| BackgroundsSorafenib, an oral drug developed as an inhibitor of Ras/Raf/ERK pathway, is the firstand only drug that has been approved by the FDA for the treatment of advancedhepatocellular carcinoma (HCC). However, it has been reported that the response rate ofsorafenib is actually quite low, and the disease-stabilizing effect normally lasts only for afew months. Only23%of HCC cases were pERK positive in tumor tissues, and thesuppression of RAS/RAF/ERK signaling pathway by sorafenib may generate thecompensatory activation of PI3K/AKT siganling pathway. Therefore, it is necessary toexamine the activation status of both pathways in HCC before sorafenib treatment, andmonitor and evaluate the efficacy during and after sorafenib treatment. Considering thatcirculating tumor cells (CTCs) are a readily accessible real-time liquid biopsy of tumors toreplace metastatic tissue biopsies, we detected phospho-AKT (pAKT) and phospho-ERKexpressions in CTCs from HCC patients, and evaluate whether they are used as therapeuticbiomarkers that may predict the response to sorafenib.Methods1. Several HCC cell lines were used in cell-spiking experiment to establish amulti-color immunofluorescent detecion model of CTCs, utilizing CKs as an identifyingmarker, and simultaneously detecting the expression of pAKT and pERK.2. Serial tissue sections of18HCC patients were examined by immunohistochemistryof pAKT/pERK expression.3. A total of30patients were invovled. Five milliliters of the whole blood was drawnfrom each subject, and mononuclear cells were enriched by Ficoll density gradient.4. The separated HCC CTCs were centrifuged on a glass slide, and cell cytospinswere subsequently triple stained by CKs antibody along with pAKT and pERK antibodies. 5. The results obtained form CTCs and from HCC tissues were compared, andanalized for correlations.6. Among30patients,8patients received sorafenib treatment, another5milliliters ofblood samples were collected2weeks after treatment, and HCC CTC numbers weremeasured. Also, clinical data were collected and survival data were followed up forefficacy evaluation.Results1. The expression of pAKT and pERK diversifies in the tissues from18HCC patients.Accordingly, they can be divided into four types as pAKT(+)/pERK(+), pAKT(+)/pERK(-),pAKT(-)/pERK(+), and pAKT(-)/pERK(-). They were2cases (11.1%),13cases (72.2%),2cases (11.1%) and1case (5.6%), respectively. Among them,15cases were pAKT+(83.3%), and4cases were pERK+(22.2%).2. CTCs were identified in28out of30HCC patients, averaged31±18per5milliliters peripheral blood. CTCs with four different phenotypes of pAKT/pERK werefound in HCC patients. They were pAKT(+)/pERK(+), pAKT(+)/pERK(-),pAKT(-)/pERK(+) and pAKT(-)/pERK(-).3. The numbers and ratio of these four kinds of CTCs varies in different patients.According to pAKT and pERK expression status in CTCs, all the patients could be dividedinto four types: pAKT(+)/pERK(+), pAKT(+)/pERK(-), pAKT(-)/pERK(+) andpAKT(-)/pERK(-). They were5cases (17.9%),18cases (64.3%),3cases (10.7%), and2cases (7.1%) respectively. pAKT (+) CTCs were found in23patients (82.1%), and pERK(+) CTCs in8(28.6%).4. The phenotypes of CTCs varies even in individuals. In some patients, only onephenotype of CTCs were detected; In others,2to4of all4phenotypes of CTCs weredetected. The numbers of each phenotype were different.5. The pAKT/pERK status of HCC patients detected in CTCs was in concordancewith that detected in tumor tissue (16/17,94%).6. Among8patients received sorafenib treatment,2patients were pAKT(+)/pERK(+),3pAKT(+)/pERK(-),2pAKT(-)/pERK(+) and1pAKT(-)/pERK(-). After sorafenibteatment,2cases with pAKT(-)/pERK(+) CTCs had a sharp reduced CTCs numbers, from26and27to5and7, respectively.2case with pERK(+)/pAKT(+) CTCs exhibited a comparatively slighter decline in CTCs numbesr, while3pAKT(+)/pERK(-) and1pAKT(-)/pERK(-) cases showed very little change in CTCs numbers.ConclusionsThe current results indicate that pERK and pAKT expression status in HCC CTCswould be helpful to predict the response to sorafenib, and could be used as therapeuticbiomarkers to guide the individualized therapy for HCC patients undergoing sorafenibtreatment. |
PDF Full Text Request