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Background: Late distant recurrence is a challenge for the treatment of invasive lobular carcinoma (ILC) of the breast. Despite in-depth characterisation of primary ILC, the molecular landscape of metastatic ILC is still only partially understood., Methods: We retrospectively identified 38 ILC patients from the tissue banks of six European institutions. DNA extracted from patient matched primary and metastatic FFPE tissue blocks was whole genome sequenced to compute somatic copy number aberrations. This, in turn, was used to infer the evolutionary history of these patients., Findings: The data show different metastatic seeding patterns, with both an early and late divergence of the metastatic lineage observed in ILC. Additionally, cascading dissemination from a metastatic precursor was a dominant rule. Alterations in key cancer driver genes, such as TP53 or CCND1, were acquired early while additional aberrations were present only in the metastatic branch. In about 30% of the patients, the metastatic lineage harboured less aberrations than the primary tumour suggesting a period of tumour dormancy or prolonged adaptation at the distant site. This phenomenon was mostly observed in tumours from de novo metastatic patients., Interpretation: Our results provide insights into ILC evolution and offer potential paths for optimised ILC care., Funding: This work has received financial support from Les Amis de l'Institut Bordet, MEDIC, the Breast Cancer Research Foundation (BCRF) and the Belgian Fonds National de la Recherche Scientifique (F.R.S-FNRS)., Competing Interests: Declaration of interests C.S.: advisory board (receipt of honoraria or consultations fees): Astellas, Cepheid, Vertex, Seattle genetics, Puma, Amgen. Participation in company sponsored speaker's bureau: Eisai, Prime Oncology, Teva, Foundation Medicine. Other support (travel, accommodation expenses): Roche, Genentech, Pfizer (outside the submitted work). C.M. has received personal consultancy fees from Bayer, Roche, AstraZeneca, Daiichi Sankyo outside the scope of the submitted work; Grants from Italian Association for Cancer Research (AIRC); honoraria for lectures, presentations from Novartis. R.S. reports funding for research not related to the current manuscript from Breast Cancer Research Foundation; honoraria for lectures unrelated to the current manuscript from Bristol Myers Squibb; congress registration and travel not related to the current manuscript from Merck and Bristol Myers Squibb; participation on advisory boards unrelated to the current manuscript from Roche and Bristol Myers Squibb. R.S. has no conflicts of interests related to this project. G.E. reports consulting fees as general medical affairs consultancy from Thermofisher; role as secretary on the board of the scientific working group of the Belgian Society of Pathology and board member of the Belgian Society of Pathology not reimbursed. All other authors have no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The JAK/STAT3 signaling pathway may be aberrantly activated and have various and conflicting roles in breast cancer. The current study explored prognostic implications of activated STAT3 in human epidermal growth factor receptor 2 (HER2)-positive primary breast cancers in the context of a large prospective study (ALTTO). Activated STAT3 was determined by immunohistochemical analysis of STAT3 phosphorylation (Y705) performed on the primary tumors. This analysis evaluated whether patients with activated STAT3 had disease-free survival (DFS) and overall survival (OS) different from patients without activated STAT3. A total of 5694 patients out of the 8381 patients enrolled in ALTTO were included in this analysis (67.9%), and 2634 of them (46%) had evidence of STAT3 activation (minimum tumor Allred score ≥2). The median follow-up was 6.93 years (6.85-6.97 years), at the end of which 1035 (18.18%) and 520 (9.13%) patients experienced DFS and OS events, respectively. Patients with STAT3 activation experienced improved DFS compared to those without it (multivariable hazard ratio [HR], 0.84; 95% confidence interval [CI] 0.74-0.95; P = .006). There were no group differences in OS (multivariable HR, 0.92; 95% CI 0.78-1.10; P = .37). This effect was limited to ER-positive tumors. In conclusion, these findings support the role of STAT3 activation as a marker of favorable outcome in ER-positive/HER2-positive breast cancer patients., (© 2020 Union for International Cancer Control.)

Adipocytes and cancer-associated adipocytes (CAAs) are poorly investigated cells in the tumor microenvironment. Different image analysis software exist for identifying and measuring these cells using scanned hematoxylin and eosin (H&E)-stained slides. It is however unclear which one is the most appropriate for breast cancer (BC) samples. Here, we compared three software (AdipoCount, Adiposoft, and HALO®). HALO® outperformed the other methods with regard to adipocyte identification, (> 96% sensitivity and specificity). All software performed equally good with regard to area and diameter measurement (concordance correlation coefficients > 0.97 and > 0.96, respectively). We then analyzed a series of 10 BCE samples (n = 51 H&E slides) with HALO®. Distant adipocytes were defined >2 mm away from cancer cells or fibrotic region, whereas CAAs as the first three lines of adipocytes close to the invasive front. Intra-mammary heterogeneity was limited, implying that measuring a single region of ∼500 adipocytes provides a reliable estimation of the distribution of their size features. CAAs had smaller areas (median fold-change: 2.62) and diameters (median fold-change: 1.64) as compared to distant adipocytes in the same breast (both p = 0.002). The size of CAAs and distant adipocytes was associated with the body mass index (BMI) of the patient (area: rho = 0.89, p = 0.001; rho = 0.71, p = 0.027, diameter: rho = 0.87 p = 0.002; rho = 0.65 p = 0.049, respectively). To conclude, we demonstrate that quantifying adipocytes in BC sections is feasible by digital pathology using H&E sections, setting the basis for a standardized analysis of mammary adiposity in larger series of patients., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: In breast cancer (BC), axillary lymph node (ALN) involvement is one of the strongest adverse prognostic factors. However, it is unclear whether loco-regional lymph node deposits are effectively the root of secondary metastases or only an indicator of competence of the primary tumour to spread to distant organs., Methods: Here, we investigated the evolutionary trajectories of primary tumour, ALN and distant metastasis samples from 16 estrogen-receptor (ER)-positive lymph node-positive BC patients. Low-pass whole genome sequencing was performed to infer somatic copy number aberrations and the phylogenetic profiles for all patients were obtained., Findings: We show that lymph nodes and distant metastases shared a common origin in only 25% of the cases highlighting that the predominant route of metastatic dissemination is the direct seeding of tumour cells from the primary tumour to distant organs, independently of lymph node metastasis. Noticeably, patients sharing a common origin significantly have worse prognosis., Interpretation: Our results shed light on the routes on which tumour cells metastasize and their role in disease progression in ER-positive BC., Funding: This work has received financial support from Les Amis de l'Institut Bordet, MEDIC, the Breast Cancer Research Foundation (BCRF), the Belgian Fonds National de la Recherche Scientifique (F.R.S-FNRS) and from a grant of the Région Wallonne., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: In the neoadjuvant treatment (NAT) setting, dual HER2-targeted therapy is associated with increased pathologic complete response (pCR) rates compared with each therapy alone. Biomarkers allowing to predict treatment response during NAT are needed. We aim to evaluate whether circulating tumor DNA (ctDNA) is associated with response to anti-HER2-targeted therapy., Experimental Design: Plasma DNA collected before NAT, at week 2, and before surgery from patients enrolled in the NeoALTTO trial was assessed using digital PCR for PIK3CA and TP53 mutation detection., Results: A total of 69 of 455 (15.2%) patients had a PIK3CA and/or TP53 mutation detected in the baseline tumor sample and evaluable ctDNA results from baseline samples. CtDNA was detected in 41%, 20%, and 5% patients before NAT, at week 2, and before surgery, respectively. ctDNA detection before NAT was significantly associated with older age and ER-negative status. ctDNA detection before NAT was associated with decreased odds of achieving pCR (OR = 0.15; 95% CI, 0.034-0.7; P = 0.0089), but not with event-free survival (EFS). Analyses for EFS were underpowered. Interestingly, the patients with HER2-enriched subtype tumors and undetectable ctDNA at baseline had the highest pCR rates. In contrast, patients with persistent ctDNA detection at baseline and week 2 had the lowest rate of pCR., Conclusions: ctDNA detection before neoadjuvant anti-HER2 therapies is associated with decreased pCR rates. Interestingly, patients with HER2-enriched tumors and undetectable ctDNA at baseline had the highest pCR rates, therefore appearing as the best candidates for treatment deescalation strategies., (©2019 American Association for Cancer Research.)

Background: Invasive lobular breast cancer (ILC) is the second most common histological subtype of breast cancer after invasive ductal cancer (IDC). Here, we aimed at evaluating the prevalence, levels, and composition of tumor-infiltrating lymphocytes (TILs) and their association with clinico-pathological and outcome variables in ILC, and to compare them with IDC., Methods: We considered two patient series with TIL data: a multicentric retrospective series (n = 614) and the BIG 02-98 study (n = 149 ILC and 807 IDC). We compared immune subsets identified by immuno-histochemistry in the ILC (n = 159) and IDC (n = 468) patients from the Nottingham series, as well as the CIBERSORT immune profiling of the ILC (n = 98) and IDC (n = 388) METABRIC and The Cancer Genome Atlas patients. All ILC/IDC comparisons were done in estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative tumors. All statistical tests were two-sided., Results: TIL levels were statistically significantly lower in ILC compared with IDC (fold-change = 0.79, 95% confidence interval = 0.70 to 0.88, P < .001). In ILC, high TIL levels were associated with young age, lymph node involvement, and high proliferative tumors. In the univariate analysis, high TIL levels were associated with worse prognosis in the retrospective and BIG 02-98 lobular series, although they did not reach statistical significance in the latter. The Nottingham series revealed that the levels of intratumoral but not total CD8+ were statistically significantly lower in ILC compared with IDC. Comparison of the CIBERSORT profiles highlighted statistically significant differences in terms of immune composition., Conclusions: This study shows differences between the immune infiltrates of ER-positive/HER2-negative ILC and IDC in terms of prevalence, levels, localization, composition, and clinical associations.

In the present study, in order to investigate the role of signal transducer and activator of transcription 3 (STAT3) in estrogen receptor (ER)-positive breast cancer prognosis, we evaluated the phosphorylated STAT3 (p-STAT3) status and investigated its effect on the outcome in a pooled analysis and in a large prospective adjuvant trial. By using the TCGA repository, we developed gene signatures that reflected the level of p-STAT3. Using pooled analysis of the expression data from luminal breast cancer patients, we assessed the effects of the p-STAT3 expression signature on prognosis. We further validated the p-STAT3 prognostic effect using immunohistochemistry (IHC) and immunofluorescence staining of p-STAT3 tissue microarrays from a large randomised prospective trial. Our analysis demonstrated that p-STAT3 expression was elevated in luminal A-type breast cancer (Kruskal-Wallis test, P<10e-10) and was significantly associated with a good prognosis (log-rank, P<10e-10). Notably, the p-STAT3 expression signature identified patients with a good prognosis irrespective of the luminal subtype (log-rank: luminal A, P=0.026; luminal B, P=0.006). p-STAT3 staining by IHC in the stroma or tumour was detected in 174 out of 610 ER-positive samples (28.5%) from the BIG 2-98 randomised trial. With a median follow-up of 10.1 years, p-STAT3 was associated with a reduced risk of recurrence in ER-positive/HER2-negative breast cancer (Cox univariate HR, 0.66; 95% CI, 0.44-0.98; P=0.04). On the whole, our data indicate that p-STAT3 is associated with an improved outcome in ER-positive breast cancer.

Several studies using genome-wide molecular techniques have reported various degrees of genetic heterogeneity between primary tumours and their distant metastases. However, it has been difficult to discern patterns of dissemination owing to the limited number of patients and available metastases. Here, we use phylogenetic techniques on data generated using whole-exome sequencing and copy number profiling of primary and multiple-matched metastatic tumours from ten autopsied patients to infer the evolutionary history of breast cancer progression. We observed two modes of disease progression. In some patients, all distant metastases cluster on a branch separate from their primary lesion. Clonal frequency analyses of somatic mutations show that the metastases have a monoclonal origin and descend from a common 'metastatic precursor'. Alternatively, multiple metastatic lesions are seeded from different clones present within the primary tumour. We further show that a metastasis can be horizontally cross-seeded. These findings provide insights into breast cancer dissemination.

Purpose: Invasive lobular breast cancer (ILBC) is the second most common histologic subtype after invasive ductal breast cancer (IDBC). Despite clinical and pathologic differences, ILBC is still treated as IDBC. We aimed to identify genomic alterations in ILBC with potential clinical implications., Methods: From an initial 630 ILBC primary tumors, we interrogated oncogenic substitutions and insertions and deletions of 360 cancer genes and genome-wide copy number aberrations in 413 and 170 ILBC samples, respectively, and correlated those findings with clinicopathologic and outcome features., Results: Besides the high mutation frequency of CDH1 in 65% of tumors, alterations in one of the three key genes of the phosphatidylinositol 3-kinase pathway, PIK3CA, PTEN, and AKT1, were present in more than one-half of the cases. HER2 and HER3 were mutated in 5.1% and 3.6% of the tumors, with most of these mutations having a proven role in activating the human epidermal growth factor receptor/ERBB pathway. Mutations in FOXA1 and ESR1 copy number gains were detected in 9% and 25% of the samples. All these alterations were more frequent in ILBC than in IDBC. The histologic diversity of ILBC was associated with specific alterations, such as enrichment for HER2 mutations in the mixed, nonclassic, and ESR1 gains in the solid subtype. Survival analyses revealed that chromosome 1q and 11p gains showed independent prognostic value in ILBC and that HER2 and AKT1 mutations were associated with increased risk of early relapse., Conclusion: This study demonstrates that we can now begin to individualize the treatment of ILBC, with HER2, HER3, and AKT1 mutations representing high-prevalence therapeutic targets and FOXA1 mutations and ESR1 gains deserving urgent dedicated clinical investigation, especially in the context of endocrine treatment., (© 2016 by American Society of Clinical Oncology.)

Introduction: Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement., Methods: CellSearch® images (N = 272) of either CTCs or white blood cells or artifacts from 109 non-metastatic (M0) and 22 metastatic (M1) breast cancer patients from reported studies were sent to 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories. Each image was scored as No CTC vs CTC HER2- vs CTC HER2+. The 8 Veridex readers were summarized to a Veridex Consensus (VC) to compare each academic reader using % agreement and kappa (κ) statistics. Agreement was compared according to disease stage and CTC counts using the Wilcoxon signed rank test., Results: For CTC definition (No CTC vs CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range 0.37 to 0.93). Lower agreement was observed in images from M0 (median 91%, range 70 to 96%) compared to M1 (median 98%, range 64 to 100%) patients (P < 0.001) and from M0 and <3CTCs (median 87%, range 66 to 95%) compared to M0 and ≥3CTCs samples (median 95%, range 77 to 99%), (P < 0.001). For CTC HER2 expression (HER2- vs HER2+), the median agreement was 87% (range 51 to 95%) with a median κ of 0.74 (range 0.25 to 0.90)., Conclusions: The inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts. Continuous training and independent image review are required.

Current evaluation of histological sections of breast cancer samples remains unsatisfactory. The search for new predictive and prognostic factors is ongoing. Infrared spectroscopy and its potential to probe tissues and cells at the molecular level without requirement for contrast agents could be an attractive tool for clinical and diagnostic analysis of breast cancer. In this study, we report the successful application of FTIR (Fourier transform infrared) imaging for breast tissue component characterization. We show that specific FTIR spectral signatures can be assigned to the major tissue components of breast tumor samples. We demonstrate that a tissue component classifier can be built based on a spectral database of well-annotated tissues and successfully validated on independent breast samples. We also demonstrate that spectral features can reveal subtle differences within a tissue component, capturing for instance lymphocytic and stromal activation. By investigating in parallel lymph nodes, tonsils and wound healing tissues, we prove the uniqueness of the signature of both lymphocytic infiltrate and tumor microenvironment in the breast disease context. Finally, we demonstrate that the biochemical information reflected in the epithelial spectra might be clinically relevant for the grading purpose, suggesting potential to improve breast cancer management in the future.

Purpose: Previous preclinical and clinical data suggest that the immune system influences prognosis and response to chemotherapy (CT); however, clinical relevance has yet to be established in breast cancer (BC). We hypothesized that increased lymphocytic infiltration would be associated with good prognosis and benefit from immunogenic CT-in this case, anthracycline-only CT-in selected BC subtypes., Patients and Methods: We investigated the relationship between quantity and location of lymphocytic infiltrate at diagnosis with clinical outcome in 2009 node-positive BC samples from the BIG 02-98 adjuvant phase III trial comparing anthracycline-only CT (doxorubicin followed by cyclophosphamide, methotrexate, and fluorouracil [CMF] or doxorubicin plus cyclophosphamide followed by CMF) versus CT combining doxorubicin and docetaxel (doxorubicin plus docetaxel followed by CMF or doxorubicin followed by docetaxel followed by CMF). Readings were independently performed by two pathologists. Disease-free survival (DFS), overall survival (OS), and interaction with type of CT associations were studied. Median follow-up was 8 years., Results: There was no significant prognostic association in the global nor estrogen receptor (ER) -positive/human epidermal growth factor receptor 2 (HER2) -negative population. However, each 10% increase in intratumoral and stromal lymphocytic infiltrations was associated with 17% and 15% reduced risk of relapse (adjusted P = .1 and P = .025), respectively, and 27% and 17% reduced risk of death in ER-negative/HER2-negative BC regardless of CT type (adjusted P = .035 and P = .023), respectively. In HER2-positive BC, there was a significant interaction between increasing stromal lymphocytic infiltration (10% increments) and benefit with anthracycline-only CT (DFS, interaction P = .042; OS, P = .018)., Conclusion: In node-positive, ER-negative/HER2-negative BC, increasing lymphocytic infiltration was associated with excellent prognosis. Further validation of the clinical utility of tumor-infiltrating lymphocytes in this context is warranted. Our data also support the evaluation of immunotherapeutic approaches in selected BC subtypes.

Breast cancer is a molecularly, biologically and clinically heterogeneous group of disorders. Understanding this diversity is essential to improving diagnosis and optimizing treatment. Both genetic and acquired epigenetic abnormalities participate in cancer, but the involvement of the epigenome in breast cancer and its contribution to the complexity of the disease are still poorly understood. By means of DNA methylation profiling of 248 breast tissues, we have highlighted the existence of previously unrecognized breast cancer groups that go beyond the currently known 'expression subtypes'. Interestingly, we showed that DNA methylation profiling can reflect the cell type composition of the tumour microenvironment, and in particular a T lymphocyte infiltration of the tumours. Further, we highlighted a set of immune genes having high prognostic value in specific tumour categories. The immune component uncovered here by DNA methylation profiles provides a new perspective for the importance of the microenvironment in breast cancer, holding implications for better management of breast cancer patients., (Copyright © 2011 EMBO Molecular Medicine.)

Purpose: Circulating Tumor Cells (CTCs) detection and phenotyping are currently evaluated in Breast Cancer (BC). Tumor cell dissemination has been suggested to occur early in BC progression. To interrogate dissemination in BC, we studied CTCs and HER2 expression on CTCs across the spectrum of BC staging., Methods: Spiking experiments with 6 BC cell lines were performed and blood samples from healthy women and women with BC were analyzed for HER2-positive CTCs using the CellSearch®., Results: Based on BC cell lines experiments, HER2-positive CTCs were defined as CTCs with HER2 immunofluorescence intensity that was at least 2.5 times higher than the background. No HER2-positive CTC was detected in 42 women without BC (95% confidence interval (CI) 0-8.4%) whereas 4.1% (95%CI 1.4-11.4%) of 73 patients with ductal/lobular carcinoma in situ (DCIS/LCIS) had 1 HER2-positive CTC/22.5 mL, 7.9%, (95%CI 4.1-14.9%) of 101 women with non metastatic (M0) BC had ≥1 HER2-positive CTC/22.5 mL (median 1 cell, range 1-3 cells) and 35.9% (95%CI 22.7-51.9%) of 39 patients with metastatic BC had ≥1 HER2-positive CTC/7.5 mL (median 1.5 cells, range 1-42 cells). In CTC-positive women with DCIS/LCIS or M0 BC, HER2-positive CTCs were more commonly detected in HER2-positive (5 of 5 women) than HER2-negative BC (5 of 12 women) (p = 0.03)., Conclusion: HER2-positive CTCs were detected in DCIS/LCIS or M0 BC irrespective of the primary tumor HER2 status. Nevertheless, their presence was more common in women with HER2-positive disease. Monitoring of HER2 expression on CTCs might be useful in trials with anti-HER2 therapies.

Purpose: Optimal management of breast ductal carcinoma in situ (DCIS) is controversial, and many patients are still overtreated. The local death of myoepithelial cells (MECs) is believed to be a pre-requisite to tumor invasion. We thus hypothesized that loss of CD10 expression, a MEC surface peptidase, would signify basement membrane disruption and confer increased risk of relapse in DCIS. The aim of our study was to retrospectively evaluate the prognostic value of CD10 in DCIS., Experimental Design: CD10 expression was evaluated by quantitative RT-PCR and immunohistochemistry using paraffin-embedded samples of normal breast tissue (n = 11); of morphologically normal ducts associated with DCIS (n = 10); and of DCIS without an invasive component (n = 154)., Results: CD10 immunostaining was only observed in MECs in normal tissue and in DCIS. Normal tissue showed high mRNA expression levels of CD10, whereas DCIS showed a variable range. After a median follow-up of 6 years, DCIS with CD10 expression below the levels observed in normal tissue (71%) demonstrated a higher risk of local relapse (HR = 1.88; [95CI:1.30-2.70], p = 0.001) in univariate analysis. No relapse was observed in patients expressing high CD10 mRNA levels (29%) similar to the ones observed in normal tissue. In multivariate analysis including known prognostic factors, low CD10 mRNA expression remained significant (HR = 2.25; [95%CI:1.24-4.09], p = 0.008), as did the recently revised Van Nuys Prognostic Index (VNPI) score (HR = 2.03; [95%CI:1.23-3.35], p = 0.006)., Conclusion: The decrease of CD10 expression in MECs is associated with a higher risk of relapse in DCIS; this knowledge has the potential to improve DCIS management.

Background: Proliferation and tumor differentiation captured by the genomic grade index (GGI) are important prognostic indicators in breast cancer (BC) especially for the estrogen receptor positive (ER+) disease. The aims of this study were to convert this microarray index to a qRT-PCR assay (PCR-GGI), which could be realized on formalin fixed paraffin embedded samples (FFPE), and to assess its prognostic performance and predictive value of clinical benefit in early and advanced ER+ BC patients treated with tamoxifen., Methods: The accuracy and concordance of the PCR-GGI with the GGI was assessed using BC patients for which frozen and FFPE tissues as well as microarray data were available (n = 19). The evaluation of the prognostic value of the PCR-GGI was assessed on FFPE material using a consecutive series of 212 systemically treated early BC patients. The predictive performance for tamoxifen benefit was assessed using two ER+ BC populations treated either with adjuvant tamoxifen only (n = 77+139) or first-line tamoxifen for advanced disease (n = 270)., Results: The PCR-GGI is based on the expression of 8 genes (4 representative of the GGI and 4 reference genes). A significant correlation was observed between the microarray-derived GGI and the qRT-PCR assay using frozen (rho = 0.95, p < 10E-06) and FFPE material (rho = 0.89, p < 10E-06). The prognostic performance of the PCR-GGI was confirmed on FFPE samples (HRunivar. = 1.89; [95CI:1.01-3.54], p = 0.05). The PCR-GGI further identified two subgroups of patients with statistically different time to distant metastasis free survival (DMFS) across the two cohorts of ER+ BC patients treated with adjuvant tamoxifen. Additionally, the PCR-GGI was associated with response to tamoxifen in the advanced setting (HRunivar. = 1.98; [95CI:1.51-2.59], p = 6.9E-07)., Conclusion: PCR-GGI recapitulates in an accurate and reproducible manner the performances of the GGI using frozen and FFPE samples.

The farnesoid X receptor (FXR, NR1H4), a member of the nuclear receptor superfamily of ligand-dependent transcription factors, is normally produced in the liver and the gastrointestinal tract, where it acts as a bile acid sensor. It has been recently detected in breast cancer cell lines and tissue specimens. The expression of FXR was scored (0-8) by immunohistochemistry on 204 breast cancer samples and correlated with established cancer biomarkers. Moreover, the effect of the FXR activator chenodeoxycholic acid (CDCA) was determined on cell proliferation and estrogen receptor regulation/activation in breast cancer cell lines. FXR was detected in 82.4% of samples with a high median expression score of 5. FXR expression significantly correlated with estrogen receptor (ER) expression (P = 0.009) and luminal-like markers. In ER-positive tumors, FXR expression was significantly correlated with the proliferation marker Ki-67 (P < 0.001) and the nodal status (P = 0.028), but only so in postmenopausal women, suggesting that lack of estrogens may disclose the association between FXR and cell proliferation. In vitro experiments confirmed clinical data since CDCA stimulated the proliferation of ER-positive cells only in steroid-free medium, a stimulation inhibited upon siRNA-silencing of FXR expression as well as ER blockade by antiestrogens. Moreover, co-immunoprecipitation experiments revealed that CDCA activated-FXR interacted with ER. These results suggest that ER-positive breast tumors could be stimulated to proliferate via a crosstalk between FXR and ER, particularly in a state of estrogen deprivation (menopause, aromatase inhibitors).

Approximately 50% of patients with estrogen receptor (ER)-positive breast cancer (BC) and 70%-80% of patients with ER-positive and progesterone receptor (PgR)-positive BC respond to hormonal therapy. Additional predictive markers are needed. A group of 287 patients with ER- and/or PgR-positive tumors was selected from 804 patients previously enrolled in a multicenter phase III trial. Bcl-2 expression was evaluated and correlated with response to adjuvant tamoxifen and survival. Estrogen receptor and PgR were determined by biochemical means and Bcl-2 by immunohistochemistry. With a median follow-up of 76 months (95 relapses and 60 deaths), of the 287 patients with, 187 (65%) had Bcl-2-positive tumors and 78 of these patients received tamoxifen. Of the 100 patients with Bcl-2-negative disease, 51 received tamoxifen and 49 regular follow-up. Using patients treated with tamoxifen as a reference, a univariate analysis of disease-free interval for patients who did not receive tamoxifen showed a hazard ratio (HR) of 1.42 (95% CI, 0.82-2.44; P = 0.21) for patients with Bcl-2-positive disease and a HR of 1.05 (95% CI, 0.55-1.99; P = 0.89) for patients with Bcl-2-negative disease (P = 0.48). After adjusting for number of positive lymph nodes, degree of receptor and PgR positivity, and type of surgery, the HRs were 1.54 (95% CI, 0.87-2.73; P = 0.14) for Bcl-2-positive disease and 1.05 (95% CI, 0.52-2.11; P = 0.88) for Bcl-2-negative disease. Despite its being a retrospective nonrandomized study with a relatively low number of patients, our results suggest that Bcl-2 deserves further evaluation as a predictive factor of sensitivity to tamoxifen.

Purpose: The predictive value of topoisomerase-II alpha (topo-II) has been evaluated in advanced breast cancer patients randomly treated with single-agent doxorubicin or docetaxel., Experimental Design: Primary tumor samples from patients enrolled in a randomized, phase III clinical trial comparing single-agent doxorubicin (75 mg/m(2) q3wks) with docetaxel (100 mg/m(2) q3wks) were collected and topo-II status was evaluated by immunohistochemistry (clone KiS1)., Results: Topo-II status was evaluated in 108 samples, 55 (51%) in the doxorubicin arm and 53 (49%) in the docetaxel arm. An increment of 10% in cells expressing topo-II is associated with a statistically significant odds ratio (OR; 95% confidence interval) of 1.09 (1.03-1.15; P = 0.002) for overall response to doxorubicin versus 1.002 (0.94-1.07; P = 0.95) in the docetaxel arm. With increasing topo-II, the favorable OR for overall response to docetaxel compared with doxorubicin decreases to become not significant in patients with topo-II tumor content >10%. In a multivariate analysis, (a) HER-2 status seems positively correlated with overall response to chemotherapy (OR, 2.34; 95% confidence interval, 0.87-6.27; P = 0.09). (b) Overall response to doxorubicin is significantly lower than overall response to docetaxel (OR, 0.17; 95% confidence interval, 0.04-0.64; P = 0.009) but with a significant interaction term for doxorubicin-treated patients with topo-II tumor content >10% (OR, 8.31; 95% confidence interval, 1.86-37.03; P = 0.05)., Conclusions: (a) Topo-II overexpression confers a higher probability of response in the doxorubicin arm only. (b) Despite being a small retrospective study, this study is in line with previously reported studies and the hypotheses raised are now being tested in a prospective neoadjuvant trial.

This study investigated the degree of interlaboratory agreement when HER-2/neu was evaluated by immunohistochemistry (IHC) on archival primary breast cancer samples. IHC for HER-2/neu was performed on the same archival tissue sections from 394 invasive primary breast cancers in two different laboratories. Both laboratories used the primary antibody NCL-CB11; however, different methods of immunostaining (antigen retrieval procedure and manual processing or no antigen retrieval and autostainer processing) as well as different scoring systems were used. Fluorescence in situ hybridization (FISH), considered as the correlation method for HER-2/neu status determination, was performed using the PathVysion kit and compared to the IHC results. Forty-eight of 394 analyzed tumors (12.2%) were scored as HER-2/neu positive in one laboratory, and 109 (27.7%) in the other laboratory where antigen retrieval was performed. Complete concordance in categorization of HER-2/neu status between the two laboratories was achieved in 333 of 394 cases (84.5%). FISH performed in 248 formalin-fixed samples revealed HER-2/neu gene amplification in 55/248 (22.2%). Concordance of FISH and IHC was found in 211/248 cases (85.1%) and 220/248 cases (88.7%) when the CB11 antibody was used without and with antigen retrieval, respectively. Both IHC methods generated similar rates of false results, but with different positive predictive values. Our data demonstrate that HER-2/neu evaluation by IHC is not a reproducible technique if there is no standardization of the procedure.

Background: HER-2/neu status was determined by immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) methods in more than 300 paraffin-embedded primary breast cancer samples., Materials and Methods: HER-2/neu status was determined by FISH using the PathVysion kit (Vysis) and by IHC using either a monoclonal antibody CB11 or a cocktail of antibodies: the monoclonal TAB250 and the polyclonal pAb1., Results: Of the 324 cases evaluable by IHC, 65 out of 318 (20%) and 24 out of 324 (7%) were scored as positive when using the antibody cocktail and the CB11, respectively. HER-2/neu gene amplification occured in 64 out of 324 cases (20%). Concordance of FISH and IHC was found in 285 out of 318 cases (90%) and 278 out of 324 cases (86%) using the cocktail and the CB11, respectively., Conclusion: The cost-effectiveness analysis revealed that the use of a sensitive IHC method followed by confirmation of positive results by FISH considerably decreased the FISH costs and may become standard practice for HER-2/neu evaluation.

Purpose: The purpose of this study is to evaluate HER-2 and topoisomerase IIalpha (topo IIalpha) as candidates for predicting the activity of anthracyclines in the adjuvant treatment of breast cancer patients., Experimental Design: In this study, we evaluated HER-2 and topo IIalpha gene aberrations by fluorescence in situ hybridization in a series of 430 primary breast cancer samples. Samples came from node-positive breast cancer patients randomly treated either with one of two anthracycline-based regimens [full-dose epirubicin-cyclophosphamide (HEC) and moderate-dose epirubicin-cyclophosphamide (EC)] or with cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) in the context of a Phase III adjuvant therapy trial. Event-free survival comparisons were performed between the three study arms in the subgroups of HER-2-amplified and nonamplified tumors. An explorative analysis was also performed to evaluate the predictive value of topo IIalpha in the cohort of HER-2-amplified patients., Results: HER-2 amplification was observed in 73 of the 354 evaluable samples (21%), whereas topo IIalpha amplification was found in 23 of the 61 evaluable HER-2-amplified tumors (38%). The three event-free survival comparisons were CMF versus HEC, CMF versus EC, and EC versus HEC. Hazard ratios (HRs) and 95% confidence intervals (CIs) were as follows: (a) CMF versus HEC, HR = 1.42 for HER-2-amplified tumors (95% CI, 0.54-3.76; P = 0.48) and 0.84 for HER-2-nonamplified tumors (95% CI, 0.49-1.44; P = 0.53); (b) CMF versus EC, HR = 1.65 for HER-2-amplified tumors (95% CI, 0.66-4.13; P = 0.29) and 0.66 for HER-2-nonamplified tumors (95% CI, 0.39-1.10; P = 0.11); and (c) EC versus HEC, HR = 0.93 for HER-2-amplified tumors (95% CI, 0.31-2.77, P = 0.90) and 1.33 for HER-2-nonamplified tumors (95% CI, 0.82-2.14; P = 0.25). Observed HRs suggest that the anthracycline-based therapy could be more effective than CMF in the subgroup of HER-2-amplified patients, whereas treatments could be equally active in the HER-2-nonamplified cohort. topo IIalpha evaluation suggests that the superiority of anthracyclines over CMF in HER-2-amplified patients could be confined to the subgroup of topo IIalpha-amplified tumors., Conclusions: HER-2 could have a predictive value for the activity of anthracycline-based regimens in the adjuvant therapy of breast cancer patients. The predictive value of HER-2 would most likely be related to the concomitant amplification of the topo IIalpha gene.