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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., (© 2024. The Author(s).)

Introduction: Breast cancer is still the most common malignancy among women worldwide. The Prospective Breast Cancer Biobank (PBCB) collects blood and urine from patients with breast cancer every 6 or 12 months for 11 years from 2011 to 2030 at two university hospitals in Western Norway. The project aims to identify new biomarkers that enable detection of systemic recurrences at the molecular level. As blood represents the biological interface between the primary tumour, the microenvironment and distant metastases, liquid biopsies represent the ideal medium to monitor the patient's cancer biology for identification of patients at high risk of relapse and for early detection systemic relapse.Including patient-reported outcome measures (PROMs) allows for a vast number of possibilities to compare PROM data with biological information, enabling the study of fatigue and Quality of Life in patients with breast cancer., Methods and Analysis: A total of 1455 patients with early-stage breast cancer are enrolled in the PBCB study, which has a one-armed prospective observational design. Participants consent to contribute liquid biopsies (i.e., peripheral blood and urine samples) every 6 or 12 months for 11 years. The liquid biopsies are the basis for detection of circulating tumour cells, circulating tumour DNA (ctDNA), exosomal micro-RNA (miRNA), miRNA in Tumour Educated Platelet and metabolomic profiles. In addition, participants respond to 10 PROM questionnaires collected annually. Moreover, a control group comprising 200 women without cancer aged 25-70 years will provide the same data., Ethics and Dissemination: The general research biobank PBCB was approved by the Ministry of Health and Care Services in 2007, by the Regional Ethics Committee (REK) in 2010 (#2010/1957). The PROM (#2011/2161) and the biomarker study PerMoBreCan (#2015/2010) were approved by REK in 2011 and 2015 respectively. Results will be published in international peer reviewed journals. Deidentified data will be accessible on request., Trial Registration Number: NCT04488614., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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.

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.

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.

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., Competing Interests: Jan P.A. Baak is a member of the PLOS ONE Editorial Board and acts as an independent researcher representing his own company; namely “Dr. Med. Jan Baak AS”. Assuredly, this does not alter the authors' adherence to PLOS ONE Editorial policies and criteria, and the authors declare that no other competing interests exist.

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.

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.

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.

Aims: The proliferation factor Ki67 is prognostic in breast cancer and included in international therapy guidelines, but measurement procedures differ between laboratories. We compared the reproducibility and prognostic value of different Ki67 sampling and measurement methods., Methods and Results: In 237 T(1,2) N(0) M(0) breast cancers without adjuvant systemic treatment, strictly standardized section thickness, automated antigen retrieval and immunohistochemistry were used. The percentages of Ki67-positive nuclei were assessed using (i) a 'quick-scan rapid estimate', (ii) ocular-square-guided counts by independent pathologists, (iii) computerized point-grid-sampling interactive morphometry (CIM) and (iv) automated digital image analysis (DIA). Quick-scan rapid estimates were poorly reproducible. The optimal prognostic thresholds of Ki67 counts by two pathologists differed greatly (4%, 14%; kappa: 0.36), with many therapeutic differences. CIM-Ki67 and DIA-Ki67 were strongly prognostic (P<0.0001) and reproducible. DIA-Ki67 (threshold: 6.5%) was the strongest and most robust prognosticator (the threshold could vary from 4 to 15% without significant prognostic loss). Ki67 was prognostically strongest in the periphery of the tumour., Conclusion: In node-negative breast cancer without adjuvant systemic treatment, Ki67% by DIA, but not subjective counts, is reproducible and prognostically strong. This casts serious doubt on therapeutic guidelines using subjective counts of Ki67., (© 2012 Blackwell Publishing Limited.)

There is a need for new biomarkers to more correctly identify node-negative breast cancer patients with a good or bad prognosis. Myristoylated alanine-rich C kinase substrate like-1 (MARCKSL1) is a membrane-bound protein that is associated with cell spreading, integrin activation and exocytosis. Three hundred and five operable T(1,2)N(0)M(0) lymph node-negative breast cancer patients (median follow-up time 121 months, range 10-178 months) were evaluated for MARCKSL1 expression by immunohistochemistry and quantitative real-time PCR. The results were compared with classical prognosticators (age, tumor diameter, grade, estrogen receptor, and proliferation), using single (Kaplan-Meier) and multivariate survival analysis (Cox model). Forty-seven patients (15 %) developed distant metastases. With single and multivariate analysis of all features, MARCKSL1 protein expression was the strongest prognosticator (P < 0.001, HR = 5.1, 95 % CI = 2.7-9.8). Patients with high MARCKSL1 expression (n = 23) showed a 44 % survival versus 88 % in patients with low expression at 15-year follow-up. mRNA expression of MARCKSL1 in formalin fixed paraffin-embedded tissue was also prognostic (P = 0.002, HR = 3.6, 95 % CI = 1.5-8.3). However, the prognostic effect of high and low was opposite from the protein expression, i.e., low expression (relative expression ≤ 0.0264, n = 76) showed a 79 % survival versus 92 % in those with high expression of MARCKSL1 mRNA. Multivariate analysis of all features with distant metastases free survival as the end-point showed that the combination of MARCKSL1 protein and phosphohistone H3 (PPH3) has the strongest independent prognostic value. Patients with high expression (≥13) of PPH3 and high MARCKSL1 protein had 45 % survival versus 78 % survival for patients with low MARCKSL1 protein expression and high expression (≥13) of PPH3. In conclusion, MARCKSL1 has strong prognostic value in lymph node-negative breast cancer patients, especially in those with high proliferation.

Introduction: Although lymph node negative (LN-) breast cancer patients have a good 10-years survival (∼85%), most of them still receive adjuvant therapy, while only some benefit from this. More accurate prognostication of LN- breast cancer patient may reduce over- and under-treatment. Until now proliferation is the strongest prognostic factor for LN- breast cancer patients. The small molecule microRNA (miRNA) has opened a new window for prognostic markers, therapeutic targets and/or therapeutic components. Previously it has been shown that miR-18a/b, miR-25, miR-29c and miR-106b correlate to high proliferation., Methods: The current study validates nine miRNAs (miR-18a/b miR-25, miR-29c, miR-106b, miR375, miR-424, miR-505 and let-7b) significantly correlated with established prognostic breast cancer biomarkers. Total RNA was isolated from 204 formaldehyde-fixed paraffin embedded (FFPE) LN- breast cancers and analyzed with quantitative real-time Polymerase Chain Reaction (qPCR). Independent T-test was used to detect significant correlation between miRNA expression level and the different clinicopathological features for breast cancer., Results: Strong and significant associations were observed for high expression of miR-18a/b, miR-106b, miR-25 and miR-505 to high proliferation, oestrogen receptor negativity and cytokeratin 5/6 positivity. High expression of let-7b, miR-29c and miR-375 was detected in more differentiated tumours. Kaplan-Meier survival analysis showed that patients with high miR-106b expression had an 81% survival rate vs. 95% (P = 0.004) for patients with low expression., Conclusion: High expression of miR-18a/b are strongly associated with basal-like breast cancer features, while miR-106b can identify a group with higher risk for developing distant metastases in the subgroup of Her2 negatives. Furthermore miR-106b can identify a group of patients with 100% survival within the otherwise considered high risk group of patients with high proliferation. Using miR-106b as a biomarker in conjunction to mitotic activity index could thereby possibly save 18% of the patients with high proliferation from overtreatment.

Phosphohistone H3 assessed proliferation has strong prognostic value. Lymph vessel invasion by D2-40 is also prognostic, but D2-40+ myoepithelial expression in small ducts completely filled by solid-pattern ductal carcinoma in situ can mimic lymphovascular invasion. As myoepithelial cells are also p63 positive, we have investigated whether lymph vessel invasion identified by combined D2-40/p63 is stronger prognostically than by D2-40 alone, and whether it has independent prognostic value to phosphohistone H3. In 240 operable T(1-2)N(0)M(0) node negative invasive breast cancer patients <71 years, phosphohistone H3 was determined by quantitative immunohistochemistry and lymph vessel invasion by D2-40/p63 double immunostaining. Correlation analysis between the clinico-pathologic factors and lymph vessel invasion, and univariate and multivariate prognostic survival analysis were performed. With median 117 (range: 12-192) months follow-up, 36 patients (15%) developed and 28 (12%) died of distant metastases. Ten of the 61 patients (16%) with cancer cells surrounded by D2-40 were p63 positive and none of these 'false lymph vessel invasion' recurred. D2-40+/p63- lymph vessel invasion occurred in 51/239 (21%) cases and correlated with grade, mitotic activity index, phosphohistone H3, ER, cytokeratin14, and HER2. D2-40+/p63- lymph vessel invasion was strongly prognostic, but far more in women ≥55 than those <55 years (P<0.0001 and 0.04). With multivariate analysis, phosphohistone H3 proliferation was the strongest single prognosticator. Lymph vessel invasion had additional prognostic value to phosphohistone H3 only in women ≥55. This group of patients, without/with lymph vessel invasion, had 10-year survival rates of 83 and 50%, respectively (hazard ratio-lymph vessel invasion=3.0, P=0.04; hazard ratio-phosphohistone H3=6.9, P=0.002). Where age was <55 years, only phosphohistone H3 had independent prognostic value. Combinations of other features had no additional value. In conclusion, T(1-2)N(0)M(0) invasive breast cancer patients ≥55 years with phosphohistone H3≥13, D2-40+/p63- defined lymph vessel invasion identifies a subgroup with a high risk of distant metastases.

Purpose: In breast cancer, different tools are used for prognostication and adjuvant systemic therapy selection. We compared the accuracy of the online program Adjuvant!, the Norwegian Breast Cancer Group (NBCG) guidelines, and the proliferation factor mitotic activity index (MAI) in patients with lymph node (LN) -negative disease (pN0)., Patients and Methods: Adjuvant! and MAI thresholds were set to 90% to 95% breast cancer-specific survival (BCSS) rates. These thresholds were 95% for Adjuvant!, 3 for MAI, and as follows for NBCG: pT1 grade 1 + pT1a-b grade 2 to 3; all pN0M0 and estrogen receptor/progesterone receptor positive versus all others. In 516 patients younger than age 55 years (T1-3N0M0) without adjuvant systemic therapy, univariable and multivariable 10-year BCSS rates were estimated., Results: Median follow-up time was 118 months. The concordance between MAI and Adjuvant! or NBCG was fair (κ = 0.35 and κ = 0.29, respectively). Adjuvant!, NBCG, and MAI were all prognostically significant (P ≤ .001). In the univariable analysis, the 10-year BCSS of MAI less than 3 versus ≥ 3 was 95% v 71%, respectively, with a hazard ratio of 7.0. In multivariable analysis, MAI was superior to Adjuvant! and NBCG. The 10-year survival of Adjuvant! ≥ 95% versus less than 95% was 91% v 74%, respectively, but stratification by MAI identified subgroups with different prognosis. Similar results occurred for NBCG and MAI. Adjuvant! and NBCG were not prognostic to each other., Conclusion: MAI is superior to Adjuvant! and NBCG in prognostication of patients with LN-negative breast cancer younger than age 55 years.

Background: DAX-1 inhibits oestrogen receptor (ER) activities and aromatase P450 expression. The prognostic effect of DAX-1 expression in breast cancer was investigated., Patients and Methods: DAX-1, apolipoprotein D (ApoD), mitotic activity index (MAI) and other prognosticators were evaluated, by quantitative immunohistochemistry (IHC) and/or real-time reverse-transcription polymerase chain reaction (qRT-PCR) analysis, in 103 invasive node-negative breast carcinomas., Results: With median 122 months follow-up, 24 patients developed distant metastases, of whom, 21 (20%) died. Low DAX-1 expression both by qRT-PCR and IHC was associated with poor survival. Combined strong DAX-1 and Apo D expression identified a subgroup with excellent survival. The most favourable prognostic combination was MAI <3 or (MAI ≥ 3 combined with DAX-1 and ApoD high expression) versus MAI ≥ 3 with either low ApoD and/or low DAX-1 expression) (p<0.0001, HR=6.1)., Conclusion: In operable node-negative breast cancer, strong DAX-1 expression is associated with excellent survival.

Breast cancer is a heterogeneous disease. Different subgroups can be recognized on the basis of the steroid receptors, HER-2, cytokeratin expression and proliferation patterns. As a result of mRNA-profiling studies, five major groups can be recognized, of which the triple-negative and basal-like tumors have the worst prognosis. Many of these tumors have a high proliferation that has the strongest prognostic value in node negative breast cancer. In the current study we analyzed the microRNA pattern in 103 lymph node negative breast cancers and compared these profiles with different biological characteristics and clinicopathological features. Unsupervised hierarchical cluster analysis divides the patients into four main groups, of which the basal-like/triple-negative group is the most prominent (11% of all cases), the luminal A cancers containing the Her2 negative and estrogen receptor/progesterone receptor-positive tumors is the largest group (57%), and the group of luminal B (32%) is more heterogeneous and contains the Her2 positive/estrogen receptor-negative patients as well. The highest overall classification values by analysis of variance followed by cross validation (leave one sample out and reselect genes) were found for cytokeratin 5 and 6, triple-negative and estrogen receptor, with 97, 90 and 90% accuracy, respectively. MiR-106b gene is prominent in all of these signatures and correlates strongest with high proliferation. Other interesting observations are the presence of several microRNAs (miR532-5p, miR-500, miR362-5p, and miR502-3p) located at Xp11.23 in cancers with a triple-negative signature, and the upregulation of several miR-17 cluster members in estrogen receptor-negative tumors. The current study shows that estrogen receptor negativity and cytokeratin 5 and 6 expression are important, and specific biological processes in lymph node negative breast cancer, as microRNA signatures are strongest in these subgroups.

Evaluation of prognostic factors in lymph node negative (LNneg) invasive lobular cancers (ILCs). Prospective analysis of proliferation and other prognosticators in 121 LNneg ILCs (119 months median follow-up, range 19-181), without adjuvant chemotherapy. ILC subtype was assessed in accordance with WHO-2003 criteria. Immunohistochemical E-cadherin and estrogen receptor were used. With a median follow up time of 83 months (range 19-181), 30 of the 121 (25%) ILC patients developed distant metastases and 27 (22%) died. None of the cases classified as solid/pleomorphic lobular were E-cadherin or estrogen receptor positive, contrasting the other ILCs. The solid/alveolar ILCs (n = 17) had a worse survival (50%) than the other ILCs (n = 104; 83%, P < 0.0001). Mitotic activity index (MAI) (but not nuclear grade or tubule formation) was prognostic with a threshold 0-5 versus >5 (=MAI-5) (contrasting MAI < 10 vs. > or = 10 in breast cancers in general; 85 and 54% survival, P < 0.0001). In multivariate analysis only subtype and MAI but none of the other characteristics had independent prognostic value. Histologic subtype and MAI have independent prognostic value in node negative invasive lobular cancers.

Independent studies have shown that in node negative breast cancer patients less than 71 years, the proliferation marker mitotic activity index (MAI) is the strongest, most well reproducible prognosticator and chemotherapy success predictor. The MAI overshadows the prognostic value of tubule formation, nuclear atypia and thereby grade. An often used crude mitotic impression is much less prognostic than the MAI; strict adherence to the MAI protocol is therefore important. The prognostic value of the MAI is age dependent: although patients with a MAI > or = 10 always have a poor prognosis irrespective of age, a low MAI (<10) loses its favourable prognostic association in women >70 years. PPH3 counts are prognostically stronger than the MAI, and markers such as Cyclin-B and E2FR are promising, but must be validated. Compared with commercial prognostic gene expression signatures, the MAI is at least as strong prognostically, has far fewer false positive results and as such should be included as an independent feature in any node negative breast cancer pathology report.

We validated and compared the prognostic value of the proliferation marker phosphohistone H3 (PPH3) with classical variables in 241 T(1-2)N(0)M(0) breast cancer patients less than 71 years old with long-term follow-up (median 117 months) and without adjuvant treatment. PPH3 was measured by automated digital image analysis. Thirty-seven patients (15%) developed distant metastases and 29 (12%) died. The previously established PPH3 prognostic threshold H3 <13 (n = 157; 65% of all cases) vs. >or=13 (n = 84; 35% of all cases) was the strongest prognostic threshold exceeding all other characteristics, with 10-year recurrence-free survival of distant metastases of 96 and 64%, respectively (P = < 0.0001, hazard ratio = 7.8, 95% confidence interval = 3.4-17.9). PPH3 is robust as it showed high inter-observer reproducibility and was prognostic over wide range of thresholds around 13 and is the strongest prognostic variable in invasive node-negative breast cancer patients less than 71 years old.

Apolipoprotein D (ApoD) is a lipocalin involved in various cellular processes, including cytoprotection. We retrospectively evaluated its prognostic impact in 272 women (median age 63 (range 21-89) years, with operable breast cancer (Stage I or IIa) treated according to national guidelines, stratified by age and menopausal status, with a median long-term follow-up of 12 years. ApoD, estrogen receptor-alpha (ERalpha), and progesterone receptor (PR) positivity were determined by quantitative immunohistochemistry in tissue microarrays taken from the invasive front of the tumors and in whole tumor sections. ApoD was expressed in 50% of the tumors. Diffuse nuclear and cytoplasmic (ApoD(CN)), but not granular cytoplasmic ApoD expression in the invasive front was a detrimental factor for elderly patients over 70 years of age, (n = 83), but not for younger and pre-menopausal patients. ApoD(CN )expression correlated with lymph node metastases (P = 0.04). Moreover, a correlation between ApoD(CN )localization and age over 70 years was observed (P = 0.03). In the elderly, ApoD(CN) positive tumors had both a significantly shorter relapse-free survival (all locations) (P = 0.02) and a decreased breast cancer-specific survival (P < 0.0001). Moreover, the prognostic importance of ApoD was dose dependent (P = 0.002) Multivariate analysis confirmed the prognostic value of ApoD(CN). When the elderly patients were stratified by nodal status, ApoD showed prognostic importance only in the node positive patients (n = 30) (HR = 9.6; 95% CI = 2.3-40.4). The potential relevance of ApoD in this increasingly frequent group of breast cancer patients should be further explored.

Background: Estrogen receptor-alpha (ERalpha) is an important prognostic and predictive marker in breast cancer. ERalpha signaling normally down-regulates expression of Apolipoprotein D (ApoD), a lipocalin that binds, transports or chelates lipophilic ligands, including tamoxifen (TAM). Hence, the co-expression of ApoD may therefore identify clinical relevant subgroups of ERalpha positive breast cancer patients., Material and Methods: ApoD, ERalpha, and progesterone receptor (PR) protein expressions were determined by immunohistochemistry (IHC) in primary tumors of 290 patients with operable breast cancer. The median follow-up was 12 years. Patients were stratified according to age, nodal stage and the expression of ERalpha and the combined cytoplasm and nuclear staining of ApoD (ApoD(CN))., Results: In elderly women (> or =70 years) (n = 76), ApoD(CN) expression identified different prognostic subgroups in ERalpha positive patients (Trend: p < 0.0001). Multivariate analysis in this age group (n = 72), showed that the ERalpha-positive /ApoD(CN)-negative subgroup had a better breast cancer specific survival (BCSS) compared with the ERalpha-positive/ApoD(CN)-positive group (hazard ratio (HR) = 4.3; 95% CI = 1.6-11.9; p = 0.005). This difference was predominantly seen in the node positive patients (n = 30) (HR = 10.5; 95% CI = 2.3-47.6; p = 0.002). In a subset of postmenopausal ERalpha-positive/node positive patients (n = 60) previously enrolled in a trial on 2 year adjuvant TAM 20 mg vs. placebo, a better BCSS was observed in ApoD(CN) negative patients compared to placebo (p = 0.02). In ApoD(CN) positive patients, adjuvant TAM did not provide any survival benefit., Discussion: ERalpha and ApoD(CN) co-expression seems to be of prognostic importance in node positive elderly patients with operable breast cancer. In addition, we hypothesize that ApoD(CN) expression may be a novel marker and/or mechanism of TAM resistance in postmenopausal node positive patients. Thus, when targeting the ERalpha pathway in these patients, the ApoD status of the tumor may be of clinical relevance.

Purpose: Prognostic comparison of phosphohistone-H3 (PPH3) with Cytokeratin 5/6 and/or 14 positive (=basal-CK), triple (ER, PR, HER2)-negative (=TNP) and basal-like (=TNP and basal-CK positive) phenotype in invasive breast cancers., Patients and Methods: Classical variables, PPH3, ER, PR, basal-CK and HER2 in 240 T1-2N0M0 patients under 71 years., Results: TNP and basal-like cancers had higher PPH3 expression than the other cancers (mean 48 versus 11, P<0.001). Fifteen percent of the patients in the whole group, but 32-38% of TNP and basal-like cancers recurred. With multivariate analysis, PPH3<13 (n=156) versus >or=13 (n=84=35% of all cases) was the strongest and only prognosticator (10-year survival 96% and 64%, P

Background: The mitotic activity index (MAI) is a strong prognosticator in node-negative invasive breast cancer patients. Recently, a correlation between the MAI and specific chromosomal aberrations at chromosome 1p was described., Methods: Analysis of MAI, immunohistochemical staining patterns for proliferation-associated phosphohistone H3 (PPH3), phosphorylated ERK1/2, p21, cyclin E, Ki67 and cyclin D1 proteins; and prognosis in 158 adjuvant chemotherapy-treated T1-2N0M0 invasive breast cancer patients, analysis of LOH at 1p31 (including ARHI) using the dinucleotide repeats D1S207, D1S430 and D1S464 in 76 patients. Single and multivariate survival analysis was used to evaluate the importance of the various markers tested., Results: LOH at 1p31 did not correlate with MAI nor provide prognostic information. Phosphohistone H3 was the best prognosticator for patients in all age groups with 20 year distant metastasis free survival of distant metastases 93% vs. 72% respectively (p=0.004, HR=4.5). In multivariate analysis, phosphohistone H3<13 vs. > or =13 exceeded the prognostic value of the mitotic activity index., Conclusions: LOH at 1p31 is common in breast cancer, and correlates with loss of proliferation-associated proteins, but not with MAI, PPH3 or prognosis. PPH3 is the best prognosticator in this study group of adjuvant chemotherapy-treated lymph node-negative breast cancer patients.

Background and Objectives: Androgen receptor (AR) is relevant for prognostication in breast cancer. Different determination methods and cut-off levels hamper interpretation and comparisons of studies. Long-term prognostic evaluation of different AR assays in patients comprising operable breast cancers is scarce., Methods: AR was evaluated in 120 primary tumors using the dextran-coated charcoal method (charc-AR), and quantitative immunohistochemistry (IHC) on whole sections (WS) and tissue microarrays (TMA). Nuclear and cytoplasmic-AR localization was determined, and the prognostic importance of AR assays was assessed. Comparisons and correlations with the mitotic activity index (MAI), estrogen receptor (ERalpha), progesterone receptor (PR), HER-2, and histological grade (WHO I-III) were made., Results: Nuclear-AR in WS, but not charc-AR, strongly correlated with MAI (P = 0.001). However, prognostic information appeared in univariate survival analyses only. Nuclear-AR in TMA was not prognostic. Charc-AR was independent prognostic in node positives both for relapse free survival (RFS) and breast cancer specific survival (BCSS). Both charc-AR and IHC cytoplasmic-AR provided independent prognostic survival information for BCSS in women <55 years., Conclusion: Methods that can detect AR localized in the cytoplasm yield important prognostic information, and further studies in patients with operable breast cancer are warranted., ((c) 2008 Wiley-Liss, Inc.)

The proliferation marker mitotic activity index is the strongest prognostic indicator in lymph node-negative breast cancer. The World Health Organization (WHO) 2003-defined procedure for determining WHO-mitotic activity index is often replaced by a quick scan mitotic impression. We evaluated the prognostic consequences of this practice in 433 T(1-3)N(0)M(0) lymph node-negative invasive ductal type breast cancers with long-term follow-up (median, 112 months; range, 12-187 months). Twenty-seven percent of the studied cases developed distant metastases, and 25% died of disease. Agreement between WHO-mitotic activity index (0-5 = 1, 6-10 = 2, >10 = 3) and mitotic impression (1, 2, 3) categories was 66% (kappa = 0.41), including 85% for category 1, 26% for category 2, and 52% for category 3. The WHO-mitotic activity index was a much stronger prognosticator than the mitotic impression, and the 10-year survival rates of the same categories (eg, mitotic activity index and mitotic impression category both 2) differed greatly. When grade was assessed by combining WHO-mitotic activity index or mitotic impression with the same values for tubular formation and nuclear atypia, grades disagreed in 18% of the cases. Deviation from the formal WHO-mitotic activity index assessment guidelines in breast cancer often results in erroneous prognosis estimations with therapeutic consequences and may explain why the prognostic value of proliferative activity in breast cancer is not always confirmed.

Background: Apolipoprotein D (ApoD) is a promising prognostic and predictive factor in breast cancer, but the analysis methods and results vary., Patients and Methods: Determination of ApoD content by immunoelectrophoresis in tumour cytosol (EPC), immunohistochemistry (IHC) in whole sections (WS) and tissue micro arrays (TMA) were compared in 283 breast carcinomas., Results: With EPC, 45% and with IHC, 71% of the tumours were ApoD-positive. Correlation between the degrees of ApoD positivity by ECP and IHC was poor (R2=0.04), caused by higher sensitivity of the IHC (resulting in many ECP negative carcinomas being IHC positive) and ApoD positivity of normal tissues and cysts (resulting in ApoD positivity by ECP in up to 33% of the IHC negative cases). Discrepancies between WS and TMA were considerable due to tumour heterogeneity., Conclusion: In breast cancer, IHC ApoD determination is superior to EPC analysis, but intratumor heterogeneity must be carefully considered when using TMA technology.

The proliferation factor mitotic activity index is the strongest prognostic factor in early breast cancer, but it may lack reproducibility. We analyzed the prognostic value of phosphohistone H3, a marker of cells in late G(2) and M phase, measuring highly standardized immunohistochemical nuclear phosphohistone H3 expression by subjective counts and digital image analysis. Expression was compared with classical clinico-pathologic prognostic variables and the mitotic activity index in 119 node-negative invasive breast cancers in patients less than 55 years old treated with adjuvant systemic chemotherapy with long-term follow-up (median 168 months). Nineteen patients (16%) developed distant metastases and 16 (13%) died. Strong phosphohistone H3 expression occurred preferentially in the peripheral growing front; counts were highly reproducible between observers (R=0.92) and highly consistent with digital image analysis (R=0.96). Phosphohistone H3 correlated (P<0.05) with tumor diameter, estrogen receptor, carcinoma grade, and mitotic activity index. Phosphohistone H3 values were systematically (80%) higher than the mitotic activity index. Receiver-operating curve analysis objectively showed that phosphohistone H3 <13 (n=53; 45% of all cases) vs phosphohistone H3> or =13 (n=66; 55% of all cases) was the strongest prognostic threshold, with 20-year recurrence-free survival of distant metastases of 96 and 58%, respectively (P=0.0002, HR=9.6). Mitotic activity index was the second strongest prognostic variable (P=0.003, HR=3.9). In multivariate analysis, phosphohistone H3 <13 vs> or =13 exceeded the prognostic value of the mitotic activity index. None of the other classical prognostic factors examined offered prognostic value additional to phosphohistone H3. Phosphohistone H3 is by far the strongest prognostic variable in early invasive node-negative breast cancer patients less than 55 years old with long-term follow-up.

In lymph-node-negative invasive breast cancer patients<55 years, the proliferation marker mitotic activity index (MAI) has previously been shown to be the strongest prognosticator. In studies without age definition, MAI was not strongly prognostic. We investigated the age dependency of the prognostic value of proliferation for distant metastasis-free (MFS) and overall cancer-related survival (OS) in 1004 histologically diagnosed T1-3N0M0 invasive breast cancers (n=516, <55 years; n=322, 55-70 years; n=166, >70 years) without systemic adjuvant therapy and long follow-up (median: 108 months). The MAI decreases with age and the prognostic value of MAI varied by age group. For patients<55 years, hazard ratios (HR) for MAI>or=10 versus<10 for MFS and OS were 3.1 and 4.4, respectively (P<.0001 for both), but only 1.9 and 1.9 (P=.004 and .006) for patients aged 55-70 years, while over 70 years, MAI was not significant (P=.11). The prognostic value of proliferation was age-dependent. Prognostic breast cancer studies must clearly indicate the age group being studied.