Search

Purpose Predictive biomarkers are needed to aid the individualization of radiotherapy (RT) in breast cancer. Cancer-associated fibroblasts have been implicated in tumor radioresistance and can be identified by platelet-derived growth factor receptor-beta (PDGFRb). This study aims to analyze how PDGFRb expression affects RT benefit in a large randomized RT trial. Methods PDGFRb was assessed by immunohistochemistry on tissue microarrays from 989 tumors of the SweBCG91RT trial, which enrolled lymph node-negative, stage I/IIA breast cancer patients randomized to RT after breast-conserving surgery. Outcomes were analyzed at 10 years for ipsilateral breast tumor recurrence (IBTR) and any recurrence and 15 years for breast cancer specific death (BCSD). Results PDGFRb expression correlated with estrogen receptor negativity and younger age. An increased risk for any recurrence was noted in univariable analysis for the medium (HR 1.58, CI 95% 1.11–2.23, p = 0.011) or PDGFRb high group (1.49, 1.06–2.10, p = 0.021) compared to the low group. No differences in IBTR or BCSD risk were detected. RT benefit regarding IBTR risk was significant in the PDGFRb low (0.29, 0.12–0.67, p = 0.004) and medium (0.31, 0.16–0.59, p p = 0.110) in multivariable analysis. Likewise, risk reduction for any recurrence was less pronounced in the PDGFRb high group. No significant interaction between RT and PDGFRb-score could be detected. Conclusion A higher PDGFRb-score conferred an increased risk of any recurrence, which partly can be explained by its association with estrogen receptor negativity and young age. Reduced RT benefit was noted among patients with high PDGFRb, however without significant interaction.

Purpose The study aimed to investigate expression of epithelial-to-mesenchymal transition (EMT)-related proteins and phenotypes during breast cancer progression and to relate this to patient outcome. Methods Protein expression patterns of E-cadherin, N-cadherin, twist, and vimentin were examined by immunohistochemistry on formalin-fixed paraffin-embedded samples from primary tumors (PTs) (n = 419), synchronous lymph node metastases (LNMs) (n = 131) and recurrences (n = 34) from patients included in an observational prospective primary breast cancer study. Markers were evaluated individually and combined as defined EMT phenotypes (epithelial, mesenchymal, partial EMT, and negative). EMT profiles were compared between matched tumor progression stages, and related to clinicopathological data and distant recurrence-free interval (DRFi). Results N-cadherin-positivity, vimentin-positivity, mesenchymal and partial EMT phenotypes were associated with more aggressive tumor characteristics such as triple-negative subtype. Single EMT markers and phenotype discordance rates between paired tumor samples were observed in the range of 2–35%. Non-epithelial phenotypes were more frequently identified in recurrences compared to PTs, however, no skewness of expression or phenotype was detected between PTs and matched LNMs or between PTs and matched recurrences (Exact McNemar test). Interestingly, patients with a twist positive PT had shorter DRFi, compared to patients with a twist negative PT (hazard ratio (HR) 2.4, 95% confidence interval (CI) 1.2–5.1, P = 0.02). Essentially, the same effect was seen in multivariable analysis (HR 2.5, 95% CI 0.97–6.6, P = 0.06). Conclusion The epithelial phenotype was indicated to be lost between PTs and recurrences as a reflection of tumor progression. Twist status of the PT was related to long-term prognosis warranting further investigation in larger cohorts.

Purpose According to the 2017 St Gallen surrogate definitions of the intrinsic subtypes, Ki67, progesterone receptor (PR) and Nottingham histological grade (NHG) are used for prognostic classification of estrogen receptor (ER) positive/HER2-negative breast cancer into luminal A- or luminal B-like. The aim of the present study was to investigate if additional biomarkers, related to endocrine signaling pathways, e.g., amplified in breast cancer 1 (AIB1), androgen receptor (AR), and G protein-coupled estrogen receptor (GPER), can provide complementary prognostic information in a subset of ER-positive/HER-negative invasive lobular carcinoma (ILC). Methods Biomarkers from 224 patients were analyzed immunohistochemically on tissue microarray. The primary endpoint was breast cancer mortality (BCM), analyzed with 10- and 25-year follow-up (FU). In addition, the prognostic value of gene expression data for these biomarkers was analyzed in three publicly available ILC datasets. Results AIB1 (high vs. low) was associated to BCM in multivariable analysis (adjusted for age, tumor size, nodal status, NHG, Ki67, luminal-like classification, and adjuvant systemic therapy) with 10-year FU (HR 6.8, 95% CI 2.3–20, P = 0.001) and 25-year FU (HR 3.0, 95% CI 1.1–7.8, P = 0.03). The evidence of a prognostic effect of AIB1 could be confirmed by linking gene expression data to outcome in independent publicly available ILC datasets. AR and GPER were neither associated to BCM with 10-year nor with 25-year FU (P > 0.33). Furthermore, Ki67 and NHG were prognostic for BCM at both 10-year and 25-year FU, whereas PR was not. Conclusions AIB1 is a new putative prognostic biomarker in ER-positive/HER2-negative ILC. Electronic supplementary material The online version of this article (10.1007/s10549-019-05138-7) contains supplementary material, which is available to authorized users.

Use of archived formalin-fixed paraffin-embedded (FFPE) tissue is a standard method for evaluation of proposed prognostic and predictive tumour markers. However, little is known of the preservation of biomarker expression in old FFPE tumour blocks. We investigate the quality of immunohistochemical (IHC) oestrogen (ER) and progesterone receptor (PR) evaluation in FFPE tissue over time (1978-2000) using a large breast cancer tissue microarray (N = 573) with access to receptor analyses in cytosol (CYT) at diagnosis, coexpression of other biomarkers and follow-up data. We found a good correlation between ER analysed with CYT at diagnosis and ER analysed with IHC in archived FFPE tissue from the same tumour. ER evaluation did not seem to be affected by tissue storage time. Nor was there any time-dependent difference in ERIHC correlation with other biomarkers (HER2, Ki67) or survival. Discordant cases were more often classified as ER-positive with IHC than with CYT. For PR, however, we found an increased correlation between methods in more recent time periods. This may possibly be explained by more reliable PRIHC results in newer samples, although other explanations may also contribute. Our results indicate stable ER expression in FFPE tissue archived for up to 40 years.

Background One third of patients with stage T1 urothelial carcinoma (UC) progress to muscle-invasive disease requiring radical surgery. Thus, reliable tools are needed for risk stratification of stage T1 UC. Objective To investigate the extent to which stratification of stage T1 tumours into previously described molecular pathologic UC subtypes can provide improved information on tumour progression. Design, setting, and participants A population-based cohort of 167 primary stage T1 UCs was characterised by immunohistochemistry and classified into the molecular subtypes urobasal (Uro, 32%), genomically unstable (GU, 58%), and squamous-cell-carcinoma–like (SCCL, 10%). Outcome measurements and statistical analysis Progression-free survival using univariate and multivariate models. Results and limitations Subtype classification was validated using nine additional markers with known subtype-specific expression. Analysis of mRNA expression of progression biomarkers revealed a strong association with molecular subtype. Kaplan-Meier analyses showed that the risk of progression was low for Uro tumours and high for GU/SCCL tumours. High progression risk scores were found only for GU/SCCL tumours. Clinical risk factors such as multifocality, concomitant carcinoma in situ, invasion depth, lymphovascular invasion, and high CD3 + lymphocyte infiltration were observed almost exclusively in GU/SCCL cases. Conclusions Molecular subtypes Uro, GU, and SCCL were identified in an independent population-based cohort of stage T1 UCs. Biomarkers and clinical risk factors for progression were associated with molecular subtype. Rapidly progressing T1 tumours were of subtype GU or SCCL and had either a high progression risk score or an elevated CD3 + cell count. Patient summary We show that classification of stage T1 urothelial carcinoma into molecular subtypes can improve the identification of patients with progressing tumours.

Aim A contralateral breast cancer (CBC) is today treated as an independent primary tumour, although recent data suggest risk and prognosis of CBC to be influenced by characteristics of and treatment given for the first tumour (BC1). We hereby investigate phenotypical and prognostic features of the second tumour (BC2) in relation to prior endocrine treatment and radiotherapy. Methods From a well-defined population-based cohort of CBC-patients, we have constructed a unique tissue-microarray including 600 pairs of primary tumours and CBCs. Breast cancer mortality was primary end-point for prognosis. Results Both oestrogen receptor (ER) status and stage was strongly correlated between BC1 and BC2 within CBC-pairs. Although BC2 had the highest prognostic impact, BC1 continued to influence prognosis after diagnosis of CBC. Patients diagnosed with two high stage tumours within a short time-interval had a particularly bad prognosis. Prior endocrine therapy and radiotherapy both correlated to ER-negativity of BC2. An ER-negative BC2 was associated with an inferior prognosis compared to an ER-positive BC2 regardless of ER-status of BC1 or prior endocrine therapy. Conclusions Our results suggest that both the residual prognostic impact of BC1, the possibility of contralateral metastasis, as well as prior treatment given, need to be considered when determining appropriate diagnostic work-up and treatment of CBC. In addition, radiation to the contralateral breast and risk of inducing CBC with an aggressive ER-negative phenotype should be considered when establishing new radiation treatment techniques. This study indicates loss of ER-expression as an important ‘endocrine treatment escape mechanism’, although further studies are warranted.

AimsAldehyde dehydrogenase family 1 member A1 (ALDH1A1) is a putative marker of breast cancer stem cells (CSCs) with prognostic implications when expressed in cancer or stroma. However, previous results are contradictory and we therefore aimed to further evaluate the impact of ALDH1A1 on distant disease-free survival (DDFS) and correlation with clinicopathological variables in breast cancer, specifically by evaluating different cut-offs.MethodsTwo breast cancer cohorts (N=216 and N=210) were evaluated with immunohistochemistry for ALDH1A1 on tissue microarrays with three different cut-offs in cancer cells and in stromal cells. The association of ALDH1A1 with DDFS and other clinicopathological variables was assessed. As further validation, gene expression levels of ALDH1A1 and association with survival were analysed in one of the cohorts and a separate cohort.ResultsALDH1A1 expression in cancer cells was associated with either a better or a worse prognosis, depending on cut-off. Considering weakly stained cancer cells as positive, ALDH1A1+ was associated with a better prognosis in both cohorts. Considering only strongly stained cells as positive, ALDH1A1+ was associated with oestrogen receptor and progesterone receptor negativity in both cohorts and worse prognosis in one of the cohorts. Stromal ALDH1A1 staining was associated with improved DDFS in one cohort. Gene expression analysis showed that a high ALDH1A1 expression was associated with a better prognosis.ConclusionsALDH1A1 is associated with DDFS and clinicopathological variables, both in cancer cells and stroma, but is highly cut-off dependent. Only the strongly ALDH1A1-stained cells show a more aggressive phenotype typical for CSCs.

The primary objective was to estimate serum thymidine kinase 1 (TK1) activity, reflecting total body cell proliferation rate including cancer cell proliferation, in women with loco regional inoperable or metastatic breast cancer participating in a prospective and randomized study. Secondary objectives were to analyze TK1 in relation to progression-free survival (PFS), overall survival (OS), therapy response and other tumour characteristics, including CA 15-3, widely used as a standard serum marker for disease progression. TK1 and CA 15-3 were analysed in 198 serum samples collected prospectively from women included in the randomized TEX trial between December 2002 and June 2007. TK1 activity was determined by the ELISA based DiviTum™ assay, and CA 15-3 analyses was generated with the electrochemiluminescence immunoassay Cobas Elecsys CA 15-3 II. High pre-treatment TK1 activity predicted shorter PFS (10 vs. 15 months p = 0.02) and OS (21 vs. 38 months, p

BACKGROUND: The need for completion axillary lymph node dissection (ALND) in breast cancer patients with micrometastases in the sentinel nodes (SNs) is controversial. The aim of this retrospective observational study is to determine if the method of detection of early breast cancer is predictive for additional positive nodes in patients with micrometastases in the SNs. METHODS: Between 2001 and 2011 a total of 1993 women with primary unilateral breast cancer had surgery at Skane University Hospital, Lund. Of 1993 patients, 1458 had an SN biopsy and nearly all patients with micro- and macrometastases had ALND. RESULTS: Micrometastases defined as >0.2 mm/>200 cells and ≤2.0 mm were found in 62 of 757 screen-detected patients and in 81 of 701 patients with symptomatic breast cancer. Only 3 of the screen-detected patients with micrometastases, all with tumour size >15 mm (range 18-39 mm), had metastases in the completion ALND whereas this was found in 18 of the symptomatic patients with micrometastases (p = 0.01), (tumour size, range 10-30 mm). Logistic regression analysis adjusted for method of detection, tumour size and histological grade showed 5 times higher odds for further metastases in ALND in patients with symptomatic presentation vs. screen-detected breast cancer. CONCLUSION: Despite the small number of patients with micrometastases in this large cohort of breast cancer patients, these results support the contention that completion ALND can safely be omitted in screen-detected breast cancer patients with micrometastases in the SNs.

Background The steroid-receptor coactivator amplified in breast cancer one (AIB1) is implicated to be a prognostic factor, although the results are not unanimous. Recently its effect was suggested to be modified by paired box 2 gene product (PAX2). Patients and methods Using immunohistochemistry (IHC) AIB1 and PAX2 were investigated in two cohorts of early breast cancer, including systemically untreated premenopausal lymph-node-negative women and pre- and postmenopausal women receiving tamoxifen. Results AIB1 scores were available for 490 patients and PAX2 scores were available for 463 patients. High AIB1 was a negative prognostic factor for distant disease-free survival (DDFS, P = 0.02) and overall survival (OS, P < 0.001) in systemically untreated women, while no prognostic effect was seen in the tamoxifen-treated cohort, indicating AIB1 to be a predictor of tamoxifen response. In systemically untreated patients, PAX2 was not a prognostic factor, nor did it modify the effect of AIB1. However, in ER-positive patients receiving tamoxifen, PAX2 appeared to be a positive prognostic factor in premenopausal patients, while a negative factor in postmenopausal. The interaction between the menopausal status and PAX2 was significant (P = 0.01). Conclusions In an independent cohort of low-risk premenopausal patients, we validate AIB1 as a negative prognostic factor, indicating AIB1 to be an interesting target for new anti-cancer therapies. The effect of PAX2 warrants further studies.

Polycomb repressive complex 2 (PRC2) and its core member enhancer of zeste homolog 2 (EZH2) mediate the epigenetic gene silencing mark: trimethylation of lysine 27 on histone 3 (H3K27me3). H3K27me3 is characteristic of the chromatin at genes involved in developmental regulation in undifferentiated cells. Overexpression of EZH2 has been found in several cancer types such as breast, prostate, melanoma and bladder cancer. Moreover, overexpression is associated with highly proliferative and aggressive types of breast and prostate tumors. We have analyzed the abundance of EZH2 and H3K27me3 using immunohistochemistry in two large and well-characterized breast tumor data sets encompassing more than 400 tumors. The results have been analyzed in relation to the molecular subtypes of breast tumors (basal-like, luminal A, luminal B, HER2-enriched and normal-like), as well as in subtypes defined by clinical markers (triple negative, ER+/HER2-/Ki67low, ER+/HER2-/Ki67high and HER2+), and were validated in representative breast cancer cell lines by western blot. We found significantly different expression of both EZH2 and H3K27me3 across all subtypes with high abundance of EZH2 in basal-like, triple negative and HER2-enriched tumors, and high H3K27me3 in luminal A, HER2-enriched and normal-like tumors. Intriguingly, the two markers show an inverse correlation, particularly for the basal-like and triple negative tumors. Consequently, high expression of EZH2 was associated with poor distant disease-free survival whereas high expression of H3K27me3 was associated with better survival. Additionally, none of 182 breast tumors was found to carry a previously described EZH2 mutation affecting Tyr641. Our observation that increased expression of EZH2 does not necessarily correlate with increased abundance of H3K27me3 supports the idea that EZH2 can have effects beyond epigenetic silencing of target genes in breast cancer.

Objective. Being able to predict the recurrence or progression of non-muscle-invasive bladder cancer would facilitate effective planning of treatments and follow-up. Biomarkers are needed that can supply prognostic information beyond that provided by clinical and pathological parameters. Tissue microarray (TMA)-based analysis of Ta bladder tumours was used to investigate the prognostic value of expression of several proteins involved in bladder carcinogenesis. Material and methods. Tumour tissue from 52 patients with Ta bladder cancer was investigated. At least three 0.6 mm punch cores from each tumour were placed in a paraffin array block. Tumour expression of tumour protein 53 (TP53), CDH1 (E-cadherin), proliferating cell nuclear antigen (PCNA), cyclooxygenase-2 (COX2), fibroblast growth factor receptor-3 (FGFR3) and epidermal growth factor receptor (EGFR) was quantified by immunohistochemistry (IHC) and correlated with time to recurrence. Median follow-up time was 3.1 years. Whole-section IHC analysis was performed to validate significant findings. Results. Of all patients, 69% (36/52) experienced recurrence. In univariate analysis, recurrence was associated with multifocality, number of earlier recurrences and a low quantity score for EGFR. In a multivariate model, a low EGFR quantity score was correlated with early recurrence (hazard ratio = 5.5, p = 0.003). However, whole-section IHC results for EGFR differed markedly from the TMA findings (κ = 0.07) and no association with time to recurrence was found (p = 0.65). Conclusions. Expression of EGFR measured by TMA-IHC, but not by whole-section IHC, was associated with early recurrence. The results suggest that the proteins assessed have no predictive value for recurrences. Concerns are raised regarding the methodology and generalization of results obtained with TMA-IHC. (Less)

The retinoblastoma tumor suppressor (RB) is important for retaining cell cycle control and loss of RB function is commonly observed in various malignancies. Experimental and animal studies have shown that RB knockdown in ER+ (estrogen receptor) cell lines and xenografts leads to resistance to tamoxifen, indicating that RB-inactivation could be linked to impaired response to specific cancer treatments. To address this issue, we utilized a unique randomized trial including 500 premenopausal breast cancer patients receiving either two years of adjuvant tamoxifen treatment or no treatment after surgery, and defined the tamoxifen response in RB-subgroups. Non-functional RB tumors were defined by lack of concordance between RB-phosphorylation and proliferation, in comparison to RB-functional tumors displaying comparable RB-phosphorylation and proliferation. In the ER+ tumors harboring a functional RB pathway (N=204), patients benefited from adjuvant tamoxifen with fewer breast cancer recurrences (HR=0.53, 95% CI 0.34-0.81, P=0.003). In the small subgroup of ER+ and RB non-functional tumors there was no benefit of tamoxifen (HR=2.28, 95% CI 0.51-10.3, P=0.28). In a multivariate analysis, the interaction between status of the RB pathway and treatment was significant (P=0.010), validating that despite being a small subgroup of ER+ breast cancer, RB functional status appears to be linked to response to tamoxifen treatment. These findings are in line with earlier experimental data altogether suggesting that analyses of RB status in breast cancer have the potential to be one among other future predictive factors that needs to be analyzed in order to successfully identify patients that will benefit from tamoxifen treatment.

Purpose Human epidermal growth factor receptor 2 (HER2) gene amplification or protein overexpression (HER2 positivity) defines a clinically challenging subgroup of patients with breast cancer (BC) with variable prognosis and response to therapy. We aimed to investigate the heterogeneous biologic appearance and clinical behavior of HER2-positive tumors using molecular profiling. Patients and Methods Hierarchical clustering of gene expression data from 58 HER2-amplified tumors of various stage, histologic grade, and estrogen receptor (ER) status was used to construct a HER2-derived prognostic predictor that was further evaluated in several large independent BC data sets. Results Unsupervised analysis identified three subtypes of HER2-positive tumors with mixed stage, histologic grade, and ER status. One subtype had a significantly worse clinical outcome. A prognostic predictor was created based on differentially expressed genes between the subtype with worse outcome and the other subtypes. The predictor was able to define patient groups with better and worse outcome in HER2-positive BC across multiple independent BC data sets and identify a sizable HER2-positive group with long disease-free survival and low mortality. Significant correlation to prognosis was also observed in basal-like, ER-negative, lymph node–positive, and high-grade tumors, irrespective of HER2 status. The predictor included genes associated with immune response, tumor invasion, and metastasis. Conclusion The HER2-derived prognostic predictor provides further insight into the heterogeneous biology of HER2-positive tumors and may become useful for improved selection of patients who need additional treatment with new drugs targeting the HER2 pathway.

Prognostic and treatment-predictive biomarkers in primary breast cancer are routinely analyzed in the primary tumor, whereas metastatic tumor cells in lymph node metastases are not characterized. The present study aimed to define the concordance between biomarkers in matched pairs of breast cancers and lymph node metastases and to relate their expression to clinical outcome. Patients with primary breast cancer treated with adjuvant tamoxifen for 2 years were included. A tissue microarray of primary tumors and lymph node metastases was constructed, and estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki67 were analyzed immunohistochemically in 262, 257, 104, and 101 patients, respectively. Five years’ distant disease-free survival (DDFS) was used as the primary end point. The concordance for biomarker expression in primary tumors and corresponding lymph node metastases was 93% for ER, 84% for PR, 97% for HER2, and 85% for Ki67. The discordant cases for HER2 status were all negative in the tumor but positive in the node. ER positivity was a significant independent predictor of improved 5-year DDFS when analyzed in the primary tumor as well as in the lymph node metastases. Ki67 positivity analyzed in both locations correlated with shortened DDFS. HER2 positivity at both locations was an indicator of early relapse. The concordance for the biomarkers analyzed in matched pairs of primary tumors and lymph node metastases was high. Moreover, survival analyses showed that the expression of biomarkers in lymph node metastases can provide prognostic information when no analyses of the primary tumor can be done. Treatment selection based on biomarkers in the lymph node is a topic for further studies.

Triple-negative breast cancer (TNB) has poor prognosis and moreover patients with TNB do not benefit from established targeted drugs with endocrine therapy or trastuzumab. The aim of the study was to analyze the prevalence of candidate biomarkers in tumors from patients with TNB. Tissue microarrays were prepared from primary tumors from premenopausal breast cancer patients (500/564) randomized to adjuvant tamoxifen or no adjuvant treatment. Immunohistochemical (IHC) staining included ER, PR, HER2, epidermal receptor growth factor (EGFR), vascular endothelial growth factor A (VEGF-A), and vascular endothelial growth factor receptor 2 (VEGFR2). EGFR and HER2 gene copy number was defined by fluorescence in situ hybridization (FISH). All patients were included in the descriptive analysis, but only untreated patients in the survival analysis. TNB was diagnosed in 96 patients and correlated significantly to low age, Nottingham histological grade (NHG) III, high Ki67-index, T2 tumors, node negativity, EGFR positivity, increased EGFR gene copy number and high VEGFR2 expression. TNB was an independent prognostic factor for decreased 5-year breast cancer specific survival (BCSS) (HR 2.0 (95% CI 1.1-3.6), P = 0.01), but not for 10-year BCSS. High VEGFR2 expression was significantly correlated to decreased BCSS in TNB patients. TNB was associated with decreased BCSS and clinicopathological characteristics of an aggressive tumor type. High VEGFR2 expression, EGFR expression, and EGFR gene copy number were significantly correlated to TNB, supporting their role as putative candidate biomarkers for selection of targeted therapy in TNB.

Clinical trials implicate the estrogen receptor (ER) coactivator amplified in breast cancer 1 (AIB1) to be a prognostic and a treatment-predictive factor, although results are not unanimous. We have further investigated this using a controlled randomised trial of tamoxifen versus control.A total of 564 premenopausal women were entered into a randomised study independent of ER status. Using a tissue microarray, AIB1 and ER were analysed by immunohistochemistry.AIB1 scores were obtained from 349 women. High AIB1 correlated to factors of worse prognosis (human epidermal growth factor receptor 2, Nottingham histological grade 3, and lymph node metastases) and to ER negativity. In the control arm, high AIB1 was a negative prognostic factor for recurrence-free survival (RFS) (P = 0.02). However, ER-positive patients with high AIB1 responded significantly to tamoxifen treatment (P = 0.002), increasing RFS to the same level as for systemically untreated patients with low AIB1. Although ER-positive patients with low AIB1 had a better RFS from the beginning, this was not further improved by tamoxifen (P = 0.8).In the control group, high AIB1 was a negative prognostic factor. However, ER-positive patients with high AIB1 responded significantly to tamoxifen. This implicates high AIB1 to be an independent predictive factor of improved response to tamoxifen and not, as has previously been discussed, a factor predicting tamoxifen resistance.

The aim of this study was to evaluate the prognostic value of Ki67 in relation to established prognostic factors in lymph node-negative breast cancer, and furthermore, whether the prognostic impact was dependent on estrogen receptor (ER) status and histological grade. In 200 premenopausal patients, with 5 years of follow-up, Ki67 was determined on tissue microarrays. In univariate analysis, Ki67 (or = 20 vs20%) was a prognostic factor for distant disease-free survival (hazard ratio: 2.7, 95% confidence interval: 1.3-5.4, P=0.005) and overall survival (hazard ratio: 4.9, 95% confidence interval: 1.7-14, P=0.003). When stratifying for ER status and histological grade, Ki67 was a significant prognostic factor for distant disease-free survival and overall survival only in the ER-positive group, and only in patients with histological grade 2, respectively. In multivariate analysis, human epidermal growth factor receptor 2 and age were independent prognostic factors for distant disease-free survival, whereas Ki67, histological grade, and tumor size were not. Ki67 was, however, an independent prognostic factor in the 87% of the patients who had not received adjuvant medical treatment. Agreement between the three readers was very good (kappa-values: 0.83-0.88). Furthermore, Ki67 was a significant prognostic factor for all three investigators (hazard ratio: 2.7-3.2). This study shows that Ki67 is a prognostic factor in node-negative breast cancer. It is noteworthy that the prognostic information of Ki67 is restricted to ER-positive patients, and to patients with histological grade 2. Taken together, Ki67, as an easily assessed and reproducible proliferation factor, may be an alternative or complement to histological grade as a prognostic tool and for selection of adjuvant treatment.

We investigate the prognostic significance of the pro-angiogenic cytokine vascular endothelial growth factor (VEGF), urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1), and S-phase fraction (SPF) for distant disease free survival (DDFS) in 219 premenopausal patients with node-negative breast cancer (NNBC). In univariate analysis significantly shorter DDFS was observed for patients with high VEGF (p = 0.006), high uPA (p = 0.001). and high SPF (p < 0.001). The prognostic significance of VEGF varied over time being very, strong for early relapses (0-2.25 years follow-up) (HR = 7.9: p = 0.006) while no difference was seen in the subsequent follow-up period (HR = 1.3: p = 0.62). In a series of bivariate analyses VEGF provided prognostic information during the whole observation period (0-72 months) in addition to age, tumor size, oestrogen receptor (ER), progesterone receptor (PgR), and uPA. Also this effect was more pronounced during the first follow-up period suggesting VEGF as a marker of early recurrences. (C) 2008 Elsevier Ltd. All rights reserved. (Less)

The driving force behind oncoproteomics is to identify biomarker signatures associated with a particular malignancy. Here, we have for the first time used large-scale recombinant scFv antibody microarrays in an attempt to classify metastatic breast cancer versus healthy controls, based on differential protein expression profiling of whole serum samples. Using this multiplexed and miniaturised assay set-up providing pM range sensitivities, breast cancer could be classified with a specificity and sensitivity of 85% based on 129 serum analytes. However, by adopting a condensed 11 analyte biomarker signature, composed of nine non-redundant serum proteins, we were able to distinguish cancer versus healthy serum proteomes with a 95% sensitivity and specificity, respectively. When a subgroup of patients, not receiving anti-inflammatory drugs, was analysed, a novel eight analyte biomarker signature with a further improved predictive power was indicated. In a longer perspective, antibody microarray analysis could provide a tool for the development of improved diagnostics and intensified biomarker discovery for breast cancer patients.

SELDI-TOF MS is a mass spectrometric technique which has been extensively used for biomarker discovery. In this study, we show that in-source decay is an important source for the generation of additional spectral peaks with this technique, both for pure proteins and proteins in serum samples. Thus, SELDI-TOF MS could be used to gain sequence information from proteins, but the results also question the uncritical use of SELDI-TOF MS as a general method for the detection of biomarkers.

Breast cancer is a heterogenous disease and it is of importance to select patients with regard to different prognosis and treatment sensitivity to individualize treatment regimes. In this study we successfully adapted a protein extraction protocol from mRNA extracted tumor samples enabling two-dimensional gel electrophoresis (2-DE) analysis of samples previously analyzed by cDNA microarray. The aim was to find candidate proteins that distinguish breast cancer patients with or without recurrences after adjuvant CMF (cyclophosphamide, methotrexate and 5-FU) treatment within four years to follow-up. We identified several proteins distinguishing the recurrence group from the non-recurrence group, especially in the ER and PgR positive subgroup (n = 7). The induced proteins were involved in translation/folding, iron ion binding, and protease inhibition, whereas proteins involved in signaling, ubiquitination, and splicing were decreased in expression. These results show that it is possible to use 2-DE to separate high abundant proteins in breast cancer tissue and to find discriminating proteins to identify patients with different prognosis after adjuvant CMF treatment.

A large proportion of breast cancer patients are treated with adjuvant chemotherapy after the primary operation, but some will recur in spite of this treatment. In order to achieve an improved and more individualised therapy, our knowledge in mechanisms for drug resistance needs to be increased. We have investigated to what extent cDNA microarray measurements could distinguish the likelihood of recurrences after adjuvant CMF (cyclophosphamide, methotrexate and 5-fluorouracil) treatment of premenopausal, lymph node positive breast cancer patients, and have also compared this with the corresponding performance when using conventional clinical variables. We tried several gene selection strategies, and built classifiers using the resulting gene lists. The best performing classifier with odds ratio (OR) = 6.5 (95% confidence interval (CI) = 1.4-62) did not outperform corresponding classifiers based on clinical variables. For the clinical variables, calibrated on the samples, either using all the clinical parameters or the Nottingham Prognostic Index (NPI) parameters, the areas under the receiver operating characteristics (ROC) curve were 0.78 and 0.79, respectively. The ORs at 90% sensitivity were 15 (95% CI = 3.1-140) and 10 (95% CI = 2.1-97), respectively. Our data have provided evidence for a comparable prediction of clinical outcome in CMF-treated breast cancer patients using conventional clinical variables and gene expression based markers. (c) 2006 Elsevier Ltd. All rights reserved.

The rate of ipsilateral local recurrence after ductal carcinoma in situ (DCIS) varies (between 5% and 30%) and depends on the type of operation (mastectomy vs. breast-conserving operation), and whether postoperative radiotherapy has been used. Ipsilateral local recurrence can either emanate from the primary lesion or be a new primary tumour. Contralateral lesions may also develop after DCIS. We compared histopathological and cell biological characteristics in 37 subsequent ipsilateral tumours (25 DCIS and 12 invasive cancers) and 13 subsequent contralateral invasive breast cancers with their corresponding primary DCIS. The histopathological parameters were re-evaluated and the cell biological factors were analysed using conventional immunohistochemical techniques in paraffin-embedded material. The concordance rate for high grade (nuclear grade 3) vs. non-high grade (nuclear grades 1+2) between the primary DCIS and the subsequent ipsilateral tumour was higher than between the primary DCIS and the subsequent contralateral invasive cancer (68% vs. 31%). Similar patterns in the concordance rates between the primary DCIS and ipsilateral vs. contralateral tumours were also found in the oestrogen receptor status (83% vs. 50%) and the progesterone receptor status (87% vs. 58%). The pattern persisted in the other factors examined (p53, c-erbB2, bcl-2 and Ki67), although it was less pronounced. The overall high rate of concordance in the characteristics between the primary DCIS and the subsequent ipsilateral tumours suggests that, in most cases, they represent true local recurrences. Subsequent contralateral tumours are more likely to be new primary cancers.

We compared the power of gene expression measurements with that of conventional prognostic markers, i.e., clinical, histopathological, and cell biological parameters, for predicting distant metastases in breast cancer patients using both established prognostic indices (e.g., the Nottingham Prognostic Index (NPI)) and novel combinations of conventional markers. We used publicly available data on 97 patients, and the performance of metastasis prediction was represented by receiver operating characteristic (ROC) areas and Kaplan-Meier plots. The gene expression profiler did not perform noticeably better than indices constructed from the clinical variables, e.g., the well established NPI. When analysing separately subgroups, according to the oestrogen receptor (ER) status both approaches could predict clinical outcome more easily for the ER-positive than for the ER-negative cohort. Given the time it may take before microarray processing is used worldwide, particularly due to the costs and the lack of standards, it is important to pursue research using conventional markers. Our analysis suggests that it might be possible to improve the combination of different conventional prognostic markers into one prognostic index.

We utilized the high-throughput tissue microarray method to characterize immunohistochemical expression patterns with correlations to prognosis in rectal cancer. Immunostaining for the markers Ki-67, Bcl-2, p53, EGFR, E-cadherin, beta-catenin, MLH1 and MSH2 was performed in 269 rectal cancers. Expression profiles were correlated to metastasis-free survival. Immunostaining revealed frequent upregulation and/or aberrant staining patterns for several of the markers, but Ki-67, p53, Bcl-2 and EGFR did not show any correlation to prognosis. However, reduced membranous staining for beta-catenin (p = 0.04), lack of cytoplasmic staining for beta-catenin (p = 0.04), reduced membranous staining for E-cadherin (p = 0.02) and lack of cytoplasmic staining for E-cadherin (p = 0.02) correlated with metastatic disease. Multivariate analysis including the factors Dukes' stage and tumor differentiation grade demonstrated increased risk of metastatic disease in tumors with lack of cytoplasmic staining for beta-catenin (HR = 3.1, p = 0.02), reduced membranous staining for beta-catenin (HR = 1.7, p = 0.06) and reduced membranous staining for E-cadherin (HR = 2.1, p = 0.06). Loss of MMR protein expression was confirmed to be a rare event in rectal cancer with loss of MLH1 staining in 3% and MSH2 in 1% of the tumors. The lack of prognostic information contributed by most of these markers suggests that single markers for prognosis may be of limited value in rectal cancer. However, altered expression of beta-catenin and E-cadherin correlated with metastatic disease, and these markers may have prognostic importance in rectal cancer.

The urokinase plasminogen activator (uPA) system is involved in tumor growth and metastasis. We assayed the components of the uPA system in homogenates of 64 primary epithelial ovarian tumors and 5 metastases and evaluated the association of these parameters to prognosis in the 51 malignant cases. The levels of uPA, PAI-2 and the uPA:PAI-1 complex increased with progressive loss of histological differentiation (p(trend)