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).

In stage IV pulmonary adenocarcinoma patients, treatment with Traditional Chinese Medicine alone gives prognostically superior results to treatment with Platinum-Based Chemotherapy alone.

BACKGROUND: About 30% of pulmonary stage IV adenocarcinomas die within 3 months of diagnosis. Western medical treatments with Platinum-Based Chemotherapy=PBC and tyrosine-kinase inhibitors Targeted Therapy=TT can improve prognosis. In China, Traditional Chinese Medicine herbal treatments (TCM) are often used in addition to PBC and TT. A considerable number of patients refuse Western medical treatments and use TCM alone. However, the survival impact of the latter is unknown. HYPOTHESES TESTED: Treatment with TCM alone is prognostically superior to PBC alone. Addition of PBC or TT or both TT to TCM improves survival. METHODS: In this prospective observational, non-interventional study of 1017 consecutive first-onset stage IV NSCLC patients with up to 10 years follow-up, 261 who Died of Disease (DOD) within 3 months were omitted, as they never got the optimal Western medical therapies. All 218 non-adenocarcinomas were also omitted, leaving 538 stage IV adenocarcinomas treated by TCM alone (n = 29), PBC alone (N = 19) and TCM and other Western medical combinations (299 TCM and PBC, 50 TCM and TT, 141 TCM and PBC and TT) with 3 - 120 months follow-up. Survivals were compared using Alive with Disease (AWD) and DOD as endpoints. RESULTS: The patients treated only with TCM had 7 months better median survival than those that received PBC alone (17 and 10 months). The patients that received TCM and PBC had a better median survival (24 months) than TCM alone and much better than PBC alone. None of the patients that received TCM alone survived > 54 months, whereas 18% of TCM and PBC patients survived much longer. Over the observation period of 3 - 120 months, survivals of TCM and TT, TCM and PBC and TT, and TCM and PBC were not different and therefore grouped as TCM and Western medicines. Median survival times of PBC alone and TCM alone were lower than that of TCM and Western medical treatments (p < 0.0001, 10, 17 and 27 months). CONCLUSIONS: Pulmonary stage IV adenocarcinoma patients with at least 3 months survival, treated with TCM alone have a significantly better survival than those treated with PBC alone. Adding Western PBC, TT or both to TCM further improves prognosis.

Supersaturation and Precipitation Applicated in Drug Delivery Systems: Development Strategies and Evaluation Approaches.

Supersaturation is a promising strategy to improve gastrointestinal absorption of poorly water-soluble drugs. Supersaturation is a metastable state and therefore dissolved drugs often quickly precipitate again. Precipitation inhibitors can prolong the metastable state. Supersaturating drug delivery systems (SDDS) are commonly formulated with precipitation inhibitors, hence the supersaturation is effectively prolonged for absorption, leading to improved bioavailability. This review summarizes the theory of and systemic insight into supersaturation, with the emphasis on biopharmaceutical aspects. Supersaturation research has developed from the generation of supersaturation (pH-shift, prodrug and SDDS) and the inhibition of precipitation (the mechanism of precipitation, the character of precipitation inhibitors and screening precipitation inhibitors). Then, the evaluation approaches to SDDS are discussed, including in vitro, in vivo and in silico studies and in vitro-in vivo correlations. In vitro aspects involve biorelevant medium, biomimetic apparatus and characterization instruments; in vivo aspects involve oral absorption, intestinal perfusion and intestinal content aspiration and in silico aspects involve molecular dynamics simulation and pharmacokinetic simulation. More physiological data of in vitro studies should be taken into account to simulate the in vivo environment. The supersaturation theory should be further completed, especially with regard to physiological conditions.

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.

Clinical and Biological Interpretation of Survival Curves of Cancer Patients, Exemplified With Stage IV Non-Small Cell Lung Cancers With Long Follow-up.

Worldwide, 18.1 million new invasive cancers and 9.9 million cancer deaths occurred in 2020. Lung cancer is the second most frequent (11.4%) and, with 1.8 million deaths, remains the leading cause of cancer mortality. About 1.7 million of lung cancers are of the non-small cell lung cancer (NSCLC) subtype, and of these, 60%-70% are in advanced stage IV at the time of diagnosis. Thus, the annual worldwide number of new NSCLC stage IV patients is about 1 million, and they have a very poor prognosis. Indeed, 25%-30% die within 3 months of diagnosis. However, the survival duration of the remaining 700,000 new patients per year surviving >3 months varies enormously. Surprisingly, little research has been done to explain these survival differences, but recently it was found that classical patient, tumour and treatment features cannot accurately distinguish short- and very long-term survivors. What then are the causes of these bewildering survival variations amongst "the same cancers"? Clonality, proliferation differences, neovascularization, intra-tumour heterogeneity, genetic inhomogeneity and other cancer hallmarks play important roles. Considering each of these, single or combined, can greatly improve our understanding. Another technique is analysis of the survival curve of a seemingly homogeneous group of cancer patients. This can give valuable information about the existence of subgroups and their biological characteristics. Different basic survival curves and what their shapes tell about the biological properties of these invasive cancers are discussed. Application of this analysis technique to the survival curve of 690 stage IV NSCLC patients with a 3.2-120.0-month survival suggests that this seemingly homogeneously group of patients probably consists of 4-8 subgroups with a very different survival. A subsequent detailed mathematical analysis shows that a model of 8 subgroups gives a very good match with the original survival curve of the whole group. In conclusion, the survival curve of a seemingly homogeneous group of cancer patients can give valuable information about the existence of subgroups and their biological characteristics. Application of this technique to 690 NSCLC Stage IV patients makes it probable that 8 different subgroups with very different survival rates exist in this group of cancers.

Traditional Chinese Medicine Aconiti Radix Cocta Improves Rheumatoid Arthritis via Suppressing COX-1 and COX-2.

According to Traditional Chinese Medicine (TCM), Aconiti Radix Cocta (AC) is clinically employed to expel wind, remove dampness, and relieve pain. We evaluated the antirheumatoid arthritis (RA) activities and underlying mechanisms of AC. The chemical constituents of AC were analyzed by high-performance liquid chromatography (HPLC) using three reference compounds (benzoylaconitine, benzoylmesaconine, and benzoylhypacoitine). The anti-RA effects of AC were evaluated in adjuvant-induced arthritis (AIA) rats by hind paw volume and histopathological analysis. The effects of AC on inflammatory cytokines (IL-1ß and IL-17A) were determined by enzyme-linked immunosorbent assay. The regulation of cyclooxygenases (COX-1 and/or COX-2) was determined by Western blot and real-time quantitative reverse transcription polymerase chain reaction analyses. AC significantly reduced paw swelling, attenuated the inflammation and bone destruction in joint tissues, and reduced IL-1ß and IL-17A in the serum. Moreover, AC downregulated the expression of COX-1 and COX-2 in the synovial tissues. We also identified that AC possesses significant anti-RA activities on AIA, which may be ascribed to the regulation of inflammatory cytokines IL-1ß and IL-17, as well as to the inhibition of arachidonic acid signaling pathways. Our findings provide theoretical support for AC as an effective nature-derived therapeutic agent for RA treatment.

Major bioactive chemical compounds in Astragali Radix samples from different vendors vary greatly.

The worldwide traditional Chinese medicine (TCM) herbs sales figures have increased considerably to 50 billion US$ (2018). Astragali Radix (AR) is amongst the most often sold TCM herbs; sales in the European Union (EU) need European Medicines Agency (EMA) approval. However, comparisons of characteristic bioactive molecules concentrations in AR from different EU vendors are lacking. This study uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) with standard addition to evaluate the influence of different sample and preparation types and ammonia treatment on bioactive molecules concentrations in AR. We also compare AR samples from different EU-vendors. Astragaloside IV (AG-IV), ononin and calycosin 7-O-ß-D-glucoside concentrations were higher in root powder samples when extracted with boiled water than with ultrasonication using 70% methanol. AG-IV content was by far the highest in granulates from vendor 1 (202 ± 35 µg/g) but very low in hydrophilic concentrates from vendor 1 (32 ± 7 µg/g) and granulates from vendor 4 (36 ± 3 µg/g). Ammonia-treatment significantly increased AG-IV concentrations in all samples (e.g., to 536 ± 178 µg/g in vendor 1 granulates). Comparable effects were found for most other bioactive molecules. AG-IV and other bioactive molecules concentrations differed strongly depending on sample types, extraction processes, ammonia treatment-or-not and especially between different vendors samples. Ammonia-treatment is debatable, as it is supposed to convert other astragalosides, to AG-IV. The results indicate that routine quantitative analysis of major bioactive compounds present in AR, helps in quality control of AR and to guarantee the quality of commercial products.

"How Long Have I Got?" in Stage IV NSCLC Patients With at Least 3 Months Up to 10 Years Survival, Accuracy of Long-, Intermediate-, and Short-Term Survival Prediction Is Not Good Enough to Answer This Question.

BACKGROUND: Most lung cancer patients worldwide [stage IV nonsmall cell lung cancer (NSCLC)] have a poor survival: 25%-30% die <3 months. Yet, of those surviving >3 months, 10%-15% (70,000-105,000 new patients worldwide per year) survive (very) long. Surprisingly, little scientific attention has been paid to the question, which factors cause the good prognosis in these NSCLC stage IV long survivors. Therefore, "How long do I still have?" currently cannot be accurately answered. We evaluated in a large group of 737 stage IV NSCLC patients surviving 3.2-120.0 months, the accuracies of short- and long-term survival predictive values of baseline factors, radiotherapy (RT), platinum-based chemotherapy (PBT), and tyrosine kinase inhibitor targeted therapy (TKI-TT). METHODS: This is a noninterventional study of 998 consecutive first-onset stage IV NSCLC patients. A total of 737 (74%) survived 3.2-120.0 months, 47 refused RT, PBT, and TKI-TT. Single and multivariate survival analysis and receiver operating curve (ROC) analysis were used with dead of disease (DOD) or alive with disease (AWD) as endpoints. RESULTS: The median survival (16.1 months) of 47 patients who refused PBT, RT, and TKI-TT was significantly worse than those with RT, PBT, and/or TKI-TT (23.3 months, HR = 1.60, 95% CI = 1.06-2.42, p = 0.04). Of these latter 690 patients, 42% were females, 58% males, median age 63 years (range 27-85), 1-, 2-, 5-, and 10-year survival rates were 74%, 49%, 16%, and 5%. In total, 16% were alive with disease (AWD) at the last follow-up. Pathology subtype (adenocarcinoma vs. all others), performance score, TNM substage, the number of PBT cycles and TKI-TT had independent predictive value. However, with the multivariate combination of these features, identification results of short-term nonsurvivors and long-term survivors were poor. CONCLUSIONS: In stage IV NSCLC patients with >3 months survival, baseline features, and systemic therapeutic modalities have strong survival predictive value but do not accurately identify short- and long-term survivors. The predictive value of other features and interventions discussed should be investigated in the worldwide very large group of stage IV NSCLC patients with >3 months survival.

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.

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.

Prognostic value and reproducibility of different microscopic characteristics in the WHO grading systems for pTa and pT1 urinary bladder urothelial carcinomas.

BACKGROUND: European treatment guidelines for pTa and pT1 urinary bladder urothelial carcinoma depend highly on stage and WHO-grade. Both the WHO73 and the WHO04 grading systems show some intra- and interobserver variability. The current pilot study investigates which histopathological features are especially sensitive for this undesired lack of reproducibility and the influence on prognostic value. METHODS: Thirty-eight cases of primary non-muscle invasive urothelial carcinomas, including thirteen cases with stage progression, were reviewed by three pathologists. Thirteen microscopic features were extracted from pathology textbooks and evaluated separately. Reproducibility was measured using Gwet's agreement coefficients. Prognostic ability regarding progression was estimated by the area under curve (AUC) of the receiver operating characteristics (ROC) function. RESULTS: The best reproducible features (Gwet's agreement coefficient above 0.60) were papillary architecture, nuclear polarity, cellular maturation, nuclear enlargement and giant nuclei. Nucleoli was the strongest prognostic feature, and the only feature with an AUC above 0.70 for both grading systems, but reproducibility was not among the strongest. Nuclear polarity also had prognostic value with an AUC of 0.70 and 0.67 for the WHO73 and WHO04, respectively. The other features did not have significant prognostic value. CONCLUSIONS: The reproducibility of the histopathological features of the different WHO grading systems varied considerably. Of all the features evaluated, only nuclear polarity was both prognostic and significantly reproducible. Further validation studies are needed on these features to improve grading of urothelial carcinomas.

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.

In Metastatic Non-small cell Lung Cancer Platinum-Based Treated Patients, Herbal Treatment Improves the Quality of Life. A Prospective Randomized Controlled Clinical Trial.

Background: According to clinical experience, Traditional Chinese Medicine (TCM) herbs added to platinum-based therapy (PBT) improve the Quality of Life (QOL) in metastatic non-small cell lung cancer (NSCLC) patients, but this must be prospectively validated. Patients and Methods: Based on clinical impressions regarding the effect of adding TCM herbs to platinum-based chemotherapy, we anticipated that 2 × 21 patients would be sufficient to obtain significant results with an α < 0.05 and power (1 - ß) of 90%. To be on the safe side, we enrolled at least 28 patients in each group. In a prospective randomized controlled trial, 61 uniquely defined consecutive patients (PBT+PLACEBO, N = 32; PBT+TCM, N = 29) with stage IIIB-IV, Eastern Cooperative Oncology Group (ECOG) performance scores (PS) = 0-1 and TCM syndrome combined Qi-Yin deficiency were enrolled. These 61 patients were selected from originally 154 consecutive stage IIIB-IV lung cancer patients in the enrollment period. Patients were hospitalized and strictly controlled/surveyed during the entire 2-month treatment period, to guarantee use of or abstinence from TCM herbal and placebo fluids. Occurrence of nausea-vomiting, QOL by Functional Assessment of Cancer Therapy-Lung (FACT-L) scales and changes in ECOG "improved and stable rates" were compared before and after two treatment cycles. Results: Before treatment, the clinico-pathologic and QOL features in PBT+PLACEBO and PBT+TCM patients did not differ (P > 0.10). The only side effects attributed by some of the patients to the TCM herbs were transient, mild gastric/abdominal heaviness in the first 2 weeks, but these also occurred amongst the PBT+PLACEBO patients (17 and 13%, P > 0.10). The incidence rates of nausea during treatment were 17% in PBT+TCM versus 75% in PBT+PLACEBO; vomiting rates were 14 and 56% (P < 0.0001 and 0.002). Moreover, ECOG "improved and stable rates" were 90% in the PBT+TCM versus 69% in the PBT+PLACEBO group (P = 0.04). In PBT+TCM patients, FACT-L social/familial and functional subscales were better after 2 months' treatment (P = 0.02 and 0.03). Contrarily, in PBT+PLACEBO patients, the QOL variables total score, physical and emotional subscales were worse after PBT treatment (P = 0.03, 0.0001, and 0.003). Conclusion: In stage IIIB-IV ECOG-PS = 0-1 NSCLC patients with Qi-Yin deficiency and platinum-based chemotherapy, adding TCM herbal medication improves the QOL. As this category of patients constitutes 40% of all metastatic NSCLCs, these results could have significant clinical impact.

Prediction of the anti-inflammatory mechanisms of curcumin by module-based protein interaction network analysis.

Curcumin, the medically active component from Curcuma longa (Turmeric), is widely used to treat inflammatory diseases. Protein interaction network (PIN) analysis was used to predict its mechanisms of molecular action. Targets of curcumin were obtained based on ChEMBL and STITCH databases. Protein-protein interactions (PPIs) were extracted from the String database. The PIN of curcumin was constructed by Cytoscape and the function modules identified by gene ontology (GO) enrichment analysis based on molecular complex detection (MCODE). A PIN of curcumin with 482 nodes and 1688 interactions was constructed, which has scale-free, small world and modular properties. Based on analysis of these function modules, the mechanism of curcumin is proposed. Two modules were found to be intimately associated with inflammation. With function modules analysis, the anti-inflammatory effects of curcumin were related to SMAD, ERG and mediation by the TLR family. TLR9 may be a potential target of curcumin to treat inflammation.

Prediction of spontaneous regression of cervical intraepithelial neoplasia lesions grades 2 and 3 by proteomic analysis.

Regression of cervical intraepithelial neoplasia (CIN) 2-3 to CIN 1 or less is associated with immune response as demonstrated by immunohistochemistry in formaldehyde-fixed paraffin-embedded (FFPE) biopsies. Proteomic analysis of water-soluble proteins in supernatants of biopsy samples with LC-MS (LTQ-Orbitrap) was used to identify proteins predictive of CIN2-3 lesions regression. CIN2-3 in the biopsies and persistence (CIN2-3) or regression (≤CIN1) in follow-up cone biopsies was validated histologically by two experienced pathologists. In a learning set of 20 CIN2-3 (10 regressions and 10 persistence cases), supernatants were depleted of seven high abundance proteins prior to unidimensional LC-MS/MS protein analysis. Mean protein concentration was 0.81 mg/mL (range: 0.55-1.14). Multivariate statistical methods were used to identify proteins that were able to discriminate between regressive and persistent CIN2-3. The findings were validated in an independent test set of 20 CIN2-3 (10 regressions and 10 persistence cases). Multistep identification criteria identified 165 proteins. In the learning set, zinc finger protein 441 and phospholipase D6 independently discriminated between regressive and persistent CIN2-3 lesions and correctly classified all 20 patients. Nine regression and all persistence cases were correctly classified in the validation set. Zinc finger protein 441 and phospholipase D6 in supernatant samples detected by LTQ-Orbitrap can predict regression of CIN2-3.

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.