The role of the epithelial-mesenchymal transition (EMT) in cancer has been studied extensively in vitro, but involvement of the EMT in tumorigenesis in vivo is largely unknown. We investigated the potential of microRNAs as clinical markers and analyzed participation of the EMT-associated microRNA-200–ZEB–E-cadherin pathway in cancer progression. Expression of the microRNA-200 family was quantified by real-time RT-PCR analysis of fresh-frozen and microdissected formalin-fixed paraffin-embedded primary colorectal tumors, normal colon mucosa, and matched liver metastases. MicroRNA expression was validated by in situ hybridization and after in vitro culture of the malignant cells. To assess EMT as a predictive marker, factors considered relevant in colorectal cancer were investigated in 98 primary breast tumors from a treatment-randomized study. Associations between the studied EMT-markers were found in primary breast tumors and in colorectal liver metastases. MicroRNA-200 expression in epithelial cells was lower in malignant mucosa than in normal mucosa, and was also decreased in metastatic compared to non-metastatic colorectal cancer. Low microRNA-200 expression in colorectal liver metastases was associated with bad prognosis. In breast cancer, low levels of microRNA-200 were related to reduced survival and high expression of microRNA-200 was predictive of benefit from radiotheraphy. MicroRNA-200 was associated with ER positive status, and inversely correlated to HER2 and overactivation of the PI3K/AKT pathway, that was associated with high ZEB1 mRNA expression. Our findings suggest that the stability of microRNAs makes them suitable as clinical markers and that the EMT-related microRNA-200 – ZEB – E-cadherin signaling pathway is connected to established clinical characteristics and can give useful prognostic and treatment-predictive information in progressive breast and colorectal cancers.

Trastuzumab has markedly improved the treatment and long-term prognosis of patients with HER2-positive breast cancer. A frequent clinical challenge in patients with relapsing and/or metastatic disease is de novo or acquired trastuzumab resistance, and to date no predictive biomarkers for palliative trastuzumab have been established. In the present study, the prognostic values of factors involved in the HER2-associated PI3K/Akt signalling pathway were explored. The first 46 consecutive patients treated at the Department of Oncology, Linköping University Hospital between 2000 and 2007 with trastuzumab for HER2-positive metastatic breast cancer were retrospectively included. The gene copy number variation and protein expression of several components of the PI3K/Akt pathway were assessed in the tumour tissue and biopsy samples using droplet digital polymerase chain reaction and immunohistochemistry. Patients with tumours displaying a high-grade ERBB2 (HER2) amplification level of ≥6 copies had a significantly improved overall survival hazard ratio [(HR)=0.4; 95%, confidence interval (CI): 0.2–0.9] and progression-free survival (HR=0.3; 95% CI: 0.1–0.7) compared with patients with tumours harbouring fewer ERBB2 copies. High-grade ERBB2 amplification was significantly associated with the development of central nervous system metastases during palliative treatment. Copy gain (≥3 copies) of the gene encoding the tyrosine phosphatase PTPN2 was associated with a shorter overall survival (HR=2.0; 95% CI: 1.0–4.0) and shorter progression-free survival (HR=2.1; 95% CI: 1.0–4.1). In conclusion, high ERBB2 amplification level is a potential positive prognostic factor in trastuzumab-treated HER2-positive metastatic breast cancer, whereas PTPN2 gain is a potential negative prognostic factor. Further studies are warranted on the role of PTPN2 in HER2 signalling.

IntroductionSince its introduction in standard of care, trastuzumab has revolutionized the treatment of patients with early and late stages of HER2-positive breast cancer. While the initial clinical trials were convincing and lead to major changes in practice, more knowledge on the long-term outcome and tolerability is needed. The present study was designed to assess the survival, prognostic factors and relapse patterns after the implementation of trastuzumab in a real-world cohort.MethodsAll cases of HER2-positive breast cancer diagnosed between 2006 and 2014 in the Southeast Healthcare Region of Sweden were retrospectively identified. Medical records were thoroughly reviewed with regard to clinicopathological parameters, treatments, relapse pattern and adverse events.Results643 patients were identified and 599 were eligible for analysis. Breast cancer specific survival, distant recurrence free survival and local recurrence free survival were 93.4%, 89.7% and 98.0% for trastuzumab treated patients and 87.4%, 81.6% and 87.4% in patients not treated with trastuzumab, respectively. ER status, nodal status and trastuzumab treatment were all independent prognostic factors in multivariable analysis. No new safety concerns were discovered.ConclusionThe real-world outcome of trastuzumab-treated patients with early HER2-positive breast cancer is similar to what has been previously reported in long-term follow up of prospective clinical trials. ER status, nodal status and trastuzumab treatment are independent prognostic factors for breast cancer specific mortality rate, distant recurrence rate and locoregional recurrence rate in HER2-positive patients in the trastuzumab era.

The majority of all cancer deaths are caused by metastases rather than the primary tumour. The process by which cells can develop migratory properties is suggested to occur through an epithelial-mesenchymal transition (EMT). The microRNA (miR) 200 family have been associated with EMT and acts negatively on ZEB1 which is a repressor of CDH1 (E-cadherin) thereby supports the epithelial maintenance. Our aim was to investigate the expression of the EMT markers ZEB1, CDH1, miR-200c and miR141 in metastatic colorectal cancer and primary breast tumours and to explore their prognostic significance as well as their correlations to clinicopathological factors. Matched tissue from primary tumour and liver metastasis of 10 colorectal cancer patients and another 15 tissues from CR liver metastasis were used in this study. Moreover, primary tumours from 90 stage II breast tumours from postmenopausal patients, randomized to radiotherapy vs chemotherapy as well as tamoxifen or no endocrine treatment, were included. microRNA and gene expression was studied by quantitative real-time PCR. Correlations between the mir-200 family and CDH1 and inverse correlations to ZEB were found in 15 CR liver metastases as well as in the breast cancer material. In the breast cancer material the gene expression of CDH1 was confirmed by positive correlation to CNV of the CDH1 gene. In the liver metastases low CDH1 was associated with recurrence. A reduction in the mir-200 family was seen in matched normal CR mucosas, primary CRC tumours and liver metastasis and lower expression was found in metastatic CRC primary tumours compared to non-metastatic. In the breast cancer material, wild-type p53 was associated with high expression of the miR cluster which is in agreement with recently presented data of p53 as a suggested regulator of the EMT pathway, via the miR-200 family. In addition, low miR-141/200 expression was associated with activation of the PI3K/AKT pathway, especially in combination with a wild-type p53. miR-141 correlated with positive oestrogen receptor (ER) status, whereas miR200c negatively correlated with HER2 status, suggesting that these miRs may play different roles in different subtypes of breast cancer. A low expression of the miR cluster was associated with a worse outcome in terms of a decreased distant recurrence-free survival. In addition, the levels of miR141/200c were associated with the response to radiation and chemotherapy. Among patients carrying tumours with low miR-141/200c expression, chemotherapy significantly reduced the risk of distant metastases, whereas a high expression of miR141/200c predicted good benefit from radiation, using local or distant recurrence-free survival as an endpoint. These findings support the role of miR141/200c in metastatic progression, and indicate that these markers of EMT could be useful prognostic and treatment predictive factors in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 451. doi:1538-7445.AM2012-451

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