Breast cancer hormone receptor levels and benefit from adjuvant tamoxifen in a randomized trial with long-term follow-up.

BACKGROUND: Hormone receptor positivity predicts benefit from endocrine therapy but the knowledge about the long-term survival of patients with different tumor receptor levels is limited. In this study, we describe the 25 years outcome of tamoxifen (TAM) treated patients. PATIENTS AND METHODS: Between 1983 and 1992, a total of 4,610 postmenopausal patients with early-stage breast cancer were randomized to receive totally 2 or 5 years of TAM therapy. After 2 years, 4,124 were alive and free of breast cancer recurrence. Among these, 2,481 had demonstrated estrogen receptor positive (ER+) disease. From 1988, the Abbot enzyme immunoassay became available and provided quantitative receptor levels for 1,210 patients, for which our analyses were done. RESULTS: After 5 years of follow-up, when all TAM treatment was finished, until 15 years of follow-up, breast cancer mortality for patients with ER+ disease was significantly reduced in the 5-year group as compared with the 2-year group (hazard ratios [HR] 0.67, 95% confidence intervals [CI] 0.55-0.83, p < 0.001). After 15 years, the difference between the groups remained but did not increase further. A substantial benefit from prolonged TAM therapy was only observed for the subgroup of patients with ER levels below the median (HR = 0.62, 95% CI 0.46-0.84, p = 0.002). Similarly, patients with progesterone receptor negative (PR-) disease did benefit from prolonged TAM treatment. For patients with progesterone receptor positive (PR+) disease, there was no statistically significant benefit from more than 2 years of TAM.  Interpretation: As compared with 2 years of adjuvant TAM, 5 years significantly prolonged breast cancer-specific survival. The benefit from prolonged TAM therapy was statistically significant for patients with ER levels below median or PR-negative disease. There was no evident benefit from prolonged TAM for patients with high ER levels or with PR+ tumors.

Hormone receptor mRNA and protein levels as predictors of premenopausal tamoxifen benefit.

BACKGROUND AND PURPOSE: Tamoxifen remains an important adjuvant treatment in premenopausal patients with hormone receptor-positive breast cancer. Thus, determination of hormone receptors is important. Here, we compare cytosol-based methods, immunohistochemistry (IHC), and gene expression (GEX) analysis for determining hormone receptor status in premenopausal breast cancer patients from a randomised tamoxifen trial, to evaluate their performance in identifying patients that benefit from tamoxifen. PATIENTS AND METHODS: Premenopausal patients (n=564) were randomised to 2 years of tamoxifen or no systemic treatment. Estrogen receptor (ER) and progesterone receptor (PR) status by protein expression measured by cytosol-based methods and IHC, and mRNA by GEX analysis were compared in 313 patients with available data from all methods. Kaplan Meier estimates and Cox regression were used to evaluate the treatment-predictive value for recurrence-free interval (RFi) and overall survival (OS). Median follow-up for event-free patients was 26 (RFi) and 33 (OS) years. RESULTS: The mRNA data of ESR1 and PGR distributed bimodally, patterns confirmed in an independent cohort. Kappa-values between all methods were 0.76 and 0.79 for ER and PR, respectively. Tamoxifen improved RFi in patients with ER-positive (ER+) or PR-positive (PR+) tumours (Hazard Ratio [HR] and 95% confidence interval [CI]), cytosol-ER+ 0.53 [0.36-0.79]; IHC-ER+ 0.55 [0.38-0.79]; GEX-ER+ 0.54 [0.37-0.77]; cytosol-PR+ 0.49 [0.34-0.72]; IHC-PR+ 0.58 [0.40-0.85]; GEX-PR+ 0.55 [0.38-0.80]). Results were similar for OS. INTERPRETATION: These methods can all identify patients that benefit from 2 years of tamoxifen with equal performance, indicating that GEX data might be used to guide adjuvant tamoxifen therapy.

Tamoxifen-predictive value of gene expression signatures in premenopausal breast cancer: data from the randomized SBII:2 trial.

BACKGROUND: Gene expression (GEX) signatures in breast cancer provide prognostic information, but little is known about their predictive value for tamoxifen treatment. We examined the tamoxifen-predictive value and prognostic effects of different GEX signatures in premenopausal women with early breast cancer. METHODS: RNA from formalin-fixed paraffin-embedded tumor tissue from premenopausal women randomized between two years of tamoxifen treatment and no systemic treatment was extracted and successfully subjected to GEX profiling (n = 437, NanoString Breast Cancer 360™ panel). The median follow-up periods for a recurrence-free interval (RFi) and overall survival (OS) were 28 and 33 years, respectively. Associations between GEX signatures and tamoxifen effect were assessed in patients with estrogen receptor-positive/human epidermal growth factor receptor 2-negative (ER+ /HER2-) tumors using Kaplan-Meier estimates and Cox regression. The prognostic effects of GEX signatures were studied in the entire cohort. False discovery rate adjustments (q-values) were applied to account for multiple hypothesis testing. RESULTS: In patients with ER+/HER2- tumors, FOXA1 expression below the median was associated with an improved effect of tamoxifen after 10 years with regard to RFi (hazard ratio [HR]FOXA1(high) = 1.04, 95% CI = 0.61-1.76, HRFOXA1(low) = 0.30, 95% CI = 0.14-0.67, qinteraction = 0.0013), and a resembling trend was observed for AR (HRAR(high) = 1.15, 95% CI = 0.60-2.20, HRAR(low) = 0.42, 95% CI = 0.24-0.75, qinteraction = 0.87). Similar patterns were observed for OS. Tamoxifen was in the same subgroup most beneficial for RFi in patients with low ESR1 expression (HRRFi ESR1(high) = 0.76, 95% CI = 0.43-1.35, HRRFi, ESR1(low) = 0.56, 95% CI = 0.29-1.06, qinteraction = 0.37). Irrespective of molecular subtype, higher levels of ESR1, Mast cells, and PGR on a continuous scale were correlated with improved 10 years RFi (HRESR1 = 0.80, 95% CI = 0.69-0.92, q = 0.005; HRMast cells = 0.74, 95% CI = 0.65-0.85, q < 0.0001; and HRPGR = 0.78, 95% CI = 0.68-0.89, q = 0.002). For BC proliferation and Hypoxia, higher scores associated with worse outcomes (HRBCproliferation = 1.54, 95% CI = 1.33-1.79, q < 0.0001; HRHypoxia = 1.38, 95% CI = 1.20-1.58, q < 0.0001). The results were similar for OS. CONCLUSIONS: Expression of FOXA1 is a promising predictive biomarker for tamoxifen effect in ER+/HER2- premenopausal breast cancer. In addition, each of the signatures BC proliferation, Hypoxia, Mast cells, and the GEX of AR, ESR1, and PGR had prognostic value, also after adjusting for established prognostic factors. Trial registration This trial was retrospectively registered in the ISRCTN database the 6th of December 2019, trial ID: https://clinicaltrials.gov/ct2/show/ISRCTN12474687 .

Breast cancer hypoxia in relation to prognosis and benefit from radiotherapy after breast-conserving surgery in a large, randomised trial with long-term follow-up.

BACKGROUND: Breast-conserving surgery followed by radiotherapy is part of standard treatment for early-stage breast cancer. Hypoxia is common in cancer and may affect the benefit of radiotherapy. Cells adapt to hypoxic stress largely via the transcriptional activity of hypoxia-inducible factor (HIF)-1α. Here, we aim to determine whether tumour HIF-1α-positivity and hypoxic gene-expression signatures associated with the benefit of radiotherapy, and outcome. METHODS: Tumour HIF-1α-status and expression of hypoxic gene signatures were retrospectively analysed in a clinical trial where 1178 women with primary T1-2N0M0 breast cancer were randomised to receive postoperative radiotherapy or not and followed 15 years for recurrence and 20 years for breast cancer death. RESULTS: The benefit from radiotherapy was similar in patients with HIF-1α-positive and -negative primary tumours. Both ipsilateral and any breast cancer recurrence were more frequent in women with HIF-1α-positive primary tumours (hazard ratio, HR0-5 yrs1.9 [1.3-2.9], p = 0.003 and HR0-5 yrs = 2.0 [1.5-2.8], p < 0.0001). Tumour HIF-1α-positivity is also associated with increased breast cancer death (HR0-10 years 1.9 [1.2-2.9], p = 0.004). Ten of the 11 investigated hypoxic gene signatures correlated positively to HIF-1α-positivity, and 5 to increased rate/risk of recurrence. CONCLUSIONS: The benefit of postoperative radiotherapy persisted in patients with hypoxic primary tumours. Patients with hypoxic primary breast tumours had an increased risk of recurrence and breast cancer death.

Prognostic and predictive impact of stroma cells defined by PDGFRb expression in early breast cancer: results from the randomized SweBCG91RT trial.

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 < 0.001) groups but not the PDGFRb high group (0.64, 0.36-1.11, 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.

Tumour-infiltrating lymphocytes as a prognostic and tamoxifen predictive marker in premenopausal breast cancer: data from a randomised trial with long-term follow-up.

BACKGROUND: Tumour-infiltrating lymphocytes (TILs) are of important prognostic and predictive value in human epidermal growth factor receptor 2-positive (HER2+) breast cancer (BC) and triple-negative breast cancer (TNBC), but their clinical relevance in oestrogen receptor-positive/HER2-negative (ER+/HER2-) remains unknown. The primary study aim was to analyse the prognostic effect of TILs on the BC-free interval (BCFi) in premenopausal patients stratified by BC subtypes. The secondary aim was to investigate if TILs are predictive of tamoxifen (TAM) benefit. METHODS: Archival tissues from primary breast tumours were collected from patients from the SBII:2pre trial, in which 564 premenopausal women were randomised to 2 years of adjuvant TAM or no systemic treatment, regardless of hormone receptor status. TILs were scored on whole tissue sections from 447 patients with available ER status. Tumours were divided into ER+/HER2-, HER2+ and TNBC subtypes by immunohistochemistry and in situ hybridisation. The prognostic value of TILs was analysed in systemically untreated patients (n = 221); the predictive information was investigated in the ER+ subgroup (n = 321) by cumulative incidence curves and Cox regression analyses. The median follow-up was 28 years. RESULTS: High (≥ 50%) infiltration of TILs was a favourable prognostic factor in terms of BCFi (univariable analysis: hazard ratioBCFi (HRBCFi) 0.40; 95% confidence interval (CI) 0.22-0.71; P = 0.002). Similar effects were observed across all BC subtypes. The effect of adjuvant TAM was stronger in patients with ER+ tumours and TILs < 50% (HRBCFi 0.63; 95% CI 0.47-0.84; P = 0.002) than in patients with high immune infiltration (≥ 50%) (HRBCFi 0.84; 95% CI (0.24-2.86); P = 0.77). However, evidence for differential effects of TAM in categories of TILs, i.e. interaction, was weak. CONCLUSIONS: We demonstrate a long-term favourable prognostic value of high infiltration of TILs in a cohort of premenopausal BC patients and the positive prognostic effect was extended to the ER+/HER2- subgroup. A beneficial effect of TAM in ER+ patients was observed in patients with tumours of low TIL infiltration, but evidence for a treatment predictive effect was weak. TRIAL REGISTRATION: This trial is registered in the ISRCTN database, trial ID: ISRCTN12474687 .

Expression of epithelial-mesenchymal transition-related markers and phenotypes during breast cancer progression.

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.

Refinement of breast cancer molecular classification by miRNA expression profiles.

BACKGROUND: Accurate classification of breast cancer using gene expression profiles has contributed to a better understanding of the biological mechanisms behind the disease and has paved the way for better prognostication and treatment prediction. RESULTS: We found that miRNA profiles largely recapitulate intrinsic subtypes. In the case of HER2-enriched tumors a small set of miRNAs including the HER2-encoded mir-4728 identifies the group with very high specificity. We also identified differential expression of the miR-99a/let-7c/miR-125b miRNA cluster as a marker for separation of the Luminal A and B subtypes. High expression of this miRNA cluster is linked to better overall survival among patients with Luminal A tumors. Correlation between the miRNA cluster and their precursor LINC00478 is highly significant suggesting that its expression could help improve the accuracy of present day's signatures. CONCLUSIONS: We show here that miRNA expression can be translated into mRNA profiles and that the inclusion of miRNA information facilitates the molecular diagnosis of specific subtypes, in particular the clinically relevant sub-classification of luminal tumors.

The estrogen receptor coactivator AIB1 is a new putative prognostic biomarker in ER-positive/HER2-negative invasive lobular carcinoma of the breast.

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.

Agreement between molecular subtyping and surrogate subtype classification: a contemporary population-based study of ER-positive/HER2-negative primary breast cancer.

PURPOSE: Oestrogen receptor-positive (ER+) and human epidermal receptor 2-negative (HER2-) breast cancers are classified as Luminal A or B based on gene expression, but immunohistochemical markers are used for surrogate subtyping. The aims of this study were to examine the agreement between molecular subtyping (MS) and surrogate subtyping and to identify subgroups consisting mainly of Luminal A or B tumours. METHODS: The cohort consisted of 2063 patients diagnosed between 2013-2017, with primary ER+/HER2- breast cancer, analysed by RNA sequencing. Surrogate subtyping was performed according to three algorithms (St. Gallen 2013, Maisonneuve and our proposed Grade-based classification). Agreement (%) and kappa statistics (κ) were used as concordance measures and ROC analysis for luminal distinction. Ki67, progesterone receptor (PR) and histological grade (HG) were further investigated as surrogate markers. RESULTS: The agreement rates between the MS and St. Gallen 2013, Maisonneuve and Grade-based classifications were 62% (κ = 0.30), 66% (κ = 0.35) and 70% (κ = 0.41), respectively. PR did not contribute to distinguishing Luminal A from B tumours (auROC = 0.56). By classifying HG1-2 tumours as Luminal A-like and HG3 as Luminal B-like, agreement with MS was 80% (κ = 0.46). Moreover, by combining HG and Ki67 status, a large subgroup of patients (51% of the cohort) having > 90% Luminal A tumours could be identified. CONCLUSIONS: Agreement between MS and surrogate classifications was generally poor. However, a post hoc analysis showed that a combination of HG and Ki67 could identify patients very likely to have Luminal A tumours according to MS.

Identification and validation of single-sample breast cancer radiosensitivity gene expression predictors.

BACKGROUND: Adjuvant radiotherapy is the standard of care after breast-conserving surgery for primary breast cancer, despite a majority of patients being over- or under-treated. In contrast to adjuvant endocrine therapy and chemotherapy, no diagnostic tests are in clinical use that can stratify patients for adjuvant radiotherapy. This study presents the development and validation of a targeted gene expression assay to predict the risk of ipsilateral breast tumor recurrence and response to adjuvant radiotherapy after breast-conserving surgery in primary breast cancer. METHODS: Fresh-frozen primary tumors from 336 patients radically (clear margins) operated on with breast-conserving surgery with or without radiotherapy were collected. Patients were split into a discovery cohort (N = 172) and a validation cohort (N = 164). Genes predicting ipsilateral breast tumor recurrence in an Illumina HT12 v4 whole transcriptome analysis were combined with genes identified in the literature (248 genes in total) to develop a targeted radiosensitivity assay on the Nanostring nCounter platform. Single-sample predictors for ipsilateral breast tumor recurrence based on a k-top scoring pairs algorithm were trained, stratified for estrogen receptor (ER) status and radiotherapy. Two previously published profiles, the radiosensitivity signature of Speers et al., and the 10-gene signature of Eschrich et al., were also included in the targeted panel. RESULTS: Derived single-sample predictors were prognostic for ipsilateral breast tumor recurrence in radiotherapy-treated ER+ patients (AUC 0.67, p = 0.01), ER+ patients without radiotherapy (AUC = 0.89, p = 0.02), and radiotherapy-treated ER- patients (AUC = 0.78, p < 0.001). Among ER+ patients, radiotherapy had an excellent effect on tumors classified as radiosensitive (p < 0.001), while radiotherapy had no effect on tumors classified as radioresistant (p = 0.36) and there was a high risk of ipsilateral breast tumor recurrence (55% at 10 years). Our single-sample predictors developed in ER+ tumors and the radiosensitivity signature correlated with proliferation, while single-sample predictors developed in ER- tumors correlated with immune response. The 10-gene signature negatively correlated with both proliferation and immune response. CONCLUSIONS: Our targeted single-sample predictors were prognostic for ipsilateral breast tumor recurrence and have the potential to stratify patients for adjuvant radiotherapy. The correlation of models with biology may explain the different performance in subgroups of breast cancer.

Assessment of early response biomarkers in relation to long-term survival in patients with HER2-negative breast cancer receiving neoadjuvant chemotherapy plus bevacizumab: Results from the Phase II PROMIX trial.

Pathologic complete response (pCR) is a predictor for favorable outcome after neoadjuvant treatment in early breast cancer. Modulation of gene expression may also provide early readouts of biological activity and prognosis, offering the possibility for timely response-guided treatment adjustment. The role of early transcriptional changes in predicting response to neoadjuvant chemotherapy plus bevacizumab was investigated. One-hundred-and-fifty patients with large, operable and locally advanced HER2-negative breast cancer received epirubicin and docetaxel, with the addition of bevacizumab. Patients underwent tumor biopsies at baseline, after Cycle 2 and at the time of surgery. The primary end point, pCR, and its relation with the secondary endpoints event-free survival (EFS), overall survival (OS) and gene expression profiles, are reported. The pCR rate was 13% (95% CI 8.6-20.2), with significantly more pCRs among triple-negative [28% (95% CI 14.8-45.4)] than among hormone receptor positive (HR+) tumors [9% (95% CI 4.6-16.3); (OR = 3.9 [CI = 1.5-10.3])]. pCR rates were not associated with EFS or OS. PAM50 subtypes significantly changed after Cycle 2 (p = 0.03) and an index of absolute changes in PAM50 correlations between these time-points was associated with EFS [HR = 0.62 (CI = 0.3-1.1)]. In univariable analyses, signatures for angiogenesis, proliferation, estrogen receptor signaling, invasion and metastasis, and immune response, measured after Cycle 2, were associated with pCR in HR+ tumors. Evaluation of changes in molecular subtypes and other signatures early in the course of neoadjuvant treatment may be predictive of pCR and EFS. These factors may help guide further treatment and should be considered when designing neoadjuvant trials.

Immune gene expression and response to chemotherapy in advanced breast cancer.

BACKGROUND: Transcriptomic profiles have shown promise as predictors of response to neoadjuvant chemotherapy in breast cancer (BC). This study aimed to explore their predictive value in the advanced BC (ABC) setting. METHODS: In a Phase 3 trial of first-line chemotherapy in ABC, a fine needle aspiration biopsy (FNAB) was obtained at baseline. Intrinsic molecular subtypes and gene modules related to immune response, proliferation, oestrogen receptor (ER) signalling and recurring genetic alterations were analysed for association with objective response to chemotherapy. Gene-set enrichment analysis (GSEA) of responders vs non-responders was performed independently. Lymphocytes were enumerated in FNAB smears and the absolute abundance of immune cell types was calculated using the Microenvironment Cell Populations counter method. RESULTS: Gene expression data were available for 109 patients. Objective response to chemotherapy was statistically significantly associated with an immune module score (odds ratio (OR)=1.62; 95% confidence interval (CI), 1.03-2.64; P=0.04). Subgroup analysis showed that this association was restricted to patients with ER-positive or luminal tumours (OR=3.54; 95%, 1.43-10.86; P=0.012 and P for interaction=0.04). Gene-set enrichment analysis confirmed that in these subgroups, immune-related gene sets were enriched in responders. CONCLUSIONS: Immune-related transcriptional signatures may predict response to chemotherapy in ER-positive and luminal ABC.

Non-linear transformations of age at diagnosis, tumor size, and number of positive lymph nodes in prediction of clinical outcome in breast cancer.

BACKGROUND: Prognostic factors in breast cancer are often measured on a continuous scale, but categorized for clinical decision-making. The primary aim of this study was to evaluate if accounting for continuous non-linear effects of the three factors age at diagnosis, tumor size, and number of positive lymph nodes improves prognostication. These factors will most likely be included in the management of breast cancer patients also in the future, after an expected implementation of gene expression profiling for adjuvant treatment decision-making. METHODS: Four thousand four hundred forty seven and 1132 women with primary breast cancer constituted the derivation and validation set, respectively. Potential non-linear effects on the log hazard of distant recurrences of the three factors were evaluated during 10 years of follow-up. Cox-models of successively increasing complexity: dichotomized predictors, predictors categorized into three or four groups, and predictors transformed using fractional polynomials (FPs) or restricted cubic splines (RCS), were used. Predictive performance was evaluated by Harrell's C-index. RESULTS: Using FP-transformations, non-linear effects were detected for tumor size and number of positive lymph nodes in univariable analyses. For age, non-linear transformations did, however, not improve the model fit significantly compared to the linear identity transformation. As expected, the C-index increased with increasing model complexity for multivariable models including the three factors. By allowing more than one cut-point per factor, the C-index increased from 0.628 to 0.674. The additional gain, as measured by the C-index, when using FP- or RCS-transformations was modest (0.695 and 0.696, respectively). The corresponding C-indices for these four models in the validation set, based on the same transformations and parameter estimates from the derivation set, were 0.675, 0.700, 0.706, and 0.701. CONCLUSIONS: Categorization of each factor into three to four groups was found to improve prognostication compared to dichotomization. The additional gain by allowing continuous non-linear effects modeled by FPs or RCS was modest. However, the continuous nature of these transformations has the advantage of making it possible to form risk groups of any size.

Histological grade provides significant prognostic information in addition to breast cancer subtypes defined according to St Gallen 2013.

BACKGROUND: The St Gallen surrogate definition of the intrinsic subtypes of breast cancer consist of five subgroups based on estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor type 2 (HER2), and Ki-67. PgR and Ki-67 are used for discriminating between the 'Luminal A-like' and 'Luminal B-like (HER2-negative)' subtypes. Histological grade (G) has prognostic value in breast cancer; however, its relationship to the St Gallen subtypes is not clear. Based on a previous pilot study, we hypothesized that G could be a primary discriminator for ER-positive/HER2-negative breast cancers that were G1 or G3, whereas Ki-67 and PgR could provide additional prognostic information specifically for patients with G2 tumors. To test this hypothesis, a larger patient cohort was examined. PATIENTS AND METHODS: Six hundred seventy-one patients (≥35 years of age, pT1-2, pN0-1) with ER-positive/HER2-negative breast cancer and complete data for PgR, Ki-67, G, lymph node status, tumor size, age, and distant disease-free survival (DDFS; median follow-up 9.2 years) were included. RESULTS: 'Luminal A-like' tumors were mostly G1 or G2 (90%) whereas 'Luminal B-like' tumors were mostly G2 or G3 (87%) and corresponded with good and poor DDFS, respectively. In 'Luminal B-like' tumors that were G1 (n = 23), no metastasis occurred, whereas 14 of 40 'Luminal A-like' tumors that were G3 metastasized. In the G2 subgroup, low PgR and high Ki-67 were associated with an increased risk of distant metastases, hazard ratio (HR) and 95% confidence interval (CI) 1.8 (0.95-3.4) and 1.5 (0.80-2.8), respectively. CONCLUSIONS: Patients with ER-positive/HER2-negative/G1 breast cancer have a good prognosis, similar to that of 'Luminal A-like', while those with ER-positive/HER2-negative/G3 breast cancer have a worse prognosis, similar to that of 'Luminal B-like', when assessed independently of PgR and Ki-67. Therapy decisions based on Ki-67 and PgR might thus be restricted to the subgroup G2.

Contralateral breast cancer can represent a metastatic spread of the first primary tumor: determination of clonal relationship between contralateral breast cancers using next-generation whole genome sequencing.

INTRODUCTION: By convention, a contralateral breast cancer (CBC) is treated as a new primary tumor, independent of the first cancer (BC1). Although there have been indications that the second tumor (BC2) sometimes may represent a metastatic spread of BC1, this has never been conclusively shown. We sought to apply next-generation sequencing to determine a "genetic barcode" for each tumor and reveal the clonal relationship of CBCs. METHODS: Ten CBC patients with detailed clinical information and available fresh frozen tumor tissue were studied. Using low-coverage whole genome DNA-sequencing data for each tumor, chromosomal rearrangements were enumerated and copy number profiles were generated. Comparisons between tumors provided an estimate of clonal relatedness for tumor pairs within individual patients. RESULTS: Between 15-256 rearrangements were detected in each tumor (median 87). For one patient, 76 % (68 out of 90) of the rearrangements were shared between BC1 and BC2, highly consistent with what has been seen for true primary-metastasis pairs (>50 %) and thus confirming a common clonal origin of the two tumors. For most of the remaining cases, BC1 and BC2 had similarly low overlap as unmatched randomized pairs of tumors from different individuals, suggesting the CBC to represent a new independent primary tumor. CONCLUSION: Using rearrangement fingerprinting, we show for the first time with certainty that a contralateral BC2 can represent a metastatic spread of BC1. Given the poor prognosis of a generalized disease compared to a new primary tumor, these women need to be identified at diagnosis of CBC for appropriate determination of treatment. Our approach generates a promising new method to assess clonal relationship between tumors. Additional studies are required to confirm the frequency of CBCs representing metastatic events.

Highly reproducible results of breast cancer biomarkers when analysed in accordance with national guidelines - a Swedish survey with central re-assessment.

BACKGROUND: Biomarkers are crucial for decisions regarding adjuvant therapy in primary breast cancer, and their correct assessment is therefore of the utmost importance. AIMS: To investigate the concordance between Swedish pathology departments and a reference laboratory, for routine analysis of oestrogen receptor (ER), progesterone receptor (PR), Ki67, and human epidermal growth factor receptor 2 (HER2), alone, and in combination (St Gallen subtypes). METHODS: This survey included 27 of the 28 pathology laboratories in Sweden, covering 98% of cases of primary breast cancer surgery in Sweden. Paraffin-embedded tumour blocks (n = 270) were collected and sent to the central reference laboratory, together with the originally stained slides, for re-analysis. The primary evaluations were previously performed according to national Swedish guidelines, without any knowledge of the subsequent central assessment. RESULTS: The agreement for ER, PR, and Ki67 was 99% [kappa value (κ) = 0.95], 95% (κ = 0.85), and 85% (κ = 0.70), respectively. The agreement for HER2 (0/1 + vs. 2+/3+) was 85% (κ = 0.64), but when equivocal tumours were further analysed with in situ hybridisation, only one discrepancy was observed. Discrepancies between results for ER and PR seem to be explained by analytical differences, whereas the interpretation of staining seems to be more critical for Ki67 and HER2 immunohistochemistry. The agreement between the results from the Swedish laboratories and the reference laboratory, based on the St Gallen subtypes, was 88% (κ = 0.81). CONCLUSIONS: When applying national guidelines, highly reproducible results were obtained in routine assessment of breast cancer biomarkers, and the results of this study confirm the clinical utility of these markers for decisions regarding the treatment of primary breast cancer.

Grading breast cancer tissues using molecular portraits.

Tumor progression and prognosis in breast cancer patients are difficult to assess using current clinical and laboratory parameters, where a pathological grading is indicative of tumor aggressiveness. This grading is based on assessments of nuclear grade, tubule formation, and mitotic rate. We report here the first protein signatures associated with histological grades of breast cancer, determined using a novel affinity proteomics approach. We profiled 52 breast cancer tissue samples by combining nine antibodies and label-free LC-MS/MS, which generated detailed quantified proteomic maps representing 1,388 proteins. The results showed that we could define in-depth molecular portraits of histologically graded breast cancer tumors. Consequently, a 49-plex candidate tissue protein signature was defined that discriminated between histological grades 1, 2, and 3 of breast cancer tumors with high accuracy. Highly biologically relevant proteins were identified, and the differentially expressed proteins indicated further support for the current hypothesis regarding remodeling of the tumor microenvironment during tumor progression. The protein signature was corroborated using meta-analysis of transcriptional profiling data from an independent patient cohort. In addition, the potential for using the markers to estimate the likelihood of long-term metastasis-free survival was also indicated. Taken together, these molecular portraits could pave the way for improved classification and prognostication of breast cancer.

Lack of G protein-coupled estrogen receptor (GPER) in the plasma membrane is associated with excellent long-term prognosis in breast cancer.

G protein-coupled estrogen receptor (GPER), or GPR30, is a membrane receptor reported to mediate non-genomic estrogen responses. Tamoxifen is a partial agonist at GPER in vitro. Here, we investigated if GPER expression is prognostic in primary breast cancer, if the receptor is treatment-predictive for adjuvant tamoxifen, and if receptor subcellular localization has any impact on the prognostic value. Total and plasma membrane (PM) GPER expression was analyzed by immunohistochemistry in breast tumors from 742 postmenopausal lymph node-negative patients subsequently randomized for tamoxifen treatment for 2-5 years versus no systemic treatment, regardless of estrogen receptor (ER) status, and with a median follow-up of 17 years for patients free of event. PM GPER expression was a strong independent prognostic factor for poor prognosis in breast cancer without treatment-predictive information for tamoxifen. In the tamoxifen-treated ER-positive and progesterone receptor (PgR)-positive patient subgroup, the absence of PM GPER (53 % of all ER-positive tumors) predicted 91 % 20-year distant disease-free survival, compared to 73 % in the presence of GPER (p = 0.001). Total GPER expression showed positive correlations with ER and PgR and negative correlation with histological grade, but the correlations were biphasic. On the other hand, PM GPER expression showed strong negative correlations with ER and PgR, and strong positive correlation with HER2 overexpression and high histological grade. GPER overexpression and PM localization are critical events in breast cancer progression, and lack of GPER in the PM is associated with excellent long-term prognosis in ER-positive and PgR-positive tamoxifen-treated primary breast cancer.

Toward a molecular pathologic classification of urothelial carcinoma.

We recently defined molecular subtypes of urothelial carcinomas according to whole genome gene expression. Herein we describe molecular pathologic characterization of the subtypes using 20 genes and IHC of 237 tumors. In addition to differences in expression levels, the subtypes show important differences in stratification of protein expression. The selected genes included biological features central to bladder cancer biology, eg, cell cycle activity, cellular architecture, cell-cell interactions, and key receptor tyrosine kinases. We show that the urobasal (Uro) A subtype shares features with normal urothelium such as keratin 5 (KRT5), P-cadherin (P-Cad), and epidermal growth factor receptor (EGFR) expression confined to basal cells, and cell cycle activity (CCNB1) restricted to the tumor-stroma interface. In contrast, the squamous cell cancer-like (SCCL) subtype uniformly expresses KRT5, P-Cad, EGFR, KRT14, and cell cycle genes throughout the tumor parenchyma. The genomically unstable subtype shows proliferation throughout the tumor parenchyma and high ERBB2 and E-Cad expression but absence of KRT5, P-Cad, and EGFR expression. UroB tumors demonstrate features shared by both UroA and SCCL subtypes. A major transition in tumor progression seems to be loss of dependency of stromal interaction for proliferation. We present a simple IHC/histology-based classifier that is easy to implement as a standard pathologic evaluation to differentiate the three major subtypes: urobasal, genomically unstable, and SCCL. These three major subtypes exhibit important prognostic differences.