EGFR T790M Mutation as a Possible Target for Immunotherapy; Identification of HLA-A*0201-Restricted T Cell Epitopes Derived from the EGFR T790M Mutation

Treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, has achieved high clinical response rates in patients with non–small cell lung cancers (NSCLCs). However, over time, most tumors develop acquired resistance to EGFR-TKIs, which is associated with the secondary EGFR T790M resistance mutation in about half the cases. Currently there are no effective treatment options for patients with this resistance mutation. Here we identified two novel HLA-A*0201 (A2)-restricted T cell epitopes containing the mutated methionine residue of the EGFR T790M mutation, T790M-5 (MQLMPFGCLL) and T790M-7 (LIMQLMPFGCL), as potential targets for EGFR-TKI-resistant patients. When peripheral blood cells were repeatedly stimulated in vitro with these two peptides and assessed by antigen-specific IFN-γ secretion, T cell lines responsive to T790M-5 and T790M-7 were established in 5 of 6 (83%) and 3 of 6 (50%) healthy donors, respectively. Additionally, the T790M-5- and T790M-7-specific T cell lines displayed an MHC class I-restricted reactivity against NSCLC cell lines expressing both HLA-A2 and the T790M mutation. Interestingly, the NSCLC patients with antigen-specific T cell responses to these epitopes showed a significantly less frequency of EGFR-T790M mutation than those without them [1 of 7 (14%) vs 9 of 15 (60%); chi-squared test, p  =  0.0449], indicating the negative correlation between the immune responses to the EGFR-T790M-derived epitopes and the presence of EGFR-T790M mutation in NSCLC patients. This finding could possibly be explained by the hypothesis that immune responses to the mutated neo-antigens derived from T790M might prevent the emergence of tumor cell variants with the T790M resistance mutation in NSCLC patients during EGFR-TKI treatment. Together, our results suggest that the identified T cell epitopes might provide a novel immunotherapeutic approach for prevention and/or treatment of EGFR-TKI resistance with the secondary EGFR T790M resistance mutation in NSCLC patients.     

Discovery of novel selective inhibitors for EGFR-T790M/L858R

Through a receptor-based and ligand-based combined virtual screening protocol, 21 novel compounds covering 15 scaffolds were identified as novel inhibitors for EGFR-T790M/L858R, among which, 12 of them were identified as selective inhibitors for EGFR-T790M/L858R to wild-type EGFR, and 5 of them exhibited 'dual-effective' to wild-type and mutant EGFR. Meanwhile, their antiproliferative effects toward EGFR high-expressing human lung cancer cell (A549), epidermoid carcinoma cell (A431), and the mutant EGFR-dependent cell (NCI-H1975) were also evaluated.     

Gefitinib Inhibits Invasive Phenotype and Epithelial-Mesenchymal Transition in Drug-Resistant NSCLC Cells with MET Amplification

Despite the initial response, all patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) eventually develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). The EGFR-T790M secondary mutation is responsible for half of acquired resistance cases, while MET amplification has been associated with acquired resistance in about 5-15% of NSCLCs. Clinical findings indicate the retained addiction of resistant tumors on EGFR signaling. Therefore, we evaluated the molecular mechanisms supporting the therapeutic potential of gefitinib maintenance in the HCC827 GR5 NSCLC cell line harbouring MET amplification as acquired resistance mechanism. We demonstrated that resistant cells can proliferate and survive regardless of the presence of gefitinib, whereas the absence of the drug significantly enhanced cell migration and invasion. Moreover, the continuous exposure to gefitinib prevented the epithelial-mesenchymal transition (EMT) with increased E-cadherin expression and down-regulation of vimentin and N-cadherin. Importantly, the inhibition of cellular migration was correlated with the suppression of EGFR-dependent Src, STAT5 and p38 signaling as assessed by a specific kinase array, western blot analysis and silencing functional studies. On the contrary, the lack of effect of gefitinib on EGFR phosphorylation in the H1975 cells (EGFR-T790M) correlated with the absence of effects on cell migration and invasion. In conclusion, our findings suggest that certain EGFR-mutated patients may still benefit from a second-line therapy including gefitinib based on the specific mechanism underlying tumor cell resistance.     

Recent Development of the Second and Third Generation Irreversible Epidermal Growth Factor Receptor Inhibitors

Recent reports suggested that essential directions for new lung cancer, breast carcinoma therapies, as well as the roomier realm of targeted cancer therapies were provided through targeting the epidermal growth factor receptor (EGFR). Patients who carrying non‐small cell lung carcinoma (NSCLC) with activating mutations in EGFR initially respond well to the EGFR inhibitors erlotinib and gefitinib, which were located the active site of the EGFR kinase and designed to act as competitive inhibitors of combining with the ATP. However, patients who were treated with the erlotinib and gefitinib will relapse because of the emergence of drug‐resistant mutations, with T790M mutations accounting for approximately 60% of all resistance. In order to overcome drug resistance, Pharmaceutical chemistry experts recently devoted great endeavors to the development of second‐generation irreversible selective inhibitors which covalently modify Cys797 or Cys773 at the ATP binding cleft. Nevertheless, these inhibitors have not reached ideal effect of experts in patients with T790M positive mutation and apparently because of the dose‐limiting toxicities associated with inhibition of wild type EGFR. A novel class of ‘third generation’ EGFR TKIs have been developed that is sensitising and T790M mutant‐specific whilst sparing WT EGFR, representing a significant breakthrough in the treatment in NSCLC patients with acquired resistance harboring these genotypes. Herein, we provides an overview of the second and third generation inhibitors currently approved, in clinical trial and also encompasses novel structures of discovery. This review mainly focuses on drug resistance, their mechanisms of action, development of structure–activity relationships and binding modes.     

Using the MCF10A/MCF10CA1a Breast Cancer Progression Cell Line Model to Investigate the Effect of Active,Mutant Forms of EGFR in Breast Cancer Development and Treatment Using Gefitinib

Background

Basal-like and triple negative breast cancer (TNBC) share common molecular features, poor prognosis and a propensity for metastasis to the brain. Amplification of epidermal growth factor receptor (EGFR) occurs in ~50% of basal-like breast cancer, and mutations in the epidermal growth factor receptor (EGFR) have been reported in up to ~ 10% of Asian TNBC patients. In non-small cell lung cancer several different mutations in the EGFR tyrosine kinase domain confer sensitivity to receptor tyrosine kinase inhibitors, but the tumourigenic potential of EGFR mutations in breast cells and their potential for targeted therapy is unknown.

Materials and Methods

Constructs containing wild type, G719S or E746-A750 deletion mutant forms of EGFR were transfected into the MCF10A breast cells and their tumorigenic derivative, MCF10CA1a. The effects of EGFR over-expression and mutation on proliferation, migration, invasion, response to gefitinib, and tumour formation in vivo was investigated. Copy number analysis and whole exome sequencing of the MCF10A and MCF10CA1a cell lines were also performed.

Results

Mutant EGFR increased MCF10A and MCF10CA1a proliferation and MCF10A gefitinib sensitivity. The EGFR-E746-A750 deletion increased MCF10CA1a cell migration and invasion, and greatly increased MCF10CA1a xenograft tumour formation and growth. Compared to MCF10A cells, MCF10CA1a cells exhibited large regions of gain on chromosomes 3 and 9, deletion on chromosome 7, and mutations in many genes implicated in cancer.

Conclusions

Mutant EGFR enhances the oncogenic properties of MCF10A cell line, and increases sensitivity to gefitinib. Although the addition of EGFR E746-A750 renders the MCF10CA1a cells more tumourigenic in vivo it is not accompanied by increased gefitinib sensitivity, perhaps due to additional mutations, including the PIK3CA H1047R mutation, that the MCF10CA1a cell line has acquired. Screening TNBC/basal-like breast cancer for EGFR mutations may prove useful for directing therapy but, as in non-small cell lung cancer, accompanying mutations in PIK3CA may confer gefitinib resistance.     

BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations

Background

Epidermal growth factor receptor (EGFR) mutations are present in the majority of patients with non-small cell lung cancer (NSCLC) responsive to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. These EGFR-dependent tumors eventually become TKI resistant, and the common secondary T790M mutation accounts for half the tumors with acquired resistance to gefitinib. However, the key proapoptotic proteins involved in TKI-induced cell death and other secondary mutations involved in resistance remain unclear. The objective of this study was to identify the mechanism of EGFR TKI-induced apoptosis and secondary resistant mutations that affect this process.

Methods and Findings

To study TKI-induced cell death and mechanisms of resistance, we used lung cancer cell lines (with or without EGFR mutations), Ba/F3 cells stably transfected with EGFR mutation constructs, and tumor samples from a gefitinib-resistant patient. Here we show that up-regulation of the BH3-only polypeptide BIM (also known as BCL2-like 11) correlated with gefitinib-induced apoptosis in gefitinib-sensitive EGFR-mutant lung cancer cells. The T790M mutation blocked gefitinib-induced up-regulation of BIM and apoptosis. This blockade was overcome by the irreversible TKI CL-387,785. Knockdown of BIM by small interfering RNA was able to attenuate apoptosis induced by EGFR TKIs. Furthermore, from a gefitinib-resistant patient carrying the activating L858R mutation, we identified a novel secondary resistant mutation, L747S in cis to the activating mutation, which attenuated the up-regulation of BIM and reduced apoptosis.

Conclusions

Our results provide evidence that BIM is involved in TKI-induced apoptosis in sensitive EGFR-mutant cells and that both attenuation of the up-regulation of BIM and resistance to gefitinib-induced apoptosis are seen in models that contain the common EGFR T790M and the novel L747S secondary resistance mutations. These findings also suggest that induction of BIM may have a role in the treatment of TKI-resistant tumors.     

Absence of evidence for epidermal growth factor receptor and human homolog of the Kirsten rat sarcoma-2 virus oncogene mutations in breast cancer

Aims: The epidermal growth factor receptor (EGFR) is an available target of effective anti-EGFR therapy for human breast cancer. KRAS, the human homolog of the Kirsten rat sarcoma-2 virus oncogene, encodes a main downstream signaling molecule in the EGFR pathway. The aim of this study was to assess the presence of EGFR and KRAS gene mutations in breast cancer. Materials and methods: EGFR and KRAS gene mutations were investigated in formalin-fixed, paraffin-embedded tissues from 143 Chinese female patients with breast cancer by means of real-time quantitative polymerase chain reaction (RT-PCR). Results: Based on RT-PCR, 2/143 (1.4%) samples and 1/143 (0.7%) had EGFR and KRAS gene mutations, respectively. Overall, none of the cases was identified with mutations of both of these two genes. Conclusions: In this study, both EGFR and KRAS mutations were present rarely in this cohort of samples with breast cancer. This suggested that mutation analyses for EGFR and KRAS are not useful as screening tests for sensitivity to anti-EGFR therapy for breast carcinomas.     

第 3 代 EGFR 抑制剂 Osimertinib Mesylate

Osimertinib mesylate 为阿斯利康公司研发的第 3 代 EGFR 抑制剂,不可逆地抑制EGFR- T790M,2015 年 11 月 13 日获美国 食品药品监督管理局（FDA）批准用于非小细胞肺癌治疗。介绍 osimertinib mesylate 的化学合成、临床前药理学研究、临床研究及专利 保护情况等,为抗肿瘤药物研发提供参考。     

Osimertinib for lung cancer cells harboring low-frequency EGFR T790M mutation

Osimertinib, a third-generation EGFR tyrosine kinase inhibitor, shows significant benefit among patients with EGFR T790M mutation at disease progression. We analyzed the whole exome sequence of 48 samples obtained from 16 lung cancer patients with a longitudinal follow-up: treatment-naïve-baseline primary tumors positive for EGFR activating-mutations, paired re-biopsies upon disease progression but negative for EGFR T790M mutation based on qPCR, and their matched normal blood samples. Our Next generation sequencing (NGS) analysis identified an additional set of 25% re-biopsy samples to harbor EGFR T790M mutation occurring at a low-allele frequency of 5% or less, undetectable by conventional qPCR-based assays. Notably, the clinical utility of osimertinib among patients harboring low-allele frequency of EGFR T790M in tissue biopsy upon disease progression remains less explored. We established erlotinib-resistant PC-9R cells and twenty single-cell sub-clones from erlotinib-sensitive lung cancer PC-9 cells using in vitro drug-escalation protocol. NGS and allele-specific PCR confirmed the low-allele frequency of EGFR T790M present at 5% with a 100-fold higher resistance to erlotinib in the PC-9R cells and its sub-clones. Additionally, luciferase tagged PC-9, and PC-9R cells were orthotopically injected through the intercostal muscle into NOD-SCID mice. The orthotopic lung tumors formed were observed by non-invasive bioluminescence imaging. Consistent with in vitro data, osimertinib, but not erlotinib, caused tumor regression in mice injected with PC-9R cells, while both osimertinib and erlotinib inhibited tumors in mice injected with PC-9 cells. Taken together, our findings could extend the benefit of osimertinib treatment to patients with low EGFR T790M mutation allele frequency on disease progression.     

Structural insights into how irreversible inhibitors can overcome drug resistance in EGFR

Resistance to kinase-targeted cancer drugs has recently been linked to a single point mutation in the ATP binding site of the kinase. In EGFR, the crucial Thr790 gatekeeper residue is mutated to a Met and prevents reversible ATP competitive inhibitors from binding. Irreversible 4-(phenylamino)quinazolines have been shown to overcome this drug resistance and are currently in clinical trials. In order to obtain a detailed structural understanding of how irreversible inhibitors overcome drug resistance, we used Src kinase as a model system for drug resistant EGFR-T790M. We report the first crystal structure of a drug resistant kinase in complex with an irreversible inhibitor. This 4-(phenylamino)quinazoline inhibits wild type and drug resistant EGFR in vitro at low nM concentrations. The co-crystal structure of drug resistant cSrc-T338M kinase domain provides the structural basis of this activity.     

Insight into binding mechanisms of EGFR allosteric inhibitors using molecular dynamics simulations and free energy calculations

Abstract

Lung cancer is the leading cause of cancer death, and epidermal growth factor receptor (EGFR) kinase domain mutations are a common cause of non-small-cell lung cancer (NSCLC), a major subtype of lung cancers. Patients harboring most of these mutations respond well to the EGFR inhibitors Gefitinib and Erlotinib initially, but soon develop resistance to them due to the emergence of the gatekeeper mutation T790M. The new-generation inhibitors such as AZD9291, HM61713, CO-1686 and WZ4002 can overcome T790M through covalent binding to Cys 797, but ultimately lose their efficacy upon the emergence of the C797S mutation that abolishes the covalent bonding. Allosteric inhibitors EAI001 and EAI045 are a new type of EGFR inhibitors that bind to EGFR away from the ATP-binding site and not relying on Cys 797. In this study, molecular dynamics simulations and free energy calculations were carried out on EAI001 and EAI045 in complex with EGFR, revealing the detailed inhibitory mechanism of EAI001 and EAI045 as EGFR allosteric inhibitor, which was expected to provide a basis for rational drug design of the EGFR allosteric inhibitors.

Communicated by Ramaswamy H. Sarma     

Integrin beta1 over-expression associates with resistance to tyrosine kinase inhibitor gefitinib in non-small cell lung cancer

The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) such as gefitinib and erlotinib have been widely used in treating patients with advanced non-small cell lung cancer (NSCLC). However, acquired resistance to EGFR TKI almost occurs in every patient eventually. To identify its potential mechanism, we established a human NSCLC cell line PC9/AB2 which was 576-fold decrease in gefitinib sensitivity compared with its parental PC9 cell lines. No EGFR-T790M mutation or abnormal expression of c-Met protein was found in PC9/AB2 cells. Over-expression of integrin β1 was found, accompanied with increase of the cells' adhesion and migration. To further confirm the role of integrin β1 in gefitinib acquired resistance, we transferred its siRNA-expressing plasmid and its whole cDNA expressing plasmid into PC9/AB2 and into PC9 cells, respectively. The sensitivity of NSCLC cells to gefitinib was negatively correlated with integrin β1 expression levels. All these data suggest that up-regulation of integrin β1 might be an important factor for gefitinib resistance in NSCLC cell line PC9/AB2.     

Identification of novel drug-resistant EGFR mutant inhibitors by in silico screening using comprehensive assessments of protein structures

EGFR is a target protein for the treatment of non small cell lung cancer (NSCLC). The mutations associated with the activation of EGFR kinase activity, such as L858R and G719S, destabilize the inactive conformation of EGFR and are closely linked with the development of NSCLC. The additional T790M mutation reportedly causes drug resistance against the commercially available EGFR inhibitors, gefitinib and erlotinib. In this study, we searched for novel G719S/T790M EGFR inhibitors by a new in silico screening strategy, using two datasets. The results of in silico screening using protein-ligand docking are affected by the selection of 3D structure of the target protein. As the first strategy, we chose the 3D structures for in silico screening by test dockings using the G719S/T790M crystal structure, its molecular dynamics snapshots, and known inhibitors of the drug-resistant EGFR. In the second strategy, we selected the 3D structures by test dockings using all of the EGFR structures, regardless of the mutations, and all of the known EGFR inhibitors. Using each of the 3D structures selected by the strategies, 1000 compounds were chosen from the 71,588 compounds. Kinase assays identified 15 G719S/T790M EGFR inhibitors, including two compounds with novel scaffolds. Analyses of their structure-activity relationships revealed that interactions with the mutated Met790 residue specifically increase the inhibitory activity against G719S/T790M EGFR.     

EGFR基因在非小细胞肺癌、乳腺癌中突变的研究

表皮生长因子受体(EGFR)基因酪氨酸激酶域体细胞突变与非小细胞肺癌(NSCLC)患者对酪氨酸激酶抑制剂吉非替尼敏感性密切相关。文章分析和检测本院75例非小细胞肺癌、10例乳腺癌患者石蜡包埋标本EGFR基因突变状况。采用PCR技术进行EGFR基因19和21外显子突变分析。结果显示:75例NSCLC患者中有13例(13/75,17.33%)酪氨酸激酶域存在体细胞突变。其中7例(7/75,9.33%)为19外显子缺失突变,6例(6/75,8%)为21外显子替代突变(2573T>G,L858R)。病理分型显示,腺癌突变率高于其他几种类型NSCLC。乳腺癌患者均为免疫组化HER-2阳性女性,EGFR基因的19、21外显子中未见突变发生。中国非小细胞肺癌患者总突变率高于高加索人种,女性患者较男性患者突变率高,提示肺腺癌的患者突变率高可能在吉非替尼的治疗中获益。     

用于非小细胞肺癌治疗的第三代 EGFR-TKIs 研究进展

肺癌是全球致死率最高的恶性肿瘤,其中非小细胞肺癌(NSCLC)占全部肺癌病例的 80% 左右。相较于传统化疗,表皮生长因 子受体酪氨酸激酶抑制剂(EGFR-TKIs)用于 EGFR 突变型 NSCLC 患者,可获得更为卓越的疗效。但是,EGFR-TKIs 长期使用会产生 获得性耐药,主要机制为 EGFR T790M 突变。临床研究表明,以奥希替尼、rociletinib 和 HM61713 为代表的第三代 EGFR-TKIs 对 EGFR T790M 突变阳性 NSCLC 具有较好的疗效,为 EGFR T790M 突变阳性患者的有效治疗带来了新的希望。综述第三代 EGFR-TKIs 的最新临床研究及耐药机制研究进展。     

Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer

Background

Triple Negative Breast Cancer (TNBC) accounts for 12–24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20–40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data.

Materials and Methods

PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components.

Results

PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC.

Conclusions

Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.     

Tumor cell killing mechanisms of epidermal growth factor receptor (EGFR) antibodies are not affected by lung cancer-associated EGFR kinase mutations

The epidermal growth factor receptor (EGFR) serves as a molecular target for novel cancer therapeutics such as tyrosine kinase inhibitors (TKI) and EGFR Abs. Recently, specific mutations in the EGFR kinase domain of lung cancers were identified, which altered the signaling capacity of the receptor and which correlated with clinical response or resistance to TKI therapy. In the present study, we investigated the impact of such EGFR mutations on antitumor cell activity of EGFR Abs. Thus, an EGFR-responsive cell line model was established, in which cells with tumor-derived EGFR mutations (L858R, G719S, delE746-A750) were significantly more sensitive to TKI than wild-type EGFR-expressing cells. A clinically relevant secondary mutation (T790M) abolished TKI sensitivity. Significantly, antitumor effects of EGFR Abs, including signaling and growth inhibition and Ab-dependent cellular cytotoxicity, were not affected by any of these mutations. Somatic tumor-associated EGFR kinase mutations, which modulate growth inhibition by TKI, therefore do not impact the activity of therapeutic Abs in vitro.     

Extracellular vesicles shed from gefitinib‐resistant nonsmall cell lung cancer regulate the tumor microenvironment

Epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitors (TKIs), including gefitinib, are the first‐line treatment of choice for nonsmall cell lung cancer patients who harbor activating EGFR mutations, however, acquired resistance to EGFR‐TKIs is inevitable. The main objective of this study was to identify informative protein signatures of extracellular vesicles (EV) derived from gefitinib‐resistant nonsmall cell lung cancer cells using proteomics analysis. Nano‐LC–MS/MS analysis identified with high confidence (false discovery rate < 0.05, fold change ≥2) 664 EV proteins enriched in PC9R cells, which are resistant to gefitinib due to EGFR T790M mutation. Computational analyses suggested components of several signal transduction mechanisms including the AKT (also PKB, protein kinase B)/mTOR (mechanistic target of rapamycin) pathway are overrepresented in EV from PC9R cells. Treatment of recipient cells with EV harvested from PC9R cells increased phosphorylation of signaling molecules, and enhanced proliferation, invasion, and drug resistance to gefitinib‐induced apoptosis. Dose‐ and time‐dependent pharmaceutical inhibition of AKT/mTOR pathway overcame drug resistance of PC9R cells and those of H1975 exhibiting EGFR T790M mutation. Our findings provide new insight into an oncogenic EV protein signature regulating tumor microenvironment, and will aid in the development of novel diagnostic strategies for prediction and assessment of gefitinib resistance.     

BRCA2 mutations and triple-negative breast cancer

Recently, BRCA1 germline mutations were found in a high proportion (14-34%) of patients with triple-negative breast cancer (TNBC). BRCA2 was either not analyzed or showed much lower mutation frequencies. Therefore, we screened a group of TNBC patients (n = 30) of white European descent for mutations in BRCA2 as well as in BRCA1. Cases were unselected for age of disease-onset (median age at breast cancer diagnosis was 58 years, ranging from 37 to 74 years), family history of cancer and BRCA1 and BRCA2 mutation status. Half of the patients (15/30) showed a family history of breast and/or ovarian cancer. A high frequency of deleterious germline mutations was observed in BRCA2 (5/30; 16.7%), and only one case showed a BRCA1 mutation (3.3%). Although the study group was small, these results point to BRCA2 mutations being important in TNBC.