An Interobserver Reproducibility Analysis of Ki67 Visual Assessment in Breast Cancer

Background

Ki67 labeling index (LI) is used as a predictive marker and is associated with prognosis in breast cancer. However, standardised methodologies for measurement are lacking which has limited its application in clinical practice. In this study, we evaluated the interobserver concordance of visual assessment of Ki67 LI in breast cancer.

Methods

Ki67- immunostained slides of 160 cases of primary invasive breast cancer were visual assessed by five breast pathologists with two different methods to choose the scoring fields: (1) hot-spot score, (2) average score. Proportions of positive invasive tumor cells at 10 % intervals were scored. The intra-class correlation coefficient (ICC) was used to assess the interobserver reproducibility.

Results

(1) A perfect concordance of Ki67 LI was demonstrated according to both score methods (P<0.0001). Average score method (ICC, 0.904) demonstrated a better correlation than hot-spot score method (ICC, 0.894). (2) By respective means according to two score methods, all cases were classified into three groups (≤10%, 11%-30% and >30% Ki-67 LI). The concordance was relatively low in intermediate Ki67 LI group compared with low and high Ki67 LI groups. (3) All cases were classified into three groups by paired-difference (d) between means of hot-spot score and average score (d<5, 5≤d<10, d≥10). The consistency was observed to decrease with increasing paired-difference according to both methods.

Conclusions

Visual assessment of Ki67 LI at 10 % intervals is a candidate for a standard method in breast cancer clinical practice. Average score and hot-spot score of visual assessment both demonstrated a perfect concordance, and an overall average assessment across the whole section including hot spots may be a better method. Interobserver concordance of intermediate Ki67 LI in which most cutoffs are located for making clinical decisions was relatively low.     

Proliferative activity in human breast cancer: Ki-67 automated evaluation and the influence of different Ki-67 equivalent antibodies

Background

Ki67 labeling index (Ki67 LI), the percentage Ki67 immunoreactive cells, is a measure of tumor proliferation, with important clinical relevance in breast cancer, and it is extremely important to standardize its evaluation.

Aim

To test the efficacy of computer assisted image analysis (CAIA) applied to completely digitized slides and to assess its feasibility in routine practice and compare the results obtained using two different Ki67 monoclonal antibodies.

Materials and methods

315 consecutive breast cancer routinely immunostained for Ki-67 (223 with SP6 and 92 with MM1 antibodies previously examined by an experienced pathologist, have been re-evaluated using Aperio Scanscope Xs.

Results

Mean human Ki67 LI values were 36%± 14.% and 28% ± 18% respectively for SP6 and MM1 antibodies; mean CAM Ki67 LI values were 31%± 19% and 22% ± 18% respectively for SP6 and MM1. Human and CAIA evaluation are statistically highly correlated (Pearson: 0.859, p<0.0001), although human LI are systematically higher. An interobserver variation study on CAIA performed on 84 cases showed that the correlation between the two evaluations was linear to an excellent degree.

Discussion

Our study shows that a) CAIA can be easily adopted in routine practice, b) human and CAIA Ki67 LI are highly correlated, although human LI are systematically higher, c) Ki67 LI using different evaluation methods and different antibodies shows important differences in cut-off values.

     

Evaluation of Ki67 Expression across Distinct Categories of Breast Cancer Specimens: A Population-Based Study of Matched Surgical Specimens,Core Needle Biopsies and Tissue Microarrays

Introduction

Tumor cell proliferation in breast cancer is strongly prognostic and may also predict response to chemotherapy. However, there is no consensus on counting areas or cut-off values for patient stratification. Our aim was to assess the matched level of proliferation by Ki67 when using different tissue categories (whole sections, WS; core needle biopsies, CNB; tissue microarrays, TMA), and the corresponding prognostic value.

Methods

We examined a retrospective, population-based series of breast cancer (n = 534) from the Norwegian Breast Cancer Screening Program. The percentage of Ki67 positive nuclei was evaluated by visual counting on WS (n = 534), CNB (n = 154) and TMA (n = 459).

Results

The median percentage of Ki67 expression was 18% on WS (hot-spot areas), 13% on CNB, and 7% on TMA, and this difference was statistically significant in paired cases. Increased Ki67 expression by all evaluation methods was associated with aggressive tumor features (large tumor diameter, high histologic grade, ER negativity) and reduced patient survival.

Conclusion

There is a significant difference in tumor cell proliferation by Ki67 across different sample categories. Ki67 is prognostic over a wide range of cut-off points and for different sample types, although Ki67 results derived from TMA sections are lower compared with those obtained using specimens from a clinical setting. Our findings indicate that specimen specific cut-off values should be applied for practical use.     

Digital immunohistochemistry wizard: image analysis-assisted stereology tool to produce reference data set for calibration and quality control

Background

Digital image analysis (DIA) enables better reproducibility of immunohistochemistry (IHC) studies. Nevertheless, accuracy of the DIA methods needs to be ensured, demanding production of reference data sets. We have reported on methodology to calibrate DIA for Ki67 IHC in breast cancer tissue based on reference data obtained by stereology grid count. To produce the reference data more efficiently, we propose digital IHC wizard generating initial cell marks to be verified by experts.

Methods

Digital images of proliferation marker Ki67 IHC from 158 patients (one tissue microarray spot per patient) with an invasive ductal carcinoma of the breast were used. Manual data (mD) were obtained by marking Ki67-positive and negative tumour cells, using a stereological method for 2D object enumeration. DIA was used as an initial step in stereology grid count to generate the digital data (dD) marks by Aperio Genie and Nuclear algorithms. The dD were collected into XML files from the DIA markup images and overlaid on the original spots along with the stereology grid. The expert correction of the dD marks resulted in corrected data (cD). The percentages of Ki67 positive tumour cells per spot in the mD, dD, and cD sets were compared by single linear regression analysis. Efficiency of cD production was estimated based on manual editing effort.

Results

The percentage of Ki67-positive tumor cells was in very good agreement in the mD, dD, and cD sets: regression of cD from dD (R²=0.92) reflects the impact of the expert editing the dD as well as accuracy of the DIA used; regression of the cD from the mD (R²=0.94) represents the consistency of the DIA-assisted ground truth (cD) with the manual procedure. Nevertheless, the accuracy of detection of individual tumour cells was much lower: in average, 18 and 219 marks per spot were edited due to the Genie and Nuclear algorithm errors, respectively. The DIA-assisted cD production in our experiment saved approximately 2/3 of manual marking.

Conclusions

Digital IHC wizard enabled DIA-assisted stereology to produce reference data in a consistent and efficient way. It can provide quality control measure for appraising accuracy of the DIA steps.

     

New Insight into Ki67 Expression at the Invasive Front in Breast Cancer

Purpose

To investigate the distribution of Ki67+ cells in breast cancer in relation to clinical-pathological parameters and prognosis.

Materials and Methods

Ki67 expression status was detected in 1,086 breast cancer specimens using immunohistochemistry staining and examining the relationship between the Ki67+ cells'' location. Subsequently, clinical-pathological parameters and prognosis were determined.

Results

In total, Ki67 protein expression was found in 781 (71.92%) of the 1,086 breast cancer specimens. Among the 781 Ki67+ cases, 461 were defined as diffuse type and 320 were defined as borderline type. After universal correlation analysis, significant differences were observed in age, histological grade, metastatic nodes, postoperative distant metastasis, and molecular subtype between Ki67+ and Ki67− cases (P = 0.01, 0.001, 0.001, 0.001, and 0.001, respectively). After subgroup analysis, the borderline cases were found to be characterized by a high distant metastasis rate compared to the diffuse cases as well as the Ki67− cases (P = 0.001). No differences were observed between diffuse type or Ki67− cases (P = 0.105). Multivariate analysis showed that age, tumor size, histological grade, lymph node metastasis, molecular subtype, and the Ki67 distribution pattern were observed to be related to postoperative distant metastasis (all P<0.05). Furthermore, borderline type was shown to attain a significantly more distant bone and liver metastasis and worse disease-specific survival than the other types (P = 0.001). In the Cox regression test, the Ki67 distribution pattern was detected as an independent prognostic factor (P = 0.001).

Conclusion

The distribution pattern of Ki67 may be a new independent prognostic factor for breast cancer.     

Risk Factors Associated with Discordant Ki-67 Levels between Preoperative Biopsy and Postoperative Surgical Specimens in Breast Cancers

Purpose

The Ki-67 labelling index is significant for the management of breast cancer. However, the concordance of Ki-67 expression between preoperative biopsy and postoperative surgical specimens has not been well evaluated. This study aimed to find the correlation in Ki-67 expression between biopsy and surgical specimens and to determine the clinicopathological risk factors associated with discordant values.

Patients and Methods

Ki-67 levels were immunohistochemically measured using paired biopsy and surgical specimens in 310 breast cancer patients between 2008 and 2013. ΔKi-67 was calculated by postoperative Ki-67 minus preoperative levels. The outliers of ΔKi-67 were defined as [lower quartile of ΔKi-67–1.5 × interquartile range (IQR)] or (upper quartile + 1.5 × IQR) and were evaluated according to clinicopathological parameters by logistic regression analysis.

Results

The median preoperative and postoperative Ki-67 levels were 10 (IQR, 15) and 10 (IQR, 25), respectively. Correlation of Ki-67 levels between the two specimens indicated a moderately positive relationship (coefficient = 0.676). Of 310 patients, 44 (14.2%) showed outliers of ΔKi-67 (range, ≤-20 or ≥28). A significant association with poor prognostic factors was found among these patients. Multivariate analysis determined that significant risk factors for outliers of ΔKi-67 were tumor size >1 cm, negative progesterone receptor (PR) expression, grade III cancer, and age ≤35 years. Among 171 patients with luminal human epidermal growth factor receptor 2-negative tumors, breast cancer subtype according to preoperative or postoperative Ki-67 levels discordantly changed in 46 (26.9%) patients and a significant proportion of patients with discordant cases had ≥1 risk factor.

Conclusion

Ki-67 expression showed a substantial concordance between biopsy and surgical specimens. Extremely discordant Ki-67 levels may be associated with aggressive tumor biology. In patients with luminal subtype disease, clinical application of Ki-67 values should be cautious considering types of specimens and clinicopathological risk factors.     

Long-Term Prognostic Performance of Ki67 Rate in Early Stage,pT1-pT2, pN0, Invasive Breast Carcinoma

Background

Molecular signatures may become of use in clinical practice to assess the prognosis of breast cancers. However, although international consensus conferences sustain the use of these new markers in the near future, concerns remain about their degree of discordance and cost-effectiveness in different international settings. The present study aims to validate Ki67 as prognostic factor in a large cohort of early-stage (pT1–pT2, pN0) breast cancer patients.

Methods

456 patients treated in 1995–1996 were identified in the Institut Curie database. Ki67 (MIB1) was retrospectively assessed by immunohistochemistry for all cases. The prognostic value of this index was compared to that of histological grade (HG), Estrogen receptor (ER) and HER2 status. Distant disease free interval, loco-regional recurrence, time-lapse from first metastatic diagnosis to death were analyzed.

Results

All 456 patients were treated by lumpectomy plus axillary dissection and radiotherapy. 27 patients (5.9%) received systemic treatment. Tumors were classified as HG1 in 35%, HG2 in 42% and HG3 in 23% of cases. ER was expressed in 86% of the tumors, HER2 in 5% and 14% were triple negative. The median follow-up was 151 [5–191] months. Distant and loco-regional disease recurrences were observed in 16% and 18%, respectively. High (>20%) Ki67 rate [HR = 3 (1.8–4.8), p<10e−06] and HG3 [HR = 4.4 (2.2–8.6), p = 0.00002] were associated with an increased rate of distant relapse. In multivariate analysis, the Ki67 remained the only significant prognostic factor in the subgroups of ER positive HER2 negative [HR = 2.6 (1.5–4.6), p = 0.0006] and ER positive HER2 negative HG2 tumors [HR = 2.2 (1.01–4.8), p = 0.04].

Conclusions

We validate the prognosis value of the Ki67 rate in small size node negative breast cancer. We conclude that Ki67 is a potential cost-effective decision marker for adjuvant therapy in early-stage HG2, pT1–pT2, pN0, breast cancers.     

Comparative analysis of different approaches to investigate cell kinetics

The potential of different methods to investigate proliferative activity of cell populations was analysed for non-Hodgkin's lymphomas. Cells in S phase and all cycling cells were determined on cell suspensions obtained from fresh lymph node material by [3H]-thymidine autoradiography [( 3H]TdR LI), a monoclonal antibody to bromodeoxyuridine (BrdU LI), and the monoclonal antibody Ki67. A good correlation was observed between the values of [3H]TdR LI and BrdU LI (rs = 0.90; P less than 0.01), [3H]TdR LI and S phase (rs = 0.62; P less than 0.01) and [3H]TdR LI and Ki67 (rs = 0.64; P less than 0.01) in individual lymphomas. Using the median values obtained from the different approaches as cut-off points to define slowly and rapidly proliferating tumours, the best agreement was observed between [3H]TdR LI and BrdU LI (91%) and poorer agreements, even though statistically significant, were observed between [3H]TdR LI and S phase (73%) or Ki67 (76%). In conclusion, the kinetic information derived from different approaches was more or less concordant and newly proposed approaches should be directly and carefully verified for their prognostic relevance before using them as alternatives to conventional methods.     

Added Value of HER-2 Amplification Testing by Multiplex Ligation-Dependent Probe Amplification in Invasive Breast Cancer

Background

HER-2 is a prognostic and predictive marker, but as yet no technique is perfectly able to identify patients likely to benefit from HER-2 targeted therapies. We aimed to prospectively assess the added value of first-line co-testing by IHC, and multiplex ligation-dependent probe amplification (MLPA) and chromogenic in situ hybridization (CISH).

Methods

As local validation, HER-2 MLPA and CISH were compared in 99 breast cancers. Next, we reviewed 937 invasive breast cancers, from 4 Dutch pathology laboratories, that were prospectively assessed for HER-2 by IHC and MLPA (and CISH in selected cases).

Results

The validation study demonstrated 100% concordance between CISH and MLPA, if both methods were assessable and conclusive (81.8% of cases). Significant variation regarding percentages IHC 0/1+ and 2+ cases was observed between the laboratories (p<0.0001). Overall concordance between IHC and MLPA/CISH was 98.1% (575/586) (Kappa = 0.94). Of the IHC 3+ cases, 6.7% failed to reveal gene amplification, whereas 0.8% of the IHC 0/1+ cases demonstrated gene amplification. Results remained discordant after retrospective review in 3/11 discordant cases. In the remaining 8 cases the original IHC score was incorrect or adapted after repeated IHC staining.

Conclusions

MLPA is a low-cost and quantitative high-throughput technique with near perfect concordance with CISH. The use of MLPA in routinely co-testing all breast cancers may reduce HER-2 testing variation between laboratories, may serve as quality control for IHC, will reveal IHC 0/1+ patients with gene amplification, likely responsive to trastuzumab, and identify IHC 3+ cases without gene amplification that may respond less well.     

Quantum Dots-Based Quantitative and In Situ Multiple Imaging on Ki67 and Cytokeratin to Improve Ki67 Assessment in Breast Cancer

Background

As a marker for tumor cell proliferation, Ki67 has important impacts on breast cancer (BC) prognosis. Although immunohistochemical staining is the current standard method, variations in analytical practice make it difficult for pathologists to manually measure Ki67 index. This study was to develop a fluorescent spectrum-based quantitative analysis of Ki67 expression by quantum-dots (QDs) multiple imaging technique.

Methods

A QDs-based in situ multiple fluorescent imaging method was developed, which stained nuclear Ki67 as red signal and cytoplasmic cytokeratin (CK) as green signal. Both Ki67 and CK signals were automatically separated and quantified by professional spectrum analysis software. This technique was applied to tissue microarrays from 240 BC patients. Both Ki67 and CK values, and Ki67/CK ratio were obtained for each patient, and their prognostic value on 5-year disease free survival was assessed.

Results

This method simultaneously stains nuclear Ki67 and cytoplasmic CK with clear signal contrast, making it easy for signal separation and quantification. The total fluorescent signal intensities of both Ki67 sum and CK sum were obtained, and Ki67/CK ratio calculated. Ki67 sum and Ki67/CK ratio were each attributed into two grades by X-tile software based on the best P value principle. Multivariate analysis showed Ki67 grade (P = 0.047) and Ki67/CK grade (P = 0.004) were independent prognostic factors. Furthermore, area under curve (AUC) of ROC analysis for Ki67/CK grade (AUC: 0.683, 95%CI: 0.613–0.752) was higher than Ki67 grade (AUC: 0.665, 95%CI: 0.596–0.734) and HER-2 gene (AUC: 0.586, 95%CI: 0.510–0.661), but lower than N stage (AUC: 0.760, 95%CI: 0.696–0.823) and histological grade (AUC: 0.756, 95%CI: 0.692–0.820) on predicting the risk for recurrence.

Conclusions

A QDs-based quantitative and in situ multiple imaging on Ki67 and CK was developed to improve Ki67 assessment in BC, and Ki67/CK grade had better performance than Ki67 grade in predicting prognosis.     

Improvement of Left Ventricular Function under Cardiac Resynchronization Therapy Goes along with a Reduced Incidence of Ventricular Arrhythmia

Objectives

The beneficial effects of cardiac resynchronization therapy (CRT) are thought to result from favorable left ventricular (LV) reverse remodeling, however CRT is only successful in about 70% of patients. Whether response to CRT is associated with a decrease in ventricular arrhythmias (VA) is still discussed controversially. Therefore, we investigated the incidence of VA in CRT responders in comparison with non-responders.

Methods

In this nonrandomized, two-center, observational study patients with moderate-to-severe heart failure, LV ejection fraction (LVEF) ≤35%, and QRS duration >120 ms undergoing CRT were included. After 6 months patients were classified as CRT responders or non-responders. Incidence of VA was compared between both groups by Kaplan-Meier analysis and Cox regression analysis. ROC analysis was performed to determine the aptitude of LVEF cut-off values to predict VA.

Results

In total 126 consecutive patients (64±11years; 67%male) were included, 74 were classified as responders and 52 as non-responders. While the mean LVEF at baseline was comparable in both groups (25±7% vs. 24±8%; P = 0.4583) only the responder group showed an improvement of LVEF (36±6% vs. 24±7; p<0.0001) under CRT. In total in 56 patients VA were observed during a mean follow-up of 28±14 months, with CRT responders experiencing fewer VA than non-responders (35% vs. 58%, p<0.0061). Secondary preventive CRT implantation was associated with a higher likelihood of VA. As determined by ROC analysis an increase of LVEF by >7% was found to be a predictor of a significantly lower incidence of VA (AUC = 0.606).

Conclusions

Improvement of left ventricular function under cardiac resynchronization therapy goes along with a reduced incidence of ventricular arrhythmia.     

Comparative Analysis of Different Approaches to Investigate Cell Kinetics

Abstract. The potential of different methods to investigate proliferative activity of cell populations was analysed for non-Hodgkin's lymphomas. Cells in S phase and all cycling cells were determined on cell suspensions obtained from fresh lymph node material by [³H]-thymidine autoradiography ([³H]TdR LI), a monoclonal antibody to bromodeoxyuridine (BrdU LI), and the monoclonal antibody Ki67. A good correlation was observed between the values of [³H]TdR LI and BrdU LI ( r _s= 0.90; P < 0.01), [³H]TdR LI and S phase ( r _s= 0.62; P < 0.01) and [³H]TdR LI and Ki67 ( r _s= 0.64; P < 0.01) in individual lymphomas. Using the median values obtained from the different approaches as cut-off points to define slowly and rapidly proliferating tumours, the best agreement was observed between [³H]TdR LI and BrdU LI (91%) and poorer agreements, even though statistically significant, were observed between [³H]TdR LI and S phase (73%) or Ki67 (76%). In conclusion, the kinetic information derived from different approaches was more or less concordant and newly proposed approaches should be directly and carefully verified for their prognostic relevance before using them as alternatives to conventional methods.     

External Validation and Evaluation of Reliability and Validity of the S-ReSC Scoring System to Predict Stone-Free Status after Percutaneous Nephrolithotomy

Objectives

The Seoul National University Renal Stone Complexity (S-ReSC) scoring system was developed to predict the stone-free rate (SFR) after single-tract percutaneous nephrolithotomy (PCNL). This study is an external validation of this scoring system.

Materials and methods

A retrospective review included 327 patients who underwent PCNL at 2 tertiary referral centers. The S-ReSC score was assigned from 1 to 9 based on the number of sites involved. The stone free status was defined as either complete clearance or clinically insignificant residual fragments <4 mm in size at 1 month follow-up imaging. Inter-observer and test-retest reliabilities were evaluated. The statistical performance of the prediction model was assessed by its predictive accuracy, predictive probability, and clinical usefulness.

Results

The overall SFR was 65.4%. SFRs were 83.9%, 47.6%, and 21.4% in low (1–2), intermediate (3–4), and high (5–9) score groups, respectively, with significant differences (P<0.001). Inter-observer and test-retest reliabilities revealed almost perfect agreements. External validation of the S-ReSC scoring system revealed an AUC of 0.731 (95% CI 0.675–0.788). The AUC of 3-titered S-ReSC score groups was 0.691 (95% CI, 0.629–0.753). The calibration plot showed that the predicted probability of SFR had a concordance comparable to that of the observed frequency. The Hosmer–Lemeshow goodness-of-fit statistic revealed an adequate performance of the predictive model (P = 0.10). Inter-observer and test-retest reliability showed a good level of agreement.

Conclusions

The S-ReSC scoring system is useful in predicting the post-PCNL SFR and in describing the complexity of renal stones.     

The CIN4 Chromosomal Instability qPCR Classifier Defines Tumor Aneuploidy and Stratifies Outcome in Grade 2 Breast Cancer

Purpose

Quantifying chromosomal instability (CIN) has both prognostic and predictive clinical utility in breast cancer. In order to establish a robust and clinically applicable gene expression-based measure of CIN, we assessed the ability of four qPCR quantified genes selected from the 70-gene Chromosomal Instability (CIN70) expression signature to stratify outcome in patients with grade 2 breast cancer.

Methods

AURKA, FOXM1, TOP2A and TPX2 (CIN4), were selected from the CIN70 signature due to their high level of correlation with histological grade and mean CIN70 signature expression in silico. We assessed the ability of CIN4 to stratify outcome in an independent cohort of patients diagnosed between 1999 and 2002. 185 formalin-fixed, paraffin-embedded (FFPE) samples were included in the qPCR measurement of CIN4 expression. In parallel, ploidy status of tumors was assessed by flow cytometry. We investigated whether the categorical CIN4 score derived from the CIN4 signature was correlated with recurrence-free survival (RFS) and ploidy status in this cohort.

Results

We observed a significant association of tumor proliferation, defined by Ki67 and mitotic index (MI), with both CIN4 expression and aneuploidy. The CIN4 score stratified grade 2 carcinomas into good and poor prognostic cohorts (mean RFS: 83.8±4.9 and 69.4±8.2 months, respectively, p = 0.016) and its predictive power was confirmed by multivariate analysis outperforming MI and Ki67 expression.

Conclusions

The first clinically applicable qPCR derived measure of tumor aneuploidy from FFPE tissue, stratifies grade 2 tumors into good and poor prognosis groups.     

Apparent Diffusion Coefficient (ADC) Value: A Potential Imaging Biomarker That Reflects the Biological Features of Rectal Cancer

Objective

We elected to analyze the correlation between the pre-treatment apparent diffusion coefficient (ADC) and the clinical, histological, and immunohistochemical status of rectal cancers.

Materials and Methods

Forty-nine rectal cancer patients who received surgical resection without neoadjuvant therapy were selected that underwent primary MRI and diffusion-weighted imaging (DWI). Tumor ADC values were determined and analyzed to identify any correlations between these values and pre-treatment CEA or CA19-9 levels, and/or the histological and immunohistochemical properties of the tumor.

Results

Inter-observer agreement of confidence levels from two separate observers was suitable for ADC measurement (k  =  0.775). The pre-treatment ADC values of different T stage tumors were not equal (p  =  0.003). The overall trend was that higher T stage values correlated with lower ADC values. ADC values were also significantly lower for the following conditions: tumors with the presence of extranodal tumor deposits (p  =  0.006) and tumors with CA19-9 levels ≥ 35 g/ml (p  =  0.006). There was a negative correlation between Ki-67 LI and the ADC value (r  =  −0.318, p  =  0.026) and between the AgNOR count and the ADC value (r  =  −0.310, p  =  0.030).

Conclusion

Significant correlations were found between the pre-treatment ADC values and T stage, extranodal tumor deposits, CA19-9 levels, Ki-67 LI, and AgNOR counts in our study. Lower ADC values were associated with more aggressive tumor behavior. Therefore, the ADC value may represent a useful biomarker for assessing the biological features and possible relationship to the status of identified rectal cancers.     

Quantitative Apparent Diffusion Coefficient Measurements Obtained by 3-Tesla MRI Are Correlated with Biomarkers of Bladder Cancer Proliferative Activity

Purpose

To investigate the association between Apparent Diffusion Coefficient (ADC) values and cell cycle and proliferative biomarkers (p53, p21, Ki67,) in order to establish its potential role as a noninvasive biomarker for prediction of cell cycle, proliferative activity and biological aggressiveness in bladder cancer.

Materials and Methods

Patients with bladder cancer who underwent 3,0 Tesla DW-MRI of the bladder before TUR-B or radical cystectomy were eligible for this prospective IRB-approved study. Histological specimen were immunohistochemically stained for the following markers: p53, p21 and ki67. Two board-certified uropathologists reviewed the specimens blinded to DW-MRI results. Histological grade and T-stage were classified according to the WHO 2004 and the 2009 TNM classification, respectively. Nonparametric univariate and multivariate statistics including correlation, logistic regression and ROC analysis were applied.

Results

Muscle invasive bladder cancer was histologically confirmed in 10 out of 41 patients. All examined tissue biomarkers were significantly correlated with ADC values (p<0.05, respectively). Based on multivariate analysis, p53 and ADC are both independent prognostic factors for muscle invasiveness of bladder cancer (>/ = T2). (p = 0.013 and p = 0.018).

Conclusion

ADC values are associated with cell cycle and proliferative biomarkers and do thereby reflect invasive and proliferative potential in bladder cancer. ADC and p53 are both independent prognostic factors for muscle invasiveness in bladder cancer.     

Quantification of Estrogen Receptor-Alpha Expression in Human Breast Carcinomas With a Miniaturized,Low-Cost Digital Microscope: A Comparison with a High-End Whole Slide-Scanner

Introduction

A significant barrier to medical diagnostics in low-resource environments is the lack of medical care and equipment. Here we present a low-cost, cloud-connected digital microscope for applications at the point-of-care. We evaluate the performance of the device in the digital assessment of estrogen receptor-alpha (ER) expression in breast cancer samples. Studies suggest computer-assisted analysis of tumor samples digitized with whole slide-scanners may be comparable to manual scoring, here we study whether similar results can be obtained with the device presented.

Materials and Methods

A total of 170 samples of human breast carcinoma, immunostained for ER expression, were digitized with a high-end slide-scanner and the point-of-care microscope. Corresponding regions from the samples were extracted, and ER status was determined visually and digitally. Samples were classified as ER negative (<1% ER positivity) or positive, and further into weakly (1–10% positivity) and strongly positive. Interobserver agreement (Cohen’s kappa) was measured and correlation coefficients (Pearson’s product-momentum) were calculated for comparison of the methods.

Results

Correlation and interobserver agreement (r = 0.98, p < 0.001, kappa = 0.84, CI95% = 0.75–0.94) were strong in the results from both devices. Concordance of the point-of-care microscope and the manual scoring was good (r = 0.94, p < 0.001, kappa = 0.71, CI95% = 0.61–0.80), and comparable to the concordance between the slide scanner and manual scoring (r = 0.93, p < 0.001, kappa = 0.69, CI95% = 0.60–0.78). Fourteen (8%) discrepant cases between manual and device-based scoring were present with the slide scanner, and 16 (9%) with the point-of-care microscope, all representing samples of low ER expression.

Conclusions

Tumor ER status can be accurately quantified with a low-cost imaging device and digital image-analysis, with results comparable to conventional computer-assisted or manual scoring. This technology could potentially be expanded for other histopathological applications at the point-of-care.     

Prognostic Impact of Jab1, p16, p21, p62, Ki67 and Skp2 in Soft Tissue Sarcomas

Purpose

The purpose of this study is to clarify the prognostic significance of expression of Jab1, p16, p21, p62, Ki67 and Skp2 in soft tissue sarcomas (STS). Optimised treatment of STS requires better identification of high risk patients who will benefit from adjuvant therapy. The prognostic significance of Jab1, p16, p21, p62, Ki67 and Skp2 in STS has not been sufficiently investigated.

Experimental Design

Tissue microarrays from 193 STS patients were constructed from duplicate cores of viable and representative neoplastic tumor areas. Immunohistochemistry was used to evaluate the expression of Jab1, p16, p21, p62, Ki67 and Skp2.

Results

In univariate analyses, high tumor expression of Ki67 (P = 0.007) and Skp2 (P = 0.050) correlated with shorter disease-specific survival (DSS). In subgroup analysis, a correlation between Skp2 and DSS was seen in patients with malignancy grade 1 or 2 (P = 0.027), tumor size >5 cm (P = 0.018), no radiotherapy given (P = 0.029) and no chemotherapy given (P = 0.017). No such relationship was apparent for Jab1, p16, p21 and p62; but p62 showed a positive correlation to malignancy grade (P = 0.019). Ki67 was strongly positively correlated to malignancy grade (P = 0.001). In multivariate analyses, Skp2 was an independent negative prognostic factor for DSS in women (P = 0.009) and in patients without administered chemotherapy or radiotherapy (P = 0.026).

Conclusions

Increased expression of Skp2 in patients with soft tissue sarcomas is an independent negative prognostic factor for disease-specific survival in women and in patients not administered chemotherapy or radiotherapy. Besides, further studies are warranted to explore if adjuvant chemotherapy or radiotherapy improve the poor prognosis of STS with high Skp2 expression.     

Verbal Autopsy: Reliability and Validity Estimates for Causes of Death in the Golestan Cohort Study in Iran

Background

Verbal autopsy (VA) is one method to obtain valid estimates of causes of death in the absence of valid medical records. We tested the reliability and validity of a VA questionnaire developed for a cohort study in Golestan Province in northeastern Iran.

Method

A modified version of the WHO adult verbal autopsy was used to assess the cause of death in the first 219 Golestan Cohort Study (GCS) subjects who died. The GCS cause of death was determined by two internists who independently reviewed all available medical records. Two other internists (“reviewers”) independently reviewed only the VA answers and classified the cause of death into one of nine general categories; they repeated this evaluation one month later. The reliability of the VA was measured by calculating intra-reviewer and inter-reviewer kappa statistics. The validity of the VA was measured using the GCS cause of death as the gold standard.

Results

VA showed both good validity (sensitivity, specificity, PPV, and NPV all above 0.81) and reliability (kappa>0.75) in determining the general cause of death independent of sex and place of residence. The overall multi-rater agreement across four reviews was 0.84 (95%CI: 0.78–0.89). The results for identifying specific cancer deaths were also promising, especially for upper GI cancers (kappa = 0.95). The multi-rater agreement in cancer subgroup was 0.93 (95%CI: 0.85–0.99).

Conclusions

VA seems to have good reliability and validity for determining the cause of death in a large-scale adult follow up study in a predominantly rural area of a middle-income country.