- Email: [email protected]
In Silico Analyses Reveals Nuclear Asymmetry of Spongiocytes and Compact Cells of Adrenocorticotrophic Hormone-Independent Macronodular Adrenocortical Hyperplasia
BASIC INVESTIGATION
In Silico Analyses Reveals Nuclear Asymmetry of Spongiocytes and Compact Cells of Adrenocorticotrophic Hormone-Independent Macronodular Adrenocortical Hyperplasia Qian Zhang, MD, Jingtao Dou, MD, Weijun Gu, MD, Guoqing Yang, MD, Yiming Mu, MD and Juming Lu, MD
Abstract: Background: Little information is available about the risk of progression of seemingly benign adrenocortical hyperplasias to carcinomatous conditions. Using in silico approaches of digitally archived tissue sections, the nuclear morphometric parameters were compared to assess nuclear asymmetry as an index for nuclear atypia. Methods: Four groups of nuclei were used for the current study: spongiocytes and compact cells obtained from adrenocorticotropic hormone (ACTH)– independent macronodular hyperplasia, which were hypothesized to be high risk for nuclear asymmetry, and primary pigmented nodular adrenocortical disease and micronodular adrenocortical hyperplasia samples were used as internal controls. Results: Analyses reveal high nuclear irregularity index of spongiocytes and shape factor abnormalities of both spongiocytes and compact cells of ACTH–independent macronodular adrenal hyperplasia compared with the other 2 groups (high F values and very low P values after analyses of variances), thus confirming the hypothesis that ACTH–independent macronodular adrenal hyperplasia present with subtle morphometric features of nuclear atypia. Conclusions: This probably puts this class of adrenocortical tumors at risk of dysplastic progression, and more studies are needed to test the hypothesis. Key Indexing Terms: Image analyses; Morphometry; Nucleus; Atypia; Asymmetry. [Am J Med Sci 2014;347(5):400–405.]
A
lthough Cushing’s syndrome is a relatively rare condition and can occur in all age groups, very little is known about adrenocortical tumorigenic conditions that coexist with Cushing’s syndrome. Numerous morphological variants are identified currently that are associated with Cushing’s syndrome without stimulation from an external source of adrenocorticotropic hormone (ACTH). Although these conditions, commonly referred to as ACTH-independent macronodular adrenal hyperplasia (AIMAH), are generally considered to be multifocal benign adrenal tumors,1 no pathological information is available regarding the propensity of these tumors into progression to adrenocortical carcinoma. Although multiplicity of tumor loci cannot reliably predict the biological nature of these tumors,2 systematic examination of tumor pathology may provide us information regarding the nature of these tumors as to their nuclear plasticity. For example, for a long time, it is well known
From the Department of Endocrinology (QZ, JD, WG, GY, YM, JL), Chinese PLA General Hospital, Beijing, China; and Department of Endocrinology (QZ), General Hospital of Beijing Military Command, Beijing, China. Submitted April 16, 2013; accepted in revised form April 23, 2013. The authors have no financial or other conflicts of interest to disclose. Correspondence: Juming Lu, MD, Department of Endocrinology, Chinese PLA General Hospital, No. 28 Fuxing Road, Haidian District, Beijing 100853, China (E-mail: [email protected]).
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that morphological parameters of nuclei correspond well with the issues of whether the tumor mass is dysplastic in nature.3,4 In the current study, we have aimed to examine the morphological parameters of nuclei in ACTH-independent macronodular hyperplasia to estimate the biological propensity of this condition to progress to frank carcinoma. Although it is known that adrenocortical carcinoma histopathological examinations show evidences of nuclear atypia,1 such information is not available for AIMAH. The goal of the current study was to collate available evidences of digitized histopathological slides of AIMAH and examine the nuclear morphometric features to evaluate for any evidence of nuclear atypia.5–11 As internal controls, we used microscopic slides of micronodular adrenocortical hyperplasia and primary pigmented nodular adrenocortical disease (PPNAD),12–15 which are well known to represent benign adrenocortical tumorigenic conditions. In AIMAH, we consistently found evidence of reports of 2 different kinds of cell populations, viz, rounded clear spongiocytes that represented the major mass of a field of view and highly eosinophilic regions with incomplete cellular boundaries, but impacted nuclear masses placed in a semi-regular fashion, and which were identified as “compact cells.” Although cellular boundaries were not discernible in these populations, we used only nuclei for comparisons, and as such, we had no requirement for tracing cellular outlines.
MATERIALS AND METHODS Collated Histopathological Slides Slides representing AIMAH that contained both spongiocytes and compact cells were downloaded for image analyses. For internal controls, slides representing micronodular hyperplasia and PPNAD (belonging to the Carney’s triad) were downloaded as high-resolution tiff (tagged image file format) files. We ensured that all the downloaded images had similar aspect ratios, as comparisons relied on this important initial step. Image Analysis Parameters for Assessing Nuclear Dysmorphometry We evaluated the following parameters for estimating whether AIMAH nuclear parameters predicted any evidence for nuclear atypia not easily discernible by a superficial microscopic examination but may be estimated by stereologic means. PPNAD and micronodular hyperplasia, known to be benign lesions, were used as controls for comparison. A. Area of nuclei: Area of nuclei represent an easily obtainable morphometric parameter, but one which is informationdense, as increased nuclear sizes are reflective of nuclear metabolic activity and can be presumed to be representative of nuclear dysplasia from a biopsy of a tumor.
The American Journal of the Medical Sciences
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Statistics Four groups were analyzed for the current study: spongiocytes from AIMAH, compact cells from AIMAH, micronodular hyperplasia and PPNAD. In general AIMAH, although benign, were hypothesized to represent the class of adrenocortical tumor that presented with increased propensity to progress to a dysplastic condition. This assumption solely relied on the multifocal nature of the tumor. Micronodular hyperplasia and PPNAD were considered as benign lesions and served as internal controls. For trend analysis, we performed Ó 2013 Lippincott Williams & Wilkins
0.0001 0.0009 0.0001 0.0001 0.0001 47.62 5.63 59 36.13 11.31 (23.41–30.76) (0.7974–0.8505) (7.090–8.028) (1.311–3.113) (1.581–1.82) 1.05 0.06 0.14 0.03 0.04 6 6 6 6 6 27.09 0.82 7.56 2.21 1.73 (18.18–24.79) (0.8322–0.8799) (5.989–6.832) (1.269–2.888) (1.441–1.655) 0.85 0.04 0.13 0.01 0.02 6 6 6 6 6 21.48 0.86 6.41 2.08 1.98 (14.98–20.21) (0.8011–0.8389) (6.02–6.687) (0.6492–1.931) (1.85–2.02) 0.81 0.09 0.12 0.02 0.05 6 6 6 6 6
F PPNAD (mean 6 SEM) (95% CI of mean)
AIMAH, adrenocorticotropic hormone–independent macronodular adrenal hyperplasia; ANOVA, analysis of variance; CI, confidence interval; NII, nuclear irregularity index; SEM, standard error of mean.
Data Entry All datasets were entered into Excel and add-ins were used to perform statistical analyses.
17.59 0.82 6.35 1.29 1.69
Image Analyses Methods Image analyses were done using the NIH image analyses software ImageJ. As earlier mentioned, images were downloaded, keeping attention to maintaining to equalized pixel aspect ratios during downloading. Details of ImageJ analyses software are available from their website. Due to very few reported conditions, all available images were used to obtain nuclear morphometric parameters. Thresholding was used to accurately define nuclear boundaries, and thereafter, absolute pointing methods were used to obtain the numerous parameters.
(35.70–40.54) (0.7773–0.8123) (8.801–9.419) (4.981–6.166) (1.745–1.902)
where dFmax is maximum Feret diameter.
38.12 6 1.83 0.79 6 0.01 9.1 6 0.22 5.57 6 0.5 1.82 6 0.03
SFpf 5 Af =Ameasured ;
Area of nuclei Circularity Feret diameter NII Perimeter-free shape factor (SFpf)
Af 5 p ðdFmax =2Þ2
TABLE 1. Cumulative nuclear morphometry parameters in the 4 different classes of examined nuclei AIMAH_spongiocytes AlMAH_compact cells Micronodular hyperplasia 6 Nuclear morphometry (mean 6 SEM) (95% CI (mean 6 SEM) (95% CI (mean 6 SEM) 6 (95% CI parameters of mean) of mean) of mean)
B. Circularity of nuclei: Usually, nuclear shapes of cells in normal cell cycle are symmetric. Nuclei may be pleomorphic, but its shape is almost nearly representative of a sphere in 3 dimensions. We made the hypothesis that any deviation from this is reflective of nuclear dysmorphometry and subtle indication for viewing the condition as nuclear dysplasia. C. Nuclear irregularity indices (NII): Next, we went a step ahead of circularity, and using a modification of a report that examined nuclear irregularity, we estimated nuclear irregularities in sections of AIMAH, PPNAD, and micronodular hyperplasia. We used the following formula: AR + roundedness. AR represents the aspect ratio, the ratio of width of the nuclear particle to the height. Roundedness is related to asymmetry at the membrane, and we hypothesized that a metabolically active nucleus or one that progressed far into cell cycle will have increased asymmetry. D. Feret diameter: We opined that if a shape is irregular, it cannot be boxed into a uniform circle. Normally, a rounded nucleus may be restricted in a sphere that is simply overlaid on its membrane. However, for an increasingly irregular spheroid, such an attempt is not possible. Under such circumstances, chord line like Nassenstein diameter or Martin’s diameter may be used. We aimed to estimate the Feret diameter, the mean distance between pairs of parallel tangents drawn to the projected outline of the particle. E. Perimeter-free shape factor (SFpf): Because nuclei are small structures, usually composed of less than 30 pixel per particle because of the lower magnifications used for obtaining wide fields of view, we decided to use a shape factor that is not delimited by digitization of the image. For these reasons, we ignored perimeter measurements. Instead, we measured perimeter-free shape factor, using the following formula:
P (ANOVA)
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FIGURE 1.
Comparisons of nuclear areas. The left panel shows mean data and right panel is a box plot showing data swarm and 1s deviation.
FIGURE 2. Comparisons of nuclear circularity. The box plot shows data swarm and 1s deviation.
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FIGURE 3. Comparisons of nuclear irregularity indices (NII). The box plot shows data swarm and 1s deviation. Note the high NII of nuclei is obtained from spongiocytes of AIMAH. Volume 347, Number 5, May 2014
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FIGURE 4. Correlation plots of NII with mean areas of nuclei obtained from spongiocytes and compact cells of AIMAH, as compared with PPNAD and micronodular hyperplasia.
unpaired Student’s t test to compare means, although we do not report the results here. Because we have 4 different groups, 2 of which were hypothesized to possess nuclear asymmetry and the other 2, although tumorigenic, were considered benign lesions,
we performed analysis of variance (ANOVA) to obtain F statistics and P values. The cumulative values are reported and data swarms are presented to represent the overall findings, and interpretations were inferred based on such observations. For linear correlation analysis, Pearson’s coefficients were computed.
RESULTS
FIGURE 5. Box plot showing distributions of Feret diameter of nuclei obtained from spongiocytes and compact cells of AIMAH as compared with PPNAD and micronodular hyperplasia. Feret diameter is a measure of asymmetry of a quasispheroidal structure. Ó 2013 Lippincott Williams & Wilkins
Table 1 reports cumulative data representing mean, standard error, 95% confidence interval of the means and F statistics following ANOVA of the following parameters: area of nuclei, circularity, Feret diameter, NII and perimeter-free shape factors. The 4 classes of nuclei that were examined are as follows: spongiocytes (predominant cells) of AIMAH, compact cells (that occur as islands) in sections obtained from AIMAH, micronodular hyperplasia and PPNAD (primary pigmented nodular adrenocortical adenoma). Figure 1 represents significantly increased nuclear area of spongiocytes (of AIMAH) in comparison with nuclear areas of compact cells of AIMAH, as well as micronodular hyperplasia and PPNAD (F 5 47.62, P 5 0.0001, ANOVA). Note that the data swarms show a huge variance in the nuclear areas of spongiocytes. Figure 2 represents significantly decreased circularity of nuclei of spongiocytes in comparison with PPNAD and micronodular hyperplasia. Note that the compact cells of AIMAH have comparable circularity to nuclei of micronodular hyperplasia. This indicates nuclear asymmetry of spongiocytes in comparison with other nuclei of seemingly benign adrenocortical adenomas. Figure 3 represents that nuclei of spongiocytes of AIMAH have very high nuclear irregularities in comparison with nuclei of either compact cells of AIMAH or micronodular hyperplasia and PPNAD (F 5 36.13, P 5 0.0001). Figure 4 shows a correlative analysis between nuclear irregularity indices and areas of nuclei. Note the positive correlation between the spongiocytes nuclei analyzed from AIMAH and their high NII (r 5 0.96, Pearson’s coefficient). Figure 5 shows significantly increased Feret diameter in comparison with the other groups. This again provided evidence for the nuclear asymmetry of spongiocytes in AIMAH sections. Figure 6 shows distribution of shape factors of all analyzed nuclei in all groups. Table 1 shows the high levels of
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FIGURE 6. Distribution plot of perimeter-free shape factors of nuclei obtained from spongiocytes (A) and compact cells (B) of AIMAH, and PPNAD (D) and micronodular hyperplasia (C). Note that while all the previous analyses showed nuclear structural irregularities of spongiocytes, this distribution plot reveals that nuclei of compact cells also have asymmetry. A box plot of data distribution is shown to the right. The formulae for computing shape factor are presented in the Methods section.
significance of the difference between these means. The black line shows the polynomial trend, which roughly overlaps the distribution of the nuclei of sections obtained from micronodular hyperplasia. Note that this analysis actually reveals that not only spongiocytes but also compact cells actually show numerous outliers, indicating that despite their smaller nuclear profiles, some of them have morphometric parameters suggestive of nuclear asymmetry.
DISCUSSION
This is the first analysis of nuclear morphometry for prediction of atypia of seemingly benign adrenocortical hyperplasias. We examined digital archives of sections of ACTH-independent macronodular hyperplasia in comparison with micronodular adrenocortical hyperplasia and PPNAD. Our analyses initially revealed increased nuclear areas of the spongiocytes population of AIMAH. The compact cells of AIMAH had normal or slightly decreased nuclear areas, giving an impression of metabolically quiescent nuclei. However, circularity showed nuclear asymmetry, providing evidence of asymmetry of spongiocytic nuclei of AIMAH. Recent studies have used nuclear irregularity indices as measures of nuclear asymmetry. We used a linear adaptation of such measures by simply adding aspect ratios and roundedness and examined whether the asymmetry revealed by the circularity indices matched when we reexamined the correlation matrices of NII of different groups of nuclei. Our analyses revealed huge nuclear irregularities of spongiocytes. Surprisingly, compact cells, which also belong to AIMAH, show much lower NII scores, again in coherence to our earlier interpretations of close-faced nuclei. Stereological methods have been numerously used to analyze morphological parameters in medical research, including prediction of cellular parameters and estimation of tumorigenic propensity to progression to dysplasia.3,16 Using concepts from shape analysis,17–20 our study for the first time applies this to
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nuclear analyses. Because nuclei are small structures, we used estimates of Feret diameter and used a modification to derive shape factor, commonly used in metallurgy and ceramic particle analyses.21 Shape factor analysis surprisingly revealed numerous nuclear asymmetry, not only of the spongiocytes but also compact cells of AIMAH. Thus, our in silico analysis for the first time demonstrates that AIMAH nuclei shows asymmetry. Further analyses should be performed to examine whether risk of dysplasia occurs for AIMAH. Our study also reveals the importance of digitizing and archiving histopathological slides for subsequent reexamination to examine risk of dysplasia and progression to cancer in apparently benign tumorigenic conditions like AIMAH. REFERENCES 1. Stratakis CA. Cushing syndrome caused by adrenocortical tumors and hyperplasias (corticotropin-independent Cushing syndrome). Endocr Dev 2008;13:117–32. 2. Correa P, Piazuelo MB. The gastric precancerous cascade. J Dig Dis 2012;13:2–9. 3. Filippi-Chiela EC, Oliveira MM, Jurkovski B, et al. Nuclear morphometric analysis (NMA): screening of senescence, apoptosis and nuclear irregularities. PLoS One 2012;7:e42522. 4. Veltri RW, Christudass CS, Isharwal S. Nuclear morphometry, nucleomics and prostate cancer progression. Asian J Androl 2012;14:375–84. 5. Iwata M, Oki Y, Okazawa T, et al. A rare case of adrenocorticotropic hormone (ACTH)-independent macroadrenal hyperplasia showing ectopic production of ACTH. Intern Med 2012;51:2181–7. 6. Kobayashi T, Miwa T, Kan K, et al. Usefulness and limitations of unilateral adrenalectomy for ACTH-independent macronodular adrenal hyperplasia in a patient with poor glycemic control. Intern Med 2012; 51:1709–13. 7. Obata Y, Yamada Y, Baden MY, et al. Long-term efficacy of trilostane for Cushing’s syndrome due to adrenocorticotropin-independent bilateral macronodular adrenocortical hyperplasia. Intern Med 2011;50:2621–5.
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8. Chapman A, Durand J, Ouadi L, et al. Identification of genetic alterations of AXIN2 gene in adrenocortical tumors. J Clin Endocrinol Metab 2011;96:E1477–81.
15. Louiset E, Gobet F, Libé R, et al. ACTH-independent Cushing’s syndrome with bilateral micronodular adrenal hyperplasia and ectopic adrenocortical adenoma. J Clin Endocrinol Metab 2010;95:18–24.
9. Yoshida M, Hiroi M, Imai T, et al. A case of ACTH-independent macronodular adrenal hyperplasia associated with multiple endocrine neoplasia type 1. Endocr J 2011;58:269–77.
16. Chaudhury A, Shariff A, Srinivas M, et al. Changes in nitrergic innervation of def unctionalized rat colon after diversion colostomy. Neurogastroenterol Motil 2004;16:475–87.
10. Assie G, Louiset E, Sturm N, et al. Systematic analysis of G proteincoupled receptor gene expression in adrenocorticotropin-independent macronodular adrenocortical hyperplasia identifies novel targets for pharmacological control of adrenal Cushing’s syndrome. J Clin Endocrinol Metab 2010;95:E253–62.
17. Haug H. On the proper use of point-counting and semi-automatic procedures in stereology. Microsc Acta 1981;85:141–52.
11. de Groot JW, Links TP, Themmen AP, et al. Aberrant expression of multiple hormone receptors in ACTH-independent macronodular adrenal hyperplasia causing Cushing’s syndrome. Eur J Endocrinol 2010;163:293–9. 12. Tung SC, Hwang DY, Yang JW, et al. An unusual presentation of Carney complex with diffuse primary pigmented nodular adrenocortical disease on one adrenal gland and a nonpigmented adrenocortical adenoma and focal primary pigmented nodular adrenocortical disease on the other. Endocr J 2012 [Epub ahead of print].
18. Toygar M, Demirel B, Karslioglu Y, et al. Nuclear morphometry in epidermal changes due to electrical current and thermal energy: trial for usage of image analysis in histological sections. Am J Forensic Med Pathol 2010;31:227–31. 19. Nygaard JV, Andersen MØ, Howard KA, et al. Investigation of particle-functionalized tissue engineering scaffolds using X-ray tomographic microscopy. Biotechnol Bioeng 2008;100:820–9.
13. Manipadam MT, Abraham R, Sen S, et al. Primary pigmented nodular adrenocortical disease. J Indian Assoc Pediatr Surg 2011;16:160–2.
20. Pektas ZO, Keskin A, Günhan O, et al. Evaluation of nuclear morphometry and DNA ploidy status for detection of malignant and premalignant oral lesions: quantitative cytologic assessment and review of methods for cytomorphometric measurements. J Oral Maxillofac Surg 2006;64:628–35.
14. Almeida MQ, Stratakis CA. Carney complex and other conditions associated with micronodular adrenal hyperplasias. Best Pract Res Clin Endocrinol Metab 2010;24:907–14.
21. Exner HE. Stereology and 3D microscopy: useful alternatives or competitors in the quantitative analyses of microstructures? Image Anal Stereol 2004;23:73–82.
Ó 2013 Lippincott Williams & Wilkins
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In Silico Analyses Reveals Nuclear Asymmetry of Spongiocytes and Compact Cells of Adrenocorticotrophic Hormone-Independent Macronodular Adrenocortical Hyperplasia Qian Zhang, MD, Jingtao Dou, MD, Weijun Gu, MD, Guoqing Yang, MD, Yiming Mu, MD and Juming Lu, MD
Abstract: Background: Little information is available about the risk of progression of seemingly benign adrenocortical hyperplasias to carcinomatous conditions. Using in silico approaches of digitally archived tissue sections, the nuclear morphometric parameters were compared to assess nuclear asymmetry as an index for nuclear atypia. Methods: Four groups of nuclei were used for the current study: spongiocytes and compact cells obtained from adrenocorticotropic hormone (ACTH)– independent macronodular hyperplasia, which were hypothesized to be high risk for nuclear asymmetry, and primary pigmented nodular adrenocortical disease and micronodular adrenocortical hyperplasia samples were used as internal controls. Results: Analyses reveal high nuclear irregularity index of spongiocytes and shape factor abnormalities of both spongiocytes and compact cells of ACTH–independent macronodular adrenal hyperplasia compared with the other 2 groups (high F values and very low P values after analyses of variances), thus confirming the hypothesis that ACTH–independent macronodular adrenal hyperplasia present with subtle morphometric features of nuclear atypia. Conclusions: This probably puts this class of adrenocortical tumors at risk of dysplastic progression, and more studies are needed to test the hypothesis. Key Indexing Terms: Image analyses; Morphometry; Nucleus; Atypia; Asymmetry. [Am J Med Sci 2014;347(5):400–405.]
A
lthough Cushing’s syndrome is a relatively rare condition and can occur in all age groups, very little is known about adrenocortical tumorigenic conditions that coexist with Cushing’s syndrome. Numerous morphological variants are identified currently that are associated with Cushing’s syndrome without stimulation from an external source of adrenocorticotropic hormone (ACTH). Although these conditions, commonly referred to as ACTH-independent macronodular adrenal hyperplasia (AIMAH), are generally considered to be multifocal benign adrenal tumors,1 no pathological information is available regarding the propensity of these tumors into progression to adrenocortical carcinoma. Although multiplicity of tumor loci cannot reliably predict the biological nature of these tumors,2 systematic examination of tumor pathology may provide us information regarding the nature of these tumors as to their nuclear plasticity. For example, for a long time, it is well known
From the Department of Endocrinology (QZ, JD, WG, GY, YM, JL), Chinese PLA General Hospital, Beijing, China; and Department of Endocrinology (QZ), General Hospital of Beijing Military Command, Beijing, China. Submitted April 16, 2013; accepted in revised form April 23, 2013. The authors have no financial or other conflicts of interest to disclose. Correspondence: Juming Lu, MD, Department of Endocrinology, Chinese PLA General Hospital, No. 28 Fuxing Road, Haidian District, Beijing 100853, China (E-mail: [email protected]).
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that morphological parameters of nuclei correspond well with the issues of whether the tumor mass is dysplastic in nature.3,4 In the current study, we have aimed to examine the morphological parameters of nuclei in ACTH-independent macronodular hyperplasia to estimate the biological propensity of this condition to progress to frank carcinoma. Although it is known that adrenocortical carcinoma histopathological examinations show evidences of nuclear atypia,1 such information is not available for AIMAH. The goal of the current study was to collate available evidences of digitized histopathological slides of AIMAH and examine the nuclear morphometric features to evaluate for any evidence of nuclear atypia.5–11 As internal controls, we used microscopic slides of micronodular adrenocortical hyperplasia and primary pigmented nodular adrenocortical disease (PPNAD),12–15 which are well known to represent benign adrenocortical tumorigenic conditions. In AIMAH, we consistently found evidence of reports of 2 different kinds of cell populations, viz, rounded clear spongiocytes that represented the major mass of a field of view and highly eosinophilic regions with incomplete cellular boundaries, but impacted nuclear masses placed in a semi-regular fashion, and which were identified as “compact cells.” Although cellular boundaries were not discernible in these populations, we used only nuclei for comparisons, and as such, we had no requirement for tracing cellular outlines.
MATERIALS AND METHODS Collated Histopathological Slides Slides representing AIMAH that contained both spongiocytes and compact cells were downloaded for image analyses. For internal controls, slides representing micronodular hyperplasia and PPNAD (belonging to the Carney’s triad) were downloaded as high-resolution tiff (tagged image file format) files. We ensured that all the downloaded images had similar aspect ratios, as comparisons relied on this important initial step. Image Analysis Parameters for Assessing Nuclear Dysmorphometry We evaluated the following parameters for estimating whether AIMAH nuclear parameters predicted any evidence for nuclear atypia not easily discernible by a superficial microscopic examination but may be estimated by stereologic means. PPNAD and micronodular hyperplasia, known to be benign lesions, were used as controls for comparison. A. Area of nuclei: Area of nuclei represent an easily obtainable morphometric parameter, but one which is informationdense, as increased nuclear sizes are reflective of nuclear metabolic activity and can be presumed to be representative of nuclear dysplasia from a biopsy of a tumor.
The American Journal of the Medical Sciences
Volume 347, Number 5, May 2014
Statistics Four groups were analyzed for the current study: spongiocytes from AIMAH, compact cells from AIMAH, micronodular hyperplasia and PPNAD. In general AIMAH, although benign, were hypothesized to represent the class of adrenocortical tumor that presented with increased propensity to progress to a dysplastic condition. This assumption solely relied on the multifocal nature of the tumor. Micronodular hyperplasia and PPNAD were considered as benign lesions and served as internal controls. For trend analysis, we performed Ó 2013 Lippincott Williams & Wilkins
0.0001 0.0009 0.0001 0.0001 0.0001 47.62 5.63 59 36.13 11.31 (23.41–30.76) (0.7974–0.8505) (7.090–8.028) (1.311–3.113) (1.581–1.82) 1.05 0.06 0.14 0.03 0.04 6 6 6 6 6 27.09 0.82 7.56 2.21 1.73 (18.18–24.79) (0.8322–0.8799) (5.989–6.832) (1.269–2.888) (1.441–1.655) 0.85 0.04 0.13 0.01 0.02 6 6 6 6 6 21.48 0.86 6.41 2.08 1.98 (14.98–20.21) (0.8011–0.8389) (6.02–6.687) (0.6492–1.931) (1.85–2.02) 0.81 0.09 0.12 0.02 0.05 6 6 6 6 6
F PPNAD (mean 6 SEM) (95% CI of mean)
AIMAH, adrenocorticotropic hormone–independent macronodular adrenal hyperplasia; ANOVA, analysis of variance; CI, confidence interval; NII, nuclear irregularity index; SEM, standard error of mean.
Data Entry All datasets were entered into Excel and add-ins were used to perform statistical analyses.
17.59 0.82 6.35 1.29 1.69
Image Analyses Methods Image analyses were done using the NIH image analyses software ImageJ. As earlier mentioned, images were downloaded, keeping attention to maintaining to equalized pixel aspect ratios during downloading. Details of ImageJ analyses software are available from their website. Due to very few reported conditions, all available images were used to obtain nuclear morphometric parameters. Thresholding was used to accurately define nuclear boundaries, and thereafter, absolute pointing methods were used to obtain the numerous parameters.
(35.70–40.54) (0.7773–0.8123) (8.801–9.419) (4.981–6.166) (1.745–1.902)
where dFmax is maximum Feret diameter.
38.12 6 1.83 0.79 6 0.01 9.1 6 0.22 5.57 6 0.5 1.82 6 0.03
SFpf 5 Af =Ameasured ;
Area of nuclei Circularity Feret diameter NII Perimeter-free shape factor (SFpf)
Af 5 p ðdFmax =2Þ2
TABLE 1. Cumulative nuclear morphometry parameters in the 4 different classes of examined nuclei AIMAH_spongiocytes AlMAH_compact cells Micronodular hyperplasia 6 Nuclear morphometry (mean 6 SEM) (95% CI (mean 6 SEM) (95% CI (mean 6 SEM) 6 (95% CI parameters of mean) of mean) of mean)
B. Circularity of nuclei: Usually, nuclear shapes of cells in normal cell cycle are symmetric. Nuclei may be pleomorphic, but its shape is almost nearly representative of a sphere in 3 dimensions. We made the hypothesis that any deviation from this is reflective of nuclear dysmorphometry and subtle indication for viewing the condition as nuclear dysplasia. C. Nuclear irregularity indices (NII): Next, we went a step ahead of circularity, and using a modification of a report that examined nuclear irregularity, we estimated nuclear irregularities in sections of AIMAH, PPNAD, and micronodular hyperplasia. We used the following formula: AR + roundedness. AR represents the aspect ratio, the ratio of width of the nuclear particle to the height. Roundedness is related to asymmetry at the membrane, and we hypothesized that a metabolically active nucleus or one that progressed far into cell cycle will have increased asymmetry. D. Feret diameter: We opined that if a shape is irregular, it cannot be boxed into a uniform circle. Normally, a rounded nucleus may be restricted in a sphere that is simply overlaid on its membrane. However, for an increasingly irregular spheroid, such an attempt is not possible. Under such circumstances, chord line like Nassenstein diameter or Martin’s diameter may be used. We aimed to estimate the Feret diameter, the mean distance between pairs of parallel tangents drawn to the projected outline of the particle. E. Perimeter-free shape factor (SFpf): Because nuclei are small structures, usually composed of less than 30 pixel per particle because of the lower magnifications used for obtaining wide fields of view, we decided to use a shape factor that is not delimited by digitization of the image. For these reasons, we ignored perimeter measurements. Instead, we measured perimeter-free shape factor, using the following formula:
P (ANOVA)
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FIGURE 1.
Comparisons of nuclear areas. The left panel shows mean data and right panel is a box plot showing data swarm and 1s deviation.
FIGURE 2. Comparisons of nuclear circularity. The box plot shows data swarm and 1s deviation.
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FIGURE 3. Comparisons of nuclear irregularity indices (NII). The box plot shows data swarm and 1s deviation. Note the high NII of nuclei is obtained from spongiocytes of AIMAH. Volume 347, Number 5, May 2014
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FIGURE 4. Correlation plots of NII with mean areas of nuclei obtained from spongiocytes and compact cells of AIMAH, as compared with PPNAD and micronodular hyperplasia.
unpaired Student’s t test to compare means, although we do not report the results here. Because we have 4 different groups, 2 of which were hypothesized to possess nuclear asymmetry and the other 2, although tumorigenic, were considered benign lesions,
we performed analysis of variance (ANOVA) to obtain F statistics and P values. The cumulative values are reported and data swarms are presented to represent the overall findings, and interpretations were inferred based on such observations. For linear correlation analysis, Pearson’s coefficients were computed.
RESULTS
FIGURE 5. Box plot showing distributions of Feret diameter of nuclei obtained from spongiocytes and compact cells of AIMAH as compared with PPNAD and micronodular hyperplasia. Feret diameter is a measure of asymmetry of a quasispheroidal structure. Ó 2013 Lippincott Williams & Wilkins
Table 1 reports cumulative data representing mean, standard error, 95% confidence interval of the means and F statistics following ANOVA of the following parameters: area of nuclei, circularity, Feret diameter, NII and perimeter-free shape factors. The 4 classes of nuclei that were examined are as follows: spongiocytes (predominant cells) of AIMAH, compact cells (that occur as islands) in sections obtained from AIMAH, micronodular hyperplasia and PPNAD (primary pigmented nodular adrenocortical adenoma). Figure 1 represents significantly increased nuclear area of spongiocytes (of AIMAH) in comparison with nuclear areas of compact cells of AIMAH, as well as micronodular hyperplasia and PPNAD (F 5 47.62, P 5 0.0001, ANOVA). Note that the data swarms show a huge variance in the nuclear areas of spongiocytes. Figure 2 represents significantly decreased circularity of nuclei of spongiocytes in comparison with PPNAD and micronodular hyperplasia. Note that the compact cells of AIMAH have comparable circularity to nuclei of micronodular hyperplasia. This indicates nuclear asymmetry of spongiocytes in comparison with other nuclei of seemingly benign adrenocortical adenomas. Figure 3 represents that nuclei of spongiocytes of AIMAH have very high nuclear irregularities in comparison with nuclei of either compact cells of AIMAH or micronodular hyperplasia and PPNAD (F 5 36.13, P 5 0.0001). Figure 4 shows a correlative analysis between nuclear irregularity indices and areas of nuclei. Note the positive correlation between the spongiocytes nuclei analyzed from AIMAH and their high NII (r 5 0.96, Pearson’s coefficient). Figure 5 shows significantly increased Feret diameter in comparison with the other groups. This again provided evidence for the nuclear asymmetry of spongiocytes in AIMAH sections. Figure 6 shows distribution of shape factors of all analyzed nuclei in all groups. Table 1 shows the high levels of
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Zhang et al
FIGURE 6. Distribution plot of perimeter-free shape factors of nuclei obtained from spongiocytes (A) and compact cells (B) of AIMAH, and PPNAD (D) and micronodular hyperplasia (C). Note that while all the previous analyses showed nuclear structural irregularities of spongiocytes, this distribution plot reveals that nuclei of compact cells also have asymmetry. A box plot of data distribution is shown to the right. The formulae for computing shape factor are presented in the Methods section.
significance of the difference between these means. The black line shows the polynomial trend, which roughly overlaps the distribution of the nuclei of sections obtained from micronodular hyperplasia. Note that this analysis actually reveals that not only spongiocytes but also compact cells actually show numerous outliers, indicating that despite their smaller nuclear profiles, some of them have morphometric parameters suggestive of nuclear asymmetry.
DISCUSSION
This is the first analysis of nuclear morphometry for prediction of atypia of seemingly benign adrenocortical hyperplasias. We examined digital archives of sections of ACTH-independent macronodular hyperplasia in comparison with micronodular adrenocortical hyperplasia and PPNAD. Our analyses initially revealed increased nuclear areas of the spongiocytes population of AIMAH. The compact cells of AIMAH had normal or slightly decreased nuclear areas, giving an impression of metabolically quiescent nuclei. However, circularity showed nuclear asymmetry, providing evidence of asymmetry of spongiocytic nuclei of AIMAH. Recent studies have used nuclear irregularity indices as measures of nuclear asymmetry. We used a linear adaptation of such measures by simply adding aspect ratios and roundedness and examined whether the asymmetry revealed by the circularity indices matched when we reexamined the correlation matrices of NII of different groups of nuclei. Our analyses revealed huge nuclear irregularities of spongiocytes. Surprisingly, compact cells, which also belong to AIMAH, show much lower NII scores, again in coherence to our earlier interpretations of close-faced nuclei. Stereological methods have been numerously used to analyze morphological parameters in medical research, including prediction of cellular parameters and estimation of tumorigenic propensity to progression to dysplasia.3,16 Using concepts from shape analysis,17–20 our study for the first time applies this to
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nuclear analyses. Because nuclei are small structures, we used estimates of Feret diameter and used a modification to derive shape factor, commonly used in metallurgy and ceramic particle analyses.21 Shape factor analysis surprisingly revealed numerous nuclear asymmetry, not only of the spongiocytes but also compact cells of AIMAH. Thus, our in silico analysis for the first time demonstrates that AIMAH nuclei shows asymmetry. Further analyses should be performed to examine whether risk of dysplasia occurs for AIMAH. Our study also reveals the importance of digitizing and archiving histopathological slides for subsequent reexamination to examine risk of dysplasia and progression to cancer in apparently benign tumorigenic conditions like AIMAH. REFERENCES 1. Stratakis CA. Cushing syndrome caused by adrenocortical tumors and hyperplasias (corticotropin-independent Cushing syndrome). Endocr Dev 2008;13:117–32. 2. Correa P, Piazuelo MB. The gastric precancerous cascade. J Dig Dis 2012;13:2–9. 3. Filippi-Chiela EC, Oliveira MM, Jurkovski B, et al. Nuclear morphometric analysis (NMA): screening of senescence, apoptosis and nuclear irregularities. PLoS One 2012;7:e42522. 4. Veltri RW, Christudass CS, Isharwal S. Nuclear morphometry, nucleomics and prostate cancer progression. Asian J Androl 2012;14:375–84. 5. Iwata M, Oki Y, Okazawa T, et al. A rare case of adrenocorticotropic hormone (ACTH)-independent macroadrenal hyperplasia showing ectopic production of ACTH. Intern Med 2012;51:2181–7. 6. Kobayashi T, Miwa T, Kan K, et al. Usefulness and limitations of unilateral adrenalectomy for ACTH-independent macronodular adrenal hyperplasia in a patient with poor glycemic control. Intern Med 2012; 51:1709–13. 7. Obata Y, Yamada Y, Baden MY, et al. Long-term efficacy of trilostane for Cushing’s syndrome due to adrenocorticotropin-independent bilateral macronodular adrenocortical hyperplasia. Intern Med 2011;50:2621–5.
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Nuclear Asymmetry and AIMAH
8. Chapman A, Durand J, Ouadi L, et al. Identification of genetic alterations of AXIN2 gene in adrenocortical tumors. J Clin Endocrinol Metab 2011;96:E1477–81.
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