An international multicenter study to evaluate reproducibility of automated scoring for assessment of Ki67 in breast cancer.

The nuclear proliferation biomarker Ki67 has potential prognostic, predictive, and monitoring roles in breast cancer. Unacceptable between-laboratory variability has limited its clinical value. The International Ki67 in Breast Cancer Working Group investigated whether Ki67 immunohistochemistry can be analytically validated and standardized across laboratories using automated machine-based scoring. Sets of pre-stained core-cut biopsy sections of 30 breast tumors were circulated to 14 laboratories for scanning and automated assessment of the average and maximum percentage of tumor cells positive for Ki67. Seven unique scanners and 10 software platforms were involved in this study. Pre-specified analyses included evaluation of reproducibility between all laboratories (primary) as well as among those using scanners from a single vendor (secondary). The primary reproducibility metric was intraclass correlation coefficient between laboratories, with success considered to be intraclass correlation coefficient >0.80. Intraclass correlation coefficient for automated average scores across 16 operators was 0.83 (95% credible interval: 0.73-0.91) and intraclass correlation coefficient for maximum scores across 10 operators was 0.63 (95% credible interval: 0.44-0.80). For the laboratories using scanners from a single vendor (8 score sets), intraclass correlation coefficient for average automated scores was 0.89 (95% credible interval: 0.81-0.96), which was similar to the intraclass correlation coefficient of 0.87 (95% credible interval: 0.81-0.93) achieved using these same slides in a prior visual-reading reproducibility study. Automated machine assessment of average Ki67 has the potential to achieve between-laboratory reproducibility similar to that for a rigorously standardized pathologist-based visual assessment of Ki67. The observed intraclass correlation coefficient was worse for maximum compared to average scoring methods, suggesting that maximum score methods may be suboptimal for consistent measurement of proliferation. Automated average scoring methods show promise for assessment of Ki67 scoring, but requires further standardization and subsequent clinical validation.

Human papillomavirus type 16 and 18 in primary lung cancers--a meta-analysis.

Lung cancer is the leading cause of cancer mortality worldwide. A possible carcinogenic role of human papillomavirus (HPV) has been investigated for >20 years and has major clinical and public health implications. We performed a meta-analysis to assess the prevalence of HPV16 and HPV18 in primary lung cancers (2435 subjects from 37 published studies). The overall HPV prevalence ranged from 0.0 to 78.3% with large heterogeneity across geographic regions and histological tissue types. A higher proportion, 50% (7/14), of the European studies reported low or no HPV prevalence (0-10%) compared with the Asian studies, 22% (4/18). When the analysis was limited to HPV16 and HPV18 prevalence, a higher prevalence in Asia (HPV16 = 11.6% and HPV18 = 8.8%) than in Europe (HPV16 = 3.5% and HPV18 = 3.6%) was observed. Studies using HPV-specific primers resulted in higher prevalence rates than consensus HPV primers (HPV16: Asia = 13% and Europe = 6%; HPV18: Asia = 13% and Europe = 5%). Further studies are needed to elucidate the role of HPV in lung carcinogenesis with careful thought given to study design and laboratory detection methods for a more accurate assessment of HPV status in lung tumors.

Use of automated image analysis in evaluation of Mesothelioma Tissue Microarray (TMA) from National Mesothelioma Virtual Bank.

The National Mesothelioma Virtual Bank (NMVB) was established to provide annotated biospecimens to the mesothelioma research community. The resource provides tissue microarrays (TMA) to evaluate the biomarkers along with a variety of other resected mesothelioma specimens. In this manuscript, we describe the immunohistochemical evaluation of the mesothelioma TMA with three key antibodies that are used in making the diagnosis of mesothelioma, and compared the immunohistochemical assessment between manual scoring and image analysis. The TMA was assessed for the immunohistochemical expression of calretinin (N=39), cytokeratin (CK) 5/6 (N=33), and D2-40 (N=37). Immunohistochemistry was evaluated by semi-quantitative (manual) scoring using light microscope (MS) and by automated image analysis (AS). Calretinin staining was seen in both cytoplasmic and nuclear locations. CK5/6 stain was localized to the cytoplasm. D2-40 stain showed only membranous expression in our cases. • Based on the pathologist's scores, calretinin was positive in 31 of the 39 cases (80%), CK 5/6 in 15 of the 33 cases (46%) and D2-40 in 18 of the 37 cases (49%). • The percent-positive agreement between manual scores and image analysis was 90% (35/39), 94% (31/33), and 95% (35/37) for calretinin, CK 5/6, and D2-40, respectively. There was a substantial agree-ment between manual and automated scores for calretinin (kappa = 0.614) and an almost perfect agreement for CK5/6 (kappa = 0.879) and D2-40 (kappa = 0.892). Our study confirms that the immunohistochemical staining pattern of mesotheliomas in the NMVB UPMC TMA is similar to other studies. Our findings also show that automated image analysis provides similar results to manual scoring by pathologists, and provides a reproducible, objective, and accurate platform for immunohistochemical assessment of biomarker expression.

Diagnostic utility of p63/P501S double sequential immunohistochemical staining in differentiating urothelial carcinoma from prostate carcinoma.

BACKGROUND: Distinguishing urothelial carcinoma (UC) from prostate carcinoma (PC) is important due to potential therapeutic and prognostic implications. However, this can be a diagnostic challenge when there is limited tissue and in poorly differentiated tumors. We evaluated the diagnostic utility of a dual immunohistochemical stain comprising p63 and P501S (prostein), applied sequentially on a single slide and visualized by double chromogen reaction, in differentiating these two cancers. Thus far, there have been no previous studies assessing the diagnostic utility of p63 and P501S combined together as a dual immunostain in distinguishing between these two cancers. METHODS: p63/P501S dual-color sequential immunohistochemical staining was performed on archival material from 132 patients with high-grade UC and 23 patients with PC, and evaluated for p63 (brown nuclear) and P501S (red cytoplasmic) expression. Both the staining intensity and percentage of positive tumor cells were assessed. RESULTS: p63 was positive in 119/132 of UC and negative in PC. P501S was positive in 22/23 of PC and negative in UC. The p63+/P501S- immunoprofile had 90% sensitivity and 100% specificity for UC. The p63-/P501S+ immunoprofile had 96% sensitivity and 100% specificity for PC. CONCLUSION: Our results indicate that double sequential immunohistochemical staining with p63 and P501S is highly specific and can be a useful tool in distinguishing UC from PC especially when there is limited diagnostic tissue as it can be performed on a single slide.

Nuclear vitamin D receptor expression is associated with improved survival in non-small cell lung cancer.

Vitamin D has been shown to have anti-proliferative effects in a wide variety of cancers including lung cancer. The anticancer effects of vitamin D are mediated primarily by its active metabolite, 1,25-dihydroxyvitamin D (calcitriol), through vitamin D receptor (VDR) signaling. However, thus far there have been no studies evaluating the association between VDR expression and survival outcome in lung cancer. Using immunohistochemical analysis, we evaluated VDR expression, separately in the nucleus and cytoplasm, in lung cancer samples from 73 non-small cell lung carcinoma (NSCLC) patients with no prior therapy, and investigated the association between VDR expression and overall survival (OS). Cox proportional hazard models were used for our primary analyses. There were 44 deaths during a median follow-up of 51 months (range 13-93 months). High nuclear VDR expression was associated with improved OS after adjusting for age, gender, stage, smoking status, and histology (adjusted hazard ratio, 0.36; 95% confidence interval, 0.17-0.79). There was no association between cytoplasmic VDR expression and OS. Our results suggest that nuclear VDR status may be a prognostic marker in NSCLC. Future large studies to replicate our findings and to assess the impact of VDR gene polymorphisms on VDR expression are required as therapies targeting the vitamin D signaling pathway may be influenced by VDR status in the target lung cancer tissue.