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4043 Background: FOLFOX plus nivolumab (nivo) has become standard of care for first-line therapy of patients (pts) with esophagogastric adenocarcinomas. The aim of Moonlight trial is to generate signals for whether (a) dual checkpoint inhibition or (b) a triplet chemotherapy is beneficial in the context of nivolumab therapy in this setting. Methods: The AIO-STO-0417 trial (Moonlight) is a four-arm investigator-initiated trial. Pts were randomized to FOLFOX alone (Arm B) or FOLFOX plus nivo 240 mg; q2w and ipilimumab (ipi) 1 mg/kg; q6w administered in parallel (Arm A/A1) or sequentially (Arm A2) or pts were treated in a non-randomized fashion with FLOT plus nivo 240 mg; q2w (Arm C). PD-L1 expression was centrally assessed. Primary endpoint is progression-free survival (PFS). Results: The study completed enrollment with 260 pts. Arms A1/A2 and C started later and will be analyzed in Mar 2022 and presented at the meeting. The abstract, therefore, focuses on Arms A (n = 60) vs B (n = 60). Baseline characteristics were: median age 62.5y, GEJ, 55%, intestinal type, 36%. Forty-one percent had PD-L1 CPS≥1 (available in 79% of pts). Pts received a median of 10 and 9 cycles Arms A and B. Adverse events of grade 3/4 were seen in 86% for Arm A and 60% for Arm B, respectively, and serious adverse events (SAE) in 78% in Arm A and 50% in Arm B. Median follow-up was 9.7 mo. No difference in PFS (5.7 and 6.6 mo), OS (10 vs. 12 mo) or objective response rate (45% and 48%) was seen in Arms A and B, respectively. The results were similar in the PD-L1+ group. Conclusions: FOLFOX plus dual checkpoint inhibition administered in parallel is associated with an increase in toxicity but not activity. This portion of the moonlight trial does not generate a signal for further trials on FOLFOX plus nivo and ipi for adenocarcinoma of stomach and GEJ. Clinical trial information: NCT03647969.

Purpose To evaluate the association of germ-line polymorphisms of genes that may impact treatment outcome of platinum and fluorouracil combination chemotherapy in advanced gastric cancer (AGC). Patients and Methods Blood samples of 156 patients enrolled onto a phase III study comparing fluorouracil, leucovorin, and oxaliplatin with fluorouracil, leucovorin, and cisplatin were collected. Polymorphisms within genes of TS, MTHFR, MTR, OPRT, XPD, ERCC1, XRCC1, XPA, GSTP1, GSTT1, and GSTM1 were genotyped using polymerase chain reaction–based techniques. Results Median overall survival (OS) was 11.8 months (95% CI, 9.75 to 13.79 months) and median progression-free survival (PFS) was 5.8 months (95% CI, 4.99 to 6.61 months). The TS-3R/+6 haplotype (P = .004), the GSTT1 deletion polymorphism (P = .015), and genotypes of OPRT-Gly213Ala (P = .003) and XRCC1-Arg399Gln (P = .023) could be identified as independent predictors of OS. For PFS analyses, the TS-3R/+6 haplotye (P = .003) and MTR-A2756G (P = .01) were identified as independent positive predictors. The association between the GSTT1 deletion polymorphism and PFS showed only borderline significance (P = .053). Treatment related hematotoxicity in terms of grade 3/4 leukopenia was lowest among TS-3R/+6 haplotype carriers (P = .037). Grade 3/4 neutropenia was directly associated with the MTR-2756G/G genotype (P = .011), GSTP1-105Ile/Ile genotype (P = .02), and with the ERCC1-118T/8092C-haplotype (P = .042). In addition, significant associations between GSTP1-105Ile/Ile genotype and neurotoxicity and between the XPD-Asn312/751Gln haplotype and nephrotoxicity could be identified (P = .028 and P = .005, respectively). Conclusion These findings underline the hypothesis that germ-line polymorphisms may play an important role in individualizing chemotherapy in AGC and deserve further prospective evaluation in AGC patients.

Purpose This randomized study assessed whether the best overall response rate (ORR) of cetuximab combined with oxaliplatin, leucovorin, and fluorouracil (FOLFOX-4) was superior to that of FOLFOX-4 alone as first-line treatment for metastatic colorectal cancer. The influence of KRAS mutation status was investigated. Patients and Methods Patients received cetuximab (400 mg/m2 initial dose followed by 250 mg/m2/wk thereafter) plus FOLFOX-4 (oxaliplatin 85 mg/m2 on day 1, plus leucovorin 200 mg/m2 and fluorouracil as a 400 mg/m2 bolus followed by a 600 mg/m2 infusion during 22 hours on days 1 and 2; n = 169) or FOLFOX-4 alone (n = 168). Treatment was continued until disease progression or unacceptable toxicity. KRAS mutation status was assessed in the subset of patients with assessable tumor samples (n = 233). Results The confirmed ORR for cetuximab plus FOLFOX-4 was higher than with FOLFOX-4 alone (46% v 36%). A statistically significant increase in the odds for a response with the addition of cetuximab to FOLFOX-4 could not be established (odds ratio = 1.52; P = .064). In patients with KRAS wild-type tumors, the addition of cetuximab to FOLFOX-4 was associated with a clinically significant increased chance of response (ORR = 61% v 37%; odds ratio = 2.54; P = .011) and a lower risk of disease progression (hazard ratio = 0.57; P = .0163) compared with FOLFOX-4 alone. Cetuximab plus FOLFOX-4 was generally well tolerated. Conclusion KRAS mutational status was shown to be a highly predictive selection criterion in relation to the treatment decision regarding the addition of cetuximab to FOLFOX-4 for previously untreated patients with metastatic colorectal cancer.

Purpose This study was designed to compare fluorouracil, leucovorin, and oxaliplatin with fluorouracil, leucovorin, and cisplatin in patients with advanced gastric cancer. Patients and Methods Patients with previously untreated advanced adenocarcinoma of the stomach or esophagogastric junction were randomly assigned to receive either fluorouracil 2,600 mg/m2 via 24-hour infusion, leucovorin 200 mg/m2, and oxaliplatin 85 mg/m2 (FLO) every 2 weeks or fluorouracil 2,000 mg/m2 via 24-hour infusion, leucovorin 200 mg/m2 weekly, and cisplatin 50 mg/m2 every 2 weeks (FLP). The primary end point was progression-free survival (PFS). Results Two hundred twenty patients (median age, 64 years; metastatic, 94%) were randomly assigned. FLO was associated with significantly less (any grade) anemia (54% v 72%), nausea (53% v 70%), vomiting (31% v 52%), alopecia (22% v 39%), fatigue (19% v 34%), renal toxicity (11% v 34%), thromboembolic events (0.9% v 7.8%), and serious adverse events related to the treatment (9% v 19%). FLP was associated with significantly less peripheral neuropathy (22% v 63%). There was a trend toward improved median PFS with FLO versus FLP (5.8 v 3.9 months, respectively; P = .077) and no significant difference in median overall survival (10.7 v 8.8 months, respectively). However, in patients older than 65 years (n = 94), treatment with FLO resulted in significantly superior response rates (41.3% v 16.7%; P = .012), time to treatment failure (5.4 v 2.3 months; P < .001), and PFS (6.0 v 3.1 month; P = .029) and an improved OS (13.9 v 7.2 months) as compared with FLP, respectively. Conclusion FLO reduced toxicity as compared with FLP. In older adult patients, FLO also seemed to be associated with improved efficacy.

e21527 Background: HEH is a rare tumor of the liver with an unpredictable malignant potential. Surgical resection or liver transplantation is recommended in locally advanced disease and has been successfully performed in selected cases with extrahepatic manifestations. Systemic therapy, however, is not standardized. Small cohorts have been treated with interferon, chemotherapy and angiogenesis inhibitors such as thalidomide with various results. Case report: A 33-year old caucasian previously healthy male was admitted to hospital with newly diagnosed suspicious lesions in liver and spleen. Further analyses showed additional pulmonal nodules. Biopsy revealed a tumor with predominant epithelioid cells, positive for CD31 and CD34 and negative for CD117, HHV8, AFP and CEA, classified as a HEH. Disease progressed during first line treatment with adriamycin and ifosfamide (2 cycles given). Based on a case report with encouraging results with thalidomide we treated our patient with lenalidomide, which is known to have less side effects while being more effective in the myeloma setting. After 6 courses of lenalidomide 25mg daily (21/28), regression of the splenic lesions was detected. Retrospective analysis after 9 cycles demonstrated stable disease in comparison to the recent investigation, but an overall progression of 22% according to RECIST criteria in the liver was observed. Due to the excellent tolerance we increased the daily dose to 30 mg (21/28) and 6 months later a slight regression in the lung and overall stable disease in the liver was observed. The splenic lesion disappeared completely. The patient was listed for liver transplantation and after another 4 months on lenalidomide 30 mg, he was successfully transplanted recently. Because of the remaining extrahepatic lesions it is planned to continue lenalidomide therapy in combination with the immunosuppressant rapamycine. Conclusions: We report the first case of successful therapy with lenalidomide in HEH. Higher doses than 25mg seem to be more effective. We grateful thank Celgene for offering us lenalidomide for this off label use. No significant financial relationships to disclose.

18105 Background: An emerging notion for the treatment of advanced solid malignancies in adults is to manage it as a chronic disease and aim to keep tumor burden and associated symptoms at the lowest possible level. One of the most promising strategies to achieve this is to target the tumor vasculature using modified, “metronomic”, dosing schedules. We conducted a pilot study of weekly docetaxel in combination with daily trofosfamide as a metronomic second-line regimen in patients (pts) with metastatic non-small cell lung cancer (NSCLC). Methods: From March 2003 to November 2005, 21 pts with stage IV disease who had one prior chemotherapy were enrolled. Pts received docetaxel 25 mg/m2 on days 1, 8 and 15, every 4 weeks plus trofosfamide 50mg per day. Results: Median age: 60 years; gender: 18 male (85.7%), 3 female (14.3%); median Karnofsky PS: 80%; histologic subtypes: 8 squamous cell carcinomas (38.1%), 7 adenocarcinomas (33.3%), 6 large cell carcinomas (28.6%); previous chemotherapy: platinum-based in 15 pts (71.4%), platinum-free in 6 pts (28.6%). A total of 62 chemotherapy cycles were administered. The median number of cycles per pt was 3 (range 1 - 8). The intent-to-treat overall response rate was 19% (95% CI 5.5 - 41.9) 1 CR (4.8%), 2 PR (14.3%), 9 SD (42.9%), 8 PD (38.1%). The median overall survival from onset of second-line therapy was 6.9 months (95% CI 5.0 - 13.9 months), the median progression-free survival 2.9 months (95% CI 1.58 - 6.28 months), the 1-year survival rate 28.6% (95% CI 14.5 - 56.2%), and the 2-year survival rate 7.1% (95% CI 1.26 - 40.6%). Hematological and non-hematological toxicities were mild. No grade 4 toxicity was observed. Conclusions: To the best of our knowledge, this is the first trial that combines docetaxel and trofosfamide for the treatment of NSCLC. Our results suggest that the concept of metronomic chemotherapy is a valuable addition to the treatment of NSCLC. The combination of docetaxel and trofosfamide is active and well tolerated as second-line therapy in pts with metastatic NSCLC. No significant financial relationships to disclose.

15172 Background: Cholangiocarcinoma or gallbladder cancer are often diagnosed at an advanced stage with limited treatment options. Methods: Between 1994 and 2004, 94 patients (pts) (47 male, 47 female) with advanced biliary tract cancer were treated at the Department of Oncology and Hematology, University Hospital Hamburg-Eppendorf. Clinical and histopathological characteristics, response to chemotherapy, and survival were investigated in a retrospective analysis. Median age was 59 years (range 30–80) and median Karnofsky performance status was 90%. Predominant histologic type was adenocarcinoma (94.7%). Primary tumor sites were extrahepatic bile duct (29.9%), gallbladder (28.7%), intrahepatic bile duct (10.6%), ampulla of Vater (2.1%), not specified (28.7%). Predominant localizations of metastases were liver (73 pts (77.7%)), lymph nodes (49 pts (52.1%)) and the peritoneum (14 pts (14.9%)). 33 pts (35.1%) underwent surgery of the primary tumor at time of diagnosis. Results: 72 of 94 pts (76.6%) received a first-line chemotherapy, all together 10 different chemotherapy regimens were used. The median number of cycles was 2.5 (range 1 - 12). A single agent chemotherapy with gemcitabine was the most often adminstered regimen (23 pts (31.9%)), followed by carboplatin and etoposide plus whole body hyperthermia (12 pts (16.7%)) and 5- fluorouracil and folic acid (10 pts (13.9%)). The overall response rate was 8.3% (95% CI 3.1 - 17.3) (34.7% SD, 47.2% PD, 9.7% not evaluable). Second-line chemotherapy was given in 27 patients, which induced no tumor response, but a stable disease rate of 22.2%. Median time to follow- up was 44.8 months. Survival was calculated for all 94 pts since time of diagnosis. Median overall survival was 12.2 months and median progression-free survival 9.2 months. The median overall survival time for the 72 pts who were treated with chemotherapy was 14.0 months, and for the 22 pts who did not receive chemotherapy 10.7 months (p=0.2). Conclusions: Our analysis showed a poor prognosis for patients with advanced biliary tract cancer. Response rate to chemotherapy was low. Therefore, well tolerated cytotoxic agents should be used and new treatment strategies (including molecular targeted therapy) should be further investigated. No significant financial relationships to disclose.

10531 Background: Src-family tyrosine kinases are known to be involved in signal transduction pathways of growth factors and cytokines in hematopoetic cells. While the role of other src family members has been studied widely, only few data exist about c-src in leukemia. The actual study was performed to analyze src mutations in leukemic cells and to determine the role of pp60src in leukemic cell proliferation. Methods: AML cell lines and primary samples were analyzed for expression and activation of src by RT-PCR and Western blot analyses. Mutational analyses were performed by sequencing of C-terminal cDNA from 60 AML samples. The effects of src inhibition were studied by src-specific inhibitors PP1 and PP2 or by siRNA transfection. Effetcs of src inhibition were monitored in proliferation assays and analyzes of signalling through Erk1/2 and apoptosis by annexin V staining and DNA laddering. Results: In all 60 patients analyzed expression of c-src mRNA was detected by RT-PCR. Western blot analyses confirmed strong expression of src on the protein level and revealed a robust activation of the protein as determined by tyrosine phosphorylation. Incubation of leukemic cells with PP1 and PP2 caused significant inhibition of proliferation in a dose dependent manner. Similar results were observed after transfection with specific siRNAs. Src inhibition blocked phosphorylation of pp60src, Erk1/2 and induced apoptosis in leukemic cells. Mutational analyses as performed by SSCP/heteroduplex and bi-directionally sequencing revealed wildtype sequence in cell lines and 60 clinical samples. Conclusions: In summary, pp60src is highly expressed and activated in cell lines and clinical samples of human AML. Moreover, phosphorylation of src is essential for leukemic cell proliferation. Underlying mutations in the coding sequence of c-src causing constitutive activation could be excluded. These data suggest that pp60src plays a crucial role in AML and src inhibition by targeted therapy might offer a useful new approach in the treatment of AML. No significant financial relationships to disclose.

15648 Background: Combinations of cytotoxic drugs lead to increased activity and minimize resistance compared to single agents in tumor therapy. Similarly, antiangiogenic treatment could be improved by combinations targeting different pathways. We investigated a combination of endogenous inhibitors using endostatin (ES), soluble Neuropilin-1 (sNP-1), and thrombospondin-2 (TSP-2) in a model of renal cell carcinoma. Methods: Porcine aortic endothelial cells have been engineered for stable production of angiogenic inhibitors by lipofection and were encapsulated in sodium alginate microbeads. Proliferation of human umbilical vein endothelial cells or Renca renal carcinoma cells was examined after incubation with different microbeads. Similarly, effects of inhibitors on endothelial cell function were tested in tube formation and in vitro wound assays. Microbeads were implanted into SCID mice with subcutaneously growing tumors derived from Renca cells or in mice developing lung metastases after intravenous injection of tumor cells. Results: Factors released from microbeads inhibited endothelial cell function but had no effect on tumor cell proliferation in vitro. In vivo, subcutaneous tumor growth was inhibited similarly by each angiogenic inhibitor alone. After 30 days mean tumor weight was 1.3 g in controls and 0.17, 0.18, 0.18g in ES, sNP-1, and TSP-2 treated mice, respectively. Tumor weight in mice treated with all three inhibitors was further reduced to 0.03g. Histological analyses confirmed antiangiogenic activity by inhibition of microvessel density in treated tumors. In a metastastic model treatment with angiogenic inhibitors induced a significant reduction in size and number of lung metastases with additive effects when factors were used in combination. Conclusions: We conclude that combination therapy targeting multiple angiogenic pathways has synergistic activity and could help to avoid resistance to single inhibitors in tumor treatment. No significant financial relationships to disclose.

20010 Background: Bone marrow derived endothelial progenitor cells (ECPs) contribute to neoangiogenesis in cancer patients. Elevated numbers of circulating endothelial cells and EPCs can be detected in the peripheral blood of tumor patients. Recently, proliferating lymphatic vessels have been described in human cancers such as melanoma and head and neck tumors. Therefore, we investigated whether circulating lymphatic endothelial cells can be detected in cancer patients. Methods: We developed a sensitive immunocytochemical approach using a monoclonal antibody against the lymphendothelial specific hyaluronic receptor LYVE. After enrichment by Ficoll density gradient centrifugation, 7 × 10(5) peripheral blood mononuclear cells (PBMNCs) from 23 patients with metastatic cancer (6 gastrointestinal, 4 lung, 3 thymus, 3 thyroid, 1 mamma, 1 pancreas, 2 renal, 1 adrenal, 2 urothelial cancer, 1 PNET, 1 NET) and healthy individuals (n = 7) were spun onto glass slides. Two million PBMNs from each patient were stained for LYVE using the APAP technique. Isotype antibodies were used as controls. Cytospins were analyzed with the automated cellular imaging system (ACIS; ChromaVision Medical Systems). The method was validated with spiked blood samples. Results: Circulating LYVE+ lymphatic endothelial cells could be detected in 4 of 7 healthy subjects (57%) and in 16 of 23 patients (69%) with metastatic cancer. The mean number of lymphendothelial cells was significantly higher in cancer patients (15 cells/1 × 10(6) PBMNCs [range 0–276] vs. 1.0 cells/1 × 10(6) PBMNCs [range 0–2]). As a control, circulating tumor cells were enumerated after staining with a cytokeratin antibody (A45-B/B3). Circulating tumor cells could not be detected in healthy controls but in 13 of 23 of cancer patients (mean 1.4 cells/1 × 10(6) PBMNCs range [0–9]). Conclusion: Circulating lymphendothelial cells can be detected in patients with metastatic cancer and healthy subjects. Tumor patients have higher levels of circulating lymphendothelial cells than normal controls. These cells may participate in the generation of lymphatic vessels within tumor manifestations. No significant financial relationships to disclose.

7199 Background: Magic Roundabout (MR; ROBO 4) is a recently discovered gene which encodes for a protein that possess structural homology with the roundabout family of proteins. Genes of the rounda...