The Ki67 dilemma: investigating prognostic cut-offs and reproducibility for automated Ki67 scoring in breast cancer.

PURPOSE: Quantification of Ki67 in breast cancer is a well-established prognostic and predictive marker, but inter-laboratory variability has hampered its clinical usefulness. This study compares the prognostic value and reproducibility of Ki67 scoring using four automated, digital image analysis (DIA) methods and two manual methods. METHODS: The study cohort consisted of 367 patients diagnosed between 1990 and 2004, with hormone receptor positive, HER2 negative, lymph node negative breast cancer. Manual scoring of Ki67 was performed using predefined criteria. DIA Ki67 scoring was performed using QuPath and Visiopharm® platforms. Reproducibility was assessed by the intraclass correlation coefficient (ICC). ROC curve survival analysis identified optimal cutoff values in addition to recommendations by the International Ki67 Working Group and Norwegian Guidelines. Kaplan-Meier curves, log-rank test and Cox regression analysis assessed the association between Ki67 scoring and distant metastasis (DM) free survival. RESULTS: The manual hotspot and global scoring methods showed good agreement when compared to their counterpart DIA methods (ICC > 0.780), and good to excellent agreement between different DIA hotspot scoring platforms (ICC 0.781-0.906). Different Ki67 cutoffs demonstrate significant DM-free survival (p < 0.05). DIA scoring had greater prognostic value for DM-free survival using a 14% cutoff (HR 3.054-4.077) than manual scoring (HR 2.012-2.056). The use of a single cutoff for all scoring methods affected the distribution of prediction outcomes (e.g. false positives and negatives). CONCLUSION: This study demonstrates that DIA scoring of Ki67 is superior to manual methods, but further study is required to standardize automated, DIA scoring and definition of a clinical cut-off.

Proliferation and immunohistochemistry for p53, CD25 and CK20 in predicting prognosis of non-muscle invasive papillary urothelial carcinomas.

Non-muscle invasive papillary urothelial carcinoma is a prevalent disease with a high recurrence tendency. Good prognostic and reproducible biomarkers for tumor recurrence and disease progression are lacking. Currently, WHO grade and tumor stage are essential in risk stratification and treatment decision-making. Here we present the prognostic value of proliferation markers (Ki67, mitotic activity index (MAI) and PPH3) together with p53, CD25 and CK20 immunohistochemistry (IHC). In this population-based retrospective study, 349 primary non-muscle invasive bladder cancers (NMIBC) were available. MAI and PPH3 were calculated manually according to highly standardized previously described methods, Ki-67 by the semi-automated QPRODIT quantification system, p53 and CD25 by the fully automated digital image analysis program Visipharm® and CK20 with the help of the semi-quantitative immunoreactive score (IRS). Survival analyses with log rank test, as well as univariate and multivariate Cox regression analyses were performed for all investigated variables. Age and multifocality were the only significant variables for tumor recurrence. All investigated variables, except gender, were significantly associated with stage progression. In multivariate analysis, MAI was the only prognostic variable for stage progression (p<0.001).

Automated Prognostic Assessment of Endometrial Hyperplasia for Progression Risk Evaluation Using Artificial Intelligence.

Endometrial hyperplasia is a precursor to endometrial cancer, characterized by excessive proliferation of glands that is distinguishable from normal endometrium. Current classifications define 2 types of EH, each with a different risk of progression to endometrial cancer. However, these schemes are based on visual assessments and, therefore, subjective, possibly leading to overtreatment or undertreatment. In this study, we developed an automated artificial intelligence tool (ENDOAPP) for the measurement of morphologic and cytologic features of endometrial tissue using the software Visiopharm. The ENDOAPP was used to extract features from whole-slide images of PAN-CK+-stained formalin-fixed paraffin-embedded tissue sections from 388 patients diagnosed with endometrial hyperplasia between 1980 and 2007. Follow-up data were available for all patients (mean = 140 months). The most prognostic features were identified by a logistic regression model and used to assign a low-risk or high-risk progression score. Performance of the ENDOAPP was assessed for the following variables: images from 2 different scanners (Hamamatsu XR and S60) and automated placement of a region of interest versus manual placement by an operator. Then, the performance of the application was compared with that of current classification schemes: WHO94, WHO20, and EIN, and the computerized-morphometric risk classification method: D-score. The most significant prognosticators were percentage stroma and the standard deviation of the lesser diameter of epithelial nuclei. The ENDOAPP had an acceptable discriminative power with an area under the curve of 0.765. Furthermore, strong to moderate agreement was observed between manual operators (intraclass correlation coefficient: 0.828) and scanners (intraclass correlation coefficient: 0.791). Comparison of the prognostic capability of each classification scheme revealed that the ENDOAPP had the highest accuracy of 88%-91% alongside the D-score method (91%). The other classification schemes had an accuracy between 83% and 87%. This study demonstrated the use of computer-aided prognosis to classify progression risk in EH for improved patient treatment.

Digital Image Analysis of the Proliferation Markers Ki67 and Phosphohistone H3 in Gastroenteropancreatic Neuroendocrine Neoplasms: Accuracy of Grading Compared With Routine Manual Hot Spot Evaluation of the Ki67 Index.

Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) are rare epithelial neoplasms. Grading is based on mitotic activity or the percentage of Ki67-positive cells in a hot spot. Routine methods have poor intraobserver and interobserver consistency, and objective measurements are lacking. This study aimed to evaluate digital image analysis (DIA) as an objective assessment of proliferation markers in GEP-NENs. A consecutive cohort of patients with automated DIA measurement of Ki67 (DIA Ki67) and phosphohistone H3 (DIA PHH3) on immunohistochemical slides was analyzed using Visiopharm image analysis software (Hoersholm, Denmark). The results were compared with the Ki67 index from routine pathology reports (pathology Ki67). The study included 159 patients (57% males). The median pathology Ki67 was 2.0% and DIA Ki67 was 4.1%. The interclass correlation coefficient of the DIA Ki67 compared with the pathology Ki67 showed an excellent agreement of 0.96 [95% confidence interval (CI): 0.94-0.96]. The observed kappa value was 0.86 (95% CI: 0.81-0.91) when comparing grades based on the same methods. PHH3 was measured in 145 (91.2%) cases. The observed kappa value was 0.74. (95% CI: 0.65-0.83) when comparing grade based on the DIA PHH3 and the pathology Ki67. The DIA Ki67 shows excellent agreement with the pathology Ki67. The DIA PHH3 measurements were more varied and cannot replace other methods for grading GEP-NENs.

A template to quantify the location and density of CD3 + and CD8 + tumor-infiltrating lymphocytes in colon cancer by digital pathology on whole slides for an objective, standardized immune score assessment.

BACKGROUND: In colon cancer, the location and density of tumor-infiltrating lymphocytes (TILs) can classify patients into low and high-risk groups for prognostication. While a commercially available 'Immunoscore®' exists, the incurred expenses and copyrights may prevent universal use. The aim of this study was to develop a robust and objective quantification method of TILs in colon cancer. METHODS: A consecutive, unselected series of specimens from patients with colon cancer were available for immunohistochemistry and assessment of TILs by automated digital pathology. CD3 + and CD8 + cells at the invasive margin and in tumor center were assessed on consecutive sections using automated digital pathology and image analysis software (Visiopharm®). An algorithm template for whole slide assessment, generated cell counts per square millimeters (cells/mm2), from which the immune score was calculated using distribution volumes. Furthermore, immune score was compared with clinical and histopathological characteristics to confirm its relevance. RESULTS: Based on the quantified TILs numbers by digital image analyses, patients were classified into low (n = 83, 69.7%), intermediate (n = 14, 11.8%) and high (n = 22, 18.5%) immune score groups. High immune score was associated with stage I-II tumors (p = 0.017) and a higher prevalence of microsatellite instable (MSI) tumors (p = 0.030). MSI tumors had a significantly higher numbers of CD3 + TILs in the invasive margin and CD8 + TILs in both tumor center and invasive margin, compared to microsatellite stable (MSS) tumors. CONCLUSION: A digital template to quantify an easy-to-use immune score corresponds with clinicopathological features and MSI in colon cancer.

Digital Image Analysis of Ki-67 Stained Tissue Microarrays and Recurrence in Tamoxifen-Treated Breast Cancer Patients.

PURPOSE: The proliferation marker Ki-67 has been used as a prognostic marker to separate low- and high-risk breast cancer subtypes and guide treatment decisions for adjuvant chemotherapy. The association of Ki-67 with response to tamoxifen therapy is unclear. High-throughput automated scoring of Ki-67 might enable standardization of quantification and definition of clinical cut-off values. We hypothesized that digital image analysis (DIA) of Ki-67 can be used to evaluate proliferation in breast cancer tumors, and that Ki-67 may be associated with tamoxifen resistance in early-stage breast cancer. PATIENTS AND METHODS: Here, we apply DIA technology from Visiopharm using a custom designed algorithm for quantifying the expression of Ki-67, in a case-control study nested in the Danish Breast Cancer Group clinical database, consisting of stages I, II, or III breast cancer patients of 35-69 years of age, diagnosed during 1985-2001, in the Jutland peninsula, Denmark. We assessed DIA-Ki-67 score on tissue microarrays (TMAs) from breast cancer patients in a case-control study including 541 ER-positive and 300 ER-negative recurrent cases and their non-recurrent controls, matched on ER-status, cancer stage, menopausal status, year of diagnosis, and county of residence. We used logistic regression to estimate odds ratios and associated 95% confidence intervals to determine the association of Ki-67 expression with recurrence risk, adjusting for matching factors, chemotherapy, type of surgery, receipt of radiation therapy, age category, and comorbidity. RESULTS: Ki-67 was not associated with increased risk of recurrence in tamoxifen-treated patients (ORadj =0.72, 95% CI 0.54, 0.96) or ER-negative patients (ORadj =0.85, 95% CI 0.54, 1.34). CONCLUSION: Our findings suggest that Ki-67 digital image analysis in TMAs is not associated with increased risk of recurrence among tamoxifen-treated ER-positive breast cancer or ER-negative breast cancer patients. Overall, our findings do not support an increased risk of recurrence associated with Ki-67 expression.

Mitotic activity index and CD25+ lymphocytes predict risk of stage progression in non-muscle invasive bladder cancer.

In urothelial cell type non-muscle invasive urinary bladder carcinoma, TNM stage and WHO grade are widely used to classify patients into low and high­risk groups for prognostic and therapeutic decision-making. However, stage and grade reproducibility and prediction accuracy are wanting. This may lead to suboptimal treatment. We evaluated whether proliferation features, nuclear area of the epithelial cancer cells and the composition of stromal and tumor infiltrating lymphocytes have independent prognostic value. In 183 primary non-muscle invasive bladder cancer patients with long follow-up (median for stage progression cohort: 119 months, range 5-173; median for tumor recurrence cohort: 82, range 3-165) proliferation features Ki67, PPH3 and Mitotic Activity Index (MAI), Mean Nuclear Area (MNA), lymphocyte subsets (CD8+, CD4+, CD25+) and plasma cells (CD138+) were assessed on consecutive sections. Post-resection instillation treatments (none, mitomycin, BCG) were strictly standardized during the intake period. Risk of recurrence was associated with expression of Ki67 (≤ 39 vs. > 39) and Multifocality (p = 0.01). Patients with low Ki67 had a higher recurrence rate than those with high Ki67. Lymphocyte composition did not predict recurrence. Stage progression was strongly associated with high values for MAI (>15) and CD25+ (>0.2%). In a multivariate analysis the combination of MAI and CD25+ was the single most prognostic feature (p<0.001). Validation of these results in additional, independent studies is warranted.

MiR-18a and miR-18b are expressed in the stroma of oestrogen receptor alpha negative breast cancers.

BACKGROUND: Previously, we have shown that miR-18a and miR-18b gene expression strongly correlates with high proliferation, oestrogen receptor -negativity (ER-), cytokeratin 5/6 positivity and basal-like features of breast cancer. METHODS: We investigated the expression and localization of miR-18a and -18b in formalin fixed paraffin embedded (FFPE) tissue from lymph node negative breast cancers (n = 40), by chromogenic in situ hybridization (CISH). The expression level and in situ localization of miR-18a and -18b was assessed with respect to the presence of tumour infiltrating lymphocytes (TILs) and immunohistochemical markers for ER, CD4, CD8, CD20, CD68, CD138, PAX5 and actin. Furthermore, in two independent breast cancer cohorts (94 and 377 patients) the correlation between miR-18a and -18b expression and the relative quantification of 22 immune cell types obtained from the CIBERSORT tool was assessed. RESULTS: CISH demonstrated distinct and specific cytoplasmic staining for both miR-18a and miR-18b, particularly in the intratumoural stroma and the stroma surrounding the tumour margin. Staining by immunohistochemistry revealed some degree of overlap of miR-18a and -18b with CD68 (monocytes/macrophages), CD138 (plasma cells) and the presence of high percentages of TILs. CIBERSORT analysis showed a strong correlation between M1-macrophages and CD4+ memory activated T-cells with mir-18a and -18b. CONCLUSIONS: Our study demonstrates that miR-18a and miR-18b expression is associated with ER- breast tumours that display a high degree of inflammation. This expression is potentially associated specifically with macrophages. These results suggest that miR-18a and miR-18b may play a role in the systemic immunological response in ER- tumours.

Prevalence of PD-L1 expression is associated with EMAST, density of peritumoral T-cells and recurrence-free survival in operable non-metastatic colorectal cancer.

INTRODUCTION: Microsatellite instability (MSI) predict response to anti-PD1 immunotherapy in colorectal cancer (CRC). CRCs with MSI have higher infiltration of immune cells related to a better survival. Elevated Microsatellite Alterations at Tetranucleotides (EMAST) is a form of MSI but its association with PD-L1 expression and immune-cell infiltration is not known. METHODS: A consecutive, observational cohort of patients undergoing surgery for CRC. EMAST and clinicopathological characteristics were investigated against PD-L1, as well as CD3 and CD8 expression in the invasive margin or tumour centre (Immunoscore). Difference in survival between groups was assessed by log rank test. RESULTS: A total of 149 stage I-III CRCs patients, with a median follow up of 60.1 months. Patients with PD-L1+ tumours (7%) were older (median 79 vs 71 years, p = 0.045) and had EMAST+ cancers (OR 10.7, 95% CI 2.2-51.4, p = 0.001). Recurrence-free survival was longer in cancers with PD-L1+ immune cells (HR 0.35, 95% CI 0.16-0.76, p = 0.008, independent of EMAST) and high Immunoscore (HR 0.10, 95% CI 0.01-0.72, p = 0.022). Patients expressing PD-L1 in immune cells had longer disease-specific survival (HR 0.28, 95% CI 0.10-0.77, p = 0.014). CONCLUSIONS: Higher Immunoscore (CD3/CD8 cells) and expression of tumour PD-L1 is found in CRCs with EMAST. Lymphocytic infiltrate and peritumoral PD-L1 expression have prognostic value in CRC.

Correlation Between Digital and Manual Determinations of Ki-67/MIB-1 Proliferative Indices in Human Meningiomas.

Objective. Proliferative activity in tumor tissues is assessed as the percentage of Ki-67/MIB-1-positive cells, or the proliferative index (PI). The PI is routinely assessed manually. However, the subjectivity of manual assessments might result in poor reproducibility. We hypothesized that digital assessments might reduce the error. Method. In our study, we assessed Ki-67/MIB-1 PIs, both manually and digitally, with tissue microarrays constructed from 141 human meningioma samples. Spearman-rank correlation and κ statistics were applied for correlation and agreement analyses, respectively. Mann-Whitney U tests were used to compare MIB-1 PIs between groups. Prognostic ability was assessed with Kaplan-Meier and Cox regression analyses. Results. We found a significant, high correlation (Spearman ρ = 0.832, P < .01) and moderate agreement (κ coefficient = 0.617, observed agreement = 80.9%) between the 2 methods. Both methods found significantly different Ki-67/MIB-1 PIs for different World Health Organization grades (P < .05). Neither method showed significant prognostic value. Conclusion. Digital determinations of Ki-67/MIB-1 PIs in human meningiomas are feasible for the daily routine.

Metabolic consequences of perioperative oral carbohydrates in breast cancer patients - an explorative study.

BACKGROUND: The metabolic consequences of preoperative carbohydrate load in breast cancer patients are not known. The present explorative study investigated the systemic and tumor metabolic changes after preoperative per-oral carbohydrate load and their influence on tumor characteristics and survival. METHODS: The study setting was on university hospital level with primary and secondary care functions in south-west Norway. Serum and tumor tissue were sampled from a population-based cohort of 60 patients with operable breast cancer who were randomized to either per-oral carbohydrate load (preOp™; n = 25) or standard pre-operative fasting (n = 35) before surgery. Magnetic resonance (MR) metabolomics was performed on serum samples from all patients and high-resolution magic angle spinning (HR-MAS) MR analysis on 13 tumor samples available from the fasting group and 16 tumor samples from the carbohydrate group. RESULTS: Fourteen of 28 metabolites were differently expressed between fasting and carbohydrate groups. Partial least squares discriminant analysis showed a significant difference in the metabolic profile between the fasting and carbohydrate groups, compatible with the endocrine effects of insulin (i.e., increased serum-lactate and pyruvate and decreased ketone bodies and amino acids in the carbohydrate group). Among ER-positive tumors (n = 18), glutathione was significantly elevated in the carbohydrate group compared to the fasting group (p = 0.002), with a positive correlation between preoperative S-insulin levels and the glutathione content in tumors (r = 0.680; p = 0.002). In all tumors (n = 29), glutamate was increased in tumors with high proliferation (t-test; p = 0.009), independent of intervention group. Moreover, there was a positive correlation between tumor size and proliferation markers in the carbohydrate group only. Patients with ER-positive / T2 tumors and high tumor glutathione (≥1.09), high S-lactate (≥56.9), and high S-pyruvate (≥12.5) had inferior clinical outcomes regarding relapse-free survival, breast cancer-specific survival, and overall survival. Moreover, Integrated Pathway Analysis (IPA) in serum revealed activation of five major anabolic metabolic networks contributing to proliferation and growth. CONCLUSIONS: Preoperative carbohydrate load increases systemic levels of lactate and pyruvate and tumor levels of glutathione and glutamate in ER-positive patients. These biological changes may contribute to the inferior clinical outcomes observed in luminal T2 breast cancer patients. TRIAL OF REGISTRATION: ClinicalTrials.gov; NCT03886389. Retrospectively registered March 22, 2019.

Influence of pre-operative oral carbohydrate loading vs. standard fasting on tumor proliferation and clinical outcome in breast cancer patients ─ a randomized trial.

BACKGROUND: Conflicting results have been reported on the influence of carbohydrates in breast cancer. OBJECTIVE: To determine the influence of pre-operative per-oral carbohydrate load on proliferation in breast tumors. DESIGN: Randomized controlled trial. SETTING: University hospital with primary and secondary care functions in South-West Norway. PATIENTS: Sixty-one patients with operable breast cancer from a population-based cohort. INTERVENTION: Per-oral carbohydrate load (preOp™) 18 and 2-4 h before surgery (n = 26) or standard pre-operative fasting with free consumption of tap water (n = 35). MEASUREMENTS: The primary outcome was post-operative tumor proliferation measured by the mitotic activity index (MAI). The secondary outcomes were changes in the levels of serum insulin, insulin-c-peptide, glucose, IGF-1, and IGFBP3; patients' well-being, and clinical outcome over a median follow-up of 88 months (range 33-97 months). RESULTS: In the estrogen receptor (ER) positive subgroup (n = 50), high proliferation (MAI ≥ 10) occurred more often in the carbohydrate group (CH) than in the fasting group (p = 0.038). The CH group was more frequently progesterone receptor (PR) negative (p = 0.014). The CH group had a significant increase in insulin (+ 24.31 mIE/L, 95% CI 15.34 mIE/L to 33.27 mIE/L) and insulin c-peptide (+ 1.39 nM, 95% CI 1.03 nM to 1.77 nM), but reduced IGFBP3 levels (- 0.26 nM; 95% CI - 0.46 nM to - 0.051 nM) compared to the fasting group. CH-intervention ER-positive patients had poorer relapse-free survival (73%) than the fasting group (100%; p = 0.012; HR = 9.3, 95% CI, 1.1 to 77.7). In the ER-positive patients, only tumor size (p = 0.021; HR = 6.07, 95% CI 1.31 to 28.03) and the CH/fasting subgrouping (p = 0.040; HR = 9.30, 95% CI 1.11 to 77.82) had independent prognostic value. The adverse clinical outcome of carbohydrate loading occurred only in T2 patients with relapse-free survival of 100% in the fasting group vs. 33% in the CH group (p = 0.015; HR = inf). The CH group reported less pain on days 5 and 6 than the control group (p <  0.001) but otherwise exhibited no factors related to well-being. LIMITATION: Only applicable to T2 tumors in patients with ER-positive breast cancer. CONCLUSIONS: Pre-operative carbohydrate load increases proliferation and PR-negativity in ER-positive patients and worsens clinical outcome in ER-positive T2 patients. TRIAL REGISTRATION: CliniTrials.gov; NCT03886389. Retrospectively registered March 22, 2019.

Validation study of MARCKSL1 as a prognostic factor in lymph node-negative breast cancer patients.

Protein expression of Myristoylated alanine-rich C kinase substrate like-1 (MARCKSL1) has been identified as a prognostic factor in lymph-node negative (LN-) breast cancer patients. We aim to validate MARCKSL1 protein expression as a prognostic marker for distant metastasis-free survival (DMFS) in a new cohort of LN- breast cancer patients. MARCKSL1 expression was evaluated in 151 operable T1,2N0M0 LN- breast cancer patients by immunohistochemistry. Median follow-up time was 152 months, range 11-189 months. Results were compared with classical prognosticators (age, tumor diameter, grade, estrogen receptor, and proliferation) using single (Kaplan-Meier) and multivariate (Cox model) survival analysis. Thirteen patients (9%) developed distant metastases. With both single and multiple analysis of all features, MARCKSL1 did not show a significant prognostic value for DMFS (p = 0.498). Of the assessed classical prognosticators, only tumor diameter showed prognostic value (hazard ratio 9.3, 95% confidence interval 2.8-31.0, p <0.001). MARCKSL1 expression could not be confirmed as a prognostic factor in this cohort. Possible reasons include changes in diagnostic and treatment guidelines between the discovery and validation cohorts. Further studies are needed to reveal the potential biological role of this protein in breast cancer.

Assessing the prognostic value of PAX2 and PTEN in endometrial carcinogenesis.

In order to avoid the consequences of over- and under-treatment of endometrial hyperplasia, diagnostic accuracy and progression risk assessment must be improved. The aim of this study was to assess whether PAX2 or PTEN expression could predict progression-free survival in endometrial intraepithelial neoplasia (EIN) and endometrial endometrioid carcinoma (EEC). Immunohistochemistry for detection of PAX2 and PTEN was performed on 348 endometrial samples; 75 proliferative endometrium (PE), 36 EIN and 237 EEC. Cases classified as PTEN null (1 or more glands negatively stained) were more prevalent in EEC than in PE and EIN (64% EEC vs 11% PE/EIN). A progressive decrease in PAX2 expression was observed from PE to EIN to EEC. Long-term clinical follow-up (6-310 months, median: 126) was available for 62 PE cases, all 36 EIN cases and 178 EEC cases. No patients with PE demonstrated progression to EIN or EEC. Progression of disease was observed in 10 (28%) EIN patients. These patients had significantly lower PAX2 expression than those that regressed (P = 0.005). Progression-free survival analysis revealed that EIN patients with a high-risk PAX2 expression score (H-score ≤75) had a higher probability of progression of disease in comparison to those with a low-risk score (H-score >75). PAX2 expression was not prognostic in EEC nor was PTEN status of prognostic value in either EIN or EEC. PAX2 expression analysis by means of H-score has prognostic potential for the identification of high-risk progression cases in EIN but needs to be validated in a larger cohort.

Expression profiling and intracellular localization studies of the novel Proline-, Histidine-, and Glycine-rich protein 1 suggest an essential role in gastro-intestinal epithelium and a potential clinical application in colorectal cancer diagnostics.

BACKGROUND: The primary function of the intestines is the absorption of water and nutrients. Although our knowledge about these processes on the cellular level is extensive, a number of important intracellular elements remain unknown. Here, we characterize the novel proline-, histidine-, glycine-rich 1 (PHGR1) mRNA and protein on the molecular level and propose a functional role of the PHGR1 protein in the intestinal and gastric epithelium. METHODS: PHGR1 mRNA and protein expression in human tissues and cell lines were characterized by quantitative RT-PCR, in situ hybridization, Northern blotting, Western blotting, and immunohistochemistry. Glycosylation was assessed by a chemical deglycosylation assay, whereas intracellular localization was studied by immunofluorescent staining of cell line cells. PHGR1 mRNA levels in HT29 cells was reduced by RNA interference and the resulting global changes in gene expression assessed by microarray hybridization. RESULTS: PHGR1 mRNA and protein were found to be expressed specifically in epithelial cells of intestinal mucosa, with the highest expression in the most mature and differentiated cells. PHGR1 protein was found to be glycosylated and to localize to both the cytoplasm and nucleus. Transcript profiling and gene ontology analysis of HT29 cells subjected to PHGR1 knockdown suggested a functional relationship with transport and metabolic processes. Examination of PHGR1 mRNA and protein levels in lymph nodes with known colorectal cancer metastases indicated that they may serve as biomarkers for detection of such metastases. CONCLUSIONS: Functional analyses of the novel PHGR1 mRNA and protein suggest an essential role in gastrointestinal epithelium and a clinical application in detection of colorectal cancer lymph node metastases.

Prognostic comparison of proliferation markers and World Health Organization 1973/2004 grades in urothelial carcinomas of the urinary bladder.

European treatment guidelines of non-muscle-invasive urothelial carcinoma of the urinary bladder are strongly dependent on grade, but grading reproducibility is wanting. Protocolized proliferation features such as Mitotic Activity Index (MAI), Ki-67, and phosphohistone H3 are prognostic and reproducible. The objective of this population-based study was to compare proliferation biomarkers with each other and with World Health Organization (WHO) 1973/2004 grades with regard to prediction of stage progression. A total of 193 primary non-muscle-invasive urothelial carcinomas were analyzed using WHO73/04 grades and measurement of the proliferation markers mentioned above. Sensitivities, specificities, and positive and negative predictive values with confidence intervals (CIs) were estimated with regard to progression prediction. Kaplan-Meier survival curves were made, and the hazard ratio and Harrell's C-index with 95% CIs, P values, and adjusted C-index for stage progression or not of WHO73, WHO04, and the proliferation markers were calculated. The median follow-up time was 75 months (range, 1-127). A total of 111 patients (52%) experienced recurrence within 5 years, and 14 patients (7%) progressed. High values of MAI predicted stage progression with a positive predictive value of 0.22 (95% CI, 0.12-0.37). The positive predictive value of Ki-67 and phosphohistone H3 were 0.15 (both 95% CIs, 0.07-0.29) and comparable to that of the WHO04. The prognostic value of MAI was strongest, exceeding that of the other proliferation markers and the WHO grading systems. In conclusion, in non-muscle-invasive urinary bladder urothelial carcinomas, proliferation biomarkers have prognostic value, possibly exceeding that of the WHO classifications.

Prognostic value of gene signatures and proliferation in lymph-node-negative breast cancer.

INTRODUCTION: The overall survival rate is good for lymph-node-negative breast cancer patients, but they still suffer from serious over- and some undertreatments. Prognostic and predictive gene signatures for node-negative breast cancer have a high number of genes related to proliferation. The prognostic value of gene sets from commercial gene-expression assays were compared with proliferation markers. METHODS: Illumina WG6 mRNA microarray analysis was used to examine 94 fresh-frozen tumour samples from node-negative breast cancer patients. The patients were divided into low- and high-risk groups for distant metastasis based on the MammaPrint-related genes, and into low-, intermediate- and high-risk groups based on the recurrence score algorithm with genes included in Oncotype DX. These data were then compared to proliferation status, as measured by the mitotic activity index, the expressions of phosphohistone H3 (PPH3), and Ki67. RESULTS: Kaplan-Meier survival analysis for distant-metastasis-free survival revealed that patients with weak and strong PPH3 expressions had 14-year survival rates of 87% (n=45), and 65% (n=49, p=0.014), respectively. Analysis of the MammaPrint classification resulted in 14-year survival rates of 80% (n=45) and 71% (n=49, p=0.287) for patients with low and high risks of recurrence, respectively. The Oncotype DX categorization yielded 14-year survival rates of 83% (n=18), 79% (n=42) and 68% (n=34) for those in the low-, intermediate- and high-risk groups, respectively (p=0.52). Supervised hierarchical cluster analysis for distant-metastasis-free survival in the subgroup of patients with strong PPH3 expression revealed that the genes involved in Notch signalling and cell adhesion were expressed at higher levels in those patients with distant metastasis. CONCLUSION: This pilot study indicates that proliferation has greater prognostic value than the expressions of either MammaPrint- or Oncotype-DX-related genes. Furthermore, in the subgroup of patients with high proliferation, Notch signalling pathway genes appear to be expressed at higher levels in patients who develop distant metastasis.

The prognostic value of mitotic activity index (MAI), phosphohistone H3 (PPH3), cyclin B1, cyclin A, and Ki67, alone and in combinations, in node-negative premenopausal breast cancer.

Proliferation, either as the main common denominator in genetic profiles, or in the form of single factors such as Ki67, is recommended for clinical use especially in estrogen receptor-positive (ER) patients. However, due to high costs of genetic profiles and lack of reproducibility for Ki67, studies on other proliferation factors are warranted. The aim of the present study was to evaluate the prognostic value of the proliferation factors mitotic activity index (MAI), phosphohistone H3 (PPH3), cyclin B1, cyclin A and Ki67, alone and in combinations. In 222 consecutive premenopausal node-negative breast cancer patients (87% without adjuvant medical treatment), MAI was assessed on whole tissue sections (predefined cut-off ≥10 mitoses), and PPH3, cyclin B1, cyclin A, and Ki67 on tissue microarray (predefined cut-offs 7th decile). In univariable analysis (high versus low) the strongest prognostic proliferation factor for 10-year distant disease-free survival was MAI (Hazard Ratio (HR)=3.3, 95% Confidence Interval (CI): 1.8-6.1), followed by PPH3, cyclin A, Ki67, and cyclin B1. A combination variable, with patients with MAI and/or cyclin A high defined as high-risk, had even stronger prognostic value (HR=4.2, 95%CI: 2.2-7). When stratifying for ER-status, MAI was a significant prognostic factor in ER-positive patients only (HR=7.0, 95%CI: 3.1-16). Stratified for histological grade, MAI added prognostic value in grade 2 (HR=7.2, 95%CI: 3.1-38) and grade 1 patients. In multivariable analysis including HER2, age, adjuvant medical treatment, ER, and one proliferation factor at a time, only MAI (HR=2.7, 95%CI: 1.1-6.7), and cyclin A (HR=2.7, 95%CI: 1.2-6.0) remained independently prognostic. In conclusion this study confirms the strong prognostic value of all proliferation factors, especially MAI and cyclin A, in all patients, and more specifically in ER-positive patients, and patients with histological grade 2 and 1. Additionally, by combining two proliferation factors, an even stronger prognostic value may be found.

Can proliferation biomarkers reliably predict recurrence in World Health Organization 2003 defined endometrial stromal sarcoma, low grade?

An estimated 1500-3000 invasive Endometrial Stromal Sarcomas (ESS) cases annually occur worldwide. Before 2003, ESS was divided as low and high grade ESS based on mitotic activity. In 2003 the WHO changed the names, excluded mitoses and made nuclear atypia and necrosis the essential diagnostic criteria to distinguish ESS, Low Grade (ESS-LG, recurrence-free survival >90%) and Undifferentiated Endometrial Sarcoma (UES, poor prognosis). We have evaluated in WHO2003 defined ESS-LG whether proliferation biomarkers predict recurrence. Using survival analysis, the prognostic value of classical mitosis counts (Mitotic Activity Index, MAI) in haematoxyllin-eosin (H&E) sections, and immunohistochemical proliferation biomarkers (Ki-67 and PhosphoHistone-3 (PPH3)) were examined in 24 invasive endometrial stromal sarcomas. Three of 24 (12.5%) ESS-LG recurred. The MAI, PPH3 and Ki-67 were all prognostic (P = 0.001, 0.002 and 0.03). MAI values were >3 in the recurrent cases, but never exceeded 10 (the classical threshold for low and high grade). Non-recurrent cases had 0 ≤ MAI ≤ 3. PPH3 and Ki67 counts can be easier to perform than MAI and therefore helpful in the diagnosis of ESS, Low Grade. In conclusion, in this small study of WHO2003 defined ESS-LG, high levels of proliferation as measured by MAI, PPH3 and Ki-67 are predictive of recurrence. Larger studies are required to confirm these results.

Prognostic comparison of the proliferation markers (mitotic activity index, phosphohistone H3, Ki67), steroid receptors, HER2, high molecular weight cytokeratins and classical prognostic factors in T1₋2N0M0 breast cancer.

The proliferation factors: mitotic activity index (MAI), phosphohistone H3 (PPH3) and Ki67 have strong prognostic value in early breast cancer but their independent value to each other and other prognostic factors has not been evaluated. In 237 T1₋2N0M0 breast cancers without systemic adjuvant treatment, formalized MAI assessment and strictly standardized, fully automated quantitative immunohistochemistry (IHC) for Ki67, PPH3, estrogen (ER) and progesterone receptor (PR), HER2, cytokeratins-5/6 and -14, and automated digital image analysis (DIA) for measuring PPH3 and Ki67 were performed. Section thickness was measured to further control IHC measurements. All features were measured in the periphery of tumors. The different proliferation assessments and other well-established clinicopathological and biomarker prognostic factors were compared. DIA-Ki67 added prognostically to PPH3. None of the other biomarkers or clinicopathological variables added prognostically to this PPH3/Ki67 combination. However, when PPH3 is replaced by MAI the prognostic value is nearly the same. In early operable node negative breast cancer without adjuvant systemic treatment, Ki67 with a threshold of 6.5% assessed by digital image analysis in the periphery of the tumor is prognostically strong. The combination of either PPH3/Ki67 or MAI/Ki67 overshadowed the prognostic value of all other features including Ki67 alone.