Development and validation of molecular methodologies to assess PALB2 expression in sporadic breast cancer.

OBJECTIVES: Recent reports have included PALB2 (Partner and localizer of BRCA2) in the growing list of hereditary cancer genes. PALB2 mutations confer a moderate breast cancer risk in heterozygotes and Fanconi anemia in biallelic mutation carriers. PALB2 protein co-localizes with BRCA2 and BRCA1 in nuclear structures and enables error-free homologous recombination repair of double-stranded DNA breaks. This important contribution could be severely diminished if affected by epigenetic mechanisms such as promoter CpG island methylation. The aim of our study was to develop molecular methodologies in order to assess accurately PALB2 expression in breast cancer tissues. DESIGN AND METHODS: DNA and RNA were extracted from 91 sporadic fresh-frozen breast tissues with known histopathological data. DNA was subjected to sodium bisulfite conversion reaction and the CpG island of the PALB2 promoter was analyzed by pyrosequencing. RNA was converted to cDNA and analyzed by a newly developed and validated RT-qPCR assay based on a hydrolysis probe (TaqMan) in the Light Cycler. RESULTS: PALB2 promoter was not methylated in any of the samples tested. 87 out of 91 (95.6%) primary tumors were positive for PALB2 expression, as checked at the mRNA level. When levels of PALB2 mRNA were compared to histopathological data (tumor size, grade, lymph node involvement, metastasis, hormone receptors and HER2 overexpression), no significant statistical correlation was found. CONCLUSIONS: DNA methylation is an unlike mechanism for PALB2 transcriptional regulation. PALB2 mRNA expression does not seem be a promising prognostic biomarker for sporadic breast cancer.

Development of a novel PTT assay for mutation detection in PALB2 large exons and PALB2 screening in medullary breast cancer.

Beyond BRCA1 and BRCA2 genes, PALB2 (Partner and localizer of BRCA2) emerges as the third breast cancer susceptibility gene due to its role in the same DNA repair pathway: homologous recombination. In most populations studied so far, PALB2 mutations are detected in 1-2% of BRCA negative female patients. PALB2 gene contains 13 exons; exons 4 and 5 consist 65% of the coding area. We developed a protein truncation test (PTT) for quick screening of truncating pathogenic mutations of these two large exons. Specific primers were de novo, in silico designed and the PTT-PCR products were translated in the presence of biotinylated lysine and detected colorimetrically. The assay was initially tested in 30 patients with hereditary breast cancer, negative for BRCA mutations and then, in 17 patients with the rare medullary breast cancer subtype. Small PALB2 exons were screened with high-resolution melting curve analysis (HRMA) and the large DNA rearrangements with multiplex ligation-dependent probe amplification (MLPA). Any alterations detected were verified by Sanger DNA Sequencing. The developed PTT methodology is highly specific for clinical significant mutations; positive control samples that produce truncated PALB2 peptides were correctly identified and the method was accurate when compared to DNA sequencing. We did not detect any deleterious PALB2 mutation in both groups of patients. HRMA and MLPA were also negative for all tested samples. However, our novel, fast and cost-effective PTT method for pathogenic mutation detection of the two large PALB2 exons can be applied in screening of a large number of breast cancer patients.

TLR-4 and CD14 Genotypes and Soluble CD14: Could They Predispose to Coronary Atherosclerosis?

BACKGROUND: Inflammatory mechanisms are key to the pathogenesis of atherosclerosis. Functional polymorphisms of TLR-4, Asp299Gly and Thr399Ile, CD14 promoter area C260T polymorphism and plasma levels of soluble CD14 are studied in subjects with Coronary Artery Disease (CAD). METHODS: DNA was obtained from 100 human paraffin-embedded aortic specimens, from cadavers with known coronary atheromatosis (Group A) and 100 blood samples from patients with CAD, as detected by cardiac Multi-Detector-row-Computed-Tomography (MDCT) (Group B). Our control group consisted of 100 healthy individuals (Group C). Genotyping was performed by Restriction Fragment Length Polymorphism-Polymerase Chain Reaction (RFLP-PCR). Plasma levels of sCD14 were measured with ELISA. RESULTS: For TLR-4 Asp299Gly and Thr399Ile polymorphisms, no statistically significant differences were observed. Regarding the C260T polymorphism, frequencies of T allele were significantly higher in the control group compared to the case group (p = 0.05). The Odds Ratio (OR) showed statistically significant association of TT genotype with healthy individuals (OR 0.25, 95% Confidence Interval CI 0.10-0.62, p = 0.0017). Plasma levels of sCD14 in patients with CAD (mean value = 1.35 µg/mL) were reduced when compared to reference value. CONCLUSIONS: The studied polymorphisms ofTLR-4 showed no association with CAD. Conversely, the functional polymorphism of CD14 has a statistically significant difference in expression between healthy and affected by CAD individuals.

Development of novel real-time PCR methodology for quantification of COL11A1 mRNA variants and evaluation in breast cancer tissue specimens.

BACKGROUND: Collagen XI is a key structural component of the extracellular matrix and consists of three alpha chains. One of these chains, the α1 (XI), is encoded by the COL11A1 gene and is transcribed to four different variants at least (A, B, C and E) that differ in the propensity to N-terminal domain proteolysis and potentially in the way the extracellular matrix is arranged. This could affect the ability of tumor cells to invade the remodeled stroma and metastasize. No study in the literature has so far investigated the expression of these four variants in breast cancer nor does a method for their accurate quantitative detection exist. METHODS: We developed a conventional PCR for the general detection of the general COL11A1 transcript and real-time qPCR methodologies with dual hybridization probes in the LightCycler platform for the quantitative determination of the variants. Data from 90 breast cancer tissues with known histopathological features were collected. RESULTS: The general COL11A1 transcript was detected in all samples. The developed methodologies for each variant were rapid as well as reproducible, sensitive and specific. Variant A was detected in 30 samples (33 %) and variant E in 62 samples (69 %). Variants B and C were not detected at all. A statistically significant correlation was observed between the presence of variant E and lymph nodes involvement (p = 0.037) and metastasis (p = 0.041). CONCLUSIONS: With the newly developed tools, the possibility of inclusion of COL11A1 variants as prognostic biomarkers in emerging multiparameter technologies examining tissue RNA expression should be further explored.

Hereditary breast cancer: beyond BRCA genetic analysis; PALB2 emerges.

Abstract Despite the initial enthusiasm following the discovery of the association of BRCA germline mutations with hereditary breast and/or ovarian cancer, in many families affected by the syndrome no pathogenic mutations were detected in the two genes, although exhaustively searched. Many other genes have also been implicated due to their role in the same pathway of DNA repair where the BRCA1/2 genes are involved: homologous recombination (HR). Among them, PALB2 clearly emerges as the third breast cancer susceptibility gene. Its mutations have been detected in most populations investigated so far, albeit rarely: in 1%-4% of families negative for BRCA mutations, with either partial or complete penetrance. In some populations, PALB2 recurrent mutations have been identified and the estimated hazard risks are comparable to those of BRCA mutations. Since new effective targeted therapeutic options are becoming available ("synthetic lethality" with novel PARP inhibitors, etc.) that are applicable to all those patients with a defect in HR pathway, it is imperative to detect all these candidate patients. Data obtained from laboratory tests in the tumor (simple immunohistochemistry, gene expression analysis, etc.) can assist in the recognition of a specific pattern (BRCA1ness, HRless) so that even patients that look "sporadic" could benefit from these targeted therapies. Therefore, a genetic analysis algorithm is proposed, although with the advent of Next Generation Sequencing it is predicted that in the future most germline genetic alterations and also somatic or epigenetic events in the tumor of these genes will be detected.

PIK3CA hotspot mutation scanning by a novel and highly sensitive high-resolution small amplicon melting analysis method.

Somatic mutations in the PIK3CA gene have been discovered in many human cancers, and their presence correlates to therapy response. Three "hotspot" mutations within the PIK3CA gene are localized in exons 9 and 20. High-resolution melting analysis (HRMA) is a highly sensitive, robust, rapid, and cost-effective mutation analysis technique. We developed a novel methodology for the detection of hotspot mutations in exons 9 and 20 of the PIK3CA gene that is based on a combination of PCR and HRMA. The PIK3CA HRMA assay was evaluated by performing repeatability, sensitivity, and comparison with DNA sequencing studies and was further validated in 129 formalin-fixed paraffin-embedded breast tissue samples: 99 tumors, 20 noncancerous, and 10 fibroadenomas. The developed methodology was further applied in a selected group of 75 breast cancer patients who underwent Trastuzumab treatment. In sensitivity studies, the assay presented a capability to detect as low as 1% of mutated dsDNA in the presence of wtDNA for both exons. In the 99 tumor samples (validation group), 12/99 (12.1%) exon 9 mutations and 20/99 (20.2%) exon 20 mutations were found. No mutations were found in noncancerous tissues. In fibroadenomas, we report one PIK3CA mutation for the first time. In the selected group, 30/75 (40%) samples were detected as mutants. The PIK3CA HRMA assay is highly sensitive, reliable, cost-effective, and easy-to-perform, and therefore can be used as a screening test in a high-throughput pharmacodiagnostic setting.