Identification of BRCA1-like triple-negative breast cancers by quantitative multiplex-ligation-dependent probe amplification (MLPA) analysis of BRCA1-associated chromosomal regions: a validation study.

BACKGROUND: Triple-negative breast cancer (TNBC) with a BRCA1-like molecular signature has been demonstrated to remarkably respond to platinum-based chemotherapy and might be suited for a future treatment with poly(ADP-ribose)polymerase (PARP) inhibitors. In order to rapidly assess this signature we have previously developed a multiplex-ligation-dependent probe amplification (MLPA)-based assay. Here we present an independent validation of this assay to confirm its important clinical impact. METHODS: One-hundred-forty-four TNBC tumor specimens were analysed by the MLPA-based "BRCA1-like" test. Classification into BRCA1-like vs. non-BRCA1-like samples was performed by our formerly established nearest shrunken centroids classifier. Data were subsequently compared with the BRCA1-mutation/methylation status of the samples. T-lymphocyte infiltration and expression of the main target of PARP inhibitors, PARP1, were assessed on a subset of samples by immunohistochemistry. Data acquisition and interpretation was performed in a blinded manner. RESULTS: In the studied TNBC cohort, 63 out of 144 (44 %) tumors were classified into the BRCA1-like category. Among these, the MLPA test correctly predicted 15 out of 18 (83 %) samples with a pathogenic BRCA1-mutation and 20 of 22 (91 %) samples exhibiting BRCA1-promoter methylation. Five false-negative samples were observed. We identified high lymphocyte infiltration as one possible basis for misclassification. However, two falsely classified BRCA1-mutated tumors were also characterized by rather non-BRCA1-associated histopathological features such as borderline ER expression. The BRCA1-like vs. non-BRCA1-like signature was specifically enriched in high-grade (G3) cancers (90 % vs. 58 %, p = 0.0004) and was also frequent in tumors with strong (3+) nuclear PARP1 expression (37 % vs. 16 %; p = 0.087). CONCLUSIONS: This validation study confirmed the good performance of the initial MLPA assay which might thus serve as a valuable tool to select patients for platinum-based chemotherapy regimens. Moreover, frequent PARP1 upregulation in BRCA1-like tumors may also point to susceptibility to treatment with PARP inhibitors. Limitations are the requirement of high tumor content and high-quality DNA.

Clinical value of protein expression of kallikrein-related peptidase 7 (KLK7) in ovarian cancer.

Expression of the kallikrein-related peptidase 7 (KLK7) is dysregulated in ovarian cancer. We assessed KLK7 expression by ELISA and quantitative immunohistochemistry and analyzed its association with clinicopathological parameters and patients' outcome. KLK7 antigen concentrations were determined in tumor tissue extracts of 98 ovarian cancer patients by ELISA. For analysis of KLK7 immunoexpression in ovarian cancer tissue microarrays, a manual quantitative scoring system as well as a software tool for quantitative high-throughput automated image analysis was used. In immunohistochemical analyses, expression levels of KLK7 were not associated with patients' outcome. However, in multivariate analyses, KLK7 antigen levels in tumor tissue extracts were significantly associated with both overall and progression-free survival: ovarian cancer patients with high KLK7 levels had a significantly, 2-fold lower risk of death [hazard ratio (HR)=0.51, 95% confidence interval (CI)=0.29-0.90, p=0.019] or relapse [HR=0.47, 95% CI=0.25-0.91, p=0.024), as compared with patients who displayed low KLK7 levels. Our results indicate that - in contrast to earlier findings - high KLK7 antigen levels in tumor tissue extracts may be associated with a better prognosis of ovarian cancer patients.

A novel method for the in vivo isolation of circulating tumor cells from peripheral blood of cancer patients using a functionalized and structured medical wire.

The isolation of circulating tumor cells (CTCs) from the blood of patients afflicted with solid malignant tumors becomes increasingly important as it may serve as a 'liquid biopsy' with the potential of monitoring the course of the cancer disease and its response to cancer therapy, with subsequent molecular characterization. For this purpose, we functionalized a structured medical Seldinger guidewire (FSMW), normally used to obtain safe access to blood vessels and other organ cavities, with a chimeric monoclonal antibody directed to the cell surface expressed epithelial cell surface adhesion molecule (EpCAM). This medical device was optimized in vitro and its biocompatibility was tested according to the regulations for medical devices and found to be safe with no noteworthy side effects. Suitability, specificity and sensitivity of the FSMW to catch and enrich CTCs in vivo from circulating peripheral blood were tested in 24 breast cancer or non-small cell lung cancer (NSCLC) patients and in 29 healthy volunteers. For this, the FSMW was inserted through a standard venous cannula into the cubital veins of healthy volunteers or cancer patients for the duration of 30 min. After removal, CTCs were identified by immuno-cytochemical staining of EpCAM and/or cytokeratins and staining of their nuclei and counted. The FSMW successfully enriched EpCAM-positive CTCs from 22 of the 24 patients, with a median of 5.5 (0-50) CTCs in breast cancer (n=12) and 16 (2-515) CTCs in NSCLC (n=12). CTCs could be isolated across all tumor stages, including early stage cancer, in which distant metastases were not yet diagnosed, while no CTCs could be detected in healthy volunteers. In this observatory study, no adverse effects were noted. Evidently, the FSMW has the potential to become an important device to enrich CTCs in vivo for monitoring the course of the cancer disease and the efficacy of anticancer treatment.

Distribution of the cellular retinoic acid binding protein CRABP-I in the developing chick optic tectum.

Vitamin A is a major morphogen for the visual system. Most of its effects are mediated by retinoic acid (RA), whose developmental functions include pattern formation, neuronal differentiation and possibly axonal guidance. Although RA has been suggested to regulate development of the retina and its central projection, little is known about the distribution of retinoid receptors and binding proteins in the optic tectum, which in birds is the direct target of most retinofugal axons. We investigated the spatial and temporal distribution of the cellular retinoic acid binding protein-I (CRABP-I) in the chick midbrain. While the precise role of CRABP-I is still unknown, this is an intracellular transport protein for RA, which tends to be expressed in cells that are responsive to retinoic acid. Our data show immunoreactivity of CRABP-I in the tectal anlage at E2.5 and during the entire period of embryonic development. It was found in differentiating neurons of the generative zone, in migrating cells of the prospective stratum griseum et fibrosum superficiale and in mature neurons in this layer. In addition, we detected retinoid receptors RARalpha, RARbeta, RXRalpha, RXRbeta and RXRgamma in the developing tectum. Cell culture experiments demonstrate CRABP-I expression in a subpopulation of tectal neurons as they differentiate in vitro. These results are consistent with a regulatory role of RA in tectal neurogenesis and physiology.