Accepted 2 November 2011 Published online 13 January 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI /hed.22909

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1 ORIGINAL ARTICLE Preoperative prediction of papillary thyroid carcinoma prognosis with the assistance of computerized morphometry of cytology samples obtained by fine-needle aspiration: Preliminary report Shyang Rong Shih, MD, 1,2 * Yi Cheng Chang, MD, 3 Hung Yuan Li, MD, 2 Jau Yu Liau, MD, 4 Chia Yen Lee, BSc, 1 Chung Ming Chen, PhD, 1 Tien Chun Chang, MD, PhD 2 1 Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan, 2 Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan, 3 Academia Sinica, Taiwan, 4 Department of Pathology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan. Accepted 2 November 2011 Published online 13 January 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI /hed ABSTRACT: Background. The purpose of this study was to analyze the relationship between cytologic features, clinical features, and recurrence of papillary thyroid cancer (PTC). We hoped to predict prognosis preoperatively. Methods. We retrospectively studied cytologic features by using computerized morphometry and clinical data of 118 patients with usualtype PTC without initial metastasis, including 34 patients with cancer recurrence in 10 years after surgery and 84 patients who did not have recurrence for more than 10 years after surgery. Another 24 patients were recruited for validation. Results. Multivariate logistic analysis indicated that nucleus-to-cell ratio, variation of nuclear area, tumor size, and patient age were significantly related to recurrence. Cox regression analysis showed that hazard ratios were 3.34, 1.53, 1.77, and 2.6, respectively. Conclusion. Cytologic features of PTC analyzed with computerized morphometry significantly correlated with recurrence. It helped to predict prognosis preoperatively and may be helpful for planning further treatment. VC 2012 Wiley Periodicals, Inc. Head Neck 35: 28 34, 2013 KEY WORDS: papillary thyroid carcinoma, computerized morphometry Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy worldwide, accounting for 50% to 90% of differentiated follicular thyroid cancers. 1 The influence of clinicopathologic features on the prognosis of PTC has been examined and used to develop several prognostic scoring systems. 1 7 However, these scoring systems are applicable only after surgery has been performed and the tumor has been resected; they are not useful in the early phase of designing individual treatment protocols, including determination of specifics of the operative procedure and choice and details of adjunctive treatments such as radioactive iodine therapy. There is disagreement about the optimal extent of thyroidectomy and the indications for routine or selective lymphadenectomy Surgical treatment of papillary microadenomas is even more widely debated, and some experts have suggested follow-up (watchful waiting) without surgical resection The purpose of this study was to identify the relationship of the patients prognosis with their demographic data and the *Corresponding author: S.-R. Shih. srshih@ntu.edu.tw Contract grant sponsor: This work was supported by the National Taiwan University Hospital, Yun Lin Branch. T.-C. Chang and C.-M. Chen are co-corresponding authors. cytologic features of specimens obtained by fine-needle aspiration (FNA). This finding might prove useful in the early phase of designing individual treatment protocols. We had proposed a prognostic scoring system based on the cytologic features of samples obtained by FNA, and the score was derived by assigning the following values for the factors present at diagnosis: light staining of nuclei and apparent nucleoli, 1; irregular-shaped nuclei in cells away from the fibrous cord which means the aggregation of fibrous tissue and vessels which are surrounded by follicular cells, 1.5; high nucleus-to-cytoplasm ratio, 1; and nuclear atypia, Higher prognostic scores were related to a higher probability of cancer recurrence. Sensitivity and specificity of outcome prediction by using a cutoff point of 2.5 were 83.33% and 77.55%, respectively. 18 However, such detailed assessment of cytologic features is subjective and may differ among cytologists. Computerized morphometry is a computer-assisted image analysis technique that enables quantitative estimation of selected parameters in individual cells. 19 It is a potentially powerful tool for objective malignancy grading. 20 Thus, another purpose of this study was to determine whether computerized morphometric analysis of cytologic features of samples obtained by FNA had prognostic significance. We hope that the results of this study will help in modifying the previous prognostic scoring system to make it more objective. 28 HEAD & NECK DOI /HED JANUARY 2013

2 PROGNOSIS OF PAPILLARY THYROID CARCINOMA TABLE 1. Basic demographic data of patients (N 5 118). Recurrence in 10 y No recurrence in 10 y Variable No. of patients % No. of patients % p value Patient no Sex.3 Male Female Age, y <.05 Average Range Availability of all surgical and pathological records Primary tumor Average tumor size (cm; range) 3.4 ( ) 2.4 ( ) <.05 Lymph node metastasis Unilateral Bilateral Single Multiple Extrathyroid extension Recurrence site At thyroid bed At lymph node At thyroid bed and lymph node Distant metastasis MATERIALS AND METHODS This study was approved by the institutional review board of the National Taiwan University Hospital and was registered on Clinicaltrials.gov with the protocol number of NCT This study also complied with the Declaration of Helsinki. We retrospectively reviewed the medical records and cytology samples of patients admitted to our institution, the National Taiwan University Hospital, during 1977 to 2000; the patients had the usual type of PTC without initial presentation of distant metastasis. These PTCs were diagnosed by using samples obtained with FNA, which was performed percutaneously with a 22-gauge needle fitted to a 20-mL syringe; the obtained cells were then airdried and stained with Riu s stain for microscopic evaluation. The cancer was surgically treated with total or neartotal thyroidectomy We recruited patients who showed cancer recurrence at the thyroid bed or lymph nodes within 10 years of the surgery and patients who did not show recurrence during follow-up for more than 10 years. A total of 118 patients of usual (classical) type of PTC were recruited. Experienced pathologists reviewed the pathological specimens to confirm the diagnosis of usual type of PTC. Among these patients, 34 had cancer recurrence at an average of 44.2 months (range, months) after surgery. The remaining 84 patients did not have cancer recurrence for more than 10 years after surgery (Table 1). Table 1 shows the basic demographic data of all patients. Computerized morphometry software (Image Pro Plus, version 5.0; Media Cybernetics; Silver Springs, MD) was used to analyze the digital images of these cytology slides, which were photographed using a camera connected to a microscope. The fields of view that showed dispersed follicular cells were selected because the nuclear and cytoplasmic areas would be inaccurately measured if the cells were crowded together or if they overlapped. On an average, 26 cells (range, cells) were analyzed from each patient. The nucleus-to-cell ratio (NCR), nuclear area, and nuclear roundness were calculated using the morphometry software. NCR was defined as the percentage of the area of the nucleus in the whole cell and was calculated using the following formula: (nuclear area)/(cell area). Variation of nuclear area (VNA) was determined by the following formula: (SD of nuclear area)/(mean nuclear area). Nuclear roundness was a measure of how circular an object is (the expected perimeter of a circular object divided by the actual perimeter). It was determined by the following formula: (perimeter ^2)/(4 p area). Variation of nuclear roundness was determined by the following formula: (SD of nuclear roundness)/(mean nuclear roundness). Figures 1 and 2 show examples of NCR and VNA obtained using computerized morphometry software. For validation, we retrospectively reviewed the medical records and cytology samples of another 24 patients (4 men and 20 women) with usual type of PTCs. Their cancers were surgically treated with total or near-total thyroidectomy. The diagnosis of usual type of PTCs was pathologically confirmed by experienced pathologists. Two of the patients showed cancer recurrence within 10 years of the surgery and the other 22 patients did not show recurrence during follow-up for more than 10 years. Statistical analyses Data were represented as numbers and percentages. The variables, including NCR, VNA, nuclear roundness, tumor size, and age, were divided into 2 or 3 groups according to HEAD & NECK DOI /HED JANUARY

3 SHIH ET AL. FIGURE 1. Nucleus-to-cell ratio. Left: Cytology smear of papillary thyroid cancer (Riu s stain). Right: Computerized morphometry. (A) Large nucleus-to-cell ratio (70.89%). (B) Small nucleus-to-cell ratio (31.61%). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] their distribution in the study population. Univariate and multivariate logistic analyses were performed using the above-mentioned variables as independent variables and cancer recurrence or nonrecurrence as the dependent variable (Table 3 and 4) Kaplan Meier survival curves were used to determine the relationship between cancer recurrence and the different variables. Stata/SE 9.0 for Windows (StataCorp LP, College Station, TX) was used for statistical analyses. A p value of <.05 was considered statistically significant. RESULTS In the study group, age and tumor size differed significantly between patients with and without recurrence in 10 years (Table 1). NCR, VNA, and nuclear roundness were determined using the computerized morphometry software (Figures 1 and 2). The variables, including NCR, VNA, nuclear roundness, tumor size, and age, were divided into 2 or 3 groups according to their distribution in the study population. NCR was divided into 3 groups: small (NCR, <35%; labeled as 0), medium (NCR, 35% and <60%; labeled as 1), and large (NCR, 60%; labeled as 2). Tumor size was divided into 3 groups: small (size, 2 cm; labeled as 1), medium (size, >2 cm and 4 cm; labeled as 2), and large (size, >4 cm; labeled as 3). Age was divided into 2 groups: young (age, <50 years; labeled as 0) and old (age, 50 years; labeled as 1). VNA was divided into 3 groups: small (VNA, <12%; labeled as 0), medium (VNA, 12% and <25%; labeled as 1), and large (VNA, 25%; labeled as 2). Nuclear roundness was divided into 2 groups: rounder (mean of nuclear roundness, <1.125, labeled as 0), and more irregular (mean of nuclear roundness, 1.125, labeled as 1). Variation of nuclear roundness is divided into 2 groups: small (variation, <3.3%, labeled as 0), and large (variation, 3.3%, labeled as 1). Univariate logistic analysis showed that NCR and VNA were positively correlated with tumor recurrence after correction for age and sex (p ¼.037 and.059, respectively; Table 2 and 3). Nuclear roundness was not significantly correlated with tumor recurrence (Table 3). Tumor size was positively correlated with tumor recurrence. We put variables that were significantly correlated with recurrence together with age and sex into multivariate logistic analysis using tumor recurrence as dependent variable. Multivariate logistic analysis showed that NCR, VNA, tumor size, and age were significantly correlated with prognosis (p ¼.002,.044,.009, and.001, respectively; Table 4). In Kaplan Meier survival analysis, patients with large and medium NCRs had significantly higher rates of cancer recurrence in 10 years (hazard ratios, 3.44 and 2.89, respectively) than patients with small NCR (Figure 3). Large and medium-sized primary tumors recurred significantly (hazard ratios, 4.94 and 2.46, respectively) compared to small tumors (Figure 4). Older patients had significantly higher recurrence rates in 10 years (hazard ratio, 2.92) than younger patients (Figure 5). Tumors of cells with large and medium VNA had recurred more often than tumors of cells with small VNA, but this finding was not statistically significant (Figure 6). According to the coefficients of the dependent variables in the multivariate logistic regression model (Table 4), FIGURE 2. Variation of nuclear area, determined by the following formula: SD of nuclear area/mean nuclear area. Left: Cytology smear of papillary thyroid cancer (Riu s stain). Right: Computerized morphometry. (A) Large variation of nuclear area (27.28%). (B) Small variation of nuclear area (11.55%). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] 30 HEAD & NECK DOI /HED JANUARY 2013

4 PROGNOSIS OF PAPILLARY THYROID CARCINOMA TABLE 2. Cytologic features and 10-year outcome in patients (N 5 118). Recurrence in 10 y (n ¼ 34) No recurrence in 10 y (n ¼ 84) Variable Mean Minimum Maximum Mean Minimum Maximum p value Nucleus-to-cell ratio (%) Variation of nuclear area (%)xxxxxxxxxxxxxx Small Medium Large Small Medium Large p Value Nucleus-to-cell ratio*, no. of patients (%) 2 (5.9) 26 (76.5) 6 (17.6) 13 (15.5) 67 (79.8) 4 (4.8).038 Variation of nuclear area, no. of patients (%) 0 (0) 23 (67.6) 11 (32.4) 8 (9.5) 58 (69.0) 18 (21.4).110 * Small, medium, and large nucleus-to-cell ratios are defined as nucleus-to-cell ratios of <35%, 35%, <60%, and 60%, respectively. Small, medium, and large variations of nuclear area are defined as variations of <12%, 12%, <25%, and 25%, respectively. we generate a prognostic score (PS). PS was defined as follows: 2.25 NCR þ 1.03 VNA þ 1.1 tumor size þ 1.88 age 0.4 sex. Patients with a higher PS had a higher possibility of cancer recurrence and vice versa. The area under the receiver operating characteristic curve was 82.13%. The optimal cutoff for PS was 6.63 with a sensitivity and specificity of 65.63% and 89.33%, respectively. In the validation group consisting of another 24 patients, the performance of PS was good, with the area under the receiver operating characteristic curve of 85.23%. The average of PS was 8.67 in patients with cancer recurrence and was 5.53 in patients without cancer recurrence in 10 years. As shown in Table 5, all of the patients (100%) in the recurrence group had their PS 6.63, and 7 patients (31.8%) in the nonrecurrence group had their PS The sensitivity and specificity of the PS for predicting cancer recurrence in 10 years is 100% and 68.2%, respectively, in this validation group. DISCUSSION Computerized morphometry is a useful tool for assessing tumors, and it can provide reliable parameters for diagnosis and prognosis. 19 In gastrointestinal and breast epithelial tumors, nuclear morphometry can help in differentiating benign tumors from malignant ones In malignant melanoma, renal cell carcinoma, and rhabdomyosarcoma, morphometry can help in predicting prognosis Various researchers have also shown the usefulness of morphometry as an objective method for histologic grading in thyroid neoplasms. 19,31 33 In thyroid follicular neoplasms, nuclear parameters, including mean nuclear area, mean nuclear perimeter, ratio of the largest to the smallest diameters of the nuclei, coefficient of variation of the nuclear area, and circular rate, have been helpful in differential diagnosis of follicular adenoma and carcinoma. 19,33,34 Morphometry of lymphoid cells of jugular and paratracheal lymph nodes collected by biopsy and autopsy was used for rapid diagnosis of regional metastases in papillary thyroid cancer. 31 Quantitative changes in intrathyroid lymphoid cells, determined by morphometry, were used as markers of thyroid malignancy. 32 Morphometry is difficult in histology specimens because cell boundaries are often crowded. This is less of a problem in cytology specimens because cells are more spread out. 35 Our study showed that NCR and VNA of FNA samples of PTC had prognostic significance. Nuclear area has been shown to be correlated to prognosis in many carcinomas It may be related to cellular activities of duplication and protein production. 18 In this study, we used NCR instead of nuclear area because NCR is more TABLE 3. Relationship between tumor recurrence, cytologic features, tumor size, age, and sex. Tumor recurrence or not in 10 years was used as the dependent variable in univariate logistic regression models. Nucleus-to-cell ratio* Variation of nuclear area Nuclear roundness Variation of nuclear roundness Tumor size Age** Sex Crude Coefficient p value Adjusted by age and sex Coefficient p value Note: Tumor recurrence was labeled as 1. Tumor without recurrence in 10 years was labeled as 0. * Nucleus-to-cell ratio is divided into 3 groups: small (ratio, <35%; labeled as 0), medium (ratio, 35% and <60%; labeled as 1), and large (60%; labeled as 2). Variation of nuclear area is divided into 3 groups: small (variation, <12%; labeled as 0), medium (variation, 12% and <25%; labeled as 1), and large (variation, 25%; labeled as 2). Nuclear roundness is divided into 2 groups: rounder (mean of nuclear roundness, <1.125, labeled as 0), and more irregular (mean of nuclear roundness, 1.125, labeled as 1). Variation of nuclear roundness is divided into 2 groups: small (variation, <3.3%, labeled as 0), and large (variation, 3.3%, labeled as 1). Tumor size is divided into 3 groups: small (size, 2 cm; labeled as 1), medium (size, >2 cm and 4 cm; labeled as 2), and large (size, >4 cm; labeled as 3). ** Age is divided into 2 groups: young (age, <50 years; labeled as 0) and old (age, 50 years; labeled as 1). Sex: male is labeled as 1 and female as 0. HEAD & NECK DOI /HED JANUARY

5 SHIH ET AL. TABLE 4. Multivariate logistic regression model and Cox proportional hazard model for the cytologic and clinical features to predict tumor recurrence (no. of patients 5 83). Multivariate logistic analysis Variable Coefficient p value Cox regression analysis Hazard ratio p value Nucleus-to-cell ratio* Variation of nuclear area Tumor size Age Sex * Nucleus-to-cell ratio is divided into 3 groups: small (ratio, <35%; labeled as 0), medium (ratio, 35% and <60%; labeled as 1), and large (60%; labeled as 2). Variation of nuclear area is divided into 3 groups: small (variation, <12%; labeled as 0), medium (variation, 12% and <25%; labeled as 1), and large (variation, 25%; labeled as 2). Tumor size is divided into 3 groups: small (size, 2 cm; labeled as 1), medium (size, >2 cm and 4 cm; labeled as 2), and large (size, >4 cm; labeled as 3). Age is divided into 2 groups: young (age, <50 years; labeled as 0) and old (age, 50 years; labeled as 1). Sex: male is labeled as 1 and female as 0. FIGURE 4. Kaplan Meier survival estimates of tumor sizes determined using recurrence as endpoint. *HR: hazard ratio, compared with the group with small tumor size after adjustment for age and sex. simply calculated. In addition, a study showed that the nuclear area of PTC cells was significantly related to patient s age, 36 which may cause statistical deviation. Our study showed that NCR was not significantly related to patient s age (p ¼.716), which makes it suitable for prognosis prediction. The coefficient of variation of nuclear area represents different ploidy statuses and DNA contents and is related to prognosis in PTC and breast cancer. 37,38 In addition to the usual type of PTC, several variants of the carcinoma have been described, such as follicular, tall cell, columnar cell, solid, diffuse sclerosing, and trabecular PTCs. 5 Tall cell and columnar cell variants are more aggressive, and controversy exists regarding the outcomes for the diffuse sclerosing variant. 11,39 In addition to considering the different variants, certain histopathologic features have been found to be of significant importance for patient survival. 40 The current histologic grading system is based on nuclear atypia, tumor necrosis, and vascular invasion (VAN score). Furthermore, patient age, extrathyroidal invasion, initial distant metastasis, large primary tumor size, and extent of initial tumor resection are also related to outcome. 1 4,6,7 The TNM staging system; patient age, tumor grade, tumor extent, and tumor size (AGES) scoring system; and metastasis, age, completeness of resection, invasion, and size (MACIS) scoring system were developed accordingly, but these scores can be determined only after surgical resection 3 5 and, thus, are not useful in deciding the extent of primary surgery. More data should be collected, by using patient age, tumor size (determined by echographic measurement), and cytology specimens, and a new preoperative prognostic scoring system should be developed to help doctors make decisions regarding individual treatment options. In previous prognostic scoring systems, mortality was used as an endpoint. We studied recurrence as an outcome because only a few patients (about 5%) had causespecific mortality compared with those who had relapse of disease (about 15%). 5 The patients quality of life may improve if conservative surgical resection is performed when the preoperative prognostic score is low. The method used for cell fixation is an important factor in quantitative analysis. Different fixatives affect cell FIGURE 3. Kaplan Meier survival estimates of nucleus-to-cell ratio determined using recurrence as endpoint. *HR: hazard ratio, compared with the group with small nucleus-to-cell ratio after adjustment for age and sex. FIGURE 5. Kaplan Meier survival estimates of age determined using recurrence as endpoint. (Hazard ratio ¼ 2.92; p ¼.002, after adjustment for age and sex). 32 HEAD & NECK DOI /HED JANUARY 2013

6 PROGNOSIS OF PAPILLARY THYROID CARCINOMA In conclusion, computerized morphometry of cytologic features of samples obtained by FNA is an objective and easy method of predicting prognosis before operation, which is nonexistent currently. It is also easier for nonpathology doctors and students to observe cytologic features. It may be helpful for planning the course of treatment and for learning. FIGURE 6. Kaplan Meier survival estimates of variation of nuclear area determined using recurrence as endpoint. (Hazard ratio ¼ 1.53; p ¼.186, after adjustment for age and sex). morphology in histology sections and can influence the results of morphometry. 41,42 Each cell fixative changes the cell shape; so the challenge is to choose one that minimally affects the cells. Some authors suggest that for standardization of the fixation procedure, the cytology sample should be fixed immediately after obtaining it by using Merckofix spray (Merck, Darmstadt, Germany). 35 The spray fixative is an aqueous-alcoholic solution containing polyethylene glycol. It is sprayed quickly onto moist slides to build up a protective film of polyglycol and stops the cells from drying out. Then the specimens were stained or stored. Specimens prepared in this manner remain stable for several weeks. The major limitation of this study was the small patient number and the varied number of cells analyzed in each patient. This variation occurred because we chose fields where follicular cells were dispersed, and cell numbers in these fields varied depending on the number of cells aspirated and the skill of the person preparing the smear. Theoretically, the result would be more representative if more cells were analyzed. 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