BRCA1 promoter hypermethylation, 53BP1 protein expression and PARP-1 activity as biomarkers of DNA repair deficit in breast cancer.

BACKGROUND: Poly(adenosine diphosphate-ribose) polymerase 1 (PARP-1) and the balance between BRCA1 and 53BP1 play a key role in the DNA repair and cell stress response. PARP inhibitors show promising clinical activity in metastatic triple negative (TN) or BRCA-mutated breast cancer. However, a comprehensive analysis of PARP-1 activity, BRCA1 promoter methylation and 53BP1 expression in tumours without known BRCA1 mutation has not yet been carried out. METHODS: We investigated cytosolic PARP-1 activity, 53BP1 protein levels and BRCA1 promoter methylation in 155 surgical breast tumour samples from patients without familial breast cancer history or known BRCA1 mutations who were treated between January 2006 and November 2009 and evaluated their statistical association with classical predictive and prognostic factors. RESULTS: The mitotic count score was the only parameter clearly associated with PARP-1 activity. BRCA1 promoter hypermethylation (15.4% of all cancers) was significantly associated with uPA and PAI-1 levels, tumour grade, mitotic count score, hormone receptor and HER2 negative status and TN profile (29% of TN tumours showed BRCA1 promoter hypermethylation compared to 5% of grade II-III hormone receptor-positive/HER2-negative and 2% of HER2-positive tumours). No statistical association was found between BRCA1 promoter hypermethylation and PARP-1 activity. High 53BP1 protein levels correlated with lymph node positivity, hormone receptor positivity, molecular grouping, unmethylated BRCA1 promoter and PARP-1 activity. In TN tumours, BRCA1 promoter methylation was only marginally associated with age, PARP-1 activity was not associated with any of the tested clinico-pathological factors and high 53BP1 protein levels were significantly associated with lymph node positivity. Only 3 of the 14 TN tumours with BRCA1 promoter hypermethylation presented high 53BP1 protein levels. CONCLUSIONS: Breast cancers that harbour simultaneously high 53BP1 protein level and BRCA1 promoter hypermethylation and are the putative target population of drugs targeting DNA repair appear to be restricted to a small subgroup of TN tumours.

Lack of EGFR-activating mutations in European patients with triple-negative breast cancer could emphasise geographic and ethnic variations in breast cancer mutation profiles.

INTRODUCTION: Triple-negative breast cancers (TNBCs) are characterised by lack of expression of hormone receptors and epidermal growth factor receptor 2 (HER-2). As they frequently express epidermal growth factor receptors (EGFRs), anti-EGFR therapies are currently assessed for this breast cancer subtype as an alternative to treatments that target HER-2 or hormone receptors. Recently, EGFR-activating mutations have been reported in TNBC specimens in an East Asian population. Because variations in the frequency of EGFR-activating mutations in East Asians and other patients with lung cancer have been described, we evaluated the EGFR mutational profile in tumour samples from European patients with TNBC. METHODS: We selected from a DNA tumour bank 229 DNA samples isolated from frozen, histologically proven and macrodissected invasive TNBC specimens from European patients. PCR and high-resolution melting (HRM) analyses were used to detect mutations in exons 19 and 21 of EGFR. The results were then confirmed by bidirectional sequencing of all samples. RESULTS: HRM analysis allowed the detection of three EGFR exon 21 mutations, but no exon 19 mutations. There was 100% concordance between the HRM and sequencing results. The three patients with EGFR exon 21 abnormal HRM profiles harboured the rare R836R SNP, but no EGFR-activating mutation was identified. CONCLUSIONS: This study highlights variations in the prevalence of EGFR mutations in TNBC. These variations have crucial implications for the design of clinical trials involving anti-EGFR treatments in TNBC and for identifying the potential target population.

Ultra-sensitive EGFR T790M Detection as an Independent Prognostic Marker for Lung Cancer Patients Harboring EGFR del19 Mutations and Treated with First-generation TKIs.

PURPOSE: The detection of preexisting EGFR T790M subclones and the assessment of their clinical significance in the pretreatment of patients with EGFR T790M non-small cell lung cancer (NSCLC) remain unclear. EXPERIMENTAL DESIGN: A total of 179 tumor samples from patients treated or not with a first-generation tyrosine kinase inhibitor (TKI) was analyzed. The presence of ultra-low levels of preexisting EGFRT790M mutation was evaluated using ultra-sensitive droplet digital PCR (ddPCR) and the clinical implication of these mutations on first-generation TKI efficiency assessed. RESULTS: With a ddPCR linear performance of 0.999 and an analytical sensitivity of approximately 0.001%, we observed a 66% (99/150) overall incidence of ultra-low EGFR T790M mutation. Among 82 patients harboring EGFR activating mutations, the presence of a preexisting EGFR T790M mutation prior to any treatment was significantly associated with a longer progression-free survival (PFS; P = 0.009; log-rank test). Interestingly, longer PFS was linked to concomitant EGFR del19 and ultra-low EGFR T790M mutations. Moreover, the presence of both EGFR del19 and ultra-low EGFR T790M mutations was identified as the best fit for predicting the clinical outcome of patients treated with TKI compared with an ultra-low EGFR T790M mutation status or an activating mutation alone (P = 0.042 and P = 0.0071, respectively). CONCLUSIONS: We demonstrate that the detection of the ultra-low EGFR T790M mutation in TKI-naïve patients is not a rare event. We suggest that ddPCR should be used in clinical practice to distinguish patients who may respond to first- or third-generation TKIs.

Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens.

Genotyping BRAF in melanoma samples is often challenging. The presence of melanin greatly interferes with thermostable DNA polymerases and/or nucleic acids in traditional polymerase chain reaction (PCR)-based methods. In the present work, we evaluated three easy-to-use strategies to improve the detection of pigmented DNA refractory to PCR amplification. These pre-PCR processing methods include the addition of bovine serum albumin (BSA), the dilution of DNA, and the purification of DNA using the NucleoSpin® gDNA Clean-up XS Kit. We found that BRAF genotyping in weakly and moderately pigmented samples was more efficient when the sample was processed with BSA or purified with a NucleoSpin® gDNA Clean-up XS Kit prior to PCR amplification. In addition, the combination of both methods resulted in successful detection of BRAF mutation in pigmented specimens, including highly pigmented samples, thereby increasing the chance of patients being elicited for anti-BRAF treatment. These solutions to overcome melanin-induced PCR inhibition are of tremendous value and provide a simple solution for clinical chemistry and routine laboratory medicine.