The prognostic value of tumor-stroma ratio in tumor-positive axillary lymph nodes of breast cancer patients.

The tumor-stroma ratio (TSR) has previously been found to be a strong prognostic parameter in primary breast cancer tumors. Since the presence of tumor cells in lymph nodes is important for clinical decision making, the influence of TSR in the primary breast tumor combined with the TSR in tumor-positive lymph nodes on prognosis was evaluated. Women with invasive breast cancer without distant metastasis who underwent an axillary lymph node dissection between 1985 and 1994 at the Leiden University Medical Center were retrospectively analyzed. TSR assessment was performed on hematoxylin and eosin stained tissue slides. In total, 87 (45.5%) primary tumors were scored as stroma-low and 104 (54.5%) as stroma-high. Patients with a high stromal percentage in the primary tumors had a statistically significant worse relapse free period (RFP) compared to stroma-low tumors (HR 1.97, 95% CI 1.37-2.82, p < 0.001). A total number of 915 lymph nodes were assessed for TSR. In 101 (52.9%) patients, heterogeneity was observed between stroma percentage category in primary tumor and lymph nodes. The combination of TSR of the primary tumor combined with TSR of tumor-positive lymph nodes strengthened each other as independent prognostic parameter for RFP (p = 0.019). Patients with primary tumor stroma-low/lymph nodes stroma-low tumors showed strongly improved RFP rates compared to patients with primary tumor stroma-high/lymph node stroma-high tumors with 10-year percentages of 58 versus 8%, respectively. Assessing the TSR on tumor-positive lymph nodes can provide additional prognostic information. Stromal activation strongly differs between primary tumors and lymph node metastasis.

High nuclear expression levels of histone-modifying enzymes LSD1, HDAC2 and SIRT1 in tumor cells correlate with decreased survival and increased relapse in breast cancer patients.

BACKGROUND: Breast cancer is a heterogeneous disease with a highly variable clinical outcome in which both genetic and epigenetic changes have critical roles. We investigated tumor expression levels of histone-modifying enzymes LSD1, HDAC2 and SIRT1 in relation with patient survival and tumor relapse in a retrospective cohort of 460 breast cancer patients. Additionally, we correlated expression levels with tumor differentiation and tumor cell proliferation. METHODS: Immunohistochemical staining for LSD1, HDAC2 and SIRT1 was performed on tissue microarrays of tumor and corresponding normal formalin-fixed paraffin-embedded tissues from breast cancer patients. Median nuclear expression levels in tumor tissues were used to divide the patients into low and high expression categories. In combined expression analyses, patients were divided into four subgroups: 1, all enzymes below-median; 2, one enzyme above-median; 3, two enzymes above-median; 4, all three enzymes above-median. The Cox proportional hazard model was used for univariate and multivariate survival analyses. The Pearson Chi-square method was used to assess correlation of combined expression levels with tumor cell proliferation and tumor differentiation. RESULTS: Expression of LSD1 and SIRT1, but not of HDAC2, was significantly increased in tumor tissues compared to their normal counterparts (both p < 0.001). Multivariate survival analyses identified SIRT1 as independent prognostic factor for relapse-free survival (RFS) with a hazard ratio (HR) of 1.34 (95% CI = 1.04-1.74, p = 0.02). For overall survival (OS), no significant differences were found when the individual enzymes were analyzed. Analyses of combined expression levels of the three histone-modifying enzymes correlated with OS (p = 0.03) and RFS (p = 0.006) with a HR of respectively 1.49 (95% CI = 1.07-2.08) and 1.68 (95% CI = 1.16-2.44) in multivariate analyses and were also related to tumor differentiation (p < 0.001) and tumor cell proliferation (p = 0.002). CONCLUSIONS: When the combined expression levels were analyzed, high expression of LSD1, HDAC2 and SIRT1 showed shorter patient survival time and shorter time to tumor relapse and correlated with poor tumor differentiation and a high level of tumor cell proliferation. Expression of these histone-modifying enzymes might therefore be involved in breast cancer pathogenesis.

Comparison of frequencies and prognostic effect of molecular subtypes between young and elderly breast cancer patients.

PURPOSE: To compare the distribution and prognostic effect of the breast cancer molecular subtypes in young and elderly breast cancer patients. PATIENTS AND METHODS: Our study population (n = 822) consisted of all early breast cancer patients primarily treated with surgery in our center between 1985 and 1996. A total of 142/822 fresh frozen tissues were available with good quality RNA and analyzed by gene expression microarray. Gene expression molecular subtypes were determined by correlation to the expression centroids of 534 "intrinsic" genes. Sections of a tissue micro array containing formalin-fixed paraffin-embedded tumor tissue of 714/822 patients were immunohistochemically (IHC) stained for Ki67, EGFR, CK5/6. Tumor expression of ER, PR, HER2 was previously determined. IHC molecular subtypes were defined based on expression of these markers: Luminal A: ER+ and/or PR+, HER2- and Ki67-; Luminal B: ER+ and/or PR+ and ki67+; ERBB2: ER-, PR- and HER2+; Basal-like: ER-, PR-, HER2- and EGFR+ and/or CK5/6+; Unclassified: ER-, PR-, HER2-, EGFR- and CK5/6-. IHC molecular subtypes were validated against gene expression defined molecular subtypes. Assessment of distribution and prognostic effect of molecular subtypes was stratified to age (<65 versus ≥65 years). RESULTS: Validation of molecular subtypes determined by IHC against gene expression revealed a substantial agreement in classification (Cohen's kappa coefficient 0.75). A statistically significant association (p = 0.02) was found between molecular subtypes and age, where Luminal tumors were more often found in elderly patients, while ERBB2, basal-like and unclassified subtypes were more often found in young patients. Molecular subtypes showed a prognostic association with outcome in young patients concerning relapse-free period (RFP) (p = 0.01) and relative survival (RS) (p < 0.001). No statistically significant prognostic effect was found for molecular subtypes in elderly patients (RFP p = 0.5; RS p = 0.1). Additional analyses showed that no molecular subtypes showed a statistically significant difference in outcome for elderly compare to young patients. CONCLUSION: We have shown that molecular subtypes have a different distribution and prognostic effect in elderly compared to young breast cancer patients, emphasizing the fact that biomarkers may have different distributions and prognostic effects and therefore different implications in elderly compared to their younger counterparts. Our results support the premise that breast cancer clinical behavior is significantly affected by patient age. We suggest that competing risks of death in elderly patients, ER-driven differences and micro-environmental changes in biology are underlying these age-dependent variations in patient prognosis.

Tumor immune subtypes distinguish tumor subclasses with clinical implications in breast cancer patients.

There is strong evidence that the host's cellular immune response is linked to tumor progression, however its impact on patient outcome in breast cancer is poorly understood. The purpose of this study is to define tumor immune subtypes, focusing on cellular immune responses and investigate their prognostic effect in breast cancer patients. Our training (n = 440) and validation cohort (n = 382) consisted of all early breast cancer patients primarily treated with surgery in our center between 1985 and 1996. Tumor tissue sections were immunohistochemically stained for CD8 (CTL) and PEN5 (NK cells). Tumor expression of classical and non-classical human leukocyte antigen class I, and tumor-infiltrating Tregs were previously determined. Tumor immune subtypes were constructed based on quantification of these markers and biological rationale. High, intermediate, and low immune susceptible tumor immune subtypes were found, respectively, in 16, 63, and 20 % of patients in the training cohort and 16, 71, and 13 % in the validation cohort. The subtypes showed to be statistically significant prognostic in multivariate analyses for relapse free period (RFP) [p < 0.0001, intermediate versus high: hazard ratio (HR) 1.95; low versus high HR 2.98] and relative survival (RS) (p = 0.006, intermediate versus high HR 3.84; low versus high: HR 4.26). Validation of these outcome analyses confirmed the independent prognostic associations: RFP (p = 0.025) and RS (p = 0.040). The tumor immune subtypes that we present represent a prognostic profile with solid underlying biological rationale and with high discriminative power confirmed in an independent validation cohort. Our results emphasize the importance of tumor immune surveillance in the control of tumor development and, therefore, in determining patient prognosis. Tumor immune subtype profiling is promising for prognosis prediction and the achievement of tailored treatment for breast cancer patients.

The prognostic value of apoptotic and proliferative markers in breast cancer.

Increasing ability of early breast cancer (BC) diagnosis leading to more early stage detection, better survival, and low relapse marks one of the milestones achieved over the decades. Foregoing poses a challenge for clinicians regarding optimal treatment, in which over- and under-treatment should be avoided. Classical prognostic and predictive factors fall short for individualized adjuvant therapy selection in this patient group. The key to better characterization may be found in the biology underlying individual tumors. We hypothesized that markers related to cellular proliferation and apoptosis and the balance between these two processes in tumor development will be predictive for clinical outcome. Our study population (N = 822) consisted of all early stage BC patients primarily treated with surgery in our center between 1985 and 1996. Sections of available tumor tissue (87 %, 714/822) were immunohistochemically stained for expression of p53, active-caspase-3, and Ki67. In 43 % (304/714) and 18 % (126/714) of this cohort, respectively, a biochemical C2P(®) risk prediction and caspase-3 assay were performed. Expression data of the mentioned markers, single, or combined, were analyzed. Results showed that both the single and combined markers, whether of apoptotic or proliferative origin had associations with clinical outcome. An additive effect was seen for the hazard ratios when data on p53, active caspase-3, and Ki67 status were combined. The assembled prognostic apoptotic-proliferative subtype showed significant association for both the overall survival (p = 0.024) and relapse-free period (p = 0.001) in the multivariate analyses of grade I breast tumors. Combined markers of tumor cell apoptosis and proliferation represent tumor aggressiveness. The apoptotic-proliferative subtypes that we present in this study represent a clinical prognostic profile with solid underlying biological rationale and pose a promising method for accurate identification of grade I BC patients in need of an aggressive therapeutic approach, thus contributing to precision medicine in BC disease.

TRAF4 promotes TGF-ß receptor signaling and drives breast cancer metastasis.

TGF-ß signaling is a therapeutic target in advanced cancers. We identified tumor necrosis factor receptor-associated factor 4 (TRAF4) as a key component mediating pro-oncogenic TGF-ß-induced SMAD and non-SMAD signaling. Upon TGF-ß stimulation, TRAF4 is recruited to the active TGF-ß receptor complex, where it antagonizes E3 ligase SMURF2 and facilitates the recruitment of deubiquitinase USP15 to the TGF-ß type I receptor (TßRI). Both processes contribute to TßRI stabilization on the plasma membrane and thereby enhance TGF-ß signaling. In addition, the TGF-ß receptor-TRAF4 interaction triggers Lys 63-linked TRAF4 polyubiquitylation and subsequent activation of the TGF-ß-activated kinase (TAK)1. TRAF4 is required for efficient TGF-ß-induced migration, epithelial-to-mesenchymal transition, and breast cancer metastasis. Elevated TRAF4 expression correlated with increased levels of phosphorylated SMAD2 and phosphorylated TAK1 as well as poor prognosis among breast cancer patients. Our results demonstrate that TRAF4 can regulate the TGF-ß pathway and is a key determinant in breast cancer pathogenesis.

Alternatively spliced tissue factor promotes breast cancer growth in a ß1 integrin-dependent manner.

Full-length tissue factor (flTF), the coagulation initiator, is overexpressed in breast cancer (BrCa), but associations between flTF expression and clinical outcome remain controversial. It is currently not known whether the soluble alternatively spliced TF form (asTF) is expressed in BrCa or impacts BrCa progression. We are unique in reporting that asTF, but not flTF, strongly associates with both tumor size and grade, and induces BrCa cell proliferation by binding to ß1 integrins. asTF promotes oncogenic gene expression, anchorage-independent growth, and strongly up-regulates tumor expansion in a luminal BrCa model. In basal BrCa cells that constitutively express both TF isoforms, asTF blockade reduces tumor growth and proliferation in vivo. We propose that asTF plays a major role in BrCa progression acting as an autocrine factor that promotes tumor progression. Targeting asTF may comprise a previously unexplored therapeutic strategy in BrCa that stems tumor growth, yet does not impair normal hemostasis.

Hypomethylation of LINE-1 in primary tumor has poor prognosis in young breast cancer patients: a retrospective cohort study.

Long interspersed element 1 (LINE-1), a non-coding genomic repeat sequence, methylation status can influence tumor progression. In this study, the clinical significance of LINE-1 methylation status was assessed in primary breast cancer in young versus old breast cancer patients. LINE-1 methylation index (MI) was assessed by absolute quantitative assessment of methylated alleles (AQAMA) PCR assay. Initially, LINE-1 MI was assessed in a preliminary study of 235 tissues representing different stages of ductal breast cancer development. Next, an independent cohort of 379 primary ductal breast cancer patients (median follow-up 18.9 years) was studied. LINE-1 hypomethylation was shown to occur in DCIS and invasive breast cancer. In primary breast cancer it was associated with pathological tumor stage (p = 0.026), lymph node metastasis (p = 0.022), and higher age at diagnosis (>55, p < 0.001). In multivariate analysis, LINE-1 hypomethylation was associated with decreased OS (HR 2.19, 95 % CI 1.17-4.09, log-rank p = 0.014), DFS (HR 2.05, 95 % CI 1.14-3.67, log-rank p = 0.016) and increased DR (HR 2.83, 95 % CI 1.53-5.21, log-rank p = 0.001) in younger (≤55 years), but not older patients (>55 years). LINE-1 analysis of primary breast cancer demonstrated cancer-related age-dependent hypomethylation. In patients ≤55 years, LINE-1 hypomethylation portends a high-risk of DR.

NKG2D ligand tumor expression and association with clinical outcome in early breast cancer patients: an observational study.

BACKGROUND: Cell surface NKG2D ligands (NKG2DL) bind to the activating NKG2D receptor present on NK cells and subsets of T cells, thus playing a role in initiating an immune response. We examined tumor expression and prognostic effect of NKG2DL in breast cancer patients. METHODS: Our study population (n = 677) consisted of all breast cancer patients primarily treated with surgery in our center between 1985 and 1994. Formalin-fixed paraffin-embedded tumor tissue was immunohistochemically stained with antibodies directed against MIC-A/MIC-B (MIC-AB), ULBP-1, ULBP-2, ULBP-3, ULBP-4, and ULBP-5. RESULTS: NKG2DL were frequently expressed by tumors (MIC-AB, 50% of the cases; ULBP-1, 90%; ULBP-2, 99%; ULBP-3, 100%; ULBP-4, 26%; ULBP-5, 90%) and often showed co-expression: MIC-AB and ULBP-4 (p = 0.043), ULBP-1 and ULBP-5 (p = 0.006), ULBP-4 and ULBP-5 (p < 0.001). MIC-AB (p = 0.001) and ULBP-2 (p = 0.006) expression resulted in a statistically significant longer relapse free period (RFP). Combined expression of these ligands showed to be an independent prognostic parameter for RFP (p < 0.001, HR 0.41). Combined expression of all ligands showed no associations with clinical outcome. CONCLUSIONS: We demonstrated for the first time that NKG2DL are frequently expressed and often co-expressed in breast cancer. Expression of MIC-AB and ULBP-2 resulted in a statistically significant beneficial outcome concerning RFP with high discriminative power. Combination of all NKG2DL showed no additive or interactive effect of ligands on each other, suggesting that similar and co-operative functioning of all NKG2DL can not be assumed. Our observations suggest that among driving forces in breast cancer outcome are immune activation on one site and tumor immune escape on the other site.

Age determines the prognostic role of the cancer stem cell marker aldehyde dehydrogenase-1 in breast cancer.

BACKGROUND: The purpose of this study was to compare the expression and the prognostic effect of the breast cancer stem cell marker aldehyde dehydrogenase-1 (ALDH1) in young and elderly breast cancer patients. METHODS: The study population (N = 574) consisted of all early breast cancer patients primarily treated with surgery in our center between 1985 and 1994. Median follow-up was 17.9 years (range: 0.1 to 23.5). Tissue microarray slides were immunohistochemically stained for ALDH1 expression and quantified by two independent observers who were blinded to clinical outcome. Assessment of the prognostic effect of ALDH1 expression was stratified according to age and systemic treatment. RESULTS: Complete lack of expression of ALDH1 was found in 40% of tumors. With increasing age more tumors showed complete absence of ALDH1 expression (P < .001). In patients aged > 65 years, ALDH1 status was not associated with any clinical outcome. Conversely, in patients aged < 65 years, ALDH1 positivity was an independent risk factor of worse outcome for relapse free period (hazard ratio = 1.71 (95% CI, 1.09 to 2.68); P = .021) and relative survival (relative excess risks of death = 2.36 (95% CI, 1.22 to 3.68); P = .016). Ten-year relative survival risk was 57% in ALDH1-positive patients compared to 83% in ALDH1-negative patients. CONCLUSION: ALDH1 expression and its prognostic effect are age-dependent. Our results support the hypothesis that breast cancer biology is different in elderly patients compared to their younger counterparts and emphasizes the importance of taking into consideration age-specific interactions in breast cancer research.

Primary tumor classification according to methylation pattern is prognostic in patients with early stage ER-negative breast cancer.

Breast cancer patients with similar clinical stage may experience different disease outcomes. Aberrant DNA methylation of primary breast tumors can have impact on the clinical outcome. This study aimed to assess clinical utility of tumor-specific methylated sequences (MINT17, 31) and tumor-related gene (RARß2) methylation classification in primary breast tumors. Absolute quantitative assessment of methylated alleles (AQAMA) was used to determine the methylation index (MI) of MINT17, MINT31, and RARß2 in 242 primary tumors of early stage breast cancer patients. Patients were classified into three methylation groups: meth-N, with normal methylation levels of all biomarkers; meth-L, with one biomarker hypermethylation; and meth-H, with hypermethylation of >1 biomarker. Disease outcome of methylation groups was compared during follow-up. MI of all biomarkers was successfully obtained in 237 tumors of which 79 (33%) were classified as meth-N, 86 (36%) as meth-L, and 72 (30%) as meth-H. Meth-H status was a risk factor for distant recurrence (DR) (log-rank P = 0.007) and shorter disease-free survival (DFS) (log-rank P = 0.039). Methylation classification had strongest prognostic value for patients with ER-negative tumors. In multivariate analysis (n = 222), ER-negative meth-H patients had a 4.1-fold increased risk of DR (95% CI 1.80-9.59; meth-N HR 1.0, P = 0.001), a 4.2-fold increased risk of overall recurrence (OR) (95% CI 1.88-9.47; meth-N HR 1.0, P = 0.001), and a 3.1-fold shorter DFS (95% CI 1.57-5.98; meth-N HR 1.0, P = 0.003). Methylation classification of primary breast cancer is an independent prognostic factor for disease outcome in patients with ER-negative tumors. The study's findings will have to be confirmed in an independent dataset.

Co-expression of SNAIL and TWIST determines prognosis in estrogen receptor-positive early breast cancer patients.

Epithelial mesenchymal transition (EMT) plays an important role in the development of metastases. One of the hallmarks of EMT is loss of E-cadherin and gain of N-cadherin expression, which are regulated by transcription factors, such as SNAIL, SLUG, and TWIST. We examined the prognostic value of these factors as well as E-cadherin and N-cadherin, in a well-described large cohort of breast cancer patients treated with primary surgery. Analyses were stratified by estrogen receptor (ER) status, because of its crucial role in the regulation of these transcription factors. SNAIL, SLUG, and TWIST expression were examined on a TMA containing 575 breast tumors using immunohistochemistry. Nuclear expression was quantified using a weighted histoscore and classified as high versus low expression, based on the median histoscore. High expression of SNAIL, SLUG, and TWIST was seen in 54, 50, and 50% of tumors, respectively. The level of SNAIL (P = 0.014) and TWIST (P = 0.006) expression was associated with a worse patient relapse-free period, specifically in patients with ER-positive tumors (interaction Cox proportional hazards P = 0.039). Combining both factors resulted in an independent prognostic factor with high discriminative power (both low versus either high: HR 1.15; both low versus both high HR 1.84; P = 0.010). Co-expression of SNAIL-TWIST was associated with low-E-cadherin and high-N-cadherin expression, especially in ER-positive tumors (P = 0.009), suggesting that, through interactions with ER, SNAIL and TWIST may regulate E- and N-cadherin expression, thereby inducing EMT. Our results are indicative that SNAIL and TWIST play a crucial role in EMT through regulation of E- and N-cadherin expression, exclusively in ER-positive breast cancer patients.

Allele-specific regulation of FGFR2 expression is cell type-dependent and may increase breast cancer risk through a paracrine stimulus involving FGF10.

INTRODUCTION: SNPs rs2981582 and rs2981578, located in a linkage disequilibrium block (LD block) within intron 2 of the fibroblast growth factor receptor 2 gene (FGFR2), are associated with a mildly increased breast cancer risk. Allele-specific regulation of FGFR2 mRNA expression has been reported previously, but the molecular basis for the association of these variants with breast cancer has remained elusive to date. METHODS: mRNA levels of FGFR2 and three fibroblast growth factor genes (FGFs) were measured in primary fibroblast and epithelial cell cultures from 98 breast cancer patients and correlated to their rs2981578 genotype. The phosphorylation levels of downstream FGFR2 targets, FGF receptor substrate 2α (FRS2α) and extracellular signal-regulated kinases 1 and 2 (ERK1/2), were quantified in skin fibroblasts exposed to FGF2. Immunohistochemical markers for angiogenesis and lymphocytic infiltrate were semiquantitatively assessed in 25 breast tumors. RESULTS: The risk allele of rs2981578 was associated with increased FGFR2 mRNA levels in skin fibroblasts, but not in skin epithelial cell cultures. FGFR2 mRNA levels in skin fibroblasts and breast fibroblasts correlated strongly in the patients from whom both cultures were available. Tumor-derived fibroblasts expressed, on average, eight times more FGFR2 mRNA than the corresponding fibroblasts from normal breast tissue. Fibroblasts with higher FGFR2 mRNA expression showed more FRS2α and ERK1/2 phosphorylation after exposure to FGF2. In fibroblasts, higher FGFR2 expression correlated with higher FGF10 expression. In 25 breast tumors, no associations between breast tumor characteristics and fibroblast FGFR2 mRNA levels were found. CONCLUSIONS: The influence of rs2981578 genotypes on FGFR2 mRNA expression levels is cell type-dependent. Expression differences correlated well with signaling levels of the FGFR2 pathway. Our results suggest that the increased breast cancer risk associated with SNP rs2981578 is due to increased FGFR2 signaling activity in stromal fibroblasts, possibly also involving paracrine FGF10 signaling.

COX2 expression in prognosis and in prediction to endocrine therapy in early breast cancer patients.

In breast cancer, the prognostic impact of COX2 expression varies widely between studies. We examined the prognostic value of COX2 expression in a large cohort of breast cancer patients treated with primary surgery between 1985 and 1994 and explained the variable results of COX2 expression found in the literature. A tissue microarray was constructed of available tumour material, and ER, PgR, HER2, Ki67 and COX2 were examined by immunohistochemistry. Median follow-up was 19 years. Fifty-five percent (n = 369/677) of patients received no systemic treatment. COX2 was scored using a weighted histoscore. Analysis of COX2 expression in two groups based on the median (148; below vs. above) showed an increased hazard ratio (HR) of 1.35 (95% CI 1.05-1.75, P = 0.021) for disease-free survival (DFS) and of 1.39 (95% CI 1.03-1.82, P = 0.016) for overall survival (OS). However, COX2 did not remain independent in multivariate analysis. In patients with hormone receptor positive tumours, COX2 expression had a negative influence on outcome (low vs. high: DFS: HR 1.37, 95% CI 1.07-1.76, P = 0.013). This effect disappeared when endocrine therapy was administered (low vs. high: DFS: HR 0.93, 95% CI 0.51-1.70, P = 0.811) while it remained statistically significant when endocrine therapy was omitted (low vs. high: DFS: HR 1.48, 95% CI 1.12-1.94, P = 0.005). Our results show that COX2 plays a role in hormonal pathways. Our results can explain the results found in previously published studies.

Tumor-stroma ratio in the primary tumor is a prognostic factor in early breast cancer patients, especially in triple-negative carcinoma patients.

Stroma tissue surrounding cancer cells plays an important role in tumor development and behavior. In colorectal cancer, it has been found that the amount of stroma within the primary tumor is of prognostic value. We therefore have evaluated the prognostic value of this tumor-stroma ratio for breast cancer. A cohort of 574 early breast cancer patients, primarily treated with surgery between 1985 and 1994 was analyzed for the tumor-stroma ratio. The percentage of stroma was visually estimated on Haematoxylin-Eosin (H&E) stained histological sections. Patients with more than 50% intra-tumor stroma were quantified as stroma rich and patients with less than 50% as stroma poor. For the total group of patients, stroma-rich tumors had a shorter relapse-free period (RFP) (P = 0.001) and overall survival (OS) (P = 0.025) compared to stroma-poor tumors. Tumor-stroma ratio was an independent prognostic parameter for the total group of patients (P < 0.001) and also in stratified analysis based on systemic treatment. Importantly, in the triple-negative cancer subpopulation, patients with stroma-rich tumors had a 2.92 times higher risk of relapse (P = 0.006) compared to those with stroma-poor tumors, independently of other clinico-pathological parameters. Five-year RFP-rates for triple-negative cancer patients with stroma-rich compared to stroma-poor tumors were 56 and 81%, respectively. Tumor-stroma ratio has proven to be an independent prognostic factor for RFP in breast cancer patients and especially in the triple-negative cancer subpopulation. Tumor-stroma ratio could be easily implemented in routine daily pathology diagnostics, as it is simple to determine, reproducible, and performed in quick time.

HLA-E and HLA-G expression in classical HLA class I-negative tumors is of prognostic value for clinical outcome of early breast cancer patients.

Nonclassical HLAs, HLA-E and HLA-G, are known to affect clinical outcome in various tumor types. We examined the clinical impact of HLA-E and HLA-G expression in early breast cancer patients, and related the results to tumor expression of classical HLA class I. Our study population (n = 677) consisted of all early breast cancer patients primarily treated with surgery in our center between 1985 and 1995. Tissue microarray sections of arrayed tumor and normal control material were immunohistochemically stained for HLA-E and HLA-G. For evaluation of HLA-E and HLA-G and the combined variable, HLA-EG, a binary score was used. Expression of classical HLA class I molecules was determined previously. HLA-E, HLA-G, and HLA-EG on breast tumors were classified as expression in 50, 60, and 23% of patients, respectively. Remarkably, only in patients with loss of classical HLA class I tumor expression, expression of HLA-E (p = 0.027), HLA-G (p = 0.035), or HLA-EG (p = 0.001) resulted in a worse relapse-free period. An interaction was found between classical and nonclassical HLA class I expression (p = 0.002), suggestive for a biological connection. We have demonstrated that, next to expression of classical HLA class I, expression of HLA-E and HLA-G is an important factor in the prediction of outcome of breast cancer patients. These results provide further evidence that breast cancer is immunogenic, but also capable of evading tumor eradication by the host's immune system, by up- or downregulation of HLA class Ia and class Ib loci.

The predictive value of HLA class I tumor cell expression and presence of intratumoral Tregs for chemotherapy in patients with early breast cancer.

PURPOSE: We hypothesized that T-cell immune interaction affects tumor development and thus clinical outcome. Therefore, we examined the clinical impact of human leukocyte antigen (HLA) class I tumor cell expression and regulatory T-cell (Treg) infiltration in breast cancer. EXPERIMENTAL DESIGN: Our study population (N = 677) is consisted of all early breast cancer patients primarily treated with surgery in our center between 1985 and 1994. Formalin-fixed, paraffin-embedded tumor tissue was immunohistochemically stained using HCA2, HC10, and Foxp3 monoclonal antibodies. RESULTS: HLA class I expression was evaluated by combining results from HCA2 and HC10 antibodies and classified into three groups: loss, downregulation, and expression. Remarkably, only in patients who received chemotherapy, both presence of Treg (P = 0.013) and higher HLA class I expression levels (P = 0.002) resulted in less relapses, independently of other variables. Treg and HLA class I were not of influence on clinical outcome in patients who did not receive chemotherapy. CONCLUSIONS: We showed that HLA class I and Treg affect prognosis exclusively in chemotherapy-treated patients and are therefore one of the few predictive factors for chemotherapy response in early breast cancer patients. Chemotherapy may selectively eliminate Treg, thus enabling CTLs to kill tumor cells that have retained HLA class I expression. As a consequence, HLA class I and Treg can predict response to chemotherapy with high discriminative power. These markers could be applied in response prediction to chemotherapy in breast cancer patients.