Whole-transcriptome analysis links trastuzumab sensitivity of breast tumors to both HER2 dependence and immune cell infiltration.

While results thus far demonstrate the clinical benefit of trastuzumab, some patients do not respond to this therapy. To identify a molecular predictor of trastuzumab benefit, we conducted whole-transcriptome analysis of primary HER2+ breast carcinomas obtained from patients treated with trastuzumab-containing therapies and correlated the molecular portrait with treatment benefit. The estimated association between gene expression and relapse-free survival allowed development of a trastuzumab risk model (TRAR), with ERBB2 and ESR1 expression as core elements, able to identify patients with high and low risk of relapse. Application of the TRAR model to 24 HER2+ core biopsies from patients treated with neo-adjuvant trastuzumab indicated that it is predictive of trastuzumab response. Examination of TRAR in available whole-transcriptome datasets indicated that this model stratifies patients according to response to trastuzumab-based neo-adjuvant treatment but not to chemotherapy alone. Pathway analysis revealed that TRAR-low tumors expressed genes of the immune response, with higher numbers of CD8-positive cells detected immunohistochemically compared to TRAR-high tumors. The TRAR model identifies tumors that benefit from trastuzumab-based treatment as those most enriched in CD8-positive immune infiltrating cells and with high ERBB2 and low ESR1 mRNA levels, indicating the requirement for both features in achieving trastuzumab response

Characterization of the Microflow Through 3D Synthetic Niche Microenvironments Hosted in a Millifluidic Bioreactor

Background: Development of new medicines is a lengthy process with high risk of failure since drug efficacy measured in vitro is difficult to confirm in vivo. Intended to add a new tool aiding drug discovery, the MOAB-NICHOID device was developed: a miniaturized optically accessible bioreactor (MOAB) housing the 3D engineered scaffold NICHOID. The aim of our study was to characterize the microflow through the 3D nichoid microenvironment hosted in the MOAB-NICHOID device. Methods: We used computational fluid dynamics (CFD) simulations to compute the flow field inside a very fine grid resembling the scaffold microenvironment. Results: The microflow inside the multi-array of nichoid blocks is fed and locally influenced by the mainstream flow developed in the perfusion chamber of the device. Here we have revealed a low velocity, complex flow field with secondary, backward, or local recirculation micro-flows induced by the intricate architecture of the nichoid scaffold. Conclusion: Knowledge of the microenvironment inside the 3D nichoids allows planning of cell experiments, to regulate the transport of cells towards the scaffold substrate during seeding or the spatial delivery of nutrients and oxygen which affects cell growth and viability

HER2 splice variants and their relevance in breast cancer

The HER2 gene amplification occurs in 20-30% of breast cancer and is correlated with a poorer prognosis compared to HER2-negative disease due to increased proliferation and metastatic potential. Two major types of receptor inhibitors have been developed for therapy and one for each categories is currently used in clinic: i) the humanized monoclonal antibody trastuzumab, directed against the HER2 extracellular domain; and ii) the EGFR/HER2 dual tyrosine kinase inhibitor lapatinib. However, patients may develop resistance to drugs and show disease progression. Several resistant mechanisms have been explored and are still under investigation. Here, we focus our attention on the role played by the alternative splicing forms of HER2 in mediating HER2 oncogenic activity and in conditioning the response to HER2 therapies. Three HER2 splice variants have been described so far; the p100 and the herstatin gave raised to two secreted proteins of 100 kd and 68 kd, respectively that act as cell growth inhibitors. Herstatin has been described for its ability to interrupt the constitutive HER2 activation, but also for its capacity to hamper HER2 dimerization with the others HER receptors. Interestingly, herstatin, present as mRNA and protein in non cancerous tissue in areas adjacent to breast carcinoma, is absent as protein in 75% of mammary tumors, which indicates that cancer cells are protected by some intrinsic mechanism against the putative growth-inhibitory effects of this naturally occurring molecule. The third splice form of HER2 gene is the Δ16HER2, encoding for a receptor lacking exon16, whose absence determines a constitutive active dimers with transforming activity in vitro and in vivo. The Δ16HER2 binds to trastuzumab to a less extend, due to conformational changes of the extracellular domain. The Δ16HER2 accounts for almost 9% of the total HER2 transcripts in human breast cancers and, additionally, Δ16HER2 levels are supposed to increase proportionally at the increasing of the HER2 wild-type copy numbers in human primary breast cancers. The availability of a specific assay to determine and quantify the expression levels of this splicing form and the availability of Δ16HER2 transgenic mice models made this variant as the most promising for the development of biodrugs. Finally, HER2 carboxy-terminal fragments (CTFs), generated by alternative initiation of translation, were observed in breast cancer patients. In particular, 611-CTF was described to activate multiple signaling pathways since it is expressed as a constitutively active homodimer. Expression of 611-CTF led to development of aggressive and invasive mammary tumors and it was suggested to be a potent oncogene capable of promoting mammary tumor progression and metastasis.</span

Tyrosine Phosphorylation of Grb2: Role in Prolactin/Epidermal Growth Factor Cross Talk in Mammary Epithelial Cell Growth and Differentiation▿

Characterizing mechanisms regulating mammary cell growth and differentiation is vital, as they may contribute to breast carcinogenesis. Here, we examine a cross talk mechanism(s) downstream of prolactin (PRL), a primary differentiation hormone, and epidermal growth factor (EGF), an important proliferative factor, in mammary epithelial cell growth and differentiation. Our data indicate that EGF exerts inhibitory effects on PRL-induced cellular differentiation by interfering with Stat5a-mediated gene expression independent of the PRL-proximal signaling cascade. Additionally, our data show that PRL is a potent inhibitor of EGF-induced cell proliferation. We identify tyrosine phosphorylation of the growth factor receptor-bound protein 2 (Grb2) as a critical mechanism by which PRL antagonizes EGF-induced cell proliferation by attenuating the activation of the Ras/mitogen-activated protein kinase (MAPK) pathway. Together, our results define a novel negative cross-regulation between PRL and EGF involving the Jak2/Stat5a and Ras/MAPK pathways through tyrosine phosphorylation of Grb2

Whole-transcriptome analysis links trastuzumab sensitivity of breast tumors to both HER2 dependence and immune cell infiltration.

While results thus far demonstrate the clinical benefit of trastuzumab, some patients do not respond to this therapy. To identify a molecular predictor of trastuzumab benefit, we conducted whole-transcriptome analysis of primary HER2+ breast carcinomas obtained from patients treated with trastuzumab-containing therapies and correlated the molecular portrait with treatment benefit. The estimated association between gene expression and relapse-free survival allowed development of a trastuzumab risk model (TRAR), with ERBB2 and ESR1 expression as core elements, able to identify patients with high and low risk of relapse. Application of the TRAR model to 24 HER2+ core biopsies from patients treated with neo-adjuvant trastuzumab indicated that it is predictive of trastuzumab response. Examination of TRAR in available whole-transcriptome datasets indicated that this model stratifies patients according to response to trastuzumab-based neo-adjuvant treatment but not to chemotherapy alone. Pathway analysis revealed that TRAR-low tumors expressed genes of the immune response, with higher numbers of CD8-positive cells detected immunohistochemically compared to TRAR-high tumors. The TRAR model identifies tumors that benefit from trastuzumab-based treatment as those most enriched in CD8-positive immune infiltrating cells and with high ERBB2 and low ESR1 mRNA levels, indicating the requirement for both features in achieving trastuzumab response