SPARC in cancer-associated fibroblasts is an independent poor prognostic factor in non-metastatic triple-negative breast cancer and exhibits pro-tumor activity.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and lacks specific targeted therapeutic agents. The current mechanistic evidence from cell-based studies suggests that the matricellular protein SPARC has a tumor-promoting role in TNBC; however, data on the clinical relevance of SPARC expression/secretion by tumor and stromal cells in TNBC are limited. Here, we analyzed by immunohistochemistry the prognostic value of tumor and stromal cell SPARC expression in 148 patients with non-metastatic TNBC and long follow-up (median: 5.4 years). We also quantified PD-L1 and PD-1 expression. We detected SPARC expression in tumor cells (42.4%), cancer-associated fibroblasts (CAFs; 88.1%), tumor-associated macrophages (77.1%), endothelial cells (75.2%) and tumor-infiltrating lymphocytes (9.8%). Recurrence-free survival was significantly lower in patients with SPARC-expressing CAFs. Multivariate analysis showed that SPARC expression in CAFs was an independent prognostic factor. We also detected tumor and stromal cell SPARC expression in TNBC cytosols, and in patient-derived xenografts and cell lines. Furthermore, we analyzed publicly available single-cell mRNA sequencing data and found that in TNBC, SPARC is expressed by different CAF subpopulations, including myofibroblasts and inflammatory fibroblasts that are involved in tumor-related processes. We then showed that fibroblast-secreted SPARC had a tumor-promoting role by inhibiting TNBC cell adhesion and stimulating their motility and invasiveness. Overall, our study demonstrates that SPARC expression in CAFs is an independent prognostic marker of poor outcome in TNBC. Patients with SPARC-expressing CAFs could be eligible for anti-SPARC targeted therapy.

A 9-kDa matricellular SPARC fragment released by cathepsin D exhibits pro-tumor activity in the triple-negative breast cancer microenvironment.

Rationale: Alternative therapeutic strategies based on tumor-specific molecular targets are urgently needed for triple-negative breast cancer (TNBC). The protease cathepsin D (cath-D) is a marker of poor prognosis in TNBC and a tumor-specific extracellular target for antibody-based therapy. The identification of cath-D substrates is crucial for the mechanistic understanding of its role in the TNBC microenvironment and future therapeutic developments. Methods: The cath-D substrate repertoire was investigated by N-Terminal Amine Isotopic Labeling of Substrates (TAILS)-based degradome analysis in a co-culture assay of TNBC cells and breast fibroblasts. Substrates were validated by amino-terminal oriented mass spectrometry of substrates (ATOMS). Cath-D and SPARC expression in TNBC was examined using an online transcriptomic survival analysis, tissue micro-arrays, TNBC cell lines, patient-derived xenografts (PDX), human TNBC samples, and mammary tumors from MMTV-PyMT Ctsd-/- knock-out mice. The biological role of SPARC and its fragments in TNBC were studied using immunohistochemistry and immunofluorescence analysis, gene expression knockdown, co-culture assays, western blot analysis, RT-quantitative PCR, adhesion assays, Transwell motility, trans-endothelial migration and invasion assays. Results: TAILS analysis showed that the matricellular protein SPARC is a substrate of extracellular cath-D. In vitro, cath-D induced limited proteolysis of SPARC C-terminal extracellular Ca2+ binding domain at acidic pH, leading to the production of SPARC fragments (34-, 27-, 16-, 9-, and 6-kDa). Similarly, cath-D secreted by TNBC cells cleaved fibroblast- and cancer cell-derived SPARC at the tumor pericellular acidic pH. SPARC cleavage also occurred in TNBC tumors. Among these fragments, only the 9-kDa SPARC fragment inhibited TNBC cell adhesion and spreading on fibronectin, and stimulated their migration, endothelial transmigration, and invasion. Conclusions: Our study establishes a novel crosstalk between proteases and matricellular proteins in the tumor microenvironment through limited SPARC proteolysis, revealing a novel targetable 9-kDa bioactive SPARC fragment for new TNBC treatments. Our study will pave the way for the development of strategies for targeting bioactive fragments from matricellular proteins in TNBC.

Co-Expression of Androgen Receptor and Cathepsin D Defines a Triple-Negative Breast Cancer Subgroup with Poorer Overall Survival.

BACKGROUND: In the triple-negative breast cancer (TNBC) group, the luminal androgen receptor subtype is characterized by expression of androgen receptor (AR) and lack of estrogen receptor and cytokeratin 5/6 expression. Cathepsin D (Cath-D) is overproduced and hypersecreted by breast cancer (BC) cells and is a poor prognostic marker. We recently showed that in TNBC, Cath-D is a potential target for antibody-based therapy. This study evaluated the frequency of AR/Cath-D co-expression and its prognostic value in a large series of patients with non-metastatic TNBC. METHODS: AR and Cath-D expression was evaluated by immunohistochemistry in 147 non-metastatic TNBC. The threshold for AR positivity (AR+) was set at ≥1% of stained cells, and the threshold for Cath-D positivity (Cath-D+) was moderate/strong staining intensity. Lymphocyte density, macrophage infiltration, PD-L1 and programmed cell death (PD-1) expression were assessed. RESULTS: Scarff-Bloom-Richardson grade 1-2 and lymph node invasion were more frequent, while macrophage infiltration was less frequent in AR+/Cath-D+ tumors (62.7%). In multivariate analyses, higher tumor size, no adjuvant chemotherapy and AR/Cath-D co-expression were independent prognostic factors of worse overall survival. CONCLUSIONS: AR/Cath-D co-expression independently predicted overall survival. Patients with TNBC in which AR and Cath-D are co-expressed could be eligible for combinatory therapy with androgen antagonists and anti-Cath-D human antibodies.

BRCA1 Promoter Hypermethylation is Associated with Good Prognosis and Chemosensitivity in Triple-Negative Breast Cancer.

The aberrant hypermethylation of BRCA1 promoter CpG islands induces the decreased expression of BRCA1 (Breast Cancer 1) protein. It can be detected in sporadic breast cancer without BRCA1 pathogenic variants, particularly in triple-negative breast cancers (TNBC). We investigated BRCA1 hypermethylation status (by methylation-specific polymerase chain reaction (MS-PCR) and MassARRAY® assays), and BRCA1 protein expression using immunohistochemistry (IHC), and their clinicopathological significance in 248 chemotherapy-naïve TNBC samples. Fifty-five tumors (22%) exhibited BRCA1 promoter hypermethylation, with a high concordance rate between MS-PCR and MassARRAY® results. Promoter hypermethylation was associated with reduced IHC BRCA1 protein expression (p = 0.005), and expression of Programmed death-ligand 1 protein (PD-L1) by tumor and immune cells (p = 0.03 and 0.011, respectively). A trend was found between promoter hypermethylation and basal marker staining (p = 0.058), and between BRCA1 expression and a basal-like phenotype. In multivariate analysis, relapse-free survival was significantly associated with N stage, adjuvant chemotherapy, and histological subtype. Overall survival was significantly associated with T and N stage, histology, and adjuvant chemotherapy. In addition, patients with tumors harboring BRCA1 promoter hypermethylation derived the most benefit from adjuvant chemotherapy. In conclusion, BRCA1 promoter hypermethylation is associated with TNBC sensitivity to adjuvant chemotherapy, basal-like features and PD-L1 expression. BRCA1 IHC expression is not a good surrogate marker for promoter hypermethylation and is not independently associated with prognosis. Association between promoter hypermethylation and sensitivity to Poly(ADP-ribose) polymerase PARP inhibitors needs to be evaluated in a specific series of patients.

PTPN13 induces cell junction stabilization and inhibits mammary tumor invasiveness.

Clinical data suggest that the protein tyrosine phosphatase PTPN13 exerts an anti-oncogenic effect. Its exact role in tumorigenesis remains, however, unclear due to its negative impact on FAS receptor-induced apoptosis. Methods: We crossed transgenic mice deleted for PTPN13 phosphatase activity with mice that overexpress human HER2 to assess the exact role of PTPN13 in tumor development and aggressiveness. To determine the molecular mechanism underlying the PTPN13 tumor suppressor activity we developed isogenic clones of the aggressive human breast cancer cell line MDA-MB-231 overexpressing either wild type or a catalytically-inactive mutant PTPN13 and subjected these to phosphoproteomic and gene ontology analyses. We investigated the PTPN13 consequences on cell aggressiveness using wound healing and Boyden chamber assays, on intercellular adhesion using videomicroscopy, cell aggregation assay and immunofluorescence. Results: The development, growth and invasiveness of breast tumors were strongly increased by deletion of the PTPN13 phosphatase activity in transgenic mice. We observed that PTPN13 phosphatase activity is required to inhibit cell motility and invasion in the MDA-MB-231 cell line overexpressing PTPN13. In vivo, the negative PTPN13 effect on tumor invasiveness was associated with a mesenchymal-to-epithelial transition phenotype in athymic mice xenografted with PTPN13-overexpressing MDA-MB-231 cells, as well as in HER2-overexpressing mice with wild type PTPN13, compared to HER2-overexpressing mice that lack PTPN13 phosphatase activity. Phosphoproteomic and gene ontology analyses indicated a role of PTPN13 in the regulation of intercellular junction-related proteins. Finally, protein localization studies in MDA-MB-231 cells and HER2-overexpressing mice tumors confirmed that PTPN13 stabilizes intercellular adhesion and promotes desmosome formation. Conclusions: These data provide the first evidence for the negative role of PTPN13 in breast tumor invasiveness and highlight its involvement in cell junction stabilization.

Quantification of HER1, HER2 and HER3 by time-resolved Förster resonance energy transfer in FFPE triple-negative breast cancer samples.

BACKGROUND: Triple-negative breast cancer (TNBC) has a worse prognosis compared with other breast cancer subtypes, and biomarkers to identify patients at high risk of recurrence are needed. Here, we investigated the expression of human epidermal receptor (HER) family members in TNBC and evaluated their potential as biomarkers of recurrence. METHODS: We developed Time Resolved-Förster Resonance Energy Transfer (TR-FRET) assays to quantify HER1, HER2 and HER3 in formalin-fixed paraffin-embedded (FFPE) tumour tissues. After assessing the performance and precision of our assays, we quantified HER protein expression in 51 TNBC specimens, and investigated the association of their expression with relapse-free survival. RESULTS: The assays were quantitative, accurate, and robust. In TNBC specimens, HER1 levels ranged from ≈4000 to more than 2 million receptors per cell, whereas HER2 levels varied from ≈1000 to 60,000 receptors per cell. HER3 expression was very low (less than 5500 receptors per cell in all samples). Moderate HER2 expression was significantly associated with higher risk of recurrence (HR = 3.93; P = 0.003). CONCLUSIONS: Our TR-FRET assays accurately quantify HER1, HER2 and HER3 in FFPE breast tumour specimens. Moderate HER2 expression may represent a novel prognostic marker in patients with TNBC.

Immunotherapy of triple-negative breast cancer with cathepsin D-targeting antibodies.

BACKGROUND: Triple-negative breast cancer (TNBC) treatment is currently restricted to chemotherapy. Hence, tumor-specific molecular targets and/or alternative therapeutic strategies for TNBC are urgently needed. Immunotherapy is emerging as an exciting treatment option for TNBC patients. The aspartic protease cathepsin D (cath-D), a marker of poor prognosis in breast cancer (BC), is overproduced and hypersecreted by human BC cells. This study explores whether cath-D is a tumor cell-associated extracellular biomarker and a potent target for antibody-based therapy in TNBC. METHODS: Cath-D prognostic value and localization was evaluated by transcriptomics, proteomics and immunohistochemistry in TNBC. First-in-class anti-cath-D human scFv fragments binding to both human and mouse cath-D were generated using phage display and cloned in the human IgG1 λ format (F1 and E2). Anti-cath-D antibody biodistribution, antitumor efficacy and in vivo underlying mechanisms were investigated in TNBC MDA-MB-231 tumor xenografts in nude mice. Antitumor effect was further assessed in TNBC patient-derived xenografts (PDXs). RESULTS: High CTSD mRNA levels correlated with shorter recurrence-free survival in TNBC, and extracellular cath-D was detected in the tumor microenvironment, but not in matched normal breast stroma. Anti-cath-D F1 and E2 antibodies accumulated in TNBC MDA-MB-231 tumor xenografts, inhibited tumor growth and improved mice survival without apparent toxicity. The Fc function of F1, the best antibody candidate, was essential for maximal tumor inhibition in the MDA-MB-231 model. Mechanistically, F1 antitumor response was triggered through natural killer cell activation via IL-15 upregulation, associated with granzyme B and perforin production, and the release of antitumor IFNγ cytokine. The F1 antibody also prevented the tumor recruitment of immunosuppressive tumor-associated macrophages M2 and myeloid-derived suppressor cells, a specific effect associated with a less immunosuppressive tumor microenvironment highlighted by TGFß decrease. Finally, the antibody F1 inhibited tumor growth of two TNBC PDXs, isolated from patients resistant or not to neo-adjuvant chemotherapy. CONCLUSION: Cath-D is a tumor-specific extracellular target in TNBC suitable for antibody-based therapy. Immunomodulatory antibody-based strategy against cath-D is a promising immunotherapy to treat patients with TNBC.

Prognostic value of androgen receptor and FOXA1 co-expression in non-metastatic triple negative breast cancer and correlation with other biomarkers.

BACKGROUND: In luminal androgen receptor (AR) tumours, FOXA1 may direct AR to sites occupied by ER in luminal tumours, thus stimulating proliferation. METHODS: AR and FOXA1 expression were evaluated by immunohistochemistry in 333 non-metastatic triple-negative breast cancers (TNBC). Positivity threshold was set at ≥ 1% staining. Lymphocytic infiltration, PD-L1expression, PIK3CA mutations, PTEN defects and BRCA1 promoter methylation were assessed. RESULTS: AR + /FOXA1 + tumours (42.4%) were more frequently: found in older patients, lobular, of lower nuclear grade, with more frequently PIK3CA mutations; exhibited less frequently BRCA1 promoter methylation, defects of PTEN and PD-L1 expression than others. Recurrence-free and overall survivals were significantly lower for AR + /FOXA1 + TNBC (median follow-up: 7.8 years). CONCLUSIONS: AR + /FOXA1 + expression defines a luminal-like TNBC subgroup affected with a worse outcome compared to other TNBC and a higher risk of late recurrences. This subgroup appears enriched in PIK3CA mutations, suggesting a role for PI3K inhibitors in this subgroup.

PKCθ-induced phosphorylations control the ability of Fra-1 to stimulate gene expression and cancer cell migration.

The AP-1 transcription factor Fra-1 is aberrantly expressed in a large number of cancers and plays crucial roles in cancer development and progression by stimulating the expression of genes involved in these processes. However, the control of Fra-1 transactivation ability is still unclear and here we hypothesized that PKCθ-induced phosphorylation could be necessary to obtain a fully active Fra-1 protein. Using MCF7 stable cells overexpressing equivalent levels of unphosphorylated Fra-1 or PKCθ-phosphorylated Fra-1, we showed that PKCθ-induced phosphorylation of Fra-1 was crucial for the stimulation of MMP1 and IL6 expression. Consistently, we found a significant positive correlation between PRKCQ (coding for PKCθ) and MMP1 mRNA expression levels in human breast cancer samples. PKCθ-induced phosphorylations, in part at T217 and T227 residues, strongly and specifically increased Fra-1 transcriptional activity through the stimulation of Fra-1 transactivation domain, without affecting JUN factors. More importantly, these phosphorylations were required for Fra-1-induced migration of breast cancer cells and phosphorylated Fra-1 expression was enriched at the invasion front of human breast tumors. Taken together, our findings indicate that PKCθ-induced phosphorylation could be important for the function of Fra-1 in cancer progression.

[Neoadjuvant treatment of breast cancer: implications for the pathologist]. / Place du pathologiste dans la prise en charge néoadjuvante des cancers du sein.

These past few years, neoadjuvant strategy has taken an increasing place in the management of breast cancer patients. This strategy is mainly indicated to obtain a tumour bulk regression allowing a breast conserving surgery in patients that otherwise would have undergone mastectomy. Of note, development of new chemotherapy agents and targeted therapies has critically helped in the progress of neoadjuvant strategy as it is currently associated with better pathological response rates. In this context, the pathologist is at the crossroad of this multidisciplinary process. First, he provides on the initial core needle biopsy the tumour pathological characteristics that are critical for the choice of treatment strategy, i.e. histological type, histological grade, proliferative activity (mitotic count and Ki67/MIB1 index labeling), hormone receptor status (oestrogen receptor and progesterone receptor) and HER2 status. Secondly, the pathologist evaluates the pathological response and the status of surgical margins with regards to the residual tumour on the surgical specimen after neoadjuvant treatment. These parameters are important for the management of the patient, since it has been shown that complete pathological response is associated with improved disease free survival. Several grading systems are used to assess the pathological response in breast and axillary lymph nodes. The most frequently used in France are currently the systems described by Sataloff et al. and Chevallier et al. In this review, we detail the different steps involving the pathologist in neoadjuvant setting, with special regards to the quality process and future perspectives such as emerging predictive biomarkers.

Increased expression of fatty acid synthase and progesterone receptor in early steps of human mammary carcinogenesis.

Progestins increase the risk of breast cancer in the hormone therapy of menopause, and progesterone receptor-induced fatty acid synthase (FAS) is a potential therapeutical target of breast cancer. In a first attempt to specify in which lesions at risk of breast cancer progestins might be acting, we have compared the progesterone receptor (PR) and FAS expression in preinvasive breast lesions and in adjacent "normal" mammary glands. We used archive paraffin-embedded tissues from 116 patients, with 164 lesions of increasing histological risk from nonproliferative "benign" breast disease (BBD) to in situ breast carcinomas. Immunostaining using our FAS antibody and a PR antibody from Dako was quantified as continuous variables by computer-assisted image analysis. FAS level increased (p < 10(-3) by the Kruskall-Wallis test) in all lesions, starting from nonproliferative BBD, and was maximal in in situ carcinoma. The % of PR-positive cells increased from nonproliferative BBD and was higher in proliferative atypia (p < 10(-3)). It was very low in high-grade DCIS corresponding to a likely different carcinogenesis pathway. There was a trend for a positive correlation between FAS and PR in normal glands. However, the 2 markers increased independently in BBD and were negatively correlated in in situ carcinomas. FAS and PR were positively correlated with Ki67 in BBD. The increased PR level in premalignant steps of mammary carcinogenesis suggests an early increased responsiveness to progestins. The increased FAS expression, in lesions parallel to their increased breast cancer risk, suggests further studies to develop new markers of high-risk lesions and to prevent breast cancer.

Increased estrogen receptor betacx expression during mammary carcinogenesis.

Identification of proteins that markedly vary during early steps of mammary carcinogenesis may help to understand its pathophysiology and to develop a prevention strategy. The expression of total estrogen receptor beta (ERbeta) protein and of its COOH-terminally spliced variant ERbetacx (or ERbeta2) was compared in 43 invasive breast cancers and in 39 adjacent normal mammary glands and 26 ductal carcinoma in situ (DCIS). Thirty-six breast cancers were ER positive by radioligand binding assay. The analysis was done by immunohistochemistry on adjacent sections of formalin-fixed, paraffin-embedded tumors using polyclonal anti-ERbeta 503 IgY and sheep polyclonal ERbetacx antibodies that were previously validated. Nuclear staining was quantified using a computerized image analyzer in selected areas of normal and cancer epithelial cells. Total ERbeta expression was high in normal glands, decreased in DCIS (P = 0.0004), and increased from DCIS to invasive tumors (P = 0.029). In contrast, the ERbetacx expression was low in normal glands, increased significantly in DCIS (P = 0.0014), and continued to increase in invasive carcinomas (P = 0.0027) in both ERalpha-positive and ERalpha-negative tumors. This is the first study showing a significant increase of the ERbetacx variant protein in DCIS and invasive breast cancer compared with adjacent normal glands. This contrasts with the decrease of the total ERbeta level in the same patients and indicates different mechanisms to explain these variations during mammary carcinogenesis. It also suggests a role of the ERbetacx variant in carcinogenesis opposite to the protective effect of the wild-type ERbeta1.

Neoadjuvant percutaneous 4-hydroxytamoxifen decreases breast tumoral cell proliferation: a prospective controlled randomized study comparing three doses of 4-hydroxytamoxifen gel to oral tamoxifen.

PURPOSE: Two chemoprevention randomized studies using tamoxifen showed drug efficacy; however, adverse effects such as hot flushes, endometrial cancer, and above all, thromboembolism, remain a problem. 4 hydroxytamoxifen (4-OHT) is a very active metabolite of tamoxifen. This randomized study was designed to analyze if 4-OHT gel, administered percutaneously on the breast skin, can inhibit the proliferation of malignant breast cells to the same extent as orally administered tamoxifen. PATIENTS AND METHODS: Fifty-five postmenopausal women with an invasive estrogen receptor-positive breast cancer were randomly assigned to receive (for 2 to 3 weeks) either 4-OHT gel (0.5, 1, or 2 mg/d) or oral tamoxifen (20 mg/d) or no treatment. Response was evaluated using proliferation markers (Ki-67, proliferating cell nuclear antigen) and apoptosis markers in tissue samples obtained by Tru-cut biopsy before treatment, and at surgery after treatment. RESULTS: Administration of 4-OHT gel resulted in reductions in tumor tissue proliferation indexes (Ki-67 and PCNA), with approximate equivalence between the 1.0 mg/d or 2.0 mg/d 4-OHT dose, and oral tamoxifen, but had no effect on apoptotic markers. Plasma levels of 4-OHT were consistently higher in the oral tamoxifen group than in the gel groups. No dose-related pattern was shown for estrogen or progesterone receptor levels, and topical 4-OHT gel appeared to be generally well tolerated. Hot flushes are as common in the two higher gel doses as with tamoxifen. CONCLUSION: Percutaneous 4-OHT gel has a local impact on tumor proliferation. It could be tested in future prospective trials of chemoprevention or ductal carcinoma in situ adjuvant hormonotherapy.

Estrogen receptor beta (ER beta) level but not its ER beta cx variant helps to predict tamoxifen resistance in breast cancer.

The antiestrogen tamoxifen, a major endocrine therapy of estrogen receptor (ER)-positive breast cancer, is nevertheless inefficient in 30 to 40% of cases for unknown reasons. We retrospectively studied 50 ER-positive primary breast carcinomas. All of the patients had received tamoxifen as the only adjuvant therapy. They were divided into two groups depending on whether they relapsed within 5 years (16 tamoxifen-resistant cases) or did not relapse within 5 years (34 tamoxifen-sensitive cases). The expression of total ER beta protein, and of ER beta cx protein, was estimated anonymously in formalin-fixed, paraffin-embedded tumor sections, by using specific antibodies and quantifiying nuclear immunostaining with a computer image analyzer. All of the tumors were found to be HER-2/neu-negative by immunohistochemistry. Univariate analysis showed that Scarff-Bloom-Richardsson grade modified by Elston (SBR grade; P < 0.001), tumor size (P = 0.042), and MIB-1 proliferation index (P = 0.02) were significantly higher in tamoxifen-resistant tumors. A low level of total ER beta, whether in percentage of positive cells or in quantitative immunocytochemical (QIC) score, was also associated with tamoxifen resistance (P = 0.004). ER beta cx expression and lymph node status were similar between the two groups. The expression of ER beta in the total population was positively correlated with ER beta cx (r = 0.63, P < 0.001), and was independent of the other parameters. In a multivariate analysis, ER beta expression was the most important variable (P = 0.001), followed by SBR grade (I+II versus III; P = 0.008), and MIB-1 (P = 0.016). To conclude, tamoxifen resistance is associated with classical variables of aggressive tumors (high SBR grade, proliferation index, and tumor size) but not with node invasiveness. Low ER beta level is an additional independent marker, better than ER alpha level, to predict tamoxifen resistance.

Spontaneous apoptosis in the intra-ductal component's stroma of breast invasive carcinoma.

Spontaneous apoptosis by in situ detection of DNA fragmentation (DNAf) was investigated in breast invasive ductal carcinoma (IDC) frozen samples removed from 61 untreated patients. The incidence of DNAf was low in carcinoma cells and was mainly detected in the stroma. In the stroma at a distance from carcinoma cells, DNAf was inversely related to estradiol plasma level variations (p=0.01), indicating that it probably remained under physiological hormonal regulation. In the stroma adjacent to carcinoma cells, DNAf was correlated to tumor progression parameters such as the presence of a comedo intra ductal carcinoma (DCIS) component (p=0.001) and axillary lymph node metastasis (p=0.002), suggesting that this stromal compartment more probably represented a tumoral component closely associated to epithelial tumor cells. Therefore, the detection of DNAf in the adjacent stroma of breast carcinoma could help to predict progression in non invasive tumors and also in invasive tumors in those patients without lymph node invasion.

Immunohistochemical assessment of the peripheral benzodiazepine receptor in breast cancer and its relationship with survival.

PURPOSE: The peripheral benzodiazepine receptor (PBR) expression has been shown dramatically increased in neoplastic tissues and tumor cell lines originated from ovary, liver, colon, breast, or brain relative to untransformed tissues. Its expression has been also associated with tumor progression and aggressiveness. To explore whether PBR expression level could be of prognostic value in invasive breast cancer, we studied a series of 117 patients who underwent surgery for primary breast carcinomas and were followed-up for 8 years. EXPERIMENTAL DESIGN: Using an immunohistochemical approach, we first compared PBR expression in normal and tumoral tissues, then we studied PBR expression together with clinicopathological variables (histological type, histological grade, lymph node, estrogen and progesterone receptor status), and biological markers such as BclII, Ki-67, and HER2/Neu. RESULTS: Our results revealed a significant increase of PBR expression in tumoral versus normal breast cells. We found a negative correlation between PBR expression and estrogen receptor status (P = 0.03) as well as a positive correlation between PBR and Ki-67 (P = 0.044). Although the disease-free survival was not affected by PBR in the whole population, high PBR expression level was significantly correlated with a shorter disease-free survival in the lymph node-negative patients, P = 0.038. CONCLUSIONS: As the axillary lymph node-negative status is generally considered as a good prognosis factor, the high expression of PBR in this patient subgroup may be used to identify a new high risk population, for which a more specific therapy would be beneficial.

Immunohistochemical assessment of the peripheral benzodiazepine receptor in human tissues.

Exhaustive analysis of the location of the peripheral benzodiazepine receptor (PBR) both at the subcellular and the tissue level is warranted to gain a better understanding of its biological roles. To date, many studies have been performed in animal models, such as rat, mouse, and pig, that yielded important information. However, only a few reports were dedicated to the analysis of PBR expression in humans. To enlarge on previous studies, we investigated PBR expression in different human organs using the monoclonal antibody 8D7 that specifically recognized the human PBR. First, we performed electron microscopic analysis that for the first time unambiguously demonstrated the localization of the PBR on the outer mitochondrial membrane. Second, focusing our analysis on human tissues for which information on PBR expression is sparse (lung, stomach, small intestine, colon, thyroid, adrenal gland, pancreas, breast, prostate, ovary), we found that PBR exhibits selective localization. This characterization of PBR localization in human tissues should provide important insights for the understanding of PBR functions.

Identification and pharmacological characterization of SRBP-2: a novel SR31747A-binding protein.

SR31747A is a sigma ligand with potent antiproliferative activity against tumor cells and for which three binding proteins have been identified to date: (a) SRBP-1 (also called sigma 1); (b) HIS; and (c) sigma 2. In this study, we characterized an additional SR31747A binding site, i.e., SRBP-2 (SR31747A-binding protein 2). Using an in silico screening approach, we identified this novel sequence, which exhibits 41% homology with HSI. The 1142-bp cDNA was found to encode a 206 amino acid protein not related to SRBP-1. Northern blot analysis of SRBP-2 mRNA expression revealed a single 1.1-kb transcript that was widely expressed in organs; the liver was particularly enriched, and the brain showed the lowest abundance. A murine homologue that exhibited a similar expression pattern was also characterized. Subcellular localization analysis using specific polyclonal antibodies revealed that SRBP-2 had the same nuclear membrane and endoplasmic reticulum localization as other members of the SR31747A-binding protein family. Considering SRBP-2-binding properties, pharmacological analysis clearly highlighted that SRBP-2 was distinct from sigma 2. Scatchard plot analysis revealed K(d) values of 10 and 3 nM for SR31747A and Tamoxifen, respectively. In contrast with HSI, the protein also did not exhibit detectable isomerase activity. When analyzing SRBP-2 expression in human breast cancer biopsies, we obtained evidence that SRBP-2 expression, together with SRBP-1 and HSI, may be of interest as a prognostic marker. These findings demonstrated that SRBP-2 represents an additional molecular target for SR31747A, which could help to understand the immunosuppressive and antiproliferative effects of the molecule.

Oxidant-antioxidant status in relation to survival among breast cancer patients.

The role of plasma oxidant-antioxidant status in survival after breast cancer surgery was investigated in a cohort of patients (n = 363) hospitalized in Southern France between 1989 and 1992. The median follow-up was 8 years after surgery for histologically confirmed breast cancer. Plasma analyses were performed after diagnosis and before surgery and adjuvant therapy. We found an inverse relationship between plasma lipoperoxides (MDA) and tumor size at diagnosis, together with higher lipoperoxide levels in node-negative tumors than in node-positive ones (TNM). The longitudinal approach revealed an increased risk of recurrence for patients with plasma lipoperoxides in the highest tertile of the sample (RR = 2.1, 95% CI 1.1-4.0). In addition, the risk of recurrence increased (RR = 1.7, 95%CI 1.0-3.0), after adjustment for the known prognostic factors (TNM), for patients with plasma lipid-adjusted vitamin E levels of over 22 micromol/l. The risk of breast cancer death was twice as great for patients with plasma lipid-adjusted vitamin E levels above this value. Excesses of plasma lipoperoxides and vitamin E appear to be factors in poor prognosis for breast cancer-specific survival (OVS) and disease-free survival (DFS), respectively, independent of tumor characteristics at diagnosis. Several hypotheses are advanced to explain the possible role of plasma vitamin E as a factor in poor prognosis for survival.