Thomas Bachleitner-Hofmann

Dendritic Cell-Based Vaccination in Solid Cancer

PURPOSE Dendritic cell (DC)-based immunotherapy is rapidly emerging as a viable tool in cancer treatment. This approach has been used mostly in patients in the presence of defined tumor antigens such as melanoma. In this study, cancer patients with advanced disease that lacks defined tumor antigens were vaccinated with tumor lysate-pulsed DCs. PATIENTS AND METHODS Twenty patients (pancreatic, hepatocellular, cholangiocellular, and medullary thyroid carcinoma) with stage IV disease were enrolled in the study. In 3-week intervals, freshly isolated autologous CD14 magnetic bead-selected monocytes were cultured in granulocyte-macrophage colony-stimulating factor and interleukin-4 to obtain immature DCs. These cells were pulsed with autologous tumor lysate and matured with tumor necrosis factor alpha. Mature DCs were applied into a groin lymph node, under ultrasound guidance. Adjuvant interleukin-2 (20,000 U/kg) was given subcutaneously daily, for 12 days, after each vaccination. Toxicity, tumor marker profile, immune response, and clinical response were determined. RESULTS Vaccination was well tolerated. No physical signs of autoimmunity were detected. DC vaccination induced delayed-type hypersensitivity reactivity in 18 patients. Tumor marker responses were observed in eight patients. In addition, in three patients the generation of interferon gamma-positive T cells was induced during the vaccination. Objective changes in measurable lesions or tumor markers were evident in seven of 20 assessed patients. None of the patients was found to meet the criteria for partial or complete responses. CONCLUSION These data indicate that vaccination with autologous tumor-pulsed DCs generated from peripheral blood is safe and can induce tumor-specific cellular cytotoxicity. Clinical responses are achievable, even in patients with advanced disease.

HGF rescues colorectal cancer cells from EGFR inhibition via MET activation

Purpose: Cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), is active in colorectal cancer (CRC). However, response rates range from only 10% to 20%. Here, we investigate hepatocyte growth factor (HGF)-dependent mesenchymal-epithelial transition factor (MET) activation as a mediator of cetuximab resistance through signal diversification in CRC cell lines. Experimental Design: DiFi, GEO, and LIM1215 cells were treated with varying concentrations and combinations of EGF, HGF, cetuximab, and PHA-665752 (a highly specific MET kinase inhibitor). Biological end points included proliferation, cell cycle arrest, and apoptosis. Proliferation was measured using WST-1 assays and synergy investigated via isobolograms. Expression and signaling were examined using immunoblotting. Results: EGFR and MET are coexpressed in these CRC cell lines, and dual receptor activation synergistically increased proliferation. Cetuximab inhibited cell growth by 60%–80% with an associated dephosphorylation of EGFR, MAPK, and/or AKT. Addition of HGF to cetuximab-treated cells phosphorylated MET, but not EGFR or ErbB3, restimulated the MAPK and AKT pathways, restored cell proliferation, and rescued cells from G1 arrest and apoptosis. Importantly, this effect could be abrogated by inhibiting MET activation with PHA-665752 or by downregulating MET expression with RNAi. Conclusions: HGF-induced MET activation is a novel mechanism of cetuximab resistance in CRC. Inhibition of the HGF-MET pathway may improve response to EGFR inhibitors in CRC, and combination therapy should be further investigated. Clin Cancer Res; 17(3); 472–82. ©2010 AACR.

Enhancement of arsenic trioxide‐mediated apoptosis using docosahexaenoic acid in arsenic trioxide‐resistant solid tumor cells

It has been shown that the polyunsaturated fatty acid docosahexaenoic acid (DHA) can sensitize various tumor cells to reactive oxygen species (ROS)‐inducing anticancer agents. Recently, we demonstrated that DHA also enhances the apoptotic effect of clinically achievable concentrations (1–2 μM) of arsenic trioxide (As2O3) in several As2O3‐resistant human leukemic cell lines via a ROS‐dependent mechanism. The aim of the present study was to evaluate whether this combined effect of As2O3 and DHA is also applicable to As2O3‐resistant solid tumor cells. We have tested 12 different tumor cell lines, including MDA‐MB‐468, SK‐BR‐3, MCF‐7 (breast cancer), ES‐2, SKOV‐3 (ovarian cancer), HT‐29, SW‐620, LS‐174T (colon cancer), PC‐3 (prostate cancer), HeLa (cervical cancer), PANC‐1 (pancreatic cancer) and one primary melanoma cell line. With the exception of MDA‐MB‐468 and ES‐2, all cells were resistant to treatment with either As2O3 or DHA alone. However, combined treatment with As2O3 and DHA significantly reduced viability in 7 of the 10 As2O3‐resistant solid tumors tested. The cytotoxic effect of As2O3 and DHA was associated with the induction of apoptosis and a concomitant increase of intracellular lipid peroxidation products. Importantly, the combined effect of As2O3 and DHA was selectively toxic for malignant cells since no cytotoxic effect was observed in normal skin fibroblasts, human microvascular endothelial cells and peripheral blood mononuclear cells derived from healthy donors. Our data indicate that DHA may help to extend the therapeutic spectrum of As2O3 in the treatment of solid tumors since it may overcome de novo or acquired resistance to As2O3.

Antitumor Activity of SNX-2112, a Synthetic Heat Shock Protein-90 Inhibitor, in MET-Amplified Tumor Cells with or without Resistance to Selective MET Inhibition

Purpose: Heat shock protein-90 (HSP-90), a molecular chaperone required by numerous oncogenic kinases [e.g., HER-2, epidermal growth factor receptor (EGFR), Raf-1, v-Src, and AKT] for conformational stability, has attracted wide interest as a novel target for cancer therapy. HSP-90 inhibition induces degradation of HSP-90 client proteins, leading to a combinatorial inhibition of multiple oncogenic signaling pathways with consecutive growth arrest and apoptosis. MET, a tyrosine kinase that is constitutively active in tumor cells with MET oncogene amplification, has recently been identified as another HSP-90 client. Experimental Design: The aim of our study was to assess the efficacy of SNX-2112, a synthetic HSP-90 inhibitor, in 3 different MET-amplified tumor cell lines (GTL-16, MKN-45, and EBC-1) as well as PR-GTL-16 cells, a GTL-16 subline selected for resistance to the highly selective MET kinase inhibitor PHA-665752. Results: In all cell lines, SNX-2112 led to degradation of MET, HER-2, EGFR, and AKT, as well as abrogation of Ras/Raf/MEK/MAPK and PI3K/AKT signaling, followed by complete cell cycle arrest. SNX-5542, an orally bioavailable prodrug of SNX-2112, displayed significant antitumor efficacy in vivo in nude mice bearing MET-amplified tumor xenografts. Importantly, HSP-90 inhibition maintained its antitumor efficacy in PR-GTL-16 cells both in vitro and in vivo, suggesting that HSP-90 inhibition could be a particularly valuable strategy in MET-amplified tumors that have acquired resistance to MET kinase inhibition. Conclusions: Our study provides evidence for the efficacy of HSP-90 inhibition in MET-amplified cancer cells, particularly when MET kinase inhibitor resistance has emerged. Clin Cancer Res; 17(1); 122–33. ©2011 AACR.

Genomic classifier ColoPrint predicts recurrence in stage II colorectal cancer patients more accurately than clinical factors.

BACKGROUND Approximately 20% of patients with stage II colorectal cancer will experience a relapse. Current clinical-pathologic stratification factors do not allow clear identification of these high-risk patients. ColoPrint (Agendia, Amsterdam, The Netherlands, http://www.agendia.com) is a gene expression classifier that distinguishes patients with low or high risk of disease relapse. METHODS ColoPrint was developed using whole-genome expression data and validated in several independent validation cohorts. Stage II patients from these studies were pooled (n = 416), and ColoPrint was compared with clinical risk factors described in the National Comprehensive Cancer Network (NCCN) 2013 Guidelines for Colon Cancer. Median follow-up was 81 months. Most patients (70%) did not receive adjuvant chemotherapy. Risk of relapse (ROR) was defined as survival until first event of recurrence or death from cancer. RESULTS In the pooled stage II data set, ColoPrint identified 63% of patients as low risk with a 5-year ROR of 10%, whereas high-risk patients (37%) had a 5-year ROR of 21%, with a hazard ratio (HR) of 2.16 (p = .004). This remained significant in a multivariate model that included number of lymph nodes retrieved and microsatellite instability. In the T3 microsatellite-stable subgroup (n = 301), ColoPrint classified 59% of patients as low risk with a 5-year ROR of 9.9%. High-risk patients (31%) had a 22.4% ROR (HR: 2.41; p = .005). In contrast, the NCCN clinical high-risk factors were unable to distinguish high- and low-risk patients (15% vs. 13% ROR; p = .55). CONCLUSION ColoPrint significantly improved prognostic accuracy independent of microsatellite status or clinical variables, facilitating the identification of patients at higher risk who might be considered for additional treatment.

Intermediate Monocytes but Not TIE2-Expressing Monocytes Are a Sensitive Diagnostic Indicator for Colorectal Cancer

We have conducted the first study to determine the diagnostic potential of the CD14++CD16+ intermediate monocytes as compared to the pro-angiogenic subset of CD14++CD16+TIE2+ TIE2-expressing monocytes (TEMs) in cancer. These monocyte populations were investigated by flow cytometry in healthy volunteers (N = 32) and in colorectal carcinoma patients with localized (N = 24) or metastatic (N = 37) disease. We further determined blood levels of cytokines associated with monocyte regulation. The results revealed the intermediate monocyte subset to be significantly elevated in colorectal cancer patients and to show the highest frequencies in localized disease. Multivariate regression analysis identified intermediate monocytes as a significant independent variable in cancer prediction. With a cut-off value at 0.37% (intermediate monocytes of total leukocytes) the diagnostic sensitivity and specificity ranged at 69% and 81%, respectively. In contrast, TEM levels were elevated in localized cancer but did not differ significantly between groups and none of the cytokines correlated with monocyte subpopulations. Of interest, in vitro analyses supported the observation that intermediate monocytes were more potently induced by primary as opposed to metastatic cancer cells which may relate to the immunosuppressive milieu established in the advanced stage of metastatic disease. In conclusion, intermediate monocytes as compared to TIE2-expressing monocytes are a more sensitive diagnostic indicator of colorectal cancer.

Breast-conserving surgery for T3/T4 breast cancer: an analysis of 196 patients

IntroductionBreast conservation therapy (BCT) increases quality of life and self-esteem of breast cancer patients. In special cancer centers up to 90% of patients are treated with BCT. T3/T4 breast cancer is one of the few contraindications for BCT. However, retrospective data suggest that BCT may be eligible in selected cases of T3/T4 breast cancer.MethodWe analyzed retrospectively 196 breast cancer patients (operated between 1995 and 2004) suffering from T3/T4 tumors and compared BCT and radiotherapy with mastectomy in these patients in terms of overall survival (OS), local recurrence free-survival (LRFS) and breast cancer-related death (BCRD).ResultDemographic data showed no significant differences in prognostic factors between patients treated with mastectomy compared with BCT. Kaplan-Meier curves demonstrated no significant difference for OS, LRFS and BCRD between the two groups.DiscussionOur data strongly suggest that BCT with R0 resection followed by radiotherapy is feasible in patients with T3/T4 breast cancer. Prospective studies have to be performed to further investigate this issue.

Plasma HMGB‐1 after the initial dose of epirubicin/docetaxel in cancer

The response of breast cancer patients to neoadjuvant chemotherapy (NCT) is highly heterogeneous, and reliable predictive instruments remain to be defined. High‐mobility group box‐1 (HMGB‐1) protein is a cell death marker, which is easily detectable in plasma. We hypothesized that the initial dose of NCT with epirubicin/docetaxel induces changes in plasma HMGB‐1 which could allow for an early prediction of response to therapy.

Modulation of plasma complement by the initial dose of epirubicin/docetaxel therapy in breast cancer and its predictive value

Sir, We appreciate the comments provided by Garantziotis (2011) on our recent publication in British Journal of Cancer (Michlmayr et al, 2010). In this study, we investigated whether the protein expression profile in plasma samples from breast cancer patients changes within a few days in response to the initial dose of epirubicin/docetaxel therapy. The expression of several plasma proteins was found to be modulated by the therapy, including inter-a-trypsin inhibitor (IaI) and different members of the complement cascade. We focused our attention on the complement components and might have underestimated the potential importance of increased IaI levels. IaI proteins are a family of structurally related serine protease inhibitors with hyaluronan-binding capacities, assembled from a light chain and one of five homologous heavy chains (H1–H5). In correspondence, separation of IaI by two-dimensional gel electrophoresis results in different spots with a molecular weight of B80, 125 and 250 kDa (Josic et al, 2006). In our study, we found that the abundance of several 125 kDa IaI spots (388, 393, 397, 405, 406 and 407) is influenced by the epirubicin/docetaxel therapy (see Figure 1A). All these spots reacted nearly identical to the treatment. Figure 1B shows the expression level of the IaI spot 405 before and after the initial dose of epirubicin/docetaxel (time a and b, respectively). The abundance of this spot increased in nearly all patients (n1⁄4 22; increase1⁄4 32±28%). Garantziotis referred in his letter to a recently found interaction of IaI with the complement system. In an experimental study, he could show that IaI attenuates in vitro complement activation and reduces in vivo complement-induced lung injury (Garantziotis et al, 2007). This is of special interest with respect to our findings. Our study revealed that the plasma level of total C3 decreases in response to epirubicin/docetaxel therapy (Figure 1C). Comparing the abundances of IaI (spot 405) with the total plasma C3 revealed that both parameters correlate negatively with each other (Spearman’s Rho r1⁄4 0.49, Po0.01, n1⁄4 44). The total plasma

Omega-3 and omega-6 polyunsaturated fatty acids enhance arsenic trioxide efficacy in arsenic trioxide-resistant leukemic and solid tumor cells.

Recently we showed that the polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) sensitizes arsenic trioxide (As2O3)-resistant tumor cells to a clinically achievable concentration (1 microM) of As2O3 via a reactive oxygen species (ROS)-dependent mechanism. The aim of the present study was to evaluate, whether this combined effect of As2O3 and DHA is also applicable to other PUFAs [i.e., eicospentaenoic acid (EPA), arachidonic acid (AA), and gamma-linolenic acid (GLA)]. Fourteen tumor cell lines were incubated with As2O3 (1 microM), PUFA (25-100 microM), or the combination thereof (+/- vitamin E). Cell viability (colorimetric), apoptosis (bivariate annexin V/propidium iodide staining, detection of hypodiploid DNA), and thiobarbituric acid reactive substances (TBARS) were evaluated. Twelve of 14 As2O3-resistant cell lines tested were resistant to PUFA monotherapy. However, combined treatment with As2O3 and either PUFA significantly reduced cell viability in a dose-dependent manner with AA being the most potent As2O3 enhancer. The combined cytotoxic effect of As2O3/AA treatment was due to induction of apoptosis, preceded by increased intracellular TBARS and was abolished by the antioxidant vitamin E. Importantly, the combined effect of As2O3 and AA was selectively toxic for malignant cells because no cytotoxic effect was observed in normal skin fibroblasts and human microvascular endothelial cells. In conclusion, our study shows that also other PUFAs than DHA-and in particular the omega-6-PUFA AA--can be used as effective modulators of tumor cell chemosensitivity to clinically achievable concentrations of As2O3. Enhanced lipid peroxidation most likely constitutes the key mechanism for the combined effect.