with presumably minimum sun exposure, from skin of the forearm with no visible sun damage, from skin of the forearm with visible sun damage and from normal-appearing skin from the forearm of individuals who had sun exposure that had resulted in actinic keratosis (AK) lesions. In addition, a data set of nuclei from AKs was recorded., Results: In histopathologically normal skin, monotonically increasing damage was observed in individuals with increased exposure to solar radiation., Conclusion: Karyometry can detect and statistically secure changes in skin due to solar exposure at a stage at which the skin is histopathologically determined to be normal.

Objective: The study summarizes results of karyometric measurements in epithelial cells of the colorectal mucosa to document evidence of a field effect of preneoplastic development among patients with colorectal adenocarcinoma or adenoma., Methods: Karyometric analyses were done on high-resolution images of histologic sections from 48 patients with colorectal adenocarcinomas and 44 patients with adenomas and on images from matching normal-appearing mucosa directly adjacent to such lesions, at a 1-cm and 10-cm distance from the lesions or from the rectal mucosa of adenoma patients, as well as from 24 healthy normal controls with no family history of colonic disease., Results: The nuclei recorded in the histologically normal-appearing mucosa of patients with either colorectal adenoma or adenocarcinoma exhibited differences in karyometric features in comparison with nuclei recorded in rectal mucosa from patients who were free of a colonic lesion. These differences were expressed to the same extent in tissue adjacent to the lesions and in normal-appearing tissue as distant as the rectum., Conclusions: The nuclear chromatin pattern may serve as an integrating biomarker for a preneoplastic development. The field effect might provide an end point in chemopreventive intervention trials.

Objective: To determine whether karyometric measurements taken in biopsies from histologically normal-appearing rectal mucosa could serve as a biomarker for the risk of recurrence of polyps., Materials and Methods: Biopsies were taken from the rectal mucosa of cases with a prior history of colonic polyps at the baseline of the study. In 57 cases recurrent polyps occurred (R cases); in 72 cases no recurrent disease was found at the end of the study (NR cases). From each biopsy 100 nuclei were recorded at high resolution. After segmentation, feature extraction and selection of a discriminating subset of features, a number of discriminant functions were derived. Also, measures of nuclear abnormality were computed., Results: The differences in karyometricfeature values for nuclei from biopsies of cases with recurrent or nonrecurrent disease were very small and not notably expressed in the majority of nuclei. It was possible by focusing on nuclei showing clear deviations from normal to derive a discriminant function that exhibited a shift for the NR and R data sets. The distributions of discriminant function scores were then subjected to a second-order discriminant analysis to separate cases according to recurrence status. This function showed a statistically highly significant correlation with recurrence. At one extreme of its score distribution were 11 of 57 cases that had a recurrence, and at the other extreme were 8-10 of 72 cases that had no recurrence. The distributions of nuclear abnormality values for these subsets of cases were drastically different, with an average value of 1.72 for the group that may be at high risk for another recurrence and 1.02 for the group possibly at low risk. All cases with a prior history of colonic polyps showed a nuclear abnormality deviating from normal., Conclusion: Measurement of a sample of 100 nuclei from the rectal mucosa will suggest, for approximately 10% of cases, that a high risk for recurrence of adenomatous polyps exists and, for a slightly lower proportion, confirm that the nuclei deviate only slightly from those from individuals with no history of colonic polyps. For the majority of cases with a prior history of adenoma, the nuclei in the biopsy show a notable deviation from normal, but the deviation is practically the same for cases that had a recurrence and those that did not. However, a tentative discriminant function (DF I,3) derived from the characteristics of the extreme cases correctly classified approximately 64% of nonrecurrent and 83% of recurrent cases using a Bayesian decision boundary.

Corynebacterium pseudotuberculosis phospholipase D (PLD) significantly affected viability of ovine neutrophils after 24 hours' exposure. This effect was more marked in cells that ingested PLD emulsified in oil. Treatment of neutrophils with PLD significantly (P < 0.05) reduced the ability of these cells to migrate toward activated sheep serum. The PLD was not chemotactic, but it activated normal sheep serum, producing factors that were chemotactic for neutrophils.

After intravenous infection with Candida albicans, rats received daily doses of itraconazole for 3 days. All rats receiving 2.5 mg kg-1 day-1 survived while all rats receiving sham-treatment died. With subsequent reduction of the itraconazole dose to 0.63 mg kg-1 day-1, no survival occurred and mortality rates equalled those of the control group.

To assess the relationships of various symptoms and other early findings to the diagnosis of primary coccidioidomycosis, we devised a 40-question survey that was completed by 556 college students seeking medical care for illness possibly due to Coccidioides immitis. The results of routine laboratory studies on these patients were also compiled. Of 269 who had coccidioidal antibody determinations and other diagnostic tests, coccidioidomycosis was diagnosed in 36 (13%). By logistic regression procedures, an elevated erythrocyte sedimentation rate, male gender, "red lumps on shins," recent arrival to an endemic area, acuteness of symptoms, and decreased total peripheral blood lymphocyte counts were independent factors positively associated with infection (P less than .05). Relative risk analysis indicated that 60% of patients with four or more of these factors were found to have coccidioidomycosis. Other significantly but not independently associated factors were an increased total leukocyte count, chest pain with breathing, fever, an absence of hoarseness, and an abnormal chest roentgenogram.