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Although hyperthermia offers clinical appeal to sensitize cells to chemotherapy, this approach has been limited in terms of longterm outcome as well as economic and technical burden. Thus, a more detailed knowledge about how hyperthermia exerts its effects on chemotherapy may illuminate ways to improve the approach. Here, we asked whether hyperthermia alters the response to chemotherapy-induced DNA damage and whether this mechanism is involved in its sensitizing effect in BRCAcompetent models of ovarian and colon cancer. Notably, we found that hyperthermia delayed the repair of DNA damage caused by cisplatin or doxorubicin, acting upstream of different repair pathways to block histone polyADP-ribosylation (PARylation), a known effect of chemotherapy. Furthermore, hyperthermia blocked this histone modification as efficiently as pharmacologic inhibitors of PARP (PARPi), producing comparable delay in DNA repair, induction of double-strand breaks (DSB), and cell cytotoxicity after chemotherapy. Mechanistic investigations indicated that inhibiting PARylation by either hyperthermia or PARPi induced lethal DSB upon chemotherapy treatment not only by reducing DNA repair but also by preventing replication fork slowing. Overall, our work reveals how PARP blockade, either by hyperthermia or small-molecule inhibition, can increase chemotherapy-induced damage in BRCA-competent cells.

The objective of this one-institutional study was to determine the number of large-core needle biopsies (LCNB), under threedimensional ultrasound (3D-US) validation, that are sufficient to obtain a reliable histological diagnosis of a sonographically detectable breast lesion. Over an 28-month period, 962 sonographically guided LCNB were performed under 3D-US validation to assess 962 breast lesions. All biopsies were carried out with an automated core biopsy device fitted with 14-gauge (22 mm excursion) needles. Data of 962 biopsied breast lesions were gathered. Surgical follow-up was available for 659 lesions. Breast malignancies were diagnosed by ultrasound-guided LCNB with a sensitivity of 98.2% by performing three cores per lesion. In few cases, the open surgical specimen revealed the presence of invasive carcinomas in contrast to initial LNCB-based classification as ductal carcinomas in situ (DCIS, 11 lesions), lobular carcinoma in situ (one lesion), and atypical ductal hyperpasia (one lesion). Owing to disagreement between classification based on breast-imaging and histological findings, eight of these tumours were subsequently excised. Of the lesions that were removed at the patients' requests despite benign LCNB diagnosis, two were infiltrating carcinoma and one a DCIS. We demonstrate that three 3D-US-guided percutaneous core specimens are sufficient to achieve tissue for a reliable histological assessment of sonographically detectable breast lesions and allow the detection of malignancies with high sensitivity and low rate of false-negative diagnoses.

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Recombinant adeno-associated virus (AAV) is a promising non-pathogenic vector in the emerging field of gene therapy. For AAV serotype 2 (AAV-2) infection, experimental evidence points to an involvement of heparan sulphate proteoglycans (HSPG), but also to the existence of additional receptors. We investigated a potential role of the tetraspanin CD9 in AAV-2 infection of breast cancer cells mainly because it binds to the heparin-binding EGF-like growth factor, suggesting that it may also interact with a heparin-binding virus. Among breast cancer cell lines, expression of HSPG or potential AAV-2 (co)-receptors was not found to correlate with transduction efficiency. In complete accordance with the role of CD9, blocking with anti-CD9 antibodies resulted in drastically decreased AAV-2 transduction efficiencies in cell lines with low expression of HSPG. Furthermore, specific inhibition of CD9 expression with siRNA resulted in fewer transgene-positive cells, whereas overexpression of CD9 in the breast cancer cell line T47D as well as in BT8Ca and BT12Ca rat glioma cells (with low background expression of HSPG and CD9) increased the number of AAV-transduced cells. The minimal epitope recognized by antibody 72F6, which most efficiently blocked AAV-mediated transgene expression, was deduced from the specific binding to peptides immobilized on colour-encoded microspheres consisting of the amino acid sequence PKKDV located in the large extracellular loop of CD9. Our results clearly point to an involvement of CD9 in the attachment, uptake or processing of AAV-2 by target cells expressing low amounts of HSPG, which may help to define cell populations accessible in AAVbased therapeutic applications.

Purpose: Identification of molecular characteristics that are useful to define subgroups of patients fitting into differential treatment schemes is considered a most promising approach in cancer research. In this first study of such type, we therefore investigated the potential of multiplexed sandwich immunoassays to define protein expression profiles indicative of clinically relevant properties of malignant tumors. Experimental Design: Lysates prepared from large core needle biopsies of 113 invasive breast carcinomas were analyzed with bead-based miniaturized sandwich immunoassays specific for 54 preselected proteins. Results: Five protein concentrations [fibroblast growth factor-2 (FGF-2), Fas, Fas ligand, tissue inhibitor of metalloproteinase-1, and RANTES] were significantly different in the groups of patients with or without axillary lymph node metastasis. All 15 protein parameters that resulted in P values <0.2 and other diagnostic information [estrogen receptor (ER) status, tumor size, and histologic grading] were analyzed together by multivariate logistic regression.This yielded sets of five (FGF-2, Fas, Fas ligand, IP10, and PDGF-AB/BB) or six (ER staining intensity, FGF-2, Fas ligand, matrix metalloproteinase-13, PDGF-AB/BB, and IP10) parameters for which receiveroperator characteristic analyses revealed high sensitivities and specificities [area under curve (AUC) = 0.75 and AUC = 0.83] to predict the nodal status. A similar analysis including all identified parameters of potential value (15 proteins, ER staining intensity, T) without selection resulted in a receiver-operator characteristic curve with an AUC of 0.87. Conclusion: We clearly showed that this approach can be used to quantify numerous proteins from breast biopsies accurately in parallel and define sets of proteins whose combined analyses allow the prediction of nodal involvement with high specificity and sensitivity.