P4-07-05: Comparison of PIK3CA Hot Spot Mutations in the Primary Tumor or Metastases with PIK3CA Mutations or PIK3CA Over-Expression in Circulating Tumor Cells of Metastatic Breast Cancer Patients under Sequential Palliative Therapy.
Authors: Aktas B , Kasimir-Bauer S , Kasper S , Derks C , Kimmig R , Schuler M , Tewes M 
Published: June 2012

Abstract
Background: Stem cell like tumor cells have been implied as the active source of metastatic spread in primary tumors. To disseminate and metastasize, these cells may undergo phenotypic changes, known as epithelial-mesenchymal transition (EMT). The PI3K/AKT signaling pathway has been identified as one of the most important and most frequently mutated pathways involved in these processes. Assuming that metastasis requires a dissemination of tumor stem cells or tumor cells showing EMT, we studied 174 blood samples of 43 metastatic breast cancer patients under follow-up of palliative chemo-, antibody - or antihormonal therapy for the presence of sternness like circulating tumor cells (slCTCs). Further, we correlated these data with the occurrence of PIK3CA mutations in slCTCs and with the detection of hot spot mutations as well as PTEN loss in primary tumors or metastases.
Materials and Methods: All blood samples underwent immunomagnetic enrichment using the ***AdnaTest ***BreastCancerSelect (AdnaGen AG, Germany). RNA was recovered and reverse transcribed for analysis using the AdnaTest EMT (multiplex RT-PCR for TWIST, AKT2, PI3K), and separately for the stem cell marker ALDH1 applying the AdnaTest TumorStemCell. The identification of EMT markers was considered positive if at least one of the three markers was detected in the sample. The expression of CD34 was analyzed in a subset of samples to exclude potential interference of normal hematopoietic stem cells. The analysis of PCR products was performed by capillary electrophoresis on the Agilent Bioanalyzer 2100. The PIK3CA 3140 G/A mutation in slCTC-derived cDNA was identified by direct sequencing. PIK3CA hot spot mutations in primary tumors or metastases were identified by direct sequencing from microdissected FFPE tumor tissue. PTEN loss (as defined as &lt;10% of cells exhibiting positive staining) was detected by immunohistochemistry.
Results: During follow up, slCTCs were detected in 23/43 (53%) of the patients at least at one time point. ALDH1 was present in 18/43 (42%) patients, and at least one of the EMT markers was detected in 22/43 (51%) of the patients with the over-expression of PI3K (87%), AKT (96%) and TWIST (22%), respectively. Positivity for both, ALDH1 and EMT markers, was found in 15/23 (65%) of the patients. So far, analyzing one sample of each slCTC-positive patient during follow-up of the disease, there seems to be an occurrence of PIK3CA 3140 G/A mutation in slCTCs in about 25% of the patients. Examining the tissue samples, PTEN loss was found in 3/43 (7%) patients, in two of which slCTCs were present. DNA has been extracted from the dissected tumor tissues of all patients and is currently analyzed for PIK3CA mutations. The results will be available for presentation during the SABCS.
Conclusion: This study provides evidence for the presence of therapy-resistant breast cancer stemness like cells in the blood, possibly derived from the tumor or the metastases. Specific PI3K pathway inhibitors, alone or in combination therapy, may provide a therapeutic strategy for eliminating these cells to improve the prognosis of these patients.
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-05.