The positive is inside the negative: HER2-negative tumors can express the HER2 intracellular domain and present a HER2-positive phenotype.

Overexpression of human epithelial growth factor receptor 2 (HER2) is a poor prognostic factor in breast cancer. HER2 is a transmembrane receptor comprising an extracellular domain (ECD), a single transmembrane domain, and an intracellular domain (ICD) with tyrosine-kinase activity. Receptor dimerization triggers pivotal effector pathways in cancer, such as phosphatidylinositol 3-kinase (PI3K) signaling. Currently, screening of HER2 in breast tumors for prognostic and therapeutic purposes involves immunohistochemical (IHC) phenotyping for the ECD, in which tumors with IHC scores below 2+ are reported as HER2-negative. We used a label-free liquid chromatography-mass spectrometry (LC-MS) proteomic approach to compare plasma samples from patients with HER2-positive breast tumors and patients with HER2-negative tumors. Patients with HER2-negative tumors expressed higher circulating levels of calpain-10 than patients with HER2-positive tumors. Calpains cleave HER2, releasing its ECD and transforming phenotypically positive tumors into phenotypically negative tumors. Therefore, we investigated the expression of the ICD in HER2-negative samples that overexpressed calpain-10. We found that 16% of HER2-negative tumors were positive for HER2-ICD, which was associated with circulating HER2-ECD. HER2 gene amplification was also observed in some HER2-negative tumors. Positive staining for the PI3K pathway was observed in the HER2-negative, ICD-positive tumors, similar to the HER2-positive cohort. Microarray analysis revealed that HER2-negative, ICD-positive samples clustered between HER2-positive tumors and triple-negative tumors. Survival analysis revealed that outcome in women with HER2-negative, ICD-positive tumors was better than in women bearing HER2-negative, ICD-negative (triple negative) tumors but was quite similar to HER2-positive tumors and worse than women with luminal A tumors. Moreover, in vitro analyses revealed that MDA-MB 231, a triple negative cell line, possesses calpain-10 and HER2-p-ICD up-regulation and blockage of calpain-10 activity promoted an increase in HER2-p-ICD and p-AKT levels, suggesting an increase in these pathways signaling. These data indicate that HER2-negative tumors with HER2-ICD positivity exhibit clinical behavior closer to that of HER2-positive tumors. This indicates a need for HER2-ICD screening when determining the molecular profile of breast tumors. These findings further indicate that lapatinib should be investigated as a target therapy for HER2-ICD-positive breast tumors.

CXCL12, CXCR4 and IFNγ genes expression: implications for proinflammatory microenvironment of breast cancer.

Signals from the microenvironment have a profound influence on the maintenance or progression of breast cancer. In the present study, the frequency of CXCL12 rs1801157 polymorphism in peripheral blood and the expression of CXCL12, CXCR4 and IFNγ mRNA in normal and mammary gland tumor tissues were assessed in breast cancer patients. Genotyping was performed by polymerase chain reaction followed by restriction fragment length polymorphism and expression analyses by quantitative RT-PCR. A lower CXCL12 mRNA relative expression was observed among allele A carriers when compared to GG carriers (p = 0.012). ER-positive breast cancer allele A carriers showed a significantly lower expression of CXCL12 mRNA within tumor tissue than in normal breast tissue when compared to GG ER-positive patients (p = 0.016). CXCR4 mRNA (p < 0.001) and CXCL12 mRNA (p = 0.02) relative expressions were significantly correlated with relative IFNγ mRNA expression. Allele A carriers presenting high levels of IFNγ had a significantly higher expression of CXCR4 mRNA in tumor tissue than GG patients (p = 0.026). It is possible that allele A carrier hormone receptor-positive patients could be more susceptible to metastasis development, since they present a lower CXCL12 expression in tumor tissue, and tumor cells expressing CXCR4 could migrate toward CXCL12 gradient. IFNγ expression increases in order to improve immune response and could favor higher CXCR4 expression leading to migration of cells, possibly of metastatic ones, too.

Toll-like receptor 3: implications for proinflammatory microenvironment in human breast cancer.

Under many circumstances, the host constituents that are found in the tumor microenvironment support a malignancy network and provide the cancer cells with advantages in proliferation, invasiveness and metastasis establishment at remote organs. It is known that Toll like receptors (TLRs) are expressed not only on immune cells but also on cancer cells and it has suggested a deleterious role for TLR3 in inflammatory disease. Hypothesizing that altered IFNγ signaling may be a key mechanism of immune dysfunction common to cancer as well CXCR4 is overexpressed among breast cancer patients, the mRNA expression of TLR3, CXCR4 and IFNγ in breast cancer tumor tissues was investigated. No statistically significant differences in the expression of CXCR4 mRNA, IFNγ and TLR3 between healthy and tumor tissues was observed, however, it was verified a positive correlation between mRNA relative expression of TLR3 and CXCR4 (p < 0.001), and mRNA relative expression of TLR3 was significantly increased in breast cancer tumor tissue when compared to healthy mammary gland tissue among patients expressing high IFNγ (p = 0.001). Since the tumor microenvironment plays important roles in cancer initiation, growth, progression, invasion and metastasis, it is possible to propose that an overexpression of IFNγ mRNA due to the pro-inflammatory microenvironment can lead to an up-regulation of CXCR4 mRNA and consequently to an increased TLR3 mRNA expression even among nodal negative patients. In the future, a comprehensive study of TLR3, CXCR4 and IFNγ axis in primary breast tumors and corresponding healthy tissues will be crucial to further understanding of the cancer network.

TGF-ß polymorphism and its expression correlated with CXCR4 expression in human breast cancer.

The role of chemokines and the growth factors has been extensively analyzed both in cancer risk and tumor progression. The transforming growth factor beta (TGF-ß) and chemokine (C-X-C motif) receptor 4 (CXCR4) genes are implicated in several diseases, including breast cancer. Genomic DNA was obtained from 21 samples of peripheral blood or from normal tissue, previously fixed in formalin and embedded in paraffin for TGF-ß T869C polymorphism analyses. Total cellular RNA was extracted from the same 21 patients, but from fresh tissue (tumor and adjacent healthy from the same breast) for expression analysis by Real Time PCR. No significant differences were observed in genotype distribution according to clinicopathological characteristics. Transforming growth factor beta (TGF-ß) mRNA expression was assessed according to T869C polymorphism and CC patients presented a higher TGF-ß expression but not significant when compared to other genotypes (p = 0.064). A positive correlation was observed in relative mRNA expressions of CXCR4 and TGF-ß (p = 0.020). It is known that overexpression of TGF-ß by both tumor and stromal tissue can facilitate the development of metastases, mainly by TGF-ß stimulated angiogenesis and increased tumor cell motility. Our findings suggested a role of these genes as progression markers for breast carcinoma.

CXCL12 rs1801157 polymorphism and expression in peripheral blood from breast cancer patients.

The role of chemokines has been extensively analyzed both in cancer risk and tumor progression. Among different cytokines, CXCR4 and its ligand CXCL12 have been recently subjected to a closer examination. The single-nucleotide polymorphism (SNP) rs1801157 (previously known as CXCL12-A/SDF1-3'A) in the CXCL12 gene and the relative expression of mRNA CXCL12 in peripheral blood were assessed in breast cancer patients, since the chemokine CXCL12 and its receptor CXCR4 regulate leukocyte trafficking and many essential biological processes, including tumor growth, angiogenesis and metastasis of different types of tumors. Genotyping was performed by PCR-RFLP (polymerase chain reaction followed by restriction fragment length polymorphism) using MspI restriction enzyme and the expression analyses by quantitative RT-PCR. No difference in GG genotype and allele A carrier frequencies were observed between breast cancer patients and healthy blood donors and nor when CXCL12 mRNA expression was assessed among patients with different tumor stages. However a significant difference was observed when CXCL12 mRNA relative expression was analyzed in breast cancer patients in accordance to the presence or absence of the CXCL12 rs1801157 allele A. Allele A breast cancer patients presented a mRNA CXCL12 expression about 2.1-fold smaller than GG breast cancer patients. Estrogen positive patients presenting CXCL12 allele A presented a significantly lower expression of CXCL12 in peripheral blood (p=0.039) than GG hormone positive patients. Our findings demonstrated that allele A is associated with low expression of CXCL12 in the peripheral blood from ER-positive breast cancer patients, which suggests implications on breast cancer clinical outcome.

CCR5 and p53 codon 72 gene polymorphisms: implications in breast cancer development.

The aim of this study was to investigate the CCR5 gene and p53 codon 72 polymorphisms in a Brazilian population with breast cancer compared with healthy control subjects and to associate the clinical stage with these genotypes. No differences were detected for the D32 allele between breast cancer patients and the normal healthy donors (p=0.270), although this allele was more often present in blood donors. For p53 genotype analysis, breast cancer patients presented a significant (p<0.05) over-representation of p53 Arg homozygosity (55.5%) compared with the healthy control group (33.3%). Although no statistical difference occurred, a very strong tendency in breast cancer patients in stage III (p=0.0503) presenting homozygous genotype for Arg was verified. Five breast cancer patients were D32 deletion carriers and two patients presenting metastasis showed homozygous genotype for Arg. It is possible that p53 Arg homozygosity is associated with breast cancer and may represent a potential risk factor for breast tumorigenesis. In the present study, a higher percentage of breast cancer patients presented homozygous genotype for Arg and wild-type for CCR5 than the control subjects.

Plasma malondialdehyde levels and CXCR4 expression in peripheral blood cells of breast cancer patients.

INTRODUCTION: Breast cancer is one of the most common neoplasms in women and is a leading cause of cancer related deaths worldwide. Chemokines and their receptors are involved in the control of lymphocyte traffic, a critical component of systemic immunity. CXCR4 mRNA could be involved in the development of variety of diseases. Lipid peroxidation, the result of nonenzymatic autooxidation of polyunsaturated fatty acids, presents numerous harmful effects on biological systems and has been implicated in diseases like cancer. This study examined CXCR4 mRNA expression in peripheral blood cells and malondialdehyde (MDA) in plasma from blood donors and breast cancer patients. MATERIALS AND METHODS: CXCR4 expression in peripheral blood cells from 59 breast cancer patients and 76 healthy blood donors was analyzed by real-time PCR. Plasma MDA was analyzed using high-performance liquid chromatography (HPLC). CONCLUSION: In all stages, MDA levels in total breast cancer patients (1.41 +/- 0.11) were significantly higher (P < 0.01) than those in healthy subjects (0.34 +/- 0.03). No statistically significant difference occurred between CXCR4 expression in peripheral blood cells from breast cancer patients (1.69 +/- 1.05) and the normal healthy control group (1.8 +/- 0.65). However, stage II samples differed statistically (4.3 +/- 1.72) from control, total cancer patients and stages I, III and IV samples.