Michael Yozwiak

A Phase 2a Study of Topical Perillyl Alcohol Cream for Chemoprevention of Skin Cancer

The chemopreventive and antitumor properties of perillyl alcohol (POH) that were studied preclinically indicate that topical POH inhibits both UVB-induced murine skin carcinogenesis (squamous cell tumor models) and 7,12-dimethylbenz(a)anthracene–induced murine melanoma (transgenic models involving tyrosinase-driven Ras). A previous phase 1 clinical trial in participants with normal-appearing skin showed that topical POH cream was well tolerated at a dose of 0.76% (w/w). Here, we performed a 3-month, double-blind, randomized, placebo-controlled phase 2a trial of two different doses of topical POH in individuals with sun-damaged skin. Participants applied POH cream twice daily to each dorsal forearm. Baseline and end-of-study biopsies were taken from each participant to evaluate whether the topical application of POH was effective in reversing actinic damage as evidenced by normalization of quantitative skin histopathologic scores and change in nuclear chromatin pattern as measured by karyometric analysis. There was a borderline reduction in the histopathologic score of the lower-dose POH group compared with the placebo (P = 0.1), but this was not observed in the high-dose group. However, in the high-dose group, a statistically significant reduction in the proportion of nuclei deviating from normal was observed by the use of karyometric analysis (P < 0.01). There was no statistical significance shown in the lower-dose group. No changes were observed in p53 expression, cellular proliferation (by proliferating cell nuclear antigen expression), or apoptosis in either treatment group compared with the placebo group. These results suggest that whereas our karyometric analyses can detect a modest effect of POH in sun-damaged skin, improved delivery into the epidermis may be necessary. Cancer Prev Res; 3(2); 160–9

Karyometry of the Colonic Mucosa

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. (Cancer Epidemiol Biomarkers Prev 2007;16(12):2704–16)

Nuclear Morphometry Identifies a Distinct Aggressive Cellular Phenotype in Cutaneous Squamous Cell Carcinoma

By identifying aggressive cutaneous squamous cell carcinoma (cSCC) in patients who are at high risk for recurrences or second primaries after resection, intensive surveillance and therapy may decrease morbidity and mortality. We investigated the role of nuclear morphometry (karyometry) in differentiating between aggressive and nonaggressive cSCC. We retrospectively analyzed cSCC lesions from 40 male patients. Twenty-two patients had evidence of aggressive cSCC (local/regional recurrence or a second primary cSCC), and 18 patients were identified with similar ages and sites of disease as control patients with nonaggressive cSCC (no evidence of recurrence, metastasis, or second primary). We carried out karyometric analysis to identify nuclear features that discriminate between aggressive and nonaggressive cSCC nuclei. We used statistically significant differences (Kruskal–Wallis test, P < 0.0001) to compose a quantitative aggressive classification score (proportion of aggressive nuclei from 0% to 100%). For comparisons, we used Fishers exact test or Students t test. The mean age was 79 ± 7 years for aggressive cSCC and 80 ± 9 years for nonaggressive cSCC (P = 0.66). We analyzed a mean of 96 nuclei in each group. The mean classification score for aggressive cSCC was significantly higher (69% ± 6%) than for nonaggressive cSCC (28% ± 5%, P = 0.00002). Overall, the classification score accurately categorized 80% of our patients (P = 0.0004). In most patients, karyometry differentiated between aggressive and nonaggressive cSCC. We found that classification scores, which provide information on individual lesions, could be used for risk stratification. Cancer Prev Res; 4(11); 1770–7. ©2011 AACR.

Phase IIB Randomized Study of Topical Difluoromethylornithine and Topical Diclofenac on Sun-Damaged Skin of the Forearm

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. Cancer Prev Res; 9(2); 128–34. ©2015 AACR. See related article by Tsai and Hawk, p. 125

Karyometry in atypical endometrial hyperplasia: A Gynecologic Oncology Group study

OBJECTIVES Treatment for atypical endometrial hyperplasia (AEH) is based on pathologic diagnosis. About 40% of AEH is found to be carcinoma at surgery. This studys 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.

Quantitative histopathology identifies patients with thin melanomas who are at risk for metastases.

This small exploratory study was designed to test the hypothesis that thin melanoma lesions contain nuclei of two similar phenotypes, in different proportions. In lesions likely to progress to metastatic disease, one of these phenotypes predominates. Histopathological sections from 18 cases of thin melanomas which did not progress to metastasis, and from 10 cases which did progress were imaged and digitized at high resolution, with a total of 2084 and 1148 nuclei, respectively, recorded. Five karyometric features were used to discriminate between nuclei from indolent and from potentially metastatic lesions. For each case, the percentage of nuclei classified by the discriminant function as having come from a potentially metastatic lesion was determined and termed as case classification criterion. Standard histopathological criteria, such as ulceration and high mitotic index, indicated in this material the need for intensive therapy for only one of the 10 participants, as compared with 7/10 identified correctly by the karyometric measure. Using a case classification criterion threshold of 40%, the overall accuracy was 86% in the test set. The proportion of nuclei of an aggressive phenotype may lend itself as an effective prognostic clue for thin melanoma lesions. The algorithm developed in this training set appears to identify those patients at high risk for metastatic disease, and demonstrates a basis for a further study to assess the utility of prognostic clues for thin melanomas.

Limits of Detection of Chemopreventive Efficacy: Karyometry of Skin Biopsies

Objective: This study was designed to establish estimates of the smallest effects due to chemopreventive intervention detectable by karyometry in skin biopsies. Methods: Estimates of the smallest change of statistical significance and estimates of the power of the test were derived for several key features descriptive of the distribution of nuclear chromatin. Results from triplicate biopsies from the same case were used to provide estimates of the within-case, biopsy-to-biopsy variance. Results: Generally, a change in feature value due to chemopreventive intervention can be statistically secured when it amounts to 5% to 10%. In clinical trials where matched baseline and end of study biopsies from the same cases are available, paired comparison ANOVA can detect a 2% change on samples of 25 cases. Establishing efficacy in individual cases requires a change in feature values on the order of 10% to 15%. Conclusions: Karyometry provides a sensitive, quantitative method for the assessment of efficacy of chemoprevention. The effects of within-case, biopsy-to-biopsy variance need to be considered only in the evaluation of individual cases and are on the order of 5% in skin biopsies. (Cancer Epidemiol Biomarkers Prev 2008;17(7):1689–95)

Evaluating IPMN and pancreatic carcinoma utilizing quantitative histopathology

Intraductal papillary mucinous neoplasms (IPMN) are pancreatic lesions with uncertain biologic behavior. This study sought objective, accurate prediction tools, through the use of quantitative histopathological signatures of nuclear images, for classifying lesions as chronic pancreatitis (CP), IPMN, or pancreatic carcinoma (PC). Forty‐four pancreatic resection patients were retrospectively identified for this study (12 CP; 16 IPMN; 16 PC). Regularized multinomial regression quantitatively classified each specimen as CP, IPMN, or PC in an automated, blinded fashion. Classification certainty was determined by subtracting the smallest classification probability from the largest probability (of the three groups). The certainty function varied from 1.0 (perfectly classified) to 0.0 (random). From each lesion, 180 ± 22 nuclei were imaged. Overall classification accuracy was 89.6% with six unique nuclear features. No CP cases were misclassified, 1/16 IPMN cases were misclassified, and 4/16 PC cases were misclassified. Certainty function was 0.75 ± 0.16 for correctly classified lesions and 0.47 ± 0.10 for incorrectly classified lesions (P = 0.0005). Uncertainty was identified in four of the five misclassified lesions. Quantitative histopathology provides a robust, novel method to distinguish among CP, IPMN, and PC with a quantitative measure of uncertainty. This may be useful when there is uncertainty in diagnosis.

Abstract A83: Nuclear morphometry differentiates chronic pancreatitis, IPMN, and pancreatic carcinoma

Background: It can be difficult to distinguish between chronic pancreatitis (CP), IPMN, and pancreatic carcinoma (PC) on tissue biopsy. Nuclear morphometry can measure up to 93 unique nuclear features based on standard histopathology. The goal of this work is to build novel, objective, and accurate prediction tools, based on nuclear morphometric signatures in high resolution images of nuclei of histologic sections, for classifying pancreatic tissues into three distinct groups. Materials & Methods: 44 patients who underwent pancreatic resections were identified. 12 cases of CP, 16 cases of IPMN, and 16 cases of PC were utilized in this pilot study. 180 ± 22 nuclei from each lesion were imaged with high resolution microscopy. Clincodemographic data was obtained retrospectively from the medical record. Statistically significant nuclear features were determined by a fully automated penalized multinomial regression algorithm in order to determine a multi-class classifier and simultaneously identify important nuclear features. The LASSO penalty function, and associated regularization parameter, is adaptively chosen by cross validation to prevent over-fitting. In order to test the veracity of the automated algorithm, we randomly removed 25% of the cases as a training set and utilized the remaining cases as a test set; this was repeated 10 times. Results: The average age was 64 ± 15 years, with patients in the CP being slightly younger; 63% were male. Median follow-up time was 3 years in the CP group, 3 years in the IPMN group, and 5 years in PC group. The method described automatically identified 6 unique and statistically significant nuclear features (corrected overall P Conclusions: Nuclear morphometry classifies pancreatic lesions into CP, IPMN, and PC with 84.5% accuracy using a fully automated algorithm to determine statistically significant and unique nuclear features. Since the incorrectly classified lesions had a larger proportion of mixed nuclei, diagnostic uncertainty may be determined in a quantitative manner allowing for a confidence probability estimation of whether a given lesions should be classified as a CP, IPMN, or PC. Further studies will validate these results in a resected cohort as well as a cohort based on biopsied specimens alone. Citation Format: Evan S. Glazer, Hao Zhang, Kimberly A. Hill, Charmi Patel, Stephanie T. Kha, Peter H. Bartels, Michael L. Yozwiak, Hubert G. Bartels, Joseph C. Watkins, David S. Alberts, Robert S. Krouse. Nuclear morphometry differentiates chronic pancreatitis, IPMN, and pancreatic carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A83.

Abstract 4614: Karyometry in atypical endometrial hyperplasia: A Gynecologic Oncology Group study

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Treatment for atypical endometrial hyperplasia (AEH) is based on pathologic diagnosis. About 40% of AEH is found to be carcinoma at surgery. The histopathologic classification of endometrial lesions is a difficult task requiring extensive experience. The Gynecologic Oncology Group (GOG) conducted a study (GOG 167A) that was designed to estimate the frequency of endometrial cancer in hysterectomy specimens in subjects who had a biopsy diagnosis of atypical endometrial hyperplasia. This studys objective is to derive an objective characterization of nuclei from cases diagnosed as AEH or superficially invasive endometrial cancer (SIEC). 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. 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 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. AEH comprises cases which may constitute a low risk group involving 40 % of nuclei of the preneoplastic phenotype. Nuclei of the preneoplastic phenotype in AEH lesions are almost indistinguishable from nuclei in SIEC, where this percentage exceeds 60%. While karyometric assessment does not provide a distinctive diagnostic criterion for an individual patient, it does provide a measure of risk for the development of invasive disease. Studies to support the diagnostic assessment of endometrial lesions by computer aided morphometric analyses go back over 30 years. These efforts were aimed at two objectives, the diagnostic distinction between benign and cancer, and procedures that allow a stratification of patients into risk groups for the development of myometrial invasion. The percentage of nuclei of preneoplastic phenotype in AEH lesions might serve as criterion for assessment of risk for the development of invasive disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4614. doi:1538-7445.AM2012-4614