A New Genetic Risk Score to Predict the Outcome of Locally Advanced or Metastatic Breast Cancer Patients Treated With First-Line Exemestane: Results From a Prospective Study.

INTRODUCTION: Approximately 50% of locally advanced or metastatic breast cancer (MBC) patients treated with first-line exemestane do not show objective response and currently there are no reliable biomarkers to predict the outcome of patients using this therapy. The constitutive genetic background might be responsible for differences in the outcome of exemestane-treated patients. We designed a prospective study to investigate the role of germ line polymorphisms as biomarkers of survival. PATIENTS AND METHODS: Three hundred two locally advanced or MBC patients treated with first-line exemestane were genotyped for 74 germ line polymorphisms in 39 candidate genes involved in drug activity, hormone balance, DNA replication and repair, and cell signaling pathways. Associations with progression-free survival (PFS) and overall survival (OS) were tested with multivariate Cox regression. Bootstrap resampling was used as an internal assessment of results reproducibility. RESULTS: Cytochrome P450 19A1-rs10046TC/CC, solute carrier organic anion transporter 1B1-rs4149056TT, adenosine triphosphate binding cassette subfamily G member 2-rs2046134GG, fibroblast growth factor receptor-4-rs351855TT, and X-ray repair cross complementing 3-rs861539TT were significantly associated with PFS and then combined into a risk score (0-1, 2, 3, or 4-6 risk points). Patients with the highest risk score (4-6 risk points) compared with ones with the lowest score (0-1 risk points) had a median PFS of 10 months versus 26.3 months (adjusted hazard ratio [AdjHR], 3.12 [95% confidence interval (CI), 2.18-4.48]; P < .001) and a median OS of 38.9 months versus 63.0 months (AdjHR, 2.41 [95% CI, 1.22-4.79], P = .012), respectively. CONCLUSION: In this study we defined a score including 5 polymorphisms to stratify patients for PFS and OS. This score, if validated, might be translated to personalize locally advanced or MBC patient treatment and management.

Pathological complete response rates following different neoadjuvant chemotherapy regimens for operable breast cancer according to ER status, in two parallel, randomized phase II trials with an adaptive study design (ECTO II).

Sequential doxorubicin/paclitaxel (AT) followed by CMF treatment was shown to be an active neoadjuvant chemotherapy regimen in the first European Cooperative Trial in Operable Breast Cancer (ECTO I trial). The aim of the current study (ECTO II) is to assess the complete pathological response (pCR) rate following three different anthracycline and taxane-containing neoadjuvant chemotherapy regimens, with or without capecitabine (X). Patients with operable, invasive breast cancer > 2.0 cm in diameter, were randomized to AT→CMF, AT→CMX or AC→TX regimens in two parallel, randomized, open-label, phase II trials (within a single study) in patients with estrogen receptor negative (ER-) and estrogen receptor positive (ER+) diseases, respectively. Exemestane was delivered concomitantly with neoadjuvant chemotherapy in ER+ tumors. Achievement of pCR was more common in ER- than ER+ women (45.3 vs. 10.4%). Capecitabine was only associated with a higher frequency of pCR in ER+ patients receiving AT→CMX. Overall response rates (ORR) ranged from 88 to 97%, and this translated into high rates of breast-conserving surgery (67% of ER- patients and 72% of ER+ patients). All three regimens were well tolerated. Febrile neutropenia and gastrointestinal effects were the most common grade ≥ 3 adverse events. As expected, the ECTO II study showed higher pCR rates in patients with ER- disease. Substituting capecitabine for fluorouracil (± methotrexate) in anthracycline/taxane-containing regimens appeared to be beneficial only in ER+ tumors. Translational studies investigating interactions between therapeutic agents and tumor biology are warranted to refine patient selection and improve the results of neoadjuvant chemotherapy.

A liquid chromatography-tandem mass spectrometry method for the simultaneous determination of exemestane and its metabolite 17-dihydroexemestane in human plasma.

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantitation of exemestane (Exe) and its main metabolite 17-dihydroexemestane (DhExe) in human plasma. The analytes were extracted by protein precipitation with acetonitrile, containing stable 13C-labelled Exe (13C3-Exe) as internal standard, and measured by LC-MS/MS. The best chromatographic separation of the analytes from the interferences was achieved by using a Phenyl column operating under isocratic regime conditions. The total chromatographic runtime was 5.0 min and the elution of Exe and DhExe occurred at 2.5 min and 2.9 min, respectively. Quantitation was performed by employing the positive electrospray ionization (ESI) technique and multiple reaction monitoring mode (MRM). The monitored precursor to product-ion transitions for Exe, DhExe and 13C3-Exe internal standard were m/z 297.0 --> 120.8, m/z 299.1 --> 134.9 and m/z 300.0 --> 123.2, respectively. The lower limit of quantitation (LLOQ) was 0.1 ng/ml for DhExe and 0.2 ng/ml for Exe. The method was linear up to 36-51 ng/ml with r2 > or = 0.998. The intra- and inter-assay precision were < or = 7.7% and 5.1% for Exe and < or = 8.1 and 4.9% for DhExe while deviations from nominal values were in the 1.5-13.2% and - 9.0-5.8% ranges for Exe and DhExe, respectively. The analytical method resulted robust and suitable for pharmacokinetic monitoring of Exe and its main metabolite during adjuvant therapy in patients with breast cancer.

Correlation between genetic and biological aspects in primary non-metastatic breast cancers and corresponding synchronous axillary lymph node metastasis.

This study investigated the changes, if any, in the level of expression of a well defined panel of cell proliferation, differentiation and apoptosis markers between the primary breast tumor and the corresponding synchronous lymph node metastasis from a population of patients with a comparable disease status, in terms of clinical features, and natural history.Ninety pure invasive ductal carcinomas with 10 or more axillary lymph nodes involved and without evidence of distant metastasis were included in this study. Primary tumor and corresponding metastatic lymph node tissue specimens were evaluated for the expression of Cyclin B1, MMP1 metalloproteinase, ICAM-1, RARbeta, Ki67, ER, PgR, p53, bcl-2 and c-erbB2 by immunohistochemistry using standard methods. The bivariate Pearson correlation analysis demonstrated a close relationship between primary and matching corresponding metastatic node. A high grade of correlation has been maintained even when staining results where categorized as positive/negative according to each one marker cut-off level of staining expression. We report the most extensive immunohistochemical analysis of biological determinants in a well defined population of patients with invasive ductal carcinomas and involvement of 10 or more axillary nodes and no distant metastasis. We found a close correlation between the primary tumor and corresponding metastatic node in terms of the expression of all 10 of the markers investigated in this study. The not complete concordance observed could be explained by the gene expression modulation by extrinsic factors and by the microenvironment in which the cancer cells reside.

The impact of progenitor enrichment, serum, and cytokines on the ex vivo expansion of mobilized peripheral blood stem cells: a controlled trial.

The aim of this study was to verify, and possibly improve, culture conditions to expand human mobilized peripheral blood stem cells (PBSCs). We investigated the role of three parameters: A) the culture medium (serum-free versus serum-dependent); B) the initial cell population (Ficoll-separated mononucleated cells versus CD34(+)-selected cells), and C) the low concentration of recombinant cytokines, flt3 ligand, and thrombopoietin in association with a basic cocktail of stem cell factor, interleukin (IL)-6, IL-3, GM-CSF, and erythropoietin. Eighteen leukapheresis samples were monitored in static culture for 15 days. The expansion potential was assessed at day 10 and 15 by total nuclear cells, colony-forming-units (CFUs) (burst-forming units-erythroid [BFU-E], colony-forming units-granulocyte-macrophage [CFU-GM], and colony-forming units-granulocyte-erythroid-macrophage-megakaryocyte [CFU-GEMM]), and flow cytometry immunophenotyping (CD34(+)/CD38(-), CD38(+), CD33(+), CD41(+), GlyA(+) progenitor cells). The results, evaluated by multivariate analysis of variance, emphasize that some variables affected the outcome of stem and progenitor cell expansion. CD34(+) enrichment increased expansion of total nuclear cells, number of CD38(+) and CD33(+) late precursors, and number of the CFU-GM compartment. Interestingly, however, quantitative expansion of GlyA(+) and the early progenitor cells (CD34(+)/CD38(-), CFU-GEMM, BFU-E) are favored by the use of unselected mononucleated cells. Regarding the role of serum, no significant difference was observed except for expansion of total nuclear cells, CFU-GM, and BFU-E. Cytokine combinations, in particular the use of flt3 ligand, stimulated expansion of almost all the cellular subsets, reaching a statistical significance for total nuclear cells and CFU-GM. Our study indicates that progenitor and late precursor multilineage cell compartments of mobilized PBSCs may be significantly expanded in short-term cultures by well-defined experimental conditions. Furthermore, these data might be useful when evaluating ex vivo expansion of hematopoietic cells for clinical purposes.

Human fibroblasts from normal and malignant breast tissue grown in vitro show a distinct senescence profile and telomerase activity.

The telomerase activity and the senescence profile of cultured breast fibroblasts from normal human interstitial and malignant stromal tissue were studied in comparison with their proliferation and differentiation pattern. Fibroblasts were grown either in the presence or absence of a conditioned medium (CM) obtained from cultures of the oestrogen receptor-positive breast cancer MCF-7 cell line. At different passages (from the 2nd up to the 48th), fibroblasts were examined for the telomerase activity by the Telomerase Repeats Amplification Protocol (TRAP) assay, for proliferation profile by Ki-67 antigen expression, and the myofibroblast or smooth muscle cell-like differentiation pattern by immunofluorescence with monoclonal antibodies specific for smooth muscle markers. Serial passages of fibroblasts from normal or tumour breast reveal that the relationship between the levels of telomerase activity and phenotypic/proliferation profile changes with cell subcultivation in a different manner in the two cell populations. The fibroblasts from normal tissue completed 12 passages in a CM-independent way prior to senescence whereas fibroblasts from tumour stroma senescence were attained after 48 passages. These cells showed a marked decrease of telomerase activity, growth rate and smooth muscle alpha-actin expressing myofibroblasts after the 32nd passage. CM treatment of this fibroblast population induces a decline in the myofibroblast content, which precedes the changes in telomerase activity. Passaged fibroblasts from normal breast tissue can be converted to myofibroblasts upon CM treatment whereas those from tumour stroma were CM-insensitive. Taken together our data suggest that a heterogeneous fibroblast population with different life span is activated/recruited in the breast interstitium and poses the problem of a unique activation/recruitment of fibroblasts in neoplastic conditions.