Efstathios N. Stathopoulos

Akt1 and Akt2 protein kinases differentially contribute to macrophage polarization

Activated macrophages are described as classically activated or M1 type and alternatively activated or M2 type, depending on their response to proinflammatory stimuli and the expression of genetic markers including iNOS, arginase1, Ym1, and Fizz1. Here we report that Akt kinases differentially contribute to macrophage polarization, with Akt1 ablation giving rise to an M1 and Akt2 ablation resulting in an M2 phenotype. Accordingly, Akt2−/− mice were more resistant to LPS-induced endotoxin shock and to dextran sulfate sodium (DSS)-induced colitis than wild-type mice, whereas Akt1−/− mice were more sensitive. Cell depletion and reconstitution experiments in a DSS-induced colitis model confirmed that the effect was macrophage-dependent. Gene-silencing studies showed that the M2 phenotype of Akt2−/− macrophages was cell autonomous. The microRNA miR-155, whose expression was repressed in naive and in LPS-stimulated Akt2−/− macrophages, and its target C/EBPβ appear to play a key role in this process. C/EBPβ, a hallmark of M2 macrophages that regulates Arg1, was up-regulated upon Akt2 ablation or silencing. Overexpression or silencing of miR-155 confirmed its central role in Akt isoform-dependent M1/M2 polarization of macrophages.

Cytokeratin-19 mRNA-Positive Circulating Tumor Cells After Adjuvant Chemotherapy in Patients With Early Breast Cancer

PURPOSE To evaluate the prognostic significance of cytokeratin-19 (CK-19) mRNA-positive circulating tumor cells (CTCs) in peripheral blood of women with early-stage breast cancer after the completion of adjuvant chemotherapy. PATIENTS AND METHODS Blood was obtained from 437 patients with early breast cancer before the start and after the completion of adjuvant chemotherapy, and the presence of CK-19 mRNA-positive CTCs was assessed by real-time reverse transcriptase polymerase chain reaction. Interaction with known prognostic factors and association of CTCs with clinical outcome were investigated. RESULTS CK-19 mRNA-positive CTCs were detected before chemotherapy in 179 patients (41.0%). After adjuvant chemotherapy, a significant change in CK-19 status was observed, as status for 51% of patients with initially CK-19 mRNA-positive disease turned negative, and status for 22% of patients with initially CK-19 mRNA-negative disease became positive (McNemar test P = .004). The detection of CK-19 mRNA-positive CTCs postchemotherapy was associated with involvement of more than three axillary lymph nodes (P = .026). Clinical relapses and disease-related deaths were significantly increased in patients with detectable postchemotherapy CK-19 mRNA-positive CTCs (both P < .001, respectively). Disease-free and overall survival were significantly reduced in patients with detectable CK-19 mRNA-positive CTCs postchemotherapy (P < .001 and P = .001, respectively). In multivariate analysis, the detection of CK-19 mRNA-positive CTCs before and after adjuvant chemotherapy was an independent factor associated with reduced disease-free survival (P < .001) and overall survival (P = .003). CONCLUSION The detection of CK-19 mRNA-positive CTCs in the blood after adjuvant chemotherapy is an independent risk factor indicating the presence of chemotherapy-resistant residual disease.

Different Prognostic Value of Cytokeratin-19 mRNA Positive Circulating Tumor Cells According to Estrogen Receptor and HER2 Status in Early-Stage Breast Cancer

PURPOSE To examine the prognostic value of cytokeratin-19 (CK-19) mRNA-positive circulating tumor cells (CTCs) in early-stage breast cancer patients focusing on clinically relevant subgroups based on estrogen receptor (ER) and HER2 expression. PATIENTS AND METHODS CK-19 mRNA-positive CTCs were detected by real-time reverse transcriptase polymerase chain reaction in the blood of 444 consecutive, stage I-III, breast cancer patients before initiation of adjuvant chemotherapy. The association between detection of CK-19 mRNA-positive CTCs and clinical outcome was analyzed for patients with ER-positive, ER-negative, triple-negative, HER2-positive, and ER-positive/HER2-negative tumors. RESULTS CK-19 mRNA-positive CTCs were detected in 181 (40.8%) of 444 patients; 109 (41.9%) of 260 patients with ER-positive tumors; 71 (40.6%) of 175 patients with ER-negative tumors; 27 (35%) of 77 patients with triple-negative tumors; 35 (39.8%) of 88 patients with HER2-positive tumors; and 82 (44.1%) of 186 patients with ER-positive/HER2-negative tumors. After a median follow-up of 53.5 months, patients with CK-19 mRNA-positive CTCs experienced reduced disease-free survival (DFS; P < .001) and overall survival (OS; P < .001); this was mainly observed in patients with ER-negative (P < .001 and P < .001, respectively) but not ER-positive tumors (P = .172 and P = .425, respectively) and in patients with triple-negative (P = .008 and P = .001, respectively) and HER2-positive (P = .023 and P = .040, respectively) but not ER-positive/HER2-negative tumors (P = .210 and P = .578, respectively). In multivariate analysis, the interaction between CK-19 mRNA-positive CTCs and ER status was the strongest independent prognostic factor for reduced DFS (hazard ratio [HR], 3.808; 95% CI, 2.415 to 6.003; P < .001) and OS (HR, 4.172; 95% CI, 2.477 to 9.161; P < .001). CONCLUSION Detection of CK-19 mRNA-positive CTCs before adjuvant chemotherapy predicts poor clinical outcome mainly in patients with ER-negative, triple-negative, and HER2-positive early-stage breast cancer.

Prognostic Value of the Molecular Detection of Circulating Tumor Cells Using a Multimarker Reverse Transcription-PCR Assay for Cytokeratin 19, Mammaglobin A, and HER2 in Early Breast Cancer

Purpose: To investigate the prognostic value of the molecular detection of circulating tumor cells (CTCs) using three markers [cytokeratin 19 (CK19), mammaglobin A (MGB1), and HER2] in early breast cancer. Experimental Design: CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected using real-time (CK19) and nested (MGB1 and HER2) reverse transcription-PCR in the peripheral blood of 175 women with stage I to III breast cancer before the initiation of adjuvant chemotherapy. The detection of CTCs was correlated with clinical outcome. In 10 patients, immunofluorescence staining experiments were done to investigate the coexpression of cytokeratin, MGB1, and HER2 in CTCs. Results: CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected in 41.1%, 8%, and 28.6% of the 175 patients, respectively. Patients had one of the following molecular profiles: CK19mRNA+/MGB1mRNA+/HER2mRNA+ (n = 8), CK19mRNA+/MGB1mRNA+/HER2mRNA− (n = 1), CK19mRNA+/MGB1mRNA−/HER2mRNA+ (n = 42), CK19mRNA+/MGB1mRNA−/HER2mRNA− (n = 21), CK19mRNA−/MGB1mRNA+/HER2mRNA− (n = 5), and CK19mRNA−/MGB1mRNA−/HER2mRNA− (n = 98). Double-immunofluorescence experiments confirmed the following CTC phenotypes: CK+/MGB1+, CK+/MGB1−, CK−/MGB1+, CK+/HER2+, CK+/HER2−, MGB1+/HER2−, and MGB1+/HER2+. In univariate analysis, the detection of CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells was associated with shorter disease-free survival (DFS; P < 0.001, P = 0.001, and P < 0.001, respectively), whereas the detection of CK19mRNA+ and MGB1mRNA+ cells was associated with worse overall survival (P = 0.044 and 0.034, respectively). In multivariate analysis, estrogen receptor–negative tumors and the detection of CK19mRNA+ and MGB1mRNA+ cells were independently associated with worse DFS. Conclusion: The detection of peripheral blood CK19mRNA+ and MGB1mRNA+ cells before adjuvant chemotherapy predicts poor DFS in women with early breast cancer.

Comparison of EGFR and K-RAS gene status between primary tumours and corresponding metastases in NSCLC

In non-small-cell lung cancer (NSCLC), epidermal growth factor receptor (EGFR) and K-RAS mutations of the primary tumour are associated with responsiveness and resistance to tyrosine kinase inhibitors (TKIs), respectively. However, the EGFR and K-RAS mutation status in metastases is not well studied. We compared the mutation status of these genes between the primary tumours and the corresponding metastases of 25 patients. Epidermal growth factor receptor and K-RAS mutation status was different between primary tumours and corresponding metastases in 7 (28%) and 6 (24%) of the 25 patients, respectively. Among the 25 primary tumours, three ‘hotspot’ and two non-classical EGFR mutations were found; none of the corresponding metastases had the same mutation pattern. Among the five (20%) K-RAS mutations detected in the primary tumours, two were maintained in the corresponding metastasis. Epidermal growth factor receptor and K-RAS mutations were detected in the metastatic tumours of three (12%) and five (20%) patients, respectively. The expressions of EGFR and phosphorylated EGFR showed 10 and 50% discordance, in that order. We conclude that there is substantial discordance in EGFR and K-RAS mutational status between the primary tumours and corresponding metastases in patients with NSCLC and this might have therapeutic implications when treatment with TKIs is considered.

MicroRNA expression analysis in triple-negative (ER, PR and Her2/neu) breast cancer

miRNAs are small, regulatory molecules approximately 21-24 nucleotides in length. They function at the post-transcriptional level by controlling the expression of more than 50% of human protein-coding genes and play an essential role in cell signaling pathways. The objective of the present study was to explore the expression profile of oncomiRs and tumor-suppressor miRs, and to define their possible correlations in triple-negative (ER, PR and Her2/neu) primary breast cancers. Forty-nine primary triple-negative breast cancer cases, along with 34 matched tumor-associated normal samples were investigated for the expression of 9 miRNAs using qPCR. Relationships between the expression of miR-10b, miR-21, miR-122a, miR-145, miR-205, miR-210, miR-221, miR-222 and miR-296 and the pathologic features of the tumors were examined, as were the influences of miR expression on patient overall and cancer-specific survival. miR-21, miR-210 and miR-221 were significantly overexpressed, whereas miR-10b, miR-145, miR-205, miR-122a were significantly underexpressed in the triple-negative primary breast cancers. Significant correlations among all of the studied miRs were scored both in the breast cancer and control tissue. Expression of miR-222 and miR-296 did not exhibit any significant difference between the breast cancer and normal tissue. There was a non-significant trend for high expression levels of the microRNAs, miR-21, miR-210, miR-221 and miR-222, to be associated with worse patient disease-free and overall survival. miR-21, miR-210 and miR-221 expression plays a significant role in triple- negative primary breast cancers.

The human prostate cancer cell line LNCaP bears functional membrane testosterone receptors that increase PSA secretion and modify actin cytoskeleton

Recent findings have shown that, in addition to the genomic action of steroids, through intracellular receptors, short‐time effects could be mediated through binding to membrane sites. In the present study of prostate cancer LNCaP cells, we report that dihydrotestosterone and the non‐internalizable analog testosterone‐BSA increase rapidly the release of prostate‐specific antigen (PSA) in the culture medium. Membrane testosterone binding sites were identified through ligand binding on membrane preparations, flow cytometry, and confocal laser microscopy of the non‐internalizable fluorescent analog testosterone‐BSA‐FITC, on whole cells. Binding on these sites is time‐ and concentration‐dependent and specific for testosterone, presenting a Kd of 10.9 nM and a number of 144 sites/mg protein (~13000 sites/cell). Membrane sites differ immunologically for intracellular androgen receptors. The secretion of PSA after membrane testosterone receptor stimulation was inhibited after pretreatment with the actin cytoskeleton disrupting agent cytochalasin B. In addition, membrane testosterone binding modifies the intracellular dynamic equilibrium of monomeric to filamentous actin and remodels profoundly the actin cytoskeleton organization. These results are discussed in the context of a possible involvement of these sites in cancer chemotherapy.

Trastuzumab Administration Can Effectively Target Chemotherapy-Resistant Cytokeratin-19 Messenger RNA–Positive Tumor Cells in the Peripheral Blood and Bone Marrow of Patients With Breast Cancer

Purpose: The detection of disseminated occult breast cancer cells in peripheral blood and bone marrow is associated with poor prognosis. Since a high proportion of these cells express the HER-2 receptor, we evaluated the effectiveness of the anti-HER-2 antibody trastuzumab (Herceptin) administration to eliminate them. Experimental Design: Thirty patients with prior chemotherapy exposure were recruited to the study on the basis of having detectable cytokeratin-19 (CK-19) mRNA transcripts by nested reverse transcription (RT)-PCR in the peripheral blood and/or bone marrow. There were 13 patients with stage I, II, or III breast cancer and 17 with stage IV disease. They were treated in two cohorts with either 4 to 8 weekly infusions of trastuzumab at 2 mg/kg (4 mg/kg loading dose; 20 patients) or 2 to 3 infusions every 3 weeks at 6 mg/kg (8 mg/kg loading dose; 10 patients). All of the patients’ samples were also analyzed for HER-2 by nested RT-PCR, but detectable HER-2 messenger RNA (mRNA) was not required for inclusion in the study. After trastuzumab infusions, patients were closely monitored by nested RT-PCR and real-time RT-PCR for the detection of CK-19 mRNA-positive cells. Results: Before trastuzumab infusions, CK-19 mRNA-positive cells were detected in the peripheral blood (n = 10), bone marrow (n = 14), or both (n = 6). In 25 of 30 patients (83%), HER-2 mRNA expression was detected by nested RT-PCR in the pretrastuzumab CK-19–positive sample. After trastuzumab infusions, overall, 28 of 30 (93%) patients became CK-19 mRNA negative by nested RT-PCR and 20 of 30 (67%) by real-time RT-PCR. After a median follow-up of 6 months (range 2 to 22+), the median duration of CK-19 mRNA negativity by nested RT-PCR was 9, 12, and 6 months for stage I/II, III, and IV disease, respectively. Conclusions: Therapy-resistant CK-19 mRNA-positive cells in the peripheral blood and bone marrow can be effectively targeted by trastuzumab administration. Further studies are needed to evaluate the prognostic significance of the disappearance of these cells.

Impact of KRAS, BRAF, PIK3CA Mutations, PTEN, AREG, EREG Expression and Skin Rash in ≥2nd Line Cetuximab-Based Therapy of Colorectal Cancer Patients

Background To investigate the predictive significance of KRAS, BRAF, PIK3CA mutational status, AREG- EREG mRNA expression, PTEN protein expression and skin rash in metastatic colorectal cancer (mCRC) patients treated with cetuximab containing salvage chemotherapy. Methods Primary tumors from 112 mCRC patients were analyzed. The worst skin toxicity during treatment was recorded. Results KRAS, BRAF and PIK3CA mutations were present in 37 (33%), 8 (7.2%) and 11 (9.8%) cases, respectively, PTEN was lost in 21 (19.8%) cases, AREG and EREG were overexpressed in 48 (45%) and 51 (49%) cases. In the whole study population, time to tumor progression (TTP) and overall survival (OS) was significantly lower in patients with KRAS (p = 0.001 and p = 0.026, respectively) or BRAF (p = 0.001 and p<0.0001, respectively) mutant tumors, downregulation of AREG (p = 0.018 and p = 0.013, respectively) or EREG (p = 0.002 and p = 0.004, respectively) and grade 0-1 skin rash (p<0.0001 and p<0.0001, respectively). In KRAS wt patients TTP and OS was significantly lower in patients with BRAF (p = 0.0001 and p<0.0001, respectively) mutant tumors, downregulation of AREG (p = 0.021 and p = 0.004, respectively) or EREG (p = 0.0001 and p<0.0001, respectively) and grade 0-1 skin rash (p<0.0001 and p<0.0001, respectively). TTP was significantly lower in patients with PIK3CA mutations (p = 0.01) or lost PTEN (p = 0.002). Multivariate analysis revealed KRAS (Hazard Ratio [HR] 4.3, p<0.0001), BRAF mutation (HR: 5.1, p<0.0001), EREG low expression (HR: 1.6, p = 0.021) and absence of severe/moderate skin rash (HR: 4.0, p<0.0001) as independent prognostic factors for decreased TTP. Similarly, KRAS (HR 2.9, p = 0.01), BRAF mutation (HR: 3.0, p = 0.001), EREG low expression (HR: 1.7, p = 0.021), absecence of severe/moderate skin rash (HR: 3.7, p<0.0001) and the presence of undifferantited tumours (HR: 2.2, p = 0.001) were revealed as independent prognostic factors for decreased OS. Conclusions These results underscore that KRAS-BRAF mutations and EREG expression can be used as biomarkers to further select patients undergoing anti-EGFR treatment.

Detection of cytokeratin-19 mRNA-positive cells in the peripheral blood and bone marrow of patients with operable breast cancer

Background:To compare detection rates and evaluate the clinical relevance of cytokeratin-19 (CK-19) mRNA-positive cells in the peripheral blood (circulating tumour cells, CTCs) and bone marrow (disseminated tumour cells; DTCs) of patients with early breast cancer.Methods:Paired samples of peripheral blood and bone marrow were obtained from 165 patients with stage I–II breast cancer before the initiation of adjuvant chemotherapy. In 84 patients, paired blood and bone marrow samples were also available after chemotherapy. The detection of CK-19 mRNA-positive CTCs and DTCs was assessed by real-time PCR.Results:CK-19 mRNA-positive CTCs and DTCs were detected in 55.2 and 57.6% of patients before chemotherapy, respectively. After chemotherapy, CTCs and DTCs were identified in 44 (52.4%) and 43 (51.2%) of the 84 patients, respectively. There was a 93.9% (McNemar; P=0.344) and 72.6% (McNemar; P=0.999) concordance between blood and bone marrow samples before and after chemotherapy, respectively. The detection of CK-19 mRNA-positive CTCs or DTCs before chemotherapy was associated with decreased overall survival (P=0.024 and P=0.015, respectively). In addition, their simultaneous detection was also associated with an increased incidence of disease-related death and decreased overall survival (P=0.016).Conclusions:The detection of CK-19 mRNA-positive CTCs using reverse transcription-PCR (RT–PCR) both before and after chemotherapy is correlated with the detection of CK-19 mRNA-positive DTCs in patients with early-stage breast cancer. The determination of the CTC status by RT–PCR conveys clinically relevant information that is not inferior to DTC status and, owing to the ease of sampling, warrants further evaluation as a tool for monitoring minimal residual disease.