Expression of the CTLA-4 ligand CD86 on plasmacytoid dendritic cells (pDC) predicts risk of disease recurrence after treatment discontinuation in CML.

This corrects the article DOI: 10.1038/leu.2017.9.

Long-term survival of sorafenib-treated FLT3-ITD-positive acute myeloid leukaemia patients relapsing after allogeneic stem cell transplantation.

BACKGROUND: Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD)-positive acute myeloid leukaemia (AML) relapsing after allogeneic stem cell transplantation (allo-SCT) has a dismal prognosis with limited therapeutic options. FLT3-ITD kinase inhibition is a reasonable but palliative experimental treatment alternative in this situation. Information on long-term outcome is not available. METHODS: We performed a long-term follow-up analysis of a previously reported cohort of 29 FLT3-ITD-positive AML patients, which were treated in relapse after allo-SCT with sorafenib monotherapy. FINDINGS: With a median follow-up of 7.5 years, 6 of 29 patients (21%) are still alive. Excluding one patient who received a second allo-SCT, five patients (17%) achieved sustained complete remissions with sorafenib. Four of these patients are in treatment-free remission for a median of 4.4 years. INTERPRETATION: Sorafenib may enable cure of a proportion of very poor risk FLT3-ITD-positive AML relapsing after allo-SCT.

Assessment of imatinib as first-line treatment of chronic myeloid leukemia: 10-year survival results of the randomized CML study IV and impact of non-CML determinants.

Chronic myeloid leukemia (CML)-study IV was designed to explore whether treatment with imatinib (IM) at 400 mg/day (n=400) could be optimized by doubling the dose (n=420), adding interferon (IFN) (n=430) or cytarabine (n=158) or using IM after IFN-failure (n=128). From July 2002 to March 2012, 1551 newly diagnosed patients in chronic phase were randomized into a 5-arm study. The study was powered to detect a survival difference of 5% at 5 years. After a median observation time of 9.5 years, 10-year overall survival was 82%, 10-year progression-free survival was 80% and 10-year relative survival was 92%. Survival between IM400 mg and any experimental arm was not different. In a multivariate analysis, risk group, major-route chromosomal aberrations, comorbidities, smoking and treatment center (academic vs other) influenced survival significantly, but not any form of treatment optimization. Patients reaching the molecular response milestones at 3, 6 and 12 months had a significant survival advantage. For responders, monotherapy with IM400 mg provides a close to normal life expectancy independent of the time to response. Survival is more determined by patients' and disease factors than by initial treatment selection. Although improvements are also needed for refractory disease, more life-time can currently be gained by carefully addressing non-CML determinants of survival.

Expression of the CTLA-4 ligand CD86 on plasmacytoid dendritic cells (pDC) predicts risk of disease recurrence after treatment discontinuation in CML.

It is unknown, why only a minority of chronic myeloid leukemia (CML) patients sustains treatment free remission (TFR) after discontinuation of tyrosine kinase inhibitor (TKI) therapy in deep molecular remission (MR). Here we studied, whether expression of the T-cell inhibitory receptor (CTLA-4)-ligand CD86 (B7.2) on plasmacytoid dendritic cells (pDC) affects relapse risk after TKI cessation. CML patients in MR displayed significantly higher CD86+pDC frequencies than normal donors (P<0.0024), whereas TFR patients had consistently low CD86+pDC (n=12). This suggested that low CD86+pDC might be predictive of TFR. Indeed, in a prospective analysis of 122 patients discontinuing their TKI within the EURO-SKI trial, the one-year relapse-free survival (RFS) was 30.1% (95% CI 15.6-47.9) for patients with >95 CD86+pDC per 105 lymphocytes, but 70.0% (95% CI 59.3-78.3) for patients with <95 CD86+pDC (hazard ratio (HR) 3.4, 95%-CI: 1.9-6.0; P<0.0001). Moreover, only patients with <95 CD86+pDC derived a significant benefit from longer (>8 years) TKI exposure before discontinuation (HR 0.3, 95% CI 0.1-0.8; P=0.0263). High CD86+pDC counts significantly correlated with leukemia-specific CD8+ T-cell exhaustion (Spearman correlation: 0.74, 95%-CI: 0.21-0.92; P=0.0098). Our data demonstrate that CML patients with high CD86+pDC counts have a higher risk of relapse after TKI discontinuation.

[Molecular tumour therapy]. / Molekulare Tumortherapie.

In recent years, molecular tumour therapy has dramatically improved the treatment of various types of cancer. Molecular therapy is expected to attack malignant cells more specifically with fewer side effects than conventional chemotherapy. Both kinase inhibitors and monoclonal antibodies are key components of today's molecular cancer therapy. These substances cannot fully overcome the major tumour-biological problems, e.g. therapy resistance. However, as can be vividly seen with the example of immune checkpoint inhibitors, the rational design of molecularly designed drugs will considerably change therapeutic practice in oncology, albeit at the expense of exponentially growing health care costs.

Ruxolitinib in corticosteroid-refractory graft-versus-host disease after allogeneic stem cell transplantation: a multicenter survey.

Despite major improvements in allogeneic hematopoietic cell transplantation over the past decades, corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) cause high mortality. Preclinical evidence indicates the potent anti-inflammatory properties of the JAK1/2 inhibitor ruxolitinib. In this retrospective survey, 19 stem cell transplant centers in Europe and the United States reported outcome data from 95 patients who had received ruxolitinib as salvage therapy for SR-GVHD. Patients were classified as having SR-aGVHD (n=54, all grades III or IV) or SR-cGVHD (n=41, all moderate or severe). The median number of previous GVHD-therapies was 3 for both SR-aGVHD (1-7) and SR-cGVHD (1-10). The overall response rate was 81.5% (44/54) in SR-aGVHD including 25 complete responses (46.3%), while for SR-cGVHD the ORR was 85.4% (35/41). Of those patients responding to ruxolitinib, the rate of GVHD-relapse was 6.8% (3/44) and 5.7% (2/35) for SR-aGVHD and SR-cGVHD, respectively. The 6-month-survival was 79% (67.3-90.7%, 95% confidence interval (CI)) and 97.4% (92.3-100%, 95% CI) for SR-aGVHD and SR-cGVHD, respectively. Cytopenia and cytomegalovirus-reactivation were observed during ruxolitinib treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. Ruxolitinib may constitute a promising new treatment option for SR-aGVHD and SR-cGVHD that should be validated in a prospective trial.

NFATc1 as a therapeutic target in FLT3-ITD-positive AML.

Internal tandem duplications (ITD) in the Fms-related tyrosine kinase 3 receptor (FLT3) are associated with a dismal prognosis in acute myeloid leukemia (AML). FLT3 inhibitors such as sorafenib may improve outcome, but only few patients display long-term responses, prompting the search for underlying resistance mechanisms and therapeutic strategies to overcome them. Here we identified that the nuclear factor of activated T cells, NFATc1, is frequently overexpressed in FLT3-ITD-positive (FLT3-ITD+) AML. NFATc1 knockdown using inducible short hairpin RNA or pharmacological NFAT inhibition with cyclosporine A (CsA) or VIVIT significantly augmented sorafenib-induced apoptosis of FLT3-ITD+ cells. CsA also potently overcame sorafenib resistance in FLT3-ITD+ cell lines and primary AML. Vice versa, de novo expression of a constitutively nuclear NFATc1-mutant mediated instant and robust sorafenib resistance in vitro. Intriguingly, FLT3-ITD+ AML patients (n=26) who received CsA as part of their rescue chemotherapy displayed a superior outcome when compared with wild-type FLT3 (FLT3-WT) AML patients. Our data unveil NFATc1 as a novel mediator of sorafenib resistance in FLT3-ITD+ AML. CsA counteracts sorafenib resistance and may improve treatment outcome in AML by means of inhibiting NFAT.

Interferon alpha 2 maintenance therapy may enable high rates of treatment discontinuation in chronic myeloid leukemia.

A minority of chronic myeloid leukemia (CML) patients is capable of successfully discontinuing imatinib. Treatment modalities to increase this proportion are currently unknown. Here, we assessed the role of interferon alpha 2a (IFN) on therapy discontinuation in a previously reported cohort of 20 chronic phase CML patients who were treated upfront with IFN alpha plus imatinib followed by IFN monotherapy to maintain cytogenetic or molecular remission (MR) after imatinib discontinuation. After a median follow-up of 7.9 years (range, 5.2-12.2), relapse-free survival was 73% (8/11 patients) and 84% (5/6 patients) for patients who discontinued imatinib in major MR (MMR) and MR4/MR4.5, respectively. Ten patients discontinued IFN after a median of 4.5 years (range, 0.24-9.3). After a median of 2.8 years (range, 0.7-5.1), nine of them remain in ongoing treatment-free remission with MR5 (n=6) and MR4.5 (n=3). The four patients who still administer IFN are in stable MR5, MR4.5, MR4, and MMR, respectively. In conclusion, an IFN/imatinib induction treatment followed by a temporary IFN maintenance therapy may enable a high rate of treatment discontinuation in CML patients in at least MMR when stopping imatinib.

Safety and efficacy of imatinib in CML over a period of 10 years: data from the randomized CML-study IV.

Tyrosine kinase inhibitors (TKI) have changed the natural course of chronic myeloid leukemia (CML). With the advent of second-generation TKI safety and efficacy issues have gained interest. The randomized CML - Study IV was used for a long-term evaluation of imatinib (IM). 1503 patients have received IM, 1379 IM monotherapy. After a median observation of 7.1 years, 965 patients (64%) still received IM. At 10 years, progression-free survival was 82%, overall survival 84%, 59% achieved MR(5), 72% MR(4.5), 81% MR(4), 89% major molecular remission and 92% MR(2) (molecular equivalent to complete cytogenetic remission). All response levels were reached faster with IM800 mg except MR(5). Eight-year probabilities of adverse drug reactions (ADR) were 76%, of grades 3-4 22%, of non-hematologic 73%, and of hematologic 28%. More ADR were observed with IM800 mg and IM400 mg plus interferon α (IFN). Most patients had their first ADR early with decreasing frequency later on. No new late toxicity was observed. ADR to IM are frequent, but mostly mild and manageable, also with IM 800 mg and IM 400 mg+IFN. The deep molecular response rates indicate that most patients are candidates for IM discontinuation. After 10 years, IM continues to be an excellent initial choice for most patients with CML.

Immune regulatory plasmacytoid dendritic cells selectively accumulate in perithyroidal lymph nodes of patients with Graves disease: implications for the understanding of autoimmunity.

UNLABELLED: Endocrine orbitopathy (EO) is the most common extrathyroidal manifestation of Graves disease (GD) but the involvement of the underlying immunological dysregulations remains largely unknown. The major source for IFNa-production, plasmacytoid dendritic cells (PDC) are fundamentally involved in the integration of TH1 and TH2 immune responses but also implicated in the pathogenesis of autoimmune diseases such as lupus. AIM: To establish whether PDC may play a role in GD autoimmune reaction and in the pathogenesis of EO. MATERIAL AND METHODS: In a series of six sequential patients with GD as well as six further patients with multinodular goiter, cervical lymph nodes (LN) were sampled and preserved in the setting of thyroid resection. In parallel, peripheral blood samples were collected. The frequency of PDC from the peripheral blood and lymph nodes were determined. Mononuclear cells (MC) were enriched from lymph nodes and peripheral blood using Fiquoll-hypaque density gradient centrifugation. Mononuclear cells were stained using BDCA-2-PE and CD123-FITC monoclonal antibodies and their frequency subsequently analyzed using fluorescence activated cell sorting (FACS). Dead cells were excluded from analysis by appropriate gating strategies and propidium iodide (PI) staining. RESULTS: In all patients with GD (with or without EO), PDC frequency was significantly increased in perithyroidal LN as compared to LN of patient not suffering form autoimmune diseases, e. g. patients with multinodular goiter (p < 0.01). The number of PDC infiltrating lymph nodes (LN-PDC) was also higher when compared to peripheral blood PDC (pB-PDC) of patients with multinodular goiter (p < 0.05). Finally, LN-PDC counts in perithyroidal lymph nodes of patients with GD were substantially increased when compared to pB-PDC of the same patients (p < 0.01) indicating a migration and accumulation of PDC in the draining LN. CONCLUSIONS: We found evidence that PDC selectively accumulate in perithyoidal LN of patients with GD, but not other thyroid diseases such as multinodular goiter. Based on their central importance in the pathogenesis of autoimmune processes such as in lupus erythematoides, we suggest that the migration and accumulation of PDC in an anatomical joining point between the thyroid gland and orbita may be of critical importance for the initiation and maintenance of a chronic autoimmune stimulation. This implies a so far unknown role for PDC in GD and as putative cellular targets for new therapeutic approaches.

Toxicity-reduced, myeloablative allograft followed by lenalidomide maintenance as salvage therapy for refractory/relapsed myeloma patients.

Relapse after dose-reduced allograft in advanced myeloma patients remains high. To reduce the risk of relapse, we investigated a myeloablative toxicity-reduced allograft (aSCT) consisting of i.v. BU and CY followed by lenalidomide maintenance therapy in 33 patients with multiple myeloma (MM) who relapsed following an autograft after a median of 12 months. The cumulative incidence of non-relapse mortality at 1 year was 6% (95% confidence interval (CI): 0-14). After a median interval of 168 days following aSCT, 24 patients started with a median dose of 5 mg (r, 5-15) lenalidomide without dexamethasone. During follow-up, 13 patients discontinued lenalidomide owing to progressive disease (n=6), GvHD (n=3), thrombocytopenia (n=2), or fatigue (n=2). Major toxicities of lenalidomide were GvHD II-III (28%), viral reactivation (16%), thrombocytopenia (III-IV°,16%), neutropenia (III/IV°, 8%), peripheral neuropathy (I/II°, 16%), or other infectious complication (8%). Cumulative incidence of relapse at 3 years was 42% (95% CI: 18-66). The 3-year estimated probability of PFS and OS was 52% (95% CI: 28-76) and 79% (95% CI: 63-95), respectively. Toxicity-reduced myeloablative allograft followed by lenalidomide maintenance is feasible and effective in relapsed patients with MM, but the induction of GvHD should be considered.

High activity of sorafenib in FLT3-ITD-positive acute myeloid leukemia synergizes with allo-immune effects to induce sustained responses.

Preliminary evidence suggests that the multikinase inhibitor sorafenib has clinical activity in FLT3-ITD-positive (FLT3-ITD) acute myeloid leukemia (AML). However, the quality and sustainability of achievable remissions and clinical variables that influence the outcome of sorafenib monotherapy are largely undefined. To address these questions, we evaluated sorafenib monotherapy in 65 FLT3-ITD AML patients treated at 23 centers. All but two patients had relapsed or were chemotherapy-refractory after a median of three prior chemotherapy cycles. Twenty-nine patients (45%) had undergone prior allogeneic stem cell transplantation (allo-SCT). The documented best responses were: hematological remission in 24 patients (37%), bone marrow remission in 5 patients (8%), complete remission (with and without normalization of peripheral blood counts) in 15 patients (23%) and molecular remission with undetectable FLT3-ITD mRNA in 10 patients (15%), respectively. Seventeen of the patients without prior allo-SCT (47%) developed sorafenib resistance after a median treatment duration of 136 days (range, 56-270 days). In contrast, allo-SCT patients developed sorafenib resistance less frequently (38%) and significantly later (197 days, range 38-225 days; P=0.03). Sustained remissions were seen exclusively in the allo-SCT cohort. Thus, sorafenib monotherapy has significant activity in FLT3-ITD AML and may synergize with allogeneic immune effects to induce durable remissions.

Differential diagnosis of pericardial effusion after stem cell transplantation in acute myeloic leukemia.

The pathology underlying a pericardial effusion in a 24-year-old patient, who had suffered from acute myeloic leukemia 5 years previously and undergone chemotherapy followed by whole body radiation prior to allogeneic stem cell transplantation, could be identified by the careful analysis of pericardial cytology and epicardial biopsy guided by flexible pericardioscopy. Molecular, histological, cytochemical and immunological examination of the effusion and the epicardial biopsy for a viral or bacterial infection despite known CMV reactivation, or an effusion induced by radiation or graft-versus-host reaction, could be ruled out as possible causes of pericardial tamponade. The infiltration of CD 117-positive cells in the biopsied cardiac tissue revealed recurrent acute myeloic leukemia now also affecting the heart and the pericardium. An intrapericardial instillation of 1000 mg triamcinolone acetate at day 1 and 50 mg/m(2) cisplatin at day 3 effectively prevented the recurrence of tamponade, but could not prevent a lethal outcome 3 weeks later.

[Chronic myeloid leukemia. Diagnostics, therapy and future strategy]. / Chronische myeloische Leukämie. Diagnostik, Therapie und Zukunftsstrategie.

Survival of patients with chronic myeloid leukemia (CML) has dramatically improved with the introduction of the BCR-ABL-specific tyrosine kinase inhibitor imatinib. As a rule patients on therapy with imatinib achieve permanent complete cytogenetic and molecular remission. Patients who are primarily refractive to imatinib or lose remission achieved using imatinib are in the minority. This group has a poor prognosis. This article gives a transparent review of the diagnostics necessary when CML is primarily diagnosed and for assessment of the response during the course of the therapy. The guidelines developed for this procedure by the European leukemia network on the type and frequency of surveillance controls as well as the diagnostic criteria for imatinib resistance or suboptimal response will be presented. The indications for allogenic stem cell transplantation and the administration of second generation BCR-ABL inhibitors will be discussed as therapeutic alternatives in cases of imatinib failure in a stage-specific manner. Finally a view on therapy targets and forms of future first-line therapy of CML will be given.

[Sorafenib in relapsed and refractory FLT3-ITD positive acute myeloid leukemia: a novel treatment option]. / Sorafenib bei rezidivierter und therapierefraktärer FLT3-ITD-positiver akuter myeloischer Leukämie: eine neue Behandlungsoption.

BACKGROUND: The therapeutic options for relapsed or refractory FLT3-ITD positive AML are limited, particularly in case of a prior allogenic stem cell transplantation (SCT) or poor performance status. The clinical value of a targeted intervention using the FLT3-ITD-specific inhibitor sorafenib in this situation is largely unknown. PATIENTS AND METHODS: Between 2007 and 2010 eight patients (4 men, 4 women; age 40-75 years) with relapsed or refractory FLT3-ITD positive acute myeloid leukemia (AML) before (n=4) and after allogenic SCT (n=5) were treated off-label with sorafenib. RESULTS: All patients showed rapid hematological responses. There were three complete molecular remissions when sorafenib was given after allogenic SCT. Two of them are ongoing for 12 and 15 months, respectively. Long-term remissions after prior allogenic SCT were associated with the re-establishment of a chronic graft versus host reaction. Side effects could be controlled by dose reduction. CONCLUSION: Sorafenib is apparently an effective treatment alternative for patients with relapsed or refractory FLT3-ITD positive AML. In the context of a prior allogenic SCT it may have curative potential via inducing a synergism between targeted inhibition of FLT3-ITD and anti-leukemic immunity.

Differential effects of cetuximab and AEE 788 on epidermal growth factor receptor (EGF-R) and vascular endothelial growth factor receptor (VEGF-R) in thyroid cancer cell lines.

This study evaluated the role of EGF and the effects of EGF-targeting drugs (Cetuximab, AEE 788) on growth, apoptosis, and autocrine VEGF-secretion of thyroid cancer (TC) cells. Autocrine activation of the epidermal growth factor receptor (EGF-R) is commonly regarded to contribute to the malignant phenotype of TC cells and may therefore represent a rational therapeutic target. Out of a number of TC cell lines two anaplastic (Hth74, C643), one follicular (FTC133), and one papillary thyroid cancer cell line (TPC1) were analyzed in depth for VEGF-R-and EGF-R-expression, basal and EGF-stimulated (1-100 ng/ml) VEGF protein secretion and proliferation. Subsequently the antiprolifereative and antiangiogenic effect of cetuximab (Erbitux), a monoclonal antibody that blocks the EGF-R and AEE 788, a novel dual-kinase inhibitor of EGF-R and VEGF-R were assessed, and the downstream EGF-R signal transduction was analyzed by means of detecting phosphorylated pEGF-R, pVEGF-R, pAkt, and p-MAPK. EGF stimulated VEGF-mRNA expression and protein secretion in all TC cell lines. The EGF-R antagonist Cetuximab consistently decreased VEGF secretion in all TC cell lines (min. 15%, n.s. in C643 cells and max. 90% in Hth74 cells, P < 0.05), but did not affect tumor cell proliferation in vitro. In contrast, the EGF-R- and VEGF-R-kinase inhibitor AEE 788 not only reduced VEGF secretion (min. 55%, P < 0.05 in C643 and max. 75%, P < 0.05, in FTC133), but also exhibited a dose-dependent inhibition of tumor cell proliferation (min. 75%, P < 0.05 in C643 and max. 95%, P < 0.05 in Hth74) and was a potent inductor of apoptosis in two of four TC cell lines. These effects were always accompanied by reduced levels of pEGF-R, pVEGF-R, pAkt, and pMAPK. Although inhibition of the EGF-receptor by Cetuximab potently disrupts autocrine secretion of VEGF, only the concurrent inhibition of the VEGF- and EGF receptor, e.g., by AEE 788 induces reduced proliferation and apoptosis in vitro. This suggests a particular rationale for the use of tyrosine kinase inhibitors with dual modes of action such as AEE 788 in thyroid cancer.

Imatinib mesylate and nilotinib (AMN107) exhibit high-affinity interaction with ABCG2 on primitive hematopoietic stem cells.

The majority of chronic phase chronic myeloid leukemia (CML) patients treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate maintain durable responses to the drug. However, most patients relapse after withdrawal of imatinib and advanced stage patients often develop drug resistance. As CML is considered a hematopoietic stem cell cancer, it has been postulated that inherent protective mechanisms lead to relapse in patients. The ATP binding-cassette transporters ABCB1 (MDR-1; P-glycoprotein) and ABCG2 are highly expressed on primitive hematopoietic stem cells (HSCs) and have been shown to interact with TKIs. Herein we demonstrate a dose-dependent, reversible inhibition of ABCG2-mediated Hoechst 33342 dye efflux in primary human and murine HSC by both imatinib and nilotinib (AMN107), a novel aminopyrimidine inhibitor of BCR-ABL. ABCG2-transduced K562 cells were protected from imatinib and nilotinib-mediated cell death and from downregulation of P-CRKL. Moreover, photoaffinity labeling revealed interaction of both TKIs with ABCG2 at the substrate binding sites as they compete with the binding of [(125)I] IAAP and also stimulate the transporter's ATPase activity. Therefore, our evidence suggests for the role of ABC transporters in resistance to TKI on primitive HSCs and CML stem cells and provides a rationale how TKI resistance can be overcome in vivo.

Targeting the EGF/VEGF-R system by tyrosine-kinase inhibitors--a novel antiproliferative/antiangiogenic strategy in thyroid cancer.

AIM: In thyroid cancer (TC), endothelial growth factor (EGF) has been associated with dedifferentiation, tumor cell proliferation, and angiogenesis. Vascular endothelial growth factor (VEGF) has been documented to be the main stimulator of angiogenesis in the thyroid gland. Patients with undifferentiated thyroid cancer are in desperate need of new therapeutic strategies because common protocols of therapy usually fail. The aim of this study, therefore, was to evaluate two tyrosine-kinase inhibitors (TKI, ZD 1839 gefitinib and ZD 6474 vandetanib), directed against the EGF/VEGF receptor for possible antitumor therapy in thyroid cancer. METHODS: EGF/VEGF-R was documented in anaplastic (Hth74, C643), follicular (FTC133), and papillary (TPC1) thyroid cancer cell lines by Western blot analysis. The antiproliferative effect of two TKI (0.1-10 microM) on thyroid cancer cell lines in vitro was quantified by MTT assay, the antiangiogenic effect by assessing secretion of VEGF by enzyme-linked immunosorbent assay (R&D Systems). ZD 1839 is mainly directed against EGF-R and ZD 6474 against VEGF-R (AstraZeneca, UK), single applications and combinations of compounds were evaluated. RESULTS: EGF-R and VEGF-R as well as the phosphorylated receptor were documented in all of the cell lines. Administration of ZD1839 led to an up to 90% reduction of cell number in Hth74, 80% in C643, 50% in FTC133, and 90% in TPC1 (p < 0.05). ZD1839 induced a decrease of VEGF secretion between 30% in C643 and 90% in Hth74. Administration of ZD6474 led to an up to 95% reduction of cell number in Hth74, 85% in C643, 90% in FTC133, and 90% in TPC1 (p < 0.05). The ZD6474 induced decrease of VEGF secretion ranged between 20% (FTC133) and 60% (TPC1). Combinations of IC50 concentrations of TKI showed synergistic effects, resulting in additional inhibition of proliferation between 50 and 90% compared to single drug administration. CONCLUSION: The EGF/EGF-R system resembles a powerful VEGF-stimulating pathway in all histiotypes of TC and can be inhibited by TKI. TKI directed against EGF-R as well as VEGF-R inhibit tumor cell proliferation and VEGF secretion in vitro. Combinations of TKI are more effective than strategies using single agents. It is suggested that targeting EGF-R/VEGF-R-mediated pathways may have therapeutic potential in some undifferentiated thyroid cancers.