Renaissance of Radiotherapy in Intestinal Lymphoma? 10-Year Efficacy and Tolerance in Multimodal Treatment of 134 Patients: Follow-up of Two German Multicenter Consecutive Prospective Phase II Trials.

PURPOSE: This article reports on the long-term impact of radiotherapy adapted to stage, histology, and previous resection in a large cohort of patients with intestinal lymphoma (iL) treated with definitive or adjuvant curative-intent radiation therapy (RT) ± chemotherapy (CHOP, MCP, or COP). PATIENTS AND METHODS: In two consecutive prospective study designs, 134 patients with indolent (stage IE-IIE) or aggressive (stage IE-IVE) iL were referred to 61 radiotherapeutic institutions between 1992 and 2003. Patients with indolent iL received extended field (EF) 30 Gy (+10 Gy boost in definitive treatment); patients with aggressive iL received involved field (IF) (EF) 40 Gy by means of stage-, histology-, and operation-adapted radiation fields. RESULTS: The patients had median age 58 years and were predominantly male (2:1). Histology showed aggressive prevalence (1.6:1), stage IE-to-stage IIE ratio of iL 1.04:1, and localized stages-to-advanced stages ratio of aggressive lymphoma 23:1. Median follow-up was in total 11.7 years: 10.0 years in the first study, GIT (GastroIntestinal-Tract) 1992, and 11.8 years in the second study, GIT 1996. Lymphoma involvement was predominantly a single intestinal lesion (82.1%). Decrease of radiation field size from EF to IF in stage I aggressive iL from GIT 1992 to GIT 1996 resulted in a nonsignificant partial reduction of chronic toxicity while maintaining comparable survival rates (5-year overall survival 87.9 vs. 86.7%, 10-year overall survival 77.4 vs. 71.5%) with nonsignificant difference in event-free survival (5-year event-free survival 82.6 vs. 86.7%, 10-year event-free survival 69.7 vs. 71.5%) and lymphoma-specific survival (5-year lymphoma-specific survival 90.1 vs. 91.9%, 10-year lymphoma-specific survival 87.6% vs. 91.9%). Comparative dose calculation of two still available indolent duodenal lymphoma computed tomography scans revealed lower radiation exposure to normal tissues from applying current standard involved site RT (ISRT) 30 Gy in both cases. CONCLUSION: RT adapted to stage, histology, and resection in multimodal treatment of iL, despite partially decreasing field size (EF to IF), achieves excellent local tumor control and survival rates. The use of modern RT technique and target volume with ISRT offers the option of further reduction of normal tissue complication probability. IMPLICATIONS FOR PRACTICE: Although patients with intestinal lymphoma (iL) are heterogeneous according to histology and subtype, they benefit from radiotherapy. Prospective study data from 134 patients with indolent iL (stage IE-IIE) or aggressive iL (stage IE-IVE) show 100% tumor control after definitive or adjuvant curative-intent radiation therapy ± chemotherapy. Radiation treatment was applied between 1992 and 2003. Median follow-up in total was 11.7 years. No radiotherapy-associated death occurred. Relapse developed in 15.7% of the entire cohort; distant failure was more frequent than local (4:1). Normal tissue complication probability can be further improved using modern involved site radiation therapy techniques.

Prognostic factors for acute myeloid leukaemia in adults--biological significance and clinical use.

Acute myeloid leukaemia (AML) is a heterogeneous disease. Prognosis of AML is influenced both by patient-specific as well as disease-specific factors. Age is the most prominent patient-specific risk factor, while chromosomal aberrations are the strongest disease-specific risk factors. For patients with cytogenetically normal AML, prognosis can be specified by mutational status of the genes NPM1, FLT3 and CEBPA. A growing number of recurrent mutations in additional genes have recently been identified, for which the prognostic effect yet has to be determined. Performance status, geriatric assessment, secondary leukaemia following myelodysplastic syndrome or cytotoxic treatment, common laboratory parameters, leukaemic stem cell frequency, bone marrow microenvironment, gene expression levels, epigenetic changes, micro-RNA's as well as kinetics and depth of response to treatment influence prognosis of AML patients. Despite the high number of established risk factors, only few predictive markers exist which can truly aid therapy decisions in patients with AML.

Osteopontin is a prognostic factor for survival of acute myeloid leukemia patients.

Osteopontin (OPN) is a glycoprotein that is secreted by osteoblasts and hematopoietic cells. OPN suppresses the proliferation of hematopoietic stem cells in vitro and may regulate the hematopoietic stem cell pool. Increased serum OPN concentrations occur in chronic myeloid leukemia, multiple myeloma, and acute myeloid leukemia (AML). In the present study, we analyzed the prognostic impact of OPN in AML by investigating the expression and relevance of OPN in newly diagnosed AML patients from 2 large study groups (the German AML Cooperative Group and the Dutch-Belgian Hematology Oncology Cooperative group). IHC (n = 84), ELISAs of blood/BM sera (n = 41), and microarray data for mRNA levels (n = 261) were performed. Expression of OPN protein was increased in AML patients both in BM blasts (IHC) and in BM serum (ELISA) compared with healthy controls. Patients expressing high levels of OPN within the BM (IHC) experienced shortened overall survival (OS; P = .025). Multivariate analysis identified karyotype, blast clearance (day 16), and the level of OPN expression as independent prognostic factors for OS. This prompted us to analyze microarray data from 261 patients from a third cohort. The analysis confirmed OPN as a prognostic marker. In summary, high OPN mRNA expression indicated decreased event-free survival (P = .0002) and OS (P = .001). The prognostic role of OPN was most prominent in intermediate-risk AML. These data provide evidence that OPN expression is an independent prognostic factor in AML.

POEMS syndrome treated with melphalan high-dose therapy and autologous blood stem cell transplantation: a single-institution experience.

The acronym POEMS syndrome stands for a rare multi-system disorder, comprised of polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes. Here, we present a single-center report of a series of five POEMS patients treated with melphalan high-dose therapy (HDT) with subsequent autologous blood stem cell transplantation (ABSCT). After a median follow-up of 52 months from time of diagnosis (range, 15-192) and a median follow-up of 18 months after ABSCT (range, 11-120), all patients were alive. Overall, no severe transplantation-associated complications such as engraftment syndrome or peri- or post-transplant death were noted. In two cases, HDT followed by ABSCT resulted in a complete hematologic response; in the additional three cases, partial responses (PR) were achieved including one very good hematologic PR. Only one patient with initial PR developed progressive disease nearly 2.5 years after transplantation. Consequently, a second HDT with ABSCT was successfully applied resulting in clinical improvement and hematologic PR. In line with previous single-center reports, melphalan HDT followed by ABSCT proved to be a first-line treatment option with tolerable side effects in severely affected POEMS patients with progressing symptoms.

Efficacy and toxicity of a rituximab and methotrexate based regimen (GMALL B-ALL/NHL 2002 protocol) in Burkitt's and primary mediastinal large B-cell lymphoma.

There have been several attempts to improve treatment and outcome of patients with primary mediastinal B-cell lymphoma (PMBL) and Burkitt's lymphoma (BL). In recent years, chemotherapy dose intensification and the addition of rituximab have led to a remarkable progress and have developed into integral parts of treatment for both entities of lymphoma [1­4]. Here, we report our monocenter results of a high-dose methotrexate based alternating regimen with rituximab (B-ALL/NHL 2002 protocol) in 15 patients with PMBL and 28 patients with sporadic BL. Since the early 1980s, protocols of GMALL have been continuously adapted and in the meantime they have become reference treatment for BL and B-ALL in Germany. The latest changes comprised the additional use of rituximab, standardized G-CSF support,implementation of high-dose cytarabine, intrathecal triple therapy,and age-adjusted stratification. Furthermore, we additionally amended supportive care with palifermin as it reduced severity and prevalence of mucositis [5].

Targeting Tissue Factor to Tumor Vasculature to Induce Tumor Infarction.

Besides its central functional role in coagulation, TF has been described as being operational in the development of malignancies and is currently being studied as a possible therapeutic tool against cancer. One of the avenues being explored is retargeting TF or its truncated extracellular part (tTF) to the tumor vasculature to induce tumor vessel occlusion and tumor infarction. To this end, multiple structures on tumor vascular wall cells have been studied at which tTF has been aimed via antibodies, derivatives, or as bifunctional fusion protein through targeting peptides. Among these targets were vascular adhesion molecules, oncofetal variants of fibronectin, prostate-specific membrane antigens, vascular endothelial growth factor receptors and co-receptors, integrins, fibroblast activation proteins, NG2 proteoglycan, microthrombus-associated fibrin-fibronectin, and aminopeptidase N. Targeting was also attempted toward cellular membranes within an acidic milieu or toward necrotic tumor areas. tTF-NGR, targeting tTF primarily at aminopeptidase N on angiogenic endothelial cells, was the first drug candidate from this emerging class of coaguligands translated to clinical studies in cancer patients. Upon completion of a phase I study, tTF-NGR entered randomized studies in oncology to test the therapeutic impact of this novel therapeutic modality.

Polatuzumab vedotin as a salvage and bridging treatment in relapsed or refractory large B-cell lymphomas.

The antibody-drug conjugate polatuzumab vedotin (pola) has recently been approved in combination with bendamustine and rituximab (pola-BR) for patients with refractory or relapsed (r/r) large B-cell lymphoma (LBCL). To investigate the efficacy of pola-BR in a real-world setting, we retrospectively analyzed 105 patients with LBCL who were treated in 26 German centers under the national compassionate use program. Fifty-four patients received pola as a salvage treatment and 51 patients were treated with pola with the intention to bridge to chimeric antigen receptor (CAR) T-cell therapy (n = 41) or allogeneic hematopoietic cell transplantation (n = 10). Notably, patients in the salvage and bridging cohort had received a median of 3 prior treatment lines. In the salvage cohort, the best overall response rate was 48.1%. The 6-month progression-free survival and overall survival (OS) was 27.7% and 49.6%, respectively. In the bridging cohort, 51.2% of patients could be successfully bridged with pola to the intended CAR T-cell therapy. The combination of pola bridging and successful CAR T-cell therapy resulted in a 6-month OS of 77.9% calculated from pola initiation. Pola vedotin-rituximab without a chemotherapy backbone demonstrated encouraging overall response rates up to 40%, highlighting both an appropriate alternative for patients unsuitable for chemotherapy and a new treatment option for bridging before leukapheresis in patients intended for CAR T-cell therapy. Furthermore, 7 of 12 patients with previous failure of CAR T-cell therapy responded to a pola-containing regimen. These findings suggest that pola may serve as effective salvage and bridging treatment of r/r LBCL patients.

Lymphangiogenesis in cancer: current perspectives.

Although the lymphatic system has been initially described in the sixteenth century, basic research has been limited. Despite its importance for the maintenance of tissue fluid homeostasis and for the afferent immune response, research of the molecular mechanisms of lymphatic vessel formation and function has for a long time been hampered. One reason could be because of the difficulties of visibility due to the lack of lymphatic markers. But since the discovery of several molecules specifically expressed in lymphatic endothelial cells, a rediscovery of the lymphatic vasculature has taken place. New scientific insights has facilitated detailed analysis of the nature and organization of the lymphatic system in physiological and pathophysiological conditions, such as in chronic inflammation and metastatic cancer spread. Knowledge about the molecules that control lymphangiogenesis and tumor-associated lymphangiogenesis is now expanding, allowing better opportunities for the development of drugs interfering with the relevant signaling pathways. Advances in our understanding of the mechanisms have translated into a number of novel therapeutic studies.

Angiogenesis inhibition in cancer therapy: platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) and their receptors: biological functions and role in malignancy.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen in vitro and an angiogenic inducer in a variety of in vivo models. VEGF gene transcription is induced in particular in hypoxic cells. In developmental angiogenesis, the role of VEGF is demonstrated by the finding that the loss of a single VEGF allele results in defective vascularization and early embryonic lethality. Substantial evidence also implicates VEGF as a mediator of pathological angiogenesis. In situ hybridization studies demonstrate expression of VEGF mRNA in the majority of human tumors. Platelet-derived growth factor (PDGF) is mainly believed to be an important mitogen for connective tissue, and also has important roles during embryonal development. Its overexpression has been linked to different types of malignancies. Thus, it is important to understand the physiology of VEGF and PDGF and their receptors as well as their roles in malignancies in order to develop antiangiogenic strategies for the treatment of malignant disease.

S100A2 induces metastasis in non-small cell lung cancer.

PURPOSE: S100 proteins are implicated in metastasis development in several cancers. In this study, we analyzed the prognostic role of mRNA levels of all S100 proteins in early stage non-small cell lung cancer (NSCLC) patients as well as the pathogenetic of S100A2 in the development of metastasis in NSCLC. EXPERIMENTAL DESIGN: Microarray data from a large NSCLC patient cohort was analyzed for the prognostic role of S100 proteins for survival in surgically resected NSCLC. Metastatic potential of the S100A2 gene was analyzed in vitro and in a lung cancer mouse model in vivo. Overexpression and RNAi approaches were used for analysis of the biological functions of S100A2. RESULTS: High mRNA expression levels of several S100 proteins and especially S100A2 were associated with poor survival in surgically resected NSCLC patients. Upon stable transfection into NSCLC cell lines, S100A2 did not alter proliferation. However, S100A2 enhanced transwell migration as well as transendothelial migration in vitro. NOD/SCID mice injected s.c. with NSCLC cells overexpressing S100A2 developed significantly more distant metastasis (64%) than mice with control vector transfected tumor cells (17%; P < 0.05). When mice with S100A2 expressing tumors were treated i.v. with shRNA against S100A2, these mice developed significantly fewer lung metastasis than mice treated with control shRNA (P = 0.021). CONCLUSIONS: These findings identify S100A2 as a strong metastasis inducer in vivo. S100A2 might be a potential biomarker as well as a novel therapeutic target in NSCLC metastasis.

Tumor immune escape by the loss of homeostatic chemokine expression.

The novel keratinocyte-specific chemokine CCL27 plays a critical role in the organization of skin-associated immune responses by regulating T cell homing under homeostatic and inflammatory conditions. Here we demonstrate that human keratinocyte-derived skin tumors may evade T cell-mediated antitumor immune responses by down-regulating the expression of CCL27 through the activation of epidermal growth factor receptor (EGFR)-Ras-MAPK-signaling pathways. Compared with healthy skin, CCL27 mRNA and protein expression was progressively lost in transformed keratinocytes of actinic keratoses and basal and squamous cell carcinomas. In vivo, precancerous skin lesions as well as cutaneous carcinomas showed significantly elevated levels of phosphorylated ERK compared with normal skin, suggesting the activation of EGFR-Ras signaling pathways in keratinocyte-derived malignancies. In vitro, exogenous stimulation of the EGFR-Ras signaling pathway through EGF or transfection of the dominant-active form of the Ras oncogene (H-RasV12) suppressed whereas an EGFR tyrosine kinase inhibitor increased CCL27 mRNA and protein production in keratinocytes. In mice, neutralization of CCL27 led to decreased leukocyte recruitment to cutaneous tumor sites and significantly enhanced primary tumor growth. Collectively, our data identify a mechanism of skin tumors to evade host antitumor immune responses.

Combined anti-PDGFRalpha and PDGFRbeta targeting in non-small cell lung cancer.

Activation of the platelet-derived growth factor (PDGF)-receptors is critically involved into various stromal cell functions including recruitment of stromal cells and vascular endothelial growth factor (VEGF) induction in tumor and perivascular cells. To evaluate the effects of combined PDGFRalpha and -beta inhibition in a non-small cell lung cancer model, we stably transfected A549 lung cancer cells with the PDGF-A mutant PDGF-0. PDGF-0 has been generated by substituting amino acids in the binding region of PDGF-A with the corresponding VEGF-A region, leading to a decreased receptor-binding affinity and activation. Compared with control vector transfected cells, transfection with PDGF-0 had no impact on monolayer growth and apoptosis in vitro, but significantly impaired the number of colony formation in soft agar. After subcutaneous injections, all mice developed tumors within 5 days. While control vector transfected A549 cells were characterized by constant tumor growth, PDGF-0 transfected A549 revealed a reduced tumor mass (p < 0.001) with no further growth beyond 14 days (2 months observation time) and complete regressions in 7 of 13 cases. Immunohistochemical analyses revealed that PDGF-0 transfected tumors demonstrated decreased recruitment of periendothelial cells, while the tumor invasion zone was similar to control vector transfectants. Similarly, conditioned medium from PDGF-0 transfected cells induced significantly less migration of smooth muscle cells and fibroblasts in vitro. Interestingly, in PDGF-0 transfectants, neither total vessel count nor VEGF expression were significantly altered. These studies demonstrate that combined inhibition of PDGFRalpha and -beta results in markedly decreased tumor growth in vivo because of impaired recruitment of periendothelial cells.

Six versus eight cycles of bi-weekly CHOP-14 with or without rituximab in elderly patients with aggressive CD20+ B-cell lymphomas: a randomised controlled trial (RICOVER-60).

BACKGROUND: Cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP) is used to treat patients with non-Hodgkin lymphoma. Interval decrease from 3 weeks of treatment (CHOP-21) to 2 weeks (CHOP-14), and addition of rituximab to CHOP-21 (R-CHOP-21) has been shown to improve outcome in elderly patients with diffuse large B-cell lymphoma (DLBCL). This randomised trial assessed whether six or eight cycles of R-CHOP-14 can improve outcome of these patients compared with six or eight cycles of CHOP-14. METHODS: 1222 elderly patients (aged 61-80 years) were randomly assigned to six or eight cycles of CHOP-14 with or without rituximab. Radiotherapy was planned to sites of initial bulky disease with or without extranodal involvement. The primary endpoint was event-free survival; secondary endpoints were response, progression during treatment, progression-free survival, overall survival, and frequency of toxic effects. Analyses were done by intention to treat. The trial is registered on National Cancer Institute website, number NCT00052936 and as EU-20243. FINDINGS: 3-year event-free survival was 47.2% after six cycles of CHOP-14 (95% CI 41.2-53.3), 53.0% (47.0-59.1) after eight cycles of CHOP-14, 66.5% (60.9-72.0) after six cycles of R-CHOP-14, and 63.1% (57.4-68.8) after eight cycles of R-CHOP-14. Compared with six cycles of CHOP-14, the improvement in 3-year event-free survival was 5.8% (-2.8-14.4) for eight cycles of CHOP-14, 19.3% (11.1-27.5) for six cycles of R-CHOP-14, and 15.9% (7.6-24.2) for eight cycles of R-CHOP-14. 3-year overall survival was 67.7% (62.0-73.5) for six cycles of CHOP-14, 66.0% (60.1-71.9) for eight cycles of CHOP-14, 78.1% (73.2-83.0) for six cycles of R-CHOP-14, and 72.5% (67.1-77.9) for eight cycles of R-CHOP-14. Compared with treatment with six cycles of CHOP-14, overall survival improved by -1.7% (-10.0-6.6) after eight cycles of CHOP-14, 10.4% (2.8-18.0) after six cycles of R-CHOP-14, and 4.8% (-3.1-12.7) after eight cycles of R-CHOP-14. In a multivariate analysis that used six cycles of CHOP-14 without rituximab as the reference, and adjusting for known prognostic factors, all three intensified regimens improved 3-year event-free survival (eight cycles of CHOP-14: RR [relative risk] 0.76 [0.60-0.95], p=0.0172; six cycles of R-CHOP-14: RR 0.51 [0.40-0.65], p<0.0001; eight cycles of R-CHOP-14: RR 0.54 [0.43-0.69], p<0.0001). Progression-free survival improved after six cycles of R-CHOP-14 (RR 0.50 [0.38-0.67], p<0.0001), and eight cycles of R-CHOP-14 (RR 0.59 [0.45-0.77], p=0.0001). Overall survival improved only after six cycles of R-CHOP-14 (RR 0.63 [0.46-0.85], p=0.0031). In patients with a partial response after four cycles of chemotherapy, eight cycles were not better than six cycles. INTERPRETATION: Six cycles of R-CHOP-14 significantly improved event-free, progression-free, and overall survival over six cycles of CHOP-14 treatment. Response-adapted addition of chemotherapy beyond six cycles, though widely practiced, is not justified. Of the four regimens assessed in this study, six cycles of R-CHOP-14 is the preferred treatment for elderly patients, with which other approaches should be compared.

Expression of VEGF-C and its receptor VEGFR-3 in the bone marrow of patients with acute myeloid leukaemia.

Vascular endothelial growth factor-C (VEGF-C) has been shown to promote survival and resistance to chemotherapy of AML-cells in vitro. We investigated the expression of VEGF-C/VEGFR-3 in the bone marrow and pretherapeutic plasma levels of VEGF-C in patients with newly diagnosed AML. Expression of VEGF-C/VEGFR-3 was significantly higher in AML patients than in controls, while circulating levels did not differ. However, VEGF-C/VEGFR-3 expression was not able to predict clinical outcome. In conclusion, AML is associated with an increased expression of VEGF-C/VEGFR-3. Although expression levels display no prognostic significance in our study, strategies targeting the VEGF-C/VEGFR-3-pathway might be a promising treatment approach.

Generation of fusion proteins for selective occlusion of tumor vessels.

Selective activation of blood coagulation in tumor vessels with subsequent thrombosis and tumor infarction is a promising strategy in cancer therapy. To this end, different fusion proteins consisting of the extracellular domain of tissue factor (truncated tissue factor, tTF) were fused to the peptides GRGDSP (abbr. RGD), GNGRAHA (abbr. NGR) or cyclic derivates of these peptides, which selectively target alpha(v)-integrins or aminopeptidase N (CD13), respectively. Rationale for this strategy is the fact that these surface receptors are preferentially expressed on tumor endothelial cells. The tTF constructs were expressed in Escherichia coli BL21 (DE3). The integrity of the fusion proteins was evaluated by SDS-PAGE, immunoblotting and mass spectrometry. The screening process for the activity contained coagulation assays as well as purified receptor binding assays. The fusion proteins which retained their thrombogenic and binding activity were evaluated further. In vivo studies in nude mice bearing established different malignant human tumors revealed that i.v. administration of tTF-RGD or tTF-NGR induced partial or complete thrombotic occlusion of tumor vessels, which was demonstrated by histological analysis. Furthermore, treatment studies showed that the targeted tTF fusion proteins but not untargeted tTF proteins induced significant tumor growth retardation in human adenocarcinoma of the breast in a nude mice model without apparent side effects such as thrombosis in liver, kidney, heart or lung at therapeutic dose levels. Finally, we illustrate the upscaling process of fusion protein fabrication in order to produce the amounts needed for clinical studies. Thus, generation and screening of active fusion proteins, which induce selective thrombosis in the tumor vasculature, may be a promising strategy for the development of new drugs as cancer therapeutics.

Lymphangiogenesis in development and disease.

The lymphatic vascular system plays an important role in the maintenance of fluid homeostasis, in the afferent immune response, in the intestinal lipid uptake and in the metastatic spread of malignant cells. The recent discovery of specific markers and growth factors for lymphatic endothelium and the establishment of genetic mouse models with impairment of lymphatic function have provided novel insights into the molecular control of the lymphatic system in physiology and in embryonic development. They have also identified molecular pathways whose mutational inactivation leads to human diseases associated with lymphedema. Moreover, the lymphatic system plays a major role in chronic inflammatory diseases and in transplant rejection. Importantly, malignant tumors can directly promote lymphangiogenesis within the primary tumor and in draining lymph nodes, leading to enhanced cancer metastasis to lymph nodes and beyond. Based upon these findings, novel therapeutic strategies are currently being developed that aim at inhibiting or promoting the formation and function of lymphatic vessels in disease.

Anticancer therapy by tumor vessel infarction with polyethylene glycol conjugated retargeted tissue factor.

tTF-NGR consists of the extracellular domain of tissue factor and the peptide GNGRAHA, a ligand of the surface protein aminopeptidase N and of integrin αvß3. Both surface proteins are upregulated on endothelial cells of tumor vessels. tTF-NGR shows antitumor activity in xenografts and inhibition of tumor blood flow in cancer patients. We performed random TMS(PEG)12 PEGylation of tTF-NGR to improve the antitumor profile of the molecule. PEGylation resulted in an approximately 2-log step decreased procoagulatory activity of the molecule. Pharmacokinetic studies in mice showed a more than 1-log step higher mean area under the curve. Comparison of the LD10 values for both compounds and their lowest effective antitumor dose against human tumor xenografts showed an improved therapeutic range (active/toxic dose in mg/kg body weight) of 1/5 mg/kg for tTF-NGR and 3/>160 mg/kg for TMS(PEG)12 tTF-NGR. Results demonstrate that PEGylation can significantly improve the therapeutic range of tTF-NGR.

The miR-290-295 cluster suppresses autophagic cell death of melanoma cells.

We compared the expression levels of 307 miRNAs in six different B16F1 melanoma cell lines of differing malignant properties and found that the miR-290-295 cluster showed a strong upregulation in the more malignant B16F1 daughter cell lines. Its overexpression in B16F1 cells had no major effects on cell proliferation, migration or anchorage-independent growth, but conferred resistance to glucose starvation. This was mediated by miR-290-295-induced downregulation of several essential autophagy genes, including Atg7 and ULK1, which resulted in inhibition of autophagic cell death induced by glucose starvation. Similar effects were observed after knockdown of Atg7 or ULK1 in B16F1 melanoma cells, and after treatment with two chemical inhibitors of autophagy. Together, these results indicate that autophagy mediates cell death of melanoma cells under chronic nutrient deprivation, and they reveal an unanticipated role of the miR-290-295 cluster in conferring a survival advantage to melanoma cells by inhibiting autophagic cell death.

Induced lymphatic sinus hyperplasia in sentinel lymph nodes by VEGF-C as the earliest premetastatic indicator.

Research on tumor-induced lymphangiogenesis has predominantly focused on alterations and abnormal growth of peritumoral and intratumoral lymphatic vessels. However, recent evidence indicates that lymphangiogenesis of sentinel lymph nodes might also contribute to cancer progression. In clinical oncology, the sentinel lymph nodes play an important role in diagnosis, staging and management of disease. The prognostic value that may be placed in the analysis of various parameters in tumor-free lymph nodes is still under debate. We, therefore, chose to investigate genetically fluorescent MDA-MB-435/green fluorescent protein human cancer cells transfected to overexpress VEGF-C in a nude mouse model and investigated metastasis, lymph node lymphangiogenesis, lymph node angiogenesis and size of sentinel lymph nodes. The nature of MDA-MB-435, identified as a breast cancer cell line for several decades, has recently been reidentified as being from melanoma origin. Vascular endothelial growth factor-C overexpression induced early metastasis and significantly increased the lymphatic vessel area in sentinel lymph nodes even before the tumor metastasis. At early time-points, expansion of the lymphatic network was observed even though no difference of blood vessel area and lymph node size was detected. These results suggest that primary tumors -via secretion of VEGF-C- can induce hyperplasia of the sentinel lymph node lymphatic vessel network and thereby promote their further spread. In cases of tumor-free lymph nodes the increased lymphatic network of sentinel lymph nodes is a very early premetastatic sign and may provide a new prognostic indicator and target for aggressive diseases.

Analysis of a novel highly metastatic melanoma cell line identifies osteopontin as a new lymphangiogenic factor.

Tumor cell invasion and metastasis are hallmarks of malignancy. Despite recent advances in the understanding of lymphatic spread, the mechanisms by which tumors metastasize to sentinel/distant lymph nodes and beyond are poorly understood. To gain new insights into this complex process, we established highly metastatic melanoma cell lines by in vivo passaging the B16 parental cell line through the lymphatic system. In this study we characterized morphology, rate of cell proliferation, colony formation, migration, tumorigenicity, lymph flow, and capacities to induce tumor- and sentinel lymph node-lymphangiogenesis. Furthermore, microarray-based comparative analysis between parental and passaged cell lines was performed to identify specific gene expression profiles. The most differentially expressed gene was SPP (osteopontin), a secreted glycophosphoprotein which is known to be involved in cancer metastasis. Overexpression of osteopontin in B16 F1-variant was confirmed by western blot analysis and quantitative RT-PCR. Treatment of cultured lymphatic endothelial cells (LECs) with osteopontin promoted cell migration mediated by the integrin α9 pathway. Our results identify osteopontin as a novel lymphangiogenic factor.