Similar response profile to neoadjuvant chemotherapy, but different survival, in inflammatory versus locally advanced breast cancers.

Inflammatory breast cancer (IBC) is a very aggressive form of breast cancer, as compared to locally advanced breast cancer (LABC). Neoadjuvant chemotherapy followed by surgery is the standard treatment in both cases. Whether IBC is less chemosensitive than LABC remains unclear. We retrospectively compared the rate of pathological complete response (pCR) to neoadjuvant chemotherapy in IBC and LABC. METHODS: Patients with IBC or LABC treated with neoadjuvant anthracycline-based chemotherapy followed by surgery were selected from our institutional database. The primary endpoint was the pCR rate, defined as absence of invasive tumor in breast and axillary lymph nodes. RESULTS: A total of 450 patients were included, 144 with IBC and 306 with LABC. The pCR rate was similar between the two groups, in the whole population (31%) and in each molecular subtype separately. Univariate analyses for pCR in IBC and LABC separately identified the same predictive variables, except the pathological type that was associated with pCR in LABC only, but not in IBC. IBC patients displayed shorter 5-year metastasis-free survival and overall survival than LABC patients in the whole population (57% and 69% versus74% and 88% respectively), and in each molecular subtype separately. The IBC phenotype was an independent prognostic feature. Similarly, IBC patients displayed shorter 5-year loco-regional relapse-free survival than LABC patients (86% versus 95%). CONCLUSIONS: Similar pCR rates to chemotherapy were found in IBC and LABC, suggesting that IBC is not less chemosensitive than LABC. Survival was shorter in IBC, suggesting that the corresponding poorer prognosis is more due to a higher metastatic risk and/or other feature(s) than to a lesser chemosensitivity.

MARCKS protein overexpression in inflammatory breast cancer.

BACKGROUND: Inflammatory breast cancer (IBC) is the most aggressive form of locally-advanced breast cancer. Identification of new therapeutic targets is crucial. We previously reported MARCKS mRNA overexpression in IBC in the largest transcriptomics study reported to date. Here, we compared MARCKS protein expression in IBC and non-IBC samples, and searched for correlations between protein expression and clinicopathological features. RESULTS: Tumor samples showed heterogeneity with respect to MARCKS staining: 18% were scored as MARCKS-positive (stained cells ≥ 1%) and 82% as MARCKS-negative. MARCKS expression was more frequent in IBC (36%) than in non-IBC (11%; p = 1.4E-09), independently from molecular subtypes and other clinicopathological variables. We found a positive correlation between protein and mRNA expression in the 148/502 samples previously analyzed for MARCKS mRNA expression. MARCKS protein expression was associated with other poor-prognosis features in the whole series of samples such as clinical axillary lymph node or metastatic extension, high pathological grade, ER-negativity, PR-negativity, HER2-positivity, and triple-negative and HER2+ statutes. In IBC, MARCKS expression was the sole tested variable associated with poor MFS. MATERIALS AND METHODS: We retrospectively analyzed MARCKS protein expression by immunohistochemistry in 502 tumors, including 133 IBC and 369 non-IBC, from Tunisian and French patients. All samples were pre-therapeutic clinical samples. We searched for correlations between MARCKS expression and clinicopathological features including the IBC versus non-IBC phenotype and metastasis-free survival (MFS). CONCLUSIONS: MARCKS overexpression might in part explain the poor prognosis of IBC. As an oncogene associated with poor MFS, MARCKS might represent a new potential therapeutic target in IBC.

A whole-genome sequence and transcriptome perspective on HER2-positive breast cancers.

HER2-positive breast cancer has long proven to be a clinically distinct class of breast cancers for which several targeted therapies are now available. However, resistance to the treatment associated with specific gene expressions or mutations has been observed, revealing the underlying diversity of these cancers. Therefore, understanding the full extent of the HER2-positive disease heterogeneity still remains challenging. Here we carry out an in-depth genomic characterization of 64 HER2-positive breast tumour genomes that exhibit four subgroups, based on the expression data, with distinctive genomic features in terms of somatic mutations, copy-number changes or structural variations. The results suggest that, despite being clinically defined by a specific gene amplification, HER2-positive tumours melt into the whole luminal-basal breast cancer spectrum rather than standing apart. The results also lead to a refined ERBB2 amplicon of 106 kb and show that several cases of amplifications are compatible with a breakage-fusion-bridge mechanism.

Cytokeratin 7 as a predictive factor for response to concommitant radiochemotherapy for locally advanced cervical cancer: a preliminary study.

BACKGROUND: The role of completion surgery after concurrent radiochemotherapy (CCRC) for advanced cervical cancer remains controversial. Individual predictive factors of CCRC response and survival are mandatory for treatment adaptation and to determine a population who would take interest in completion surgery after CCRC. The aim of this study was to evaluate the ability of biomarkers to predict the response to CCRC. PATIENTS AND METHODS: Between 1996 and 2008, in 58 patients with advanced cervical cancer for whom pre-therapeutic cone biopsy was available, we tested several biomarkers (ALDH1, CD44, CD24, IDO, Ki67, P63, CK7, p-Stat3, Foxp3 and IDO). RESULTS: Residual disease was found in 49.1% of cases (n=26). We found a significant association between progression-free survival and residual disease on completion hysterectomy (p=0.044). Univariate analysis of the different factors showed that negativity for cytokeratin 7 expression was a strong predictor for the presence of residual tumor (p=0.001). CONCLUSION: These results are encouraging and CK7 could be used as a predictive factor of response to CCRC.

Candidate luminal B breast cancer genes identified by genome, gene expression and DNA methylation profiling.

Breast cancers (BCs) of the luminal B subtype are estrogen receptor-positive (ER+), highly proliferative, resistant to standard therapies and have a poor prognosis. To better understand this subtype we compared DNA copy number aberrations (CNAs), DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes, in 32 luminal B tumors with those observed in 156 BCs of the other molecular subtypes. Frequent CNAs included 8p11-p12 and 11q13.1-q13.2 amplifications, 7q11.22-q34, 8q21.12-q24.23, 12p12.3-p13.1, 12q13.11-q24.11, 14q21.1-q23.1, 17q11.1-q25.1, 20q11.23-q13.33 gains and 6q14.1-q24.2, 9p21.3-p24,3, 9q21.2, 18p11.31-p11.32 losses. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs) presented a deregulated expression in relation with their CNAs, including 11 genes previously reported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. A total of 100 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 of these was associated with poor survival in luminal tumors. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, KCNB2 mutations were associated with luminal B cases while candidate TSGs MDN1 (6q15) and UTRN (6q24), were mutated in this subtype. In conclusion, we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype.

Instant-quality fluorescence in-situ hybridization as a new tool for HER2 testing in breast cancer: a comparative study.

AIMS: HER2 instant-quality fluorescence in-situ hybridization (IQFISH) is a new fluorescence in-situ hybridization (FISH) assay developed with a non-toxic buffer that reduces the hybridization time to 1-2 h, enabling a turnaround time of 3 h 30 min from dewax to counting. The aim of this study was to compare assessment of HER2 status using IQFISH and assessment using standard FISH. METHODS AND RESULTS: We selected 160 breast cancer samples according to their HER2 status as determined by immunohistochemistry (IHC) in a retrospective multicentre cohort (40 cases in each scoring category, i.e. 0/1+/2+/3+). Each participating site (n = 5) constructed its tissue microarray (TMA) of 32 archival cases and sent it to the central site (site 1). HER2 IHC, HER2 FISH and HER2 IQFISH were performed blindly at site 1. IQFISH provided excellent quality signals without any background staining, thus allowing excellent reading conditions even on TMA. Statistical analysis showed almost perfect agreement between IQFISH and FISH (99.3%, κ = 0.98). The only discordant case was an equivocal one with an HER2/CEP17 ratio near the ASCO/CAP cut-off. CONCLUSIONS: The highly concordant data support IQFISH as a useful alternative to FISH, allowing reliable assessment of HER2 status. Use of this method could lead to reporting of HER status to the oncologist within a day.

ALDH1-positive cancer stem cells predict engraftment of primary breast tumors and are governed by a common stem cell program.

Cancer stem-like cells (CSC) have been widely studied, but their clinical relevance has yet to be established in breast cancer. Here, we report the establishment of primary breast tumor-derived xenografts (PDX) that encompass the main diversity of human breast cancer and retain the major clinicopathologic features of primary tumors. Successful engraftment was correlated with the presence of ALDH1-positive CSCs, which predicted prognosis in patients. The xenografts we developed showed a hierarchical cell organization of breast cancer with the ALDH1-positive CSCs constituting the tumorigenic cell population. Analysis of gene expression from functionally validated CSCs yielded a breast CSC signature and identified a core transcriptional program of 19 genes shared with murine embryonic, hematopoietic, and neural stem cells. This generalized stem cell program allowed the identification of potential CSC regulators, which were related mainly to metabolic processes. Using an siRNA genetic screen designed to target the 19 genes, we validated the functional role of this stem cell program in the regulation of breast CSC biology. Our work offers a proof of the functional importance of CSCs in breast cancer, and it establishes the reliability of PDXs for use in developing personalized CSC therapies for patients with breast cancer.

SISH/CISH or qPCR as alternative techniques to FISH for determination of HER2 amplification status on breast tumors core needle biopsies: a multicenter experience based on 840 cases.

BACKGROUND: Until now, FISH has been the gold standard technique to identify HER2 amplification status in ambiguous cases of breast cancer. Alternative techniques have been developed to increase the capacities of investigating HER2 amplification status. The aims of this multicenter study in a large series of breast cancer patients were to prospectively compare the level of performance of CISH, SISH, and qPCR alternative techniques on paraffin-embedded core biopsies with "gold standard FISH" for evaluation of HER2 amplification status. METHODS: This study was performed on 840 cases scored by immunohistochemistry (IHC): 0=317 (38%), 1+=183 (22%), 2+=109 (13%), 3+=231 (27%). Each of the 15 French centers participating in the study analyzed 56 breast carcinoma cases diagnosed on fixed paraffin-embedded core biopsies. HER2 amplification status was determined by commercially available FISH used as the reference technique with determination of the HER2/CEN17 ratio or HER2 copy number status. The alternative techniques performed on the same cases were commercially available SISH or CISH and a common qPCR method especially designed for the study including a set of 10 primer pairs: 2 for HER2 (exons 8 and 26), 5 to evaluate chromosome 17 polysomy TAOK1, UTP6, MRM1, MKS1, SSTR2 and 3 for diploidy control TSN, LAP3 and ADAMTS16. RESULTS: The concordance between IHC and FISH was 96% to 95% based on the HER2/CEN17 ratio (n=766) or HER2 copy number (n=840), respectively. The concordance of the alternative techniques with FISH was excellent: 97% and 98% for SISH (498 and 587 cases), 98% and 75% for CISH (108 and 204 cases) and 95% and 93% (699 and 773 cases) for qPCR based on the HER2/CEN17 ratio or HER2 copy number, respectively. Similarly, sensitivity ranged from 99% to 95% for SISH, 100% to 99% for CISH and 89% to 80% for qPCR. The concordance with FISH (ratio) in the 2+ cases was 89% for SISH, 100% for CISH and 93% for qPCR. CONCLUSION: These alternative techniques showed an excellent concordance with FISH in core biopsies allowing their use in routine clinical practice. This newly designed qPCR on paraffin-embedded core biopsies deserves special attention, as it is reliable, easy to perform and less expensive than ISH tests.

Prediction of BRCA1 germ-line mutation status in patients with breast cancer using histoprognosis grade, MS110, Lys27H3, vimentin, and KI67.

Family structure, lack of reliable information, cost, and delay are usual concerns when deciding to perform BRCA analyses. Testing breast cancer tissues with four antibodies (MS110, lys27H3, vimentin, and KI67) in addition to grade evaluation enabled us to rapidly select patients for genetic testing identification. We constituted an initial breast cancer tissue microarray, considered as a learning set, comprising 27 BRCA1 and 81 sporadic tumors. A second independent validation set of 28 BRCA1 tumors was matched to 28 sporadic tumors using the same original conditions. We investigated morphological parameters and 21 markers by immunohistochemistry. A logistic regression model was used to select the minimal number of markers providing the best model to predict BRCA1 status. The model was applied to the validation set to estimate specificity and sensibility. In the initial set, univariate analyses identified 11 markers significantly associated with BRCA1 status. Then, the best multivariate model comprised only grade 3, MS110, Lys27H3, vimentin, and KI67. When applied to the validation set, BRCA1 tumors were correctly classified with a sensitivity of 83% and a specificity of 81%. The performance of this model was superior when compared to other profiles. This study offers a new rapid and cost-effective method for the prescreening of patients at high risk of being BRCA1 mutation carriers, to guide genetic testing, and finally to provide appropriate preventive measures, advice, and treatments including targeted therapy to patients and their families.

Peripheral blood NK cells from breast cancer patients are tumor-induced composite subsets.

Human NK lymphocytes are involved in antitumor immunity. The therapeutic potential of this population against cancers has stimulated their study and led to the discovery of several NK cell subsets, each of which is endowed with different immunoregulatory functions. We have previously reported that NK cell functions are profoundly altered in advanced breast cancer patients. In this study, we show that these tumor-mediated alterations also variably affect NK cell subsets. We found that in addition to the known human CD56(dim)CD16(+), CD56(bright)CD16(-), and CD56(-)CD16(+) NK cell subsets, two additional subsets, namely the CD56(bright)CD16(+) and CD56(dim)CD16(-) subsets, were increased in the peripheral blood of patients with advanced invasive breast cancers. These subsets corresponded to the main two subsets found at the tumor site. The extensive phenotype of these subsets revealed an "à la carte" pattern of expression for the various NK receptors, functional molecules, adhesion molecules, and chemokine receptors, depending on the subset. We next compared these subsets to known NK cell populations endowed with specific phenotypic characteristics, but also with functional properties. Our data show that advanced breast cancer patients have an increased proportion of more immature and noncytotoxic NK cell subsets in their peripheral blood, which might account for at least part of the low cytotoxic functions observed in these patients. They reveal a major heterogeneity and plasticity of the NK cell compartment, which are both tightly linked to the microenvironment. The identification of NK cell subsets endowed with particular functional capabilities might help monitor residual antitumor NK cell-mediated responses in breast cancer patients.

Breast metastasis of a squamous cell carcinoma of the uterine cervix mimicking inflammatory breast cancer.

Breast metastases from distant carcinoma are infrequent, and cervix carcinoma is rarely the primary lesion. We describe the first case of a cervical squamous cell carcinoma with breast metastasis mimicking an inflammatory breast cancer in a 74-year-old woman. Seventeen months after the treatment of a primary tumor, the patient developed breast lesions looking like an inflammatory breast tumor. After a 1-year delay due to the patient's refusal, pathological examination and immunohistochemistry confirmed the diagnosis of breast metastasis from a poorly differentiated squamous cell carcinoma. The volume of the breast was huge, associated with axillary lymphadenopathies and multiple lung metastases. Despite platinum-based chemotherapy, the disease progressed and the patient died rapidly, 3 months after the first chemotherapy cycle and 15 months after the first mammary symptoms. We review the literature concerning breast metastases from gynecologic cancers and, particularly, from cervical squamous cell carcinoma. Differential diagnosis of such lesions may be problematic but is essential to avoid unnecessary mutilating surgery and to institute the appropriate systemic therapy. The prognosis is poor.

Ki-67: level of evidence and methodological considerations for its role in the clinical management of breast cancer: analytical and critical review.

Clinicians can use biomarkers to guide therapeutic decisions in estrogen receptor positive (ER+) breast cancer. One such biomarker is cellular proliferation as evaluated by Ki-67. This biomarker has been extensively studied and is easily assayed by histopathologists but it is not currently accepted as a standard. This review focuses on its prognostic and predictive value, and on methodological considerations for its measurement and the cut-points used for treatment decision. Data describing study design, patients' characteristics, methods used and results were extracted from papers published between January 1990 and July 2010. In addition, the studies were assessed using the REMARK tool. Ki-67 is an independent prognostic factor for disease-free survival (HR 1.05-1.72) in multivariate analyses studies using samples from randomized clinical trials with secondary central analysis of the biomarker. The level of evidence (LOE) was judged to be I-B with the recently revised definition of Simon. However, standardization of the techniques and scoring methods are needed for the integration of this biomarker in everyday practice. Ki-67 was not found to be predictive for long-term follow-up after chemotherapy. Nevertheless, high KI-67 was found to be associated with immediate pathological complete response in the neoadjuvant setting, with an LOE of II-B. The REMARK score improved over time (with a range of 6-13/20 vs. 10-18/20, before and after 2005, respectively). KI-67 could be considered as a prognostic biomarker for therapeutic decision. It is assessed with a simple assay that could be standardized. However, international guidelines are needed for routine clinical use.

Non sentinel node involvement prediction for sentinel node micrometastases in breast cancer: nomogram validation and comparison with other models.

PURPOSE: The risk of non sentinel node (NSN) involvement varies in function of the characteristics of sentinel nodes (SN) and primary tumor. Our aim was to determine and validate a statistical tool (a nomogram) able to predict the risk of NSN involvement in case of SN micro or sub-micrometastasis of breast cancer. We have compared this monogram with other models described in the literature. METHODS: We have collected data on 905 patients, then 484 other patients, to build and validate the nomogram and compare it with other published scores and nomograms. RESULTS: Multivariate analysis conducted on the data of the first cohort allowed us to define a nomogram based on 5 criteria: the method of SN detection (immunohistochemistry or by standard coloration with HES); the ratio of positive SN out of total removed SN; the pathologic size of the tumor; the histological type; and the presence (or not) of lympho-vascular invasion. The nomogram developed here is the only one dedicated to micrometastasis and developed on the basis of two large cohorts. The results of this statistical tool in the calculation of the risk of NSN involvement is similar to those of the MSKCC (the similarly more effective nomogram according to the literature), with a lower rate of false negatives. CONCLUSION: this nomogram is dedicated specifically to cases of SN involvement by metastasis lower or equal to 2 mm. It could be used in clinical practice in the way to omit ALND when the risk of NSN involvement is low.

Diagnostic performance of one-step nucleic acid amplification for intraoperative sentinel node metastasis detection in breast cancer patients.

The purpose of this prospective multicenter study was to assess one-step nucleic acid amplification (OSNA) for intraoperative sentinel lymph node (SLN) metastasis detection in breast cancer patients, using final histology as the reference standard. OSNA results were also compared to intraoperative histology SLN evaluation and to standard clinicopathological risk markers. For this study, fresh SLNs were cut in four blocks, and alternate blocks were used for OSNA and histology. CK19 mRNA copy number was categorized as strongly positive, positive or negative. Positive histology was defined as presence of macrometastasis or micrometastasis. When discrepancies occurred, the entire SLNs were subjected to histological studies and the node lysates to additional molecular studies. Five hundred three SLN samples from 233 patients were studied. Mean time to evaluate two SLNs was 40 min. Sensitivity per patient was 91.4% (95% CI, 76.9-98.2%), specificity 93.3% (95% CI, 88.6-96.6%), positive likelihood ratio 13.7 and negative likelihood ratio 0.1. Sensitivity was 63.6% for frozen sections and 47.1% for touch imprint cytology. Both methods were 100% specific. Positive histology and positive OSNA were significantly associated with highest clinical stage, N1 status and vascular invasion; and OSNA results correlated with HER2/neu status and benefited patients with negative histology. These findings show that OSNA assay can allow detection of SLN metastasis in breast cancer patients intraoperatively with a good sensitivity, thus minimizing the need for second surgeries for axillary lymph node detection.

High expression of indoleamine 2,3-dioxygenase in the tumour is associated with medullary features and favourable outcome in basal-like breast carcinoma.

Medullary breast cancer (MBC) is a basal-like breast carcinoma (BLBC) with a favourable outcome, whereas nonmedullary BLBC has a poor prognosis. Tumour infiltrating lymphocytes (TILs) are present in both MBC and BLBC. We hypothesized that the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) could modulate the TILs effects among these tumours and explain their different outcomes. The amount of TILs and IDO expression were analysed using immunohistochemistry (IHC) in 155 BC cases including MBC (n = 17), atypical MBC (n = 13) and non-MBC (n = 125). Messenger RNA expression of the INDO gene, which encodes IDO, was measured in 262 cases from our institution. INDO mRNA expression and histoclinical data of 1,487 BC cases were collected from public databases. IDO immunostaining was present in both neoplastic and stromal cells in 100% of MBC and was associated with histological medullary features among non-MBC cases. There was a significant correlation between IDO positivity and TIL amounts. In our series including mostly grade-3 BC, IDO immunostaining was the most significant marker (p = 0.02) associated with better survival in multivariate analysis. Among our 262 analysed BC cases, INDO mRNA showed significant overexpression in BLBC as compared to luminal A tumours, and in MBC as compared to basal-like non-MBC. In the pooled series of 1,749 BC cases, INDO mRNA was overexpressed in BLBC and was the most significant predictor of better survival in this subtype using multivariate analysis (p = 0.0024). In conclusion, high IDO expression is associated with morphological medullary features and has an independent favourable prognostic value in BLBC.

Protein expression, survival and docetaxel benefit in node-positive breast cancer treated with adjuvant chemotherapy in the FNCLCC-PACS 01 randomized trial.

INTRODUCTION: The PACS01 trial has demonstrated that a docetaxel addition to adjuvant anthracycline-based chemotherapy improves disease-free survival (DFS) and overall survival of node-positive early breast cancer (EBC). We searched for prognostic and predictive markers for docetaxel's benefit. METHODS: Tumor samples from 1,099 recruited women were analyzed for the expression of 34 selected proteins using immunohistochemistry. The prognostic and predictive values of each marker and four molecular subtypes (luminal A, luminal B, HER2-overexpressing, and triple-negative) were tested. RESULTS: Progesterone receptor-negativity (HR = 0.66; 95% CI 0.47 to 0.92, P = 0.013), and Ki67-positivity (HR = 1.53; 95% CI 1.12 to 2.08, P = 0.007) were independent adverse prognostic factors. Out of the 34 proteins, only Ki67-positivity was associated with DFS improvement with docetaxel addition (adjusted HR = 0.51, 95% CI 0.33 to 0.79 for Ki67-positive versus HR = 1.10, 95% CI 0.75 to 1.61 for Ki67-negative tumors, P for interaction = 0.012). Molecular subtyping predicted the docetaxel benefit, but without providing additional information to Ki67 status. The luminal A subtype did not benefit from docetaxel (HR = 1.16, 95% CI 0.73 to 1.84); the reduction in the relapse risk was 53% (HR = 0.47, 95% CI 0.22 to 1.01), 34% (HR = 0.66, 95% CI 0.37 to 1.19), and 12% (HR = 0.88, 95% CI 0.49 to 1.57) in the luminal B, HER2-overexpressing, and triple-negative subtypes, respectively. CONCLUSIONS: In patients with node-positive EBC receiving adjuvant anthracycline-based chemotherapy, the most powerful predictor of docetaxel benefit is Ki67-positivity.

Primary leiomyosarcoma of the seminal vesicle: case report and review of the literature.

BACKGROUND: Primary leiomyosarcoma of the seminal vesicle is exceedingly rare. CASE PRESENTATION: We report a case of a 59-year-old man with tumour detected by rectal symptoms and ultrasonography. Computed tomography and magnetic resonance imaging suggested an origin in the right seminal vesicle. Transperineal biopsy of the tumour revealed leiomyosarcoma. A radical vesiculo-prostactectomy with bilateral pelvic lymphadenectomy was performed. Pathological examination showed a grade 2 leiomyosarcoma of the seminal vesicle. The patient received adjuvant radiotherapy. He developed distant metastases 29 months after diagnosis, and received chemotherapy. Metastatic disease was controlled by second-line gemcitabine-docetaxel combination. Fifty-one months after diagnosis of the primary tumour, and 22 months after the first metastases, the patient is alive with excellent performance status, and multiple asymptomatic stable lung and liver lesions. CONCLUSIONS: We report the eighth case of primary leiomyosarcoma of the seminal vesicle and the first one with a so long follow-up.

Human breast cancer cells enhance self tolerance by promoting evasion from NK cell antitumor immunity.

NK cells are a major component of the antitumor immune response and are involved in controlling tumor progression and metastases in animal models. Here, we show that dysfunction of these cells accompanies human breast tumor progression. We characterized human peripheral blood NK (p-NK) cells and malignant mammary tumor-infiltrating NK (Ti-NK) cells from patients with noninvasive and invasive breast cancers. NK cells isolated from the peripheral blood of healthy donors and normal breast tissue were used as controls. With disease progression, we found that expression of activating NK cell receptors (such as NKp30, NKG2D, DNAM-1, and CD16) decreased while expression of inhibitory receptors (such as NKG2A) increased and that this correlated with decreased NK cell function, most notably cytotoxicity. Importantly, Ti-NK cells had more pronounced impairment of their cytotoxic potential than p-NK cells. We also identified several stroma-derived factors, including TGF-ß1, involved in tumor-induced reduction of normal NK cell function. Our data therefore show that breast tumor progression involves NK cell dysfunction and that breast tumors model their environment to evade NK cell antitumor immunity. This highlights the importance of developing future therapies able to restore NK cell cytotoxicity to limit/prevent tumor escape from antitumor immunity.

Gene expression profiling and its utility in prediction of local relapse after breast-conserving therapy in early breast cancer.

BACKGROUND: Local relapse (LR) after breast-conserving therapy (BCT) is not accurately predicted by histological/clinical factors. Gene expression profiling was used here to discover LR-associated transcriptional alterations. MATERIALS AND METHODS: Gene expression profiling was carried out of 81 early breast carcinomas obtained from 30 patients who developed a LR (LR(+)) after BCT and 51 who did not (LR(-)). LR(+) and LR(-) samples were matched for known LR risk features. RESULTS: LR was not associated with a given molecular subtype. Supervised analysis identified a 212-gene signature, which was not validated in independent tumors. No gene set or biological pathway was differentially expressed between LR(+) and LR(-) groups. Twelve published prognostic expression signatures failed to distinguish these groups of carcinomas. The gene expression profiles of 9 cases of LR and the corresponding primary tumors were very similar despite the delivery of radiotherapy. CONCLUSION: In this series, the onset of LR was not predicted by gene expression alterations.

Kinome expression profiling and prognosis of basal breast cancers.

BACKGROUND: Basal breast cancers (BCs) represent ~15% of BCs. Although overall poor, prognosis is heterogeneous. Identification of good- versus poor-prognosis patients is difficult or impossible using the standard histoclinical features and the recently defined prognostic gene expression signatures (GES). Kinases are often activated or overexpressed in cancers, and constitute targets for successful therapies. We sought to define a prognostic model of basal BCs based on kinome expression profiling. METHODS: DNA microarray-based gene expression and histoclinical data of 2515 early BCs from thirteen datasets were collected. We searched for a kinome-based GES associated with disease-free survival (DFS) in basal BCs of the learning set using a metagene-based approach. The signature was then tested in basal tumors of the independent validation set. RESULTS: A total of 591 samples were basal. We identified a 28-kinase metagene associated with DFS in the learning set (N = 73). This metagene was associated with immune response and particularly cytotoxic T-cell response. On multivariate analysis, a metagene-based predictor outperformed the classical prognostic factors, both in the learning and the validation (N = 518) sets, independently of the lymphocyte infiltrate. In the validation set, patients whose tumors overexpressed the metagene had a 78% 5-year DFS versus 54% for other patients (p = 1.62E-4, log-rank test). CONCLUSIONS: Based on kinome expression, we identified a predictor that separated basal BCs into two subgroups of different prognosis. Tumors associated with higher activation of cytotoxic tumor-infiltrative lymphocytes harbored a better prognosis. Such classification should help tailor the treatment and develop new therapies based on immune response manipulation.