Molecular Prognostic and Predictive Markers in Triple - Negative Breast Cancer

Triple-negative breast cancer (TNBC) is defined as a molecular subtype of breast cancer that lacks expression of hormone receptors (oestrogen and progesterone receptor) and HER2/neu/ErbB2 protein. It accounts for 15–20% of all invasive breast cancers. The occurrence of TNBC is often associated with younger age at the time of diagnosis and pre-menopausal status, early onset of menarche, higher body mass index (BMI) in the pre-menopausal period, race and ethnicity (African, Hispanic) and the presence of germline mutation in the BRCA1/2 genes or somatic mutation in the TP53 or PTEN genes. TNBCs are specific in its aggressive biological behaviour, shorter interval to disease progression and more frequent relapse within five years (19 to 40 months). The most of TNBCs are represented by high-grade invasive carcinomas of no special type (NST) with high proliferation index measured by Ki-67 nuclear expression, followed by metaplastic carcinomas, secretory carcinomas, and adenoid cystic carcinomas. Genetical and morphological heterogeneity inside TNBC is responsible for the higher frequency of primary and secondary resistance to systemic therapy. The scope of this chapter is to summarise the potential therapeutic agents involved in regulation of cell proliferation, migration, angiogenesis, apoptosis, gene expression and DNA damage or immune response. The insight into this issue is essential for the setting of the optimal chemotherapy regimen and targeted therapeutic strategy.

Mechanism of sensitivity to TPF chemoagents and its potential alternative of erbB2 in oral cancer with GDF15 overexpression.

e18542 Background: To explore the mechanism of sensitivity to docetaxel, cisplatin, and 5-fluorouracil (TPF) chemoagents in patients with oral squamous cell carcinoma (OSCC) and growth differentiation factor 15 (GDF15) overexpression recruited in a TPF induction chemotherapy trial; and to explore potential alternative agents for patients who might not benefit from TPF induction chemotherapy. Methods: GDF15-overexpression and silenced OSCC cells were used to investigate sensitivity to TPF chemoagents in vitro and in vivo. Immunoprecipitation combined with mass spectrometry was used to screen out the possible receptor for GDF15, and associated signaling pathways were detected after intervention with the possible receptor of GDF15. The efficacy of an inhibitor of the phosphorylation of the possible GDF15 receptor was evaluated by treatment of GDF15-overexpression OSCC cells in vitro and in vivo. Results: Overexpression of GDF15 in OSCC cell lines and xenografts lead to an increase of chemotherapeutics sensitivity on apoptosis effects induced by docetaxel, cisplatin and 5-fluorouracil, through a caspase -dependent pathway. Immunoprecipitation combined with mass spectrometry revealed that the erbB2 protein was a potential binding protein of GDF15, as verified by coimmunoprecipitation. When GDF15 was overexpressed, the phosphorylation of erbB2 and downstream PI3K/AKT and MAPK/ERK pathways was significantly upregulated, but was downregulated when GDF15 expression was interfered. The erbB2 phosphorylation inhibitor CI1033 significantly inhibited the proliferation and colony-forming ability of GDF15-overexpression OSCC cells as well as the growth of xenografts. CI1033 might be used as a potential targeted drug or alternative of TPF chemoagents for OSCC patients with GDF15 overexpression. Conclusions: OSCC with GDF15 overexpression exhibit sensitivity to TPF chemoagents through a caspase-dependent pathway. ErbB2 phosphorylation inhibitors might be used as potential targeted drugs or an alternative of TPF chemoagents for OSCC patients with GDF15 overexpression.

Microscaled Proteogenomic Methods for Precision Oncology

Cancer proteogenomics integrates genomics, transcriptomics and mass spectrometry (MS)-based proteomics to gain insights into cancer biology and treatment efficacy. A proteogenomics approach was therefore developed for frozen core biopsies using tissue-sparing specimen processing with a “microscaled” proteomics workflow. For technical proof-of-principle, biopsies from ERBB2 positive breast cancers before and 48-72 hours after the first dose of neoadjuvant trastuzumab-based chemotherapy were analyzed. ERBB2 protein and phosphosite levels, as well as mTOR target phosphosites, were significantly more suppressed upon treatment in cases associated with pathological complete response, suggesting MS-based pharmacodynamics is achievable. Furthermore, integrated analyses indicated potential causes of treatment resistance including the absence of ERBB2 amplification (false-ERBB2 positive) and insufficient ERBB2 activity for therapeutic sensitivity despite ERBB2 amplification (pseudo-ERBB2 positive). Candidate resistance features in true-ERBB2+ cases, including androgen receptor signaling, mucin expression and an inactive immune microenvironment were observed. Thus, proteogenomic analysis of needle core biopsies is feasible and clinical utility should be investigated.

ErbB2 overexpression upregulates antioxidant enzymes, reduces basal levels of reactive oxygen species, and protects against doxorubicin cardiotoxicity

Levels of the HER2/ErbB2 protein in the heart are upregulated in some women during breast cancer therapy, and these women are at high risk for developing heart dysfunction after sequential treatment with anti-ErbB2/trastuzumab or doxorubicin. Doxorubicin is known to increase oxidative stress in the heart, and thus we considered the possibility that ErbB2 protein influences the status of cardiac antioxidant defenses in cardiomyocytes. In this study, we measured reactive oxygen species (ROS) in cardiac mitochondria and whole hearts from mice with cardiac-specific overexpression of ErbB2 (ErbB2 tg) and found that, compared with control mice, high levels of ErbB2 in myocardium result in lower levels of ROS in mitochondria ( P = 0.0075) and whole hearts ( P = 0.0381). Neonatal cardiomyocytes isolated from ErbB2 tg hearts have lower ROS levels and less cellular death ( P < 0.0001) following doxorubicin treatment. Analyzing antioxidant enzyme levels and activities, we found that ErbB2 tg hearts have increased levels of glutathione peroxidase 1 (GPx1) protein ( P < 0.0001) and GPx activity ( P = 0.0031) in addition to increased levels of two known GPx activators, c-Abl ( P = 0.0284) and Arg ( P < 0.0001). Interestingly, although mitochondrial ROS emission is reduced in the ErbB2 tg hearts, oxygen consumption rates and complex I activity are similar to control littermates. Compared with these in vivo studies, H9c2 cells transfected with ErbB2 showed less cellular toxicity and produced less ROS ( P < 0.0001) after doxorubicin treatment but upregulated GR activity ( P = 0.0237) instead of GPx. Our study shows that ErbB2-dependent signaling contributes to antioxidant defenses and suggests a novel mechanism by which anticancer therapies involving ErbB2 antagonists can harm myocardial structure and function.

Incomplete surgical resection of ductal carcinomas in situ results in activation of ERBB2 in residual breast cancer cells

ERBB2 amplification and consecutive overexpression is a predictor for poor prognosis in breast cancer patients. In addition, incomplete resection of ERBB2-overexpressing tumors leads to increased proliferation of residual breast cancer cells. While the local release of cytokines is thought to be responsible for the malignant behavior of remaining tumor tissue, the exact mechanism is still unknown. We have analyzed epidermal growth factor receptor (EGFR), activated (p)EGFR, and activated (p)ERBB2 protein expression in ERBB2-overexpressing and in non-ERBB2-overexpressing tumors from patients who underwent breast surgery and consecutive re-excision for involved margins, and compared expression levels by immunohistochemistry. While overall ERBB2 protein expression in the initial and the re-excised sample were comparable, we observed an increase in pERBB2 in ductal carcinomas in situ in both, ERBB2-overexpressing (16/21 vs 24/24; P=0.018, χ2 test) and non-ERBB2-overexpressing tumors (3/28 vs 5/12; P=0.025, χ2 test). pERBB2 was not increased in invasive tumors, regardless on whether the samples had been taken from a ERBB2-overexpressing (9/25 vs 6/17; P=0.261, χ2 test) or a non-ERBB2-overexpressing tumor (1/27 vs 0/8; P=0.581, χ2 test). EGFR expression was only detected in 1/47 ERBB2-overexpressing primary tumors and 2/48 non-ERBB2-overexpressing tumors, and was undetectable in re-excised specimen. Taken together, we have demonstrated an increase in ERBB2 receptor activation in incompletely resected preinvasive breast cancer. We hypothesize that receptor phosphorylation is caused by growth factor stimulation in response to intraoperative tissue damage, and perioperative inhibition of specific cytokines could become a promising therapeutic strategy.