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BackgroundBSMO 2014-01 is a published prospective phase 2 study investigating neoadjuvant weekly paclitaxel and carboplatin, followed by epirubicin and cyclophosphamide in 63 patients with triple-negative breast cancer. Pathological complete response (pCR) was observed in 54%. A secondary endpoint of the study was to correlate pCR rate to the presence of germline pathogenic variants in DNA damage response (DDR) genes or in core genes involved in Homologous Recombination (HR).MethodsPeripheral blood from 60 patients was collected for germline DNA analysis. Whole Exome Sequencing was performed; only rare variants (minor allelic frequency < 0.01) in 276 DDR genes were considered. The correlation between pCR rate and DDR or HR deficiency was analyzed using the Fisher's exact test. The same was done for the correlation between DDR gene mutations and the presence of hematologic toxicities. ResultsThirty-five out of 60 patients (58.3%) carried a protein disrupting germline mutation in a DDR gene. Twenty-four of these 35 patients (68.6%) had a pCR, while a pCR was observed in 40% without a DDR mutation (p=0.026). In 14/15 patients (93.3%) with a HR core gene mutation a pCR was obtained, while a pCR was present in 44.4% without a HR core gene mutation (p=0.0007). Eight of nine patients (88.9%) with a BRCA1 or BRCA2 pathogenic variant reached a pCR compared to 51% of the BRCA wild-type patients (p=0.035). ConclusionsThis is the first study to demonstrate that germline pathogenic variants in genes involved in HR core genes predicts for pCR after platinum-containing neoadjuvant chemotherapy.

Background: BSMO 2014-01 is a published prospective phase 2 study evaluating the efficacy of neoadjuvant EC and PC in the treatment of TNBC patients. (1) The addition of weekly carboplatin to neoadjuvant paclitaxel and dose-dense EC lead to a pathologic complete response (pCR) of 54%, comparable to prior studies. A secondary endpoint was to correlate pCR to homologous recombination (HR) and DNA damaging repair (DDR) deficiency due to germline mutations. Methods: Sixty-three patients (pts) of which nine already identified with a BRCA 1/2 mutation were considered. Peripheral blood was collected in 52 pts after obtaining informed consent for a broad genomic DNA analysis. Whole Exome Sequencing was performed, and only rare variants (M.A.F. Citation Format: Christel Fontaine, Vincent Renard, Heidi Van den Bulck, Peter Vuylsteke, Philippe Glorieux, Catherine Dopchie, Lore Decoster, Ahmad Awada, Hans Wildiers, Sylvie De Brakeleer, Erik Teugels, Jacques De Grève. Evaluation of germline whole exome sequencing of early breast cancer patients with triple negative breast cancer (TNBC) included in a prospective study of neoadjuvant chemotherapy (NAC) with epirubicin and cyclophosphamide (EC) and carboplatin-paclitaxel (PC) (BSMO 2014-01) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-05.

Two out of 41 non-small cell lung cancer patients enrolled in a clinical study were found with a somatic CRAF mutation in their tumor, namely CRAFP261A and CRAFP207S. To our knowledge, both mutations are novel in lung cancer and CRAFP261A has not been previously reported in cancer. Expression of CRAFP261A in HEK293T cells and BEAS-2B lung epithelial cells led to increased ERK pathway activation in a dimer-dependent manner, accompanied with loss of CRAF phosphorylation at the negative regulatory S259 residue. Moreover, stable expression of CRAFP261A in mouse embryonic fibroblasts and BEAS-2B cells led to anchorage-independent growth. Consistent with a previous report, we could not observe a gain-of-function with CRAFP207S. Type II but not type I RAF inhibitors suppressed the CRAFP261A-induced ERK pathway activity in BEAS-2B cells, and combinatorial treatment with type II RAF inhibitors and a MEK inhibitor led to a stronger ERK pathway inhibition and growth arrest. Our findings suggest that the acquisition of a CRAFP261A mutation can provide oncogenic properties to cells, and that such cells are sensitive to combined MEK and type II RAF inhibitors. CRAF mutations should be diagnostically and therapeutically explored in lung and perhaps other cancers. ispartof: ONCOGENE vol:38 issue:31 pages:5933-5941 ispartof: location:England status: published

Tumor infiltrating lymphocytes (TIL), programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) expression are important prognostic markers. This study aimed to investigate these markers in lung adenocarcinoma (ADC) biopsies from patients with stage IIIB or IV ADC with little or no smoking history, to investigate their prognostic value and to correlate these results with the presence of driver mutations in the tumors. TIL were retrospectively evaluated on hematoxylin and eosin stained slides from 152 tumor samples. PD-1/PD-L1 expression was retrospectively evaluated with PD-1/PD-L1 immunohistochemistry (IHC) double staining on 74 tumor samples with sufficient residual tissue. PD-L1 expression was analysed on stromal cells of the tumor compartment, the tumor cells and TIL and PD-1 on TIL. Median overall survival (OS) was longer in patients with high stromal TIL infiltration compared to patients with low stromal TIL infiltration (68 weeks vs. 35 weeks respectively; p = 0.003). This was observed most prominently in KRAS mutant tumors (95 weeks vs. 12 weeks; p = 0.003). Only PD-L1 expression on tumor stromal cells influenced OS and indicated a worse prognosis (77 weeks vs 25 weeks; p = 0.013). Stromal TIL counts nor PD-1/PD-L1 expression levels were associated with the presence of driver mutations. The results of the current study reinforce the prognostic role of TIL in lung ADC, which is most prominent in KRAS mutant cancers. The results of the PD-1/PD-L1 analysis suggest that stromal cells can effectively suppress the anti-tumor immune response via the PD-L1 pathway.

In non‐small cell lung cancer (NSCLC), activating mutations in the epidermal growth factor receptor (EGFR) induce sensitivity to EGFR tyrosine kinase inhibitors. Despite impressive clinical responses, patients ultimately relapse as a reservoir of drug‐tolerant cells persist, which ultimately leads to acquired resistance mechanisms. We performed an unbiased high‐throughput siRNA screen to identify proteins that abrogate the response of EGFR‐mutant NSCLC to EGFR‐targeted therapy. The deubiquitinase USP13 was a top hit resulting from this screen. Targeting USP13 increases the sensitivity to EGFR inhibition with small molecules in vitro and in vivo. USP13 selectively stabilizes mutant EGFR in a peptidase‐independent manner by counteracting the action of members of the Cbl family of E3 ubiquitin ligases. We conclude that USP13 is a strong mutant EGFR‐specific cotarget that could improve the treatment efficacy of EGFR‐targeted therapies., What's new? Mutations in epidermal growth factor receptor (EGFR) are present in as many as 30 percent of patients with non‐small‐cell lung cancer (NSCLC). EGFR mutations render NSCLC sensitive to EGFR tyrosine kinase inhibitors, though over time many lung tumors become resistant to these therapies. This investigation shows that receptor stabilization and drug resistance in EGFR‐mutant NSCLC is mediated at least in part by the deubiquitinase USP13. Co‐targeting EGFR and USP13 increased NSCLC sensitivity to EGFR inhibitors in vitro and in vivo. The study identifies USP13 targeted therapy as a promising means for overcoming EGFR inhibitor resistance in EGFR‐mutant NSCLC.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in cancer cells without causing damage to normal cells. However, some tumors are resistant to TRAIL monotherapy and clinical studies assessing targeted agents towards the TRAIL receptor have failed to show robust therapeutic activity. Evidence has shown that standard anti-mitotic drugs can induce synergistic apoptosis upon combination with TRAIL via cell cycle arrest. Polo like kinase-1 (PLK1) plays a critical role in different stages of cell cycle progression and mitosis. A number of investigations have demonstrated that PLK1 inhibition causes cell cycle arrest and mitotic catastrophe in non-small cell lung cancer (NSCLC), and we thus postulated that PLK1 inhibition could enhance TRAIL-induced apoptosis. We demonstrate that the combination of a TRAIL receptor agonist and a PLK1 inhibitor synergistically reduces cell viability, and strongly increases apoptosis in NSCLC cellular models. Consistent with our in vitro observations, this drug combination also significantly reduces tumor growth in vivo. Our data additionally reveal that G2/M cell cycle arrest and downregulation of Mcl-1 and signal transducer and activator of transcription 3 (STAT3) activity following PLK1 inhibition may contribute to the sensitization of TRAIL-induced apoptosis in NSCLC. Together, these data support the further exploration of combined TRAIL and PLK1 inhibition in the treatment of NSCLC.

A large fraction of somatic driver BRAF mutations in lung cancer are non-V600 and impaired-kinase. Non-V600 BRAF mutations predict sensitivity to combination of a type I RAF inhibitor, Dabrafenib, and a MEK inhibitor, Trametinib. Singly, Dabrafenib only weakly suppresses mutant BRAF-induced ERK signaling and can induce ERK paradoxical activation in CRAF-overexpressing cells. The present study compared the effects of Dabrafenib and a type II RAF inhibitor, AZ628, on ERK activity in HEK293T cells expressing several tumor-derived BRAF mutants, and in a non-V600 and impaired-kinase BRAF-mutant lung cancer cell line (H1666). Unlike Dabrafenib, AZ628 did not induce paradoxical ERK activation in CRAF-overexpressing cells and BRAF-mutant cells overexpressing CRAF were more responsive to AZ628 compared to Dabrafenib in terms of ERK inhibition. AZ628 inhibited ERK more effectively than Dabrafenib in both H1666 cells and HEK293T cells co-expressing several different BRAF-mutants with CRAF. Similarly, AZ628 plus Trametinib had better MEK-inhibitory and pro-apoptotic effects in H1666 cells than Dabrafenib plus Trametinib. Moreover, prolonged treatment of H1666 cells with AZ628 plus Trametinib produced greater inhibition of cell growth than Dabrafenib plus Trametinib. These results indicate that AZ628 has greater potential than Dabrafenib, both as a single agent and combined with Trametinib, for the treatment of non-V600 BRAF mutant lung cancer.

Lung cancer is the number one cause of cancer-related death worldwide with cigarette smoking as its major risk factor. Although the incidence of lung cancer in never smokers is rising, this subgroup of patients is underrepresented in genomic studies of lung cancer. Here, we assembled a prospective cohort of 46 never-smoking, nonsmall cell lung cancer (NSCLC) patients and performed whole-exome and low-coverage whole-genome sequencing on tumors and matched germline DNA. We observed fewer somatic mutations, genomic breakpoints and a smaller fraction of the genome with chromosomal instability in lung tumors from never smokers compared to smokers. The lower number of mutations, enabled us to identify TSC22D1 as a potential driver gene in NSCLC. On the other hand, the frequency of mutations in actionable genes such as EGFR and ERBB2 and of amplifications in MET were higher, while the mutation rate of TP53, which is a negative prognostic factor, was lower in never smokers compared to smokers. Together, these observations suggest a more favorable prognosis for never smokers with NSCLC. Classification of somatic mutations into six-substitution type patterns or into 96-substitution type signatures revealed distinct clusters between smokers and never smokers. Particularly, we identified in never smokers signatures related to aging, homologous recombination damage and APOBEC/AID activity as the most important underlying processes of NSCLC. This further indicates that second-hand smoking is not driving NSCLC pathogenesis in never smokers. ispartof: INTERNATIONAL JOURNAL OF CANCER vol:146 issue:11 pages:3207-3218 ispartof: location:United States status: published

// Amir Noeparast 1 , Erik Teugels 1 , Philippe Giron 1 , Gil Verschelden 1 , Sylvia De Brakeleer 1 , Lore Decoster 1 , Jacques De Greve 1 1 Laboratory of Molecular Oncology and Department of Medical Oncology, Oncologisch Centrum, UZ Brussel, Vrije Universiteit Brussel, Brussels, Belgium Correspondence to: Jacques De Greve, email: jacques.degreve@uzbrussel.be Keywords: non-V600 BRAF, lung cancer, impaired-kinase, Trametinib, Dabrafenib Received: October 08, 2015 Accepted: June 09, 2016 Published: August 26, 2016 ABSTRACT Approximately half of BRAF-mutated Non-small cell lung cancers (NSCLCs) harbor a non-V600 BRAF mutation, accounting for ~40,000 annual deaths worldwide. Recent studies have revealed the benefits of combined targeted therapy with a RAF-inhibitor (Dabrafenib) and a MEK-inhibitor (Trametinib) in treating V600 BRAF mutant cancers, including NSCLC. In contrast, sensitivity of non-V600 BRAF mutations to these inhibitors is not documented. Non-V600 mutations can either increase or impair BRAF kinase activity. However, impaired BRAF kinases can still activate the ERK pathway in a CRAF-dependent manner. Herein, beyond describing a cohort of BRAF mutant NSCLC patients and functionally analyzing 13 tumor-derived BRAF mutations, we demonstrate that both types of non-V600 BRAF mutations can be sensitive to clinically relevant doses of Dabrafenib and Trametinib in HEK293T cells, in lung epithelial cellular model (BEAS-2B) and in human cancer cell lines harboring non-V600 BRAF mutations. ERK activity induced by both types of these mutations is further reduced by combinatorial drug treatment. Moreover, the combination leads to more prolonged ERK inhibition and has anti-proliferative and pro-apoptotic effects in cells harboring both types of non-V600 BRAF mutations. This study provides a basis for the clinical exploration of non-V600 BRAF mutant lung cancers upon treatment with Trametinib and Dabrafenib.

Triple-negative breast cancer (TNBC) accounts for 10-20% of all breast cancers (BCs), and conventional chemotherapy is the only effective systemic treatment. Germline BRCA1/2 mutations are found in approximately 15% of TNBC patients. In the past, we have documented pathogenic mutations in BARD1, a BRCA1 interacting protein, in families at high risk for BC. In this study, we have analyzed germline DNA from 61 estrogen receptor negative patients (of which 42 were TNBC) for the presence of mutations in the BRCA1, BRCA2 and BARD1 gene. BRCA1/2 mutations were found in 8 out of 42 (19%) TNBC patients, but not in the ER-/HER2+ cohort. We also found four good candidate pathogenic BARD1 mutations in the TNBC cohort, including two protein-truncating mutations (p.Gln564Ter and p.Arg641Ter). Our data suggest that TNBC patients are enriched for pathogenic BARD1 germline mutations as compared to control samples and high BC risk families. Ten of the 42 investigated TNBC patients carry a BRCA pathway mutation (in BRCA1, BRCA2 or BARD1) rendering them susceptible to homologous recombination deficiency. These patients should become eligible for exploring the efficacy of poly (ADP-ribose) polymerase (PARP) inhibitors.

e23197 Background: During the Belgian FIELT II clinical study, two non-small cell lung cancers among 41 were found to harbor a CRAF mutation. One of these mutations (P207S) was previously found in fibrosarcoma and characterized as not activating the ERK pathway at higher levels compared to the wild-type CRAF. The other mutation (P261A) has never been reported in cancer but found in Noonan syndrome. CRAF mutations can be oncogenic thereby activation of the ERK pathway. Serine 259 is a negative regulatory site and its phosphorylation is essential for keeping the CRAF auto-inhibited. P261A CRAF prevents phosphorylation of S259 but its role in cancer and its possible response to small molecule inhibitors has yet to be uncovered. Methods: We generated recombinant CRAF expression vectors by site-directed mutagenesis and investigated the ERK pathway activation status induced by CRAF mutants, in HEK293T cells and BEAS-2B (lung epithelial) cells. We tested the effect of candidate inhibitors at a clinically relevant dose on ERK pathway activity in BEAS-2B cells expressing CRAF mutants. Results: Expression of P261A CRAF in both HEK293T and BEAS-2B cells leads to increased ERK pathway activation compared to wtCRAF, accompanied by decreased phosphorylation of S259. P207S CRAF induces ERK pathway activity at levels comparable to wtCRAF and we observed that co-expression of BRAF with P207S CRAF does not lead to increased ERK activity compared to wtCRAF/BRAF. RAF-inhibitors LY3009120 and AZD-628 suppress ERK pathway activity induced by P261A CRAF in BEAS-2B cells. LY3009120 showed stronger ERK-inhibitory effect compared to AZD-628. In contrast, Dabrafenib (RAF-inhibitor) treatment of mutant or wtCRAF expressing cells leads to paradoxical ERK activation. Combinatorial treatment of LY3009120 with Trametinib (MEK-inhibitor) leads to stronger ERK-inhibitory effects compared to either single agent treatment in cells expressing mutant CRAF. Conclusions: The P261A CRAF mutation is ERK pathway activating and predicts sensitivity to LY3009120 and Trametinib. CRAF inhibition should be clinically explored in lung cancers and perhaps other cancers with sensitizing CRAF mutations.

e14104 Background: TNF-related apoptosis-ligand (TRAIL) can selectively induce apoptosis in cancer cells without causing damage to normal cells. However, some tumors are resistant to TRAIL monotherapy and clinical studies assessing targeted agents towards the TRAIL receptor have failed to show robust therapeutic activity. A number of studies have reported that the combination of targeted agents against the TRAIL receptor with standard systemic therapy and another targeted therapy considerably increased anti-tumor activity in experimental NSCLC models. Polo like kinase 1 (PLK1) is an emerging anti-mitotic target for NSCLC with high selectivity. Depleting PLK1 has been shown to reduce cell proliferation and induce apoptosis in NSCLC cells but not in normal cells. Both PLK1 and TRAIL-induced apoptosis pathways are important in supporting the malignant phenotype. We postulated that inhibiting PLK1 could enhance TRAIL-induced apoptosis. We therefore combined human recombinant TRAIL (rhTRAIL) and the PLK1 inhibitor RO3280, and studied the effect of combinatorial treatment on NSCLC cells. Methods: Cell growth was assessed by MTS assay and apoptotic activity by FACS and western blot analysis. The effect of combinatorial treatment on MAPK/ERK, PI3K/Akt and JAK/STAT signaling pathways that play an essential role in the survival of NSCLC cells were investigated by western blot analysis. Results: In this study, we demonstrate that the combination of a rhTRAIL with RO3280 synergistically reduces cell growth and strongly increases apoptotic activity in NSCLC cells. In response to RO3280 treatment, STAT3 activity is inhibited, possibly contributing to the sensitization of NSCLC cells to TRAIL-induced apoptosis. Blockade of STAT3 activity with a STAT3 inhibitor (stattic) and siRNA-mediated knockdown of STAT3 significantly enhances TRAIL-induced apoptosis in NSCLC cells. Conclusions: Our results suggest that this synergistic effect might occur through the inhibition of STAT3 activity which in turn may further contribute to the sensitization of the NSCLC cells to TRAIL therapy. Taken together, our results support the further exploration of PLK1 inhibitors in combination with TRAIL therapy in the treatment of NSCLC.

Pathogenic Alu element insertions are rarely reported, whereas their occurrence is expected to be much higher. Alu containing alleles are usually out-competed during the PCR process and consequently undetectable with the classical screening methods. However, with the introduction of the next generation sequencing (NGS) technology in the diagnostic field, new opportunities are emerging. NGS data for a particular genomic region can be seen as the summation of all the individual sequences (reads) obtained for that region and no longer as the mean of this sum as it is the case for traditional Sanger sequencing. Because each single read covering that region is expected to be generated from a different template molecule, the presence of one single mutant read must theoretically be sufficient to identify the mutation. However, generation and identification of mutant reads bearing Alu insertions remains challenging and several wet/dry bench parameters need to be optimized. Hereby we present the proof of principle of a NGS-based mutation screening procedure allowing the detection of inherited Alu insertions within any predefined sequence by investigating 2 cases: c.1739_1740insAlu in BRCA1 and c.156_157insAlu in BRCA2.

We previously showed that theData were collected from 129In this cohort the cumulative risk of BC and OC by age 70 years was 20% and 6%, respectively. The relative risk for developing cancer was higher when using a model that included the effects of both the R1699Q variant and a residual polygenic component compared with monogenic model (for BC 3.67 vs 2.83, and for OC 6.41 vs 5.83).Our results confirm that

9 páginas, 4 figuras, 1 tabla.-- El pdf del artículo es la versión pre-print.-- et al., The c.156_157insAlu BRCA2 mutation has so far only been reported in hereditary breast/ovarian cancer (HBOC) families of Portuguese origin. Since this mutation is not detectable using the commonly used screening methodologies and must be specifically sought, we screened for this rearrangement in a total of 5,443 suspected HBOC families from several countries. Whereas the c.156_157insAlu BRCA2 mutation was detected in 11 of 149 suspected HBOC families from Portugal, representing 37.9% of all deleterious mutations, in other countries it was detected only in one proband living in France and in four individuals requesting predictive testing living in France and in the USA, all being Portuguese immigrants. After performing an extensive haplotype study in carrier families, we estimate that this founder mutation occurred 558 ± 215 years ago. We further demonstrate significant quantitative differences regarding the production of the BRCA2 full length RNA and the transcript lacking exon 3 in c.156_157insAlu BRCA2 mutation carriers and in controls. The cumulative incidence of breast cancer in carriers did not differ from that of other BRCA2 and BRCA1 pathogenic mutations. We recommend that all suspected HBOC families from Portugal or with Portuguese ancestry are specifically tested for this rearrangement., This study was supported by Ministério da Saúde (Project N8 15/2007) and Liga Portuguesa Contra o Cancro. IPATIMUP was funded by Fundaçáo para a Ciência e Tecnologia, through POCI (Programa Operacional Ciència e Inovaçáo 2010). MT and WDF are supported by the Susan G. Komen Foundation for the Cure, the Jewish General Hospital Weekend to End Breast Cancer and the Fonds de la Recherche en Santé du Québec. EAV and MD are supported by grants PI061102 (Ministerio de Ciencia e Innovación) and 200820I135 (CSIC).

Somatic mutations found within the tyrosine kinase domain (TKD) of the human epidermal growth factor (HER) family of receptors have been implicated in the development and progression of non-small cell lung cancer (NSCLC). However, no conclusive reports have described pathogenic mutations in kinase-impaired HER3. Here, we report a case of an advanced chemotherapy-resistant NSCLC, harboring a novel HER3V855A somatic mutation homologous to the EGFRL858Ractivating mutation. Co-expression of HER3V855A and wild-type HER2 enhances ligand-induced transformation of murine and human cell lines, while HER-targeted inhibitors potently suppress mutant HER3 activity. Consistent with these observations, in silico computational modeling predicts that mutant V855A alters the kinase domain and c-terminal end of the HER3 protein. Taken together, these findings provide a basis for the clinical exploration of targeted therapies in HER3 mutant NSCLC and by extrapolation, in other cancers that more frequently carry somatic HER3 mutations. ispartof: Oncotarget vol:7 issue:3 pages:3068-83 ispartof: location:United States status: published

INTRODUCTION: Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibition is the preferred first-line treatment of advanced adenocarcinoma of the lung that harbors EGFR activating tyrosine kinase domain mutations. Most data available pertain to Asian populations in which such mutations are more prevalent. We report on the long-term results of first-line treatment with erlotinib in Caucasian patients with advanced adenocarcinoma of the lung that have a somatic EGFR mutation in their tumor., SCOPUS: ar.j, info:eu-repo/semantics/published

Introduction Lung cancer remains the leading cause of cancer-related mortality worldwide, with metastatic disease frequently a prominent feature at the time of diagnosis. The role of NSCLC-derived EGFR mutations in cancer cell proliferation and survival has been widely reported, but little is known about the function of these mutations in invasive growth and metastasis. In this study, we sought to evaluate the intrinsic invasive properties of NSCLC cells with differing EGFR status and examine possible therapeutic targets that can abrogate invasive growth. Materials and methods Collagen-based assays and 3D cell cultures were used to assess morphological features, actin cytoskeleton dynamics and the invasive capacity of NSCLC cell lines with differing EGFR status. The role of the RhoA/ROCK/MYPT1 and EGFR/HER pathways in NSCLC-related invasion was investigated by pharmacological inhibition and RNA interference techniques. Results We demonstrate a positive correlation between EGFR mutational/amplification status and invasive capacity. Knockdown of wild-type and mutant EGFR leads to depletion of active and total MYPT1 levels. Combined pharmacological inhibition or genetic ablation of ROCK/EGFR suppresses the hallmarks of cancer cells and abrogates the invasive phenotype in EGFR-dependent NSCLC cells. Conclusions These observations suggest that combined targeting of the ROCK and EGFR/HER pathways may be a potential therapeutic approach in limiting invasive growth in NSCLC.

Objectives Afatinib, an oral irreversible ErbB family blocker, has demonstrated efficacy in patients with epidermal growth factor receptor (EGFR) mutation-positive advanced lung adenocarcinoma. Other potential biomarkers predicting response to afatinib, such as human epidermal growth factor receptor-2 (HER2) mutations and EGFR gene amplification, have not been validated yet. This phase II study investigated whether afatinib conferred clinical benefit in cohorts of adenocarcinoma patients with: (1) EGFR mutation and failing on erlotinib/gefitinib; or (2) increased copy number of EGFR by fluorescence in situ hybridization (FISH); or (3) HER2 mutation. Materials and methods Patients started daily afatinib 50 mg monotherapy. Upon disease progression, patients could continue, at the investigator's discretion, afatinib (40 mg) with the addition of paclitaxel (80 mg/m2 weekly for 3 weeks/4-week cycle). Endpoints included confirmed objective response (OR), progression-free survival (PFS), disease control, and safety. Results Of 41 patients treated (cohort 1: n = 32; cohort 2: n = 2; cohort 3: n = 7), 33 received afatinib monotherapy; eight subsequently received afatinib plus paclitaxel. With afatinib monotherapy, one patient achieved a confirmed OR (partial response [PR]; cohort 2). Two further patients achieved unconfirmed PRs (one each in cohort 1 and cohort 3). Disease control was achieved by 17/32 (53%), 2/2 (100%) and 5/7 (71%) patients in cohorts 1, 2 and 3, respectively. In patients receiving combination therapy (median PFS: 6.7 weeks), one (cohort 3) had confirmed PR of 41.9 weeks. The most common afatinib-related adverse events were diarrhea (95%) and rash/acne (80%). Conclusion Afatinib demonstrated signs of clinical activity in heavily pretreated patients with activating HER2 or EGFR mutations or EGFR FISH-positive tumors.

// Ijeoma Adaku Umelo 1 , Olivier De Wever 2 , Peter Kronenberger 1, 3 , Jan Van Deun 2 , Alfiah Noor 1 , Kshitiz Singh 4 , Erik Teugels 1 , Gang Chen 1 , Marc Bracke 2 , Jacques De Greve 1 1 Laboratory of Molecular Oncology and Department of Medical Oncology, Oncologisch Centrum, Universitair Ziekenhuis Brussel, Brussels, Belgium 2 Laboratory of Experimental Cancer Research and Department of Radiotherapy, Universitair Ziekenhuis Gent, Brussels, Belgium 3 Laboratory of Biotechnology, Department of Healthcare, Erasmushogeschool Brussel, Brussels, Belgium 4 Laboratory of Gene Therapy & Regenerative Medicine, Vrije Universiteit Brussel, Brussels, Belgium Correspondence to: Jacques De Greve, e-mail: Jacques.Degreve@uzbrussel.be Keywords: lung cancer, KRAS, EGFR, HER, targeted therapy Received: December 09, 2014 Accepted: May 05, 2015 Published: May 15, 2015 ABSTRACT KRAS is a frequently mutated oncogene in lung cancer and among the most refractory to EGFR targeted therapy. Recently, preclinical evidence in pancreatic cancer has demonstrated that mutant KRAS can be regulated by EGFR. However, the distinct correlation between the EGFR/HER family members and mutant KRAS has not been investigated. Here, we show that non-small cell lung cancer cell lines harboring differing isoforms of mutant KRAS, can be broadly divided into EGFR/HER dependent and EGFR/HER independent groups. Combined therapeutic targeting of EGFR, HER2 and HER3 in isoforms regulated by extracellular growth signals promotes in vitro and in vivo efficacy. We also provide evidence that depletion of EGFR via RNA interference specifically abolishes the EGFR/KRAS interaction in the dependent subset. Taken together, these findings suggest that upstream inhibition of the EGFR/HER receptors may be effective in treating a subset of KRAS mutant lung cancers.

We report a case of a mixed adenoneuroendocrine carcinoma developed in a colorectal adenocarcinoma with lymph node and liver metastases exclusively emanating from the neuroendocrine carcinoma component. The patient underwent right hemicolectomy and postoperatively received chemotherapy with cisplatin and etoposide and subsequent high-dose induction chemotherapy, followed by autologous stem cell transplantation. Following this treatment, there was a complete remission. Currently, thirty months after treatment, the patient is in unmaintained complete remission. Comparative exome sequencing of germline DNA and DNA from the two separate malignant components revealed six somatic changes in cancer consensus genes. Both components shared somatic mutations in Adenomatous polyposis coli (APC), Kirsten rat sarcoma viral oncogene homolog (KRAS), B-cell CLL/lymphoma 9 (BCL9) and Forkhead Box P1 (FOXP1) genes. Mutation in SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (SMARCA4) was only found in the neuroendocrine carcinoma component. The finding of several identical somatic mutations in both components supports a clonal relationship between the neuroendocrine carcinoma and the adenocarcinoma. We suggest that a mutation in SMARCA4 could be responsible for the transformation of the adenocarcinoma component into the neuroendocrine phenotype.

Toxicity associated with plasmid/liposome transfection of eucaryote cells has been attributed to the inherent toxicity of cationic lipid formulations and also to bacterial contaminants of plasmid DNA preparations, such as lipopolysaccharides (LPS). Certain plasmid preparations were observed to trigger apoptosis in DNA/liposome transfected OVCAR3 human epithelial ovarian cancer cells. In contrast, AZ224 and SKOV3 cells were unaffected under the same conditions. Agarose gel electrophoresis with recovery of the plasmid DNA removed the toxic component, but not purification by phenol/chloroform extraction or isopicnic CsCl ultracentrifugation. The toxicity of individual preparations correlated with the concentration of bacterial LPS. However, polymixin B could not neutralise the toxicity and neither could the effect be reproduced by the addition of bacterial LPS to non-toxic plasmid preparations. Surprisingly, the conditioned medium of OVCAR3 cells undergoing apoptosis was found to kill non-transfected OVCAR3 cells but not AZ224 or SKOV3 cells. This observation illustrates the possibility that unpredictable contaminants of bacterial plasmid preparations are able to cause cell death in the context of plasmid/liposome transfection in a cell-type specific way. It emphasizes the importance of achieving maximal plasmid DNA purity when performing DNA transfection experiments that focus on cell survival.

Aberrant expression of microRNA-146a (miR-146a) has been reported to be involved in the development and progression of various types of cancers. However, its role in non-small cell lung cancer (NSCLC) has not been elucidated. The aim of this study was to investigate the contribution of miR-146a to various aspects of the malignant phenotype of human NSCLCs. In functional experiments, miR-146a suppressed cell growth, induced cellular apoptosis and inhibited EGFR downstream signaling in five NSCLC cell lines (H358, H1650, H1975, HCC827 and H292). miR-146a also inhibited the migratory capacity of these NSCLC cells. On the other hand, miR-146a enhanced the inhibition of cell proliferation by drugs targeting EGFR, including both TKIs (gefitinib, erlotinib, and afatinib) and a monoclonal antibody (cetuximab). These effects were independent of the EGFR mutation status (wild type, sensitizing mutation or resistance mutation), but were less potent compared to the effects of siRNA targeting of EGFR. Our results suggest that these effects of miR-146a are due to its targeting of EGFR and NF-κB signaling. We also found, in clinical formalin fixed paraffin embedded (FFPE) lung cancer samples, that low expression of miR-146a was correlated with advanced clinical TNM stages and distant metastasis in NSCLC (P

Epidermal growth factor receptor (EGFR) and c-MET receptors are expressed on many non-small cell lung cancer (NSCLC) cells. Current single agent therapeutic targeting of a mutant EGFR has a high efficacy in the clinic, but is not curative. Here, we investigated the combination of targeting EGFR and c-MET pathways in NSCLC cells resistant to receptor tyrosine kinase inhibitors (TKIs), using RNA interference and inhibition by TKIs. Different NSCLC cell lines with various genomic characteristics (H358, H1650 and H1975) were transfected with EGFR-specific-siRNA, T790M-specific-siRNA, c-MET siRNA or the combination. Subsequently EGFR TKIs (gefitinib, erlotinib or afatinib) or monoclonal antibody cetuximab were combined respectively with the c-MET-specific TKI su11274 in NSCLC cell lines. The cell proliferation, viability, caspase-3/7 activity and apoptotic morphology were monitored by spectrophotometry, fluorimetry and fluorescence microscopy. The combined effect of EGFR TKIs, or cetuximab and su11274, was evaluated using a combination index. The results showed that the cell lines that were relatively resistant to EGFR TKIs, especially the H1975 cell line containing the resistance T790M mutation, were found to be more sensitive to EGFR-specific-siRNA. The combination of EGFR siRNA plus c-MET siRNA enhanced cell growth inhibition, apoptosis induction and inhibition of downstream signaling in EGFR TKI resistant H358, H1650 and H1975 cells, despite the absence of activity of the c-MET siRNA alone. EGFR TKIs or cetuximab plus su11274 were also consistently superior to either agent alone. The strongest biological effect was observed when afatinib, an irreversible pan-HER blocker was combined with su11274, which achieved a synergistic effect in the T790M mutant H1975 cells. In a conclusion, our findings offer preclinical proof of principle for combined inhibition as a promising treatment strategy for NSCLC, especially for patients in whom current EGFR-targeted treatments fail due to the presence of the T790M-EGFR-mutation or high c-MET expression.

Mutation and gene amplification of the epithelial growth factor receptor (EGFR) is one of the most common genetic alterations in glioblastoma (GB). EGFR is, therefore, an attractive molecular target for the treatment of GB. EGFR-targeted therapies however have been largely ineffective in clinical trials. In this study, we investigated the correlation between the EGFR gene amplification status, expression of the EGFR variant III (EGFRvIII) and EGFR variant IV (EGFRvIV) mutations, expression of the phosphatase and tensin homologue gene on chromosome 10 (PTEN) and mutation of the isocitrate dehydrogenase 1 (IDH1) gene and the survival of patients suffering from recurrent glioblastoma who were treated with the EGFR-targeted monoclonal antibody cetuximab in a prospective phase II clinical trial. EGFR amplification was detected in 19 out of 35 GB (54%), EGFRvIII expression in 11 (31.4%) and EGFRvIV expression in 7 (20%). The EGFRvIII and EGFRvIV mutations were exclusively found in GB with EGFR amplification and were almost mutually exclusive with IDH1 mutation (EGFRvIII mutation was found in 1 out of 11 GB with an IDH1 mutation). Patients with an EGFR amplification lacking EGFRvIII expression had a significantly superior progression free survival (PFS) and a numerical better overall survival (OS) following treatment with cetuximab [median PFS 3.03 vs. 1.63 months (p=0.006); median OS 5.57 vs. 3.97 months (p=0.12)]. Within the subgroup of patients with EGFR amplification, patients with EGFRvIII positive glioblastoma had a worse survival [median PFS 1.63 vs. 3.03 months (p=0.01); median OS 3.27 vs. 5.57 months (p=0.08)]. Our observations indicate that the type of EGFR mutation may determine the outcome of GB patients treated with cetuximab. Prospective investigation of both the EGFR amplification and mutation status in clinical trials with EGFR-targeted therapies for GB is indicated.

The aim of the study was to analyse the results of BRCA1/2 testing in a group of patients with double primary breast and ovarian cancer (DPBOC) in Slovenia. Additionally, the family history and the clinicopathologic characteristics of BRCA1/2 mutation positive and negative patients with DPBOC were analysed, comparing them to a group of untested patients with DPBOC. For these groups of patients, survival analysis was also performed. From the 52 patients who were invited to genetic counselling and testing, 20 responded positively (38% compliance). BRCA1/2 mutations were found in 60% (12/20): 45% BRCA1 and 15% BRCA2 (9 and 3 patients, respectively). There was significantly higher grade of ovarian cancer and significantly higher rate of multiple primary breast cancer in BRCA1/2 positive group. Additionaly, there was a trend towards higher rate of first-degree family history of breast cancer, a trend towards higher stage of ovarian cancer, and a trend towards breast cancer being the first cancer in BRCA1/2 positive group. According to survival analysis, the tested group was not a representative sample of the larger untested group (of 51 patients), so we estimate that the rate of BRCA1/2 mutations in DPBOC patients is probably less than 60%. Keywords: breast cancer, ovarian cancer, BRCA1 gene, BRCA2 gene, double primary cancer.

Background Real-time quantitative RT-PCR (RT-qPCR) is a "gold" standard for measuring steady state mRNA levels in RNA interference assays. The knockdown of the epidermal growth factor receptor (EGFR) gene with eight individual EGFR small interfering RNAs (siRNAs) was estimated by RT-qPCR using three different RT-qPCR primer sets. Results Our results indicate that accurate measurement of siRNA efficacy by RT-qPCR requires careful attention for the selection of the primers used to amplify the target EGFR mRNA. Conclusions We conclude that when assessing siRNA efficacy with RT-qPCR, more than one primer set targeting different regions of the mRNA should be evaluated and at least one of these primer sets should amplify a region encompassing the siRNA recognition sequence.

Background The BRCA1 and BRCA2 mutation spectrum and mutation detection rates according to different family histories were investigated in 521 subjects from 322 unrelated Slovenian cancer families with breast and/or ovarian cancer. Methods The BRCA1 and BRCA2 genes were screened using DGGE, PTT, HRM, MLPA and direct sequencing. Results Eighteen different mutations were found in BRCA1 and 13 in BRCA2 gene. Mutations in one or other gene were found in 96 unrelated families. The mutation detection rates were the highest in the families with at least one breast and at least one ovarian cancer - 42% for BRCA1 and 8% for BRCA2. The mutation detection rate observed in the families with at least two breast cancers with disease onset before the age of 50 years and no ovarian cancer was 23% for BRCA1 and 13% for BRCA2. The mutation detection rate in the families with at least two breast cancers and only one with the disease onset before the age of 50 years was 11% for BRCA1 and 8% for BRCA2. In the families with at least two breast cancers, all of them with disease onset over the age of 50 years, the detection rate was 5% for BRCA2 and 0% for BRCA1. Conclusion Among the mutations detected in Slovenian population, 5 mutations in BRCA1 and 4 mutations in BRCA2 have not been described in other populations until now. The most frequent mutations in our population were c.181T > G, c.1687C > T, c.5266dupC and c.844_850dupTCATTAC in BRCA1 gene and c.7806-2A > G, c.5291C > G and c.3978insTGCT in BRCA2 gene (detected in 69% of BRCA1 and BRCA2 positive families).

Fifteen years ago BRCA1 and BRCA2 were reported as high penetrant breast cancer predisposing genes. However, mutations in these genes are found in only a fraction of high risk families. BARD1 is a candidate breast cancer gene, but only a limited number of missense mutations with rather unclear pathogenic consequences have been reported.We screened 196 high risk breast cancer families for the occurrence of BARD1 variants. All genetic variants were analyzed using clinical information as well as IN SILICO predictive tools, including protein modeling. We found three candidate pathogenic mutations in seven families including a first case of a protein truncating mutation (p.Glu652fs) removing the entire second BRCT domain of BARD1. In conclusion, we provide evidence for an increased breast cancer risk associated to specific BARD1 germline mutations. However, these BARD1 mutations occur in a minority of hereditary breast cancer families.

A simple and sensitive real-time reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was developed to quantify threonine-to-methionine substitution at amino acid position 790 (T790M) mutant transcripts in a wild-type (wt) epidermal growth factor receptor background. The assay is based on three unmodified oligonucleotides, and both SYBR Green and a Taqman probe can be used. To increase the discrimination between mutant and wt signals, ARMS (amplification refractory mutation system) and LNA (locked nucleic acid) primers were tested, but a benefit was observed only with plasmids and not with cellular complementary DNA. The RT-qPCR assay using transcript-specific primers can detect as few as 1% T790M transcripts in a wt background and, therefore, will be useful in RNA interference studies specifically targeting mutant RNA.

Cholecystokinin (CCK) mRNA has been detected by in situ hybridization histochemistry using two different oligonucleotide probes in small to medium-sized neurons of layers II-III and X of Rexed and in large neurons of layer IX in the rat spinal cord at cervical, thoracic and lumbo-sacral levels. No labeled cells were detected in the dorsal root ganglia. This confirms the previously established distribution of CCK-like immunoreactivity in layers II-III and X and indicates, in addition, that motoneurons of layer IX may express true genuine CCK whilst conversely, dorsal root ganglia neurons do not.

Purpose of review The proportion of breast cancers directly attributable to determinant hereditary factors is estimated to be 5-10%. A number of recent findings with regard to hereditary breast cancer should affect the criteria and scope of routine genetic testing and, soon, breast cancer therapy. Recent findings The number of genes causing genetic cancer has expanded, mostly with genes that encode proteins that function in the BRCA1/2 pathways. The risk level associated with some genes is still under investigation, but is high for specific mutations. Some mutant alleles occur frequently, some are rare. High-throughput technologies will progressively allow investigating all genes involved in genetic (breast) cancer risks in all individuals for whom this information could be relevant. This and the emerging novel treatment options specific for cancers in mutation carriers will oblige us to progressively drop all currently used selection criteria such as familial phenotype for genomic testing. A major challenge remains the effective penetration of this knowledge in the professional and lay community, the broad application and financing of this high-throughput technology, and the identification of as yet unknown breast cancer predisposition genes. Summary The assessment of breast cancer predisposition genes, previously only an optional predictive genetic test, is growing in importance as it also becomes a therapeutic predictive test.

We followed with interest the articles and commentary notes about the screening of the BRCA2 c.156_157insAlu mutation, a mutation that we have detected for the first in a Portuguese patient residing in Belgium [1]. Machado et al. recently described this mutation in 20 Portuguese families [2], while Peixoto et al. reported a little bit later this same mutation in 14 Portuguese families [3]. All authors agree that this highly recurrent mutation initially occurred in a single patient (founder effect), but with some disagreement about which method to use for the molecular identification of the high breast cancer risk mutation carriers. In a letter to the editor of this journal, Machado and Vaz proposed a three step PCR assay for which they have a patent pending [4]. The mutant BRCA2 allele is unable to generate wild type full length mRNA, but a shorter messenger missing exon 3 (the target exon for the Alu repeat insertion). A shorter BRCA2 mRNA form missing exon 3 can also be generated by the wt allele, but in (much) smaller amounts when compared to the full length form. The biological significance of the BRCA2 alternative messenger missing exon 3 is actually unknown. According to Machado et al. [5] the alternative and mutant transcripts missing exon 3 are not identical, but differ at the level of the exon 10 coding sequence, a property that these authors want to exploit in their proprietary assay to detect the c.156_157insAlu associated mutant transcript. We want to comment shortly on two points. First, because of the frequent occurrence of the c.156_157insAlu mutation in Portuguese breast cancer families (while absent in a corresponding control population) and a clear genotype/phenotype correlation, we are convinced that performing the molecular diagnosis on RNA material in addition to genomic DNA is without significant use and much more cumbersome. Moreover, results obtained on RNA from leucocytes are not necessarily representative for what would occur in the breast epithelial cells. A well designed mutation specific single step gDNA PCR/sequence analysis should be sufficient for the correct characterization of high risk patients or families. Second, the mutation was first reported by us in a publication dating from 2005. As we mentioned in that article, we collected a large number of blood samples from family members residing in Portugal. Then, the team in Lisbon (Dr Vaz and colleagues) accepted to perform for us the genetic counseling for the interested family members living in Portugal. In July 2002 we provided them the clinical phenotypes and genotypes of 24 family members, eight being mutation carriers. We communicated that the mutation corresponded to an insertion of about 300 bp within exon 3 of BRCA2, resulting in the synthesis of mRNA molecules that lack exon 3. This information made it possible to easily characterize the mutation. For these reasons and aside from ethical considerations, we question the patentability of the assay for specific identification of the c.156_157insAlu mutation. We hope this specific mutation, together with all other BRCA1/2 mutations, will remain ‘public domain’ in the interest of all families concerned. E. Teugels (&) J. De Greve Laboratory of Molecular Oncology & Familial Cancer Clinic (UZ Brussel), Vrije Universiteit Brussel, Laarbeeklaan 101, 1090 Brussel, Belgium e-mail: eteugels@uzbrussel.be

New targeted treatments offer an important opening towards the improvement of the results in the treatment of lung cancer. Currently two types of therapeutic targets are developed successfully in the treatment of advanced non-small cell lung cancer: tumour angiogenesis and growth factor receptors. Therapeutic drugs are small molecules inhibitors and the monoclonal antibodies. Bevacizumab is a monoclonal antibody against vascular endothelial growth factor receptor, VEGF, an important molecule in tumour angiogenesis. The combination of bevacizumab with first line chemotherapy increases the median survival of advanced NSCLC with a few weeks. At this moment no biomarker predicting efficacy that can help in the selection of patients is unfortunately available for this expensive treatment, although it is important to select patients based on specific contra-indications. The epidermal growth factor receptor (EGFR or HER1) is activated in NSCLC by several mechanisms. The small molecules erlotinib and gefitinib are targeting the intracellular kinase domain of the EGF receptor, thus inhibiting the signal transduction cascade. Erlotinib is currently registered and reimbursed in Belgium. The concomitant use of these small molecules with chemotherapy is ineffective in non-selected NSCLC patients. On the other hand, these molecules have great activity in patients with tumours having a constitutionally activated EGFR. Changes in the kinase domain of the receptor give rise to extremely high response rates and unexpectedly improved survival duration in patients with NSCLC. Currently studies are exploring whether erlotinib should be employed in the first line treatment of NSCLC. The FIELT study is a translational academic and multicentre phase II study in Belgium and Luxemburg addressing this issue. Specific mechanisms of resistance for these agents are gradually discovered and the new drugs being able to overcome these resistances are at the horizon.

Background The status of the EGFR and HER2-neu genes has not been fully defined in ovarian cancer. An integrated analysis of both genes could help define the proportion of patients that would potentially benefit from targeted therapies. Methods We determined the tumour mutation status of the entire tyrosine kinase (TK) domain of the EGFR and HER2-neu genes in a cohort of 52 patients with invasive epithelial ovarian cancer as well as the gene copy number and protein expression of both genes in 31 of these patients by DGGE and direct sequecing, immunohistochemistry and Fluorescent in Situ Hybridisation (FISH). Results The EGFR was expressed in 59% of the cases, with a 2+/3+ staining intensity in 38%. HER2-neu expression was found in 35%, with a 2/3+ staining in 18%. No mutations were found in exons 18–24 of the TK domains of EGFR and HER2-neu. High polysomy of the EGFR gene was observed in 13% of the invasive epthelial cancers and amplification of the HER2-neu gene was found in 10% and correlated with a high expression level by immunohistochemistry. Mutations within the tyrosine kinase domain were not found in the entire TK domain of both genes, but have been found in very rare cases by others. Conclusion Genomic alteration of the HER2-neu and EGFR genes is frequent (25%) in ovarian cancer. EGFR/HER2-neu targeted therapies should be investigated prospectively and specifically in that subset of patients.

Not available

Background: The most common BRAF mutations in non-small cell lung cancer (NSCLC) are non-V600 in contrast to melanoma. BRAF pathway inhibitors have not been systematically investigated in non-V600 mutations in vitro and in the clinic. We tested the effect of two clinically available BRAF pathway inhibitors (Trametinib and Dabrafenib) on a subset of clinically identified BRAF mutations in a cohort of lung cancers enriched for adenocarcinoma in patients with no or limited smoking history. Methods: NSCLC tumor samples (FFPE) were tested for the presence of EGFR, KRAS, NRAS, HRAS and BRAF mutations by DGGE or NGS-based methods. We generated 15 BRAF expression plasmids, harboring the mutations found in the given cohort and others described in the literature. BRAF mutants were subjected to an in vitro kinase assay. BRAF constructs were also expressed in HEK293T cells (with and without wt-CRAF) to study their impact on ERK signaling and determine the effect of inhibitors. Results: Among 229 NSCLC patients, 12 patients (5.2%) were found to harbor a BRAF mutation in their tumor: V600 (25%), G469A (16.7%), G469V (8.3%), D594N (25%), D594E (8.3%), G596C (8.3%) and G466V (8.3%). Mutations were characterized as activating or kinase-impaired (in-vitro kinase assay). Kinase-impaired BRAF mutants could still activate the ERK pathway in a CRAF-dependent manner, more than wt-BRAF/wt-CRAF co-transfectant. A MEK inhibitor (Trametinib) and a selective BRAF-inhibitor (Dabrafenib) were tested at clinically relevant doses on HEK293T transfectants (either expressing BRAF mutant alone or together with CRAF). ERK signaling induced by activating mutations was reduced in response to both inhibitors separately. Trametinib inhibited the CRAF-dependent ERK signaling induced by impaired-kinase BRAF mutations. Dabrafenib activated the ERK pathway in cells expressing only CRAF as well as cells co-expressing a kinase-impaired BRAF mutation and CRAF. Conclusions: This study predicts sensitivity of activating non-V600 BRAF mutations in lung cancer to Trametinib or Dabrafenib. Targeting kinase impaired BRAF mutations which signal through CRAF using Dabrafenib will require the addition of Trametinib.

Linkage analysis suggests that mutations in the BRCA1 and BRCA2 genes are responsible for cancer predisposition in more than 80% of the families with high incidence of breast/ovarian cancer. However, pathogenic mutations in the BRCA1/2 genes are generally identified in much less than half of the families investigated in a diagnostic setting with the currently used PCR-based screening protocols. Here we report the identification of two different de novo Alu element insertions within the BRCA1/2 coding sequences in three out of the 50 families in which we found a cancer predisposing mutation, suggesting that this type of mutation is much more common than suggested by their occurrence in mutation databases. The Alu insertion in the BRCA2 gene resulted in the removal of the targeted exon from the corresponding mRNA molecule. Unexpectedly the Target Site Duplications generated by both Alu element insertions contained a specific 9 bp long segment, which might eventually serve as a recognition site for the transposition machinery. Finally, in contrast to the disease causing Alu insertions reported to date, the transposon identified in the BRCA1 gene does not belong to a “young” AluY but to an AluS subfamily, indicating that some of these “old” Alu elements, which are supposed to be non-functional fossil relics, are still able to retrotranspose in vivo. © 2005 Wiley-Liss, Inc.

1107 Background: Triple negative breast cancer (TNBC) accounts for 10-20% of all breast cancers and conventional chemotherapy is the only effective systemic treatment. Germline BRCA1/2 mutations ar...