Quantitative histopathological assessment of ocular surface squamous neoplasia using digital image analysis

Introduction/ Background Ki67 is an important immunohistochemical marker of proliferation used in grading of breast cancer and endocrine neoplasms. Ki67 proliferation indices (PI) are calculated as number of Ki67 positive tumour cells divided by total number of tumour cells. However, manual counting and calculation of Ki67 proliferation index is laborious and prone to inter-observer variability. Recently, a computerized algorithm that enables virtual alignment of two consecutive slides stained for pancytokeratin and Ki67 has been developed. Digital image analysis (DIA) based on Virtual Double Staining (VDS) enables exclusion of stromal cells and calculation of Ki67 PI in tumour cells only. Aims The purpose of this study was to validate the VDS algorithm by comparing manual counting and DIA on VDS for assessment of Ki67 PI in breast carcinomas. Methods Tissue Micro Arrays (TMA) were constructed with 158 cores of breast carcinomas. Two slides were cut from each TMA and immunohistochemically stained for pancytokeratin and Ki67. For each core, between 2-20% of the total core area were selected for exact manual counting of Ki67-positive and negative cells using the stereological principle of systematic uniformly random sampling. A minimum of 200 cells were counted. The same areas were then counted using the VDS algorithm. Additionally, the VDS algorithm was used to calculate Ki67 PI for each core. In order two analyze the importance of the distance between neighbouring slides five consecutive slides were stained for PCK. These slides were digitally fused and the percentage of overlap between stained and not stained areas calculated. Additionally, the VDS principle was used to examine differences in Ki67 PI when the immunohistochemical staining protocol was based on different antibody clones (Mib1, SP6 and 30-9) and staining platforms (Dako Autostainer, Leica Bond and Ventana Ultra). Results There was good correlation (R2 >0.90, ICC >0.95) between manual counting in systematic randomized selected areas and DIA on VDS of both the whole core and in selected areas. Comparison of the two methods using Bland-Altman plots did not reveal any skewness in certain data ranges. Overlap agreement between neighbouring sections was on average above 88%, lower for diffusely infiltrating tumours than more solid tumours. Analysis revealed significant differences in calculated Ki67 PI between the different antibodies. The Mib1 and SP6 clones (concentrated format) were comparable on the Dako and Leica platform, while the average Ki67 PI for the SP6 clone was 44% higher on the Ventana platform. For the ready-to-use formats, Mib1 and MM1 based Ki67 PIs were 28% and 36% lower than average, while the clone 30.9 based Ki67 PI was 23% higher. In c onclusion, DIA based on VDS may be an important future tool for improving accuracy and reproducibility of Ki67 PI in diagnostic and research settings.

Incidence of ocular surface squamous neoplasia in pterygium specimens

Although uncommon, medullary thyroid carcinoma (MTC) accounts for a significant proportion of thyroid cancer deaths. Recent studies have validated the two-tier International Medullary Thyroid Carcinoma Grading System (IMTCGS) to predict clinical outcomes. A 5% Ki67 proliferative index (Ki67PI) cut-off separates low-grade from high-grade MTC. In this study, we compared digital image analysis (DIA) to manual counting (MC) for determining the Ki67PI in a MTC cohort, and explored the challenges encountered. Available slides from 85 MTCs were reviewed by two pathologists. The Ki67PI was documented by immunohistochemistry for each case, scanned with the Aperio® slide scanner at 40× magnification, and quantified using the QuPath® DIA platform. The same hotspots were screenshot, printed in color, and blindly counted. For each case, over 500 MTC cells were counted. Each MTC was graded using IMTCGS criteria. In our MTC cohort (n = 85), 84.7 and 15.3% were low- and high-grade with the IMTCGS. In the entire cohort, QuPath® DIA performed well (R2 = 0.9891) but appeared to undercall compared to MC. QuPath® performed better in high-grade cases (R2 = 0.99) compared to low-grade cases (R2 = 0.7071). Overall, Ki67PI determined with either MC or DIA did not affect IMTCGS grade. Encountered DIA challenges include optimizing cell detection, overlapping nuclei, and tissue artifacts. Encountered MC challenges include background staining, morphologic overlap with normal elements, and counting time. Our study highlights the utility of DIA in quantifying Ki67PI for MTC and can serve as an adjunct for grading in conjunction with the other criteria of mitotic activity and necrosis.

To describe the pattern of ocular surface squamous neoplasia (OSSN), clinical presentations, the risk factors and treatment options. An observational case series. Khyber Institute of Ophthalmic Medical Sciences, Hayatabad Medical Complex, Peshawar, from April 2003 till August 2006. The study included 36 eyes of 35 patients with biopsy-proven ocular surface neoplasia. The details of patients regarding age, gender, laterality and risk factors were entered into a specially-designed proforma. Each patient was also assessed biomicroscopically for type and complications of ocular surface neoplasia. The frequency of OSSN was 0.37 among admitted hospital patients. Among 36 cases of OSSN, squamous cell carcinoma of the conjunctiva was the most common type of OSSN seen in 63.9%, followed by carcinoma in situ of conjunctiva in 25% and carcinoma in situ of cornea in 11.1%. Male patients outnumbered female (65.7% vs 34.3%) with 71.42% of patients above 60 years of age. The risk factors identified were: old age, ultraviolet B exposure and xeroderma pigmentosa. Treatment consisted of local resection with or without adjuvant therapy in 61.1%, exenteration in 30.5%, enucleation in 5.5% and chemo/radiotherapy in 2.7%. Intraocular invasion was seen in 5.5% and orbital spread in 30.5%. The frequency of OSSN was 0.37% among admitted patients. Identification of exact etiological factors will enable to formulate strategies that are likely to decrease the incidence of this disease and the associated morbidity and mortality.

Optimal Approaches to Grading Enteropancreatic Neuroendocrine Tumors Using Ki-67 Proliferation Index: Hotspot and Whole-Slide Digital Quantitative Analysis.

To investigate the expression of effector protease receptor-1 (EPR-1), proliferative index ki-67 and apoptosis index in patients with primary advanced gastric adenocarcinoma and to clarify the significance of EPR-1 expression and its correlationship with the proliferation and apoptosis indexes. Using immunohistochemical staining and terminal deoxynucleotidyl transferase mediated nick end labelling (TUNEL) technique, we determined the expression of EPR-1, proliferative index (Ki-67) and apoptotic index (AI) in 120 paraffin-embedded specimens of primary advanced gastric adenocarcinoma as well as lymph node metastasis and adjacent normal tissues. EPR-1 expression was distributed in the cytoplasm of normal gastric mycoderma, carcinoma cells and smooth muscle cells. The positive rate of EPR-1 expression in the primary gastric adenocarcinomas, invasion tumor node and lymph node metastasis was 65.83%, 55.29% and 68%, respectively. While the positive rate in normal gastric mycoderma and smooth muscle cells was 46.7% and 53.3%, respectively. The average positive rate of ki-67 in EPR-1-positive tumors was 7.00% which was significantly lower than that of 8.53% in EPR-1-negative tumors, but the average AI in EPR-1-positive tumors was 1.25%, which was significantly higher than that of 1.00% observed in EPR-1-negative tumors. On the other hand, the average positive labeling index for Ki-67 (ki-67) in EPR-1-positive lymph node metastasis was 7.65%, which was significantly lower than that of 9.44% observed in EPR-1-negative lymph node metastasis. However, the average AI in EPR-1-positive lymph node metastasis tumors was 0.99%, which was significantly higher than that of 0.67% observed in EPR-1-negative lymph node metastasis. The frequency of EPR-1 expression was significantly higher in primary gastric adenocarcinoma and in its lymph node metastasis than that in normal gastric mucosa. Expression of EPR-1 was significantly correlated with tumor histological subtypes and tumor differentiation. Weighted EPR-1 Score is positively correlated with apoptosis index, but is negatively related with proliferative index. Thus, Weighted EPR-1 Score and EPR-1 expression in gastric adenocarcinoma cells maybe a potential marker in clinical setting.

Urinary cytology is sensitive and specific for diagnosing and screening high-grade urothelial carcinomas (HGUC). The Paris System (TPS) for urinary cytology was introduced in 2016 to standardize reporting. According to TPS diagnostic categories of HGUC and suspicious for HGUC (SHGUC), the average nuclear-to-cytoplasm (N:C) ratio of atypical cells should be ≥0.7. The objective of the current study was to measure the N:C ratio of urine cytology specimens with HGUC and SHGUC diagnoses and biopsy-proven HGUC follow-up. A cohort of 64 cases (HGUC, 49 cases; SHGUC, 15 cases) from 57 patients was constructed. Urine cytology slides were scanned into whole-slide digital images. The nuclear and cytoplasmic areas were enumerated by digital image analysis (DIA), and the N:C ratios were measured. In total, 640 cells were analyzed by DIA (HGUC, 490 cells; SHGUC, 150 cells). For HGUC and SHGUC, the average N:C ratios were 0.57 and 0.53, respectively. The maximum average N:C ratio was 0.73 for HGUC and 0.68 for SHGUC. HGUC had higher average N:C ratio (P < .001), higher average nuclear area (P < .001), higher average maximum N:C ratio (P = .005), and higher average maximum nuclear area (P = .006) compared with SHGUC. The N:C ratios for the HGUC (0.57) and SHGUC (0.53) categories are lower than those previously suggested in TPS. The authors advocate reducing the N:C ratio below the current threshold of 0.7.

Background Oncotype DX is a 21-gene assay for node-negative, ER positive breast cancer. Results are expressed as a Recurrence Score (RS). This predicts a low, intermediate or high risk of distant relapse. Proliferation-related genes such as Ki67 impact heavily upon the calculation of the RS. The assay encompasses several histopathologic factors (ER, HER2) and is limited by cost, proprietary nature and turnaround time. We investigated whether Ki67 proliferation index (PI) and routine clinicopathologic parameters could be used to predict RS. Materials and Methods We obtained clinicopathologic details for 69 patients (diagnosed 2007–2010) who had RS available. Quantitative analysis of Ki67 immunohistochemistry was performed on primary tumour. Patients were classified as having a low (≤15%), intermediate (16-30%) or high (&amp;gt;30%) Ki67 PI. Risk of recurrence was calculated using Adjuvant! Online. Patients with categorized as having a poor, moderate, good or excellent prognosis as per the Nottingham prognostic index (NPI) and a low, intermediate or high risk of relapse, according to the St. Gallen criteria. Results Mean age at diagnosis was 51.4 years (range: 34–74 years). Mean tumour size was 1.9 cm (range: 0.8−4.1cm). Mean Ki67 PI was 13.4 % (range 0.2 to 52.6%). RS was low (0-17) in 34 cases, intermediate (18-30) in 26 cases and high (&amp;gt;30) in 9 cases. The RS was significantly correlated with Ki67 expression (Spearman's rank correlation, r = 0.472, p&amp;lt;0.001). RS was significantly higher in patients with a high Ki67 PI, when compared to patients with low and intermediate Ki67 PI (Mann Whitney U, p&amp;lt;0.05). RS was significantly higher in patients with grade 3 tumours, when compared to patients with grade 1 and 2 tumours (Mann Whitney U, p&amp;lt;0.05). Risk of recurrence as predicted by Adjuvant! Online, was significantly higher in patients with a high RS, when compared to patients with low and intermediate RS (Mann Whitney U, p&amp;lt;0.05). RS was significantly higher in patients with moderate prognosis, as determined by the NPI, when compared to patients classified as having a good/excellent prognosis (Mann Whitney U, p&amp;lt;0.05). No patients were classified by the NPI as having a poor prognosis. RS was significantly higher in patients deemed to be at high risk of relapse by the St. Gallen criteria, when compared to patients classified as low and intermediate risk (Mann Whitney U, p&amp;lt;0.05). The majority (66.7%) of cases in the high RS group had LVI present. Conclusions This pilot study indicates a trend whereby Ki67 and grade will predict whether the (Oncotype DX RS is low/intermediate or high. Adjuvant! Online, St. Gallen and NPI are also able to distinguish patients with a high RS from those with low/intermediate RS. In order to verify these findings, we are developing a statistical model using these 69 patients as a training set. As there is insufficient follow-up to correlate either RS or Ki67 with relapse, a potential test set comprised of an additional 50 LN negative, ER positive patients, with an average of 13 years follow-up, has been identified. Ki67 analysis of these cases is underway to address this issue. Acknowledgement This work is funded by SFI (SRC award, 08/SRC/B1410 to MTCI). Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-13-12.

Objective Meningiomas are known to recur frequently, even after complete resection, and some are refractory to treatment. Residual cancer stem cells (CSCs) after routine therapy may cause tumor recurrence and metastasis through self-renewal and multilineage differentiation. CSCs are increasingly realized to be necessary targets for effective cancer treatment. CD133 could be characterized as a marker of a CSC subpopulation and an invasive tumor phenotype. Aim The aim of the study was to investigate the possible role of the immunohistochemical expression of CD133 in prognosis, as well as the relationship between CD133, matrix metalloproteinase-9 (MMP-9), and Ki-67 expression in various grades of meningiomas. Materials and methods Fifty-two cases of various grades of meningiomas were tested by immunohistochemical analysis for the expression of CD133 protein, MMP-9, and Ki-67 proliferation index (PI). Results The high expression of CD133, MMP-9, and Ki-67 PI was significantly associated with higher grade meningiomas. CD133, MMP-9 expression, and Ki-67 PI in meningiomas were positively related to each other (all P Conclusion There is a close relationship between the overexpression of CD133, MMP-9, and Ki-67 PI and progression of meningiomas, which together may play important roles in aggressive growth and relapse. Thus, their expression may be of value in further understanding the biological behavior and predicting the prognosis of meningiomas.

A knowledge-based system has been used for analysis of morphometric data obtained from 218 neuroepithelial tumors. These tumors have been investigated using standardized morphometric procedures including nuclear area, shape factors, proliferation index Ki-67. It was intended to perform a comprehensive data analysis using different grouping variables (“tumortype” and “tumorgrade”). Additionally, non-discrete numerical variables (“mean nuclear area”, “proliferation index” and “patient's age”) have been tested instead of grouping variables in order to find out, whether different ranges for those variables are exhibited and show significant differences between the cases. Several important informations were provided by the implementation of the knowledge-based system, which are difficult to obtain by means of descriptive data analysis or by means of statistical analysis only. These include for instance a separation of cases with considerably high proliferation index showing a higher age of these patients. Especially, relationships between several variables could be obtained, which could not be detected in conventional statistical data analysis. The information provided by the system has a direct morphological relevance for the evaluation of the tumors. Its use for data analysis of morphometric studies has to be considered as useful, especially when many quantitative variables are under investigation.

To determine the reliability and efficiency of in vivo confocal microscopy for the diagnosis of ocular surface squamous neoplasia (OSSN). A case series with five consecutive cases of OSSN were investigated retrospectively, of which the characteristics and subspecial types had been estimated by in vivo confocal microscopy before surgery. The structure and cellular features of OSSN were analyzed with other examinations, such as anterior-segment optical coherence tomography (AS-OCT), and confirmed by histopathological biopsy. The tumors revealed red gelatinous surfaces with vascular dilatation on the ocular surface of the conjunctival and corneal epithelium in anterior segment photography. Involvement of only corneal epithelium was observed by AS-OCT in three cases, whereas the Bowman's layer and anterior stroma were also invaded in the other two cases. In vivo confocal microscopy showed cellular anisocytosis and enlarged nuclei with high nuclear to cytoplasmic ratio in three cases diagnosed as conjunctival intraepithelial neoplasia; moreover, nests were partially formed by isolated keratinized, binucleated, and actively mitotic dysmorphic epithelial cells in the other two cases diagnosed as carcinoma in situ and ocular surface squamous carcinoma (OSSC). The characteristics assessed from histopathological biopsy were similar to that revealed by in vivo confocal microscopy in all five cases. In vivo confocal microscopy analysis of cytological characteristics of OSSN is a safe, relatively noninvasive, and effective diagnostic tool in detecting characteristics of OSSN before surgical resection. Although in vivo confocal microscopy cannot replace excisional biopsy for definitive diagnosis, it can be valuable for initial diagnosis and management of patients with OSSN.

Ocular surface squamous neoplasia (OSSN) is a heterogeneous group of proliferative squamous lesions on the ocular surface with varying biologic behaviours. This study aims to report the clinical profile and pathological characteristics of cases of OSSN seen at a tertiary referral centre in North West Nigeria. A retrospective review of all cases of OSSN diagnosed over a 10-year period was done. OSSN accounted for 68 out of 91 ocular surface lesions affecting twice as many males as females and a peak incidence in the 30-39years age group. They frequently presented as higher-grade and higher-stage lesions with invasive squamous cell carcinoma being the most frequently diagnosed OSSN. They also frequently showed an association with HIV infection and a relatively long duration of symptoms before presentation. OSSN occurs in a relatively young age group in our environment. Certain clinical and epidemiological features appear to predict the occurrence of higher-grade lesions, and this may help in the clinical prediction of likely pathologic grade and/or biologic behaviour of these lesions.

Purpose To investigate a tumor proliferation marker Ki‐67 in conjunctival malignancies and to compare the results of different quantitative assessments.Methods This study was designed as a pilot study to assess nuclear proliferation protein (Ki 67) in conjunctival malignancies. Immunohistochemical staining of Ki‐67 was evaluated in 8 surgical samples of ocular surface neoplasias: 6 cases of invasive conjunctival squamous carcinoma and 2 of conjuntival melanoma. Ki‐67 counting and evaluation was assessed on selected areas using different methods: manual counting (MC) of &gt;1500 cells;digital image analysis with Nuclear V9 algorithm and digital image analysis using stereology module for ImageScope of Aperio. Ki‐67 values from manual, stereoscopic and automated analyses were compared.Results The MC, the nuclear V9 algorithm analysis and stereologic method revealed positivity of Ki‐67 in invasive conjunctival squamous carcinoma from 22 to 60%, 21.5 to 43.5 % and from 30.1 to 51.5% respectively; in conjunctival melanomas from 20 to 30 %; from 17.8 to 18%, and from 7.17 to 29.1% respectively. The strongest correlation was observed between Aperio algorithm and stereologic method in conjunctival squamous carcinoma (0.87). Weaker correlation was found between Aperio/manual (0.6) and manual/stereological (0.83) methods.Conclusion Our data demonstrate that different methods of Ki‐67 evaluation may give dissimilar results, but results of this computer assisted nuclear V9 algorithm correlates strongly with stereoscopic analysis and can be helpful in accurate quantitative evaluation of imunochistochemically stained biomarkers.

Olfactory neuroblastomas (ONBs) are rare malignant tumours that arise in the nasal vault. To date, the Hyams grade remains the only widely used histological grading system. However, it is based only on morphological criteria, and has not been updated since 1988. The objective of this study was to explore the prognostic potential of the Ki67 proliferation index (PI) and tumour-infiltrating lymphocytes (TILs) in ONB. A retrospective study was conducted on a bicentric series of 45 cases. The Ki67 PI was determined by counting at least 1000 nuclei on whole slides. TILs were evaluated with CD20, CD4 and CD8 immunohistochemical markers on whole slides. In this series, Hyams grades I, II, III and IV accounted for 13.4%, 44.4%, 20% and 22.2% of all cases, respectively. The Ki67 PI ranged from 1 to 93; the Ki67 PI was significantly higher in Hyams grade III-IV ONBs than in Hyams grade I-II ONBs (P<0.0001). A Ki67 PI of ≥25 was associated with poorer survival (P=0.02). TILs were present in both stromal and intratumoral compartments, but were located predominantly in the stromal component of the tumour. The numbers of intratumoral CD8+ cells/mm2 and CD4+ cells/mm2 were greater in high-grade ONBs than in low-grade ONBs (P=0.0015 and P=0.043, respectively). The numbers of T cells/mm2 and B cells/mm2 were not associated with survival, but a CD4/CD8 ratio of >2 was significantly associated with shorter survival (P=0.04). Our findings suggest that the Ki67 PI and TILs could be used as prognostic markers, as a potential alternative to the Hyams grade.

The characterization of nuclear area, the proliferation index, and nuclear DNA content was carried out by means of digital cell image analysis, which makes it possible to compute morphometric and densitometric features on Feulgen-stained nuclei from archival, that is, formalin-fixed, paraffin-embedded materials. The 181 meningiomas studied included 173 classic (41 meningotheliomatous, 27 fibroblastic, 82 transitional, nine psammomatous, eight angiomatous and six hemangioblastic tumors) and eight malignant meningiomas (three hemangiopericytomas and five tumors that we labeled HFM, that is, tumors exhibiting evidence of histological features of malignancy). The results reveal a strong relationship between incomplete surgical resection and recurrence on the one hand and between the probability of recurrence and histopathological type on the other. Whereas neither nuclear area nor nuclear DNA content assessments were helpful in distinguishing the six classic and the two malignant meningioma subgroups, a statistically significant increase in proliferative activity was observed in the malignant meningiomas as compared with classic ones, excepting hemangioblastomas that proliferate at the same rate as the malignant meningiomas. Furthermore, the multiple meningiomas definitely proliferated more actively than the single ones, but a similar proliferative activity was observed in the nonrecurrent and recurrent meningiomas. Proliferation analyses might be therefore helpful for determining aggressive meningiomas and for planning adjuvant therapy in these cases.