Objective Quantification of the Ki67 Proliferative Index in Neuroendocrine Tumors of the Gastroenteropancreatic System A Comparison of Digital Image Analysis With Manual Methods

Departments of *Pathology †Epidemiology & Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY ‡Department of Surgery, Yale University, School of Medicine, New Haven, CT.
The American journal of surgical pathology (Impact Factor: 5.15). 09/2012; 36(12). DOI: 10.1097/PAS.0b013e318263207c
Source: PubMed


Pathologic grading for prognostic stratification of neuroendocrine tumors (NETs) is critical but presents a challenging interpretive dilemma. Tumor cell proliferative rate is an important factor in the determination of prognosis, and immunohistochemical analysis with Ki67 is becoming more widely used to quantify the proliferative rate. However, Ki67 assessment has limitations due to lack of uniformity and consistency in quantification. These limitations are accentuated in well-differentiated NETs, as differences in the range of 1% to 5% can alter tumor grade, with potential implications for treatment. We therefore performed a concordance study to assess different Ki67 quantification techniques including: (a) digital image analysis (DIA); (b) manual counting (MC) of >2000 cells; and (c) "eyeballed" estimate (EE) of labeling percentage by pathologists (n=18), including individuals experienced in evaluating Ki67 labeling as well as others who had little prior experience assessing Ki67 percentages. Forty-five Ki67 images were selected and analyzed using the 3 methods. On the basis of the recommendations of the World health Organization (WHO) for grading NETs, MC of 2000 cells was used as the "gold standard" reference against which the other techniques were compared. Three images were presented twice, the second being inverted, to assess intraobserver consistency. Statistical analyses were performed to evaluate: (a) the concordance between methods; (b) intraobserver and interobserver consistency; and (c) correlation of NET grades on the basis of Ki67 scores by EE versus the gold standard. Agreement between scores was assessed by intraclass correlation (ICC). DIA and MC were highly concordant (ICC=0.98). The ICC between DIA and the mean EE of all observers was 0.88. However, there was discordance among individual observers on all cases quantified by EE (ICC=0.13). The ICC for intraobserver consistency was 0.39±0.26. With Ki67 in the ranges of <1%, 2% to 3%, and >20%, the mean of Ki67 by EE was, respectively, 93%±2%, 55%±7%, and 55%±15% correct against the gold standard. The κ statistics for EE exhibited low agreement (κ=0.24; 95% confidence interval, 0.23-0.25) for all WHO NET grades. Incorrect assessment by EE resulted in upgrading of all WHO G1 group tumors (n=14); in the WHO G2 group, downgrading of 41% cases occurred (n=11) when Ki67 was <5% (by DIA or MC), and upgrading of 59% cases occurred (n=16) when Ki67 was >5%. We conclude that DIA and MC are the acceptable standards for Ki67 assessment. Given the inherent discordance in determining the grade, the use of an approximate EE of the Ki67-labeling index requires critical reevaluation, especially for NETs with a labeling index straddling the cut-points between grades. Consequently, determination of therapeutic strategies should be guided by an amalgamation of clinicopathologic characteristics, including but not limited to the Ki67 index.

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    • "The utility of this marker, and others where quantification is important, may benefit from an observer independent assessment using digital imaging to allow high-throughput analysis and to overcome subjectivity and heterogeneity of expression. A recent study found that Ki-67 quantification by digital image analysis and manual counting were highly concordant [43]. It is now important to evaluate the prognostic value of PI as a continuous variable with other prognostic biomarkers to identify the most clinically informative algorithm of risk for patients with ESFT. "
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    ABSTRACT: The prognostic value of proliferation index (PI) and apoptotic index (AI), caspase-8, -9 and -10 expression have been investigated in primary Ewing's sarcoma family of tumours (ESFT). Proliferating cells, detected by immunohistochemistry for Ki-67, were identified in 91% (91/100) of tumours with a median PI of 14 (range 0–87). Apoptotic cells, identified using the TUNEL assay, were detected in 96% (76/79) of ESFT; the median AI was 3 (range 0–33). Caspase-8 protein expression was negative (0) in 14% (11/79), low (1) in 33% (26/79), medium (2) in 38% (30/79) and high (3) in 15% (12/79) of tumours, caspase-9 expression was low (1) in 66% (39/59) and high (3) in 34% (20/59), and caspase-10 protein was low (1) in 37% (23/62) and negative (0) in 63% (39/62) of primary ESFT. There was no apparent relationship between caspase-8, -9 and -10 expression, PI and AI. PI was predictive of relapse-free survival (RFS; p = 0.011) and overall survival (OS; p = <0.001) in a continuous model, whereas AI did not predict outcome. Patients with tumours expressing low levels of caspase-9 protein had a trend towards a worse RFS than patients with tumours expressing higher levels of caspase-9 protein (p = 0.054, log rank test), although expression of caspases-8, -9 and/or -10 did not significantly predict RFS or OS. In a multivariate analysis model that included tumour site, tumour volume, the presence of metastatic disease at diagnosis, PI and AI, PI independently predicts OS (p = 0.003). Consistent with previous publications, patients with pelvic tumours had a significantly worse OS than patients with tumours at other sites (p = 0.028); patients with a pelvic tumour and a PI≥20 had a 6 fold-increased risk of death. These studies advocate the evaluation of PI in a risk model of outcome for patients with ESFT.
    PLoS ONE 08/2014; 9(8):e104106. DOI:10.1371/journal.pone.0104106 · 3.23 Impact Factor
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    • "There is significant discordance between visual estimation and digital image analysis for Ki-67 immunoreactivity [15]. Thus, we performed computer-assisted cytometrical analysis using the WinRooF image-processing software, which we developed in our previous study on Ki-67 immunoreactivity [16], for grade-3 p53 basal positivity by visual estimation. "
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    ABSTRACT: The basal pattern of p53 expression, defined as its immunoreactivity confined to the basal half of the glands, is associated with early neoplastic lesions in ulcerative colitis (UC). However, their clinical utility of this finding is limited by the use of “visual estimation” (approximate immunoreactivity on the basis of scanning the stained slide, without formal counting). This study was designed to analyze the basal pattern of p53 using computer-assisted cytometry and to identify the optimal cutoff value for discriminating between UC-associated early-stage neoplasia and regenerative atypia. The specimens were obtained from eight UC patients undergoing colectomy and were classified according to the criteria by the Research Committee of Inflammatory Bowel Disease of the Ministry of Health and Welfare in Japan. Patients with classes UC-IIa (indefinite for dysplasia, probably regenerative), UC-IIb (indefinite for dysplasia, probably dysplastic), and UC-III (definitive dysplasia) were enrolled in the study. Based on the percentage of immunoreactive cells in the basal half of the crypt with visual estimation, basal positivity of p53 was classified into three categories: grade 1 (1 - 9%), grade 2 (10 - 19%), and grade 3 (≥20%). Next, crypts classified as grade 3 by visual estimation were analyzed by computer-assisted image analysis. Using visual estimation, grade-3 p53 basal positivity was observed in 46.0% of UC-IIa crypts (128 of 278), 61.9% of UC-IIb crypts (39 of 63), and 94.2% of UC-III crypts (81 of 86). Using image analysis, the median p53 basal positivities were 30.3% in UC-IIa, 52.3% in UC-IIb, and 65.4% in UC-III (P ≤0.002). A receiver operating characteristics curve was generated to determine the method’s diagnostic utility in differentiating UC-IIa from UC-III. In this cohort, the sensitivity was 0.78; the specificity was 0.98; the negative predictive value was 87.4%; the positive predictive value was 95.5%, and the accuracy was 90.2% with a cutoff value for p53 basal positivity of 46.1%. Our findings indicate that assessing p53 basal positivity by image analysis with an optimal threshold represents an alternative to visual estimation for the accurate diagnosis of UC-associated early-stage neoplasia. Virtual Slides The virtual slide(s) for this article can be found here:
    Diagnostic Pathology 05/2014; 9(1):99. DOI:10.1186/1746-1596-9-99 · 2.60 Impact Factor
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    • "In particular, computerassisted methods like the Aperio, now available in major centers, can be safely adopted thus reducing the burden of the quantitative assessment, the major criticism so far moved to Ki67-based grading systems. Of interest, similar efficacy was demonstrated for Ki67 eyeballing assessment (Tang et al. 2012). Finally, in light of previous evidence (Pelosi et al. 2005), the use of Ki67 as further grading variable will hopefully be of help in establishing the diagnosis of intermediate malignant neuroendocrine cancer (TC vs AC) in small biopsies too. "
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    ABSTRACT: Lung neuroendocrine tumors are catalogued in four categories by the World Health Organization (WHO 2004) classification. Its reproducibility and prognostic efficacy was disputed. The WHO 2010 classification of digestive neuroendocrine neoplasms is based on Ki67 proliferation assessment and proved prognostically effective. This study aims at comparing these two classifications and at defining a prognostic grading system for lung neuroendocrine tumors. The study included 399 patients who underwent surgery and with at least 1 year follow-up between 1989 and 2011. Data on 21 variables were collected, and performance of grading systems and their components was compared by Cox regression and multivariable analyses. All statistical tests were two-sided. At Cox analysis, WHO 2004 stratified patients into three major groups with statistically significant survival difference (typical carcinoid vs atypical carcinoid (AC), P=0.021; AC vs large-cell/small-cell lung neuroendocrine carcinomas, P<0.001). Optimal discrimination in three groups was observed by Ki67% (Ki67% cutoffs: G1 <4, G2 4-<25, G3 ≥25; G1 vs G2, P=0.021; and G2 vs G3, P≤0.001), mitotic count (G1 ≤2, G2 >2-47, G3 >47; G1 vs G2, P≤0.001; and G2 vs G3, P≤0.001), and presence of necrosis (G1 absent, G2 <10% of sample, G3 >10% of sample; G1 vs G2, P≤0.001; and G2 vs G3, P≤0.001) at uni and multivariable analyses. The combination of these three variables resulted in a simple and effective grading system. A three-tiers grading system based on Ki67 index, mitotic count, and necrosis with cutoffs specifically generated for lung neuroendocrine tumors is prognostically effective and accurate.
    Endocrine Related Cancer 02/2014; 21(1):1-16. DOI:10.1530/ERC-13-0246 · 4.81 Impact Factor
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