Ki-67 Score Research Articles

Expression of spindle assembly checkpoint proteins BubR1 and Mad2 expression as potential biomarkers of malignant transformation of oral leukoplakia: an observational cohort study.

Background The Spindle Assembly Checkpoint (SAC) is a surveillance mechanism essential to ensure the accuracy of chromosome segregation during mitosis. Our aim was to evaluate the expression of SAC proteins in oral carcinogenesis, and to assess their potential in predicting malignant transformation of oral leukoplakia.Material and Methods We analysed the immunoexpression of BubR1, Mad2, Bub3, and Spindly proteins in 64 oral biopsies from 52 oral leukoplakias and 12 normal tissues. Univariate and multivariate analysis were performed to evaluate predictive factors for malignant transformation (MT).Results We observed that BubR1 and Mad2 were more highly expressed in high dysplasia grade lesions than in low grade or normal tissues (P<0.05). High expression of Spindly was significantly correlated with a high Ki-67 score (P=0.004). Six (11.5%) oral leukoplakias underwent malignant transformation. In univariate analysis, the binary dysplasia grade (high grade) (P<0.001) was associated with a higher risk of malignant transformation as well as high BubR1 (P<0.001) and high Mad2 (P=0.013) expression. In multivariate analysis, high expression of BubR1 and Mad2 when combined showed an increased risk for malignant transformation (P=0.013; HR of 4.6, 95% CI of 1.4-15.1).Conclusions Our findings reveal that BubR1 and Mad2 were associated with an increased risk for malignant transformation independently of histological grade and could be potential and useful predictive risk markers of malignant transformation in oral leukoplakias. Key words:BubR1, Mad2, Spindly, Bub3, Oral Leukoplakia, epithelial dysplasia, Oral squamous cell carcinoma.

Prediction of the Ki-67 marker index in hepatocellular carcinoma based on CT radiomics features

The noninvasive detection of tumor proliferation is of great value and the Ki-67 is a biomarker of tumor proliferation. We hypothesized that radiomics characteristics may be related to tumor proliferation. To evaluate whether computed tomography (CT) radiomics feature analyses could aid in assessing the Ki-67 marker index in hepatocellular carcinoma (HCC), we retrospectively analyzed preoperative CT findings of 74 patients with HCC. The texture feature calculations were computed from MaZda 4.6 software, and the sequential forward selection algorithm was used as the selection method. The correlation between radiomics features and the Ki-67 marker index, as well as the difference between low Ki-67 (<10%) and high Ki-67 (≥10%) groups were evaluated. A simple logistic regression model was used to evaluate the associations between texture features and high Ki-67, and receiver operating characteristic analysis was performed on important parameters to assess the ability of radiomics characteristics to distinguish the high Ki-67 group from the low Ki-67 group. Contrast, correlation, and inverse difference moment (IDM) were significantly different (P < 0.001) between the low and high Ki-67 groups. Contrast (odds ratio [OR] = 0.957; 95% confidence interval [CI]: 0.926-0.990, P = 0.01) and correlation (OR = 2.5☆105; 95% CI: 7.560-8.9☆109; P = 0.019) were considered independent risk factors for combined model building with logistic regression. Angular second moment (r = −0.285, P = 0.014), contrast (r = −0.449, P < 0.001), correlation (r = 0.552, P < 0.001), IDM (r = 0.458, P < 0.001), and entropy (r = 0.285, P = 0.014) strongly correlated with the Ki-67 scores. Contrast, correlation, and the combined predictor were predictive of Ki-67 status (P < 0.001), with areas under the curve ranging from 0.777 to 0.836. The radiomics characteristics of CT have potential as biomarkers for predicting Ki-67 status in patients with HCC. These findings suggest that the radiomics features of CT might be used as a noninvasive measure of cellular proliferation in HCC.

Relationship of Oncotype Dx score with tumor grade, size, nodal status, proliferative marker Ki67 and Nottingham Prognostic Index in early breast cancer tumors in Saudi Population

BackgroundOncotype Dx is a 21-gene recurrence score, which is used as a diagnostic tool for the recurrence of breast cancer. It is also used to determine the benefit of chemotherapy for breast cancer in early stages. This study investigates the relationship of Oncotype Dx with pathological prognostic markers of protein Ki 67, Nottingham Prognostic Index (NPI) and tumor grade. MethodsData for early breast cancer patients treated at our tertiary care center was collected for statistical analysis. Data for patients from 2014 to 2018 was recorded for patient's age, ER/PR status, Ki 67, nodal status, tumor grade, NPI along with Oncotype Dx score. Metric measurements were described as mean ± SD and the non-metric data was represented by frequency (%). Chi-square or Fisher's exact tests as well as logistic regression was applied to assess the associations at 95% CI. ResultsAmong 156 breast cancer patients, the mean age was 55.7 ± 9.4 years. The tumors were classified into Grade-I (12.8%), Grade-II (67.3%) and Grade-III (19.9%). Ki67 score was 12.8 ± 12.0 and NPI score was 3.7 ± 0.8. The mean Oncotype Dx score was 17.0 ± 9.1; it was 14.1 ± 6.8 for Grade-I tumors; 15.7 ± 7.5 for grade -II tumors; and 23.2 ± 12.3 for grade-III tumors [Mean Oncotype Dx score across Tumor grades was compared by ANOVA (η = 0.121), p < 0.001]. While logistic regression analyses for the dichotomized Oncotype Dx higher score (≥25) was significantly associated with grade-III tumors odds ratio (OR) = 13.72 (95% CI: 1.62–115.89), higher Ki67 (>20) OR = 14.40, (95% CI: 1.44–143.71), average NPI score (2.41–3.40), OR = 13.60, (95% CI: 1.57–117.94) to poor NPI (>5.4). The association of Oncotype Dx with age, tumor size and nodal status was statistically not significant. ConclusionsThis study revealed that age, Ki67, tumor size and nodal status did not have a statistically significant impact on Oncotypye Dx recurrence score in the targeted patient population. There was a significant correlation of low grade node negative patients with Oncotype Dx while high grade node negative patients had poor correlations with Oncotype Dx. The use of Oncotype Dx has shown to be less cost-effective and has no noticeable association with improved life expectancy in the targeted patient population (i.e., hormone positive, node negative cases) in comparison with current clinical practices in Saudi Arabia and it is less likely to be cost-effective in this group of patients.

Impact of Dose Reduction on Efficacy: Implications of Exposure-Response Analysis of Palbociclib.

BackgroundPalbociclib is indicated for hormone receptor–positive, human epidermal growth factor receptor 2–negative advanced breast cancer (ABC).ObjectiveExposure-response analyses were conducted to evaluate efficacy in Asian versus non-Asian patients and in patients with versus without dose reduction in PALOMA-2.Patients and MethodsPALOMA-2 compared palbociclib plus letrozole versus placebo plus letrozole in patients with ABC. Population pharmacokinetic analysis provided apparent palbociclib clearance (CL/F) for each patient. The time-varying exposure metric, Cavg,t, was calculated using average dose intensity and CL/F at the time of each progression-free survival (PFS) event. A Cox proportional model characterized PFS and palbociclib Cavg,t relationships. Significant prognostic factors for PFS were identified by univariate analysis, which were subsequently included in multivariate analyses, in addition to the Cavg,t effect on PFS. PFS profiles in Asian/non-Asian patients and patients with/without dose reduction were simulated and compared using observed palbociclib exposures and established exposure-response relationships.ResultsPatients (n = 421) received palbociclib plus letrozole (Asian = 64, non-Asian = 357; no dose reduction = 272, dose reduction = 149). Based on univariate analyses, significant prognostic factors were Ki67 score, age, and baseline aspartate aminotransferase (BAST), tumor size, alkaline phosphatase, and albumin levels. In multivariate analysis, only Ki67 and BAST remained significant. Palbociclib exposure did not significantly affect PFS in either univariate (P = 0.12) or multivariate (P = 0.44) analyses.ConclusionsThis analysis suggests that palbociclib exposure has no impact on PFS when the dose reduction algorithm from palbociclib clinical trials is used. There is no difference in efficacy between Asians and non-Asians, despite the higher level of dose reductions in Asians.PfizerNCT01740427.

Primary Mantle Cell Lymphoma of the Tonsil: A Case Series

Background: Mantle cell lymphoma (MCL) is a mature B cell non- Hodgkin lymphoma (NHL) characterized by the t (11; 14) (q13; q32) translocation, which results in over-expression of cyclin D1. It comprises about 7% of NHL in the United States and Europe and its overall incidence appears to be increasing in the United States. While most patients present with advanced stage and generalized lymphadenopathy, 25% of patients with MCL present with extra-nodal disease. The most common site of involvement being gastrointestinal tract, liver and bone marrow. Isolated involvement of waldeyer's ring is rare. To date, only a few cases have been reported in the literature. Here we describe three patients with primary MCL of the tonsil, summarizing their presentation and clinical course. Method: We present a case series of MCL of the tonsils diagnosed and followed at a single center from August 2019-May 2020. Data regarding demographics, morphology, IHC stains, clinical presentation, radiographic findings, management and outcome are reviewed. Results: Three patients were diagnosed with tonsillar MCL. Clinical findings are summarized in table 1. All were men with a mean age of 63 years at diagnosis. None had B symptoms. They presented initially with swallowing difficulty and underwent tonsillectomy. They were found to have low risk pathology and clinical features suggesting the indolent form of MCL. Case 1: A 73 year-old with history of MEN-2 disorder presented with swallowing difficulty. A left tonsil mass was noted on examination and he underwent left tonsillectomy. Pathology confirmed diagnosis of MCL of the tonsil, with positive IHC stain for CD5, PAX5, cyclin D1 and SOX11, negative for CD10. The Ki-67 proliferative index was approximately 10-15%. PET-CT did not show any evidence of disease except for few non-specific level 1b and III left cervical nodes. Patient declined further staging work up, including a bone marrow biopsy and biopsy of a small left cervical lymph node to exclude other sites of involvement. He remains symptom-free on observation. Case 2: A 61 year-old presented with intermittent chocking sensation and difficulty swallowing. An enlarged right tonsil was observed on examination. He underwent right tonsillectomy and pathology was consistent with MCL, with positive IHC stain for CD5, cyclin D1 and SOX11, negative for CD 10. Ki-67 proliferative index was 30% and TP53 mutation was negative. Bone marrow evaluation showed 30% involvement with MCL, however, his peripheral cell counts were normal. PET-CT did not show any convincing evidence for other sites of involvement. We recommended watchful waiting and patient remains symptom free on close follow up. Case 3: A 55 year-old presented with dysphagia and sensation of a lump in his throat. He was noted to have bilateral tonsil enlargement. He underwent bilateral tonsillectomy. Pathology showed a population of B-cells with kappa light chain restriction and co-expression of CD5 and SOX 11 on the left tonsil only, consistent with diagnosis of MCL of left tonsil. The Ki-67 proliferative index was 20%. Bone marrow evaluation showed low level of involvement ~1% by MCL. PET-CT did not show any convincing evidence of disease elsewhere. We recommended watchful waiting and patient remains symptom free on close follow up, Conclusion: Primary MCL of the tonsils is an uncommon diagnosis. To the best of our knowledge this is the largest case series of this rare disease reported by a single institution. The demographics, histology and molecular features of our patients are similar to published data on MCL. Mainstay of therapy for MCL remains combination chemo immunotherapy which is not considered curative. Some studies have showed a prolonged median survival of 7 years in MCL patients treated nonaggressively, suggesting that a subset of patients may have an indolent course. Identification of this unique group of patients is challenging as there is no validated prognostic tool yet. In addition to MIPI, the Ki-67 score seems to be a powerful predictor of survival, with scores less than 30% showing prolonged survival. Our three patients presented with low Ki-67 score and low burden disease. They did not have B symptoms and were asymptomatic after surgery. Further studies are needed to assess whether MCL of the tonsil, without other dominant systemic disease, represents a unique clinical entity that predicts the indolent form of the disease which could be managed conservatively. Disclosures No relevant conflicts of interest to declare.

Comprehensive Analysis of Factors Predictive for Time to Transformation and Risk of Transformation in Patients (pts) with Mantle Cell Lymphoma

Comprehensive Analysis of Factors Predictive for Time to Transformation and Risk of Transformation in Patients (pts) with Mantle Cell Lymphoma

Detection of Ki67 Hot-Spots of Invasive Breast Cancer Based on Convolutional Neural Networks Applied to Mutual Information of H&amp;E and Ki67 Whole Slide Images

Ki67 hot-spot detection and its evaluation in invasive breast cancer regions play a significant role in routine medical practice. The quantification of cellular proliferation assessed by Ki67 immunohistochemistry is an established prognostic and predictive biomarker that determines the choice of therapeutic protocols. In this paper, we present three deep learning-based approaches to automatically detect and quantify Ki67 hot-spot areas by means of the Ki67 labeling index. To this end, a dataset composed of 100 whole slide images (WSIs) belonging to 50 breast cancer cases (Ki67 and H&amp;E WSI pairs) was used. Three methods based on CNN classification were proposed and compared to create the tumor proliferation map. The best results were obtained by applying the CNN to the mutual information acquired from the color deconvolution of both the Ki67 marker and the H&amp;E WSIs. The overall accuracy of this approach was 95%. The agreement between the automatic Ki67 scoring and the manual analysis is promising with a Spearman’s ρ correlation of 0.92. The results illustrate the suitability of this CNN-based approach for detecting hot-spots areas of invasive breast cancer in WSI.

The Value of P504s, 34βE12, Ki-67 and PSA in Diagnosis and Prognosis of Prostate Cancer

Background: In recent years, the incidence of prostate cancer (PCa) is increasing. Advanced PCa has a poor prognosis and high cost, which seriously affects the quality of life of patients. Therefore, how to improve the diagnosis rate and prognosis of early PCa is the focus of clinical research. This paper aims to investigate the value of P504s, 34βE12, Ki-67 and prostate-specific antigen (PSA) in the diagnosis and prognosis of PCa.&#x0D; Objective: To investigate the expression of P504s, 34βE12, Ki67 and PSA in prostate tissues and their clinical significance.&#x0D; Methods: Twenty four cases in the study group were selected from PCa confirmed by pathology in the urology department of Chengde Affiliated Hospital from October 2018 to August 2020, and 33 cases of Benign Prostate Hyperplasia (BPH) confirmed by pathology in the same period were selected as the control group. The expression of P504s, 34βE12 and Ki-67 in prostate tissues were determined by immunohistochemistry.&#x0D; Results: The positive expression rates of P504s, 34βE12 and TPSA or F/TPSA in PCa patients were 95.8%, 12.5% and 87.5%, respectively. The positive rates in BPH patients were 9.1%, 93.9% and 27.3%. The difference between the two groups was statistically significant (p&lt;0.05). PCa bone or lymph node metastasis was positively correlated with Ki-67 (r=0.423, p&lt;0.05) and Gleason score (r=0.446, p&lt;0.05), indicating a stronger correlation with Gleasonscore.&#x0D; Conclusion: The combined detection of P504s, 34βE12 and PSA is helpful for the diagnosis and differential diagnosis of PCa. High Gleason score and ki-67 expression may indicate high risk of PCa metastasis and poor prognosis.

Surface brachytherapy in Skin Cancer with High Dose Rate Remote After Loader: An Experience from NCHRC

Background: In recent years, the incidence of prostate cancer (PCa) is increasing. Advanced PCa has a poor prognosis and high cost, which seriously affects the quality of life of patients. Therefore, how to improve the diagnosis rate and prognosis of early PCa is the focus of clinical research. This paper aims to investigate the value of P504s, 34βE12, Ki-67 and prostate-specific antigen (PSA) in the diagnosis and prognosis of PCa.&#x0D; Objective: To investigate the expression of P504s, 34βE12, Ki67 and PSA in prostate tissues and their clinical significance.&#x0D; Methods: Twenty four cases in the study group were selected from PCa confirmed by pathology in the urology department of Chengde Affiliated Hospital from October 2018 to August 2020, and 33 cases of Benign Prostate Hyperplasia (BPH) confirmed by pathology in the same period were selected as the control group. The expression of P504s, 34βE12 and Ki-67 in prostate tissues were determined by immunohistochemistry.&#x0D; Results: The positive expression rates of P504s, 34βE12 and TPSA or F/TPSA in PCa patients were 95.8%, 12.5%and 87.5%, respectively. The positive rates in BPH patients were 9.1%, 93.9% and 27.3%, The difference between the two groups was statistically significant (p&lt;0.05). PCa bone or lymph node metastasis was positively correlated with Ki-67 (r=0.423, p&lt;0.05) and Gleason score (r=0.446, p&lt;0.05), indicating a stronger correlation with Gleasonscore.&#x0D; Conclusion: The combined detection of P504s, 34βE12 and PSA is helpful for the diagnosis and differential diagnosis of PCa. High Gleason score and ki-67 expression may indicate high risk of PCa metastasis and poor prognosis.

Characterization of cervical biopsies of women with HIV and HPV co-infection using p16ink4a, ki-67 and HPV E4 immunohistochemistry and DNA methylation

This study aims to characterize cervical intraepithelial neoplasia (CIN) in women living with HIV using biomarkers. Immunohistochemical (IHC) staining for human papillomavirus (HPV) E4 protein indicates CIN with productive HPV infection, whereas Ki-67 and p16ink4a indicate CIN with transforming characteristics, which may be further characterized using DNA hypermethylation, indicative for advanced transforming CIN. Cervical biopsies (n = 175) from 102 HPV positive women living with HIV were independently reviewed by three expert pathologists. The consensus CIN grade was used as reference standard. IHC staining patterns were scored for Ki-67 (0–3), p16ink4a (0–3), and E4 (0–2) and correlated to methylation levels of four cellular genes in corresponding cervical scrapes. Reference standards and immunoscores were obtained from 165 biopsies:15 no dysplasia, 91 CIN1, 31 CIN2, and 28 CIN3. Ki-67 and p16ink4a scores increased with increasing CIN grade, while E4 positivity was highest in CIN1 and CIN2 lesions. E4 positive CIN1 lesions had higher Ki-67 and p16ink4a scores and higher methylation levels compared with E4 negative CIN1 lesions. E4 positive biopsies with low cumulative Ki-67/p16 ink4a immunoscores (0-3) had significantly higher methylation levels compared with E4 negative biopsies. No significant differences in Ki-67 and p16ink4a scores and methylation levels were observed between E4 negative and positive CIN2 or CIN3 lesions. The presence of high methylation levels in scrapes of CIN lesions with IHC characteristics of both productive (E4 positive) and transforming infections (increased Ki-67/p16ink4a expression) in women living with HIV might indicate a rapid aggressive course of HPV infections towards cancer in these women.

Feasibility study of cryobiopsy for practical pathological diagnosis of primary lung cancer including immunohistochemical assessment.

Precision medicine in non-small cell lung cancer requires attainment of a sufficient amount of high-quality tumor tissue. Transbronchial cryobiopsy has emerged as a new diagnostic method for non-neoplastic lung disease with a better potential to assess morphology compared with conventional methods. However, the influence of cryobiopsy on specimen quality, particularly detection of protein expression, is unknown. We performed a comparative immunohistochemical study in specimens obtained by cryobiopsy versus conventional sampling to evaluate the feasibility of cryobiopsy for lung cancer diagnosis. Pairs of artificial biopsy specimens, collected using a cryoprobe or conventional scalpel, were obtained from 43 surgically resected primary lung tumors. Formalin-fixed, paraffin-embedded blocks were prepared in an ISO15189-certified laboratory. Immunohistochemical staining of thyroid transcription factor-1, p40, Ki67 and programmed death-ligand 1 (22C3) was performed. The H-scores for thyroid transcription factor-1 and p40, labeling index for Ki67 and tumor proportion score for programmed death-ligand 1 were assessed. Pearson's correlation coefficients between two sampling types were calculated. The thyroid transcription factor-1 and p40 H-scores showed perfect correlations between the cryobiopsy and conventional scalpel-obtained specimens (R2=0.977 and 0.996, respectively). Ki67 labeling index and PD-L1 tumor proportion score also showed strong correlations between the two sample types (R2=0.896 and 0.851, respectively). Five cases (11.6%) exhibited differences in tumor proportion score category between sample types, potentially because of intratumoral heterogeneity. Immunohistochemical expression of certain tumor markers showed a high concordance between cryobiopsy and conventional scalpel sampling. Cryobiopsy is feasible for pathological diagnostics including PD-L1 evaluation.

The Correlate between Argyrophilic Nucleolar Organizer Region Stain and Ki-67 Immnochistochemistry in Diagnostic Breast Cancer

Introduction: This experimental study gauged the value of argyrophilic nucleolar organizer region (AgNOR) staining as a possible technique for the estimation of cell kinetics in conventional histology sections, in benign and malignant breast lesions. Methods: With a silver staining technique and immunohistochemistry, we associated the numbers of AgNORs and Ki67 scores in 30 breast carcinomas and 10 benign breast lesions. Results: The mean values of Ag NORs silver stain dots count for normal, benign, grade II and III were 1.28±0.17, 2.83±0.68, 5.23±0.87 and 7.32±0.92, respectively. There was a statistically significant difference (p≤0.001) was noticed between the all individual groups, among the normal and breast lesion as well as among the GII and GIII. Immunohistochemical Results of Ki-67 protein exhibited homogenous golden-brown color in control case and a positive brown granules or diffuse dark brown color in the nuclei of both benign and malignant cases under the 400X magnify examined under the light microscope. Discussion: AgNOR counts performed on routine formalin-fixed paraffin sections could provide substantial kinetic evidence. Additionally, the difference in AgNOR counts between benign and malignant tumors is such that they may be of diagnostic worth.

181P The use of Ki67 testing and scoring in HR+, HER2- early breast cancer

Biomarkers of cellular proliferation (eg. Ki67≥20%) may be useful in estimating recurrence risk in HR+, HER2- early breast cancer (EBC: Stage I-III). The use of chemotherapy (CT) depends on multiple factors including the patient’s (pt) estimated risk of recurrence. One area of uncertainty in risk estimation is in pts with 1-3 positive nodes. This study aimed to understand the use of Ki67 testing and scoring in pts with node+, HR+, HER2- EBC and treatment (trt) patterns.

Tumor mutation burden and JARID2 gene alteration are associated with short disease-free survival in locally advanced triple-negative breast cancer.

BackgroundIn locally advanced triple-negative breast cancer (TNBC), patients who did not achieve pathologic complete response (non-pCR) after neoadjuvant chemotherapy develop rapid tumor metastasis. Tumor mutation burden (TMB) is a potential biomarker of cancer therapy, though whether it is applicable to TNBC is still unclear.MethodsA total of 14 non-pCR TNBC patients were enrolled, and tissue samples from radical operation were collected. Of these, 7 cases developed disease progression within 12 months after operation [short disease-free survival (short DFS)], while others showed longer DFS over 1 year (long DFS). Next generation sequencing (NGS) analysis targeting 422 cancer-related genes and in vitro studies were performed.ResultsA total of 72 mutations were detected within 14 patients, which ranged from 1 to 8 per patient with a median mutations number of 5. The median number of mutations in the short-DFS group was higher than that in the long-DFS group (6.0 vs. 4.3; P=0.094). Furthermore, 6 gene mutation types were detected, with missense mutations displayed in the majority (36/72, 50.0%). No correlation between mutation type and DFS was found. Among 422 cancer-related genes, alterations in 30 genes were detected. TP53 (12/14, 85.7%) was the most common mutation gene in the entire cohort. RB1 mutations significantly occurred in patients with high Ki-67 scores (P=0.013). Additionally, 4 mutations of PTPN13 (57.1%, 4/7) and 3 of JARID2 (42.9%, 3/7) were only detected in the short-DFS group, while patients with JARID2 mutation had a significantly shorter DFS period (P=0.026). Experiments in vitro confirmed that JARID2 gene was widely expressed in various breast cancer cell lines. Knockdown of JARID2 in MD-MBA-231 cells by small interfering RNA (siRNA) decreased the expression of E-cadherin, and increased the levels of vimentin, MMP7, and MMP9.ConclusionsIn non-pCR TNBC, JARID2 mutation and TMB elevated in patients with short-DFS, indicating the potential prognostic biomarkers and therapeutic molecular targets for locally advanced TNBC.

Assessment of tumour proliferation by use of the mitotic activity index, and Ki67 and phosphohistone H3 expression, in early-stage luminal breast cancer.

AimsPhosphohistone H3 (PhH3) has been proposed as a novel proliferation marker in breast cancer. This study compares the interobserver agreement for assessment of the mitotic activity index (MAI), Ki67 expression, and PhH3 in a cohort of oestrogen receptor (ER)‐positive breast cancer patients.Methods and resultsTumour samples of 159 luminal breast cancer patients were collected. MAI and PhH3 scores were assessed by three breast cancer pathologists. Ki67 scores were assessed separately by two of the three pathologists. PhH3‐positive cells were counted in an area of 2 mm2, with a threshold of ≥13 positive cells being used to discriminate between low‐proliferative and high‐proliferative tumours. Ki67 expression was assessed with the global scoring method. Ki67 percentages of <20% were considered to be low. The intraclass correlation coefficient (ICC) and Cohen's κ statistics were used to evaluate interobserver agreement. The impact on histological grading of replacing the MAI with PhH3 was assessed. Counting PhH3‐positive cells was highly reproducible among all three observers (ICC of 0.86). The κ scores for the categorical PhH3 count (κ = 0.78, κ = 0.68, and κ = 0.80) reflected substantial agreement among all observers, whereas agreement for the MAI (κ = 0.38, κ = 0.52, and κ = 0.26) and Ki67 (κ = 0.55) was fair to moderate. When PhH3 was used to determine the histological grade, agreement in grading increased (PhH3, κ = 0.52, κ = 0.48, and κ = 0.52; MAI, κ = 0.43, κ = 0.35, and κ = 0.32), and the proportion of grade III tumours increased (14%, 18%, and 27%).ConclusionPhH3 seems to outperform Ki67 and the MAI as a reproducible means to measure tumour proliferation in luminal‐type breast cancer. Variation in the assessment of histological grade might be reduced by using PhH3, but would result in an increase in the proportion of high‐grade cancers.

Abstract 5295: Impact of a multi-gene expression profile in predicting adjuvant trastuzumab outcome in HER2-positive breast cancer

Abstract Background Trastuzumab is the cornerstone of adjuvant therapy for HER2-positive breast cancer (BC), but up to 20% of patients relapse. Understanding resistance mechanisms could allow developing strategies to overcome this issue. We performed a case-control, retrospective study to compare the gene expression profiles (GEPs) of 27 patients who experienced disease relapse within 2 years from the start of adjuvant trastuzumab therapy, with those of 27 patients who were disease-free after at least 5 years. Here, we present the preliminary results on 12 cases and 12 controls, matched by age and hormone receptor status. Methods RNA was isolated from formalin-fixed paraffin-embedded primary tumors (FFPE) with AllPrep DNA/RNA FFPE Kit (Qiagen) and quantified by Nanodrop, before performing Nanostring gene expression profile. nCounter Breast Cancer 360 Panel (Nanostring Technologies) was used according to manufacturer's instructions. nSolver analysis software (Nanostring Technologies) was used to obtain normalized counts and to compare GEPs between cases and controls, considering as significant a Benjamini Yekutieli (BY) corrected p-value ≤0.05. Mann-Whitney median test was used to compare Ki67 score and Body Mass Index (BMI) between cases and controls. Results Out of 770 analyzed genes, none was significantly differentially expressed at BY p-value &amp;lt;0.05, probably due to the low number of cases. Five genes were differentially expressed with a BY p-value &amp;lt;0.1: CDKN1A (Log2 fold change -0.733, 95% CI: -1.06 − -0.409), GDF15 (Log2 fold change -1.92, 95% CI: -2.78 − -1.05), TFDP1 (Log2 fold change -0.567, 95% CI: -0.826 − -0.309) and ELF3 (Log2 fold change -1.52, 95%CI: -2.22 − -0.813) were downregulated in control patients, whereas TNFSF10 (Log2 fold change 1.27, 95% CI: 0.669 − 1.88) was upregulated. CDKN1A and TFDP1 are cell cycle regulators: the former encodes for p21, which inhibits cell cycle progression and is involved in DNA damage repair; the latter encodes a transcription factors that dimerizes with E2F, promoting cell cycle genes' transcription. GDF15 codes for a secreted ligand of the TGF-beta superfamily and acts as a pleiotropic cytokine involved in response to hypoxia, inflammation, acute injury and oxidative stress. ELF3 is a transcriptional activator involved in vascular inflammation and a critical downstream effector of HER2 pathway. TNFSF10 encodes a cytokine belonging to the TNF ligand family, inducing apoptosis in tumor cells. No differences were found between cases and controls considering Ki67 proliferation index and BMI. Conclusions Our preliminary results indicate that altered expression of genes involved in cell cycle, apoptosis, inflammation and stress response are involved in trastuzumab resistance. The validation of these results overall series is ongoing. The relations between GEPs and clinico-pathological features will be assessed. Citation Format: Sara Ravaioli, Roberta Maltoni, Francesca Pirini, Michela Palleschi, William Balzi, Elisabetta Petracci, Giovanni Martinelli, Andrea Rocca, Sara Bravaccini. Impact of a multi-gene expression profile in predicting adjuvant trastuzumab outcome in HER2-positive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5295.

WT1 Clone 6F-H2 Cytoplasmic Expression Differentiates Astrocytic Tumors from Astrogliosis and Associates with Tumor Grade, Histopathology, IDH1 Status, Apoptotic and Proliferative Indices: A Tissue Microarray Study.

Objectives:This tissue microarray (TMA) immunohistochemical (IHC) study elucidates the role of Wilms’ tumor 1 protein (WT1) in diagnosis and prognostication of astrocytic tumors. Methods:IHC was applied to 75 astrocytic lesions (18 astrogliosis and 57 astrocytic tumors) using antibodies directed against WT1 clone 6F-H2, isocitrate dehydrogenase 1(IDH1), Bcl2 and Ki67. WT1 IHC staining was evaluated and scored blindly by 2 pathologists. Bcl2 and Ki67 scores and labelling indices were assessed and IDH1 status determined. Statistical analysis was performed using the appropriate methodology. Results:WT1 cytoplasmic expression was detected in 89.5% of astrocytic tumors but not in astrogliosis. Positive WT1 differentiated astrocytic tumors (92.6% accuracy) and grade II diffuse astrocytomas (93.5% accuracy) from astrogliosis with high sensitivity, specificity and positive predictive values (p<0.001). Increased WT1 score significantly associated higher Bcl2 and Ki67 labelling indices, increasing WHO tumor grade and tumor’s histopathologic type (p<0.05) showing staining pattern variability by tumor entity and cell type. Glioblastomas, gliosarcomas and subependymal giant cell astrocytomas were the most frequently WT1 expressing tumors with frequent +3 WT1 score. About 21.4% of pilocytic astrocytomas had +3WT1 score in association with increased Bcl2 and Ki67 indices. Low WT1 scores in grade II and III diffuse astrocytomas were linked to the high frequency of IDH1 positivity, and were associated with low Bcl2 and Ki67 labelling indices. In glioblastomas, WT1 significantly associated poor prognostic variables: older age, negative-IDH1 status, high Bcl2 and Ki67 labelling indices (p=0.04, <0.001, =0.001 and <0.001 respectively). Conclusions:WT1 clone 6F-H2 is a highly accurate positive surrogate marker to differentiate astrocytic tumors notably the challenging grade II diffuse astrocytoma from astrogliosis. It significantly associates with poor prognostic variables including IDH1 negativity, high apoptotic and proliferative indices and depends on tumor’s histopathologic entity more than its grade. Evaluation of WT1 expression seems essential to tailor patient’s therapy.

Differential expression of miR-1297, miR-3191-5p, miR-4435, and miR-4465 in malignant and benign breast tumors.

Objective(s):MicroRNAs (miRs) are a class of small non-coding RNAs which are associated with tumor growth and progression. In the present study, we assessed the expression of selected miRs in malignant, benign, and adjacent normal breast tissues. Materials and Methods:The expression of miR-1297, miR-3191-5P, miR-4435, and miR-4465 were evaluated in malignant (n=50), benign (n=35), and adjacent normal breast tissues (n=20) using qRT-PCR. Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were generated for evaluating the diagnostic values of miRs. To evaluate diagnostic efficacy, miRs-based score was obtained using the logistic regression model. Results:Among malignant tumors, the expression of miR-1297, miR-3191-5p, and miR-4435 was significantly lower (P=0.024, P<0.001 and P=0.031), respectively. The expression of miR-4465 was higher (P=0.023) than that of normal tissue. The expression of these miRs was lower than those of benign tumors (P<0.01, P<0.001, P<0.0001, and P<0.01, respectively). We observed a positive correlation between miR-4465 expression levels and tumor stage (P=0.042) and a negative correlation with grade and Ki-67 score (P<0.05). The AUCs for miR-1297, miR-3191-5p, miR-4435, and miR-4465 in malignant tumors versus normal tissues were 0.784, 0.700, 0.976, and 0.865 and versus benign tumors they were 0.938, 0.857, 0.981, and 0.785, respectively. The optimal logit(P) value of 0.262 distinguished malignant from normal subjects with a sensitivity of 0.91, specificity of 0.85, and an overall accuracy of 0.89. Conclusion:The panel of these miRs are suggested as possible onco-miRs(miR-4465) or tumor suppressor-miRs (miR-3191-5P, miR-1297, miR-4435). Overall, our results indicated that these miRs could be introduced as diagnostic biomarkers in breast cancer patients.

An Advanced Automated Image Analysis Model for Scoring of ER, PR, HER-2 and Ki-67 in Breast Carcinoma

Immunohistochemistry (IHC) plays an important role in evaluating the status of ER, PR, Ki-67 and human epidermal growth factor receptor 2 (HER-2) during diagnosis of breast cancer. Although some existing automated approaches can solve the high time-consumption and inter-/intra-observer variability drawbacks to a certain extent, most of them are can’t analyze both nuclear staining and cell membrane staining using the same method. This is attributed to the difference in localization of the positive signal of immunohistochemical staining in different biological markers. The present study proposes a novel automated image analysis model for scoring and grading of ER, PR, Ki-67 and HER-2 immunohistochemical images based on whole tissue sections in breast cancer. The scoring results of the trained model and manual interpretation of ER, PR, Ki-67 and HER-2 were then finally analyzed and compared. Experimental results show that the F1-measure was 0.8450, 0.8533 and 0.7962 for nuclear recognition of Ki-67, ER/PR and HER-2 respectively. For stain grading of Ki-67, ER/PR and HER-2, the F1-measure was 0.9776, 0.8306 and 0.9573 respectively. The scoring consistency of ER/PR, Ki-67 and HER-2 between our model and expert interpretation was 0.9279, 0.9712 and 0.8046 respectively. Our results demonstrate that artificial intelligence technology is a feasible and accurate method for accurate quantitative immunohistochemical analysis that can solve the drawbacks of low repeatability and time consumption brought by manual counting. The main contribution of our proposed model is that it can recognize both nuclear staining and cell membrane staining and grade the staining intensity as a sequential learning task.

Poor Biological Factors and Prognosis of Interval Breast Cancers: Long-Term Results of Bahçeşehir (Istanbul) Breast Cancer Screening Project in Turkey.

PURPOSEThe Turkish Bahçeşehir Breast Cancer Screening Project was a 10-year, organized, population-based screening program carried out in Bahçeşehir county, Istanbul. Our aim was to examine the biologic features and outcome of screen-detected and interval breast cancers during the 10-year study period.METHODSBetween 2009 and 2019, 2-view mammograms were obtained at 2-year intervals for women aged 40 to 69 years. Clinicopathological characteristics including ER, PR, HER2-neu, and Ki-67 status were analyzed for those diagnosed with breast cancer.RESULTSIn 8,758 screened women, 131 breast cancers (1.5%) were detected. The majority of patients (82.3%) had prognostic stage 0-I disease. Contrarily, patients with interval cancers (n = 15; 11.4%) were more likely to have a worse prognostic stage (II-IV disease; odds ratio [OR], 3.59, 95% CI, 0.9 to 14.5) and high Ki-67 scores (OR, 3.14; 95% CI, 0.9 to 11.2). Interval cancers detected within 1 year were more likely to have a luminal B (57.1% v 31.9%) and triple-negative (14.3% v 1%) subtype and less likely to have a luminal A subtype (28.6% v 61.5%; P = .04). Patients with interval cancers had a poor outcome in 10-year disease-specific (DSS) and disease-free survival (DFS) compared with those with screen-detected cancers (DSS: 68.2% v 98.1%, P = .002; DFS: 78.6% v 96.5%, P = .011).CONCLUSIONOur findings suggest the majority of screen-detected breast cancers exhibited a luminal A subtype profile with an excellent prognosis. However, interval cancers were more likely to have aggressive subtypes such as luminal B subtype or triple-negative cancers associated with a poor prognosis requiring other preventive strategies.